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PROJECT TOPICS ONDETECTION AND PCR CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS ENTEROTOXIN GENESFROM MASTITIC COW MILK AMONG SOME DAIRY HERDS IN ZARIA, KADUNA STATE

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PROJECT TOPICS ON DETECTION AND PCR CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS ENTEROTOXIN GENESFROM MASTITIC COW MILK AMONG SOME DAIRY HERDS IN ZARIA, KADUNA STATE

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.

 

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).

  1. 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.

Recommended : ISOLATION AND ANTIBIOGRAM OF ESCHERICHIA COLI, STAPHYLOCOCCUS AND STREPTOCOCCUSSPECIES FROMMASTITIC COWS IN ZARIA, NIGERIA

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).

  1. 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).

PROJECT TOPICS ON DETECTION AND PCR CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS ENTEROTOXIN GENESFROM MASTITIC COW MILK AMONG SOME DAIRY HERDS IN ZARIA, KADUNA STATE

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.
  4. Determine the pattern of susceptibility of aureus to antibiotics
  5. Detect the gene encoding Staphylococcal enterotoxin A and B using PCR.
  6. Ho: Enterotoxigenic aureus is not present in bovine mastitic milk.

Ha: Enterotoxigenic S.aureus is present in bovine mastitic milk.

See Also : ISOLATION AND ANTIBIOGRAM OF ESCHERICHIA COLI, STAPHYLOCOCCUS AND STREPTOCOCCUSSPECIES FROMMASTITIC COWS IN ZARIA, NIGERIA

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