Prevalence and Distribution of Mastitis Pathogens and their Resistance Against Antimicrobial Agents in Dairy Cows in Jordan

The primary objective of this study was to determine the aetiology of bovine mastitis in ten herds of Holstein Friesian cow in Jordan, the prevalence of mastitis pathogens in dairy cows and their resistance to selected antimicrobial agents. Milk samples were collected from 220 lactating cows to determine the clinical and subclinical mastitis by white side test and confirmed by cultural tests. It was found that 138 quarters 15.7% had been diagnosed as clinical mastitis with definite gross pathological lesions and change in udder secretion, in addition to that 276 quarters 31.4% showed subclinical mastitis. The incidence of mastitis was found to be increased in older cows. Staphylococcus aurous was considered to be the most common cause of both clinical and subclinical mastitis and followed by coli forms, streptococcus spp., corynebacterium spp., proteus spp. and pseudomonas spp. Sensitivity tests was applied to different isolated strains using tertracycline, ampicillin, neomycin, erythromycin, penicillin G and sulphamethoxazol trimethoprim.


INTRODUCTION
Mastitis is considered one of the most important dairy cattle diseases and is one of the major causes of antibiotics use in dairy cows [1,2] . Over 135 different microorganisms have been isolated from bovine intramammary infection, but the majority of infections are caused by staphylococci, streptococci and gramnegative bacteria [3] . Where is staphylococcus aureus was isolated from 18% of the culture positive quarter [4] .
Mastitis known to be a complex and costly disease of dairy cows that results from the interaction of the cow and the environment including the milking machine and the microorganisms [5][6][7] , although stress and physical injuries may cause inflammation of mammary glands, infection by invading bacteria or other microorganisms (fungi, yeast and possibly viruses) is the primary cause of mastitis [8,9] .
Jordan has experienced a rapid population growth over the last three decades and an increased demand for milk and dairy products. Mastitis is considered to be the most frequent disease of dairy cattle in Jordan and has received more veterinary attention as a disease of cattle and as a serious economic problem [10,11] .
Mastitis is produced by a variety of pathogenic microorganisms. Cows and herds vary in susceptibility and extent, type and duration of infection, although some of mammary pathogens can be isolated from the environment of the cow, manure and bedding, water supplies, soil and inadequately cleaned milking machines [12,13] , thus new infection can be reduced by applying rigid herd management practices [14] . Measures to control mastitis such as improved milking hygiene have reduced streptococcus agalactia [15][16][17] . The incidence of the disease was influenced with the lactation and age of the animal; higher infection were reported in older cows [18,19] .
The aim of this paper was to determine the prevalence of mastitis pathogens in clinically and subclinically of dairy cows, a further objective was to determine the susceptibility of these bacteria to 6 antimicrobial agents that are or have been commonly used in dairy cows in Jordan.

MATERIALS AND METHODS
Milk samples were collected from 220 Holstein Friesian cows in 10 farms in Thuleil area, full history was reported for cows in all farms. The health status of the udder and the age of the cows were recorded. Prior to sampling the udder was washed and dried and the teat was disinfected. All milk samples were collected all over the year 2006 under aseptic condition. About 15 mL of the foremilk were discarded and the next fifteen mL were collected into screw-capped bottles and transferred in ice boxes to the laboratory. All samples were stored at 4°C from the time of collection until examination within 3-4 h.
All samples were centrifuged for 15 min at 3000 rpm and a loopfull was taken from sediment and inoculated separately on each of the following media, 5% sheep blood agar, Edwards and Mac Conkey's agar. The inoculated plates were then incubated at 37°C for 24, 48 and 72 h. From growing colonies smears were made and stained with Grams stain. In case of Streptococcus strains it was identified by the morphological character on blood agar and specific Edwards media. For identification of isolated Staphylococcus strains by colonial appearance on blood agar, pigments, haemolysis and for pathogenicity by using tube method of coagulase test. Coliforms, Proteus spp., Corynebacterium spp., pseudomonas spp. and the other Gram negative bacilli were identified by morphological character on sheep blood agar and by biochemical reaction when necessary.

RESULTS AND DISCUSSION
The incidence of clinical mastitis in udder quarters of Holstein Friesian cows in ten farms was found to be 15.7% Table 1. This is a little higher than that reported before in Holstein Friesian cows in Egypt 12.08% [20] . The incidence of subclinical mastitis, however, averaged 31.4% in this investigation as based on udder quarters ( Table 2).
It was evident in this study that mastitis was increased with advanced age. It is noticed in some herds the incidence of clinical and subclinical mastitis was increased with the sixth years old Table 3, Fig. 1.
The relative frequency of different types of microorganisms in this study revealed that staphylococcus aureus, Coliforms and streptococcus spp. were the chief aetiological agents responsible for both clinical and subclinical mastitis Table 4. These results agree with those presented by others [3,10,21] .
In     [22] , it was reported that the most estimates of prevalence of bacterial pathogens intramammary infections were: coagulase negative Staphylococci 16.04%, Staph. aureus 9.41%, Klebsiella spp. 6.17%, Corynebacterium bovis 5.35% and Brucella melitensis 4.52%.In U.K. the distribution of the 1457 clinical cases according to the result of the culture was E-coli 32%, strept uberis 23% staph 7%, Corynpyogens 1% mixed 10% and others 24% [23] while In Germany the mastitis pathogens were isolated from 26.4% of the milk samples and the major pathogens, staph. aureus 5.7% and streptococcus uberis 1% had the highest prevalence, at the same time streptococcus    agalactia was isolated in samples from 29% of the herds [19] . Bacteriological data from herds surveys by the Massachusetts Mastitis Loboratory were analyzed and showed that Staph. aureus and coliforms were the predominant organisms isolated from Holstein Friesian cow mastitis a result which is similar to our result [24] .
It would be of interest to note that in Sudan [25] the organisms isolated in clinical and subclinical mastitis in Zebu cattle were staph spp.50 and 68.7%. Strept spp. 15.3 and 12.6%, Coryn. spp. 11 and 00% coliforms 11 and 4.8% and mixed 10.2 and 13%. In Jordan it was reported that the most common organisms isolated from clinical and subclinical cases were staph. spp. 30% [11] .
There results of sensitivity tests of the organisms isolated to antibiotics Table 5 shown that 75% of strains were sensitive to Sulphamethaxazol Trimethoprim, 64% to Tetracycline, 54% to Peniciilin G, 52.8% to Ampicillin, 36% to Erythromycin and 21.4% to Neomycin.

Statistical analysis:
The analysis of variance for antibiotics sensitivity showed that there are differences between antibiotics treatment at 1% level of significant (Table 6). At the same time there are no significant differences between isolated organisms.

CONCLUSION
In conclusion, more efforts are needed to improve the general udder health in dairy herds as approximately 47.1% of the cows had at least one infected quarter (clinically or sub-clinically), so the recommended preventive and control measures against mastitis should include application of good sanitary and hygienic measures. In addition to that regular monitoring of the prevalence and the distribution of mastitis-causing pathogens provides, however, valuable knowledge about the effectiveness of proposed control strategies and helps in prioritizing specific mastitis control efforts.