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CLINICAL AND TREATMENT OUTCOMES IN Clarias gariepinus EXPERIMENTALLY INFECTED WITH SINGLE AND MIXED Escherichia coli AND Salmonella gallinarium STRAINS
ABSTRACT
Catfish is an important fresh water fish and is an excellent source of protein. However, it can be susceptible to microbial infection. This study therefore, was designed to determine the clinical and treatment outcomes in African Catfish experimentally infected with E. coli and S. gallinarium strains. A total of 160 juvenile African catfish were used for this study and they were randomly assigned into four groups (A, B, C, and D) of 40 fish each. Clinical isolates of E. coli and S.gallinarium were sourced from the Department of Veterinary Pathology and Microbiology, University of Nigeria, Nsukka. Fishes in groups A, B, C were infected by immersing them in water containing 1×108 colony forming units/ml of each test isolate while the fish in group D served as uninfected control. Fishes in group A were infected with E. coli alone, those in group B with S. gallinarium, those in group C with E .coli and S. gallinarium, while fishes in group D were uninfected. Haematological and biochemical parameters were measured on weeks 1 and 2 post infection. The fish were monitored daily for signs of ill-health and mortality. Body weight change and vital physicochemical properties (dissolved oxygen, ammonia and pH) of water was also determined and recorded. Treatment was instituted five weeks post infection with amoxicillin by immersion for five consecutive days. Sluggish movement, curdling together, emaciation, poor growth rate, anorexia, high morbidity and mortality were the signs of ill health observed in fishes in the experimental groups. The clinical signs were more pronounced in groups A and C when compared to group B. The uninfected control showed no obvious clinical manifestation. The body weight of the fish in groups A (51.25±4.25) and C (58.25±4.11) were significantly (p<0.05) lower than those in the control group in week 1 (74.95±1.43) and week 2 (71.50±3.62) respectively. There was no significant (p>0.05) improvement in weight changes post treatment in groups A and C. Haematology showed significant (p<0.05) decrease in RBC in group B (1.93±0.18) when compared to groups A (2.31±0.08) but was not significantly (p>0.05) different from groups C (2.28±0.14) and D (2.22±0.13). There was also a significant (p<0.05) decrease in PCV of group B (21.17±0.60) when compared with control group (23.50±0.87) unlike groups A (22.33±0.33) and C (23.00±0.29). There was no significant (p<0.05) difference in Hb concentration. There was also a significant (p<0.05) decrease in monocyte count in group C when compared to group D one week post infection unlike wbc , neutrophil and eosinophil counts which did not show any significant (p>0.05) variations. Serum biochemistry values showed that there was no significant (p>0.05) difference in creatinine, total protein and blood urea nitrogen but there was a significant (p<0.05) difference in alanine amino transferase and bilirubin two weeks post infection. Grossly, enlarged liver was observed in fish of group A and areas of focal necrosis in the liver of fish in group B. Histopathology result revealed multiple golden yellow haemosiderin depositions on the spleen of fish in group B. This study has shown that the presence of E. coli and S. gallinarium in pond can cause infection in Clarias gariepinus which can lead to adverse effect in their health status and productivity.
TABLE OF CONTENTS
Content Page
Title page … … … … … … … … i
Declaration … … … … … … … … ii
Certification and Approval … … … … … … iii
Dedication … … … … … … … … iv
Acknowledgement … … … … … … … v
Table of contents … … … … … … … vi
List of tables … … … … … … … … xi
List of figures … … … … … … … … xii
List of plates … … … … … … … … xiii
List of appendices … … … … … … … xiv
List of abbreviations … … … … … … … xv
Abstract … … … … … … … … xvi
CHAPTER ONE: INTRODUCTION … … … … … 1
1.1 Background of the study … … … … … … 1
1.2 Statement of the research problem … … … … … 4
1.3 Research questions … … … … … … … 5
1.4 Objectives of the study … … … … … … 6
CHAPTER TWO: LITERATURE REVIEW … … … … 7
2.1 Introduction … … … … … … … 7
2.2 Historical background … … … … … … 7
2.3 Fish species cultured in Nigeria … … … … … 9
2.3.1 The family Claridae … … … … … … 9
2.3.2 Physical description … … … … … … 10
2.4 Geographic range and habitat … … … … … 10
2.5 Food habits and feeding … … … … … … 11
2.6 Fish diseases … … … … … … … 11
2.6.1 Non Infectious causes of disease … … … … … 12
2.6.2 Infectious causes of disease in fish … … … … … 13
2.6.3 Signs of disease in fish … … … … … … 14
2.6.4 Fish defence against disease … … … … … 15
2.6.5 Treatment of fish disease … … … … … … 15
2.6.6 Socio-economic impact of disease to aquaculture … … … 17
2.6.7 Salmonella gallinarium and Escherichia coli … … … 19
2.6.7.1 Escherichia coli … … … … … … 19
2.6.7.2 Salmonella gallinarium … … … … … … 20
2.7 Haematology … … … … … … … 21
2.8 Pathology … … … … … … … 22
2.9 Diagnosis of disease in fish … … … … … 22
2.9.1 Laboratory diagnosis of E. coli and S. gallinarium … … … 23
2.9.2 Sensitivity test … … … … … … 2 3
CHAPTER THREE: MATERIALS AND METHOD … … … 25
3.1 Experimental animals … … … … … … 25
3.2 Materials … … … … … … … 25
3.3 Media preparation … … … … … … 26
3.3.1 Nutrient agar … … … … … … … 26
3.3.2 Nutrient broth … … … … … … … 26
3.3.3 MacConkey agar … … … … … … 26
3.3.4 Simmon’s Citrate agar … … … … … … 27
3.3.5 Triple sugar iron agar … … … … … … 27
3.3.6 Urea agar … … … … … … … 27
3.3.7 Eosine methylene blue … … … … … … 27
3.3.8 Sugar solutions … … … … … … … 28
3.4 Bacterial strains … … … … … … … 28
3.5 Infection of catfish … … … … … … 28
3.5.1 Observation for clinical outcome … … … … … 29
3.6 Body weight … … … … … … … 29
3.6.1 Absolute weight … … … … … … … 29
3.6.2 Weight variation … … … … … … … 29
3.7 Analysis of physicochemical parameters of water … … … 30
3.7.1 pH … … … … … … … … 30
3.7.2 Dissolved oxygen … … … … … … 30
3.7.3 Ammonia … … … … … … … 31
3.8 Haematology determinations … … … … … 31
3.8.1 Determination of packed cell volume … … … … 32
3.8.2 Determination of haemoglobin concentration … … … 32
3.8.3 Erythrocyte count … … … … … … 32
3.8.4 White blood cell count … … … … … … 33
3.8.5 Differential white blood cell count … … … … … 33
3.9 Serum biochemistry determination … … … … 33
3.9.1 Alanine amino transferase … … … … … 33
3.9.2 Determination of total protein … … … … … 35
3.9.3 Determination of serum urea nitrogen … … … … 35
3.9.4 Determination of creatinine … … … … … 36
3.9.5 Determination of serum total bilirubin … … … … 36
3.10 Antibiogram of the bacterial isolates … … … … 37
3.11 Treatment … … … … … … … 37
3.12 Effect of experimental infection on the pathology … … … 38
3.12.1 Gross pathology … … … … … 38
3.12.2 Histopathology … … … … … … 38
3.13 Data presentation and analysis … … … … … 39
CHAPTER FOUR: RESULT … … … … … … 40
4.1 Clinical signs … … … … … … … 40
4.2 Weight changes … … … … … … … 42
4.3 Haematological parameters … … … … … … 44
4.4 Biochemical parameters … … … … … … 48
4.5 Physicochemical parameters of water … … … … 51
4.6 Gross and histopathology lesions … … … … … 51
CHAPTER FIVE: DISCUSSION AND RECOMMENDATION … … 63
5.1 Discussion … … … … … … … 63
5.2 Conclusion … … … … … … … 67
5.3 Recommendations … … … … … … 68
REFERENCES … … … … … … … 69
APPENDICES … … … … … … … 86
CHAPTER ONE
INTRODUCTION
Background of the study
Fishes are members of the super class Pisces, but those having a conspicuous set of feelers surrounding the mouth are called Catfish (Udeze et al., 2012). All catfishes have either smooth or armoured naked bodies with bony plate. The dorsal and pectoral fins are often edged with sharp spines that are used for defence against predators (Redmond, 2010). Catfish (Clarias gariepinus) is one of the most important individual species in traditional freshwater fisheries in Africa (Skelton, 2001). It is widely distributed in Africa, where it occurs in almost any fresh water habitat, but favours floodplains, large sluggish rivers, lakes and dams (Skelton, 2001).
The importance of fish and fishery products as vital, readily available and affordable source of food and high quality protein, in job creation and general well-being to its teeming population cannot be overemphasized (Allumma and Idowu, 2011). It is widely believed that reliance on capture fisheries as a means of meeting the ever increasing demand for fish protein is not feasible (Dunham et al, 2001). Food and Agricultural organization (FAO) asserts that fish contributes about 60% of the world supply of protein and that 60% of the developing countries derive more than 30% of their animal protein from fish (FAO,1994). Fish is of importance in the diet of different countries especially in the tropics and subtropics where malnutrition is still a major problem (Alune and Andrew, 1996). The world consumption of fish rose to 115 million tonnes of fish in 2008, and demand is expected to rise as the world population increases (FAO, 2010). Consumption reached an all- time high of nearly 17kg per person, supplying over 3 billion people with at least 15 percent of their average animal protein intake (FAO, 2010). In Nigeria, fish is the preferred source of animal protein compared to poultry, beef, mutton, pork and veal (Wogu and Maduakor, 2010, Olaote and Basiru, 2013). It is comparatively cheaper and highly acceptable, with little or no religious bias, which gives it an advantage over pork or beef (Feldhusen, 2000, Wogu and Maduakor, 2010).
Fish generally allows for improved nutrition in that it has high biological value in terms of high protein retention in the body, low cholesterol level and presence of essential amino acids (Fagbenro and Arowosegbe 1998). Clarias gariepinus has high fecundity rate, grows fast, tolerate high stocking density and environmental extremes (Brutons, 1979, Edward et al., 2010). Other attributes such as resistance to disease, desiccation and ability to endure long draught and scarcity of food have endowed this fish species with one amazing capacity to survive (Dunn, 2000). Clarius gariepinus is a highly nutritious fish that contains high amount of vitamins, proteins, minerals and a little or no saturated fat and is low in carbohydrate (Lee, 1991). Clarias species are highly esteemed group of fishes in Nigeria and have a very high commercial values in the market (Huisman and Fitcher, 1987).
In recent times, there has been tremendous increase in the development of fish farming and culture attributable to the increased need for affordable animal protein especially in the tropics. Intensive production of fish increases the likelihood of and severity of parasite and disease outbreaks which constitute a major constraint to aquaculture (IIhan and IIknur, 2003). It is widely demonstrated that farmed fish are more susceptible to disease agents than their wild counterparts due to the artificial conditions posed by intensive rearing (Irene Salinas etal., 2006) The aquatic environment contains many obligate and opportunistic microbial pathogens as well as beneficial and neutral strains (Al-sunaihe et al., 2010). Bacteria are introduced to the fish farm through natural or artificial food sources and less frequently vertical transmission from brood stock (Sandaan et al., 2003; Schulze et al., 2006; Al-sunahaaeir et al., 2010) The study of bacterial disease and its effect on catfish (Clarias gariepinus) and other species of fish have been a serious concern in the fishing industry as well as a public health concern. Colonization of fishes by their various parasites from faecal source of pollution has impacted disease such as salmonellosis (Williams et al., 1989). Disease cause economic losses not only from poor growth rate, mortality but also from treatment expenses, postponement or loss of the opportunity to sell the fish. Their presence in fish intended for human consumption may constitute a potential danger not only in causing disease, but also because of the possible transfer of antibiotic resistance from aquatic bacteria to human infecting bacteria from non-aquatic sources (Olayemi et al., 1991; Ampofo and Clerk, 2010). Fish living in natural environment are known to habour pathogenic enterobacteriaceae (Pillay, 1990). Invasion of fish muscle due to the breakage of immunological barrier of fish by pathogens is likely to occur, when the fish are raised in pond with faecal coliforms (Escherichia coli and Salmonella) of greater than 103 /ml in pond water (Guzman et al., 2004).
Enterobacteraceae are a large heterogenous group and members of this group consist of medium size rods, they are Gram- negative and non spore forming. Some survive under aerobic conditions and they normally inhabit the intestine of man and animals. Some are motile while others are not. Bacteria from the genus Escherichia causes numerous infections in animal and man. Escherichia coli cause different disease syndromes in poultry, including: acute septicaemia, sub-acute fibrinopurulent synositis, yolk sac infection, cellulitis, swollen head syndrome and coligranuloma (Allan et al., 1993, Gomis et al., 2001). Escherchia coli pathogenic for poultry commonly belong to certain serogroup, particularly the serogroups 078, 01 and 02 and to some extent 015 and 055 (Gross 1994, Chart et al., 2000).
Salmonella infection caused by a variety of Salmonella species is one of the most important bacterial diseases in poultry causing heavy economic losses through mortality and reduced production (Haidet et al., 1994). Avian salmonella infection may occur in poultry either as acute or chronic form and is caused by one or more member of genus Salmonella under the family Enterobacteraceae (Hofstad et al., 1984). Avian salmonellosis is caused by Salmonella gallinarium which causes fowl typhoid in young and particularly adult birds (Shivaprasad, 1997).
Salmonella species are among the most abundant bacteria found in fresh water aquatic environment and occur in greatest number in contaminated pond (Wandili et al., 2011). Salmonella has been isolated from gills, intestine, and skin of catfish and the most potential reservoir of Salmonella species is the intestine. (Hatha, 1997, Titik et al., 2011). Udeze and Sowolu (2012) reported that Clarias gariepinus inoculated with Salmonella species showed peeling of their outermost skin and reduced appetite. Ezenwaji and Inyang (1998) also reported that some members of the family enterobacteracea which was inoculated into catfish caused shedding of skin patches and fading of colour on the skin from black to faint black. Wyatt et al (1976) found high prevalence of Salmonella in catfish which was attributed to the high temperatures in pond water, which promotes the growth of Salmonella. Chicken offal and spoiled eggs can be potential source of Salmonella species and the high risk associated with the dissemination of antibiotic resistant genes among bacteria associated with catfish and environment of aquaculture system.
1.2 Statement of the research problem
Fish takes a large number of bacteria into their gut from water segment and food (Sugita et al., 1998). It has been well known that both fresh and brackish water fishes can harbour pathogenic bacteria particularly the coliform group (Leung et al., 1990). The presence of salmonella in fish is only suggestive of a pathogenic potential (Liston, 1980); its significance in initiating disease in fish has not been sufficiently investigated. Fish and shellfish not only transmit disease to man but are themselves subject to many diseases and are capable of transmitting many of the established food borne microbial infections and intoxications. Invasion of fish muscle by these pathogenic bacteria due to the breakage of immunological barrier of fish is likely to occur and this may result to disease.
Federal Department of Fisheries (FDF) asserts that the consumption of fresh African catfish (Clariasgariepinus) and wild Tilapia fish (Oreochromisniloticus) is on the increase in both rural and urban centres in Nigeria (FDF, 2007). As a result of increase in demand, most farmers operate integrated farms where they keep birds and fishes. In some integrated poultry and fish farm, the poultry house overlays the ponds and the droppings from the birds are used to fertilize the pond. These droppings from chicken which may sometimes harbour pathogenic strains of Escherichia coli and Salmonellae may therefore serve as source of infection to the fish. Some farmers also pour poultry faeces and poultry offals, especially intestine, into the fish pond as feed for the fish. These materials are often contaminated with bacteria such as Escherichia coli and salmonellae. In addition to causing infection in catfish, presence of these enteric bacteria in the pond could also negatively affect the physicochemical properties of the pond water with attendant adverse effect on the health of the fish.
Clinical signs and lesions associated with Salmonella and Escherichia coli infection (either as a single or mixed infection) are poorly documented. There is also paucity of information on the treatment options available to fish when they are infected with either single or mixed infection with these organisms hence the need for this study.
1.3 Research questions
Based on the problems highlighted above, the following research questions guided the study.
Can Salmonella gallinarium and Escherichia coli of avian origin cause infection in (Clariasgariepinus)?
If yes, what are the clinical signs and lesions during single and mixed infection of these organisms in Clarias gariepinus?
What is the probable effect of the infection on haematology, serum biochemistry and growth rate of the Clarias gariepinus?
What are the probable effects on the physicochemical properties of the water?
What is the effect of antibacterial treatment in experimental Salmonella gallinarium and Escherichia coli infected Clarias gariepinus?
1.4 Objectives of the study
The purpose of this study was to evaluate the effect of single and mix infection of Salmonella gallinarium and Escherichia coli in Clariasgariepinus.
The specific objectives of the study were to:
determine the clinical signs and lesions associated with single and mixed experimental Escherichia coli and Salmonella gallinarium infection in Clariasgariepinus.
investigate the effect of the infection on growth rate of Clarias gariepinus
evaluate the effect of single and mixed Escherichia coli and salmonellagallinarium on the blood parameters and serum biochemistry of Clarias gariepinus.
determine the effect of the infection on the physicochemical property of the water
assess antibacterial treatment in experimental Salmonella gallinarium and Escherichia coli infected Clarias gariepinus.
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