Seroprevalance of Brucella abortus in cattle and buffaloes in district Rajanpur, Punjab, Pakistan

The current work was aimed to search out the prevalence of Brucellosis in Nili Ravi buffaloes and Dajal cattle, in tehsil Rojhan, Rajanpur and Jampur of District Rajanpur, Punjab. Animals sample size was 500 comprising of 50, 50% both buffaloes and cattle. Samples were obtained from small dairy holders and private farms. From each animal blood sample of 5ml was collected in a sterilized Vacutainer and centrifuged (2000 rpm for 5 minutes) for separation of serum. To determine the Brucella abortus antibodies with the help of Rose Bengal Plate Test (RBPT), plod plasma was stored at -20oC. The findings presented an overall prevalence of 5.2% (13/250) and 4.4% (11/250) in buffaloes and cattle, respectively (P = 0.835). For the animals having the history of abortion, Brucella abortus sero prevalence association was highly significant (P<0.001) presenting 52.6% (buffaloes) and 38.1% (cattle). Brucellosis also showed a positive correlation with other reproductive disorders i.e. metritis, repeat breeding and Retained Fetal Membranes. For confirmation of RBPT’s results, iELISA for Brucella abortus positive samples performed and results indicated an overall prevalent was 2% (buffaloes) and1.2% (cattle), respectively. Statistical analysis determined that the Brucella abortus’s overall prevalence was non-significant in district Rajanpur, for both buffaloes and cattle.


Introduction
Agriculture is the base of Economy in Pakistan and livestock share in Agriculture is more than half by contributing a 58.3% of total in agriculture, finally in the whole economy. Its contribution in GDP is about 776.5 billion rupees. In the country, entire population of buffaloes and cattle is 37.7 million and 44.4 million, respectively [1]. A major portion of our everyday necessities are obtained from these animals in the form of milk, meat, leather and bio-fertilizers. Livestock is badly affect by a large number of diseases resulting in decline of livestock's contribution in the economy and every year GDP growth of Pakistan. Brucellosis is one of the most important reproductive disorders disease that is responsible for the low infertility resulting in the decreased production rate and ultimately economic loss occur [2], causing the abortion and the endometritis [3]. World widely after rabies, it is second utmost prevalent zoonotic disease [4]. The basic bacterial species affecting the farm animals include; in goats (Brucella mellitensis), in sheep (Brucella ovis), in swines (Brucella suis) and in cattle (Brucella abortus) [2]. Birth of weak calves, the last trimester's abortion, retained fetal membrane and death of calves prepartum are major clinical signs of Brucella abortus. Aborted placenta shows grey necrotic lesions and edema. Vaginal secretions and discharge remain continue up to 2-3 months and is the major source of contamination for the water, soil and pastures. Splenomegaly, hepatomegaly, bronchopneumonia, haemorrhagic, lymph nodes swelling, fluid in thoracic cavity, may be shown in Infected fetus. Infectious conditions including of ampullae, spermatocystitis, orchitis, epididymitis, in males. Poor semen quality is observed by low motility and sperm concentration, increased morphological abnormalities and neutrophils number in semen but libido is not disturbed in affected bucks and rams [ But to be on safe side, the samples size was maintained 250 for each specie and totally 500.

Collection of blood samples
A sample of 5ml blood was collected in sterilized test tube from jugular vein of each animal aseptically. After collection the sample were allowed to settle down the blood till formation of clot and stored until centrifugation in refrigerator. The centrifugation was done for about 5 min at the rate of 2000 rpm and separated serum was stored for further examination, at -20ºC.

Serum analysis
RBPT was used to analyse the samples serum [22] and determine the brucellosis seropositivity. For confirmation of the positive samples, indirect Enzyme Linked Immuno Sorbent Assay (iELISA) was applied. Commercially available IDEXX ® Brucellosis Serum X2 test kit was used for iELISA.

Calculations
For calculations of results following formulae were applied.

Calculation of percent positivity values (PP)
All OD (optical density) values for the test samples as well as the negative control (Neg C) are related to the OD values of the positive control as follows: OD positive Control Test validity criteria The duplicate OD values of the positive control should not differ more than 25 % from the mean value of the two duplicates, to ensure validity. Moreover; the control values should be between the following limits: OD positive control > .040 OD negative control < .040 The test would be invalid if any of these criteria not fulfilled and technique may be suspected and the tests should be repeated.

Statistical analysis
The Chi-square test was used to analyse the obtained data [23]. Chi-square test was applied in specie wise sero-prevalence and correlation of seropositivity with reproductive disorders.  (Table 1). Statistically analysis also revealed that the prevalence relationship among three tehsils was also non-significant.  in Tehsil Rajanpur indicating higher prevalence than other two and 3.6%. In Tehsil Jampur, showed lowest prevalence than remaining both. (Table 3). Statistically analysis also revealed that the prevalence relationship among three tehsils was also non-significant.

Discussion
All over the world brucellosis control in animals has remained a burning issue to save economy and reduce production loss. This is only possible by maintaining a disease free stock and the isolation of infected or carrier ones. The goal is only achievable with the immediate actions, sensitive and reliable diagnostic technique to diagnose the problem. Animals' screening at farm or herd level is done by serological analysis that is a sensitive and rapid tool. For that reason in this study, serological tests like iELISA and RBPT are preferred over routine bacterial isolation and identification procedure. In routine the RBPT is used to detect Brucella antibodies in sera of various species as an initial step and among different species it may have cross reaction. Therefore to confirm the results of RBPT, iELISA is applied on the positive serum samples.
According to the present study results showed that overall brucellosis prevalence in 250 Dajal cattle is 4.4% (n=11) when checked with RBPT while it was 1.2% (n=3) when confirmed with iELISA. Overall Brucellosis prevalence was higher 2.3% when tested through iELISA in tehsil Jampur, for Dajal cattle which was more than other two tehsils; . Animals having abortion history were 3.89 times more probably to be seroprevalence positive than non-aborted ones, [36]. Approximately brucellosis seropositivity is associated with 35% in cattle and 20% in small ruminants' abortions, [37].
Though in the current work, brucellosis prevalence in cattle and buffalo and its association with many reproductive disorders has been considered to a large level, but it is appealing to further investigate the disease in other species including camels, goats and sheep to find out the Brucella abortus prevalence. Most of the developed countries have been eradicated this disease following test and slaughter policy which is impossible to follow in our country because of limited recourses. Therefore, some important measures are recommended for control of the Brucellosis viz; Public education, controlled personnel/ animals access, quarantine measures for new addition, improved management to avoid contamination by proper hygiene and disposal of aborted foetuses, eliminating Brucella positive reactors and appropriate milk sterilization before consumption.

Conclusions
Present study results determined that in the Rajanpur district, the Brucella abortus prevalence in buffaloes and cattle is fairly low but appropriate measures should be taken to control the economic losses in the form of increased calving interval lower milk yield and abortions. The current study was planned to search out the Brucella abortus seroprevalence in buffaloes and cattle in Rajanpur district. Buffaloes and cattle have a basic contribution in the economy of Pakistan. These animals in the form of meat and milk contributes a large share in GDP. In year 2016-17 the whole population of buffaloes and cattle was 37.7 million and 44.4 million, respectively. They also provided a large part of our routine necessities in the form of by products, bio fertilizers and leather. The current study was planned to search out the Brucella abortus seroprevalence in buffaloes and cattle in Rajanpur district and to identify the relationship of brucellosis with different reproductive disorders. The target animals were Nili Ravi buffalo and Dajal cattle of which home tract is the district Rajanpur. The samples collected were 250 from each specie, totally 500. Centrifugation was done to separate the serum and then Rose Bengal Plate Test (RBPT) was used for serum analysis and positive samples were subjected through iELISA for further confirmation. The findings indicated an overall Brucella abortus seroprevalence as 5.2% for buffaloes and 4.4% for cattle through RBPT. The iELISA results was slightly changed than RBPT, indicating prevalence as 1.2% in cattle and 2% in Buffalo. In cattle, the results showed a higher Brucella abortus prevalence in Tehsil Jampur than other the Rajanpur and Rojhan tehsil. When data was statistically analysed a significant relationship (P<0.05) was found between Brucella abortus prevalence and aborted animals.