DETECTION AND PCR CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS ENTEROTOXIN GENESFROM MASTITIC COW MILK AMONG SOME DAIRY HERDS IN, NIGERIA

ABSTRACT

The study is aimed to detect and PCR characterize S. aureus enterotoxin gene from mastitic cow milk within Zaria metropolis. Four hundred and three quarter milk samples were collected from 104 lactating cows in 17 dairy hreds which included Palladan, Shika, Hanwa, Wusasa and Bomo. The physicochemical characteristics (Temperature, PH, and Specific gravity and Titratable acidity) and proximate composition (Moisture content, Protein content, Fat content and Ash content of the milk were determined. The prevalence of subclinical mastitis using California mastitis test (CMT), bacteriological analysis using standard methods, antibacterial susceptibility and identification of the gene encoding staphylococcal enterotoxin A and B using PCR technique were carried out. The Temperature, PH, Specific gravity and Titratable acidity of the milk ranged from 23-37⁰C, 4.10 – 6.97, 1.023 – 1.032 and 0.14 – 0.23 respectively. The Moisture, Ash, Fat, and Protein content of the milk ranged from 86.99 – 87.67%, 0.66 – 0.69%, 3.22 – 3.81% and 3.13 – 3.45%. The prevalence of subclinical mastitis based on CMT reaction was (23.3%), 54 S.aureus were isolated from mastitic milk. The mean standard deviation ± of the total bacterial count from all the locations ranged from 6.22 ± 0.10 - 6.43 ± 0.12 and for the staphylococcal count from 2.98 ± 0.13 - 3.24 ± 0.11. The antibacterial susceptibility results shows that all the 54 S. aureus isolates were sensitive to vancomycin, norfloxacin and erythromycin (100%), 88.9% were sensitive to gentamicin and ciprofloxacin while 7.4% were sensitive to penicillin and methicillin. Out of the 15 isolates tested for enterotoxin A and B, 2 harboured gene for SEA and 5 isolates harboured gene for SEB but none of the isolates harboured both gene for SEA and SEB. The study concludes that further attention is needed to improve the hygienic and safety of milk and dairy farmers should be educated on the need to control mastitis, as it may decrease milk production and reduce the quality of the milk.

TABLE OF CONTENT

Cover Page………………………………………………………………………………………....i

Fly Leaf……………………………………………………………………………………………ii

Title Page…………………………………………………………………………........................iii

Declaration………………………………………………………………….…….......…………..iv

Certification……………...………………………………………………………………..............v

Dedication………………………………………………………………………...........................vi

Acknowledgement…………………………………………………..…………………………...vii

Abstract……………………………………………………………….…………..………..........viii

Table of Contents…………………….…………………………….………………..…………....ix

List of Tables………………………………………………………..………………..................xiv

List of Figures………………………………………………..………………………..................xv

List of Plates…………………………………………………………………………………….xvi

List of Appendices………………………………………………………………...…................xvii

CHAPTER ONE

1.0         INTRODUCTION……………………………………………………..............................1

1.1         Statement of Problem.......................................................................................................................................... 3

1.2         Justification................................................................................................................................................................. 3

1.3         Aims and Objectives….……………………………………………………………….....4

1.3.1     Aim………………………………………………………………………………………...4

1.3.2     Specific objectives…………………………………………………………………….......4

1.4         Research Questions............................................................................................................................................... 5

CHAPTER TWO

2.0         LITERATURE REVIEW................................................................................................................................ 6

2.1         Mastitis......................................................................................................................................................................... 6

2.2         Milk and Milk Products..................................................................................................................................... 7

2.2.1    Effects on milk composition............................................................................................................................... 8

2.3         Types of Mastitis……………............................................................................................. 8

2.3.1     Modes of Transmission……………………………………………………………….….11

2.4         Causative Agents of Mastitis……………………………………………………….…..10

2.5         Staphylococcus aureus as a Major Pathogen of Bovine Mastitis…………………….11

2.5.1     Identification and Morphology……………………………………………………..........12

2.5.2     Infection cause by Staphylococcus aureus ……………………………………...……….12

2.5.3    Transmission of Stapyhlococcus aureus infection……………………………………….13

2.5.4     Factors predisposing cow to mastitis…………………………………………………….13

2.6         Diagnosis of S. aureus Mastitis…………………………………………………………15

2.6.1     California Mastitis Test…………………………………………………………………..15

2.7         Control of S. aureus Mastitis……………………………………………………….…..16

2.8         Prevention of Recurrent S. aureus Infection………………………………………….23

2.9         Antimicrobial Susceptibility of Staphylococcus aureus……………………………....25

2.10      Staphylococcal Enterotoxins…………………………………………………………...26

2.10.1 Staphylococcal food poisoning………………………………………………………..…26

2.10.2 Staphylococcus aureus enterotoxins……………………………………………………..27

2.10.3 Staphylococcal Enterotoxins and Food Poisoning Outbreaks……………………...........28

CHAPTER THREE

3.0         MATERIALS AND METHODS………………………………………………………30

3.1         Sampling Plan……………………………………………………………………….......30

3.1.1     Sample size………………………………………………………………………………30

3.1.2     Study area …………………………………...…………………………………...............30

3.1.3     Collection of samples ……………….……………………………………………….…..30

3.2         Assessment of Physicochemical Quality of Milk………...……………………….…...33

3.2.1     Physical inspection of the milk…………………………………………………………..33

3.2.2     Temperature……………………………………………………………………………...33

3.2.3     PH………………………………………………………………………………………...33

3.2.4     Titratable acidity………………………………………………………………………....33

3.2.5     Specific gravity…..……………………………………………………………………....34

3.3         Proximate Composition of Milk………………………………………………………..34

3.3.1     Moisture content…………………………………………………………………………34

3.3.2     Ash content……………..………………………………………………………………..34

3.3.3     Fat content………………………………………………………………………………..35

3.3.4     Protein content…………………..……………………………………………………….35

3.4         California Mastitis Test (CMT)………………………………………………………..36

3.5         Bacteriological Analysis of Milk……………………………………………………….36

3.5.1     Total bacteria Count………………………………………………………………….......37

3.5.2     Total staphylococcal count…………………………………………………………….....37

3.5.3    Identification of Staphylococcus aureus…………………………..………….………….37

3.6         Confirmation of S. Aureus using Microgen Staph-Id Identification………………...39

3.7         Antibacterial Susceptibility Test…………...………………………………………......39

3.8         Molecular Characterization Using Polymerase Chain Reaction (PCR)…………….40

3.8.1     DNA extraction…………………………………………………………………………..40

3.8.2     PCR amplification ……………..…………………………………………………….......40

3.9         PCR Mastermix…………………………………………………………………………40

3.10     Agarose gel electrophoresis of PCR products………………………………………...40

3.11      Data Analysis……………………………………………………………………………41

CHAPTER FOUR

4.0         RESULTS……………………………………………………………………………….42

4.1         Location of Dairy Herds with number of Cows and Quarters………………………42

4.2         Milk collected at Udder Quarters of Cows..…………………………………………..42

4.3         Results of Physicochemical Quality of Milk………………………………………......42

4.3.1     Physical inspection of milk……………………………………………………………....42

4.3.2     Temperature of milk……………………………………………………………………...43

4.3.3     PH of milk………………………………………………………………………………..43

4.3.4     Titratable acidity of milk…………………………………………………………………43

4.3.5     Specific gravity of milk…………………………………………………………………..49

4.3.6     Mean scores of physicochemical quality of milk according to CMT……………………49

4.4         Results of Proximate Composition of Milk…………………………………………....49

4.5         Total Bacterial Counts in Milk…………………………..…………………………….53

4.6         Total Staphylococcal Counts in Milk……………………………………………….....53

4.7         California Mastitis Test on Milk…………………………...…………………………..53

4.8         CMT Reaction based on Anatomic Site of Udder…………………………………….53

4.9         Isolation and Identification of S. aureus………………………………………………58

4.10      Results of CMT Scores in Comparison with Frequency of Isolation of

S. aureus from Milk Sample……………………………………………………………58

4.11      Antibiotic Sensitivity Test………………………………………………………...........58

4.12      Detection of Enterotoxin Genes (A and B) by PCR…………………………………..58

CHAPTER FIVE

5.0         DISCUSSION…………………………………………………………………………..64

CHAPTER SIX

6.1         CONCLUSION AND RECOMMENDATION……………………………………….72

6.1         Conclusion……………………………………………………………………………….72

6.2         Recommendations………………………………………………………………………73

REFERENCES…………………..……………………………………………………………..74

APPENDICES………………………………..…………………………………………………88

LIST OF FIGURE

Figure                                                                                 Title                                                                                      Page

1                    Map of Zaria…………………………………………………………………………...32

LISTS OF PLATES

Plate Title                                                                                        Page

1                Amplicons of SEA gene of S.aureus with size of 102bp…………………………….........62

2                Amplicons of SEB gene of S.aureus with size of 164bp…………………………….........63

LIST OF APPENDICES

Appendix                                                                         Title                                                                                          Page

I                            Nucleotide sequence and predicted size of PCR products…………………………….88

CHAPTER ONE

1.0                                                                          INTRODUCTION

Cow milk, a fresh, clean and normal mammary secretion is a good source of animal proteins, fats, vitamins and minerals to the human body. In addition, nutritionally less useful substances like enzymes are also present in normal milk. Some of the enzymes are used as indices in screening or quality control tests. In many homes in different geographical regions of the world, milk and milk products are fed to infants and form a major component of the diets of adults (Ruuw and Berts, 2004).

Mastitis is the inflammation of mammary gland and is a complex and costly disease in dairy herds (Husain etal., 2012; Atasever, 2012). Mastitis may have a variety of causes; Bacterial being the predominant cause of mastitis among dairy cattle (Wellenberg etal., 2002).

Mastitis manifestation can be of clinical or subclinical (Eriskine, 2001). Sub clinical mastitis are those in which no visible appearance of changes in the milk or udder, but milk production decreases, bacteria are present in the secretion and composition is altered (Eriskine, 2001). Clinical cases of mastitis are characterized by the presence of one or more of symptoms such as abnormal milk, udder swelling and systemic signs including elevated temperature, lethargy and anorexia (Eriskine, 2001).

Mastitis may be caused by a large variety of bacterial pathogens and other microbes entering the gland through the teat duct (Shitandi et al., 2004). The primary cause of mastitis in cattle, goats and sheep are well recognized group of microorganisms; Streptococcus spp. Staphylococcus spp. Pasteurella spp; and Coliforms (Escherichia coli, Enterobacter spp; and Klebsiella spp.)

One of the most common type of chronic mastitis is caused by Staphylococcus aureus (Jones et al., 1998). Staphylococcus aureus is a major pathogen of bovine mastitisworldwide. It has been reported by Lafi et al. (1994) that Staphylococcus aureus occurred predominantly in both clinical and sub clinical bovinemastitis.

Mastitis caused by Staphylococcus aureus is recognized worldwide in dairy cows as subclinical and clinical intramammary infection (Akineden et al., 2001). The infection is spread at milking time when S. aureus contaminated milk from an infected gland comes in contact with an uninfected gland, and the bacteria penetrate the teat canal. Moreover Ebliny et al. (2001) reported that S. aureus causes infection of longer duration.

The number of bacteria present in a milk sample is of importance. Milk from cows infected with mastitis generally has higher total bacteria and somatic cell counts than milk from uninfected cows. Therefore bacteria counts and somatic cell count are used by dairy farmers and milk processors as indicators of milk quality. There are regulatory standards for microbial numbers (total bacteria count) as well as quality control. Infected gland usually yield more than 200 colonies of bacteria per ml of milk, with fewer counts suggesting the invasion phase before the infection is established (Blood and Radostits, 1989).

S. aureus is a Gram-positve, non spore forming spherical bacterium that belongs to the Staphylococcus genus. S. aureus produces staphylococcal enterotoxins (SE) and is responsible for almost all staphylococcal food poisoning (Montville and Mattews 2008; FDA 2012).

Staphylococcal food poisoning is an intoxication that is caused by the ingestion of food containing pre-formed SE (Argudin et al., 2010). There are several different types of SE; enterotoxin A is most commonly associated with staphylococcal food poisoning. Enterotoxins D, E and H, and to a lesser extent B, G and I, have also been associated with staphylococcal food poisoning (Seo and Bohach 2007; Pinchuk et al., 2010). Also Argudin et al (2010) reported that S. aureus enterotoxins have been divided into 5 serological ‗classical types‘ (SEA,SEB,SEC,SED,SEE), and among them SEA is considered as the main cause of SFP (Staphylococcal food poisoning) outbreaks in the United State, Japan, France, and UK.

1.1         Statement of Problem

Staphylococcus aureus is one of the important causative agent of mastitis all over the world (Cabral et al., 2004), causing both clinical and subclinical form of mastitis in cattle (Pradeep et al., 2003). S. aureus bacteria produce toxins that destroy cell membrane and can directly damage milk producing tissues which can lead to bovine mastitis (Jones et al., 1998).

S. aureus mastitis is a serious problem in dairy production and infected animals may contaminate bulk milk. Additionally, human handlers, milking equipment, the environment, and udder and teat skin of dairy animals may be other possible sources of bulk milk contamination. From a food safety perspective, this is a concern because enterotoxigenic S. aureus may be a risk of SFP (Staphylococcal food poisoning) after consumption of raw milk products (Jorgensen et al., 2005).

1.2         Justification

Milk is obtained primarily from ruminant domestic animals especially cows and goats, and it is processed either locally or in factories to derive products like powdered milk, yoghurt, ‗nono‘, cheese e.t.c. The nutrient composition of raw milk is excellent and is thus favorable for bacteria growth (Ruuw and Berts, 2004). Results of extensive investigations over many years have proved that both human and animal diseases are sometimes spread by milk and milk products.

The bacterial contamination of milk from affected cows renders it unfit for human consumption and provides a mechanism of spread of diseases.

Mastitis agents like S. aureus in milk may present a degree of risk to the consumers because of the organism‘s capacity to produce enterotoxins, which can lead to food poisoning. Carlos (1990) drew attention to the great public health significance of this organism in milk. Therefore there is a need to detect and study Staphylococcus aureus mastitic milk and suggest possible control measures.

1.3         Aim and Objectives

1.3.1     Aim

The study is therefore aimed to detect and PCR characterization of Staphylococcus aureus enterotoxin genes from mastitic cow milk among some dairy herds in Zaria.

1.3.2     Specific objectives

The specific objectives of this research were to:

1.      Determine the physicochemical and proximate composition of milk obtain from some dairy herds.

2.      Determine Total bacterial count and Staphylococcus count of the milk sample.

3.      Isolate and characterize Staphylococcus aureus present in the milk.