Pure Appl. Biol., 11(1):291-301, March, 2022 http://dx.doi.org/10.19045/bspab.2022.110031

Research Article

Epidemiological study on Brucellosis in Azi-Kheli buffalo in district Swat Khyber Pakhtoon Khwa

Tariq Aziz1, Rahmatullah Rhind2, Azhar Khan1, Farmanullah3*, Rahimullah shah4, Fasisal Anwar5, Tariq Hayat1 and Saeed Sarwar6 1. Livestock and Dairy Development department KPK, 2. Department of Microbiology Sindh Agricultural University, Tandojam, Pakistan 3. Faculty of Veterinary and Animal Sciences, National Center for Livestock Breeding Genetics and Genomics LUAWMS, Uthal, Pakistan 4. Veterinary Research Institute Peshawar, KPK, 5. College of veterinary sciences, Agriculture University Peshawar, KPK, Pakistan 6. Department of Animal Sciences Abdul Wali Khan University Mardan, Pakistan *Corresponding author’s email: [email protected] , [email protected] Citation Tariq Aziz, Rahmatullah Rhind, Azhar Khan, Farmanullah, Rahimullah Shah, Fasisal Anwar, Tariq Hayat and Saeed Sarwar. Epidemiological study on Brucellosis in Azi-Kheli buffalo in district Swat Khyber Pakhtoon Khwa. Pure and Applied Biology. Vol. 11, Issue 1, pp291-301. http://dx.doi.org/10.19045/bspab.2022.110031 Received: 24/03/2021 Revised: 18/05/2021 Accepted: 01/06/2021 Online First: 30/06/2021 Abstract An epidemiological study on the seroprevalence of brucellosis in Azi-Kheli buffaloes was carried-out. The total samples studied 109 serum samples, and 100 milk samples through Rose Bengal Plate Test (RBPT), Serum Agglutination Test (SAT) and Milk Ring Test (MRT). The overall seroprevalence of brucellosis in Azi-Kheli buffaloes was recorded as 9.17, 10.09 and 10.00% by RBPT, SAT and MRT respectively. Moreover, there were not positive sample found in male. SAT was found as the best technique for the determination antibody titer and percentage prevalence as compared to other techniques used. An area wise study was also conducted to record an epidemiological distribution which shown higher prevalence in tehsil Kabal as 11.11 and 13.33%, in tehsils Matta 9.09 and 9.09% and in tehsil Baboozai 6.66% and 6.66% by RBPT and SAT respectively. The lowest prevalence was observed in tehsil Baboozai. Similarly, an investigation was also conducted to observe the prevalence at different lactations. The prevalence of brucellosis in sera of buffaloes in their first, second, third, fourth and fifth lactations was recorded as 5.5, 16.6, 14.2, 9.09 and 0.0% respectively. The higher prevalence was observed at second while lowest at first lactation whereas at fifth, zero prevalence was recorded. The results of current study validated that brucellosis was widespread in tehsil Kabal with a relatively and significantly higher in tehsil Kabal as compared to tehsils Matta, and tehsil Baboozai. Results of the current study may support the health care organizers to formulate suitable control plans against Brucellosis in . Keywords: Azi-Kheli Buffalo; Brucellosis; MRT; RBPT; SAT Introduction and buffaloes. Brucellosis is transmitted by Brucellosis is a contagious disease of domestic exposure to birth materials or by ingesting livestock caused by the bacteria, Brucella infected milk [1]. Brucella species are small, abortus. The disease originally transmitted to Gram-negative, non-motile, non-spore- wildlife by cattle, and now is found in wild elk forming rods, considered being facultative

Published by Bolan Society for Pure and Applied Biology 291 Aziz et al. intracellular parasites that cause chronic its original home tract known as Azikhel diseases, which usually persists for life [2]. ( union council). It is an Brucellosis is considered by the FAO and important indigenous animal genetic WHO as one of the most widespread zoonotic resource of the area and got its name from in the world [3]. The bacterium Brucella its original home tract called Azikheil, one abortus is the principal cause of brucellosis in of the several tributary valleys of river Swat. buffaloes and cattle [4]. The disease is The broader home tract includes watershed characterised by late term abortion; infertility of River Swat (District Swat) and River and reduced milk production as a result of Panjkora (District Lower and Upper Dir), retained placenta, secondary endometritis, District Shangla, Bunair and Malakand excretion of the organisms in uterine agency. Pockets of the breed can also be discharges and milk. Full-term calves may die found in District Mardan, Charsadda, soon after birth. In fully susceptible herds, Nowshera and Sawabi as a result of abortion rates may vary from 30 to 80%, in transhumant migration during winter season some cases, abortions may be more incidentals from upland pastures of District Swat and [5]. Dir. The Azikheli buffalo is native breed to Buffaloes mostly belong to river type breed, the Hindukush Mountains of Northern which are five established breeds. After few Pakistan. zikhel Tehsil, the home tract of renowned buffalo breeds, the fifth breed is Azikheli buffalo [8]. Azi-Kheli, which is mainly localized in the The azikheli is known as the native breed of Swat valley in the North West Frontier swat valley. The typical animals have Province (Khyber Pakhtoon Khwa). compact body, medium sized, sickle shape Interestingly Pakistan is the second largest horns, long and wide blade ears, dairy buffalo country in the world [6]. and characteristically short tail. Body color is possesses the highest buffalo milk genetic albino; due to environmental changes potential in the world and they contribute somewhere color changes with time to light 70% of the total milk produced in the black [9]. country [7]. Population estimates for this breed is based Farmers who rear Azikheli buffalo since on the interviews with farmers and livestock long and are well aware of the breed were professionals of the area. The population of identified in the study area through Azi-Kheli buffalo is around 25,000 heads discussion with the officials of the and showing a continuously decreasing Department of Livestock and Dairy trend in population. Azi-Kheli breed is Development District Swat. Meeting with equally recognized for milk and meat, but it farmers of Azikheli buffalo were conducted could not cross the Swat valley. It is for the for identification of Azikheli buffalo breed. first time that Azi-Kheli buffalo has been Salient features of the Azikheli buffalo included in the 2006 livestock census. known in the farmers communities were Government farms in Pakistan have a few brown coat colors, short tail, blue colored thousand breeding buffaloes (along with shining eyes, with horns bent backward and their calves and young stocks) intended for upward without twisting which differentiate experimentation, but none of the it from other breed of buffalo in the country. government farms, however, has Azi-Kheli Azikheli is a buffalo breed, in Swat, and breed; as such there is no conservation acclimatized to the local conditions and is programme for this breed [10]. The present reared by farming communities. Home tract study was designed to know about the and study area The Azikheli is named after epidemiological aspects of brucellosis in

292 Pure Appl. Biol., 11(1):291-301, March, 2022 http://dx.doi.org/10.19045/bspab.2022.110031

Azi-Kheli buffaloes in district Swat (Khyber (VRS), Swat and stored at -20oC in deep Pakhtoon Khwa) on the basis of following freezer till used. major objectives such as to study the Examination of blood samples, rose seroprevalence of brucellosis in Azi-kheli bengal plate test (RBPT) antigen and buffaloes in district Swat, Khyber Pakhtoon control sera Khwa, and to compare the sensitivity of The Rose Bengal stained antigen containing Rose-Bengal, Serum Agglutination and Milk Brucella abortus cells (strain-99) was Ring Tests for the diagnosis of brucellosis in suspended in buffer at pH 3.6 as Azi-kheli buffaloes. recommended by Veterinary Research The data so generated through this research Institute (VRI), Lahore. Before conduct of will a baseline data for planning future test, the antigen and serum samples were strategies for control of brucellosis in kept at room temperature to bring them to buffaloes in Swat valley Malakand division. the normal physiological state. The Materials and Methods porcelain plate wells were labeled with The study on the seroprevalence of indelible marker according to the reference brucellosis in Azi-Kheli buffaloes was numbers of serum sample. A drop of 0.3ml carried-out during year 2009 to compare the of the serum was placed in a well of the sensitivity of Rose-Bengal, Serum porcelain plate. The other sera were placed Agglutination and Milk Ring Tests for the in wells and labeled, using sterilized diagnosis of brucellosis and to obtain a serological pipette for each sample. baseline data for planning future strategies Similarly, positive and negative control sera for control of brucellosis in this typical were placed in last two wells of the plate. buffalo breed of Swat valley. The antigen vial was shaken gently and then Collection of blood samples a drop of 0.3ml of antigen suspension was The animal is restrained manually. Hind leg taken from the vial and placed near the drop is immobilized and slight pressure may be of sera on the plate (both positive and applied gently above the knee joint. The negative controls). The serum and antigen vein is punctured using a 20G needle and were thoroughly mixed by an applicator and enough volume of blood was collected with spread in the form of circle over an area of a capillary tube or a syringe with a needle. approximately 1.5cm radius. For each serum Collection of blood specimens care as use sample a separate applicator stick used to new needle and syringe for each animal, mix the serum and antigen. The porcelain dispose needles properly, clean the puncture plate was then left for four minutes. The area with 70% alcohol, and handle animals porcelain plate was then microscopically calmly and using appropriate restraint. A examined, using magnifying glass to record total of 100 blood samples were collected and confirm the agglutination under a good from female Azi-Kheli buffaloes and 09 source of light against a dark background samples from male buffaloes (bulls) from field. Due to the positive interaction fifteen villages of three tehsils of district between antigen and serum, the granules Swat. The tehsils included were Kabal, appeared with different intensity. The Matta and Baboozai. The blood samples intensity showed the amount of antibodies in from the animals were obtained by using the serum, which indicates the specific disposable sterilized plastic syringe. On the species of bacteria in the infected animals. following day, the serum was harvested in The result of each well was matched with clean screw caped plastic bottles and positive and negative controls in the same brought to the Veterinary Research Station plate before any conclusion was made.

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Serum agglutination test (SAT) antigen Examination of milk samples and control sera Milk ring test (MRT), antigen and control The Brucella abortus agglutination milk samples concentration and phenolised suspension of Hematoxylin stained Brucella abortus Brucella abortus (strain -99) obtained from (strain -99) antigen was used for Milk Ring Veterinary Research Institute; Lahore was Test as recommended by Veterinary used as recommended by manufacturer. Five Research Institute (VRI), Lahore. The test number sterilized test tubes were placed in a tubes (sterilized) were labeled and placed in test tube rack and labeled. A 0.8ml solution the test tube rack. 1ml of each milk sample of normal saline containing 0.5% phenol was drawn into each test tube, while other was added to the first tube and then 0.5ml two test tubes were also placed in the same into the remaining tubes. A 0.2ml of the rack as negative and positive controls. A serum to be tested was added to first tube 0.05ml of Milk Ring Test stained antigen and mixed thoroughly (1/5 dilution). A (Hematoxylin) was added to each tube by 0.5ml was withdrawn from first test tube and using graduated pipette. The tubes were transferred into the second tube. After sealed with Para-film, the antigen and mixing well, 0.5ml was transferred to the samples were mixed gently and allowed to third and so on up to the last fifth tube, stand for about 2 minutes. Then the rack was where in after mixing gently, 0.5ml was placed in an incubator at 37˚C for 1 hour. withdrawn and discarded from last fifth tube After the incubation, the test tubes were (two fold dilution). A 0.5ml of the taken out from the incubator, examined and standardized Brucella abortus concentrated results were recorded as: If a deep blue antigen dilution (1:20) was added to each colouration ring on the top of milk is tube containing serum dilution giving a formed, the test is considered to be positive. series of final dilutions of 1/20, 1/40, 1/80 If no ring is formed on the top of milk and 1/160 were then mixed thoroughly from samples, the test is considered to be the highest to the lowest dilution. A series of negative. The results were matched with dilutions were also prepared for positive and control in the same row before conclusion is negative sera and kept as controls. The to be made. antigen and serum were mixed well by Results gentle shaking and then placed in an The results on the seroprevalence of incubator at 37˚C for overnight. After the brucellosis in Azi-Kheli buffaloes in Swat overnight incubation, the tubes were are given in (Table 1). The RBPT was removed and care was taken to not disturb performed using the (n=109) blood samples, the agglutination. All tubes were examined (n=10) showed agglutination and believed to using an indirect source of light against a be positive for brucellosis. Based on this dark background and antibody titers were screening, 9.17% buffaloes were found recorded. positive for brucellosis. Similarly, the SAT Collection of milk samples was also performed and (n=11) out of A total of 100 milk samples were collected (n=109) samples showed agglutination and from the same female animals as used for 10.09% was recorded to be positive for obtaining blood samples. The bottles were brucellosis. The MRT (n=100) milk samples capped tightly and brought to the laboratory of buffaloes were examined by Milk Ring of Veterinary Research Station, Swat for Test, (n=10) were found to be positive examination. reactors for Brucella antibodies.

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The seroprevalence of brucellosis was from female buffaloes were collected and recorded in buffaloes as 10%. Generally, it investigated for the seroprevalence of seems that all the test techniques produced brucellosis and it was observed from the similar percentage of prevalence of results given in the (Table 1), that the Milk brucellosis in Azi-Kheli buffaloes. However, Ring Test technique produced 10.00 % SAT showed relatively higher prevalence, brucellosis in females. It is obvious that which was recorded as 10.09% as compared irrespective of any techniques applied, a risk to Rose Bengal and Milk Ring Tests, both of higher prevalence of brucellosis is evident yielded 9.17 and 10.00% brucellosis in in lactating female buffaloes as compared to buffaloes respectively. Nevertheless, the any other category of buffaloes investigated Serum Agglutination Test (SAT) was during present survey. On the other hand, recorded as the best technique that showed when a comparison was made among the higher prevalence. While considering the test techniques, Serum Agglutination Test sensitivity for determining of seroprevalence was found to be the best technique and could of brucellosis in blood samples, the Serum be used while detecting seroprevalence of Agglutination Test (SAT) was found to be brucellosis in buffaloes. the best method that determined antibodies Milk ring test (MRT) of Brucella in the sera of Azi-Kheli The seroprevalence of brucellosis in buffaloes. buffaloes investigated at different tehsil The seroprevalence of brucellosis in male levels. and female buffaloes Three tehsils i.e. Kabal, Matta and Baboozai The data regarding the seroprevalence of were selected to determine the brucellosis in male and female buffaloes are seroprevalence of brucellosis in Azi-Kheli presented in (Table 1). In this survey (n=9) buffaloes. The data regarding the Azi-Kheli buffalo bulls were examined by seroprevalence of brucellosis in buffaloes at RBPT and SAT while (n=100) lactating different tehsils are presented in (Table 2). buffaloes were also investigated by RBPT In tehsil Kabal, 46 buffaloes were examined and SAT techniques. Moreover, 100 milk by RBPT and SAT techniques, 5 samples samples were also examined by using MRT. were found to be positive for brucellosis The prevalence of brucellosis in females was when examined by RBPT technique; while 6 recorded as 10.00, 11.00 and 10.00 % by samples were found positive for RBPT, SAT and MRT, respectively while seroprevalence of brucellosis investigated by none of the males were found to be positive using SAT technique. The prevalence of for the prevalence of brucellosis in buffaloes brucellosis was recorded as 11.11 and 13.33 by any of the techniques used for % by RBPT and SAT techniques, examination. It is concluded that prevalence respectively. The higher seroprevalence of of brucellosis was higher in female Azi- brucellosis was detected by Serum Kheli buffaloes by Serum Agglutination test Agglutination Test as compared to rest of as compared to rest of the techniques used. the techniques used. Furthermore, the Serum Agglutination Test Therefore, serum agglutination Test is also detected some variable prevalence in considered to be best the technique and females as compared to their males, because could be used for detecting brucellosis in in males both the techniques Rose Bengal buffaloes. In tehsil Matta, 33 buffaloes were Plate and Serum Agglutination Tests could examined by RBPT and SAT techniques. Of not detect prevalence of brucellosis in the examined samples, 3 samples were buffaloes. At the same time, milk samples found to be positive for brucellosis using

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RBPT technique and equally 3 samples were results further showed that the found positive for brucellosis using SAT seroprevalence of brucellosis in milk technique. The sero-prevalence of samples of Azi-Kheli buffaloes were higher brucellosis recorded in buffaloes was 9.09 in tehsil Kabal i.e., 12.195 as compared to and 9.09 % by RBPT and SAT techniques 10.34 and 6.67% seroprevalence of respectively. However, the equal brucellosis in tehsil Matta and tehsil seroprevalence of brucellosis was detected Baboozai respectively. Therefore, the in buffaloes by Rose Bengal Plate Test and highest seroprevalence of brucellosis was Serum Agglutination Test; both tests could recorded in buffaloes of tehsil Kabal while be used for reliable detection of brucellosis lowest in tehsil Baboozai of in buffaloes. In tehsil Baboozai, the blood Khyber Pakhtoon Khwa (Table 2). A total of samples from30 Azi-Kheli buffaloes were 100 blood samples from Azi-Kheli breed examined by RBPT and SAT techniques. Of during different lactations were taken and the samples examined, 2 were found to be examined by using different techniques. At positive for brucellosis by RBPT technique; first lactation, 36 samples were tested, 2 however, 2 samples were found positive for were found positive and the prevalence was brucellosis by using SAT technique. The recorded as 5.55%; while in 2nd, 3rd and 4th prevalence of brucellosis was recorded as lactations, 5, 3, and 1 sample were found 6.66 and 6.66 % by RBPT and SAT positive and the prevalence was recorded as techniques respectively. It is observed that 16.66, 14.28 and 9.09%, respectively. both the tests could be used for reliable While at 5th lactation, 2 samples were detection of seroprevalence of brucellosis in obtained and all were found negative. buffaloes. It is clear from the results that the However, the higher prevalence of seroprevalence of brucellosis in Azi-Kheli brucellosis was recorded at 2nd lactation as buffaloes was higher in tehsil Kabal i.e. compared to other lactations (Table 3). 11.11 and 13.33 % by RBPT and SAT The mean antibody titers of brucellosis in techniques respectively as compared to 9.09 the sera of Azi-Kheli buffaloes and 6.66 % seroprevalence of brucellosis in During present serological study positive tehsils Matta and Baboozai by using RBPT sera samples were examined using Serum and SAT techniques respectively. However, Agglutination Test (SAT) to determine a lower seroprevalence of brucellosis in Brucella antibody titer and results are shown buffaloes was recorded in tehsil Baboozai. in (Table 4). Eleven sera of positive reactors The seroprevalence of brucellosis in milk of buffaloes were titrated. Of the sera from samples of Azi-Khili buffaloes investigated buffaloes examined, 08 (72.72%) and 3 at different tehsil level. In tehsil Kabal, 41 (27.28%) showed a titer of 1/20 and 1/40, milk samples collected from Azi-Kheli respectively. No interaction between buffaloes were investigated by Milk Ring antibody and antigen was observed at the Test (MRT) technique. Of the samples titers of 1/80 and 1/160 in both male and examined, 5 samples were found to be female buffaloes and considered to be positive for brucellosis while 29 milk without antibody or may be having the samples collected from the buffaloes from antibodies in the least numbers that did not Matta Tehsil, 3 were found to be positive for interact with antigen and demonstrated as brucellosis by using MRT technique. negative for the Brucella antibodies. Whereas 30 milk samples collected from the However, it is concluded that brucellosis is buffaloes from Baboozai Tehsil, 2 were the evident in the area with maximum and found positive for brucellosis (Table 2). The

296 Pure Appl. Biol., 11(1):291-301, March, 2022 http://dx.doi.org/10.19045/bspab.2022.110031 relatively higher prevalence and high antibody titer in females.

Table 1. The seroprevalence of brucellosis and sex-wise seroprevalence in Azi-Kheli buffaloes No of No of Techniques used samples positive Percentage of positive samples examined samples Rose Bengal Plate 109 10 9.17 Test (RBPT) Serum Agglutination Test 109 11 10.09 (SAT) Milk Ring Test 100 10 10.00 (MRT) Techniques used Female buffaloes Male buffaloes(bulls) No. of No. of Percentage No. of No. of Percentage samples positive of positive samples positive of positive Examined samples samples examined samples samples Rose Bengal Plate 100 10 10.00 9 0 0.00 Test Serum Agglutination 100 11 11.00 9 0 0.00 Test Milk Ring Test 100 10 10.00 - - -

Table 2. The seroprevalence determined by RBPT, SAT, and MRT at different tehsil levels Tehsil Matta Technique No. of No. of Percentage of Technique No. of No. of Percentage of s used samples positive positive s used samples positive positive examined samples Samples examined samples samples RBPT 46 5 11.11 RBPT 33 03 09.9 SAT 46 06 13.13 SAT 33 03 09.9 Technique Tehsil Babozai Tehsils s used Total No. No. of Percentage No. of No. of Percentage of of milk positive of positive samples positive positive samples milk milk examined samples Samples examined samples samples RBPT 30 02 06.6 Kabal 41 5 12.19 SAT 30 02 06.6 Matta 29 3 10.34 Baboozai 30 2 6.67

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Table 3. The seroprevalence at various lactations No. of Total No. of No. of Percentage of No. of negative Percentage of Lactatio samples positive positive samples samples negative samples n examined samples 1st 36 2 5.55 34 94.44 2nd 30 5 6.66 25 83.33 3rd 21 3 4.28 18 85.71 4th 11 1 9.09 10 90.90 5th 02 00 00 2 100

Table 4. Positive reactors (%) for Brucella antibodies observed by serum agglutination test Total Positive reactors (Titres/ Dilutions) Sex of positive 1/20 dilution 1/40 dilution 1/80 dilution 1/60 dilution animals reactors No. % No. % No. % No % Female 11 8 72.72 3 27.28 0 0 0 0 Male 9 0 0 0 0 0 0 0 0

Discussion 20% prevalence of Brucella from milk In the present study, seroprevalence of samples belonged to Brucella abortus by brucellosis was examined in Azi-kheli MRT. [14] reported seroprevalence of buffaloes of Swat (Khyber Pakhtoon Khwa) 20.3% related to the presence of Brucella using Rose Bengal Plate Test (RBPT) and infection. [15] compared the sensitivity and Serum Agglutination Test (SAT) for 109 specificity of RBPT, SAT and A-B ELISA blood samples and Milk Ring Test (MRT) techniques and revealed that the sensitivity for 100 milk samples. During the study of RBPT (88.46%) was higher as compared 9.17% brucellosis prevalence was detected to SAT (46.15%), while specificity of SAT by RBPT indicating seroprevalence and (98.31%) was slightly higher than RBPT 10.09% by SAT. However, the studies (97.75%). The overall seroprevalence of conducted by [11]. who detected antibodies brucellosis was found to be 12.7% and it against Brucella abortus in the serum increased with the age of animal. [16] who samples that ranged from 55.2-68.1% was reported 31% seropositivity in water respectively, while [12] reported the buffaloes. In the present study, the prevalence of brucellosis that ranged from 1 seroprevalence of brucellosis was recorded to 57% at different buffalo farms. In the as 10% in buffaloes by MRT. The present study, SAT was used and found to seroprevalence of brucellosis in females was be the best method in determination and recorded as 10, 11 and 10% by RBPT, SAT identification of antibody titers in the sera of and MRT respectively while none of the Azi-Kheli buffaloes effectively. The positive males were found to be positive for reactors for Brucella antibodies determined brucellosis by any of the techniques used by MRT technique showed that of the 100 during present study. These results are in samples examined, 10 were found to be concurrence with those of [17] who positive for reactors of Brucella antibodies. examined buffalo calves for prevalence of Considerable research conducted by Brucella abortus and reported that the rate different authors throughout the world on of decline of titres in heifers observed by the similar aspects, [13] isolated more than SAT was much slower than in the calves.

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Vaccination with a half dose of Brucella relatively higher prevalence and high abortus did not appear to offer any antibody titer in females. These results advantage in terms of disappearance of SAT coincide with the findings of [24] who titers. Similarly, [18] tested serum samples investigated brucellosis in buffaloes and of and reported that RBPT technique is the 250 animals tested, 2% showed positive cheaper, easier to perform, more convenient reaction. Another study carried-out by [25] and gave correct results as compared to who reported the overall prevalence of ELISA. In similar kind of investigation, [19] antibodies to Brucella abortus were 12.2 identified seropositive animals to bovine and 41.9% in individual cattle and farms, brucellosis and out of 7296 and 7169 serum respectively. True rate based on the age samples examined; the seroprevalence was seroprevalence profile was estimated at 3.2 recorded as 2.61% and 2.06% respectively. per 100 cattle years-risk. Using random [20] reported 17% of the samples were effect logistic regression model as an found positive for Brucella abortus analytical method, feeding cottonseed cake, antibodies. However, results of the present sex, source of animals and levels of exotic study are not in line to the results of the [21] blood were found to be associated with who investigated the prevalence of seropositivity to Brucella abortus. During brucellosis as 37.38%, 36.45% and 40.18% present survey, the higher prevalence of in animals positive for antibodies against brucellosis was detected in buffaloes at Brucella was higher obtained by RBPT, second lactation while lower was observed SAT and A-B ELISA respectively. at fist lactation. In scientific literature no Likewise, [22] screened-out 481 buffaloes such information was available to compare from various government farms and reported the present results to other workers because 2.91% buffaloes with brucellosis as no such kind of study has been carried out determined by RBPT at government farms, by any workers before. whereas 23.70% buffaloes infected by From the present study, it is concluded that Brucella spices at private livestock farms the brucellosis is prevailing in the female detected by SAT. In another study, on the buffaloes of the area as determined by similar aspects, [23] examined blood different techniques. However, no samples for Brucella antibodies by RBPT, brucellosis was detected in the males. The SAT and ELISA, 35.30%, 32.92% and bacterial species Brucella abortus was 39.45% buffaloes were found positive identified as the only species causing respectively. brucellosis in animals. The higher incidence Positive sera were examined to determine (11%) of brucellosis was recorded by Serum Brucella antibody titer, 08 (72.72%) and 3 Agglutination Test (SAT). The serum (27.28%) samples showed titers of 1/20 and antibody titer of Brucella abortus was also 1/40, respectively. No interaction between determined which interacted with antigen at antibody and antigen was observed at the dilutions 1:20 and 1:40, however beyond titers of 1/80 and 1/160 in both male and these dilutions, no interaction between female buffaloes and considered to be antigen and serum antibody was observed. without antibody or may having the It was also concluded that the SAT antibodies in the least numbers that did not technique is highly specific, sensitive and interact with antigen and demonstrated as relatively more accurate in determining negative for the Brucella antibodies. brucellosis in animals as compared to Rose However, it is concluded that brucellosis is Bengal Plate and Milk Ring Tests which the evident in the area with maximum and sometimes produced false results. It is also

299 Aziz et al. observed that the prevalence of brucellosis 3. Zowghi E, Ebadi A & Mohseni B (1994). was higher at second lactation as compared Isolation of Brucella organisms from milk to other lactations. Keeping in view the of seronegative cows. Revi Sci Tech Office above results, the author suggests the Inter Des Epizooties 9(4): 1175-1178. following measures to be adopted for the 4. Karimuribo ED, Ngowi HA, Swai ES & control of brucellosis in Azi-Kheli buffaloes. Kambarage DM (2007). Prevalence of The Azi-Kheli buffaloes may be serological brucellosis in crossbred and indigenous investigated further and comparisons should cattle in Tanzania, Livestock Res. Rural be made with other breeds of Swat district Develop 19(10): 203-208. for the prevalence of brucellosis and the 5. Martins HBG, Bastuji F, Lima L, Flor A, suspected animals should be separated and Fonseca P & Boinas F (2009). Eradication of bovine brucellosis in the recommended for culling and slaughter. Azores, Portugal-Outcome of a 5-year When dealing with brucellosis, the programme based on test-and-slaughter veterinarian should adopt all measures and RB51 vaccination. DRDA, Vinha regarding brucellosis so as to avoid the Brava, Angra do Heroísmo 9700-861. infection in other animals. A proper 6. Ferreira AC, Cardoso R, Travassos I, awareness to the people of the area is needed Mariano I, Belo A, Rolao I, Manteigas A, regarding brucellosis in animals and Fonseca A & Correa MI (2003). infection related risk factors to human Evaluation of a modified rose bengal test health, and its economical losses. and an indirect enzyme-linked Data availability immunosorbent assay for the diagnosis of The data used to support the findings of this Brucella melitensis infection in sheep. Vet study are included within the article. Res 34: 297–305. Authors’ contributions 7. Khan MA & Niamatullah M (2010). Conceived and designed the experiments: T Buffalo versus cattle? Let us close this Aziz & R Rhind, Performed the controversy and concentrate on improving experiments: T Aziz, A Khan & R Shah, the productivity of buffalo. Proc of the 9th Analyzed the data: T Aziz, Farmanullah & F Worl Buff Cong Argent pp. 1043-1045. Anwar, Contributed materials/ analysis/ 8. Khan MA (2009). Buffalo the animal of tools: T Aziz, S Sarwar & T Hayat, Wrote future. 1st, (Edi). Published by Idara the paper: T Aziz, R Rhind, A Khan, Matbuaat-e-Sulemani.

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