Abstract: Brucella are very small, gram-negative coccobacilli that cause a zoonosis called brucellosis. These bacteria enter the body through mucous membranes. The blood carries Brucella to organs such as the liver, spleen, bone marrow and kidneys where they cause lesions. Symptoms of brucellosis include fluctuating fever, chills, sweating, headache, muscle pain and weight loss. One species of Brucella called B. abortus infects the placenta and fetus of gestating cows and causes the fetus to abort. When humans are infected by this organism they develop a severe fever. Cow and sheep production in this region is an important livelihood source. In this study, 300 milk samples were collected from villages of Toyserkan City in seasons autumn 2009 were collected randomly from cows and sheeps of different ages This milk samples were examined for detecting Brucella abortus and Brucella melitensis with Milk Ring Test (MRT). From 300 milk samples which collected from milk samples about B. abortus 21 (14%) and B. melitensis 18 (12%) showed positive reaction with Milk Ring Test.

INTRODUCTION

Brucella, a Gram-negative bacterium belonging to the a2-proteobacteriacae is a zoonotic pathogen that induces abortion and sterility in domestic mammals and a chronic fever in humans known as Malta fever (Smith and Ficht, 1990). Brucellosis continues to be a problem for animal and humans throughout the world (Bricker, 2002).

Brucellosis in cattle is usually caused by biovars of Brucella abortus. In some countries, particularly in Southern Europe and Western Asia where cattle are kept in close association with sheep and goats, infection can also be caused by B. melitensis.

This picture will be a significant zoonotic problem for cattle and human health. Refai (2002) has reported that only B. melitensis biovar 2 was responsible of brucellosis abortion cases which were taken from sheep, goat, cattle and camel in Saudi Arabia in 10 years period.

Some investigators reported that B. melitensis was regularly isolated from cattle in Southern Europe and it was considered that B. melitensis has replaced of B. abortus in cattle (Godfroid and Kasbohrer, 2002). The nature of infection and the immune response in cattle against B. melitensis is still unknown.

The transmission of the infection to human beings occurs through breaks in the skin following direct contact with tissues, blood, urine, vaginal discharges, aborted foetuses or placentas (FAO, 2003). Until recently, only the Milk Ring Test (MRT) with a sensitivity of about 89% (Hunter and Allen, 1972) was available for detection of brucella antibodies in fresh milk. Cattles production in Toyserkan City is an important livelihood source. Problems effecting cattles health is affecting their owners economically. It is hoped that this investigation with its inspection of spreading rate among the region’s cattles and presentation of the needed statistic data would help to improve milk’s quality as well as the region’s economy.

MATERIALS AND METHODS

Sources of samples: Sampling for detecting Brucella abortus and Brucella melitensis in Toyserkan cattles were taken in autumn 2009. In autumn, 300 milk samples from 30 villages are collected randomly from cows of different ages. This study was done at laboratory of Biology, Payam Noor University of Toyserkan, Iran at 2009.

Collection of samples: The teat were disinfected with alcohol and then allowed to dry. The first streak of milk was discharged into sterile tubes. These tubes were kept in ice and then transferred to laboratory.

Milk Ring Test (MRT): A modification of the procedure described by Alton et al. (1988) was used in the MRT. The test was performed in sterile tubes and the milk samples were tested within 2 h after collection. Fresh milk samples from the individual sheep and cow were thoroughly shaken and 1 mL of the milk was transferred into to a fresh tube and 30 μL of MRT antigen was added.

The tubes were mixed thoroughly and incubated at 37°C for 3 h. When the antigen precipitated in the bottom of tubes and/or the purple band occurred at the top of milk, these samples were then regarded as positive (Turutoglu et al., 2003).

Statistical analysis: The data were analyzed by one-way Analysis of Variance (ANOVA) and Tukey test. Values of p<0.05 were considered statistically significant (Steel et al., 1997).

RESULTS AND DISCUSSION

From 150 milk samples which collected about B. abortus 21 (14%) showed positive reaction and From 150 milk samples collected about B. melitensis 18 (12%) showed positive reaction.

About B. abortus 129 (86%) and B. melitensis 132 (88%) showed negative reaction with Milk Ring Test. As a total, from 300 milk samples, 39 (13%) showed positive reaction and 261 (87%) showed negative reaction. Statistical analysis revealed that there are significant statistical differences between two groups (p<0.05). In Table 1-5 where results of statistical analysis are presented values at each column for groups. All Fig. 1 and 2 were drawed using the Statistical Software Package (SPSS Version 16).

Brucellosis is endemic in Toyserkan and as elsewhere causes severe economic losses to livestock farmers and ranchers and is a serious risk to human health. Studies in various parts of the region indicate that the disease is widespread among cattle populations particularly in ranches, livestock breeding centres and dairy farms. Cattle, sheep and goats are the principal farm animals in Toyserkan.

Milk is an important source of nutrient to man and animals. Milk meant for human consumption must be free from pathogenic organisms. Brucellosis in animals causes abortion infertility, neonatal mortality and hygroma and is spread by materials contaminated by body fluids including milk. The prevention of brucellosis in human is the basis for the advocacy of milk pasteurization worldwide. Despite the existence of regulations that require milk pasteurization most (over 75%) of milk marketed in many developing countries is sold raw through local informal pathways (Staal et al., 2000).

Although, the disease prevalence in cattle has been reported to be quite high in Iran, its serological presence in cattle of Toyserkan is reported to be low in comparison. In the present study the seroprevalence of brucellosis in cattles were found to be 39 (13%).

Table 1:	The group comparisons with each other by One way Anova

Table 2:	The evaluate statistically significant (multiple comparisons)
*The mean difference is significant at the 0.05 level

Table 3:	Means for groups in homogeneous subsets are displayed

Table 4:	Report species

Table 5:	Anova

Fig. 1:	Multiple comparisons

Fig. 2:	Number of contaminated cattles to B. abortus and B. melitensis

CONCLUSION

Seroprevalence of the disease causing abort in cattles were found to be high in this region. Therefore, to eradicate such disease and to reduce economical looses more detailed studies should be made in this region to make proper and effective challenge.

ACKNOWLEDGEMENT

This study has been supported by Islamic Azad University of Aligodarz branch.