Chapter One

INTRODUCTION
Foodborne diseases constitute a budding problem triggering significant burden of

disability and mortality in most countries. Recent assessments indicate that the annual

global burden of foodborne disease is >600 million cases of illness, with almost 420,000

deaths (WHO, 2015). Africa and South East Asia have the highest occurrence of

foodborne diseases and the highest death rates among all ages (Havelaar, 2016). A tainted

food, regardless of the level of hazards, poses health threats to consumers and economic

burdens on communities and nations (Mensah et al., 2012).

Food poisoning and diarrhoea caused by foods contaminated by Citrobacter have

been reported (Doulgeraki et al., 2011). Currently, the incidence of shigellosis worldwide

is highest among children less than five years of age (Taneja and Mewara, 2016).

Escherichia coli is one of the major foodborne pathogens of foods of animal origin with

wide variability of virulence (Kobayashi et al., 2002; Johnson et al., 2005). Aeromonas

have been connected with several foodborne outbreaks and are increasingly being isolated

from patients with traveler's diarrhoea (Von Graevenitz, 2007). The true burden of

illnesses caused by Bacillus cereus is unknown probably because they commonly occur as

irregular cases, rather than in major outbreaks (Logan et al., 2011).

Foodborne infection is endemic in Nigeria with more than one million cases per

annum according to the Integrated Disease Surveillance and Response (IDSR), and most

cases are not reported (FMoH, 2014). It has been estimated that more than 200,000 deaths

occur per annum from foodborne pathogens caused by contaminated foods through

improper processing, preservation and service in Nigeria (WHO, 2009a).

1
 Regrettably, all food groups add to the burden of foodborne diseases. Meat, eggs

and seafoods cause the highest burden (Havelaar, 2016). Most people are inadvertently

exposed to microbial hazards from several sources (Okafor et al., 2017; Amini et al.,

2012), which can cause diseases that go unreported, because their symptoms are mistaken

for other illnesses such as malaria (Ajayi and Salaudeen, 2014). Mild gastroenteritis has

been reported to be common among people that consume snails regularly (Serrano et al.,

2004).

Achatina achatina is a terrestrial gastropod of the family ―Achatinidae‖ which has

been listed among edible snails (European Commission, 2004). In Africa, the edible giant

snails belong to two genera: Achatina and Archachatina. The most common species in

West Africa is Achatina achatina (Hodasi, 1984). Snail meat (also called Congo meat) is

high in protein, low in fat and a source of iron, magnesium, calcium, and zinc. The

nutritive value of snail meat is comparable to that of domestic livestock (Ademolu et al.,

2004; Malik et al., 2011; Babalola and Akinsoyinu, 2009). By the content of histidine,

glutamic acid, aspartic acid and threonine, snail meat is ahead of chicken, beef and fish

(Sando et al., 2013). It is an ideal meat for diabetics and those with vascular diseases such

as heart attack, cardiac arrest, hypertension and stroke (Fumilayo, 2008). In 2010 and

2011, world snail market recorded a turnover of 10 billion Euros per year with

consumption of about 400,000 tons of snails, of which only 50,000 tons were produced in

snail farms (Toader, 2012). There is a growing interest in heliciculture, i.e. snail rearing

for meat and sale, in Nigeria (Omole, 2001).

The identification of potential hazards and determining which hazards need to be

controlled is known as hazard analysis (Reij and van Schothorst, 2000). In this approach,

the presence of a potentially harmful agent at a detectable level in food is used as a basis

2
for legislation and/or risk management action (Barlow et al., 2015). In hazard

identification, an association between the foodborne pathogen and food is created

(Lammerding and Paoli, 1997). It aims at prevention or reduction of the presence of

pathogenic microorganisms. Such control commonly requires a food chain approach,

because end-product control is not effective (Barlow et al., 2015).

Snail meat is more prone to microbial contamination than meat from other animals,

because snails are in continuous contact with soil and debris thereby exposed to various

microorganisms. There is very limited research on the bacterial assessment of snail meat

along the supply chain from market to table/fork which makes it difficult to evaluate the

level of hazards associated with it. The literature on snails is rather replete with research

focused on the microbiological quality assessment and preservation methods (Legaspi and

Jovellanos, 1990; Tetty et al., 1997; Serrano et al., 2004; Ezeama, 2004; Kirkan et al.,

2006; Ezeama et al., 2007; Okonkwo and Anyaene, 2009; Antwi, 2009; Adegoke et al.,

2010; Omenewa et al., 2011; Adagbada et al., 2011; Ebenso et al., 2012; Emelue et al.,

2013; Nyoagbe et al., 2016).

In Nigeria, the Federal government revised the national policy on food safety, and

even mapped out its implementation strategy to, among other intents, establish an effective

early warning system that has the capacity to detect, trace and prevent outbreaks of food

borne illnesses before transmission (FMoH, 2014). The federal government desires to

achieve comprehensive, effective collaboration and coordination of food safety practices

from farm to table nationwide by adopting the integrated food management system

approach. The National Food Safety Management Committee (NFSMC) is the entity

established to implement the food safety policy.

3
 One of the goals targeted towards the implementation of this policy is to minimize

the incidence of risks associated with physical, chemical, and biological hazards in foods

and water. One of the steps proposed by NFSMC is to conduct a workshop to identify high

risk foods (FMoH, 2014). To achieve its aim, such a workshop will require data on various

hazards in foods in Nigeria. Such data is not available for many foods such as snail meat.

Statement of the Problem

Edible land snails contain bacteria that are suspected to pose public health threats

(Adegoke et al., 2010; Omenewa et al., 2011; Ebenso et al., 2012). Also, regular

consumers of snails are often plagued by mild gastroenteritis (Serrano et al., 2004). The

report on Aeromonas food poisoning that involved only a 64-year old man that consumed

already prepared snails (Achatina spp.) in northern Nigeria (Agbonlahor et al., 1982) and

other studies gauging the pathogenicity of Staphylococcus aureus isolated from edible

snails in southern Nigeria (Efuntoye et al., 2011), have provided some information on

possible bacterial hazards in edible snails.

However, information on molecular studies of presumptive pathogens in snails is

not available and it is not known if common methods of processing and cooking improve

the bacteriological quality of snails for consumption.

Hence, it has become prudent to assess the bacterial hazards in edible land snails in

order to contribute data that will promote safer snail meat along the value chain.

4
Significance of the Study
This study will provide information on important bacterial hazards associated with

edible land snails in South East, Nigeria and the efficacy of different processing methods

and cooking in eliminating these pathogens from edible snails. Such information, if made

available to handlers and consumers, will improve the levels of hygiene and adequacy of

preparing edible land snails for consumption at home. It will also provide data useful for

developing legislations that will promote the sale of safer edible snails thereby reduce the

burden of foodborne diseases in the population.

Aim
The aim of this study is to assess bacterial hazards associated with edible land

snails (Achatina achatina) from selected markets in South East, Nigeria.

Specific Objectives
1. To determine the aerobic plate count of edible land snails (Achatina achatina)

obtained from three major markets in South East, Nigeria.

2. To determine the prevalence and load of coliforms in the edible snails for sale in

the markets.

3. To identify six presumptive pathogenic bacteria from edible snails for sale in the

markets using phenotypic tests.

4. To determine the prevalence and load of six presumptive pathogenic bacteria in the

edible snails for sale in the markets.

5. To determine the prevalence of virulence potentials by phenotypic tests of

presumptive pathogenic isolates in the edible snails for sale in the markets.

5
6. To determine the prevalence of antibiotic resistance of presumptive pathogenic

isolates in the edible snails for sale in the markets.

7. To identify selected bacterial pathogens in the edible snails for sale in the markets

using 16S rRNA gene sequencing technique.

8. To detect toxin genes in selected pathogenic isolates in the edible snails for sale in

the markets using Polymerase Chain Reaction (PCR).

9. To determine the processing method that will reduce the bacterial load of snail

meat during culinary preparation.

6
 Chapter Two

LITERATURE REVIEW
2.1 Edible Snails
Snails are the largest group of mollusks. They are classified under ―Gastropoda‖

because they move with waves of muscular contraction on their foot (Antwi, 2012). Snails

have two body parts which include: Head-foot, and Visceral mass. The visceral mass is

protected by a calcerous shell secreted by mantle cavity. The organs of digestion,

excretion and reproduction are found within the visceral mass. The shell is composed of

several layers that are made of precipitated organic calcium carbonate (Toader-Williams

and Golubkina, 2009). Snails require calcium to produce the shell and a whitish fragile

calcified material known as epiphragm (Toader, 2012).

According to European Commission (2004), snails for human consumption include

the following species: Helix pomatia, H. aspersa, H. lucorum, and species of the family

―Achatinidae‖:

(a) Helix pomatia measures about 45mm across the shell. It is also called then

―Roman snail‖, ―Apple snail‖, ―Lunar‖, ―La vignaiola‖, the German

―Weinbergschnecke‖, the French ―Escargot de Bourgogne‖ or ―Burgundy snail‖ or

―Gros blanc‖. It is a native over a large part of Europe. It lives in wooded

mountains and valleys up to 2,000 meters (6,000 feet) altitude and in vineyards and

gardens. The Romans may have introduced it into Britain. Immigrants introduced it

into United States in Michigan and Wisconsin. Many consumers prefer H. pomatia

to H. aspersa for its flavor and larger size, as the ―Escargot par excellence‖.

(b) H. aspersa is also known as the French ―Petit gris‖, ―Small grey snail‖, the

―Escargot chagrine‖, or ―La zigrinata‖. The shell of a mature adult has 4-5 whorls

and measures 30 – 45mm across. It is native to the shores of Mediterranean and up

7
 the coast of Spain and Frnace. It is found on many British Isles, where the Romans

introduced it in the first century A.D. In the early 1800s, the French brought it into

California where it has become a serious pest. These snails are now common

through out the United States of America. It was introduced into several Eastern

and Gulf States even before 1850, and later introduced into other countries such as

South Africa, New Zealand, Mexico, and Argentina. H. aspersa has a life span of

2–5 years. This species is more adaptable to different climates and conditions than

many snails, and is found in woods, fields, sand dunes, and gardens. This

adaptability has increased the range of H. aspersa, and also makes its rearing easier

and less risky.

(c) H. lucorum or Turkish snail is a large, edible air-breathing land snail or escargot, a

terrestrial pulmonate gastropod mollusk in the family ―Helicidae‖. It originates

from the Black sea region, adjacent Asia Minor, today‘s western and central

Turkey. Now, it is also found on the central Balkan Peninsula (southern Romania,

Bulgaria, Thrace, as far as Albania) and Italy west of the Apennine. The species

does not occur in Germany, but has been introduced in Austria, south of Vienna; it

has also been introduced in parts of southern France and on the Iberian Peninsula.

With a shell diameter of between 30 and 60 mm, the Turkish snail is usually larger

than the Roman snail (H. pomatia). It is only active at night and after heavy rains.

The shell form of Turkish snail is similar to that of Roman snail: globular with a

depressed spire and largely rounded red-brown strips around the whorls. The shell

walls are thick and the surface is irregularly striped; the aperture looks oblique and

has a thickened rim of a reddish or brownish colour. It is not cultivated but is

collected from nature. In delicatessen shops in central Europe, cultivated Roman

snails (H. pomatia) are sometimes sold in the more colourful shells of H. lucorum.

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(d) Species of the family ―Achatinidae‖ (New Latin, from Greek ―agate‖) is a family of

medium to large sized tropical land snails, terrestrial pulmonate gastropod

mollusks from Africa. The family includes 13 genera. Well known species include:

Achatina achatina the giant African snail, and Achatina fulica the giant East

African snail. These snails are among the largest terrestrial snails and may reach

20-30cm. The shell of an agate is larger than it is wide. It is conical with an

extended body whorl and a blunt apex. Presently, giant African land snails are

found almost anywhere the natural environment is right. They are mainly

herbivores feeding on fruits and vegetables, but have been known to feed on dead

animals to provide protein. In many places, they are serious agricultural pests that

cause considerable crop damages. Also due to their large size, their slime and

faecal material create a nuisance as does the odour that occurs when something like

poisoned bait causes large numbers to die. The United States of America has made

considerable efforts to eradicate Achatina (EC, 2004).

9
2.2 Snailery in Nigeria
The habitat of edible land snails spans from the dense tropical high forest in

southern Nigeria to the fringing riparian forests of the derived Guinea Savanna (Odaibo,

1997; Fagbuaro et al., 2006). In Nigeria, edible land snails aestivate (i.e. bury themselves

in the soil or hide beneath stones in order to avoid direct solar radiation) from November

to March each year, and re-surface during the rainy season (Fagbuaro et al., 2006).

Furthermore, edible land snails are collected from the wild/nearby bushes, or

purchased from the open market, or from snail farms. The population of edible land snails

in the wild has declined considerably because of human activities such as deforestation,

pesticide use, slash and burn agriculture, bush fires and premature harvesting of snails.

Persistence of these factors will culminate in the extinction of edible land snails in Nigeria.

In addition, seasonality of supply of edible land snails from the wild limits their

use for meat on a continuous basis. All these justify rearing of edible land snails in farms

just as done for poultry, sheep and goats. Two main systems of snail farming are practiced:

indoor and out-door systems. A modification of the out-door system, in which the snails

are confined out-doors in enclosures and fed both synthetic and natural diets, fits into the

Nigerian farming system (NAERLS, 1995).

The Federal Ministry of Agriculture and Rural Development (FMARD), is a

ministry of the federal government of Nigeria that regulates agricultural research,

agricultural and natural resources, forestry and veterinary research all over the country.

The ministry relies on the membership list of Snail Farmers Association of Nigeria

(SFAN) which is a subset of All Farmers Association of Nigeria (AFAN) on the event of

workshops, sensitization campaigns and provision of loans for these farmers. The ministry

has recognized the growing interest in the rearing and marketing of snails, and has trained

10
several women and youths on snailery. Nodu et al. (2003) found consumption level of

snail meat among people of Bori (a southern city in Nigeria) to be as high as 70%.

The snail meat industry, though not well established, is projecting the snail meat as

a healthy alternative to the conventional meat and as an economically vibrant mini-

livestock. Also, there seems to be a considerable international trade and demand for snail

meat (Emslie, 1982). Data on the extent to which the snail market contributes to Nigeria‘s

economy is lacking.

The Nigeria Agricultural Quarantine Service (NAQ) has the mandate of ensuring

that all plants, animals and aquatic produce/products leaving the shores of Nigeria meet

international standards (FMoH, 2014). However, there is no legislation that lays down the

specific requirements that ensure safe snail meat from farm to table. The food supply chain

in Nigeria faces a number of challenges such as diversities in culture, lifestyles,

agricultural practices, mode of food production, handling, storage, preparation,

transportation and eating habits (FMoH, 2014).

Ebenso et al. (2012) noted the critical need for producers, retailers, processors and

consumers to take responsibility to prevent contamination, cross contamination,

mishandling as well as proper holding, storage and cooking of snail meat to eradicate

foodborne pathogenic bacteria. This can only be possible if the existing National Policy on

Food Safety and Implementation Strategy (NPFSIS) is strengthened at each enterprise

level at the federal, state and LGAs platforms (FMoH, 2014).

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2.3 Bacterial Pathogens in Snails
Unlike meat from vertebrates, snail meat is more prone to microbial contamination

which begins with the feeding habit of snails. Because formulated feeds for snails are not

available in the market, it has become common practice for snail rearers to use vegetables,

plant leaves and kitchen wastes to feed snails (Chah and Inegbedion, 2013). It has been

reported that gastropods find mammalian faeces (manure) an attractive food source

(Speiser, 2001). In addition, the regular ingestion of bacteria from the soil (Walker et al.,

1999), worsen their potential to be pathogen laden. Ebenso et al. (2012) found pathogens

(including coliforms) in snail meat sampled from various markets in Niger Delta, Nigeria

to be far above recommended microbiological limits.

Microbial contamination of snail meat inadvertently continues during collection of

edible land snails from market women who get their snails directly from the forest. This is

because snails‘ movement is characterized by continuous contact of their body with soil

and debris thereby exposing snails to various microorganisms.

The flowchart of traditional processing of fresh water snail is shown in Figure 1.

12
 Raw Shelled Snail

Shucking
(i.e. removal of shell and liquid wastes)

Drying
(Smoking, Sundrying)

Packaging in jute bags

Storage (ambient)

Figure 1: Flowchart of traditional processing of fresh water snail

(Ezeama et al., 2007)

13
 Each step of processing snail meat has the potential of influencing the microbiological

quality. Tettey et al. (1997) conducted a preliminary survey of the processing procedure, raw

materials used, handling, packaging, and storage of smoked-dry snail meat. The outcome

indicated lack of hygienically controlled processing procedure, the use of poor quality snail

meat, poor handling and storage procedures for the unpackaged smoked-dry product. Total

aerobic count/g of smoked-dry meat ranged from 1.88 x 10 5 to 2.77 x 1018 cfu/g. Food borne

illnesses due to consumption of snail meat may occur when the mollusks that contain

pathogenic microorganisms are consumed raw or improperly cooked.

Akinboade et al. (1980) and Akpavie et al. (2000) showed that various bacteria that

are potential pathogens inhabit different organs and tissues including lungs, hemocyanin,

liver, kidney, crop, and stomach of clinically healthy edible land snails. Total viable count

(log CFU/g) ranging from 6.61 to 8.29 and coliform count ranging from 5.61 to 8.50 in

Achatina spp. have been reported in Ghana (Nyoagbe et al., 2016). Total viable counts in

Cornu aspersum and Helix lucorum varieties of wild snails sampled in Greece were

determined to range from 6.0 to 8.0 log CFU/g and microbial counts enumerated in snail meat

of C. aspersum decreased following the application of processing steps (Parlapani et al.,

2014). Adegoke et al. (2010) analysed different varieties of market snails (Achatina fulica,

Limcolaria sp., and Helix pomatia) in Uyo, Akwa Ibom state and found that total bacterial

counts ranged from 8.0 to 8.1 log CFU/g, coliform count ranged from 7.1 to 7.3 log CFU/g,

Salmonella/Shigella count ranged from 7.5 to 7.8 log CFU/g. These studies show there are

differences in total bacterial counts among varieties of snails and sources of samples, but the

reasons for the differences were not explained.

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 Andrews et al. (1975) isolated Salmonella in 84 of 270 Moroccan snails (Helix

aspersa) with the use of both the pre-enrichment and direct selective enrichment procedures.

Serotyping of some Salmonella isolates in snails (Achatina achatina) revealed the presence of

S. manhattan, S. ndolo, S. reading, S. uppsala, and S. typhimurium (Obi and Nzeako, 1980).

In Ghana, Nyoagbe et al. (2016) determined Salmonella counts (log CFU/g) in market snails

(Achatina) ranged from 2.91 to 7.39 on Xylose Lactose Desoxycholate (XLD) agar. In

Greece, Salmonella was detected in wild snails (C. aspersum and H. lucorum) using selective

enrichment procedures (Parlapani et al., 2014).

In India, a study on the bacterial diversity of the gastrointestinal tract of Achatina

fulica using culture-independent and culture-dependent methods reported that an apparent

feature of bacterial communities in these snails‘ gastrointestinal tract was the abundance of

members of the genus Citrobacter (Pawar et al., 2012). Silva et al. (2013) found Citrobacter

freundii in 30% of wild fresh water snails (Biomphalaria glabrata) in Brazil. Citrobacter has

been reported to be present in Helix pomatia at Romania (Cîrlan et al., 2010).

Obi and Nzeako (1980) isolated Shigella sonnei from Achatina achatina in Eastern

part of Nigeria. Nyoagbe et al. (2016) found staphylococcal count (log CFU/g) in market

snails (Achatina) to range from 2.66 to 7.68 on Baird-Parker agar enriched with egg-yolk

emulsion. Efuntoye et al. (2011) demonstrated that some Staph. aureus isolates in snails

(Archachatina marginata, Achatina achatina and Achatina fulica) obtained from south west

Nigeria were enterotoxigenic.

Obi and Nzeako (1980) analysed Aeromonas hydrophila isolates recovered from

Achatina achatina in Eastern part of Nigeria and found 58% were enterotoxigenic, and their

15
toxin was shown to be heat labile. Agbonlahor et al. (1982) reported the first case of A.

hydrophila food poisoning in a 60-year old man after a meal of edible land snails. The snails

had been collected from a swampy forest in a village near Oshogbo in Western Nigeria. The

A. hydrophila isolate was resistant to cephalotin, ampicillin, penicillin, and sensitive to

gentamicin, tetracycline, chlorampbenicol, nalidixic acid and nitrofurantoin. Silva et al.

(2013) found A. sobria in 20% of wild fresh water snails (Biomphalaria glabrata) in Brazil.

Generally, all these reports (except Pawar et al., 2012) were not based on molecular studies.

2.4 Enterotoxins in Food

Enterotoxins are short, extracellular proteins that are water soluble. They are most

commonly described as very stable and are resistant to heat as well as degrading enzymes (Le

Loir et al., 2003). The enterotoxins, which are classified as superantigens, display common

characteristics such as pyrogenicity, immune suppression, and a mitogenic effect on T cells

(Le Loir et al., 2003).

Bacteria that produce enterotoxins by different mechanisms include: Staphylococcus

aureus, Bacillus cereus, Escherichia coli, Clostridium perfringes, and Vibrio cholerae

(Popoff, 2011). The main causes of food borne illness are bacteria (66%), chemicals (26%),

virus (4%) and parasites (4%). The two most common types of food borne illness are

intoxication and infection. Intoxication occurs when toxins produced by the pathogens cause

food poisoning, while infection is characterized by the ingestion of food containing pathogens

(CDC, 2011; Addis and Sisay, 2015).

16
 The genes encoding the different enterotoxins are carried and disseminated by

different mobile genetic elements such as plasmids and phages (Omoe et al., 2003; Schelin et

al., 2011). Convenience foods offer a suitable growth environment for toxin-producing

bacteria such as S. aureus which is able to grow and express virulence in a wide variety of

foods such as milk products, mixed foods, meat and meat products, egg and egg products,

cakes and ice cream (Adams and Moss, 2008). Staphylococcal food poisoning is often

associated with growth in protein-rich foods such as meat and dairy products (Le Loir et al.,

2003).

S. aureus can be killed through heat treatment of the food, but the enterotoxins are

very heat resistant (Le Loir et al., 2003). As little as 20ng-100ng of Staphylococcal

enterotoxin (SE) can cause food poisoning (Asao et al., 2003). The majority of reported

staphylococcal food poisoning outbreaks are associated with the classical enterotoxins: SEA ‒

SEE, with SEA being the most common cause of staphylococcal food poisoning (Kerouanton

et al., 2007).

Generally, growth of S. aureus is necessary for enterotoxin production. Some of the

factors that affect S. aureus growth and enterotoxin production include: Temperature, pH,

water activity, NaCl, oxygen, redox potential, and presence of Lactococcus lactis (Adams and

Moss, 2008; Schelin et al., 2011). Staphylococcal enterotoxins (SEs) are the toxins that

induce emesis, while the enterotoxin-like proteins (SEls) either lack emetic activity or have

not yet been tested for this (Lina et al., 2004). Twenty one SEs or SEls have been identified

and designated SEA ‒ SElV (Schelin et al., 2011). These toxins (SE and SEl) are globular

single-chain proteins with molecular weights ranging from 22 ‒ 29 KDa (Thomas et al.,

17
2007). SEA is resistant to proteolytic enzymes, thus retaining its activity in the digestive tract

after digestion (Bergdoll, 1989).

SEA and SED were found to decrease in boiled ham after a period of accumulation

(Marta et al., 2011). Schelin et al. (2011) acknowledges that some studies have reported the

disappearance of SEA in broth, minced food, raw and pasteurized milk. The apparent

decrease in enterotoxin levels could simply be an analytical artifact, such as loss of

serological recognition using immuno-based methods such as ELISA, which is a technique

commonly used to detect enterotoxins. It has also been proposed that proteases produced by

lactic acid bacteria cause the decrease in SEA levels, or that SEA becomes cell-associated and

is, therefore, not detected (Hallis et al., 1991).

Diagnosis of staphylococcal food poisoning is generally confirmed either by the

recovery of at least 105 S. aureus/g from food or by detection of SEs in food (Hennekinne et

al., 2012). Three types of methods are used to detect bacterial toxins in foods: (a)

immunological methods, (b) biossays, (c) molecular methods. Immunological methods are the

most commonly employed.

2.5 Indicator Organisms and Pathogens

The intestines of warm blooded animals contain large numbers of non-pathogenic

bacterial species, which are known as indicators of faecal pollution and also contain

pathogens such as pathogenic strains of Escherichia coli, parasites such as protozoans and

viruses (Murray et al., 2007). Indicator groups of bacteria have the advantage of being

enumerated inexpensively and easily for quantifying the performance of a production process,

18
when particular pathogens or spoilage organisms might be difficult to detect (Jordan et al.,

2006).

According to Kornacki (2011) the term indicator organism is used to describe:

(a) A (theoretical) non-pathogenic bacteria whose presence/absence indicates the possible

presence or absence of a pathogen

(b) A measure of the hygienic or sanitary conditions of surfaces in a food processing

environment

(c) An organism or test that reflects the microbiological quality of a product

(d) An organism whose presence or level indicates the potential for future spoilage

(e) As a ―surrogate‖ organism, for example those organisms that model the behaviour of

pathogens under certain conditions. Surrogate organisms are harmless microbes with

correlated survival and growth parameters to specific pathogens. They are frequently

used to validate the efficacy of critical limits for critical control points, can be

naturally occurring or artificially added and should be non-pathogenic (Kornacki,

2010; Ma et al., 2007).

Moreover, some indicators are not specific organisms but assays for groups of

organisms, such as coliforms, enterobacteriaceae, and aerobic plate count. These various uses

cause confusion and disagreement among food safety professionals when the term ―indicator‖

is used.

The criteria for a suitable ―indicator‖ organism (Medema et al., 2003) include that:

(a) It should be absent in unpolluted water and present when the source of pathogenic

microorganisms of concern is present

(b) It should not multiply in the environment

19
 (c) It should be present in greater numbers than the pathogenic microorganisms

(d) It should respond to natural environmental conditions and water treatment processes in

a manner similar to the pathogen of concern

(e) It should be easy to isolate, identify and enumerate

(f) It should be inexpensive to test thereby permitting numerous samples to be taken

(g) It should not be a pathogenic microorganism (to minimize the health risk to analysts)

The four indicator groups include: aerobic plate count (APC), total enterobacteriaceae,

total coliforms, E. coli biotype I (Jordan et al., 2006). APC is used to quantify the density of

aerobic bacteria by unit area of perhaps carcass surfaces, when a general measure of process

hygiene is required during meat production. The other three indicator groups are available for

assessing the amount of contamination on meat arising from gut contents which includes both

that originating directly from the alimentary tract and that arising indirectly through the

integument or processing environment. Coliforms are the most frequently studied indicators

due to their inclusion into drinking water regulations (Wu et al., 2011).

The occurrence of a specific pathogen is relatively infrequent with less than a few

percent of the population infected at any given time except during outbreaks or in areas where

it is endemic. An infected individual excretes a specific pathogen in the faeces for a short

period of time ranging from few days to weeks. Therefore, the occurrence of such pathogen in

the environment depends on whether a particular pathogen is circulating within the

community at the time of sampling (Payment and Locas, 2011). The occurrence of indicators

and pathogens in water will be a function of occurrence in the population, dilution of sewage,

morphology of the microorganisms, water treatments, and environmental and biotic factors

20
(Hurst et al., 2007). Bacterial indicators can predict the probable presence of pathogens in

water, but they cannot predict precisely the level of occurrence.

Variations in pathogen prevalence in population, dilution, retention, and die-off result

in conditions where relationships/correlations between any pathogen and any indicator are

random, site-specific, or time-specific (Payment and Locas, 2011). Many studies have

conflicting results with regards to quantitative relationships between indicators and pathogens.

Several studies have not found correlations among indicators and pathogens (Noble and

Fuhrman, 2001). However, several investigators have observed relationships between the

presence of traditional indicators and illnesses. E. coli in well water was found to be

significantly associated with gastrointestinal illness in family members (Raina et al., 1999).

Wu et al. (2011) concluded that indicator organisms are possibly correlated with pathogens if

sufficient data are available.

There is lack of quantitative evidence describing how the measurements collected are

related to both the occurrence of bacterial hazards in red meat or to the amount of faecal

contamination. Jordan et al. (2006) conducted analysis to provide quantitative description of

the degree of association between concentrations of different indicator organisms in red meat

and found that the correlation between coliforms and enterobacteriaceae is consistently higher

than the correlation between E. coli biotype I and enterobacteriaceae (except for sheep

carcass) and also consistently higher than correlation between E. coli biotype I and coliforms.

21
2.6 Hazard Analysis in Food Safety Assessment
Hazard analysis is one of the approaches used to ensure food safety. In this approach,

the presence of a potentially harmful agent at a detectable level in food is used as a basis for

legislation and/or risk management action (Barlow et al., 2015). It aims at prevention or

reduction of the presence of pathogenic microorganisms. Such control commonly requires a

food chain approach, because end-product control is not effective. The ineffectiveness of end-

product control stems from the following: low prevalence of contaminated products, low

concentrations of microorganisms, and the potential of microorganisms to grow.

Hazard analysis can be applied throughout the food chain from farm, through

processing, packaging, retailing to purchase by the consumer, and are often effective.

However, a zero risk approach is normally not feasible for microbiological agents as many

hazards occur ―naturally‖ at primary production (example in the soil and faeces of

production animals). Microbiological criteria define the acceptability of a product, a batch of

food stuffs, or a process, based on the absence, presence or number of microorganisms, and/or

on the quantity of their toxins/metabolites, per unit(s) of mass, volume, area or batch (CAC,

1997). They can be used as tools to assess the safety and quality of foods, but cannot

guarantee safety.

Hazard-based approach is often useful and efficient, but it can be too stringent when

the actual impact of control on the human health risk is not known. Barlow et al. (2015)

summarized the situations in which hazard-based approaches are appropriate:

(a) When exposure conditions cannot be predicted or estimated with any confidence, but

there is an immediate need for rapid communication of information on potential

hazards.
22
 (b) When no threshold for the adverse effect can be identified.

(c) When exposure is avoidable.

(d) When use in food is not permitted in law.

Currently, there is no food safety management system that ensures safe snail meat

from farm to table. This is because there is scarcity of data needed to design such system and,

as a result, consumers have been put at risk in Nigeria.

2.7 Microbiological Risk Assessment (MRA)

The major element in assuring that sound science is used to establish standards,

guidelines and other recommendations for food safety is risk assessment (Gkogka et al.,

2013). The risk of illness from consuming food that contains microbiological hazards depends

on the different types of hazards, food matrices and susceptibility of consumers (Magnusson

et al., 2012). Microbiological risk assessment is a structured, systematic approach to integrate

and evaluate information from diverse sources concerning the origin and fate of pathogens

along the food chain and to determine the magnitude of public health risks (Lammerding,

2006). The Sanitary and Phytosanitary measures Agreement specifies that decisions on

whether a food can be considered safe and fit for international trade has to be based on

science and particularly on risk assessment. The World Trade Organisation relies on the

Codex Alimentarius to specify how risk assessment should be performed (Reij and

Schothorst, 2000). According to Codex Alimentarius Commission (1999), the general

principles of MRA include:

(a.) Principle 1: MRA should be soundly based upon science.

23
 (b.)Principle 2: There should be functional separation between risk assessment and risk

management.

(c.) Principle 3: MRA should be conducted according to a structured approach that

includes: Hazard identification, Exposure assessment, Hazard characterization and

Risk characterization.

(d.)Principle 4: MRA should clearly state the purpose of the exercise, including the form

of risk estimate that will be the output.

(e.) Principle 5: The conduct of MRA should be transparent.

(f.) Principle 6: Constraint (such as cost, resources or time) that impact on MRA should be

identified and possible consequences described.

(g.)Principle 7: Description of uncertainty and source of the uncertainty during the risk.

(h.)Principle 8: Data and data collection systems should be of sufficient quality and

precision that uncertainty in the risk estimate can be determined and minimized.

(i.) Principle 9: MRA should explicitly consider the dynamics of microbiological growth,

survival and death in foods and the complexity of the interaction between human and

agent following consumption as well as the potential for further spread.

(j.) Principle 10: Wherever possible, risk estimates should be re-assessed overtime by

comparison with independent human illness data.

(k.)Principle 11: MRA may need re-evaluation as new relevant information becomes

available.

Therefore, risk assessment is concerned with the scientific evaluation of known or

potential health effects resulting from human exposure to food borne hazards and consists of

24
four steps described below (WHO, 2009b; CAC, 1999; Makita et al., 2012; Schelin et al.,

2011; Rajkovic, 2014).

(i) Hazard Identification:

It is predominantly a qualitative process. Its purpose, for microbial agents, is to

identify the microorganisms or their toxins of concern with food. Hazards can be identified

from relevant data (information) sources, such as scientific literature, databases of food

industry, government agencies, relevant international organizations and solicitation of

opinions from experts. Areas from which data can be generated are outlined below (CAC,

1999; Tuominen et al., 2001):

(a) Clinical studies

(b) Epidemiological studies and surveillance

(c) Laboratory animal studies

(d) Investigations of the characteristics of microorganisms

(e) Interaction between microorganisms and their environment through the food chain:

from primary production up to (and including) consumption

(f) Studies on analogous microorganisms and situations

(ii) Exposure Assessment:

This entails an assessment of the extent of actual or anticipated human exposure.

Exposure assessments might be based on the potential extent of food contamination by a

particular agent or its toxins, and on dietary information. It should specify the unit of food that

is of interest i.e., the portion size in most/all cases of acute illness. Microbial pathogen levels

can be dynamic and substantially increase with abused conditions. Hence, the exposure

25
assessment should describe the pathway from production to consumption. Certain factors

which must be considered in exposure assessment include the following (CAC, 1999):

(a.) The frequency of contamination of foods by the pathogenic agent and its level in those

foods overtime

(b.)Patterns of consumption

(c.) The role of the food handler as a source of contamination

(d.)The amount of hand contact with the product

(e.) Potential impact of abusive environmental time/temperature

Scenarios can be constructed to predict the range of possible exposures. Such

scenarios might reflect effects of processing, such as hygienic design, cleaning and

disinfection, as well as the time/temperature and other conditions of the food history, food

handling and consumption patterns, regulatory controls and surveillance systems. It estimates

the level, within various levels of uncertainty, of microbiological pathogens or

microbiological toxins, and the likelihood of their occurrence in foods at the time of

consumption. Qualitatively, foods can be categorized according to the likelihood of its

contamination at its source. Predictive Microbiology can be a useful tool in exposure

assessment.

(iii) Hazard Characterization:

Qualitative or quantitative description of the severity and duration of adverse effects

that may result from the ingestion of a microorganism or its toxin in food are provided in this

step. A dose-response assessment should be performed if the data are obtainable. Several

important factors that relate both to the microorganism and the human host need to be

considered in hazard characterization (CAC, 1999):

26
(a.)In relation to the microorganism: Replication capability of microorganisms,virulence

and infectivity of microorganisms depending on interaction with host and environment,

Transfer of genetic materials between microorganisms, Secondary and tertiary transmission of

microorganisms, Substantial delay of onset of clinical symptoms, Continued excretion of

microorganisms by carrier individuals and continued risk of spread, Low doses of some

microorganisms can cause severe effect, Food attributes may alter microbial pathogenicity.

(b.) In relation to the host: Genetic factors, Increased susceptibility due to breakdown of

physiological barriers, Individual host susceptibility features such as age, pregnancy,

nutrition, health and medication status, immune status, concurrent infections and previous

exposure history, Population characteristics such as access to and use of medical care,

persistence of the organism in the population and population immunity

In the absence of a known dose-response relationship, risk assessment tools such as

expert elicitations could be used to consider various factors (example infectivity) necessary to

describe hazard characterizations. Moreover, experts may be able to devise ranking systems

for characterizing severity and/or duration of disease.

(iv) Risk Characterization:

This is defined as an estimation of the probability of occurrence and severity of known

or potential adverse health effects in a population based on hazard identification, hazard

characterization and exposure assessment. It is in this step that the results of risk assessment

are presented. These results are provided in the form of risk estimates and risk descriptions

that project answers to the questions risk managers pose to risk assessors (WHO, 2009c).

Risk characterization depends on available data and expert judgements. The degree of

27
confidence in the final estimation of risk will depend on the variability, uncertainty and

assumptions identified in all previous steps (CAC, 1999).

Because sources of food contaminants are as numerous as the contaminants, the roles

of food groups and bacterial food borne pathogens in food borne infections are yet to be

established in most developing countries. Also, there is a disorganised sampling and quality

control of foodstuffs because of inadequate coordination between surveillance, food

laboratories and food inspection services. Furthermore, the emphasis is on sampling for

enforcement purposes and often there is no systematic monitoring for food contaminants as

well as no surveillance systems capable of identifying common agents of foodborne diseases

(Nguyen et al., 2014; Mensah and Julien, 2011). The risk is further aggravated by the

increase in food outlets so much that at least a meal is believed to be consumed away from

home daily (FMoH, 2014).

2.8 Antibiotic Resistance in Bacteria

Antimicrobials consist of substances that inhibit or have killing effect on

microorganisms in a clinical setting or for reducing bacterial loads in materials and surfaces

and include antibiotics and chemical biocides. Chemical biocides are used for disinfection in

the food processing environment (Mathur and Singh, 2005). However, antibiotics are used in

food animals to treat clinical diseases, to prevent and control common disease events, and to

enhance animal growth (Mc Ewen and Fedorka-Cray, 2002).

The presence of antimicrobial-resistant bacteria in food products is another important

public health issue because of the potential for the transfer of antimicrobial-resistant

foodborne pathogens to human populations. Moreover, antimicrobial-resistant bacteria may

28
represent a reservoir of resistance genes transferable to pathogenic or commensal bacteria of

the human digestive tract (Capita and Alonso-Calleja, 2013). The emergence of drug

resistance has been observed following the introduction of each new class of antibiotics and

the threat is compounded by a slow drug development pipeline (Spellberg et al., 2004; Norrby

et al., 2005).

WHO states that more than 25,000 people die each year in the European Union due to

infections caused by antibiotic-resistant bacteria (Lawley, 2013). Due to normal genetic

variations in bacterial populations, individual organisms may carry mutations that render

antibiotics ineffective, transferring a survival advantage to the mutated strain (Landers et al.,

2012). Also, advantageous mutations can be transferred through plasmid exchange within the

bacterial colony when antibiotics is present, resulting in increase of the resistance trait

(Courvalin, 2008).

Generally, antimicrobial resistance is the capacity of an organism to resist the growth

inhibitory or killing effect of an antimicrobial beyond the normal susceptibility of the

organism (Mathur and Singh, 2005). Bacteria can be resistant to antibiotics by using several

mechanisms: enzymatic degradation of antibiotics, antibiotic target modification, alteration of

the cell wall permeability, and use of alternative pathways to escape the activity (Verraes et

al., 2013). Prudent use of antibiotics with the intent of preserving their effectiveness for

serious infections have been advocated (Belongia et al., 2005).

Food may act as a vector for the transfer of antimicrobial resistant bacteria and

antimicrobial resistance genes to humans (Verraes et al., 2013). There is significant potential

threat to human health arising from inappropriate use of antibiotics in food animals. This is

because resistant pathogens propagated in these animals are bound to enter the food chain and
29
could be disseminated in food products (Cui et al., 2005; Garofalo et al., 2007). Commensal

bacteria in livestock may serve as reservoirs for resistance genes that could potentially be

transferred to pathogenic organisms in humans (Landers et al., 2012). Bergogne-Berezin

(1997) listed the routes of contamination of foods with antimicrobial resistant organisms

which included:

(a.) Faecal contamination during slaughter of animal

(b.)Faecally contaminated water used for irrigation

(c.) The environment

(d.)Handling by the butcher and consumer

A recent study estimated 1.5% as the probability of exposure to 10 3 cfu of

cephalosporin-resistant E. coli through consumption of a meal containing chicken meat

(Depoorter et al., 2012). Tetracycline resistance is prevalent in lactic acid bacteria associated

with raw meat (Gevers et al., 2003).

It has been demonstrated that sub-lethal food preservation stresses such as heat stress,

acid and salt stress can significantly alter phenotypic antimicrobial resistance in food-related

pathogens such as E. coli, Salmonella Typhimurium and Staphylococcus aureus. While sub-

lethal high temperature decreased antimicrobial resistance, increased salt or reduced pH

conditions increased the phenotypic antimicrobial resistance. It was observed that some of the

pathogens continue to express higher levels of phenotypic antimicrobial resistance after

removal of stress (Mc Mahon et al., 2007).

30
 Bacteria present in food products can survive after the application of a food processing

or preservation technique. It is possible that their growth is inhibited resulting in stressed or

sub-lethally damaged bacterial cells. Food processing and/or preservation techniques can kill

or inactivate bacteria. Such dead bacterial cells can stay intact or be lysed due to cell wall

damage. As a consequence, the bacterial DNA, including the eventual present antimicrobial

resistance genes, are released into the environment. As soon as the DNA has been released,

antimicrobial resistance genes may, theoretically, be transferred to other bacteria by

transformation. However, most food processing methods reduce bacterial load (Verreas et al.,

2013; Rajkovic et al., 2010; Wesche et al., 2009).

There is indirect evidence of the food borne transfer of Extended Spectrum β-

Lactamase (ESBL) genes of poultry to humans. Ninety-four percent of the tested chicken

meat isolates contained ESBL genes (Leverstein-van Hall et al., 2011). ESBL are hydrolytic

enzymes that mediate resistance to extended-spectrum cephalosporins. The detection of

ESBL-producing bacterium means that the bacterium possesses the therapeutic resistance to

all extended-spectrum cephalosporins, indeed to all cephalosporins, aztreonam and penicillins

(Nteimam, 2005).

The notably virulent strain of E. coli O104:H4, which caused the major fatal outbreak

of infection in Germany in 2011, was resistant to a number of antibiotics, including

ampicillin, trimethoprim, cephalosporins and tetracycline. It was also found to possess a

plasmid-borne gene for ESBL production. E. coli strains with the ESBL gene are often

resistant to a wide range of important therapeutic antibiotics, and infections are notoriously

difficult to treat. The presence of ESBL in food borne pathogens is an emerging concern

(Lawley, 2013).
31
 Agbonlahor et al. (1982) showed that Aeromonas hydrophila isolate was resistant to

cephalotin, ampicillin, penicillin, and sensitive to gentamicin, tetracycline, chlorampbenicol,

nalidixic acid and nitrofurantoin. Recently, it has been shown that ofloxacin was the most

effective antibiotic against bacterial isolates (E. coli, Enterobacter, Klebsiella pneumonia,

Pseudomonas vulgaris, P. aeruginosa, Salmonella, Shigella) recovered from snails (Achatina

achatina), while Ceftriaxone and Augmentin were the least effective on the isolates (Onifade

and Aiyenuro, 2018). Staph. aureus isolates from snails (Archachatina marginata, Achatina

achatina and Achatina fulica) have been reported to show resistance to augmentin,

cloxacillin, cefuroxime and amoxycillin-clavulanic acid and other antibiotics tested (Efuntoye

et al., 2011).

Scientific studies have come to the conclusion that soil is a major reservoir of

antibiotic resistance genes (Martinez, 2008; Finley et al., 2013; Fitzpatrick and Walsh, 2016).

2.9 National Policy on Food Safety and its Implementation Strategy

The National Policy on Food Safety and its Implementation Strategy (NPFSIS) is

expected to provide the framework for identification of National Food Safety objectives and

formulation of suitable laws, regulations and guidelines for relevant sectors of the food supply

chain needed to improve public health and trade (FMoH, 2014). It is also intended to establish

an effective early warning system that has the capacity to detect, trace and prevent outbreaks

of food borne illnesses before they spread. The desire and determination of government in the

NPFSIS is to achieve comprehensive, effective collaboration and coordination of food safety

practices from farm-to-table nationwide by adopting the Integrated Food Management System

approach. To this end, the NPFSIS proposed the establishment of National Food Safety
32
Management Committee (NFSMC) as the entity that will coordinate the National Food Safety

System, while the Inter Ministerial Committee on Food Safety (IMCFS) shall be established

as the entity that oversees the NFSMC. The composition of the IMCFS shall be the

Honourable Ministers in charge of:

(a) Health,

(b) Industry, trade and investment,

(c) Agriculture and rural development,

(d) Science and technology,

(e) Environment.

The committee shall be free to co-opt any other member from among stakeholders as

deemed necessary. On the other hand, the NFSMC shall be established by the IMCFS to

implement the NPFSIS and shall report to the IMCFS. The functions of the NFSMC shall be:

(i) Advise the IMCFS on matters related to food safety

(ii) Coordinate all programmes related to food safety

(iii) Carry out strategic planning, monitor performance and periodically evaluate

progress of NPFSIS

(iv)Facilitate the design, and coordinate training programmes for all stakeholders along

the food supply chain

(v) Coordinate risk assessment and management

(vi)Initiate and coordinate the drafting of the proposed food safety bill

(vii) Facilitate the development and/or updating of standards, regulations, guidelines, code

of practice, manuals, SOPs, etc for public and private sectors

(viii) Inform the public and private sectors about current and emerging food safety issues

33
 (ix)Coordinate programmes and seek financial and technical assistance from donor

agencies and development partners (FMoH, 2014).

The memberships of the NFSMC shall include one representative of the following

among others:

(a) Federal Ministry of Health (FMoH)

(b) Federal Ministry of Industry, trade and investment (FMITI)

(c) Federal Ministry of Agriculture and rural development (FMARD)

(d) Federal Ministry of Science and technology

(e) Federal Ministry of Environment

(f) Federal Ministry of Finance

(g) Federal Ministry of Justice

(h) Federal Departments of Agriculture

(i) Nigeria Agricultural Quarantine Services (NAQS)

(j) National Agency for Food and Drug Administration and Control (NAFDAC)

(k) Standard Organisation of Nigeria (SON)

(l) Institute of Public Analysts of Nigeria

(m)Mycotoxicology Society of Nigeria

(n) Nigerian Institute of Food Science and Technology

and other stakeholders to be invited when needed (FMoH, 2014).

The NPFSIS further states that the existing relationship between the International

Committees and the national regulatory agencies will continue to subsist. These committees

will continue with their mandates, preparation of national delegations and production of

country position on CAC, OIE, IPPC and SPS matters. Moreover, the existing multi-sectorial
34
approach to food safety where responsibilities for food safety control are shared by relevant

MDAs, States and LGAs will still continue to perform their relevant statutory mandates under

this policy. However, the statutory legislation/functions of the concerned MDAs will be re-

examined to reduce to the barest minimum overlaps and duplications of functions (FMoH,

2014).

For the successful implementation of this policy, four goals have been identified.

Goal I: To modernize the Nigerian food safety regulatory framework in line with international

best practices.

Goal II: To minimize the incidence of risks associated with physical, chemical and

biological hazards in foods and water.

Goal III: To strengthen institutional capacity for food safety.

Goal IV: To improve information and communication systems for food safety.

These goals are further defined by their objectives, strategies and activities. Goal II is

the most elaborate and has 4 objectives and 8 strategies (FMoH, 2014).

The objectives, strategies and the responsible bodies are presented (Table 1).

35
Table 1: The Objectives, Strategies and Responsible bodies for Goal II
Objectives Strategies Responsible
bodies
Identify high risk food Conduct a workshop to identify top high NFSMC
and water borne disease risk foods in Nigeria
sources
Establish a mechanism Promote the adoption of appropriate NFSMC,
to prevent food and protocols for the use and handling of food FMARD,
water borne hazards additives, processing aids, agrochemicals, NAFDAC, SON,
feed and veterinary drugs in-line with NESREA, NAQS
international best practices
Establish and implement food safety
NFSMC, SON
management control systems such as
GAP, GHP, GMP, GEP, GSP, HACCP &
ISO 22000, CAC guidelines by the
respective regulatory bodies
Ensure that all imported and exported FMITI, NFSMC,
food products are in compliance with SON
World Trade Orrganisation, Sanitary and
Phytosanitary measures agreements and
other international legislations NFSMC
Strengthen the national food defence
system to avert food borne illnesses and
hazards
Enhance food borne Strengthen sentinel sites at each of the FMoH, NFSMC
illnesses surveillance states and the FCT with up to date
and response facilities to investigate food borne
illnesses, identify causative agents and
trace outbreaks to their respective sources
Strengthen the National Food Risk NABDA
Assessment Coordinating Centre
 Conduct a detailed study of
the identified high risk NAFDAC
foods to monitor chemical
and microbial
contaminants and generate
useful data (Activity 25)
Improve inspections, Strengthen the capacity of regulatory NFSMC
compliance and inspectors, auditors and compliance
enforcement systems systems
(FMoH, 2014)

36
 Chapter Three

MATERIALS AND METHODS

3.1 Collection of Snail Samples

Three states in the South East of Nigeria were selected for this study which were

Enugu, Ebonyi and Anambra States. A central market serving as the largest platform for sale

of live edible snails in each state was selected for this study. The markets were Ogbete main

market at Enugu State, Abakiliki meat market at Ebonyi State and Nkwo Igboukwu market at

Anambra State. A total of 300 live edible snails (Achatina achatina), made up of 100 samples

from each market, were collected from three states in the south east zone of Nigeria from

May, 2016 – October, 2016 and April, 2017 – July, 2017. Edible snails (A. achatina) were

identified by the Zoology Department of Nnamdi Azikiwe University, Awka. They were

identified according to their shape, size, markings, colour, spire angle, sculpture and aperture

form (Igbinosa et al., 2016; Raut and Barker, 2002). Edible snails displayed for sale in the

market (Figure 2) were aseptically collected, with sterile hand gloves, in plastic containers

sterilized with 70% alcohol and dried with commercially available sterile paper towel.

Samples were immediately transported to the laboratory for analysis.

3.2 Sample Preparation

Shells of the snails were surface sterilized with 70% ethanol before being aseptically

shucked with a sterile iron rod to extract the meat. Lab blender was sterilized with 70%

ethanol. Fifty gram of sample was homogenized in 450 ml of Ringers solution using the lab

blender for 2 mins at medium speed. The homogenate was used for serial dilution (1:10).

Aliquot (0.1 ml) of appropriately diluted sample was used for determination of bacterial

counts.
37
Figure 2: Edible land snails (Achatina achatina).

38
3.3 Determination of Total Aerobic Plate Count

Plate count agar medium was used. It was prepared according to manufacturer‘s

instructions and maintained at 450C. Aliquot (0.1 ml) of appropriate dilution (10-6-10-8) of the

homogenate was pipetted into sterile petri dish and the molten agar media was poured into the

petri dish. The plate was swirled to mix the homogenate with the agar media. It was done in

triplicates for each sample. Plates were incubated aerobically at 370C for 24 hours after which

colonies were counted and recorded.

3.4 Determination of Coliform Count

MacConkey agar medium was used which was prepared according to manufacturer‘s

instructions. Aliquot (0.1 ml) of appropriate dilution was plated out on the agar medium. It

was done in triplicates for each sample. Plates were incubated aerobically at 37 0C for 24

hours after which pink colonies were counted and recorded.

3.5 Determination of the Prevalence of Selected Presumptive Pathogens

Salmonella-Shigella agar, Eosin Methylene Blue agar (EMB), Mannitol Salt agar

(MSA), Thiosulfate Citrate Bile salt Sucrose agar (TCBS) and Brain Heart Infusion agar

(BHI) were used and were prepared according to manufacturer‘s instructions.

Aliquot (0.1 ml) of appropriate dilution was directly plated out on appropriate agar media,

except Brain Heart Infusion agar, specific for each pathogen and incubated aerobically at

370C for 24 hours. It was done in triplicates for each sample. Typical colonies were counted

and recorded after 24 hours.

39
 Appropriate dilutions were heated in a water bath at 80 0C for 10 mins before being

plated on Brain Heart Infusion agar. It was done in triplicates for each sample. Plates were

incubated aerobically at 370C for 24 hours after which typical colonies of Bacillus cereus

were counted and recorded.

3.6 Isolation and Identification of Selected Bacterial Pathogens

The procedure for identification of the six selected pathogens were based on the

United Kingdom Standards for Microbiology Investigations as published by Public Health

England (2014 and 2015).

3.6.1 Isolation and Identification of Salmonella

Aliquot (5 ml) of the homogenate was enriched in Selenite F broth (45 ml) for 24

hours, after which a loopful was streaked on Salmonella-Shigella agar and aerobically

incubated at 370C for 24 hours. Presumptive colonies (white colonies with black centres) were

subcultured in Tryptose Soy agar and solates were subjected to further tests such as Gram

staining, catalase test, motility test, oxidase test, indole test, urease test and triple sugar iron

test.

3.6.2 Isolation and Identification of Shigella

Presumptive colonies (white colonies without black centres) on Salmonella-shigella

agar were subcultured in Tryptose Soy agar and isolates were subjected to further tests such as

Gram staining, catalase test, motility test, oxidase test, urease test and glucose fermentation

test.

40
3.6.3 Isolation and Identification of Escherichia coli

Aliquot (5 ml) of the homogenate was enriched in lactose broth (20 ml) for 18 hours,

after which a loopful was streaked on EMB agar and aerobically incubated at 37 0C for 24

hours. Presumptive colonies (blue-black colonies with green metallic sheen and dark centres)

on EMB agar were streaked on sorbitol MacConkey agar and subcultured in Tryptose Soy

agar and isolates were subjected to further tests such as Gram staining, catalase test, indole

test, urease test and citrate test.

3.6.4 Isolation and Identification of Staphylococcus aureus

Aliquot (5 ml) of the homogenate was enriched in Nutrient broth containing 3% NaCl

(20 ml) for 24 hours, after which a loopful was streaked on Mannitol Salt agar and aerobically

incubated at 370C for 24 hours. Presumptive colonies (yellow colonies) were subcultured in

Tryptose Soy agar and isolates were subjected to further tests such as Gram staining, catalase

test and coagulase test.

3.6.5 Isolation and Identification of Vibrio

Aliquot (5 ml) of the homogenate was enriched in Nutrient broth containing 3% NaCl

(20 ml) for 24 hours, after which a loopful was streaked on Thiosulfate Citrate Bile salt

Sucrose (TCBS) agar and aerobically incubated at 37 0C for 24 hours. Presumptive colonies

(yellow colonies) on TCBS agar were subcultured in Tryptose Soy agar and isolates were

subjected to further tests such as Gram staining, oxidase test and motility test.

41
3.6.6 Isolation and Identification of Bacillus cereus

Aliquot (5 ml) of the homogenate was heated in a water bath at 80 0C for 10 mins and

enriched in BHI broth (20 ml) for 24 hours, after which a loopful was streaked on BHI agar

and aerobically incubated at 37 0C for 24 hours. Presumptive colonies (raised grey colonies)

on BHI agar were subcultured in Tryptose Soy agar and isolates were subjected to further

tests such as Gram staining, spore staining and motility test.

3.7 Biochemical Tests for Identification of Isolates

3.7.1 Catalase test

A drop of 3% hydrogen peroxide was placed on a clean grease-free glass slide. A

loopful of 24 hour-culture of the isolate was emulsified on the slide. Immediate appearance of

bubbles was a positive reaction.

3.7.2 Oxidase test

A drop of freshly prepared 1% solution of oxidase reagent was placed on a piece of

filter paper. With a wireloop, a colony of the isolate was collected and rubbed on the area of

the filter paper impregnated with oxidase reagent. Change of colour to purple blue in few

seconds was a positive result.

3.7.3 Motility test

The Sulphide Indole Motility (SIM) medium was used for this test. The medium was

prepared according to the manufacturer‘s instruction. The tubes containing the semi-solid

medium were stab-inoculated with 24- hour culture of the isolates and incubated at 37 0C for

36 hours. Presence of bacterial growth diffusing away from the line of inoculation was

positive result.

42
3.7.4 Glucose fermentation test
Twenty-four hour cultures of the isolates were inoculated in tubes each containing 9ml

of peptone water and 1ml of 1% glucose solution. Phenol red was used as indicator and

Durham‘s tube was also inserted. Tubes were incubated at 37 0C for 48 hours. Colour change

from red to pink was positive and presence of gas bubbles was indicated in the Durham‘s

tube.

3.7.5 Indole test

Twenty-four hour cultures of the isolates were inoculated in tubes each containing 4ml

of peptone water and incubated at 370C for 96 hours. To view results, freshly prepared

Kovac‘s reagent (0.5 ml) was added to the tubes. Presence of red colour within seconds in the

alcohol (top) layer was positive result.

3.7.6 Urease test

Urea agar medium was used for this test. Twenty-four hour cultures of the isolates

were inoculated on urea agar slant and incubated at 37 0C. The results were viewed after 4

hours and 24 hours of incubation. Presence of purple-pink colour was positive result.

3.7.7 Triple Sugar Iron test

Triple sugar iron (TSI) agar was used for this test. Twenty-four hour cultures of the

isolates were inoculated on TSI agar slant and incubated at 37 0C for 24 hours. Results were

reported as slant/butt/gas production/hydrogen sulphide production where applicable.

43
3.7.8 Citrate Utilization test
Simmon‘s citrate agar was used for this test. Twenty-four hour cultures of the isolates

were inoculated on the agar slant and incubated at 37 0C for 24 hours. Presence of blue colour

was a positive result.

3.7.9 Coagulase test

Twenty-four hour broth cultures of the isolates (0.8 ml) were inoculated into (0.2 ml)

plasma in tubes and gently mixed before incubation for 3 hours. Presence of clotting in the

tube indicated a positive result.

3.7.10 Spore staining

A smear of the twenty-four hour broth culture was made on a clean slide and steam-

fixed on a beaker of boiling water until the underside of the slide was covered with drops of

condensed water. The smear was then flooded with 5% aqueous malachite green and warmed

for about 1 min before rinsing with cold water. Safranin (0.5%) was added for 30 secs and

rinsed. The slide was blotted dry before examining under the microscope. Spores appeared

green while vegetative cells appeared red.

3.8 Determination of Virulence Potentials of Presumptive Pathogenic Isolates

3.8.1 Haemolysis test

Blood agar supplemented with 5% human blood was used. Isolates were streaked on

the blood agar and incubated at 35 0C for 24 hours. Greenish or clear zones of haemolysis

around colonies indicated alpha and beta haemolysis respectively.

44
3.8.2 Protease test
Nutrient agar supplemented with 1% casein was used. Isolates were streaked on the

medium and incubated at 370C for 24 hours. Zone of clearing around colonies was a positive

result.

3.8.3 Starch hydrolysis test

Nutrient agar supplemented with 1% starch was used. Isolates (0.1ml) were spot

inoculated on the medium and incubated at 370C for 24 hours. Before examining plates, plates

were flooded with iodine solution. Zone of clearing around colonies was a positive result.

3.8.4 Gelatinase test

Nutrient agar supplemented with 1.2% gelatin was used. Isolates were streaked on the

medium and incubated at 370C for 24 hours. Before examining plates, plates were flooded

with acid mercuric chloride solution. Zone of clearing around colonies was a positive result.

3.8.5 Lecithinase test

Nutrient agar supplemented with 10% egg yolk emulsion was used. Isolates were spot

inoculated on the medium and incubated at 37 0C for 3-7 days. Zone of precipitation/opacity

around the colonies was positive result.

45
3.8.6 Biofilm formation assay
Brain heart infusion (BHI) agar supplemented with 5g of sucrose and 0.8g of Congo

red dye per 100ml of BHI agar was used. Isolates were streaked on the medium and incubated

at 370C for 24 hours. Black colonies with dry crystalline consistency indicated positive result.

3.9 Determination of Antibiotic Resistance in Presumptive Pathogenic Isolates

Kirby-Bauer disc diffusion method was used. The methodology in the CLSI M02-A12

Supplement (CLSI, 2015) was followed in this study. The isolates were emulsified in saline to

a 0.5 McFarland opacity standard and inoculated on Mueller Hinton agar using swab stick.

After 30 mins, discs of antibiotics (Ampicillin, Amoxycillin/Clavulanic acid, Ceftazidime,

Cefotaxime, Cephalexin, Ciprofloxacin, Gentamicin, Nalidixic acid, Pefloxacin, Septrin,

Streptomycin, Ofloxacin, Cloxacillin, Amoxycillin, Chloramphenicol, Erythromycin,

Levofloxacin, Norfloxacin and Rifampicin) were placed on the cultured plates. Plates were

incubated aerobically at 370C for 24 hours. Zones of inhibition were measured with a ruler

and recorded. The results of the antibiotic sensitivity test of the isolates were interpreted

according to the M100 supplement (CLSI, 2017).

3.10 Identification of Selected Isolates Using 16S rRNA Gene Sequencing

Ten bacterial isolates found to possess some virulence potentials were subjected to

identification by 16S rRNA gene sequencing.

3.10.1 Extraction of DNA

DNA was extracted from the bacterial isolates using ZR Fungal/Bacterial DNA

miniprepTM kit (Zymo Research, USA) in accordance with the manufacturer‘s instructions.
46
Briefly, 50-100 mg (wet weight) bacterial cells were washed and resuspended in 200 µl of

sterile phosphate buffered saline (PBS) in a ZR BashingBeadTM Lysis Tube. The cells were

then processed in a cell disruptor at maximum speed for 5 mins. A lysis solution (750 µl) was

added to help lyse cells during the mechanical lysis step. The ZR BashingBead TM Lysis Tube

containing the cells were centrifuged at 10,000 x g for 1 min. The supernatant (400 µl) of the

lysed solution was filtered into the Collection Tube using a Zymo-Spin TM IV Spin Filter and

centrifuged at 7,000 x g for 1 min. Fungal/Bacterial DNA Binding Buffer (1,200 µl) was

added to the filtrate in the Collection Tube. About 800 µl of the mixture from the last step was

transferred to a Zymo-SpinTM IIC Column in a Collection Tube and centrifuged at 10,000 x g

for 1 min. The flow through from the Collection Tube was discarded and the last step was

repeated. DNA pre-wash buffer (200 µl) was added to the Zymo-SpinTM IIC Column and

centrifuged at 10,000 x g for 1 min. After which it was washed with 500 µl Fungal/Bacterial

DNA wash buffer. Then, the column was transferred to a clean 1.5 ml microcentrifuge tube

and 100 µl of DNA Elution Buffer was added directly to the column matrix, and then

centrifuged at 10,000 x g for 30 seconds to elute the DNA.

3.10.2 PCR Amplification

Amplification of 16S rRNA gene was carried out in a thermocycler (Applied

Biosystems, USA) using the following Primers: 27F: AGAGTTTGATCMTGGCTCAG and

1525R: AAGGAGGTGWTCCARCCGCA. The PCR program was carried out in 10 µl

reaction volumes containing 2 µl DNA template (10 ng/µl), 1 µl 10xPCR buffer, 1 µl Mgcl 2

(25mM), 0.5 µl of each primer (5 pMol/µl), 0.1 µl Taq DNA polymerase (5 U/µl), 0.8 µl

dNTPs (2.5 mM/µl), 1 µl DMSO and 3.1 µl of sterile water. The PCR amplification has initial

47
DNA denaturation at 940C for 5 mins, followed by 36 cycles of denaturation at 94 0C for 30

secs, annealing at 560C for 30 secs and extension at 720C for 45 secs which was followed by a

final extension at 720C for 7 mins. The amplicon (5 µl) was analysed by electrophoresis (Bio-

Rad) in 1.5% agarose gel (InvitrogenTM) at 100 volts for 45 mins followed by staining with

1% solution of ethidium bromide (50 µl/l) and destaining with Tris-acetate-EDTA buffer for

10 mins. A 50-bp DNA ladder (NEB) was used as a molecular marker. Gels were visualized

by UV transillumination and recorded with Polaroid 667 instant film (Lane, 1991).

3.10.3 Sequencing and Analysis

Purified DNA mixture (4 µl) was added to 4 µl of Big-Dye Terminator Reaction Mix

(Applied Biosystems, USA), followed by 1.6 µl of primer and 0.4 µl of deionized water. The

sequencing reaction was then purified using ethanol/EDTA precipitation method. Sequences

of the fragments were determined using 3130xl genetic analyzer (Applied Biosystems) at

International Institute of Tropical Agriculture, Ibadan.

The sequences were edited to remove the PCR primer binding sites and manually

corrected using Finch TV version 1.4.0. The sequences of the isolates were automatically

compared, using the BLAST, against reference sequences of bacteria available in databank

with accession date: 7th July 2018 (www.ncbi.nlm.nih.gov/). The identity of each isolate was

determined based on the highest % sequence similarity to its relatives in the databank. The

16S rRNA sequence data for the bacterial isolates sequenced in this study were deposited in

the Genbank and accession numbers were obtained.

48
3.11 Detection of Specific Toxin Genes by PCR in Selected Isolates Recovered From

Snails

Some isolates (Staphylococcus and Bacillus) that were found to demonstrate some

virulence potentials, based on phenotypic tests, were selected for detection of toxin genes by

polymerase chain reaction (PCR) method. DNA was extracted from the selected bacterial

isolates using ZR Fungal/Bacterial DNA miniprep TM kit (Zymo Research, USA) in

accordance with the manufacturer‘s instructions as discussed earlier (Section 3.10.1).

Touchdown PCR was done according to Don et al. (1991) to detect the presence of

five toxin genes (hbla, hblc, nhea, nheb and cytk) in selected Bacillus isolates and two toxin

genes (sea and exhc) in selected Staphylococcus isolates. The primer sequences used in this

study for the amplification of the toxin genes are listed in Table 2. The primers were

synthesized by Integrated DNA Technologies (IDT), Belgium.

49
Table 2: Primers used in this study for the detection of toxin genes in selected bacterial
isolates recovered from snails

Target gene Primer sequence (5’-3’) Amplicon size (Base pairs)

Sea F-TAA GGA GGT GGT GCC TAT GG 180
R-CAT CGA AAC CAG CCA AAG TT
Exhc F-GAATAAATATTATGGAGTCTCTCCTGATC 525
R-CCATAGTATTTCAATCCAAAATCAGTAC
Hbla F-CAAGGTGCAGATGTTGATGC 352
R-GAACGCCCGAATATTGAG
Hblc F-AATGGTCATCGGAACTCTAT 731
R-CTCGCTGTTCTGCTGTTAAT
Nhea F-TACGCTAAGGAGGGGCA 500
R-GTTTTTATTGCTTCATCGGCT
Nheb F-CTATCAGCACTTATGGCAG 770
R-ACTCCTAGCGGTGTTCC
Cytk F-ACAGATATCGG(GT)CAAAATGC 809
R-TCCAACCCAGTT(AT)(GC)CAGTTC
(Chen et al., 2007; Nazari et al., 2014; Hansen and Hendriksen, 2001; Owusu-Kwarteng et al., 2017)

50
 For PCR amplification, the reaction mixture (25 µl) contained 3.0 µl of the appropriate

DNA template (10 ng/µl), 1 µl of primer F (5 pMol/µl), 1 µl of primer R (5 pMol/µl), 1.0 µl

Dimethyl sulphoxide, 2.0 µl of Deoxynucleoside triphosphates (2.5 Mmol/l), 2.5 µl of 10 ×

PCR buffer, 1.0 µl of MgCl2 (25 mM/l), 0.1 µl of Taq DNA polymerase (5 U/µl), and 13.4 µl

of distilled water in each reaction tube. The reaction was performed in a thermal cycler

(GeneAMP 9700, Applied Biosystems, USA) with initial denaturation at 94°C for 5 min

followed by 9 cycles of denaturation at 94°C for 15 sec, primer annealing at 55°C for 20 sec

and extension at 72°C for 30 sec, followed by 35 cycles of denaturation at 94°C for 15 sec,

primer annealing at 45°C for 20 sec and extension at 72°C for 30 sec, followed by a final

extension at 72°C for 7 min, after which a hold temperature at 10°C was maintained. The

presence of amplicons was determined by electrophoresis of the reaction products (10 µl) in a

1.5% agarose gel at 100V for 1.5 hr with 1X SB buffer (22.5 g of anhydrous boric acid/litre

and 4g of NaOH/litre) and a 50-bp DNA ladder (NEB) as a molecular marker. Gels were

stained with ethidium bromide solution (5 µl of 10 mg/ml) and documentation was done using

the Enduro gel system.

3.12 Determination of the Processing Method that Will Reduce the Bacterial Load of

Snail Meat During Culinary Preparation.

High loads of bacteria, which included pathogens, were found in samples of A.

achatina studied. This study was carried out to determine the efficacy of the different methods

used in processing and cooking snails in eliminating these bacteria in order to ensure safety of

snail meat to consumers.

51
3.12.1 Collection of samples for processing and cooking
Hundred samples of live snails (Achatina achatina) were collected from Ogbete main

market, Enugu. The weight of each of the sample before shucking ranged from 120g – 195g.

Each sample was collected into a sterile plastic container with lid and transported to the

laboratory for processing, cooking and bacteriological analysis.

3.12.2 Processing and cooking of samples

The samples were randomly divided into five batches representing different

processing methods. All utensils (bowls, spoons and knives) were washed with hot water and

detergent. Utensils were also sterilized with 70% ethanol and dried with commercially

available sterile paper towel. Water for processing and cooking was autoclaved at 121 0C for

15 mins before use. For each batch of samples, the shells were surface sterilized with 70%

ethanol and subjected aseptically to stages involved in processing and cooking which are

shucking, evisceration, desliming, washing, parboiling and cooking (Figure 3). Five different

desliming agents (potassium alum, wood ash, citrus lime, garri and cassava-retting water)

were used for each processing method. During processing, the average contact time with the

desliming agent was 5 mins. However, the desliming stage was concluded when the samples

had lost the slimy texture.

52
 SHUCKING
(Removal of shells)

EVISCERATION

DESLIMING
(Washing with desliming agent for 5 mins)

WASHING

PARBOILING
(6 mins)

COOKING
(Boiling with bitter leaf for 10 mins)

Figure 3: Stages of processing and cooking of edible snail.

53
3.12.3 Bacteriological Analysis
For each batch, samples (50g) were collected at each stage of processing and cooking

for bacteriological analysis. Each sample was homogenized in 450ml of Ringers solution

using a lab blender for 2 mins at medium speed. The homogenate was used for serial dilution

(1:10). Aliquot (0.1ml) of appropriate dilution was plated on seven different media for

enumeration of six selected presumptive pathogens namely: Salmonella, Shigella,

Staphylococcus aureus, Escherichia coli, Vibrio and Bacillus cereus. The media were: Plate

count agar, MacConkey agar, Salmonella-shigella agar, EMB agar, Mannitol salt agar, TCBS

agar and BHI agar.

Total aerobic plate count, coliform count and viable counts for each selected pathogen

were determined using the methods discussed earlier (sections 3.3 - 3.5). Typical colonies

were counted and recorded.

3.13 Data Analysis

Descriptive statistics such as means and frequencies were used to present some of the

findings. All data on plate counts were converted to logarithmic value. Analysis of variance

(ANOVA) was performed using statistical web version software package available in

Vassarstat website (http://vassarstats.net/).

Phylogenetic trees were constructed by the neighbour-joining method using web

version software package of Phylogeny.fr (http://www.phylogeny.fr/index.cgi).

54
 Chapter Four

RESULTS
4.1 Total aerobic plate count in edible land snails
The mean aerobic plate counts of samples ranged from 8.43 Log CFU/g - 9.61 Log

CFU/g. Samples from Ogbete market had the highest mean total aerobic plate count (9.32 ±

0.308 Log CFU/g) while the lowest mean count was found in Igboukwu samples (8.74 ±

0.312 Log CFU/g) (Figure 4). ANOVA (Appendix 3) revealed significant differences between

total aerobic plate counts of the following samples: Igboukwu and Abakaliki samples (p <

0.05), Igboukwu and Ogbete samples (p < 0.01), Abakaliki and Ogbete samples (p < 0.01).

4.2 Coliform count in edible land snails

Samples from Abakaliki market had the highest mean count of coliforms (7.63 ± 0.389 Log

CFU/g) followed by Ogbete samples (7.49 ± 0.358 Log CFU/g) and Igboukwu samples (7.41

± 0.191 Log CFU/g) (Figure 5). ANOVA (Appendix 4) indicated significant differences

between coliform counts of the following samples: Igboukwu and Abakaliki samples (p <

0.01), Abakaliki and Ogbete samples (p < 0.05). There was no significant difference between

Igboukwu and Ogbete samples.

4.3 Viable counts of presumptive bacterial pathogens in edible land snails

The edible land snails were analysed for viable counts of colonies characteristics of

the following pathogens: Salmonella, Shigella, E. coli, Staphylococcus, Vibrio and Bacillus

on Salmonella-Shigella agar, Eosin Methylene Blue agar (EMB), Mannitol Salt agar (MSA),

Thiosulfate Citrate Bile salt Sucrose agar (TCBS) and Brain Heart Infusion agar (BHI)

respectively.

55
 The highest mean presumptive Salmonella count was found in Abakaliki samples

(7.24 ± 0.210 Log CFU/g) followed by Igboukwu and Ogbete samples with 6.39 ± 0.114 Log

CFU/g and 6.37 ± 0.219 Log CFU/g respectively (Figure 6). ANOVA (Appendix 5) showed

that there were significant differences between presumptive Salmonella counts of the

following samples: Igboukwu and Abakaliki samples (p < 0.01), Abakaliki and Ogbete

samples (p < 0.01). There were no significant differences between Igboukwu and Ogbete

samples (p < 0.01).

Abakaliki samples were found to contain the highest mean presumptive Shigella

count (4.61 ± 0.354 Log CFU/g) followed by Igboukwu (4.43 ± 0.284 Log CFU/g) and

Ogbete samples (4.03 ± 0.571 Log CFU/g) as shown in Figure 7. ANOVA (Appendix 6)

revealed significant differences between presumptive Shigella counts of the following

samples: Igboukwu and Ogbete samples (p < 0.01), Igboukwu and Enugu samples (p < 0.01),

Abakaliki and Ogbete samples (p < 0.01).

The highest mean count of presumptive E. coli was found in Igboukwu samples

(7.14 ± 0.170 Log CFU/g) followed by Abakaliki (6.95 ± 0.179 Log CFU/g) and Ogbete

samples (5.65 ± 0.239 Log CFU/g) as shown in Figure 8. ANOVA (Appendix 7) confirmed

significant differences between all groups of samples analysed (p < 0.01).

The highest mean count of Staphylococcus was found in Abakaliki samples (4.74

± 0.192 Log CFU/g) followed by Ogbete samples (4.66 ± 0.757 Log CFU/g). Staphylococcus

was not detected in Igboukwu samples (Figure 9). There was no significant difference

between Abakaliki and Ogbete samples (Appendix 8).

56
 The highest mean count of presumptive Vibrio was found in Ogbete samples (4.80 ±

0.473 Log CFU/g) followed by Abakaliki (3.44 ± 0.197 Log CFU/g) and Igboukwu samples

(3.10 ± 0.052 Log CFU/g) (Figure 10). There were significant differences between all groups

of samples analysed (p < 0.01) (Appendix 9).

Presumptive Bacillus cereus counts revealed that Abakaliki samples had the highest

mean count of presumptive Bacillus cereus (4.50 ± 0.136 Log CFU/g) followed by Ogbete

(3.48 ± 0.135 Log CFU/g) and Igboukwu samples (3.25 ± 0.130 Log CFU/g) as shown in

Figure 11. There were significant differences between all groups of samples analysed (p <

0.01) (Appendix 10).

4.4 Prevalence of selected presumptive pathogens in edible land snails

All 300 samples of edible land snails analysed in this study were found to be

contaminated with presumptive Salmonella species irrespective of the source of the samples

(Table 3). Presumptive Shigella species were recovered from 60% of all samples analysed,

with the highest frequency occurring in Ogbete samples (80%) followed by Abakaliki

samples (60%) and Igboukwu samples (40%). Presumptive E. coli was detected in 90% of all

samples analysed. Presumptive E. coli was found in 100%, 90% and 80% of Ogbete,

Igboukwu and Abakaliki samples respectively. Staphylococcus was recovered from 36.7% of

all samples analysed, 30% and 80% of Abakaliki and Ogbete samples respectively. No

Staphylococcus was found in Igboukwu samples. Presumptive Vibrio species was recovered

from 76.6% of all samples analysed: 90%, 70% and 70% of Ogbete, Igboukwu and Abakaliki

samples were found to be contaminated by presumptive Vibrio respectively. Presumptive B.

cereus was detected in 80% of all samples analysed: 90%, 80% and 70% of Ogbete, Abakaliki

and Igboukwu samples respectively (Table 3).

57
 Igboukwu Abakaliki Ogbete

MARKETS

Figure 4: Mean Aerobic Plate Counts of bacteria in edible land snails from
three major markets in South East Nigeria.

58
 Igboukwu Abakaliki Ogbete

MARKETS

Figure 5: Mean Counts of Coliforms in edible land snails from three major
markets in South East Nigeria.

59
 Igboukwu Abakaliki Ogbete

MARKETS

Figure 6: Mean Counts of presumptive Salmonella in edible land snails

from three major markets in South East Nigeria

60
 Igboukwu Abakaliki Ogbete

MARKETS

Figure 7: Mean Counts of presumptive Shigella in edible land snails from

three major markets in South East Nigeria.

61
 Igboukwu Abakaliki Ogbete

MARKETS

Figure 8: Mean Counts of presumptive E. coli in edible land snails from

three major markets in South East Nigeria.

62
 Igboukwu Abakaliki Ogbete

MARKETS

Figure 9: Mean Counts of Staphylococcus in edible land snails from three

major markets in South East Nigeria.

63
 Igboukwu Abakaliki Ogbete

MARKETS

Figure 10: Mean Counts of presumptive Vibrio in edible land snails from
three major markets in South East Nigeria.

64
 Igboukwu Abakaliki Ogbete

MARKETS

Figure 11: Mean Counts of presumptive Bacillus cereus in edible land snails
from three major markets in South East Nigeria.

65
Table 3: Prevalence of presumptive pathogens in edible land snails from
three major markets in South East.
Presumptive Igboukwu Abakaliki Ogbete Total
Pathogen (%) (%) (%) (%)
Salmonella 100 100 100 100

Shigella 40 60 80 60

E. coli 90 80 100 90

Staphylococcus NIL 30 80 36.7

Vibrio 70 70 90 76.6

B. cereus 70 80 90 80

66
The prevalence of different levels of bacterial loads in edible snails analysed in this study

showed that all samples had total aerobic plate counts >108 CFU/g. Most samples (86.7%) had

coliform counts ranging from >106 to 108 CFU/g. Presumptive Salmonella counts ranged

from >106 to 108 CFU/g in all the samples, presumptive Shigella count was <104 CFU/g in

35% of the samples. Presumptive E. coli count was >106 to 108 CFU/g in 60% of the samples.

The presumptive staphylococci count was >10 4 to 106 CFU/g in 26.7% of the samples.

Presumptive Vibrio count was <104 CFU/g in 43.3%, presumptive Bacillus cereus count was

<104 CFU/g in 60% of the samples (Table 4).

4.5 Prevalence of virulence potentials among presumptive pathogenic isolates in edible

land snails
The results of the prevalence of virulence potentials in pathogens isolated from

Igboukwu, Abakaliki and Ogbete samples are presented in Tables 5-10.

One hundred percent and 50% of the presumptive Salmonella isolates in Abakaliki

and Ogbete samples respectively were found to be haemolytic while none of the Igboukwu

isolates were haemolytic. Protease activity was detected in 65.6%, 0.0% and 33.3% of

presumptive Salmonella isolates recovered from Igboukwu, Abakaliki and Ogbete samples

respectively. Also, Biofilm formation test was positive in 65.6%, 70.0% and 33.3% of

presumptive Salmonella isolates recovered from Igboukwu, Abakaliki and Ogbete samples

respectively (Table 5).

67
 Table 6 showed that haemolytic activity was detected in 0.0%, 33.3% and 50% of

Shigella isolates from Igboukwu, Abakaliki and Ogbete samples respectively. Protease

activity was detected only in Shigella isolates from Ogbete samples. None of the Shigella

isolates was found to be potential biofilm producers.

As shown in Table 7, haemolytic activity was detected in only E. coli isolates (40%)

recovered from Igboukwu samples. Abakaliki (50%) and Ogbete (40%) E. coli isolates were

found to be proteolytic. Moreover, Igboukwu (50%), Abakaliki (25%) and Ogbete (30%) E.

coli isolates were found to exhibit potentials for biofilm formation.

Prevalence of virulence potentials in Staphylococcus isolates from snails are presented

in Table 8. All staphylococci isolates recovered from Abakaliki samples were found to be

positive for haemolysis, gelatinase, protease, lecithinase and biofilm formation tests. Also, the

staphylococci isolates from Ogbete samples were all found to be positive for all virulence

associated tests, except biofilm formation (51.2%).

Prevalence of virulence potentials in presumptive Vibrio isolates from snails are

presented in Table 9. All presumptive Vibrio isolates from Igboukwu samples were found to

be positive for all virulence associated tests, except biofilm formation (70%). Also,

presumptive Vibrio isolates from Abakaliki and Ogbete samples were positive for lecithinase

and amylase tests. On the other hand, 50% of Abakaliki presumptive Vibrio isolates were

positive for haemolysin, while Ogbete isolates were found to be negative. About 30% and

50% of presumptive Vibrio isolates from Ogbete samples were positive for biofilm formation

and protease tests respectively. Also, 66.6% and 50% of presumptive Vibrio isolates from

Abakaliki samples were positive for biofilm formation and protease tests respectively.

68
 Prevalence of virulence potentials in presumptive Bacillus cereus isolates from market

snails are presented in Table 10. All presumptive Bacillus cereus isolates recovered from the

three states were found to be positive for gelatinase and lecithinase tests. All presumptive

Bacillus cereus isolates from Igboukwu and Abakaliki samples were positive for haemolysin

and 50% of Ogbete isolates were positive. About 51%, 50% and 33.3% of presumptive

Bacillus cereus isolates from Igboukwu, Abakaliki and Ogbete samples were positive for

biofilm formation tests respectively. Generally, > 70% of presumptive Bacillus cereus isolates

recovered from the three states exhibited proteolytic activity.

69
Table 4: Prevalence of different bacterial loads in edible land snails from three major markets in South East Nigeria.

Bacterial <104 CFU/g >104 – 106 CFU/g >106 – 108 CFU/g >108 CFU/g (%) Total (%)
b c
Counts (%) (%) (%) N=300
a
APC NIL NIL NIL 100 100

Coliforms NIL NIL 86.7 13.3 100

Salmonella NIL NIL 100 NIL 100

Shigella 35 25 NIL NIL 60

E. coli NIL 30 60 NIL 90

Staphylococcus 10 26.7 NIL NIL 36.7

Vibrio 43.3 33.3 NIL NIL 76.6

Bacillus cereus 60 20 NIL NIL 80

a
APC: Aerobic Plate Count.
b
%: Percent of samples with bacterial load.
c
N: Number of samples analysed.
NIL: Bacterial colonies not present.

70
Table 5: Prevalence of virulence potentials in presumptive Salmonella species isolated
from edible land snails in three major markets in South East Nigeria.
Virulence Igboukwu (aN=64) Abakaliki (N=50) Ogbete (N=72)
b
Property No.(%) No.(%) No.(%)
Haemolysin NIL 50 (100) 36(50)
Protease 42(65.6) NIL 24(33.3)
Biofilm formation 42(65.6) 35(70.0) 24(33.3)
a
N: Number of isolates tested.
b
No.(%): Number of positive isolates (Percent of isolates positive).
NIL: None tested positive.

71
Table 6: Prevalence of virulence potentials in Shigella species isolated from edible land
snails in three major markets in South East Nigeria.
Virulence Igboukwu (aN=25) Abakaliki (N=30) Ogbete (N=38)
b
Property No.(%) No.(%) No.(%)
Haemolysin NIL 10(33.3) 19(50)
Protease NIL NIL 13(34.2)
Biofilm formation NIL NIL NIL
a
N: Number of isolates tested.
b
No.(%): Number of positive isolates (Percent of isolates positive).
NIL: None tested positive.

72
Table 7: Prevalence of virulence potentials in E. coli isolated from edible land snails in
three major markets in South East Nigeria.
Virulence Igboukwu (aN=50) Abakaliki (N=44) Ogbete (N=70)
b
Property No.(%) No.(%) No.(%)
Haemolysin 20(40) NIL NIL
Protease NIL 22(50) 28(40)
Lecithinase NIL NIL NIL
Biofilm formation 25(50) 11(25) 21(30)
a
N: Number of isolates tested.
b
No.(%): Number of positive isolates (Percent of isolates positive).
NIL: None tested positive.

73
Table 8: Prevalence of virulence potentials in Staphylococcus isolated from edible land
snails in three major markets in South East Nigeria.
*
Virulence Abakaliki (aN=25) Ogbete (N=41)
b
Property No.(%) No.(%)
Haemolysin 25(100) 41(100)
Gelatinase 25(100) 41(100)
Protease 25(100) 41(100)
Amylase NIL 41(100)
Lecithinase 25(100) 41(100)
Biofilm formation 25(100) 21(51.2)
*
Note: Staphylococcus was not detected in samples from Igboukwu market.
a
N: Number of isolates tested.
b
No.(%): Number of positive isolates (Percent of isolates positive).
NIL: None tested positive.

74
Table 9: Prevalence of virulence potentials in presumptive Vibrio species isolated from
edible land snails in three major markets in South East Nigeria.

Virulence Igboukwu (aN=32) Abakaliki (N=40) Ogbete (N=45)

b
Property No.%) No.(%) No.(%)
Haemolysin 32(100) 20(50) NIL
Gelatinase 32(100) 25(62.5) 45(100)
Protease 32(100) 20(50) 30(66.6)
Amylase 32(100) 40(100) 45(100)
Lecithinase 32(100) 40(100) 45(100)
Biofilm formation 24(75) 20(50) 15(33.3)
a
N: Number of isolates tested.
b
No.(%): Number of positive isolates (Percent of isolates positive).
NIL: None tested positive.

75
Table 10: Prevalence of virulence potentials in Bacillus cereus isolated from edible land
snails in three major markets in South East Nigeria.

Virulence Igboukwu (aN=35) Abakaliki (N=32) Ogbete (N=66)

b
Property No.(%) No.(%) No.(%)
Haemolysin 35(100) 32(100) 33(50)
Gelatinase 35(100) 32(100) 66(100)
Protease 28(80) 32(100) 66(100)
Amylase 10(28.5) 8(25) 33(50)
Lecithinase 35(100) 32(100) 66(100)
Biofilm formation 18(51.4) 16(50) 22(33.3)
a
N: Number of isolates tested.
b
No.(%): Number of positive isolates (Percent of isolates positive).

76
4.6 Prevalence of antibiotic resistance in presumptive pathogenic isolates in edible land
snails
The prevalence of antibiotic resistance in selected pathogens isolated from edible land

snails are presented in Tables 11-16.

Results showed that among the presumptive Salmonella isolates recovered from the

three markets, Ogbete isolates were found to be resistant to most antibiotics compared to

isolates from Igboukwu and Abakaliki samples. Fifty percent, 35%, 30%, 50%, 30%, 25%

and 35% of the presumptive Salmonella isolates in Ogbete samples were found to be resistant

to ampicillin, amoxycillin/clavulanic acid, ceftazidime, ceftriaxone, gentamicin, nalidixic acid

and streptomycin respectively. Most Abakaliki presumptive Salmonella isolates (70%) were

resistant to ceftriaxone and nalidixic acid, while 80% of Igboukwu isolates were resistant to

amoxycillin/clavulanic acid. However, all presumptive Salmonella isolates from these states

were sensitive to ciprofloxacin, pefloxacin, septrin and ofloxacin (Table 11).

The prevalence of antibiotic resistance in Shigella isolates recovered from edible land

snails showed that 30%, 65%, 25% and 65% of the Ogbete Shigella isolates were resistant to

ampicillin, ceftazidime, cephalexin and ceftriaxone respectively. Abakaliki isolates (20%)

were resistant to only ceftriaxone, while Igboukwu isolates were sensitive to all antibiotics

used in this study (Table 12).

The prevalence of antibiotic resistance in E. coli isolates recovered from edible land

snails showed that most E. coli isolates recovered from Igboukwu (75%) and Abakaliki (60%)

samples were resistant to ceftazidime. Some Ogbete isolates (25%, 10% and 30%) were

resistant to amoxycillin/clavulanic acid, ceftazidime and ceftriaxone, respectively. Twenty

percent, 75%, 50%, and 45% of Igboukwu E. coli isolates were resistant to

amoxycillin/clavulanic acid, ceftazidime, ceftriaxone, and streptomycin, respectively. Thirty

77
percent, 40%, 60%, 20% and 30% of Abakaliki E. coli isolates were resistant to ampicillin,

amoxycillin/clavulanic acid, ceftazidime, ceftriaxone and streptomycin, respectively. All E.

coli isolates recovered from the three markets were sensitive to ciprofloxacin, gentamicin,

pefloxacin, septrin and ofloxacin (Table 13).

The prevalence of antibiotic resistance in Staphylococcus isolates recovered from

edible land snails showed that Abakaliki (65% and 60%) and Ogbete (40% and 50%)

Staphylococcus isolates were resistant to chloramphenicol and norfloxacin, respectively.

Thirty percent of Abakaliki isolates were found to be resistant to cloxacillin, while Ogbete

isolates were sensitive to cloxacillin. All Staphylococcus isolates were sensitive to 70% of

antibiotics used in this study (Table 14).

The prevalence of antibiotic resistance in presumptive Vibrio isolates recovered from

edible land snails showed that 40%, 26.6% and 20% of Igboukwu presumptive Vibrio isolates

were resistant to ceftazidime, ceftriaxone and nalidixic acid, respectively. Also, 25%, 50%

and 40% of Abakaliki presumptive Vibrio isolates were resistant to ampicillin, ceftazidime

and ceftriaxone, respectively. Moreover, 40%, 45% and 35% of Ogbete presumptive Vibrio

isolates were resistant to ampicillin, amoxycillin/clavulanic acid and ceftriaxone, respectively.

All isolates of presumptive Vibrio were sensitive to 70% of antibiotics used in this study

(Table 15).

The prevalence of antibiotic resistance in presumptive Bacillus cereus isolates

recovered from edible land snails showed that 10%, 5%, 65% and 60% of Ogbete

presumptive B. cereus isolates were resistant to cloxacillin, amoxycillin, chloramphenicol and

norfloxacin, respectively. Abakaliki presumptive B. cereus isolates (50% and 75%) were

78
resistant to chloramphenicol and norfloxacin, respectively. A cloxacillin-resistant presumptive

B. cereus isolate was recovered from Igboukwu samples. However, all presumptive B. cereus

isolates were found to be sensitive to 60% of antibiotics used in this study (Table 16).

Generally, some isolates were found to exhibit multidrug resistance. Antibiotic resistance

patterns detected in these isolates are presented in Table 17.

A total of 15 different antibiotic resistance patterns were detected among the multi-

resistant isolates. Among the E. coli isolates, five types of antibiotic resistance patterns (4 and

1 patterns) were found in Igboukwu and Abakaliki samples, respectively. Among the

presumptive Vibrio isolates, four types of resistance patterns (2, 1 and 1 patterns) were found

in Abakaliki, Igboukwu and Ogbete samples, respectively. Among the presumptive

Salmonella isolates, three types of resistance patterns (1, 1 and 2 patterns) were found in

Igboukwu, Abakaliki and Ogbete samples, respectively. Two resistance patterns were

detected in Shigella, Staphylococcus and Bacillus isolates recovered from Ogbete and

Abakaliki samples.

Presumptive Salmonella isolates from Igboukwu and Abakaliki samples were each

found to exhibit multi-resistance to 5 antibiotics. Furthermore, the multi-resistant isolates

were found to be most resistant to Ceftazidime > Ceftriaxone > Ampicillin > Nalidixic acid =

Streptomycin = Amoxycillin/clavulanic acid = Norfloxacin = Chloramphenicol > Cephalexin

> Cloxacillin. Multi-drug resistance was most prevalent in Abakaliki and Igboukwu isolates

tested in this study.

79
Table 11: Prevalence of antibiotic resistance in presumptive Salmonella species isolated
from edible land snails in three major markets in South East Nigeria.
Antibiotics Igboukwu (aN=20) Abakaliki (N=40) Ogbete (N=40)
b
No.(%) No.(%) No.(%)
Ampicillin 4(20) 14(35) 20(50)
Amoxy/Clav. Acid 16(80) 14(35) 14(35)
Ceftazidime 7(35) 12(30) 12(30)
Ceftriaxone 5(25) 28(70) 20(50)
Cephalexin 4(20) NIL NIL
Ciprofloxacin NIL NIL NIL
Gentamicin NIL NIL 12(30)
Nalidixic acid 4(20) 28(70) 10(25)
Pefloxacin NIL NIL NIL
Septrin NIL NIL NIL
Streptomycin NIL NIL 14(35)
Ofloxacin NIL NIL NIL
a
N: Number of isolates tested,
b
No.(%): Number of resistant isolates (Percent of isolates resistant),
NIL: None.

80
Table 12: Prevalence of antibiotic resistance in Shigella species isolated from edible land
snails in three major markets in South East Nigeria.
Antibiotics Igboukwu (aN=20) Abakaliki (N=20) Ogbete (N=20)
b
No.(%) No.(%) No.(%)
Ampicillin NIL NIL 6(30)
Amoxy/Clav. Acid NIL NIL NIL
Ceftazidime NIL NIL 13(65)
Ceftriaxone NIL 4(20) 13(65)
Cephalexin NIL NIL 5(25)
Ciprofloxacin NIL NIL NIL
Gentamicin NIL NIL NIL
Nalidixic acid NIL NIL NIL
Pefloxacin NIL NIL NIL
Septrin NIL NIL NIL
Streptomycin NIL NIL NIL
Ofloxacin NIL NIL NIL
a
N: Number of isolates tested,
b
No.(%): Number of resistant isolates (Percent of isolates resistant),
NIL: None.

81
Table 13: Prevalence of antibiotic resistance in E. coli isolated from edible land snails in
three major markets in South East Nigeria.
Antibiotics Igboukwu (aN=20) Abakaliki (N=10) Ogbete (N=20)
b
No.(%) No.(%) No.(%)
Ampicillin NIL 3(30) NIL
Amoxy/Clav. Acid 4(20) 4(40) 5(25)
Ceftazidime 15(75) 6(60) 2(10)
Ceftriaxone 10(50) 2(20) 6(30)
Cephalexin NIL NIL NIL
Ciprofloxacin NIL NIL NIL
Gentamicin NIL NIL NIL
Nalidixic acid NIL NIL NIL
Pefloxacin NIL NIL NIL
Septrin NIL NIL NIL
Streptomycin 9(45) 3(30) NIL
Ofloxacin NIL NIL NIL
a
N: Number of isolates tested,
b
No.(%): Number of resistant isolates (Percent of isolates resistant),
NIL: None.

82
Table 14: Prevalence of antibiotic resistance in Staphylococcus isolated from edible land
snails in three major markets in South East Nigeria.
Antibiotics Abakaliki (N=20) Ogbete (N=30)
No.(%) No.(%)
Cloxacillin 6(30) NIL

Amoxycillin NIL NIL

Chloramphenicol 13(65) 12(40)

Ciprofloxacin NIL NIL

Erythromycin NIL NIL

Gentamicin NIL NIL

Levofloxacin NIL NIL

Norfloxacin 12(60) 15(50)

Rifampicin NIL NIL

Streptomycin NIL NIL

Note: Staphylococcus was not detected in any sample from Anambra state.
a
N: Number of isolates tested,
b
No.(%): Number of resistant isolates (Percent of isolates resistant),
NIL: None.

83
Table 15: Prevalence of antibiotic resistance in presumptive Vibrio species isolated from
edible land snails in three major markets in South East Nigeria.
Antibiotics Igboukwu (aN=30) Abakaliki (N=20) Ogbete (N=20)
b
No.(%) No.(%) No.(%)
Ampicillin NIL 5(25) 8(40)
Amoxy/Clav. Acid NIL NIL 9(45)
Ceftazidime 12(40) 10(50) NIL
Ceftriaxone 8(26.6) 8(40) 7(35)
Cephalexin NIL NIL NIL
Ciprofloxacin NIL NIL NIL
Gentamicin NIL NIL NIL
Nalidixic acid 6(20) NIL NIL
Pefloxacin NIL NIL NIL
Septrin NIL NIL NIL
Streptomycin NIL NIL NIL
Ofloxacin NIL NIL NIL
a
N: Number of isolates tested,
b
No.(%): Number of resistant isolates (Percent of isolates resistant),
NIL: None.

84
Table 16: Prevalence of antibiotic resistance in presumptive B. cereus isolated from
edible land snails in three major markets in South East Nigeria.
Antibiotics Igboukwu (aN=20) Abakaliki (N=20) Ogbete (N=40)
b
No.(%) No.(%) No.(%)
Cloxacillin 1(5) NIL 4(10)

Amoxycillin NIL NIL 2(5)

Chloramphenicol NIL 10(50) 26(65)

Ciprofloxacin NIL NIL NIL

Erythromycin NIL NIL NIL

Gentamicin NIL NIL NIL

Levofloxacin NIL NIL NIL

Norfloxacin NIL 15(75) 24(60)

Rifampicin NIL NIL NIL

Streptomycin NIL NIL NIL

a
N: Number of isolates tested,
b
No.(%): Number of resistant isolates (Percent of isolates resistant),
NIL: None.

85
Table 17: Antibiotic resistance patterns in pathogens isolated from edible land snails in
three major markets in South East Nigeria.
Antibiotic Resistance Pattern Presumptive Pathogen Source

AMP-CEP-NA-CTX-CAZ Salmonella Igboukwu

AMP-NA-AMC-CTX-CAZ Salmonella Abakaliki

S-AMC-CTX-CAZ E. coli Abakaliki

AMP-CTX-CAZ Vibrio Abakaliki

CLX-NOR-CHL Staphylococcus Abakaliki

AMC-CTX-CAZ E. coli Igboukwu

S-CTX-CAZ E. coli Igboukwu

S-CAZ E. coli Igboukwu

NA-CAZ Vibrio Igboukwu

NA-CAZ E. coli Igboukwu

CTX-CAZ Vibrio Abakaliki

NOR-CHL B. cereus Abakaliki

NOR-CHL Staphylococcus Abakaliki

CTX-CAZ Salmonella Ogbete

CTX-CAZ Shigella Ogbete

S-AMP Salmonella Ogbete

AMP-CEP Shigella Ogbete

AMP-AMC Vibrio Ogbete

NOR-CHL B. cereus Ogbete

AMP: Ampicillin, CEP: Cephalexin, NA: Nalidixic acid, CTX: Ceftriaxone, CAZ: Ceftazidime,
AMC: Amoxy/Clav. acid, S: Streptomycin, CLX: Cloxacillin, NOR: Norfloxacin, CHL: Chloramphenicol

86
4.7 Identification of selected bacterial pathogens in edible land snails using 16S rRNA
gene sequencing

PCR amplification of the 16S rRNA genes of the selected isolates yielded good bands

after electrophoresis, except for isolate 0196AN that had no band (Lane 4, Figure 12).

Analysis of 16S rRNA gene sequence was used for identification of the ten selected

pathogens. BLAST of the nucleotide sequences of these isolates on NCBI website revealed

species of the following genera as shown in Table 18: Escherichia, Citrobacter,

Staphylococcus, Aeromonas and Bacillus, which in some cases differed from the presumptive

identity (Table 19). Phylogenetic trees showing the relationship between 16S rRNA

sequences of these isolates and sequences of other similar isolates on GenBank are presented

in Figures 13-19.

4.8 Detection of toxin genes by PCR in selected presumptive pathogenic isolates in edible
land snails.
The results of the PCR analysis to detect some toxin genes in some Staphylococcus

and Bacillus isolates are presented in Tables 20 and 21. Gel images from the PCR analysis are

presented in Appendices 11 – 17. Results showed that Sea gene was detected only in

Staphylococcus sciuri (0181EN) (Table 20). However, exhc gene was not detected in any of

the Staphylococcus isolates. The results of PCR analysis to detect toxin genes in Bacillus

isolates recovered from edible land snails are summarized in Table 21. The nheb gene was the

only gene detected in all three Bacillus isolates screened in this study, while hblc gene was

not detected in any of the isolates. Only isolate 0186EN was found to possess four ( hbla,

nhea, nheb and cytk) out of the five toxin genes used in this study. While isolate 0177EN

possessed hbla and nheb, 0197AN was found to habour nhea and nheb.

87
 M 1 2 3 4 5 6 7 8 9 10

(M) Ladder (50 bp); (1) 0163EN; (2) 0180EN; (3) 0186EN; (4) 0196AN; (5) 0184EN; (6) 0193AN; (7)
0173EN; (8) 0177EN; (9) 0181EN; (10) 0197AN.

Figure 12: Amplicons of 16S rRNA genes of selected isolates targeted for
sequencing

88
Table 18: Identity of nine selected pathogens in edible land snails determined by 16S
rRNA gene sequencing

Isolate Code Identity of Isolate Accession Number

0163EN Escherichia fergusonii MK522152
0173EN Citrobacter freundii MK526907
0177EN Bacillus cereus MK530202
0180EN Staphylococcus arlettae MK518344
0181EN Staphylococcus sciuri MK518066
0184EN Aeromonas hydrophila MK530176
0186EN Bacillus thuringiensis MK530172
0193AN Citrobacter freundii MK527109
0197AN Bacillus cereus MK530171

89
Table 19: Comparison of the Identity of nine selected pathogens determined by
phenotypic and molecular studies
Isolate Code Identity by phenotypic studies Identity by molecular studies
0163EN Escherichia coli Escherichia fergusonii
0173EN Salmonella Citrobacter freundii
0177EN Bacillus cereus Bacillus cereus
0180EN Staphylococcus Staphylococcus arlettae
0181EN Staphylococcus Staphylococcus sciuri
0184EN Vibrio Aeromonas hydrophila
0186EN Bacillus cereus Bacillus thuringiensis
0193AN Salmonella Citrobacter freundii
0197AN Bacillus cereus Bacillus cereus

90
Figure 13: Phylogenetic tree showing the relationship of Citrobacter freundii NEDU 4
and NEDU 5 to other members of the Citrobacter freundii based on 16S rRNA gene
sequences

91
Figure 14: Phylogenetic tree showing the relationship of Staphylococcus arlettae NEDU

180 to other members of the Staphylococcus arlettae based on 16S rRNA gene sequences

92
Figure 15: Phylogenetic tree showing the relationship of Staphylococcus sciuri NEDU
181 to other members of the Staphylococcus sciuri based on 16S rRNA gene sequences

93
Figure 16: Phylogenetic tree showing the relationship of Escherichia fergusonii isolate
NEDU 3 to other members of the Escherichia fergusonii based on 16S rRNA gene
sequences

94
Figure 17: Phylogenetic tree showing the relationship of Aeromonas hydrophila NEDU
184 to other members of the Aeromonas hydrophila based on 16S rRNA gene sequences

95
Figure 18: Phylogenetic tree showing the relationship of Bacillus cereus NEDU 177 and
NEDU 197 to other members of the Bacillus cereus based on 16S rRNA gene sequences

96
Figure 19: Phylogenetic tree showing the relationship of Bacillus thuringiensis isolate
NEDU 186 to other members of the Bacillus thuringiensis based on 16S rRNA gene
sequences

97
Table 20: Toxin genes detected by PCR in Staphylococcus and Bacillus isolates
recovered from edible land snails
Identity of Isolate Sea Exhc Hbla Hblc Nhea Nheb cytk
(Accession number)
Staph. arlettae - - NA NA NA NA NA
(MK518344)

Staph. Sciuri + - NA NA NA NA NA
(MK518066)

B. thuringiensis NA NA + - + + +
(MK530172)

B. cereus NA NA + - - + -
(MK530202)

B. cereus NA NA - - + + -
(MK530171)

NA: Not Applicable

98
4.9 Determination of the Processing Method that Will Reduce the Bacterial Load of
Snail Meat during Culinary Preparation.
The effect of different processing methods on the aerobic plate counts of bacteria in

five batches of snail meat showed that the initial mean counts at the end of shucking ranged

from 8.79 Log CFU/g to 9.55 Log CFU/g among batches of samples. However, mean aerobic

plate counts of samples were found to decrease along the stages of processing. At the

desliming stage, the wood ash-deslimed batch of samples had the least mean bacterial count

(5.98 Log CFU/g) followed by potassium alum-deslimed batch (6.92 Log CFU/g), cassava

water-deslimed batch (7.04 Log CFU/g), garri-deslimed batch (7.72 Log CFU/g) and lime-

deslimed batch (8.16 Log CFU/g). These represent the following reductions for each batch of

samples: 2.95 Log CFU/g, 1.3 Log CFU/g, 0.99 Log CFU/g, 0.98 Log CFU/g and 0.93 Log

CFU/g for wood ash, alum, lime, cassava water and garri deslimed batches respectively.

However, at the end of the washing stage, there were increases in the mean bacterial counts

for the batches deslimed with wood ash (6.89 Log CFU/g) and cassava water (7.35 Log

CFU/g), but their mean counts were found to be lowest (2.44 Log CFU/g and 2.67 Log

CFU/g) at the end of the cooking stage (Figure 20).

The effect of processing methods on mean coliform counts for snail meat is presented

in Figure 21. The initial mean coliform counts were above 6.85 Log CFU/g across the five

batches of samples at the end of shucking. The mean counts decreased across all batches

along the stages of processing. No coliform was recovered at the last stages of processing and

cooking.

At the desliming stage, various levels of reductions in mean counts of coliforms were

observed across batches which include: 2.78 Log CFU/g, 1.02 Log CFU/g, 0.29 Log CFU/g,

99
0.22 Log CFU/g and 0.09 Log CFU/g reductions for wood ash, alum, cassava water, lime and

garri deslimed batches, respectively. However, there was increase in mean coliform counts of

the ash-deslimed batch (5.63 Log CFU/g) at the end of washing stage, while the mean count

of the cassava water-deslimed batch (6.73 Log CFU/g) remained almost the same at this

stage.

The effect of processing methods on mean counts of Citrobacter in snail meat is

presented in Figure 22. The initial mean counts across batches of samples at the shucking

stage were above 5.47 Log CFU/g. The mean counts decreased across batches along the

stages of processing, except for the cassava water-deslimed batch which increased at the

washing stage (6.42 Log CFU/g). Citrobacter was not recovered in any batch of samples at

the end of parboiling and cooking stages. Considering the desliming stage, there were various

levels of reductions in mean Citrobacter counts across batches of samples which include: 3.85

Log CFU/g, 1.88 Log CFU/g, 0.91 Log CFU/g, 0.57 Log CFU/g and 0.35 Log CFU/g

reductions for wood ash, alum, garri, lime and cassava water deslimed batches respectively.

However, there were increases in mean counts of presumptive Citrobacter for wood ash (3.95

Log CFU/g) and alum (3.49 Log CFU/g) deslimed batches at the end of washing.

The effect of processing methods on mean counts of Shigella in snail meat is

presented in Figure 23. The initial mean counts across batches of samples at the shucking

stage ranged from 6.42 Log CFU/g to 4.43 Log CFU/g. The mean counts decreased across

batches along the stages of processing, except for the ash-deslimed batch which increased at

the washing stage (4.26 Log CFU/g). Shigella was not recovered in any batch of samples at

the end of parboiling and cooking stages. At the desliming stage, there were various levels of

reductions in mean Shigella counts across batches of samples which include: 1.68 Log CFU/g,
100
1.49 Log CFU/g, 1.49 Log CFU/g, 1.10 Log CFU/g and 0.11 Log CFU/g reductions for lime,

wood ash, alum, cassava water and garri deslimed batches respectively.

The effect of processing methods on mean counts of E. coli in snail meat is presented

in Figure 24. The initial mean counts across batches of samples at the shucking stage ranged

from 6.57 Log CFU/g to 7.59 Log CFU/g. The mean counts of E. coli decreased across

batches along the stages of processing, except for the ash-deslimed batch which increased at

the washing stage (4.90 Log CFU/g). E. coli was not recovered in any batch of samples at the

end of parboiling and cooking stages. At the desliming stage, various levels of reductions in

mean E. coli counts were observed across batches of samples which include: 3.05 Log CFU/g,

1.19 Log CFU/g, 0.92 Log CFU/g, 0.52 Log CFU/g and 0.39 Log CFU/g reductions for wood

ash, alum, cassava water, lime and garri deslimed batches respectively.

The effect of processing methods on mean counts of Staphylococcus in snail meat is

presented in Figure 25. The initial mean counts across batches of samples at the shucking

stage ranged from 5.47 Log CFU/g to 6.38 Log CFU/g. The mean counts of Staphylococcus

decreased across batches along the stages of processing, except for the ash-deslimed batch

which increased at the washing stage (4.41 Log CFU/g). Staphylococcus was not recovered in

any batch of samples at the end of parboiling and cooking stages.

At the desliming stage, various levels of reductions in mean Staphylococcus counts

were observed across batches of samples which include: 2.13 Log CFU/g, 1.22 Log CFU/g,

1.19 Log CFU/g, 0.39 Log CFU/g and 0.20 Log CFU/g reductions for wood ash, lime,

cassava water, alum and garri deslimed batches respectively.

101
 The effect of processing methods on mean counts of Aeromonas in snail meat is

presented in Figure 26. The initial mean counts across batches of samples at the shucking

stage ranged from 3.75 Log CFU/g to 5.51 Log CFU/g. The mean counts of Aeromonas

decreased across batches along the stages of processing, except for the wood ash (3.62 Log

CFU/g) and garri (4.63 Log CFU/g) deslimed batch which increased at the washing stage.

Aeromonas was not recovered in any batch of samples at the end of parboiling and cooking

stages. At the desliming stage, various levels of reductions in mean Aeromonas counts were

observed across batches of samples which include: 3.24 Log CFU/g, 1.99 Log CFU/g, 1.06

Log CFU/g, 0.69 Log CFU/g and 0.33 Log CFU/g reductions for lime, wood ash, garri,

cassava water and alum deslimed batches respectively.

The effect of processing methods on mean counts of Bacillus in snail meat is

presented in Figure 27. The initial mean counts across batches of samples at the shucking

stage ranged from 4.3 Log CFU/g to 5.54 Log CFU/g. The mean counts of Bacillus decreased

across batches along the stages of processing. Bacillus was recovered in all batches of

samples at all stages of processing and cooking. At the desliming stage, various levels of

reductions in mean Bacillus counts were observed across batches of samples which include:

1.02 Log CFU/g, 1.00 Log CFU/g, 0.44 Log CFU/g, 0.37 Log CFU/g and 0.21 Log CFU/g

reductions for lime, wood ash, cassava water, alum, and garri deslimed batches respectively.

The final mean counts of Bacillus at the end of the cooking stage ranged from 2.53 Log

CFU/g to 3.24 Log CFU/g.

102
Fig. 20: Effect of different processing methods on Mean Aerobic Plate counts of bacteria in snail meat.

103
Fig. 21: Effect of different processing methods on Mean Coliform counts in snail meat.

104
Fig. 22: Effect of different processing methods on Mean counts of presumptive Citrobacter in snail meat.

105
Fig. 23: Effect of different processing methods on Mean counts of Shigella in snail meat.

106
Fig. 24: Effect of different processing methods on Mean counts of E. coli in snail meat

107
Fig. 25: Effect of different processing methods on Mean count of Staphylococcus in snail meat.

108
Fig. 26: Effect of different processing methods on Mean counts of Aeromonas in snail meat.

109
Fig. 27: Effect of different processing methods on Mean counts of Bacillus in snail meat.

110
 Chapter five

DISCUSSION

The findings of this study have demonstrated that edible land snails for sale in three

major markets (Igboukwu market in Anambra state, Abakaliki Meat market in Ebonyi state

and Ogbete market in Enugu state) contain various levels of high loads of bacterial indicators

and pathogens. The mean aerobic plate count of samples analysed in this study ranged from

8.43 to 9.61 Log CFU/g (Figure 4). This appears close to the findings of other related studies:

Adegoke et al. (2010) reported total aerobic bacterial count in market snails at Akwa Ibom

state was 8.0 Log CFU/g. In Ghana, Nyoagbe et al. (2016) reported that total viable count

ranged from 6.61 to 8.29 Log CFU/g. However, Temelli et al. (2006) found the average total

aerobic bacterial count in live snails in Turkey to be 6.85 Log CFU/g. Also, mean aerobic

counts varied significantly (p < 0.01) between the three states from which samples were

collected (Figure 4), probably because of the difference in the nature of soil and debris present

in the natural habitats of these snails. Aerobic plate count is generally used as a means of

assessing the overall microbial quality of raw ingredients (Siragusa et al., 1998). According to

ICMSF (1986), the acceptable upper limit of total aerobic bacterial load for seafoods is 5.0

Log CFU/g and this limit has been cited in most research articles till date. It is important to

note that all snail samples analysed in this study had total aerobic plate counts >10 8 CFU/g

(Table 4). This implies that 100% of market snails analysed pose microbiological risk to

handlers and consumers. However, the use of the aerobic plate count as an indicator for the

presence of specific pathogens is generally not accepted (Siragusa et al., 1998). Snails that are

usually sold in the markets are collected from the forest. Such snails are in high demand

among consumers (Nyoagbe et al., 2016). Also, they are usually purchased alive in the market
111
and brought into homes where they are handled and prepared in the raw state in domestic

kitchens.

Coliform counts are used for assessing the amount of contamination on meat arising

from gut contents which include both those originating directly from the alimentary tract and

those arising indirectly through the integument or processing environment. Coliforms are the

most frequently studied indicators (Wu et al., 2011). The acceptable upper limit of total

coliform is 2.0 Log CFU/g (ICMSF, 1986). In this study, the coliform counts were >2.0 Log

CFU/g in all samples analysed (Figure 5 and Table 4). Similar coliform count in snails has

been previously reported (Adegoke et al., 2010; Nyoagbe et al., 2016), though the prevalence

of levels of loads of coliforms in snails is scarce in literature. It is appropriate to note that

snails discharge their faeces within their habitat (Ibom et al., 2012) and may explain the high

loads of coliforms observed in this study.

The presumptive Salmonella isolates were found to be strains of Citrobacter based on

molecular studies (Table 19). This implies that 100% of snail samples, in the present study,

were contaminated with Citrobacter rather than presumptive Salmonella as stated earlier

(Table 3). This observation is supported by another study in India on the bacterial diversity of

the gastrointestinal tract of Achatina fulica using culture-independent and culture-dependent

methods. The study also concluded that an apparent feature of bacterial communities in

snails‘ gastrointestinal tract was the abundance of members of the genus Citrobacter (Pawar

et al., 2012). The highest mean Citrobacter count was found in Ebonyi samples (7.24 ± 0.210

Log CFU/g) followed by Anambra and Enugu samples with 6.39 ± 0.114 Log CFU/g and

6.37 ± 0.219 Log CFU/g respectively (Figure 6). No significant differences were found

between Anambra and Enugu samples (p < 0.01). Citrobacter is classically considered a
112
resident commensal of the intestinal tracts of both humans and animals (Guerrant et al.,

1976). It is also prevalent in soil and water through contamination from the waste materials of

animals. A study concluded that healthy pet turtles are a potential carrier of C. freundii

(Sabrina-Hossain et al., 2017). Therefore, turtles, as sea animals, are likely to be the source of

Citrobacter around swampy environments where most snails are collected.

Ebonyi samples were found to contain the highest mean Shigella count (4.61 ± 0.354

Log CFU/g) followed by Anambra (4.43 ± 0.284 Log CFU/g) and Enugu samples (4.03 ±

0.571 Log CFU/g) (Figure 7). Most studies on snails have reported the presence of Shigella

without indicating its level of concentration (Adagbada et al., 2011). Several aquatic bodies

have been found to contain Shigella, and thus another potential source of infection may be

aquatic food which may play a role in transmission of Shigella if such food is harvested from

sewage-contaminated water (Iwamoto et al., 2010). The number of Shigella cells required to

initiate infection ranges from 101 to 104 cells/person (Dupont et al., 1989; Heymann, 2004).

Since 60% of snails analysed in the present study exceeded 10 CFU/g (Table 4), this probably

implies that such percentage of snails represent health threat to handlers and consumers.

When market snails are brought into homes, snail handlers and cooks have the tendency for

direct hand-to-mouth exposure to pathogens that may be present in these snails, and cross

contamination of the kitchen environment and ready-to-eat foods may always follow.

Currently, the incidence of shigellosis worldwide is highest among children less than five

years of age (Taneja and Mewara, 2016).

The highest mean count of E. coli was found in Anambra samples (7.14 ± 0.170 Log

CFU/g), while Enugu samples had the lowest mean count (5.65 ± 0.239 Log CFU/g) as shown

in Figure 8. Sixty percent of the market snails in this study had E. coli count > 6.0 Log CFU/g
113
(Table 4) which is within the range of counts noted for resulting in diarrhoeal diseases 6.0 to

9.0 Log CFU/g (Kornacki and Marth, 1982).

The mean Staphylococcal count in samples analysed in this study ranged from 4.66 to

4.74 Log CFU/g (Figure 9). There were no significant differences between Ebonyi and Enugu

samples (p < 0.01). Staphylococcus was not detected in Anambra samples. The only study

that quantified the level of Staphylococcus in snails reported a range between 2.66 and 7.68

Log CFU/g (Nyoagbe et al., 2016). Diagnosis of staphylococcal food poisoning is generally

confirmed by the recovery of at least 5.0 Log CFU/g from food (Halpin-Dohnalek and Marth,

1989; Hennekinne et al., 2012). It is suggested that since Staphylococcus is also present in

intestinal tract, raw meat may contain Staphylococcus due to contamination with intestinal

content during evisceration (Bhalla et al., 2007).

Presumptive Vibrio isolate was found to be a strain of Aeromonas based on molecular

studies (Table 19). The mean count of Aeromonas was found to range from 3.10 to 4.80 Log

CFU/g. There were significant differences between samples collected from different states (p

< 0.01) (Figure 10). The infectious dose of Aeromonas species in foods is not known

(Isonhood and Drake, 2012). In another study, mesophilic aeromonads were isolated from

26% of vegetable samples, 70% of meat and poultry samples, and from 72% of fish and

shrimps. Numbers of motile aeromonads present in these samples varied from < 2.0 to > 5.0

Log CFU/g (Neyts et al., 2000). In the present study, 76.6% of the snail samples contained

Aeromonas. This is important because snails feed on assortment of plant and animal species

including algae (Okafor, 1989). Aeromonas species are widely distributed in the aquatic

environment (Palumbo, 1996; Neyts et al., 2000) and their prevalence in various water and

food sources represents a significant public health threat (Wu et al., 2007).
114
 The results of this study indicate that Bacillus counts for Ebonyi samples had the

highest mean count of Bacillus (4.50 ± 0.136 Log CFU/g) followed by Enugu (3.48 ± 0.135

Log CFU/g) and Anambra samples (3.25 ± 0.130 Log CFU/g) as shown in Figure 11.

Nyoagbe et al. (2016) reported similar levels of Bacillus in snails ranging from 1.53 to 4.90

Log CFU/g. It is often noted that Bacillus cereus levels > 3.0 Log CFU/g have resulted in

illness (Harmon et al., 1992). In order to promote the consumption of products in domestic

and export markets, it is important to ensure such products are free from pathogenic

microorganisms (Dhanze et al., 2013).

Proteolysis, haemolysis and biofilm formation are the common phenotypic virulence

factors found in diarrhoea-associated Citrobacter freundii (Chen et al., 2002). In this study,

the most prevalent virulence factors among the Citrobacter isolates were biofilm formation

and haemolysin production (Table 5). In another study, which involved C. freundii isolates

from healthy pet turtles, haemolysin production was not detected among the isolates and it

was concluded that lack of haemolysis indicates the inability of the isolates to break down red

blood cells and cause hemolytic uremic syndrome in humans (Sabrina-Hossain et al., 2017).

Biofilm formation has been reported in Citrobacter isolates from clinical samples (Pardia et

al., 1980). In the present study, 100% and 70% of Ebonyi Citrobacter isolates were found to

be haemolysin and biofilm producers, which implies that these isolates demonstrate a greater

potential public health risk compared to isolates from other locations.

Absorption of congo red from an agar medium containing the dye (indirect method of

detecting biofilm formation) appears to correlate with virulence in Shigella species (Maurelli

et al., 1984). None of the Shigella isolates was found to form biofilm in this study (Table 6).

115
Haemolytic activity was only detected in 50 E. coli isolates (40%) recovered from Anambra

samples (Table 7). Bashar et al. (2011) reported that among 60 E. coli isolates from poultry

chicken faeces, 59% were found to demonstrate haemolysis. Also, several studies have

investigated haemolytic activity in E. coli isolates and their correlation with virulence (Chart

et al., 1998; Reingold et al., 1999; Bashar et al., 2011). Moreover, Bisht et al. (1977)

observed association between haemolysin and necrotoxin production among the E. coli

isolates from cases of acute gastroenteritis, chronic diarrhoea as well as healthy human

population.

All staphylococci isolates recovered from snail samples in this study were found to be

positive for haemolysis, gelatinase, protease, lecithinase and biofilm formation tests (Table 8).

These are pathogenic factors that have been reported among Staphylococcus isolates

possessing virulence genes (Bertelloni et al., 2015; Schaumburg et al., 2011; Coelho et al.,

2009; Cifrian et al., 1996; Akinjogunla et al., 2014).

Analysis of pathogenic mechanisms associated with Aeromonas has identified various

virulence factors including toxins with haemolytic, cytotoxic and protease activities among

others (Chopra et al., 2000; Singh et al., 2009; Cascon et al. 2000). From Table 9 of the

results of this study, 100% of Aeromonas isolates from Anambra samples were found to be

positive for all virulence associated tests, except biofilm formation (70%). While 50% of

Ebonyi isolates were positive for haemolysin, Enugu isolates were found to be negative. In

other studies, 94% of 767 shellfish isolates were haemolysin positive and 59% of the

haemolytic isolates were cytotoxic (Abbott et al., 1992); 86% of cat fish Aeromonas isolates

were haemolytic (Wang and Silva, 1999). It has been reported that haemolysin and cytotoxin

production were more frequent for Aeromonas species isolated from individuals with
116
diarrhoea than for those isolated from healthy individuals. However, the number of

environmental isolates that exhibited haemolysin and cytotoxin production was larger than the

number of human isolates that did (Kuhn et al., 1997). Cytotoxic activity of Aeromonas on

Vero cells has occasionally been reported and associated with haemolysin activity (Isonhood

and Drake, 2012).

All Bacillus isolates recovered from the three locations were found to be positive for

gelatinase and lecithinase tests. On the other hand, all Bacillus isolates from Anambra and

Ebonyi samples were positive for haemolysin, 50% of Enugu isolates were positive (Table

10). Haemolysin is one of the three toxins that have been implicated as aetiological agents of

the diarrhoeal disease involving Bacillus (Beecher and MacMillan, 1991; Lund and Granum,

1996; Lund et al., 2000).

The use of antibiotics is considered the most important factor promoting the

emergence, selection and dissemination of antibiotic-resistant microorganisms in both

veterinary and human medicine (Chaudhary et al., 2014). Antibiotic resistant bacteria from

foods of animal origin, are ranked as the direct causative agents of foodborne diseases and

stands for a possible source of drug resistance in human pathogens (Schlegelova et al. 2003).

In this study, most Citrobacter isolates were resistant to ceftriaxone and amoxy/clavulanic

acid compared to other antibiotics (Table 11). This corroborates with the findings from other

studies that involved Citrobacter isolates from environmental samples (Sabrina-Hossain et

al., 2017) and also from hospitalized patients (Arens et al., 1997).

The rise in drug resistance is becoming a serious problem in the treatment of

shigellosis. Such rise is comparable to the extensive uncontrolled use of antibiotics in

117
developing countries (Fauci et al., 2008). In this study, 30%, 65%, 25% and 65% of the

Enugu Shigella isolates were resistant to ampicillin, ceftazidime, cephalexin and ceftriaxone,

respectively (Table 12). Nevertheless, there is a report that nalidixic acid was introduced to

treat shigellosis caused by ampicillin and co-trimoxazole resistant strains (Iwalokun et al.,

2001). Interestingly, Shigella isolates recovered in this study were sensitive to

fluoroquinolones. These have been found effective in the treatment of shigellosis

(Bhattacharya et al., 1987; Niyogi, 2007).

E. coli isolates across the three states were found to be more resistant to 3 rd generation

cephalosporins compared to other antibiotics (Table 13). There is a report from Belgium that

about 35% of the E. coli strains isolated from live broilers are resistant to 3 rd generation

cephalosporins while over 60% of the broilers are found to be carriers of these 3 rd generation

cephalosporin-resistant E. coli after selective isolation (Depoorter et al., 2012). Furthermore,

E. coli isolates from Anambra (45%) and Ebonyi (30%) were resistant to streptomycin.

Similarly, other studies reported that more than 50% of E. coli isolates from eggs and broiler

chickens are resistant to streptomycin (Adesiyun et al., 2007; Smith et al., 2007). The possible

explanation is that the snails may have picked up these antibiotic-resistant isolates from

environments fertilized with chicken faeces and litters. This is supported by the fact that

antibiotic-resistant E. coli strains have been isolated from broiler chicken faeces and litters

(Diarrassouba et al., 2007). In addition, Johnson et al. (2007) reported that the poultry meat is

one of the major sources of transferring antibiotic resistance.

Ebonyi (> 60%) and Enugu (> 40%) Staphylococcus isolates were resistant to

chloramphenicol and norfloxacin in this study (Table 14). Thirty percent of Ebonyi isolates

were found to be resistant to cloxacillin. In a similar study in Nigeria, 46.2% of S. aureus

118
isolates from poultry meat were reported to be resistant against chloramphenicol (Otalu et al.,

2011). Efuntoye et al. (2011) found that S. aureus isolates recovered from snails had 100%

resistance to augmentin, cloxacillin, cefuroxime and amoxycillin-clavulanic acid. However, in

the present study, all Staphylococcus isolates were sensitive to 70% of antibiotics used in this

study which indicates low prevalence of resistance to several antibiotics.

In this study, Anambra Aeromonas isolates were resistant to ceftazidime, ceftriaxone

and nalidixic acid (Table 15). Also, Ebonyi isolates were resistant to ampicillin, ceftazidime

and ceftriaxone, while Enugu isolates were resistant to ampicillin, amoxycillin/clavulanic acid

and ceftriaxone. All isolates of Aeromonas were sensitive to 70% of antibiotics used in this

study (Table 15). From literature, the majority of Aeromonas appear susceptible to

tetracycline, aminoglycosides, 3rd generation cephalosporins and the quinolones (Janda and

Abbott, 1998). The resistance to quinolones and nalidixic acid is considered to be

chromosomally mediated, as a result of drug resistant isolates selective pressure (Goñi-Urriza

et al., 2000).

The least prevalence of antibiotic resistance was observed among Anambra Bacillus

cereus strains (Table 16). B. cereus is usually susceptible to aminoglycosides,

chloramphenicol among others (Luna et al., 2007). However, Ebonyi (50%) and Enugu (65%)

isolates tested in this study proved to be resistant to chloramphenicol. All Bacillus isolates

were found to be sensitive to 60% of antibiotics used in this study. This suggests that health

problems implicating B. cereus from these snails are likely to be successfully treated with

most antibiotics.

Furthermore, the findings of this present study show that bacterial pathogens in these

snails appear to possess the potential to acquire multidrug resistance as evidenced by the
119
antibiotic resistance patterns exhibited by some isolates (Table 17). These isolates may

continue to progress with this trend if the source of the antibiotic resistant genes is not

identified and checkmated. Scientific studies have come to the conclusion that soil is a major

reservoir of antibiotic resistance genes (Martinez 2008; Finley et al., 2013; Fitzpatrick and

Walsh, 2016). Another possible explanation connecting these snails and antibiotic resistant

isolates could be based on the herbivore-feeding habit of snails, primarily on vascular plants

(Rauth and Barker, 2002) and also their participation with other soil invertebrates in the

decomposition of leaf litter (Hatziioannou et al., 1994). These plants may have been

contaminated with antimicrobial resistant bacteria following sewage discharges or the use of

irrigation water contaminated with faeces of humans and animals that are victims of

antibiotics abuse (Bergogne-Berezin, 1997). This is important because formulated feeds for

snails are not available in the market and it has become common practice for snail rearers to

use vegetables, plant leaves and kitchen wastes to feed snails (Chah and Inegbedion, 2013).

The analysis of 16S rRNA gene sequences of ten selected isolates in this study

revealed them to belong to species of the following genera: Escherichia, Citrobacter,

Staphylococcus, Aeromonas and Bacillus (Table 18). Escherichia fergusonii has been shown

to cause disease in humans (Funke et al., 1993; Bain and Green, 1999; Mahapatra and

Mahapatra, 2005). It was recently isolated from vervets and baboons in South Africa and it

was portrayed as a possible emerging pathogen of zoonotic importance (Glover et al., 2017).

Also, two isolates in this study were found to be different strains of Citrobacter

freundii based on 16S rRNA gene sequence analysis (Table 18). C. freundii has been detected

in fresh beef meat samples from local markets in Nigeria (Ukut et al., 2010). In India, a study

on the bacterial diversity of the gastrointestinal tract of Achatina fulica using culture-
120
independent and culture-dependent methods reported the abundance of members of the genus

Citrobacter (Pawar et al., 2012). C. freundii is ubiquitous in humans and animals. It has also

been found to be prevalent in soil and water as a result of contamination from the waste

materials of animals (De Padua et al., 2014; Sabrina-Hossain et al., 2017). It was proposed to

be an emerging important foodborne pathogen since it has the ability to produce or acquire a

number of heat-stable enterotoxins. It has been reported in an outbreak of gastroenteritis

associated with consumption of chicken salad in the United States Air Force Academy

(Warner et al., 1991), and egg shells in Korea (Chang, 2000). Moreover, C. freundii was

detected in a sample obtained from a patient with pancreatic pseudocyst after an acute

necrotizing pancreatitis (Lozano-Leon et al., 2011).

Another isolate of interest identified by 16S rRNA gene sequencing in this study was

Staphylococcus sciuri (Table 18). It is considered one of the most ancestral and dispersed

staphylococcal species, with a wide range of habitats that includes the skin of several animals

as well as environmental reservoirs, such as soil (Kloos et al., 1976; Couto et al., 2000). Some

studies have implicated S. sciuri in different infections such as endocarditis, peritonitis, septic

shock, endophthalmitis, urinary tract infection, pelvic inflammatory disease, and wound

infections (Hedin and Widerstrom, 1998; Horii et al., 2001; Stepanovic et al., 2003; Dakic et

al., 2005a; Dakic et al., 2005b).

Two strains of Bacillus cereus were identified by 16S rRNA gene sequencing in the

present study (Table 18). B. cereus is described as being of ubiquitous presence in nature and

can be found in many types of soils, sediments, dust and plants (von Stetten et al., 1999;

Schoeni and Wong, 2005) which are all present in the natural habitat of snails. The true

121
burden of illnesses caused by B. cereus is unknown probably because they commonly occur

as sporadic cases, rather than in major outbreaks (Logan et al., 2011).

The PCR analysis to detect some toxin genes in Staphylococcus isolates revealed that

Staphylococcus sciuri (0181EN) haboured sea gene, which was not detected in

Staphylococcus arlettae (0180EN) as shown in Table 20. Although the occurrence of sea gene

in coagulase-negative staphylococci such as S. sciuri is very rare, the need to screen for such

gene among coagulase-negative staphylococci has been acknowledged in several articles

since they are suggested to be reservoir of virulence genes for other bacteria (Blaiotta et al.,

2004; Dakic et al., 2005a; Piechota et al., 2014). Staphylococcal Enterotoxin A (SEA) is the

most commonly reported enterotoxin in foods, and also considered as the main cause of

staphylococcal food poisoning, probably due to its extraordinarily high resistance to

proteolytic enzymes (Balaban and Rasooly, 2000; Zargar et al., 2014). Since SEA is toxic

even in low concentrations (0.6 ng/mL), detection of strains which harbor sea gene is

important (Holeckova et al., 2002). The predominance of SEA in most foodborne disease

outbreaks in different countries is well documented. For instance, about 90% of food

poisoning isolates were reported to contain the sea gene in Korea (Argudín et al., 2010; Cha

et al., 2006). Dakic et al. (2005b) did not detect genes encoding staphylococcal eneterotoxins

in a large panel of 48 S. sciuri group isolates. Piechota et al. (2014) detected sec genes in 5 S.

sciuri isolates obtained from cow‘s milk in Poland and reported the absence of sea gene in

these isolates. Since staphylococcal enterotoxin genes are mostly carried on mobile genetic

elements like plasmids (Fitzgerald et al., 2000), this strain of S. sciuri may have gained sea

gene through genetic transfer and could possibly transfer this gene to other strains.

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 The diarrhoeal syndrome caused by B. cereus is generally attributed to at least three

enterotoxins: haemolytic, non-haemolytic and cytotoxin K (Glasset et al., 2016; Owusu-

Kwarteng et al., 2017). In the present study, PCR analysis of Bacillus isolates for toxin genes

targeted some genes encoding haemolytic (hbla and hblc) enterotoxin complex, non-

haemolytic (nhea and nheb) enterotoxin complex and cytotoxin K (cytk). The results indicated

that nheb gene was the most frequently detected gene in all the three Bacillus isolates

screened in this study (Table 21). Simailarly, Abbas et al. (2014) found that nhea gene was

the most common gene detected in B. cereus isolated from milk and milk products in the

market. In the present study, only Bacillus isolate 0186EN (B. thuringiensis) was found to

possess four (hbla, nhea, nheb and cytk) out of the five toxin genes considered (Table 21).

These findings align with other reports indicating that enterotoxin genes appear more

predominant in B. thuringiensis than B. cereus (Hansen and Hendriksen, 2001; Prub et al.,

1999; Rivera et al., 2000). In another study, it was observed that the toxicity of B.

thuringiensis culture supernatants is generally at the same levels as those of B. cereus strains

associated with food poisoning (Rivera et al., 2000). Also, while Bacillus isolate 0177EN (B.

cereus) was found to possess hbla and nheb in this study, Bacillus isolate 0197AN (B. cereus)

was found to habour nhea and nheb.

The pathogenicity of B. cereus diarrhoeal strains is not fully understood. From

literature, the genetic studies carried out till date have been inconclusive and, regardless of the

diseases they cause, all strains seem to carry genes encoding at least one of the known

diarrhoeal toxins (Yang et al., 2005; Glasset et al., 2016). Mantynen and Lindstrom (1998)

concluded that screening for the hemolysin hbla gene by the PCR method allows faster

detection of enterotoxin production than testing with the RPLA enterotoxin test kit. It should

123
be noted that the occurrence of different combinations of enterotoxin genes in these Bacillus

strains, observed in this study, suggests that virulence varies among strains.

Because of the high prevalence of bacterial pathogens observed among snails in this

study, there was need to ascertain whether these pathogens persist along the stages of

processing as performed at home. Snail samples from Enugu state were chosen for this study

because of the high prevalence of selected pathogens recorded earlier in the present study

(Table 3).

One challenge associated with processing of snail meat is the mucus or slime secreted

by the snails (Gallo, 2002). In a nutshell, results indicated that bacterial counts in samples

decreased along the stages of processing, and that desliming agents had various degrees of

inhibitory effects on the counts of bacterial indicators and pathogens (Figures 20 – 27).

At the end of desliming stage, the highest levels of reduction in concentrations of

bacteria was achieved with wood ash, thereby improving the microbiological quality of snails:

Citrobacter counts reduced by 3.85 Log CFU/g (Figure 22), E. coli counts reduced by 3.05

Log CFU/g (Figure 24), total aerobic plate counts reduced by 2.95 Log CFU/g (Figure 20),

Coliform counts reduced by 2.78 Log CFU/g (Figure 21), Staphylococci counts reduced by

2.13 Log CFU/g (Figure 25), Aeromonas counts reduced by 1.99 Log CFU/g (Figure 26),

Shigella counts reduced by 1.49 Log CFU/g (Figure 23) and Bacillus counts reduced by 1.00

Log CFU/g (Figure 27).

Wood ash, when added to the soil, has a high acid-neutralizing capacity which in turn

increases the amount and quality of dissolved organic carbon (DOC). It has been reported that

both increased pH and DOC quality affect the bacterial community in the soil (Steenari et al.,

124
1999; Holmberg and Claesson, 2001; Jokinen et al., 2006). Therefore, all of these observed

effects on bacterial concentrations can be explained by the wood ash-induced increase in pH.

Also, the finding that wood ash had the least effect on Bacillus counts (Figure 27)

corroborates with the findings of Bang-Andreasen et al. (2017) who reported detrimental

effects on culturable bacteria at a wood ash dose of 167 t ha -1 and that spore forming bacteria

represent the majority of the bacteria capable of surviving the high wood ash dose.

Alum is a salt made of a combination of an alkali metal such as sodium, potassium or

ammonium and a trivalent metal; aluminum, iron or chromium. Potassium alum is commonly

used to remove slime from snails in Nigeria (Amadi and Ngerebara, 2017). Alum has a

property known as astringency, which refers to its ability to constrict body tissues and restrict

the flow of blood (Olmez et al., 1998). This could explain the moderate detrimental effect it

had on bacterial counts in this study (Figures 20 – 27). The results of the present study are in

agreement with that of other studies that reported broad spectrum antibacterial activity by

potassium alum (Dutta et al., 1996; Ahmed, 2011; Bnyan et al., 2014; Amadi and Ngerebara,

2017).

However, at the end of the washing stage, increase in mean bacterial counts was

observed in most cases, especially in batch samples deslimed with wood ash, water from

cassava retting and potassium alum. These findings could be because of a redistribution of

contaminants on the carcass during the washing process (Prasai et al., 1995; Bell, 1997) and

the reduction in concentration of the desliming agents at this stage . Even though B. cereus

spores are not necessarily removed by regular washing (Andersson et al., 1995; Faille et al.,

2002).

125
 In this study, the reduction observed in all bacteria after the parboiling and cooking

could be as a result of temperature reaching 850C at the center of the snail meat due to heat

applied at 1000C for 10 mins. It has been reported that for consumer safety, application of

900C heat for 1.5 mins in the center and 99 – 1000C for 3–4 mins heat are ideal for mollusk

and shellfish, respectively (Huss et al., 2000; Tzouros and Arvanitoyannis, 2000). Also a

study in Greece found that the high levels of bacterial populations were considerably reduced

after the appropriate processing of snails (Parlapani et al., 2014).

126
 CONCLUSION

This study has clearly demonstrated that edible snails displayed for sale in three

selected markets in South East, Nigeria: Igboukwu, Abakaliki and Ogbete markets are

neglected sources of bacterial pathogens in the food chain. This is because 300 samples of

edible snails from these markets contained high loads of bacterial indicators and pathogens at

high prevalence rate. Antimicrobial-resistant strains of bacterial pathogens, specifically E.

coli, were found to be most resistant to 3 rd generation cephalosporins compared to other

antibiotics.

Also, different combinations of enterotoxin genes detected in the Bacillus strains

suggests that virulence varies among strains. The use of wood ash as desliming agent can

better improve the microbiological quality of snails compared to other agents. Bacillus

isolates were found to be most resistant to the effect of any common desliming agent

compared to other bacterial isolates studied. Also, this study has provided data on bacterial

hazards associated with edible snails along the value chain which will be useful for

formulation of food safety policy.

127
 RECOMMENDATIONS

(a) There is need to identify and protect, from human and animal interferences, the natural

habitats of edible land snails.

(b) Human sewage, industrial and abattoir wastes should not be disposed in environments

for rearing edible land snails.

(c) Appropriate food safety management system targeted torwards reduction of bacterial

pathogens along the snail value chain should be formulated and implemented.

(d) Antimicrobial resistance surveillance programme should be extended to include food

animals such as edible snails.

(e) Snails should be certified safe by veterinarians before sale in the market.

(f) Snail handlers and cooks should be educated on hygiene measures for processing and

adequate cooking of edible snails to prevent dissemination of pathogens in homes.

128
 CONTRIBUTION TO KNOWLEDGE

This study has contributed to knowledge in the following ways:

(a.)The presence of important emerging pathgens in A. achatina which are Escherichia

fergusonii, Citrobacter freundii, Bacillus cereus, B. thuringiensis and Staphylococcus

sciuri has been affirmed via molecular studies,

(b.)Edible snails represent an important source of multi-drug resistant E. fergusonii and C.

freundii in the food chain.

(c.) Edible snails habour Staphylococcus sciuri, B. cereus and B. thuringiensis strains

which possess genes for enterotoxin production that are often implicated in foodborne

disease outbreaks.

(d.)Common methods for culinary preparation of edible snails do not reduce bacilli counts

in snail meat below allowable limits.

129
 REFERENCES

Abbas, B. A., Khudor, M. H. and Saeed, B. M. S. (2014). Detection of hbl, nhe and bceT
Toxin Genes in Bacillus cereus Isolates by Multiplex PCR. International Journal of
Current Microbiology and Applied Science, 3(11): 1009-1016.

Abbott, S. L., Cheung, W. K. W., Kroske-Bystrom, S., Malekzadah, T. and Janda, J. M.

(1992). Identification of Aeromonas strains to the genospecies level in the clinical
laboratory. Journal of Clinical Microbiology, 30: 1262–1266.

Adagbada, A. O., Orok, A. B. and Adesida, S. A. (2011). The prevalence and antibiotic
susceptibility pattern of entero-pathogens isolated from land snails commonly eaten in
Cross River and Akwa Ibom states, South-southern Nigeria. Asian Journal of
Pharmaceutical Health Sciences, 1(3):123-127.

Adams, M. R. and Moss, M. O. (2008). Bacterial agents of foodborne illness: Staphylococcus

aureus. Food Microbiology. Cambridge: Royal Society of Chemistry.
Addis, M. and Sisay, D. (2015). A review on major foodborne bacterial illnesses. Journal of
Tropical Diseases, 3(4): 1-7.
Adegoke, A.A., Adebayo-Tayo, B.C., Inyang, U.C., Olayinka, A.A. and Komolafe, A.O.
(2010). Snails as meat source: Epidemiological and nutritional perspectives. Journal
of Microbiology and Antimicrobials, 2(1): 001-005.
Ademolu, K.O., Idowu, A.B., Mafiana, C.F. and Osinowo, O.A. (2004). Performance,
proximate and mineral analyses of African giant snail (Archachatina marginata) fed
different nitrogenous sources. African Journal of Biotechnology, 3: 412-417.

Adesiyun, A., Offiaha, N., Seepersadsingha, N., Rodrigoa, S., Lashley, V. and Musai, L.
(2007). Antimicrobial resistance of Salmonella spp. and Escherichia coli isolated from
table eggs. Food Control, 18(4): 306-311.

Agbonlahor, D. E., Shonekan, R. A. O., Kazak, W. H. and Coker, A. O. (1982). Aeromonas

food poisoning in Nigeria: A Case Report. Central African Journal of Medicine, 28:
(2) 36-38.

130
Ahmed, K. T. (2011). Inhibition of swarming in Proteus mirabilis by Alum (Hydrated
Aluminum Potassium Sulphate). Journal of University of Anbar for Pure Science,
5(2): 20-24.

Ajayi, A.O., and Salaudeen, T. (2014). Consumer food safety awareness and knowledge in
Nigeria. Agricultural Journal, 9:191-198.

Akinboade, O. A., Adegoke, G. O., Ogunji, F. O. and Dipeolu, O. O. (1980). Bacteria isolates
from giant snail in Nigeria. Veterinary Record, 106: 48.

Akinjogunla, O. J., Ajayi, A. O. and Ekeh, N. O. (2014). Virulence factors and antibiotic
resistant Staphylococcus spp from the anterior nares of apparently healthy
undergraduate students in Uyo. American Journal of Research Communication, 2(11):
158-180.

Akpavie, S. O., Ikheloa, J. O., Odaibo, A. B. and Imevbore, E. A. (2000). Pathobiology of

land snails: Achatina achatina and Archachatina marginata. Journal of Forest
Research, 16: 160–166.

Amadi, L. O. and Ngerebara, N. N. (2017). Susceptibility Profiles of Alum on Bacteria

Isolated from Shellfish Bivalve Oyster. International Journal of Current Microbiology
and Applied Sciences, 6 (1): 941-947.

Amini, R., Abdulamir, A.S., Jahanshiri, F., Shan, L.C., Hematian, A., Amini, Y., Sekawi, Z.
and Jalilian, F.A. (2012). Isolation and identification of methicilin-resistant
Staphylococcus aureus from students‘ coins. African Journal of Biotechnology, 11:
11143-11149.

Andersson, A., Ronner, U. and Granum, P.E. (1995). What problems does the food industry
have with the spore-forming pathogens Bacillus cereus and Clostridium perfringens?
International Journal of Food Microbiology, 28:145–155.

Andrews, W.H., Wilson, C.R., Romero, A. and Poelma, P.L. (1975). The Moroccan food
snail, Helix aspersa, as a source of Salmonella. Applied Microbiology, 29(3): 328-330.

131
Antwi, S. (2009). Innovative methods for processing snail (Achatina achatina) meat powder
of high microbiological and sensory qualities. PhD thesis. Kwame Nkrumah
University of Science and Technology, Kumasi, Ghana.

Arens, S., Verhaegen, J. and Verbist, L. (1997). Differentiation and susceptibility of

Citrobacter isolates from patients in a university hospital. Clinical Microbiology and
Infection, 3(1): 53-57.

Argudín, M. A., Mendoza, M. C. and Rodicio, M. R. (2010). Food Poisoning and

Staphylococcus aureus Enterotoxins. Toxins, 2: 1751—1773.
Asao, T., Kumeda, Y., Kawai, T., Shibata, T., Oda, H., Haruki, K., Nakazawa, H. and Kozaki,
S. (2003). An extensive outbreak of staphylococcal food poisoning due to low-fat milk
in Japan: estimation of enterotoxin A in the incriminated milk and powdered skim
milk. Epidemiology of Infection, 130: 33-40.
Babalola, O.O. and Akinsoyinu, A.O. (2009). Proximate composition and mineral profile of
snail meat from different breeds of land snail in Nigeria. Pakistan Journal of
Nutrition, 8: 1842-1844.

Bain, M. S. and Green, C. C. (1999). Isolation of Escherichia fergusonii in cases clinically

suggestive of salmonellosis. Veterinary Records, 144: 511.

Balaban, N. and Rasooly, A. (2000). Staphylococcal enterotoxins. International Journal of

Food Microbiology, 61: 1–10.

Bang-Andreasen, T., Nielsen, J. T., Voriskova, J., Heise, J., Rønn, R., Kjøller, R., Hansen, H.
C. B. and Jacobsen, C. S. (2017). Wood Ash Induced pH Changes Strongly Affect
Soil Bacterial Numbers and Community Composition. Frontiers in Microbiology, 8:
1-14.

Barlow, S.M., Boobis, A.R., Bridges, J., Cockburn, A., Dekant, W., Hepburn, P., Houben,
G.F., Konig, J., Nauta, M.J., Schuermans, J. and Banati, D. (2015). The role of hazard-
and risk-based approaches in ensuring food safety. Trends in Food Science and
Technology, 46(2): 176-188.

132
Bashar, T., Rahman, M., Rabbi, F. A., Noor, R. and Rahman, M. M. (2011). Enterotoxin
profiling and antibiogram of Escherichia coli isolated from poultry faeces in Dhaka
district of Bangladesh. Stamford Journal of Microbiology, 1(1): 51-57.

Beecher, D.J. and MacMillan, J. D. (1991). Characterization of the components of hemolysin

BL from Bacillus cereus. Infection and Immunology, 59: 1778–1784.

Bell, R .G. (1997). Distribution and sources of microbial contamination on beef carcasses.
Journal of Applied Microbiology, 82: 292–300.

Belongia, E.A., Knobloch, M.J., Kieke, B.A., Davis, J.P., Janette, C. and Besser, R.E. (2005).
Impact of statewide program to promote appropriate antimicrobial drug use. Emerging
Infectious Diseases, 11: 912-920.
Bergdoll, M. S. (1989). Staphylococcus aureus. Food borne bacterial pathogens. (Doyle, M.P.
ed). Marcel Dekker Inc. Pp 463-523.

Bergogne-Berezin, E. (1997). Who or what is the source of antibiotic resistance? Journal of

Medical Microbiology, 46: 461-470.

Bertelloni, F., Fratini, F., Ebani, V. V., Galiero, A., Turchi, B. and Cerri, D. (2015). Detection
of genes encoding for enterotoxins, TSST-1, and biofilm production in coagulase-
negative staphylococci from bovine bulk tank milk. Dairy Science and Technology,
95:341–352.
Bhalla, A., Aron, D. C. and Donskey, C. J. (2007). Staphylococcus aureus intestinal
colonization is associated with increased frequency of S. aureus on skin of
hospitalized patients. BioMed Central Infectious Diseases, 7: 105-111.

Bhattacharya, S. K., Datta, P., Datta, D., Bhattacharya, M. K., Sen, D. and Saha, M. R.
(1987). Relative efficacy of trimethoprim-sulfamethoxazole and nalidixic acid for
acute invasive diarrhea. Antimicrobial Agents and Chemotherapy, 31:837.

Bisht, D., Pande, R. C., Gupta, S. C. and Mehrotra, T. N. (1977). Haemolysin and necrotoxin
production of E. coli isolated from various sources. Indian Journal of Patholology and
Microbiology, 20(4): 223-229.

133
Blaiotta, G., Ercolini, D., Pennacchia, C., Fusco, V., Casaburi, A., Pepe, O. and Villani, F.
(2004). PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp.
strains isolated from meat and dairy products. Evidence for new variants of seG and
seI in S. aureus AB-8802. Journal of Applied Microbiology, 97: 719–730.

Bnyan, I. A., Alte‘ee, A. H. and Kadhum, N. H. (2014). Antibacterial Activity of Aluminum

Potassium Sulphate and Syzygium aromaticum Extract Against Pathogenic
Microorganisms. Journal of Natural Sciences Research, 4(15): 11-14.

Capita, R. and Alonso-Calleja, C. (2013). Antibiotic-resistant bacteria: a challenge for the

food industry. Critical Reviews in Food Science and Nutrition, 53(1): 11-48.

Cascon, A., Yugueros, J., Temprano, A., Sa´nchez, M., Hernanz, C., Luengo, J.M. and
Naharro, G. (2000). A major secreted elastase is essential for pathogenicity of
Aeromonas hydrophila. Infection and Immunology, 68: 3233–3241.

Centers for Disease Control and Prevention. (C.D.C.) (2005). What is an antibiotic? In:
National Antimicrobial Resistance Monitoring System (NARMS), Department of
Health and Human Services. Frequently asked questions about antibiotic resistance.
Atlanta, USA. (http://www.cdc.gov/narms/faq_pages).

Centre for Disease Control and Prevention (CDC). (2011). Estimates of food borne illness in
the United States. https://www.cdc.gov/foodborneburden/pdfs/FACTSHEET.1-2.
Cha, J. O., Lee, J. K., Jung, Y. H., Yoo, J. I., Park, Y. K., Kim, B. S. and Lee, Y. S. (2006).
Molecular analysis of Staphylococcus aureus isolates associated with Staphylococcal
food poisoning in South Korea. Journal of Applied Microbiology, 101: 864–871.

Chah, J. M. and Inegbedion, G. (2013). Characteristics of snail farming in Edo South

Agricultural Zone of Edo State, Nigeria. Tropical Animal Health and Production,
45(2): 625-631.

Chang, Y. H. (2000). Prevalence of Salmonella spp. in poultry broilers and shell eggs in
Korea. Journal of Food Protection, 63: 655-658.

134
Chart, H., Jenkins, C., Smith, H. R., Hedges, D. and Rowe, B. (1998). Haemolysin production
by strains of verocytotoxin-producing Escherichia coli. Microbiology, 144(1): 103-
137.

Chaudhary, S., Khurana, S.K. and Mane, B.G. (2014). Escherichia coli: Animal Foods and
Public Health-Review. Journal of Microbiology, Immunology And Biotechnology, 1:
31-46.

Chen, S., Wang, Y., Chen, F., Yang, H., Gan, M. and Zheng, J. S. (2007). A Highly
Pathogenic Strain of Staphylococcus sciuri Caused Fatal Exudative Epidermitis in
Piglets. PLoS ONE, 2(1): 147-152

Chen, Y.S., Wong, W. W., Fung, C. P., Yu, K. W. and Liu, C. Y. (2002). Clinical features
and antimicrobial susceptibility trends in Citrobacter freundii bacteremia. Journal of
Microbiology, Immunology and Infection, 35: 109-114.

Chopra, A.K., Xu, X., Ribardo, D., Gonzalez, M., Kuhl, K., Peterson, J.W. and Houston,
C.W. (2000). The cytotoxic enterotoxin of Aeromonas hydrophila induces
proinflammatory cytokine production and activates arachidonic acid metabolism in
macrophages. Infection and Immunology, 68: 2808–2818.

Cicero, A., Giangrosso, G., Cammilleri, G., Macaluso, A., Curro, V., Galuppo, L., Vargetto,
D., Vicari, D. and Ferrantelli, V. (2015). Microbiological and chemical analysis of
land snails commercialized in Sicily. Italian Journal of Food Safety, 4: 66-68.

Cifrian, E., Guidry, A. J., Bramley, A. J., Norcross, N. L., Bastida-Corcuera, F. D. and
Marquardt, W. W. (1996). Effect of staphylococcal alpha toxins on the cytotoxicity,
proliferation and adherence of S. aureus to bovine mammary epithelial cells.
Veterinary Microbiology, 48: 187-198.

Cîrlan, A. F., Floriștean, V. and Șindilar, E. (2010). The characterization and identification of
some microorganisms isolated from the meat of the food snail Helix pomatia. Scientific
Works, 56: 34-38.

Clinical and Laboratory Standards Institute. (2015). Performance Standards for Antimicrobial
Disk Susceptibility Test: Approved Standards. M02-A12. 12th Ed.
135
Clinical and Laboratory Standards Institute. (2017) Performance Standards for Antimicrobial
Susceptibility Testing. CLSI supplement M100. 27th Ed.

Codex Alimentarius Commission. (1997). Recommended international code of practice -

general principles of food hygiene. CAC/GL-21.
Codex Alimentarius Commission. (1999). Principles and guidelines for the conduct of
microbiological risk assessment. CAC/GL-30.

Coelho, S. M. O., Reinoso, E., Pereira, I. A., Soares, L. C., Demo, M., Bogni, C. and Souza,
M. M. S. (2009). Virulence factors and antimicrobial resistance of Staphylococcus
aureus isolated from bovine mastitis in Rio de Janeiro. Pesq. Vet. Brasilia, 29(5): 369-
374.

Corda, A., Mara, L., Virgilio, S., Pisanu, M., Chessa, G., Parisi, A. and Cogoni, M.P. (2014).
Microbiological and chemical evaluation of Helix spp., snails from local and non-EU
markets, utilized as food in Sardinia. Italian Journal of Food Safety, 3 (2).6-7
Courvalin, P. (2008). Predictable and unpredictable evolution of antibiotic resistance. Journal
of Internal Medicine, 264: 4-16.

Couto, I., Sanches, I. S., Sa-Leao, R. and De Lencastre, H. (2000). Molecular Characterization
of Staphylococcus Sciuri Strains Isolated From Humans. Journal of Clinical
Microbiology, 38(3): 1136–1143.

Cui, S., Ge, B., Zheng, J. and Meng, J. (2005). Prevalence and antimicrobial resistance of
Campylobacter spp. and Salmonella serovars in organic chickens from Maryland retail
stores. Applied and Environmental Microbiology, 74: 4108-4111.
Dakic, I. D., Vukovic, D., Stepanovic, S., Hauschild, T., Jezek, P., Petra, P. and Morrison, D.
(2005b). Survey of Genes Encoding Staphylococcal Enterotoxins, Toxic Shock
Syndrome Toxin 1, and Exfoliative Toxins in Members of the Staphylococcus sciuri
Group. Journal of Clinical Microbiology, 43(9): 4875-4876.
Dakic, I., Morrison, D., Vukovic, D., Savic, B., Shittu, A., Jezek, P., Hauschild, T. and
Stepanovic, S. (2005a). Isolation and molecular characterization of Staphylococcus
sciuri in the hospital environment. Journal of Clinical Microbiology, 43: 2782–2785.

136
De Padua, S.B., Marques, D. P., Sebastiao, F.A., Pilarski, F., Martins, M.L. and Ishikawa,
M.M. (2014). Isolation, characterization and pathology of Citrobacter freundii
infection in native Brazilian catfish Pseudoplatystoma. Brazilian Journal of
Veterinary Pathology, 7: 151-157.

Depooter, P., Persoons, D., Uyttendaele, M., Butaye, P., De Zutter, L., Dierick, K., Herman,
L., Imberechts, H., Van Huffel, X. and Dewulf, J. (2012). Assessment of human
exposure to 3rd generation cephalosporin resistant E. coli (CREC) through
consumption of broiler meat in Belgium. International Journal of Food Microbiology,
159: 30-38.

Dhanze, H., Khurana, S.K. and Mane, B.G. (2013). Effect of Seabuckthorn leaf extract on
microbiological quality of raw chicken during extended periods of storage. Journal of
Food Quality, 36(1): 59-66.

Diarrassaouba, F., Diarra, M. S., Bach, S., Delaquis, P., Pritchard, J., Topp, E. and Skura, B.
J. (2007). Antibiotic resistance and virulence genes in commensal E. coli and
Salmonella isolates from commercial broiler chicken farms. Journal of Food
Protection, 70(6): 1316-1327.

Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K. and Mattick, J. S. (1991). ‗Touchdown‘
PCR to circumvent spurious priming during gene amplification. Nucleic Acids
Research, 19: 4008.

Doulgeraki, A. I., Paramithiotis, S. and Nychas, G. J. (2011). Characterization of the

Enterobacteriaceae community that developed during storage of minced beef under
aerobic or modified atmosphere packaging conditions. International Journal of Food
Microbiology, 145: 77–83.

DuPont, H. L. (1989). Inoculum size in shigellosis and implications for expected mode of
transmission. Journal of Infectious Diseases, 159:1126–1128.

Dutta. S., De, S. P. and Bhattacharya, S. K. (1996). In vitro antimicrobial activity of potash
alum. Indian Journal of Medical Research, 104:157-159.

137
Ebenso, I., Ekwere, A., Akpan, B., Okon, B., Inyang, U. and Ebenso, G. (2012). Occurrence
of Salmonella, Vibrio and Escherichia coli in edible land snail in Niger Delta, Nigeria.
Journal of Microbiology, Biotechnology and Food Sciences, 2(2): 610-618.
Efuntoye, M.O., Mabekoje, O.O. and Adekoya, F.A. (2011). Biochemical, enterotoxigenicity
and antibiogram profiles of Staphylococcus aureus isolated from intestines of snails.
Journal of Microbiology and Antimicrobials, 3(3): 47-50.
Emelue, G.U., Evivie, S.E. and Edmowande, P. (2013). Processing and preservation of snail
meat in Benin city, Nigeria. Nigerian Journal of Agriculture, Food and Environment,
9(2): 15-19.
Emslie, L.J. (1982). Snails and snail farming. World Animal Review, 41: 20-26.
European Commission. (2004). Regulation EC 853/2004 on Laying down specific rules for
food of animal origin.
Ezeama, C.F. (2004). Microbiological characteristics and sensory attributes of potassium
sorbate treated and untreated smoked freshwater snail (Lanistes libycus). Global
Journal of Pure and Applied Sciences, 10(3): 363-367.
Ezeama, C.F., Keke, E. and Nwachukwu, E. (2007). Influence of brine treatment, drying
methods and storage conditions on the microbial quality of freshwater snail (Lanistes
libycus) meat. Nigerian Food Journal, 25(2): 101-111.
Fagbuaro, O., Oso, J.A., Edward, J.B. and Ogunleye, R.F. (2006). Nutritional status of four
species of giant land snails in Nigeria. Journal of Zhejiang University Science B, 7(9):
686-689.
Faille, C., Jullien, C., Fontaine, F., Bellon-Fontaine, M. N., Slomianny, C. and Benezech, T.
(2002). Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: role of
surface hydrophobicity. Canadian Journal of Microbiology, 48: 728–738.

Fauci, A.S., Braunwald, E., Kasper, D.L., Hauser, S.L., Logo, D.L., Jameson, L., and
Loscalzo, J. (2008). Harrison‘s principle of internal medicine. 7th ed. USA: McGraw-
Hill Campanies.

Federal Ministry of Agriculture and Rural Development Nigeria. (2016). The official website
of the ministry: www.fmard.gov.ng. Accessed on 27th April, 2016.

138
Federal Ministry of Health, Nigeria. (2014). National Policy on Food Safety and its
Implementation Strategy.

Finley, R. L., Collignon, P. and Larsson, D. G. (2013). The scourge of antibiotic resistance:
the important role of the environment. Clinical Infections and Diseases, 57: 704–710.

Fitzgerald, J. R., Hartigan, P. J., Meaney, W. J. and Smyth, C. J. (2000). Molecular population
and virulence factor analysis of Staphylococcus aureus from bovine intramammary
infection. Journal of Applied Microbiology, 88 (6): 1028–1037.

Fitzpatrick, D. and Walsh, F. (2016). Antibiotic resistance genes across a wide variety of
metagenomes. FEMS Microbiology Ecology, 92(2):1-8.

Food and Agricultural Organization/World Health Organization (FAO/WHO). (1995).

Application of risk analysis to food standards issues. Report of the joint FAO/WHO
expert consultations.
Food and Agricultural Organization/World Health Organization. (2006). Guidance to
governments on the application of HACCP in small and/or less-developed food
businesses. FAO food and nutrition paper 86.
Fumilayo, S.M. (2008). Preliminary investigation of the growth performance of Giant land
snail (Archachatina marginata) fed with selected household wastes. African Journal
of Agricultural Research, 3(9): 647-649.

Funke, G., Hany, A. and Altwegg, M. (1993). Isolation of Escherichia fergusonii from four
different sites in a patient with pancreatic carcinoma and cholangiosepsis. Journal of
Clinical Microbiology, 31: 2201–2203.

Gallo, G. (2002). Snail breeding and Exploitation. Mundi-Prensa, Madrid, Spain. 2 nd Ed. P
184.

Garofalo, C., Vignaroli, C., Zandri, G., Aquilanti, L., Bordoni, D. and Osimani, A. (2007).
Direct detection of antibiotic resistant genes in specimens of chicken and pork meat.
International Journal of Food Microbiology, 113: 75-83.
Gevers, D., Masco, L., Baert, L., Huys, G., Debevere, J. and Swings, J. (2003). Prevalence
and diversity of tetracycline resistant lactic acid bacteria and their tet genes along the
139
 process line of fermented dry sausages. Systematic and Applied Microbiology, 26:
277-283.
Gkogka, E., Reij, M.W., Gorris, L.G.M. and Zwietering, M.H. (2013). Risk assessment
strategies as a tool in the application of the Appropriate Level of Protection (ALOP)
and Food Safety Objectives (FSO) by risk managers. International Journal of Food
Microbiology, 167: 8-28.
Glasset, B., Herbin, S., Guillier, L., Cadel-Six, S., Vignaud, M., Grout, J., Pairaud, S., Michel,
V., Hennekinne, J., Ramarao, N. and Brisabois, A. (2016). Bacillus cereus-Induced
Food-Borne Outbreaks In France, 2007–2014: Epidemiology and Genetic
Characterisation. European Surveillance, 21(48): 30413.

Glover, B., Wentzel, J., Jenkins, A. and Vuuren, M. V. (2017). The first report of Escherichia
fergusonii isolated from non-human primates in Africa. One Health, 3: 70-75.

Goñi-Urriza, M., Capdepuy, M.; Arpin, C., Raymond, N. and Caumette, P. C. (2000). Impact
of an urban effluent on antibiotic resistance of riverine Enterobacteriaceae and
Aeromonas spp. Applied and Environmental Microbiology, 66 (1): 125-132.

Guerrant, R.L., Dickens, M.D., Wenzel, R.P. and Kapikian, A.Z. (1976). Toxigenic bacterial
diarrhea: nursery outbreak involving multiple bacterial strains. Journal of Pediatrics,
89: 885–891.

Hallis, B.A., Thurston, C.F. and Mason, J. R. (1991). Glucose control of enterotoxin A
synthesis and location is mediated by cyclic AMP. FEMS Microbiology Letters, 80:
247-251.

Halpin-Dohnalek, M. I. and Marth, E. H. (1989). Staphylococcus aureus: Production of

extracellular compounds and behavior in foods - A review. Journal of Food
Protection, 52:267-282.

Hansen, B. M. and Hendriksen, N. B. (2001). Detection of Enterotoxic Bacillus cereus and

Bacillus thuringiensis strains by PCR Analysis. Applied and Environmental
Microbiology, 67(1): 185-189.

140
Harmon, S. M., Goepfert, J. M. and Bennett, R. W. (1992). Bacillus cereus. In C. Vanderzant
and D. F. Splittstoesser (eds.). Compendium of methods for the microbiological
examination of foods. American Public Health Association, Washington, D.C. pp.
593-604.

Hatziioannou, H., Eleutheriadis, N. and M. Lazaridouodim Itriadou. (1994). Food preferences

of dietary overlap by terrestrial snail in Logos area (Edessa, Macedonia, Northern
Greece). Journal of Molluscan Studies, 60:331–341.

Havelaar, A. (2016). Foodborne diseases: an ongoing global challenge. Global G. A. P.

Summit. September 27-28, 2016, Amsterdam.

Hedin, G. and M. Widerstrom. (1998). Endocarditis due to Staphylococcus sciuri. European

Journal Clinical Microbiology and Infectious Diseases, 17:673–674.

Hennekinne, J. A., De Buyser, M. L. and Dragacci, S. (2012). Staphylococcus aureus and its
food poisoning toxins: characterization and outbreak investigation. FEMS
Microbiology Review, 36: 815–836.

Heymann, D.L. (2004). The American Public Health Association. Control of Communicable
Diseases Manual: an Official Report of the American Public Health Association. 18th
edn.. Washington, DC: American Public Health Association. 700.

Hodasi, J.K.M. (1984). Some observations on the edible giant land snails of West Africa.
World Animal Review, 52: 24-28.
Holeckova, B., Holoda, E., Fotta, M., Kalinacova, V., Gondol, J., and Grolmus, J. (2002).
Occurrence of enterotoxigenic Staphylococcus aureus in food. Annals of Agriculture
and Environmental Medicine, 9 (2): 179–182.

Holmberg, S. L. and Claesson, T. (2001). Mineralogy of granulated wood ash from a heating
plant in Kalmar, Sweden. Environmental Geology, 40: 820–828.

Horii, T., Suzuki, Y., Kimura, T., Kanno, T. and Maekawa, M. (2001) Intravenous catheter-
related septic shock caused by Staphylococcus sciuri and Escherichia vulneris.
Scandinavian Journal of Infectious Diseases, 33: 930–932.

141
Hurst, C.J., Crawford, R.L., Garland, J.L., Lipson, D.A., Mills, A.L. and Stetzenbach, L.D.
(2007). Manual of Environmental Microbiology.hydrophobicity. Canadian Journal of
Microbiology, 48: 728–738.

Huss, H. H., Reilly, A. and Embarek, P. K. B. (2000). Prevention and control of hazards in
seafood. Food Control, 11: 149–156.

Ibom, L. A., Okon, B. and Bassey, B. E. E. (2012). Egg Traits, Hatchability and Survivability
of Black-Skinned, White Skinned and Crossbred Archachatina marginata Snails.
International Journal of Agricultural Science and Bioresources Engineering
Research, 1: 10-18.

Igbinosa, I. B., Isaac, C., Adamu, H. O. and Adeleke, G. (2016). Parasites of edible land snails
in Edo State, Nigeria. Helminthologia, 53(4): 331 – 335.

International Commission on Microbiological Specification for Foods. (ICMSF). (1986).

Sampling plans for fish and shellfish. In ICMSF, Microorganisms in Foods. Sampling
for Microbiological Analysis: Principles and Specific Applications. Vol. 2 (2 nd Ed).
ICMSF. University of Toronto press, Buffalo, NY, pp: 55.

Isonhood, J.H. and Drake, M. (2012). Aeromonas species in Foods. Journal of Food
Protection, 65: (3)575–582.

Iwalokun, B. A., Gbenle, G. O., Smith, S. I., Ogynledun, A., Akinside, K. A. and
Omonigbehin, E. A. (2001). Epidemiology of Shigellosis in Lagos, Nigeria: Trends in
antimicrobial resistance. Journal of Health, Population and Nutrition, 19: 183-190.

Iwamoto, M., Ayers, T., Mahon, B. E. and Swerdlow, D. L. (2010). Epidemiology of seafood-
associated infections in the United States. Clinical Microbiology Review, 23:399–411.

Janda, J. M. and Abbott, S. L. (1998). Evolving concepts regarding the genus Aeromonas: An
expanding panorama of species, disease presentations, and unanswered questions.
Clinical Infectious Diseases, 27: 332-344.

142
Johnson, J. R., Kuskowski, M. A., Smith, K., O'Bryan, T. T. and Tatini, S. (2005).

Antimicrobial-resistant and extraintestinal pathogenic Escherichia coli in retail foods.

Journal of Infectious Diseases, 191(7): 1040-1049.

Johnson, J. R., Sannes, M. R., Croy, C., Johnston, B., Clabots, C. and Kuskowski, M.A.
(2007). Antimicrobial drug–resistant Escherichia coli from humans and poultry
products, Minnesota and Wisconsin, 2002–2004. Emerging Infectious Disease, 13(6):
838-846.

Jokinen, H. K., Kiikkila, O. and Fritze, H. (2001). Exploring the mechanisms behind elevated
microbial activity after wood ash application. Soil Biology and Biochemistry, 38:
2285–2291.

Jordan, D., Philips, D., Summer, J., Morris, S. and Jenson, I. (2006). Relationships between
the density of different indicator organisms on sheep and beef carcasses and in frozen
beef and sheep meat. Journal of Applied Microbiology, 102: 57-64.
Kerouanton, A., Hennekinne, J.A., Letertre, C., Petit, L., Chesneau, O. and Brisabois, A.
(2007). Characterization of Staphylococcus aureus strains associated with food
poisoning outbreaks in France. International Journal of Food Microbiology, 115: 369-
375.
Kirkan, P.K.G., Ksoy, E.N. and Kaya, O. (2006). Detection of Listeria monocytogenes by
using PCR in Helix pomatia. Turkish Journal of Veterinary and Animal Science, 30:
375-380.
Kloos, W. E., Schleifer, K. H. and Smith, R.F. (1976). Characterization of Staphylococcus
sciuri sp. nov. and its subspecies. International Journal of Systematic Bacteriology,
26:22–37.
Kobayashi, H., Pohjanvirta, T. and Pelkonen, S. (2002). Prevalence and characteristics of
intimin- and Shigatoxin-producing Escherichia coli from gulls, pigeons and broilers in
Finland. Journal of Veterinary Medical Science, 64(11): 1071-1073.

Kornacki, J. L. and Marth, E. H. (1982). Foodborne Illness Caused by Escherichia coli: A

Review. Journal of Food Protection, 45(11): 1051-1067.

143
Kornacki, J. L. (2010). An indicator approach to enteric contamination of at risk food.
International Association for Food Protection Symposium S22.
Kornacki, J. L. (2011). Indicator organism assays: chaos, confusion and criteria. Food Safety
Magazine.

Kuhn, I., Albert, J.M., Ansaruzzaman, M., Bhuiyan, N.A., Alabi, S.A., Islam, M.S., Neogi,
P.K.B., Huys, G., Janssen, P., Kersters, K. and Mollby, R. (1997). Characterization of
Aeromonas spp. isolated from humans with diarrhea, from healthy controls and from
surfacewater in Bangladesh Journal of Clinical Microbiology, 35:369–373.

Lammerding, A.M. (2006). Modelling and risk assessment for Salmonella in meat and
poultry. Journal of AOAC International, 89(2): 543-552.
Lammerding, A.M. and Paoli, G.M. (1997). Quantitative risk assessment: an emerging tool
for emerging foodborne pathogens. Emerging Infectious Diseases, 3:483-487.
Landers, T.F., Cohen, B., Wittum, T. E. and Larson, E.L. (2012). A review of antibiotic use in
food animals: perspective, policy, and potential. Public Health Reports, 127(1): 4-22.
Lane, D.J. (1991). 16S/23S rRNA Sequencing. In Nucleic Acid Techniques in Bacterial
Systematics; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley & Sons: New York,
NY, USA. pp. 115-175.
Lawley, R. (2013). Antibiotic resistance: a global food safety problem. Food Safety Watch.

Le Loir, Y., Baron, F. and Gautier, M. (2003). Staphylococcus aureus and food poisoning.
Genetics and Molecular Research, 2(1): 63–76.

Leverstein-van Hall, M.A., Dierickx, C.M., Stuart, J.C., Voets, G.M., van den Munckhof,
M.P., van Essen-Zandbergen, A., Platteel, T., Fluit, A.C., van de Sande-Bruinsma, N.
and Scharinga, J. (2011). Dutch patients, retail chicken meat and poultry sharethe
same ESBL genes, plasmids and strains. Clinical Microbiological Infection, 17: 873-
880.
Lina, G., Bohach, G.A., Nair, S.P., Hiramatsu, K., Jouvin-Marche, E. and Mariuzza, R.
(2004). Standard nomenclature for the superantigens expressed by Staphylococcus.
Journal of Infectious Diseases, 189: 2334-2336.

144
Logan, N. A. (2011). Bacillus and relatives in foodborne illness. Journal of Applied
Microbiology, 112: 417-429.

Logan, N. A., Hoffmaster, A., Shadomy, S.V. and Stauffer, K. (2011). Bacillus and related
genera. In Manual of Clinical Microbiology ed. Versalovic, J., Carroll, K.C., Funke,
G., Jorgensen, J.H., Landry, M.L. and Warnock, D.W., 10th, edn, Vol. 1, pp. 381–402
Washington D. C: American Society for Microbiology.

Lozano-Leon, A., Iglesias-Canle, J., Iglesias-Garcia, J., Larino-Noia, J. and Mun˜oz, E. D.

(2011). Citrobacter freundii infection after acute necrotizing pancreatitis in a patient
with a pancreatic pseudocyst: a case report. Journal of Medical Case Reports, 5: 51-
54.

Luna, A. V., King, D. S., Gulledge, J., Cannons, A. C., Amuso, P. T. and Cattani, J. (2007).
Susceptibility of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus
pseudomycoides and Bacillus thuringiensis to 24 antimicrobials using Sensititre
automated microbroth dilution and Etest agar gradient diffusion methods. Journal of
Antimicrobial Chemotherapy, 60: 555–567.

Lund, T. and Granum, P.E. (1996). Characterisation of a nonhaemolytic enterotoxin complex

from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiology Letters,
141: 151–156.

Lund, T., De Buyser, M.L. and Granum, P.E. (2000). A new cytotoxin from Bacillus cereus
that may cause necrotic enteritis. Molecular Microbiology, 38: 254–261.

Ma, L., Kornacki, J.L., Zhang, G., Lin C-M. and Doyle, M.P. (2007). Development of thermal
surrogate microorganisms in ground beef for in-plant critical control point validation
studies. Journal of Food Protection, 70:952-957.
Magnusson, S.H., Gunnlaugsdottir, H., van Loveren, H., Holm, F., Kalogeras, N.and Leino,
O. (2012). State of the art in benefit-risk analysis: food microbiology. Food and
Chemical Toxicology, 50: 33-39.

Mahapatra, A. and Mahapatra, S. (2005). Escherichia fergusonii: An emerging pathogen in

South Orissa. Indian Journal of Medical Microbiology, 23:204.
145
Makita, K., Desissa, F., Teklu, A., Zewde, G. and Grace, D. (2012). Risk assessment of
staphylococcal poisoning due to consumption of informally-marketed milk and home-
made yoghurt in Debre Zeit, Ethiopia. International Journal of Food Microbiology,
153: 135-141.
Malik, A.A., Aremu, A., Bayode, G.B. and Ibrahim, B.A. (2011). A nutritional and
organoleptic assessment of the meat of the giant African land snail (Archachatina
marginata swaison) compared to the meat of other livestock. Livestock Research for
Rural Development, 23(3) 60.
Mantynen, V. and Lindstrom, K. (1998). A Rapid PCR-based DNA Test For Enterotoxic
Bacillus cereus. Applied and Environmental Microbiology, 64(5): 1634–1639.
Marta, D., Wallin-Carlquist, N., Schelin, J., Borch, E. and Radstrom, P. (2011). Extended
staphylococcal enterotoxin D expression in ham products. Food Microbiology, 28:
617-620.

Martinez, J. L. (2008). Antibiotics and antibiotic resistance genes in natural environments.

Science, 321: 365–367.

Mathur, S. and Singh, R. (2005). Antibiotic resistance in food lactic acid bacteria: a review.
International Journal of Food Microbiology, 105: 281-295.

Maurelli, A. T., Blackmon, B. and Curtiss, R. (1984). Temperature-dependent expression of

virulence genes in Shigella species. Infection and Immunology, 43:195-201.

Mc Ewen, S.A. and Fedorka-Cray, P.J. (2002). Antimicrobial use and resistance in animals.
Clinical Infectious Diseases, 34(3): 93-106.
Mc Mahon, M.A.S., Blair, I.S., Moore, J.E. and Mc Dowell, D.A. (2007). The rate of
horizontal transmission of antibiotics resistance plasmids is increased in food
preservation-stressed bacteria. Journal of Applied Microbiology, 103: 1883-1888.
Mckone, T.E. (1996). Overview of the risk analysis approach and terminology: the merging of
science, judgement and values. Food Control, 7(2). 69-76.
Medema, G.J., Payment, P., Dufour, A., Robertson, W., Waite, M., Hunter, P., Kirby, R. and
Andersson, Y. (2003). Safe drinking water: an ongoing challenge. In: Assessing
Microbial Safety of Drinking water: improving approaches and methods. Dufour, A.,

146
 Snozzi, M., Koster, W., Bartram, J., Ronchi, E., and Fewtrell, L. WHO drinking water
quality series, OECD-WHO, Paris, France. London, UK: IWA Publishing.
Mensah, L.D. and Julien, D. (2011). Implementation of food safety management systems in
the UK. Food Control, 22(8): 1216-1225.
Mensah, P., Mwamakamba, L., Mohammed, C. and Nsue-Milang, D. (2012). Public health
and food safety in the WHO Africa region. African Journal of Food, Agriculture,
Nutrition and Development,12: 6317-6335.
Murray, P. R., Baron, E.J., Jorgensen, J., Pfaller, M.A. and Landry, M.L. (2007). Manual of
Clinical Microbiology. 9th ed. ASM Press. Washington DC.
National Agricultural Extension and Research Liaison Services (NAERLS). (1995). Snail
production techniques in Nigeria. Extension Bulletin No. 108. Forestry Series No. 12.
Ahmadu Bello University, Zaria.
Nazari, R., Godarzi, H., Rahimi B. F. and Moeinrad, M. (2014). Enterotoxin gene profiles
among Staphylococcus aureus isolated from raw milk. Iranian Journal of Veterinary
Research, Shiraz University, 15(4): 409-412.

Neyts, K., Huys, G., Uyttendaele, M., Swings, J. and Debevere, J. (2000). Incidence and
Identification of Mesophilic Aeromonas Spp. from Retail Foods. Letters in Applied
Microbiology, 31: 359-363.

Nguyen, V.D., Sreenivasan, N. and Lam, E. (2014). Cholera epidemic associated with
consumption of unsafe drinking water and street-vended water-Eastern Freetown,
Sierra Leone, 2012. The American Journal of Tropical Medicine and Hygiene, 90(3):
518-523.

Niyogi, S. K. (2007). Increasing antimicrobial resistance: an emerging problem in the

treatment of shigellosis. Clinical Microbiology and Infection, 13:1141–1143.

Noble, R.T. and Fuhrman, J.A. (2001). Enteroviruses detected by reverse transcriptase
polymerase chain reaction from the coastal waters of Santa Monica Bay, California:
low correlation to bacterial indicator levels. Hydrobiologia, 460 (1-3): 175-184.

147
Nodu, M.B., Adesope, O.M. and Matthews-Njoku, E.C. (2003). Demographic characteristics
related to consumption of snail meat among inhabitants of Bori, Nigeria. African
Journal of Livestock Extension, 2: 54-57.
Norrby, S.R., Nord, C.E. and Finch, R. (2005). European Society of Clinical Microbiology
and Infectious Diseases. Lack of development of new antimicrobial drugs: a potential
serious threat to public health. Lancet Infectious Diseases, 5:115-119.
Nteimam, J. (2005). Screening for extended-spectrum beta-lactamase-producing pathogenic
enterobacteria in district general hospital. Journal of Clinical Microbiology, 43(3):
1488-1490.

Nyoagbe, L. A., Appiah, V., Nketsia- Tabiri, J., Larbi, D. and Adjei, I. (2016). Evaluation of
African giant snails (Achatina and Archachatina) obtained from markets (wild) and
breeding farms. African Journal of Food Science, 10(7): 94-104.

Obi, S. K. C. and Nzeako, B. C. (1980). Salmonella, Arizona, Shigella and Aeromonas

isolated from the snail Achatina achatina in Nigeria. Antonie van Leeuwenhoek, 46:
475-481.

Odaibo, A.B. (1997). Snail and snail farming. Nigeria Edible Land Snails. Stirling Horden
Publishers, Ibadan. 1: 1-11.
Okafor, A. C., Aquaowo, U. A., Ojiagu, K. D. and Agu, K. C. (2017). Preliminary Studies on
Processed Garri as a Source of Bacterial Hazards to Students. Immunology and
Infectious Diseases, 5(3): 25-29.

Okafor, F. C. (1989). Consumption and assimilation of food in Achatina achatina (L).

Tropical Ecology, 20(1): 52-57.

Okonkwo, T.M. and Anyaene, I.U. (2009). Meat yield and the effect of curing on the
characteristics of snail meat. Journal of Tropical Agriculture, Food, Environment and
Extension, 8(1): 66-73.

Olmez, A., Can, H., Ayhan, H. and Olur, H. (1998). Effect of an alum-containing mouthrinse
in children for plaque and salivary levels of selected oral microflora. Journal of
Clinical Paediatrics and Dentistry, 70: 191-92.

148
Omenewa, V.C., Ansa, E.J., Agokei, O.E., Uka, A. and George, O.D. (2011). Microbiological
quality of raw and processed farm reared periwinkle from brackish water eastern pond,
Buguma, Nigeria. African Journal of Food, Agriculture, Nutrition and Development,
11(2): 4621-4630.
Omoe, K., Hu, D. L., Takahashi-Omoe, H., Nakane, A. and Shinagawa, K. (2003).
Identification and characterization of a new staphylococcal enterotoxin-related
putative toxin encoded by two kinds of plasmids. Infection and Immunology, 71:
6088-6094.
Omole, A.J. (2001). Problems associated with snail farming: A paper presented at the monthly
technical review meeting of Ondo state Agricultural Development Project.

Onifade, A. K. and Aiyenuro, E. A. (2018). Antimicrobial Susceptibility Profile of

Microorganisms Isolated from the Intestine and Body Parts of the African Giant Land
Snail (Achatina achatina) Sold in Akure, Nigeria. Journal of Advances in
Microbiology, 9(4): 1-8.

Opara, L. U. (2003). Traceability in agriculture and food supply chain: a review of basic
concepts, technological implications and future prospects. Journal of Food,
Agriculture and Environment, 1(1): 101 – 106.

Oranusi, S. and Nubi, F. E. (2016). Microbiological Safety Evaluation of Ready to Eat

Shrimps and Snails Sold Along Lagos-Shagamu Expressway, Nigeria. Covenant
Journal of Physical and Life Sciences, 4(1): 20-32.

Otalu, O. J., Junaidu, K., Chukwudi, O. E. and Jarlath, U. V. (2011). Multi-drug resistant
coagulase positive S. aureus from live and slaughtered chickens in Zaria, Nigeria.
International Journal of Poultry Science, 10(11): 871-875.

Owusu-Kwarteng, J., Wuni, A., Akabanda, F., Tano-Debrah, K. and Jespersen, L. (2017).
Prevalence, virulence factor genes and antibiotic resistance of Bacillus cereus sensu
lato isolated from dairy farms and traditional dairy products. BioMedCentral
Microbiology, 17(65): 1-8.

149
Palumbo, S. A. (1996). The Aeromonas hydrophila group in food. In The Genus Aeromonas
ed. Austin, B., Altwegg, M., Gosling, P.J. and Joseph, S. Chichester, UK: Wiley and
Sons. Pp. 287-310.

Pardia, S. N., Verma, I. C., Deb, M. and Bhujwala, R. A. (1980). An outbreak of diarrhea due
to Citrobacter freundii in a neonatal special care nursery. Indian Journal of
Paediatrics, 47: 81-84.

Parlapani, F. F., Neofitou, C. and Boziaris, I. S. (2014). Microbiological quality of raw and
processed wild and cultured edible snails. Journal of the Science of Food and
Agriculture, 94:768-772.

Pawar, K. D., Banskar, S., Rane, S. D., Charan, S. S., Kulkarni, G. J., Sawant, S. S., Ghate, H.
V., Patole, M. S. and Shouche, Y. S. (2012). Bacterial diversity in different regions of
gastrointestinal tract of Giant African Snail (Achatina fulica). Microbiology Open, 1(4):
415–426.

Payment, P. and Locas, A. (2011). Pathogens in water: value and limits of correlation with
microbial indicators. Ground Water, 49(1): 4-11.
Piechota, M., Kot, B., Zdunek, E., Mitrus, J., Wicha, J., Wolska, M. K. and Sachanowicz, K.
(2014). Distribution of classical enterotoxin genes in staphylococci from milk of cows
with and without mastitis and the cowshed environment. Polish Journal of Veterinary
Sciences, 17(3): 407–411.
Popoff, M. R. (2011). Overview of bacterial enteropathogens and enterotoxins. Future
Microbiology, 6(7): 763-797.

Prasai, R. K., Phebus, R. K., Garcia Zepeda, C. M., Kastner, C. L., Boyle, A. E. and Fung, D.
Y. C. (1995). Effectiveness of trimming and/or spray washing on microbiological
quality of beef carcasses. Journal of Food Protection, 58: 1114–1117.

Prub, B. M., Dietrich, R., Nibler, B., Martbauer, E. and Scherer, S. (1999). The haemolytic
enterotoxin HBL is broadly distributed among species of the Bacillus cereus group.
Applied and Environmental Microbiology, 65: 5436-5442.

150
Public Health England. (2014). Identification of Staphylococcus species. UK Standards for
Microbiology Investigations, 3: 1-32.

Public Health England. (2015). Identification of Aeromonas and Vibrio species. UK

Standards for Microbiology Investigations, 3:1-30.

Public Health England. (2015). Identification of Bacillus species. UK Standards for

Microbiology Investigations, 3:1-27.

Public Health England. (2015). Identification of E. coli O157:H7. UK Standards for

Microbiology Investigations, 3:1-30.

Public Health England. (2015). Identification of Shigella species. UK Standards for

Microbiology Investigations, 3:1-22.
Raina, P.S., Pollari, F.L., Teare, G.F., Goss, M.J., Barry, D.A. and Wilson, J.B. (1999). The
relationship between E. coli indicator bacteria in well-water and gastrointestinal
illnesses in rural families. Canadian Journal of Public Health, 90:172–175.
Rajkovic, A. (2014). Microbial toxins and low level of foodborne exposure. Trends in Food
Science and Technology, 38(2): 149-157.

Rauth, S. K. and Barker, G. M. (2002). Achatina fulica Bowdich and other Achatinidae as a
pest in tropicultural agriculture. In L. R. Hamilton, ed. Mollusks as crop pest. KABI
International Publishing, New Zealand. P. 474.

Reij, M.W. and van Schothorst, M. (2000). Critical notes on microbiological risk assessment
of food. Brazillian Journal of Microbiology, 31(1): 1-8.

Reingold, J., Starr, N., Maurer, J. and Lee, M. D. (1999). Identification of a new Escherichia
coli She haemolysin homolog in avian E. coli. Veterinary Microbiology, 66(2): 125-
134.

Rivera, A. M. G., Granum, P. E. and Priest, F. G. (2000). Common occurrence of enterotoxin

genes and enterotoxicity in Bacillus thuringiensis. FEMS Microbiology Letters, 190:
151-155.

151
Sabrina-Hossain, S.H.M.P. Wimalasena and Gang-Joon Heo, (2017). Virulence Factors and
Antimicrobial Resistance Pattern of Citrobacter freundii Isolated from Healthy Pet
Turtles and their Environment. Asian Journal of Animal and Veterinary Advances, 12:
10-16.

Sando, D., Grujic, R. and Vujadinovic, D. (2013). Sensory attributes of snail‘s meat prepared
in different ways. Quality of Life, 4(3-4): 62-68.

Schaumburg, F., Ngoa, U. A. and Kosters, K. (2011). Virulence factors and genotypes of
Staphylococcus aureus from infection and carriage in Gabon. Clinical Microbiology
and Infection, 17: 1507–1513.

Schelin, J., Wallin-Carlquist, C.T., Marianne, L.R., Barker, C.G. and Radstrom, P. (2011).
The formation of Staphylococcus aureus enterotoxin in food environments and
advances in risk assessment. Virulence, 2(6): 580-592.

Schlegelova, J., Brychta, J., Klimova, E., Napravnikova, E. and Babak, V. (2003). The
prevalence of and resistance to antimicrobial agents of Bacillus cereus isolates from
foodstuffs. Veterinary Medicine-Czech, 11: 331–338.

Schoeni, J. L. and Wong, A. C. L. (2005). Bacillus cereus food poisoning and its toxins.
Journal of Food Protection, 68(3): 636-648.

Scott, V.N., Chen, Y., Freier, T.A., Kuehm, J., Moorman, M., Meyer, J., Morille-Hinds, T.,
Post, L., Smoot, L., Hood, S., Shebuski, J. and Banks, J. (2009). Control of Salmonella
in low moisture foods I: minimizing entry of Salmonella into a processing facility.
Food Protection Trends, 29: 342-353.

Serrano, S., Medina, L.M., Jurado, M. and Jodral, M. (2004). Microbiological quality of
terrestrial gastropods prepared for human consumption. Journal of Food Protection,
67: 1779-1781.

Silva, T. M., Melo, E. S., Lopes, A. C. S., Veras, D. L., Duarte, C. R., Alves, L. C. and
Brayne, F. A. (2013). Characterization of the bacterial microbiota of Biomphalaria
glabrata (Say, 1818) (Mollusca: Gastropoda) from Brazil. Letters in Applied
Microbiology, 57: 19–25.
152
Singh, V., Somvanshi, P., Rathore, G., Kapoor, D. and Mishra, B.N. (2009). Gene cloning,
expression and homology modeling of hemolysin gene from Aeromonas hydrophila.
Protein Experiment and Purification, 65: 1–7.

Siragusa, G. R., Dorsa, W. J., Cutter, C. N., Bennett, G. L., Keen, J. G. and Koohmaraie, M.
(1998). The Incidence of Escherichia coli on Beef Carcasses and its Association with
Aerobic Mesophilic Plate Count Categories During the Slaughter Process. Journal of
Food Protection, 61(10): 1269-1274.

Smith, J. L., Drum, D. J., Dai, Y., Kim, J. M., Sanchez, S. and Maurer, J. J. (2007). Impact of
antimicrobial usage on antimicrobial resistance in commensal Escherichia coli strains
colonizing broiler chickens. Applied and Environmental Microbiology, 73(5): 1404-
1414.

Speiser, B. (2001). Food and feeding behavior. In: the biology of terrestrial mollusks. Barker,
G.M. (ed). CABI publishing, Oxon.
Spellberg, B., Powers, J.H., Brass, E.P., Miller, L.G. and Edwards Jr., J.E., (2004). Trends in
antimicrobial drug development: implications for the future. Clinical Infectious
Diseases, 38: 1279–1286.
Sproston, E.L., Macrae, M., Ogden, I.D., Wilson, M.J. and Strachan, N.J. (2006). Slugs:
potential novel vector of Escherichia coli O157. Applied and Environmental
Microbiology, 72(1): 144-149.

Steenari, B. M., Karlsson, L. G. and Lindqvsit, O. (1999). Evaluation of the leaching

characteristics of wood ash and the influence of ash agglomeration. Biomass
Bioenergy, 16: 119–136.

Stepanovic, S., Jezek, P., Vukovic, D., Dakic, I. and Petras, P. (2003) Isolation of members of
the Staphylococcus sciuri group from urine and their relationship to urinary tract
infections. Journal of Clinical Microbiology, 41: 5262–5264.

Taneja, N. and Mewara, A. (2016). Shigellosis: Epidemiology in India. Indian Journal of

Medical Research, 143(5): 565–576.

153
Temelli, S., Dokuzlu, C. and Sen, M. K. C. (2006). Determination of microbiological
contamination sources during frozen snail meat processing stages, Food Control, 17:
22–29.

Tettey, E.C.T., Osei-Yaw, A. and Hodari-Okae, M. (1997). Studies on the quality of

traditionally smoked-dry snail meat (Achatina achatina) in Ghana. Ghana Journal of
Agricultural Science, 30: 145-150.
Thomas, D., Chou, S., Dauwalder, O. and Lina, G. (2007). Diversity in Staphylococcus
aureus enterotoxins. Chemistry, Immunology and Allergy, 93:24–41.
Toader, A. (2012). The influence of feed upon the Helix sp edible snail‘s production
performance under the bioeconomic aspect. Ph.D. thesis. University of Agricultural
science and veterinary medicine CLUJ-NAPOCA, Romania.
Toader-Williams, A. and Golubkina, N. (2009). Investigation upon the edible snail‘s potential
as source of selenium for human health and nutrition observing its food chemical
contaminant risk factor with heavy metals. Bulletin UASVM Agriculture, 66(2): 495-
499.
Tuominen, P., Hielm, S., Aarnisalo, K., Suihko, M.L., Raaska, L. and Maijala, R. (2001).
Development of a risk assessment-based software tool for the evaluation of food
industry HACCP plans. Poster. 4th International Meeting of the Noordwijk Food
Safety and HACCP Forum, Food safety - a shared responsibility. Noordwijk, the
Netherlands, March 15.-16.
Tzouros, N. E. and Arvanitoyannis, I. S. (2000). Implementation of Hazard Analysis Critical
Control Point (HACCP) system to the fish/seafood industry: A review. Food Review
International, 16: 273–325.

Ukut, I.O.E., Okonko, I.O., Ikpoh, I.S., Nkang, A.O., Udeze, A.O., Babalola, T.A., Mejeha,
O.K. and Fajobi, E.A. (2010). Assessment of bacteriological quality of fresh meats
sold in Calabar metropolis, Nigeria. Electronic Journal of Environment, Agricultural
Food Chemistry, 9: 89-100.

Verraes, C., Van Boxstael, S., Van Meervenne, E., Van Coillie, E., Butaye, P., Catry, B., de
Schaetzen, M. A., Van Huffel, X., Imberechts, H., Dierick, K., Daube, G., Saegerman,
C., De Block, J., Dewulf, J. and Herman, L. (2013). Antimicrobial resistance in the
154
 food chain: A review. International Journal of Environmental Research and Public
Health, 10:2643–2669.

Von Graevenitz, A. (2007). The role of Aeromonas in diarrhoea: a review. Infection, 35(2):
59–64.

Von Stetten, F., Mayr, R. and Scherer, S. (1999). Climatic influence on mesophilic Bacillus
cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical,
temperate and alpine soil. Environmental Microbiology, 1: 503–515.

Walker. A.J., Dmglen, A. and Shewy, P.R. (1999). Bacteria associated with the digestive
system of the slug Deroceras recticulum are not required for protein digestion. Soil
Biology and Biochemistry, 31: 387-394.

Wang, C. and Silva, J. L. (1999). Prevalence and characteristics of Aeromonas species

isolated from processed channel catfish. Journal of Food Protection, 62:30–34.

Warner, R.D., Carr, R.W., McCleskey, F.K., Johnson, P.C., Elmer, L.M. and Davison, V.E.
(1991). A large nontypical outbreak of Norwalk virus gastroenteritis associated with
exposing celery to nonpotable water and with Citrobacter freundii. Arch Internal
Medicine, 151: 2419-2424.

World Health Organisation. (2002). Use of antimicrobials outside human medicine and
resultant antimicrobial resistance in humans. 268th fact sheet. World Health
Organization, Geneva. (http://www.who.int/mediacentre/factsheets/fs268/en/).

World Health Organisation. (2006). WHO consultation to develop a strategy to estimate the
global burden of foodborne diseases: taking stock and charting the way forward,
World Health Organisation, Geneva.

World Health Organization. (2009a). Global burden of disease. Geneva.

World Health Organization. (2009b). Risk characterization of microbiological hazards in
food: Guidelines. Microbiological Risk Assessment series. No.17.
World Health Organization. (2009c). Risk assessment.
www.who.int/foodsafety/micro/riskassessment/en/index.html.

155
World Health Organization. (2013). Ensuring food safety and nutrition security to protect
consumer health: 50 years of the CAC. Bulletin of the WHO. 91:4.
World Health Organization. (2015). W.H.O. estimates of the global burden of foodborne
diseases. Foodborne disease burden epidemiology reference group 2007-2015.
Geneva.
Wu, C.J., Wu, J.J., Yan, J.J., Lee, H.C., Lee, N.Y., Chang, C.M., Shih, H.I. and Wu, H.M.
(2007). Clinical significance and distribution of putative virulence markers of 116
consecutive clinical Aeromonas isolates in southern Taiwan. Journal of Infection, 54:
151–158.
Wu, J., Long, S. C., Das, D. and Dorner, S. M. (2011). Are microbial indicators and
pathogens correlated? A statistical analysis of 40 years of research. Journal of Water
and Health, 9(2): 265-278.
Yang, I. C., Shih, D. Y-C., Huang, T-P., Huang, Y-P., Wang, J-Y. and Pan, T-M. (2005).
Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the
species in the Bacillus cereus group. Journal of Food Protection, 68(10): 2123 – 2130.
Zargar, M. H. S., Doust, R. H. and Mobarez, A. M. (2014). Staphylococcus aureus
Enterotoxin A Gene Isolated From Raw Red Meat and Poultry in Tehran, Iran.
International Journal of Enteric Pathogens, 2(3): e16085.

156
 APPENDIX 1
Descriptions of typical colonies of selected presumptive pathogens
Presumptive pathogen Description of typical colonies
Citrobacter Circular, smooth, raised, light pink colonies with black centres, 2
mm in diameter, mucoid surfaces and opaque on Salmonella-
shigella agar after 24 hours of incubation.
Shigella Circular, smooth, raised, colourless colonies, 2 mm in diameter,
mucoid surfaces and transluscent on Salmonella-shigella agar after
24 hours of incubation.
E. coli Circular, smooth, flat, greenish-metallic sheen colonies with dark
centres, semi-mucoid surfaces, 1-3 mm in diameter and opaque on
Eosin Methylene Blue agar after 24 hours of incubation.
S. aureus Circular, smooth, raised, yellow colonies with yellow zones, 2-3
mm in diameter, mucoid surfaces and opaque on Mannitol Salt
agar after 24 hours of incubation.
Aeromonas Circular, smooth, slightly flattened yellow colonies with opaque
centres and transluscent borders, 2-4 mm in diameter with mucoid
surfaces on Thiosulfate Citrate Bile salt Sucrose agar after 24
hours of incubation.
B. cereus Irregular, umbonate, entire, wrinkled, white or cream colonies
with waxy aspects, 3-7 mm in diameter, sometimes smooth and
mucoid surfaces, and opaque on Brain Heart Infusion agar after 24
hours of incubation.

157
 APPENDIX 2

Purification of Amplicon

1. Add 2 vol (20 µl) of absolute ethanol to the amplicon

2. Incubate at room temperature for 15 minutes

3. Spin down at 10,000 rpm for 15 minutes

4. Decant supernatant

5. Spin down at 10,000 rpm for 15 minutes

6. Add 2 vol (40 µl) of 70% ethanol

7. Decant supernatant

8. Air dry

9. Add about 10 µl of ultrapure water

10. Check for amplicon on 1.5% agarose

The amplicon is now ready for sequencing reaction.

158
 APPENDIX 3
DATA ANALYSIS OF AEROBIC PLATE COUNTS OF BACTERIA IN SNAILS
DATA SUMMARY ON AEROBIC PLATE COUNTS OF BACTERIA IN SNAILS
Samples
1 2 3 Total
N 100 100 100 300
X 874.4 888.3 932.2 2694.9
Mean 8.744 8.883 9.322 8.983
X2 7654.554 7900.427 8698.53 24253.511
Variance 0.0889 0.0976 0.0865 0.1513
Standard Deviation 0.2981 0.3123 0.2941 0.3889
Standard Error 0.0298 0.0312 0.0294 0.0225

STANDARD WEIGHTED-MEANS ANALYSIS

ANOVA SUMMARY
Source SS Df MS F P
Treatment 18.2042 2 9.1021 74.79 <.0001
(Between groups)
Error 24.1005 198 0.1217 ****** ******
Ss/Bl 2.9196 99 ****** ****** ******
Total 45.2243 299 ****** ****** ******
Ss/Bl = Subjects or Blocks depending on the design.

TUKEY HSD TEST

HSD[.05]=0.12; HSD[.01]=0.15
M1 vs M2 P<.05
M1 vs M3 P<.01
M2 vs M3 P<.01

M1 = Mean of Sample 1 (Anambra samples)

M2 = Mean of Sample 2 (Ebonyi samples)
M3 = Mean of Sample 3 (Enugu samples)

159
 APPENDIX 4
DATA ANALYSIS OF COLIFORM COUNTS IN SNAILS
DATA SUMMARY ON PLATE COUNTS OF COLIFORMS IN SNAILS
Samples
1 2 3 Total
N 100 100 100 300
X 741.4 763.7 749.9 2255
Mean 7.414 7.637 7.499 7.5167
X2 5500.046 5846.023 5635.057 16981.126
Variance 0.0334 0.1378 0.1167 0.1038
Standard Deviation 0.1828 0.3713 0.3417 0.3222
Standard Error 0.0183 0.0371 0.0342 0.0186

STANDARD WEIGHTED-MEANS ANALYSIS

ANOVA SUMMARY
Source SS Df MS F P
Treatment 2.5333 2 1.2666 10.93 <.0001
(Between groups)
Error 22.9514 198 0.1159 ****** ******
Ss/Bl 5.558 99 ****** ****** ******
Total 31.0427 299 ****** ****** ******
Ss/Bl = Subjects or Blocks depending on the design.

TUKEY HSD TEST

HSD[.05]=0.11; HSD[.01]=0.14
M1 vs M2 P<.01
M1 vs M3 nonsignificant
M2 vs M3 P<.05

M1 = Mean of Sample 1 (Anambra samples)

M2 = Mean of Sample 2 (Ebonyi samples)
M3 = Mean of Sample 3 (Enugu samples)

160
 APPENDIX 5
DATA ANALYSIS OF VIABLE COUNTS OF PRESUMPTIVE SALMONELLA IN
SNAILS
DATA SUMMARY ON PLATE COUNTS OF PRESUMPTIVE SALMONELLA IN SNAILS
Samples
1 2 3 Total
N 100 100 100 300
X 638.3 723.7 637.2 1999.2
Mean 6.383 7.237 6.372 6.664
2
X 4074.999 5239.733 4064.584 13379.316
Variance 0.0074 0.0234 0.0439 0.1895
Standard Deviation 0.0859 0.153 0.2095 0.4353
Standard Error 0.0086 0.0153 0.021 0.0251

STANDARD WEIGHTED-MEANS ANALYSIS

ANOVA SUMMARY
Source SS Df MS F P
Treatment 49.2554 2 24.6277 1114.38 <.0001
(Between groups)
Error 4.3693 198 0.0221 ****** ******
Ss/Bl 3.0225 99 ****** ****** ******
Total 56.6472 299 ****** ****** ******
Ss/Bl = Subjects or Blocks depending on the design.

TUKEY HSD TEST

HSD[.05]=0.05; HSD[.01]=0.06
M1 vs M2 P<.01
M1 vs M3 nonsignificant
M2 vs M3 P<.01

M1 = Mean of Sample 1 (Anambra samples)

M2 = Mean of Sample 2 (Ebonyi samples)
M3 = Mean of Sample 3 (Enugu samples)

161
 APPENDIX 6
DATA ANALYSIS OF VIABLE COUNTS OF SHIGELLA IN SNAILS
DATA SUMMARY ON PLATE COUNTS OF SHIGELLA IN SNAILS
Samples
1 2 3 Total
N 100 100 100 300
X 443.6 460 408 1311.6
Mean 4.436 4.6 4.08 4.372
X2 1972.668 2126.06 1694.468 5793.196
Variance 0.0491 0.1016 0.3013 0.1969
Standard Deviation 0.2215 0.3188 0.5489 0.4438
Standard Error 0.0222 0.0319 0.0549 0.0256

STANDARD WEIGHTED-MEANS ANALYSIS

ANOVA SUMMARY
Source SS Df MS F P
Treatment 14.1344 2 7.0672 40.73 <.0001
(Between groups)
Error 34.3569 198 0.1735 ****** ******
Ss/Bl 10.3895 99 ****** ****** ******
Total 58.8808 299 ****** ****** ******
Ss/Bl = Subjects or Blocks depending on the design.

TUKEY HSD TEST

HSD[.05]=0.14; HSD[.01]=0.17
M1 vs M2 P<.05
M1 vs M3 P<.01
M2 vs M3 P<.01

M1 = Mean of Sample 1 (Anambra samples)

M2 = Mean of Sample 2 (Ebonyi samples)
M3 = Mean of Sample 3 (Enugu samples)

162
 APPENDIX 7
DATA ANALYSIS OF VIABLE COUNTS OF E. COLI IN SNAILS
DATA SUMMARY ON PLATE COUNTS OF E. COLI IN SNAILS
Samples
1 2 3 Total
N 100 100 100 300
X 714.8 694.4 565.3 1974.5
Mean 7.148 6.944 5.653 6.5817
X2 5112.004 4825.01 3200.787 13137.801
Variance 0.0264 0.0313 0.052 0.4759
Standard Deviation 0.1625 0.1769 0.228 0.6899
Standard Error 0.0162 0.0177 0.0228 0.0398

STANDARD WEIGHTED-MEANS ANALYSIS

ANOVA SUMMARY
Source SS df MS F P
Treatment 131.4441 2 65.722 1425.64 <.0001
(Between groups)
Error 9.1339 198 0.0461 ****** ******
Ss/Bl 1.7222 99 ****** ****** ******
Total 142.3002 299 ****** ****** ******
Ss/Bl = Subjects or Blocks depending on the design.

TUKEY HSD TEST

HSD[.05]=0.07; HSD[.01]=0.09
M1 vs M2 P<.01
M1 vs M3 P<.01
M2 vs M3 P<.01

M1 = Mean of Sample 1 (Anambra samples)

M2 = Mean of Sample 2 (Ebonyi samples)
M3 = Mean of Sample 3 (Enugu samples)

163
 APPENDIX 8
DATA ANALYSIS OF VIABLE COUNTS OF STAPHYLOCOCCUS IN SNAILS
DATA SUMMARY ON PLATE COUNTS OF STAPHYLOCOCCUS IN SNAILS
Samples
1 2 3 Total
N 100 100 100 300
X 100 474.6 466.2 1040.8
Mean 1 4.746 4.662 3.4693
X2 100 2254.666 2207.85 4562.516
Variance 0 0.0224 0.3477 3.1827
Standard Deviation 0 0.1496 0.5897 1.784
Standard Error 0 0.015 0.059 0.103

STANDARD WEIGHTED-MEANS ANALYSIS

ANOVA SUMMARY
Source SS df MS F P
Treatment 914.9939 2 457.4969 4411.74 <.0001
(Between groups)
Error 20.5368 198 0.1037 ****** ******
Ss/Bl 16.1032 99 ****** ****** ******
Total 951.6339 299 ****** ****** ******
Ss/Bl = Subjects or Blocks depending on the design.

TUKEY HSD TEST

HSD[.05]=0.11; HSD[.01]=0.13
M1 vs M2 P<.01
M1 vs M3 P<.01
M2 vs M3 nonsignificant

M1 = Mean of Sample 1 (Anambra samples)

M2 = Mean of Sample 2 (Ebonyi samples)
M3 = Mean of Sample 3 (Enugu samples)

164
 APPENDIX 9
DATA ANALYSIS OF VIABLE COUNTS OF PRESUMPTIVE VIBRIO IN SNAILS
DATA SUMMARY ON PLATE COUNTS OF PRESUMPTIVE VIBRIO IN SNAILS
Samples
1 2 3 Total
N 100 100 100 300
X 310.6 341.8 480.3 1132.7
Mean 3.106 3.418 4.803 3.7757
X2 964.986 1171.866 2327.051 4463.903
Variance 0.0027 0.0363 0.2037 0.6261
Standard Deviation 0.0515 0.1905 0.4514 0.7913
Standard Error 0.0051 0.0191 0.0451 0.0457

STANDARD WEIGHTED-MEANS ANALYSIS

ANOVA SUMMARY
Source SS df MS F P
Treatment 163.1793 2 81.5896 805.43 <.0001
(Between groups)
Error 20.0621 198 0.1013 ****** ******
Ss/Bl 3.964 99 ****** ****** ******
Total 187.2054 299 ****** ****** ******
Ss/Bl = Subjects or Blocks depending on the design.

TUKEY HSD TEST

HSD[.05]=0.11; HSD[.01]=0.13
M1 vs M2 P<.01
M1 vs M3 P<.01
M2 vs M3 P<.01

M1 = Mean of Sample 1 (Anambra samples)

M2 = Mean of Sample 2 (Ebonyi samples)
M3 = Mean of Sample 3 (Enugu samples)

165
 APPENDIX 10
DATA ANALYSIS OF VIABLE COUNTS OF BACILLUS IN SNAILS
DATA SUMMARY ON PLATE COUNTS OF BACILLUS IN SNAILS
Samples
1 2 3 Total
N 100 100 100 300
X 325.7 449 348.7 1123.4
Mean 3.257 4.49 3.487 3.7447
X2 1062.035 2017.734 1217.561 4297.33
Variance 0.0124 0.0174 0.0166 0.3029
Standard Deviation 0.1115 0.132 0.1289 0.5504
Standard Error 0.0111 0.0132 0.0129 0.0318

STANDARD WEIGHTED-MEANS ANALYSIS

ANOVA SUMMARY
Source SS Df MS F P
Treatment 85.9733 2 42.9866 2669.98 <.0001
(Between groups)
Error 3.1841 198 0.0161 ****** ******
Ss/Bl 1.4141 99 ****** ****** ******
Total 90.5715 299 ****** ****** ******
Ss/Bl = Subjects or Blocks depending on the design.

TUKEY HSD TEST

HSD[.05]=0.04; HSD[.01]=0.05
M1 vs M2 P<.01
M1 vs M3 P<.01
M2 vs M3 P<.01

M1 = Mean of Sample 1 (Anambra samples)

M2 = Mean of Sample 2 (Ebonyi samples)
M3 = Mean of Sample 3 (Enugu samples)

166
 APPENDIX 11

M 1 2

(M) Ladder (50 bp); (1) 0180EN; (2) 0181EN.

Gel image from the PCR analysis of Staphylococcus isolates showing the sea gene

167
 APPENDIX 12

M 1 2

(M) Ladder (50 bp); (1) 0180EN; (2) 0181EN.

Gel image from the PCR analysis of Staphylococcus isolates for exhc gene

168
 APPENDIX 13

M 1 2 3

(M) Ladder (50 bp); (1) 0186EN; (2) 0177EN; (3) 0197AN.

Gel image from the PCR analysis of Bacillus isolates showing the hbla gene

169
 APPENDIX 14

M 1 2 3

(M) Ladder (50 bp); (1) 0186EN; (2) 0177EN; (3) 0197AN.

Gel image from the PCR analysis of Bacillus isolates for hblc gene

170
 APPENDIX 15

M 1 2 3

(M) Ladder (50 bp); (1) 0186EN; (2) 0177EN; (3) 0197AN.

Gel image from the PCR analysis of Bacillus isolates showing nhea gene

171
 APPENDIX 16

M 1 2 3

(M) Ladder (50 bp); (1) 0186EN; (2) 0177EN; (3) 0197AN.

Gel image from the PCR analysis of Bacillus isolates showing nheb genes

172
 APPENDIX 17

M 1 2 3

(M) Ladder (50 bp); (1) 0186EN; (2) 0177EN; (3) 0197AN.

Gel image from the PCR analysis of Bacillus isolates showing cytk gene

173
 APPENDIX 18

Data on Mean Aerobic Plate counts of bacteria in snail meat during processing
Desliming Shucking Evisceration Desliming Washing Parboiling Cooking
agents (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g)
Ash 9.31 8.93 5.98 6.89 2.7 2.44

Cassava water 8.79 8.02 7.04 7.35 3.09 3.12

Garri 9.4 8.65 7.72 7.22 3.68 3.45

Lime 9.56 9.15 8.16 6.81 3.6 2.67

Potassium
Alum 9.02 8.22 6.92 6.56 3.81 2.91

174
 APPENDIX 19

Data on Mean coliform counts in snail meat during processing

Desliming Shucking Evisceration Desliming Washing Parboiling Cooking
agents (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g)
Ash 7.79 7.01 4.23 5.63 0 0

Cassava water 7.32 6.94 6.65 6.73 0 0

Garri 7.3 6.8 6.71 6.07 0 0

Lime 7.62 7.06 6.84 6.04 0 0

Potassium
Alum 6.86 6.42 5.4 4.84 0 0

175
 APPENDIX 20

Data on Mean counts of Citrobacter in snail meat during processing

Desliming Shucking Evisceration Desliming Washing Parboiling Cooking
agents (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g)
Ash 7.31 6.51 2.66 3.95 0 0

Cassava water 6.82 6.27 5.92 6.42 0 0

Garri 6.83 6.37 5.46 5.09 0 0

Lime 6.48 6.15 5.58 3.2 0 0

Potassium
Alum 5.47 4.78 2.9 3.49 0 0

176
 APPENDIX 21

Data on Mean counts of Shigella in snail meat during processing

Desliming Shucking Evisceration Desliming Washing Parboiling Cooking
agents (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g)
Ash 6.42 4.77 3.28 4.26 0 0

Cassava water 4.43 3.64 2.54 2.22 0 0

Garri 4.99 3.75 3.64 2.65 0 0

Lime 4.66 4.39 2.71 0.1 0 0

Potassium
Alum 4.8 4.33 2.84 2.69 0 0

177
 APPENDIX 22

Data on Mean counts of Staphylococcus in snail meat during processing

Desliming Shucking Evisceration Desliming Washing Parboiling Cooking
agents (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g)
Ash 5.6 4.69 2.56 4.41 0 0

Cassava water 6.32 6.09 4.9 4.82 0 0

Garri 5.47 5.1 4.9 3.48 0 0

Lime 6.33 5.85 4.63 4.09 0 0

Potassium
Alum 6.38 6.19 5.8 5.3 0 0

178
 APPENDIX 23

Data on Mean counts of E. coli in snail meat during processing

Desliming Shucking Evisceration Desliming Washing Parboiling Cooking
agents (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g)
Ash 7.59 6.6 3.55 4.9 0 0

Cassava water 6.8 6.46 5.54 5.26 0 0

Garri 6.91 6.49 6.1 5.37 0 0

Lime 7.28 6.81 6.29 5.24 0 0

Potassium
Alum 6.57 6.2 5.01 3.96 0 0

179
 APPENDIX 24

Data on Mean counts of Aeromonas in snail meat during processing

Desliming Shucking Evisceration Desliming Washing Parboiling Cooking
agents (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g)
Ash 5.51 4.69 2.7 3.62 0 0

Cassava water 5.32 5.19 4.5 3.96 0 0

Garri 5.32 5.21 4.15 4.63 0 0

Lime 3.75 3.24 0.1 0.1 0 0

Potassium
Alum 3.91 3.73 3.4 2.57 0 0

180
 APPENDIX 25

Data on Mean counts of Bacillus in snail meat during processing

Desliming Shucking Evisceration Desliming Washing Parboiling Cooking
agents (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g) (Log cfu/g)
Ash 4.74 4.66 3.66 2.71 2.72 2.64

Cassava water 4.3 3.77 3.37 3.52 3.31 2.53

Garri 4.79 4.67 4.46 4.28 3.97 3.24

Lime 5.23 5.2 4.18 3.5 2.97 2.76

Potassium
Alum 5.54 5.23 4.86 4.49 4.09 3.14

181