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  $OO SUDLVHV DUH GXH WR WKH $OPLJKW\ $OODK 6XEKDQXWD¶OD WKH 2PQLSRWHQW 2PQLSUHVHQW Omniscient, and the Supreme authority of this universe, who enable the author to pursue higher education in Veterinary Science and to complete the research work and the degree of Master of Science (M. S.) in Medicine The author express her deepest sense of gratitude, sincere appreciation, profound regards and indebtedness to his teacher and research Supervisor, Dr. Md. Fazlul Hoque, Professor, Department of Medicine, Surgery and Obstetrics, Hajee Mohammad Danesh Science and Technology University, Dinajpur for his scholastic and dynamic guidance, constant inspiration, cordial consistence, affectionate feeling, utmost desire, sympathetic supervision and constructive criticism in all phases of this study and preparing of the manuscript. The author humbly desires to express his heartfelt gratitude and immense indebtedness to hishonorable and respected teacher Dr. Md. Nazrul Islam, Assistant Professor, Department of Pathology and Parasitology, and Dr. Md. Shamim Ahasan, Assistant Professor, Department of Medicine, Surgery and Obstetrics, Hajee Mohammad Danesh Science andTechnology University, Dinajpur, for there sympathy, kind co-operation, inspiration and valuable suggestions, constructive criticism and valuable advice for the completion of the research work, and preparation of the thesis. The author wants to express cordial thanks to Md. Sabirul Kibria, Microbiologist, Department of Microbiology, Zia Heart Foundation Hospital and Research Institute, Dinajpur, for his encouraging attitude and kind cooperation during the study period. The author expresses his thanks to all technicians and office staffs of the Department of Medicine, Surgery and Obstetrics, Hajee Mohammad Danesh Science and Technology University, Dinajpur for their technical assistance during the research.

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ϭϬ 

  INTRODUCTION

The cow is aptly called the 'foster mother' in the world which is serving for mankind from the time immemorial. The dairy industry began about 7000 years ago and in 3000 BC. Although no single food is perfect, but milk is the most wholesome and complete natural food. It is called 'ideal food', because it contains all the food constituents like vitamins, minerals, proteins, fats and carbohydrates, necessary for health. Moreover, it is the first food that babies consume in many species. Although milk is an almost perfect food but sound udder is of prime important for milch cow to produce such ideal food. This is more true in the case of high yielding animals as their udders are particularly prone to infection and inflammatory process because of physiological stress and strain of heavy milk production. Livestock is one of the important sources of our national economy. Cattle population in Bangladesh is about 24.50 million (FAO, 2003). Nearly, 85 percent of the populations are engaged in Agriculture and Livestock sector (Raha, 2000). The number of milking cows in Bangladesh is 3.75 million, which is 35 percent of the total cattle population in Bangladesh. The annual milk production in Bangladesh is nearly 1.62 million metric ton which is very low in respect of our demand which is nearly 9.0 million metric ton (DLS, 1998). The domestic house holders, small farmers however are facing a great problem with the diseases of udder of their animals and this has become a threat to their economy. Inflammation of udder or mastitis needs to be thoroughly studied with respect to the etiologic agents and holistic approaches. It continues to be a major economic issue for dairy producers all over the world.

ϭϭ 

The term 'mastitis' is derived from Greek word 'mastos' which means 'breast' (mammary gland) and 'itis' means inflammation, that is, inflammation of the mammary gland is called mastitis. Bovine mastitis (both clinical and subclinical) is one of the most important diseases causing considerable economic loss to the farmers and the dairy industry, and is mainly caused by bacterial infection in the udder (Abdella, 1996). The causative organisms of mastitis are ubiquitous in nature and persist long time in the cow yard or barns and there is a chance of constant udder infection under poor hygienic and management systems (Roy et al. 1988 and Radostits et al. 1994). Epidemiological studies on mastitis revealed that mastitogenic organisms are widespread on different body sites of the cows, milker's hands, milking cans and in the milk samples. Teat apices have been reported to be the most common site (Itagaki et al. 1999), from where these organisms have been isolated (Malhotra and Kapur, 1982; Prabhakar et al. 1990). The main etiological agents responsible for mastitis include Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysagalactiae, Streptococcus uberis,

Streptococcus

bovis,

Corynebacterium

bovis,

Corynebacterium

pyogenes, Escherichia coli, Enterobacter foecalis, Klebsiella pneumonia, Mycoplasma bovis etc. The clinical mastitis can be diagnosed on history and clinical findings but laboratory examinations are required to ascertain the subclinical mastitis (SCM). The prevalence of SCM has been shown to be 15 to 40 times more than the clinical mastitis (Philpot, 1984). However, no such estimation of economic loss due to mastitis is available from Bangladesh. Various indirect tests have been described (Schalm et al. 1971) to detect the SCM, but each test has got some limitations as it fails to detect SCM at all stages. The somatic cell count and bacterial load count (BLC) are three methods accepted reliably far detecting the early infection (Schalm et al. 1971). In addition, some indirect tests like ϭϮ 

Whiteside test (WST), California mastitis test (CMT), Surf field mastitis test (SFMT) have been developed for rapid screening the infection in the udder (Schalm et al. 1971; Rasool et al. 1985; Shukla and Superkar, 1984; 1986; Ali et al, 1989; Guha et al. 1989) Reduction in milk Production in subclinical mastitis (SCM) is not only responsible for great economic loss to the dairy industry but also acts as a carrier and source of infection for the healthy milch cows which is one of the biggest obstacles in the achievement of self-sufficiency in milk production in Bangladesh. The residual effects of many commonly used antibiotics, and chemotherapeutic

agents

in

treating

cases

of

mastitis,

pathogenic

microorganisms in milk (if used without boiling) can also be a potential danger to human health. So to overcome a major hurdle in the way of achieving selfsufficiency in milk production, it seems to be the prime importance to prevent the incidence of mastitis by early diagnosis of subclinical form of the disease as otherwise this subclinical form may lead to clinical form which is irreversible in most cases. Very limited research works on mastitis have been carried out in Bangladesh (Choudhury and Ali, 1975; Mahbub-E-Elahi et al., 1996; Sen et al., 1996; Rahman et al., 1997). The prevalence of subclinical mastitis in milch cows have been reported to be 16.52% with Whiteside Test (WST) and 15.77% with California Mastitis Test (CMT) from Baghabarighat, Sirajgonj district by Prodhan et al. (1996), and 18.5% with (WST) from the greater Mymensingh district by Rahman et al. (1997)Staphyiococei are the major etiological agents. Followed by Streptococci and E.coli causing subclinical mastitis in cows in India (Singh and Baxi, 1982) but only Staphylococci have been isolated from subclinical mastitis cases of cows from Bangladesh (Rahman et al., 1968). However, Mahbub-E-Elahi et al. (1996) isolated and identified Staphylococci (49.33%), Streptococci (14.0%), Corynebacterium (8.0%), E. coli (6.0%), ϭϯ 

Bacillus sp. (4.67%) and 18% unidentified bacteria from clinically affected mastitis cows of Manikgonj Milkshed area, Bangladesh Agricultural University Dairy Farm (BAUDF) and BAU Veterinary Clinic. The greatest problem in the treatment and control of mastitis is the emergence of drug resistance by pathogenic bacteria (Yadav et al., 1972; Rajangam et al., 1989; Brown and Scassera, 1990; Jha et al., 1994). The pattern of drug resistance continues to change in a particular area depending upon various epidemiological factors and indiscriminate use of antibiotics (Nag and Ghosh, 1982; Rahman and Baxi; 1982; Choudhury and Narayan, 1984; Pal et al., 1989). Successful treatment of mastitis in dairy animals depends to a great extent on the type of infection and the proper selection and administration of drugs (Biru, 1989; Bhaumik, 1997). Since the advent of the first antibiotic penicillin in 1929, in vitro sensitivity of mastitis organisms to this and other antibiotics has been extensively studied. Milunovic (1958) found resistant strains of Streptococcus agalactiae to penicillin although earlier workers had found this antibiotic almost specific in the treatment of streptococcal mastitis in cows. The emergence of resistant strains of Staphylococcus aureus to penicillin has posed serious problems in the treatment of Staphylococcal mastitis (Mists et al., 1972). A few years ago, ampicillin was found to be a good substitute for eliminating Staphylococci and Streptococci which were resistant to penicillin. Now resistant strains have developed against ampicillin and its analogues (Pal and Verma, 1988a, b). About 25 years ago, the penicillin and streptomycin have been reported to be the least effective drugs against mastitis organisms from Bangladesh (Ali and Choudhury, 1975) but no further reports on this aspect is available from this country. The indiscriminate use of antibiotics and corticosteroids for the treatment of bacterial mastitis makes the udder more susceptible to fungal infection and at the ϭϰ 

same time may lead to the development of resistant bacteria. It is therefore, important to study the sensitivity pattern of different bacteria isolated from mastitis from time to time in different geographical zones of the country in order to formulate appropriate therapeutic measures. Rapid detection of udder infection is not only a basic requirement for milk hygiene but is also decisive for the outcome of treatment to prevent the loss of milk production as well as spread of infection. Considering the above facts, these research works have been conducted with following objectives: x To find out the major bacterial profiles causing different grades of subclinical mastitis (SCM). x To isolate and identify the bacteria involved in the subclinical mastitis of dairy cows x To study the cultural and biochemical properties of the isolated bacteria. x To find out in vitro antibiotic sensitivity pattern of the bacteria isolated from milk of apparently healthy bovine udder.        ϭϱ 

     

            

ϭϲ 

  REVIEW OF LITERATURE A lot of authors have worked on different aspects of bovine mastitis. Literatures on bovine subclinical mastitis are voluminous elsewhere but limited in Bangladesh. The purpose of this chapter is to provide a selective review of the research works accomplished in relation to the present study. Literatures on bovine and caprine mastitis related with this study are reviewed: Pieperset al. (2011) conducted a study to evaluate risk factors for intramammary

infections

caused

by

coagulase-negative

staphylococci,

contagious major pathogens and environmental major pathogens in early lactating heifers at the herd, heifer and quarter levels. In total, 764 quarters of 191 dairy heifers in 20 randomly selected farms in Flanders (Belgium) were sampled. Quarter milk samples were collected between 1 and 4 days in milk and between 5 and 8 days in milk for bacteriological culture. Data were analyzed using multivariable, multilevel logistic regression analysis. Higher average herd milk somatic cell count (>200,000 cells/mL), not having an effective fly control strategy, contact with lactating cows prior to calving and moderate to severe udder edema prior to calving increased the odds of intramammary infections caused by contagious major pathogens. Poor heifer hygiene and lack of mineral/vitamin supplementation prior to calving were risk factors for intramammary infection caused by environmental major pathogens. Teat apex colonization with coagulase-negative staphylococci prior to calving seemed to protect quarters against intramammary infections caused by major pathogens. Poor heifer hygiene before calving, a non-clipped udder and not practicing of teat dipping prior to calving increased the odds of intramammary infection with coagulase-negative staphylococci. Although management is important in the prevention and control of intramammary infections in early lactating heifers, ϭϳ 

most variation in the prevalence of intramammary infections resided at the heifer and quarter levels, indicating that the susceptibility for intramammary infections around calving is mainly determined by heifer and quarter characteristics. Almeida et al. (2011) reported the emergence of strains capable of inducing chronic mastitis in dairy cows caused by Escherichia coli and that these strains adhered to and internalized into bovine mammary epithelial cells better than strains of E. coli isolated from acute mastitis. Kalmus et al. (2011) investigated 3058 clinical mastitis samples from 190 farms and 5146 subclinical mastitis samples from 274 farms. Positive results were found in 57% of the samples (4680 out of 8204), and the proportion did not differ according to year (p > 0.05). The proportion of bacteriologically negative samples was 22.3% and that of mixed growth was 20.6%. Streptococcusuberis (Str. uberis) was the bacterium isolated most frequently (18.4%) from cases of clinical mastitis, followed by Escherichia coli (E. coli) (15.9%) and Streptococcus agalactiae (Str. agalactiae) (11.9%). The bacteria that caused clinical mastitis were mainly Staphylococcus aureus (S. aureus) (20%) and coagulase-negative staphylococci (CNS) (15.4%). The probability of isolating S. aureus from milk samples was significantly higher on farms that had fewer than 30 cows, when compared with farms that had more than 100 cows (p < 0.005). A significantly higher risk of Str. agalactiae infection was found on farms with more than 600 cows (p = 0.034) compared with smaller farms. The proportion of S.aureus and CNS isolates that were resistant to penicillin was 61.4% and 38.5%, respectively. Among the E. coli isolates, ampicillin, streptomycin and tetracycline resistance were observed in 24.3%, 15.6% and 13.5%, respectively. Muhammad et al. (2010) conducted a study to evaluate a 3% solution of household detergent viz., Surf Excel (Surf field mastitis test, SFMT) vis-Ã -vis ϭϴ 

California mastitis test (CMT), Whiteside test (WST), somatic cell counts (SCC; cut off limit = 5 x 10(5) cells per milliliter) and bacteriological cultures for the detection of sub-clinical mastitis in quarter foremilk samples (n=800) of dairy cows. Rahman et al. (2009)observed that the identification of risk factors is important for the design of control programmes for mastitis in cows. Information about farms and management was collected at a farm visit. California Mastitis Test (CMT) was performed to assess sub-clinical mastitis, and cows, udder and milk were examined for clinicalmastitis. A total of 347 lactating cows from 83 farms in the dry season (November-February) and 388 lactating cows from 89 farms in the wet season (June-October) were studied. The overall prevalence

of

mastitis

was

19.9%

and

44.8%

in

dry

and

wet

seasons,respectively. The prevalence of mild mastitis was 17.3% and 40.7%, whereas that of moderate mastitis was 2.6% and 4.1% in dry and wet seasons, respectively. The prevalence of mastitis was higher (P 500,000) per ml of milk. Incidences of mastitis were significantly (p < 0.05) related to milking practices. The dominant bacterial isolates in the same order were Staphylococcus aureus, Streptococcus spp., and Escherichia coll. Other organisms isolated included Pseudomonas spp. and Klebsiefla spp. It was concluded that the high rates of sub-clinical mastitis in the area were mainly due to poor management and unhygienic milking practices. Costa et al. (2001) conducted a study to evaluate the proportion of clinical mastitis occurrence in relation to subclinical mastitis and to correlate these data with the main aetiological agents in Brazilian dairy herds. The study was conducted during 5 years in 257 dairy herds located in Sao Paulo and Minas Gerais. Diagnosis of clinical and subclinical mastitis was always performed by a veterinarian using the screening strip cup test and California mastitis test (CMT). Milk samples from all positive quarters were aseptically collected for microbiological examinations. Herds were analysed in relation to the level of clinical and subclinical mastitis and etiological agent prevalence. It was shown that the proportion of occurrence of clinical in relation to subclinical Ϯϳ 

mastitis cases ranged from 1:2 to 1:43 among the dairy herds. The lower level of clinical mastitis and the higher level of subclinical mastitis were observed when the main aetiological agent was Corynebacterium bovis while in the reverse trend the aetiological agent was coagulase positive Staphylococci. However, the difference was not statistically significant. Differences in the annual mean ranged from 1:9 to 1:4 and the 5 years mean and median was 1:7 and 1:6, respectively. Romain et al. (2000) studied on risk factors and their association with subclinical mastitis in lactating dairy cows in trinidad. Based on bulk milk samples, the farm prevalence of subclinical mastitis was 60.5% (107 of 177 farms) with a range from 33.3 to 100.0%. However, using composite milk, the farm prevalence of subclinical mastitis was 52.5% (93 of 177 farms) with a range from 21.2 to 92.9%. Subclinical mastitis was detected in 150 (45.0%) of 333 lactating cows and the farm prevalence ranged from 17.9 to 56.3%. Of 14 risk factors for mastitis studied which were related to animal husbandry, personnel, mastitis control and water, only herd size and practice of dry cow therapy were significantly associated with subclinical mastitis. Paul et al. (2000) analysed a total of 83 milk samples from cows and 49 from buffaloes, suffering from subclinical mastitis to determine the incidence of the disease in and around Chennai farms at Tamil Nadu, India and the prevalent bacteria present in the area. Antibiogram studies were also done with the characterized isolates to determine the drug of choice for the treatment of subclinical mastitis. The incidence of subclinical mastitis was higher in cows (25.78%) than in buffaloes (10.60%). The predominant bacteria in mastitis milk were Staphylococcus sp., followed by Streptococcus sp., and E. coll. Antibiogram pattern of bacterial isolates from 132 milk samples indicated that 96.20% were sensitive to neomycin, gentamicin and chloramphenicol. Oxacillin, bacitracin, penicillin and ampicillin were highly Ϯϴ 

resistant to the bacterial strains. It is concluded that antibiotics like neomycin, gentamicin, chloramphenicol, pefloxacin and norfoplaxacin are effective for controlling subclinical mastitis. It has been suggested that the antibiotic sensitivity test should be conducted prior to treatment of mastitis. Ashish-Tiwari et al. (2000) screened a total of 400 lactating cows (crossbreds, Gir and Malvi) from nearby areas of Mhow and Indore and having 1541 functional quarters to justify the incidence of subclinical mastitis in cows. The overall incidence of blind teats was 3.68%. On animal basis, the infection in Gir, Malvi and crossbred cows recorded was 66.67, 66.67 and 47.10%, respectively. On quarter basis, the incidence of subclinical mastitis in Gir, Malvi and crossbred cows was 35.43, 29.27 and 21.54%, respectively. The quarter infection rate in purebred cows, rural areas, urban areas, unorganized dairies and organized dairies was 33.01, 26.47, 21.82, 26.68 and 20.36%, respectively. The overall percentage of infected cows and quarters in Malwa region of Madhya Pradesh was 49.75 and 23.10%, respectively. Dhote et al. (1999) reported an overall 20.72% prevalence of subclinical mastitis in dairy cows. They obtained 331 isolates, of which 108 (32.62%) were Staphylococci. 138 (41.0%) Streptococci and 85 (25.67%) were gram negative bacillus. Majority of the Streptococcal and Staphylococcal isolates were sensitive to ciprofloxacin and were least sensitive to ampicillin and penicillin, respectively. The gram negative bacteria were highly sensitive to perfloxacin and least to ampicillin and cloxascillin. Itagaki et al. (1999) investigated in 150 mammary gland quarters to determine the relationship between the teat orifice abnormalities and the Soccurrence of mastitis. Subelinical mastitis was diagnosed in 49 quarters based on bacterial and cell counts. The test orifices in cows with subclinical mastitis were morphologically classified into 4 types. : smooth and well closed (type 1); Ϯϵ 

smooth and with small ring (type 2); smooth and with large ring (type 3) ; and hyperkeratotic (type 4), and the positively rates were 8.3, 23.1, 34.8 and 40.8% in types 1, 2, 3, and 4, respectively. It is suggested that the teat orifice abnormalities are closely correlated with the occurrence of subclinical mastitis in cattle. Yalcin et al. (1999) estimated minimum total cost of disease within Scottish dairy herds was $ 65.50 / cow / year. The average cost of subclinical mastitis for all high-BTSCC (bulk-tank somatic cell count) farms was $ 100 / cow / year. Joshi et al. (1998) investigated clinical mastitis in 18 cows and 56 buffaloes in Nepal. Milk samples from infected teats were subjected to bacteriological examination and an antibiotic sensitivity test. Bacterial growth were identified from 112 (88.2%) teat milk swnples, of which 76 (68.0%) had single microbial infections and 32.0% had mixed lnf&tion. Coliforms (particularly Kelebsiella pneumoniae and Escherichia toll were recorded most often (25.0%), followed by Streptococcus spp. (19.6%) and Staphylococcus spp. (9.8%). Gentamicin was the most effective (79.4% of isolates), followed by oxytetracycline (61.8%), cotrimoxazole (48.5%) and ampicillin (22.5%). Streptomycin, cloxacillin and penicillin were the least effective. A high incidence of CM was found during the first lactation of animals (64.4%), particularly during the monsoon (July to September) season (71.0%) and during the first week of frost calving (78.1%), in animals without a suckling calf (69.0%) and in animals raised under stall-fed management systems (86.3%). Jyoti et al. (1998) reported 77.77%, 63.76% and 41% prevalence of subclinical mastitis (SCM) in early, mid and late lactation respectively in an organized farm comprising 180 lactating Sahiwal x Holstein cows. Successful treatment of 72 SCM positive cows showed that there was an average increased yield of 1.38 litre of milk / cow per day but the untreated and healthy cows showed a ϯϬ 

marginally decreased milk yield. If cows were treated during early lactation, yields could be increased to about 374 litre / cow per lactation. Pushpa et al. (1998) recorded 75 bacterial isolates from 40 mastitis positive milk samples. Of the 75 isolates, 41.33% were E. coli, 22.66% Staphylococcus spp., 9.33% Corynebacterium spp., 8.0% Streptococcus and 18.66% Pseudomonas spp. They found that all of the isolates were sensitive to more than one antibiotic, most to gentamicin and kanamycin, and followed by chloramphenicol, neomycin, tetracycline, ampicillin and penicillin in decreasing order of frequency. They also observed that all the 75 isolates tested showed multiple drug resistance to 2 or more antibiotics. Shike et al. (1998) found pathogenic organisms in milk samples from 15 cows (19 quarters) with clinical signs of mastitis and 54 cows (214 quarters) with subclinical mastitis. Bacteriological examination revealed pure cultures in 7 (31.82%) and mixed cultures in 15 (68.18%) of those with subclinical infection, and from 9 (42.86%) and 12(57.14%), respectively, from the clinical cases, Staphylococcus spp. were the most commonly found pathogens. All of the isolates were sensitive to gentamicin and cephaloridine. Singh et al. (1998) examined the effect on composition of milk of 200 quarters of 50 subclinical and 60 clinically affected cows, In the samples from infected animals there were increases in the content of sodium and chlorides, and decreases in potassium. Protein content increased, while lactose content and fat content decreased, and an associated decrease in solids not fat was recorded. Bhaumik (1997) examined milk samples from 40 quarters of 23 Jersey crossbred cows, of which only 15 samples revealed bacterial growth on cultural examination of which 8 and 7 samples showed gram positive and gram negative staining characters, respectively. These bacterial isolates were subjected to antibacterial sensitivity test (AST) as per the standard disc diffusion method and ϯϭ 

on AST all the isolates were sensitive to gentamicin (100%), while doxycycline (86.66%), chloramphenicol (73.33%) streptomycin (46.66%), cephalexin (40%) cotrimoxazole (26.66%), ampicillin (26.66%) and cloxacillin (20%) exhibited some degree of resistance. Bhaumik and Bhaumik (1997) tested sensitivity of 8 antibiotics against bacterial isolates of 40 milk samples collected from 23 cows affected with clinical mastitis. Most resistance (80%) was reported with cloxacillin but there was no resistance to gentamicin and 100% recovery was reported in the animals treated with gentamicin, and 13 quarters were clinically cured after 5 days of treatment, and the other 2 quarters after treatment for a further 3 days. Devi et al. (1997) reported 75.31% animal-wise and 38.81% quarter wise overall incidence of subclinical mastitis in cows by using California Mastitis Test (CMT). They recorded breedwise incidence of 86.87% in Jersey crosses, 75.00% in Holstein Friesian crosses, 75% in Sahiwal, 57.35% in Malwi and 80% in Gir cows. Nooruddin et al. (1997)analyzed the farm records of 1082 cows and 4265 lactations to determine the distribution and association of clinical mastitis of the Central Cattle Breeding Station and Dairy Farm, Savar, Dhaka, of which 229 (21.2%) cows and 288

(6.8%) lactations, respectively affected with clinical

mastitis. Highest incidence of the disease (p