Abstract: The major route of ammonia assimilation is the reaction which is catalyzed by glutamine synthetase to give ammonia. Cell-free extracts and purified thylakoid membranes using differential centrifugation and density gradient techniques were assayed for the percentage activity of the enzyme. Glutamine synthetase was detected in all cell-free extracts. Seventy six percent of the enzyme activity was found associated with the thylakoid membranes. Using antiserum raised to the thylakoids, 78.5% inhibition of the enzyme activity was obtained.
INTRODUCTION
In recent years, antimicrobial resistance has been a growing concern worldwide. Bovine mastitis is a frequent cause of economic loss in dairy herds and is also the single most common reason for antimicrobial use in them. Bacteria involved in bovine mastitis are broadly classified as either contagious or environmental pathogens based on their epidemiological association with the disease (Rossitto et al., 2002).
Some studies showing an increasing trend over time in resistance among mastitis pathogens (Hirvela-Koski et al., 1998). Exposure to mastitis pathogens can originate from several sources, including the environment of the cow and teat skin flora (Makovec and Ruegg, 2003). Probably the most important change in mastitis epidemiology over the past decade has been the rise in importance of environmental pathogens, primarily coliforms and streptococci other than agalactiae. The objective of this study was to describe susceptibility patterns of environmental mastitis pathogens isolated from dairy herds of Chahar Mahal province in west central Iran.
MATERIALS AND METHODS
Chahar Mahal region is a semi-arid area and receives an average annual Rain fall of 400 mm. The study areas were four districts (namely Shahrekord, Broujen, Lordegan and Farsan). A two-stage random selection procedure was adopted where study herds were randomly selected from a sampling frame comprising all herds in each district in 2004.
On average, herds selected to participate in the study constituted about 10% of all herds in the study area. All lactating animals on the day of visit were listed from which 10-15 cows were randomly selected for examination and sample collection.
During farm visits, a structured questionnaire was used to collect animal-and herd-level information on knowledge on clinical and sub clinical mastitis and any antibiotic treatments related to mastitis control. Unfortunately history of past mastitis's was not available in the herd files.
All cows (155) selected in the four districts were at least one month post calving and clinically examined for evidence of clinical mastitis as manifested by visible changes in milk and in the udder.
The examination was complemented by testing milk from lactating quarters (n = 620) for sub clinical mastitis using California Mastitis Test (CMT). The results were classified as either negative, 1 +, 2 + or 3 + depending on the amount of gel formed (Esron et al., 2005).
Positive CMT Milk samples were submitted for bacteriological examination and inoculated onto blood (BA) and MacConkey (MC) agars and incubated at 37°C.
At 24 h, the plates were examined for bacterial growth. Bacterial isolates were characterized by macro- and micro-morphology, gram staining and biochemical tests using different sugars (Quinn et al., 1994).
Other tests including motility, coagulase, catalase and oxidase reactions were carried out to assist identification of different bacterial isolates.
Susceptibility to antimicrobial agents was determined for isolated bacteria by the disk diffusion method on Mueller-Hinton agar following the National Committee for Clinical Laboratory Standards guidelines (NCCLS, 2002). The selected antibiotics for antibiogram were Lincospectine, Penicillin, Colistin, Oxytetracycline, Streptomycin, Enrofloxacin, Chloramphenicole, Gentamicin, Erythromycin, Furazolidone and Kanamycin that were more common in treatment of regional bovine mastitis cases.
RESULTS
The study revealed no significant differences in the average of the herd sizes in the four districts. No clinical mastitis cases were diagnosed on days of farm visits.
Out of the 620 milk samples collected from the four districts (Table 1), 180 were CMT positive that cultured and yield, 7 (3.88%) coagulase negative Staphylococci (CNS), 15 (8.33%) Streptococci other than agalactiae and 17 (9.44%) E. coli. Also in 122 cases contagious pathogens (including coagulase positive Staphylococcus aureus and Streptococcus agalactiae) were isolated. The proportions of pathogens recovered were comparable in all four districts and also two seasons.
CNS resistance to penicillin was 14.28% while that for Streptomycin, Oxytetracycline and Colistin, were 28.57% (Table 2).
Non agalactiae Streptococci resistance to Oxytetracycline and Kanamycin were 20 and 13.33%, respectively while that for Streptomycin, Erythromycin, Gentamicin and Colistin was 6.66%.
E. coli resistance to Penicillin, Oxytetracycline, Streptomycin and Erythromycin and Colistin were 88.23, 82.35, 76.47 and 52.94%, respectively.
However some of our isolates have not criteria for inhibition zones of susceptible or resistant and were considered as medium.
| Table 1: | Distribution of herds, samples and CMT positives in four districts of Chahar Mahal province |
| Values in parenthesis shows percentage | |
| Table 2: | Antibiotic susceptibilities of 7, 15 and 17 isolates of CNS, Streptococci and E. coli, respectively |
| Values in parenthesis shows percentage | |
DISCUSSION
A number of factors have been reported to influence the occurrence of mastitis in dairy cattle. They include managerial factors particularly those related to poor milking hygiene, environmental population of mastitis pathogens, predisposing factors such as teat injuries, sores and incomplete emptying of mammary gland quarters (Jarret et al., 1981; Karimuribo et al., 2002).
In this study E. coli and Coagulase-negative Staphylococci were the most frequently isolated bacteria in agreement with other reports (Myllys et al., 1998; Watts et al., 1995).
Escherichia coli is an environmental pathogen and E. coli mastitis is a major disease in cows. Certain strains of E. coli are capable of epithelial adherence and invasion leading to chronic IMI (Intramamary infection) and recurrent clinical mastitis (Dopfer et al., 1999).
Oxytetracycline, Streptomycin, Erythromycin and Colistin resistance of E. coli reported here (82.35, 76.47, 76.47 and 52.94%, respectively) was higher than 11.5% reported by Myllys et al. (1998).
The role of CNS as a cause of bovine mastitis is not completely clear. CNS infections are associated with damage to milk secretary tissue of the mammary gland by increased connective tissue stroma (Trinidad et al., 1990).
Susceptibility of CNS isolated from bovine IMI to selected antimicrobial agents has been previously reported (Rajala-Schultz et al., 2004). Penicillin predicts susceptibility to other β-lactamase-sensitive antimicrobial agents, e.g., ampicillin. CNS β-lactamase producers will be resistant to penicillins and some cephalosporins.
A number of international reports have shown that Streptococci genus is highly sensitive to bta-lactam and some other groups of antibiotics (Myllys et al., 1998; Sobiraj et al., 1997). This was the case in this study: A high proportion of Streptococci species were sensitive to tested antibiotics (Table 2).
Although little information is available about resistance of Streptococci to enrofloxacin it is important to note that Myllys et al. (1998) found 7% of Streptococcus strains isolated from bovine mastitis in Finland were resistant. This result was not remarkably different from our record of 0.0%. Penicillin-dihydrostreptomycin combination are normally used in these herds as dry cow therapy. Unfortunately, we did not have the individual animal treatment records, but in general, the observed resistance patterns agree with the antibiotic usage patterns. The results in the present study clearly show that our country is at risk for the world hazard of bacterial resistance.