Detection of Chlamydophila abortus in ovine milk by immunomagnetic separation-polymerase chain reaction

The present study was carried out to investigate the presence of Chlamydophila abortus, the causative agent of ovine enzootic abortion, in milk samples collected from sheep flocks with and without the history of abortion in Eastern Turkey by means of immunomagnetic separation (IMS) in conjunction with the polymerase chain reaction (PCR). A total number of 201 milk samples collected from 10 flocks with abortion and four flocks without abortion were tested. In the analysis of the milk samples by IMS-PCR, correct amplification was obtained with three (1.5%) samples originating from one flock with abortion. In the digestion of PCR positive products by AluI, restriction profiles observed in all three samples were determined to be the same as C. abortus S26/3.     

Role of Chlamydophila abortus in ovine and caprine abortion in Sardinia,Italy

Between 1999–2003, 14 321 sera and 646 abortion samples (498 foetuses and 148 placentae) were analysed from 807 sheep and goat farms distributed all over the island of Sardinia. After notification of abortion in a flock, sera collected at random from adult animals were examined to detect antibodies specific to Chlamydophila (C.) abortus by ELISA, whereas foetuses and placenta were analysed by PCR assay. Specific IgG antibodies were detected in 611 (4.8%) sheep and 106 (5.8%) goats. From a total of 2050 ovine and 151 caprine fetal samples including muscle, liver, abomasum, spleen, brain and placenta, 29 (1.4%) ovine and 1 (0.6%) caprine samples were C. abortus PCR-positive. Placenta was the tissue with the highest detection rate. These results indicate that the seroprevalence of C. abortus infection in sheep and goats is very low in Sardinia, and PCR results demonstrate that C. abortus has no significant role in abortion, especially in goats.     

Detection of Chlamydophila abortus in sheep and goat flocks in southern Italy by PCR using four different primer sets

An epidemiological survey was performed to detect the presence of Chlamydophila (C.) abortus and other members of the order Chlamydiales in ovine and caprine flocks with a history of abortion in southern Italy. Four pairs of primers were compared to evaluate their ability to detect Chlamydiales using purified DNA preparations and tissue samples from aborted foetuses with suspected chlamydial infections. As expected, amplification of DNA of the reference strain C. abortus using primer pairs U23F/23Sigr, 16SF2/23R, CTU/CTL and CpsiA/CpsiB produced fragments of about 600 bp, 585 bp, 1000 bp and 300 bp, respectively. The detection limits of the four PCR tests performed on serial DNA dilutions of the C. abortus reference strain were of 10 pg, 0.1 pg, 0.1 pg and 1 fg of DNA, respectively. The most sensitive amplification of DNA extracted from the organ tissues was obtained with primer pairs CpsiA/CpsiB, which detected Chlamydophila spp. DNA in all infected tissue samples. Only C. abortus was identified during the survey. The presence of this agent was confirmed in 3 out of 27 ovine and caprine flocks included in the survey suggesting that abortion due to C. abortus is uncommon in southern Italy.     

Prevalence and molecular identification of Chlamydia abortus in commercial dairy goat farms in a hot region in Mexico

The aim of this study was to determine the seroprevalence and presence of Chlamydia abortus in Saanen breed female goats from commercial dairy goat farms under intensive production in the municipality of Guanajuato, Mexico. Sera were collected to determine the prevalence of anti-C. abortus IgG antibodies using recombinant enzyme-linked immunosorbent assay (rELISA) and cell culture. Polymerase chain reaction (PCR) was used to prove the presence of the pathogen in swab samples collected from the vagina and rectum of selected animals. Additionally, foetal tissue samples from a sudden abortion were collected. C. abortus prevalence in female goats of commercial milking farms sampled in Guanajuato, Mexico, was 4.87 % (n?=?246). Seropositive animals were found in six out of nine (66.6 %) dairy goat farms sampled, and prevalence among animals in individual farms ranged between 3.44 and 13.51 %. C. abortus was detected using PCR in spleen tissue from the aborted foetus. PCR-based detection, as well as isolation from vaginal and rectal swabs, was not possible in the present study. Isolation through cell culture was also unsuccessful from aborted foetal tissue samples. In conclusion, the results from rELISA and PCR show that C. abortus is present in dairy goat farms in the state of Guanajuato, Mexico.     

Ovine and caprine toxoplasmosis (Toxoplasma gondii) in aborted animals in Jordanian goat and sheep flocks

Two hundred and fifty-five biological samples (106 aborted foetal tissue samples and 149 blood samples from aborted sheep and goats) were collected from 188 animals during the lambing season from September 2009 to April 2010 from the Mafraq region of Jordan. The sampled animals belonged to 93 goat and sheep flocks that had cases of abortion. A total of 169 (66.3%) biological samples were collected from sheep and 86 (33.7%) from goats. Seventy-six (29.8%) biological samples (45 blood and 31 tissue samples) were positive for Toxoplasma gondii by PCR assay. The positive samples were obtained from 43 sheep and 23 goats. The overall toxoplasma-specific prevalence rate was 35.1% (66/188). Forty flocks (43%) had at least one T. gondii PCR-positive animal. The risk factors related to flock health status and farm management that are hypothesized to be associated with T. gondii PCR positivity were also assessed using multiple logistic regressions. The presence of cats (OR = 4.74), a large flock size (OR = 2.76) and the method of disposing the aborted foetuses (OR = 3.77) were all statistically significant (P < 0.05) risk factors that were positively associated with toxoplasma positivity in goat and sheep flocks.     

Isolation and sero-genomo-epidemiological studies on Brucella infection in dairy cattle in Meghalaya,India

In this study, we report the serological, bacteriological and whole genome sequencing data of a 6 years study of Brucella abortus in Meghalaya, India. Investigation of 3060 sera samples indicated overall prevalence of 6.4% by Rose Bengal Plate Test and 10.7% by ELISA. Considerably higher prevalence was observed among milk samples (17.5%, n = 362) and in blood samples (37.7%, n = 262) by direct PCR. Clinical samples (n = 94) from late abortion cases yielded 11 B. abortus isolates. Multi-locus sequence typing indicated circulation of single sequence type, ST1. Whole genome sequencing (n = 8) and phylogenomic analysis revealed close clustering of majority of isolates in two clusters alongwith genomes from other countries, indicating global relatedness among B. abortus. Taken together, the results of our study revealed the putative hotspot of infection in the dairy-dominant districts of the state and also calls for concerted One Health based action for prevention and control of this zoonotic disease.     

Brucella abortus is Prevalent in Both Humans and Animals in Bangladesh

To determine the role of different Brucella (B.) spp. in Bangladesh, 62 animal samples and 500 human sera were tested. Animal samples from cattle, goats and sheep (including milk, bull semen, vaginal swabs and placentas) were cultured for Brucella spp. Three test‐positive human sera and all animal samples were screened by Brucella genus‐specific real‐time PCR (RT‐PCR), and positive samples were then tested by IS711 RT‐PCR to detect B. abortus and B. melitensis DNA. Only B. abortus DNA was amplified from 13 human and six animal samples. This is the first report describing B. abortus as the aetiological agent of brucellosis in occupationally exposed humans in Bangladesh. Of note is failure to detect B. melitensis DNA, the species most often associated with human brucellosis worldwide. Further studies are required to explore the occurrence of Brucella melitensis in Bangladesh.     

Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR

Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell’s serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n?=?5), aborted fetuses (n?=?13), and vaginal swabs (n?=?12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.     

High prevalence and risk factors of Coxiella burnetii in milk of dairy animals with a history of abortion in Iran

Coxiella burnetii is causative agent of Q fever, which is a public health problem in most countries. The aim of this study was to study the prevalence rate of C. burnetii in raw milk of dairy animals in Iran with previous history of abortion. In this survey, milk samples were collected from different dairy animals with history of abortion from Qom province (center of Iran). Samples were tested by Nested PCR and Real-time PCR for detection of IS1111 gene of C. burnetii. In total, 34.92% (44 of 126) milk samples were positive for C. burnetii. Prevalence of C. burnetii in cattle, sheep and goat milk was 33.33%, 35.71% and 35.71%, respectively. Age was a significant risk factor for shedding of C. burnetii in cattle (P = 0.02) and goat (P = 0.05). Shedding of C. burnetii was high prevalence in milk of dairy animals with history of abortion in Iran. The high prevalence of this bacterium in milk (especially in animals with history of abortion) indicates that Excreted by milk as a potential source to spread of infection in the environment.     

Diagnosis by culture and PCR of Salmonella Abortusovis infection under clinical conditions in aborting sheep in Switzerland

Since 2003 eleven Swiss sheep flocks were affected by abortion storms due to Salmonella abortusovis, an infection which had not been reported in this country for decades although cases of salmonellosis are notifiable in Switzerland. This raised doubts about the adequacy of the currently used diagnostic tools and the origin of this infection. Therefore, PCR was tested for its potential as a more rapid and more reliable method for diagnosing S. abortusovis infections under field conditions. Fecal and vaginal samples were collected at different times after abortion and PCR was used to detect bacterial DNA. Bacteria were isolated by conventional culture techniques. For determining their origin they were analyzed by pulsed field gel electrophoresis (PFGE) and compared to isolates from Germany and France. Sequencing of randomly selected amplicons allowed confirming the specificity of the result. PCR was more sensitive because it allowed detecting S. abortusovis DNA up to three months after infection even in samples that were negative by culture. Escherichia coli from the digestive tract of sheep could inhibit the growth of S. abortusovis in vitro suggesting that the lower sensitivity of diagnosis by bacterial culture may in part be due to growth inhibition of S. abortusovis by resident bacteria. Results of PFGE indicated that the Swiss strains were closely related among themselves but distinct from German and French strains suggesting the presence of an autochthonous infection.     

Toxoplasma gondii and Chlamydophila abortus in Caprine Abortions in Tobago: a Sero‐Epidemiological Study

A sero‐epidemiological study was conducted on a goat farm that experienced an abortion epidemic in the 2005 breeding season in Tobago. Serum samples of goats (aborting and non‐aborting) and cats were collected, in addition to the use of stored sera from the farm sampled in 2003 and 2004. Farm records on the reproductive and mortality rates for year 2003, 2004 and 2005 were also reviewed. The sera were screened for Toxoplasma gondii antibodies using the latex agglutination test (LAT), Chlamydophila abortus with an enzyme‐linked immunosorbent assay (ELISA) and Brucella abortus using the buffered plate agglutination test (BPAT). Farm records revealed that for the period 2003–2005, the average kid per doe rate decreased from 2.1 to 1.5, the mortality rate increased from 6.3% in 2002 to 19.4% in 2004 and the fertility rate decreased from 98–99% (2002–2004) to 89% (2005). There was a dramatic increase in the abortion rate from <1% (2002, 2003 and 2004) to 29.2% (2005). Of a total of 161 sera tested comprising 12 from 2003, 89 from 2004 and 70 from 2005, 0 (0.0%), 21 (23.6%) and 45 (64.3%) were positive for T. gondii agglutinins (i.e. titres ≥1 : 64) and the differences were statistically significant (P < 0.05; χ²). Of all serum samples tested, only 1 (1.1%) of 89 from 2004 was positive for C. abortus while all the sera tested were negative for B. abortus. Amongst the 24 does which aborted in 2005 and were available for testing in mid‐2005, 15 (62.5%) had reciprocal titres of ≥1 : 2048, three (12.5%) each had titres of 1 : 1024, 1 : 256 and ≤1 : 16 i.e. negative. The seroprevalence and titres of does that aborted, 20 (87.0%) of 23, all with titres ≥1 : 256 suggesting current infection, were statistically significantly (P < 0.05; χ²) higher than was detected amongst does that delivered normal kids, 25 (53.25) of 47 with 22 (48.8%) having titres of ≥1 : 256. One (50.0%) of two cats caught and tested was seropositive with a reciprocal titre of 128. This is considered the first documentation of T. gondii agglutinins in caprine abortion as well the detection of C. abortus antibodies from livestock in Trinidad. It is concluded that of the three zoonotic abortifacient pathogens tested for, T. gondii appeared to have played some aetiological role in the abortion epidemic investigated.     

Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle

Background: Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level.

Objective: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle.

Methods: LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR).

Results: The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye.

Conclusion: The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.     

Letters to the Editor

Extract

Sir.—There has been some correspondence in the Velerinary Record recently concerning brucella abortion breakdowns in heifers vaccinated with Strain 19 Brucella abortus. This year we have been alarmed by the sudden occurrence of abortion storms in vaccinated heifers. Brucella abortus has been seen in large numbers in direct smears taken from cotyledons and foetal stomach contents stained by the modified Z-N technique, both in our own practice laboratory and at the Department of Agriculture laboratory. To remove any possible doubt as to the identity of the organism, it has been cultured. Blood samples have been taken from the heifers concerned and sent to the Department of Agriculture laboratory where agglutination titres to Br. abortus were observed at dilutions of up to 1 in 1,280. Our practice here serves 550 dairy farms in one of the most closely stocked areas in New, Zealand. In 18 herds more than 25% of the heifers have aborted owing to brucellosis.     

Chlamydiaceae in riverine buffalo (Bubalus bubalis) and cows (Bos taurus) in Egypt with and without signs of reproductive disease

Abstract

AIMS: To obtain information and compare the prevalence of Chlamydiaceae in riverine buffalo (Bubalus bubalis) and cows (Bos taurus) in Egypt with and without clinical signs of reproductive disease.

METHODS: Vaginal swabs and blood samples were collected from animals attending Governmental Veterinary Clinics without (buffalo n=39, cows n=20) and with (buffalo n=63, cows n=53) signs of reproductive disease. Serum samples were tested for antibodies to Chlamydiaceae using complement fixation testing (CFT). Vaginal swabs were tested for Chlamydiaceae following inoculation into Vero cells and 6-day-old embryonated chicken eggs, using modified Giménez and immunoperoxidase staining, PCR analyses targeting the omp2 gene, and Restriction Fragment Length Polymorphism PCR (RFLP-PCR) for species identification.

RESULTS: Antibodies to Chlamydiaceae were detected in 30/39 (77%) and 50/63 (79%) buffalo without and with signs of reproductive disease, respectively, and 10/20 (50%) and 39/53 (74%) of cows with and without signs of reproductive disease, respectively. Positive samples from PCR analysis were identified in 31/39 (79%) and 37/63 (59%) buffalo without and with signs of reproductive disease, respectively, and 12/20 (60%) and 46/53 (89%) of cows without and with signs of reproductive disease, respectively. Using RFLP-PCR, 57/68 (84%) of samples from buffalo, and 47/58 (81%) from cows, were identified as Chlamydophila psittaci and the reminder as Cp. abortus. From the CFT and PCR results there was no significant difference in the prevalence of positive samples between species, or between animals without or with signs of reproductive disease.

CONCLUSION: The presence of anti-Chlamydiaceae antibodies in 77% of the animals with signs of reproductive disease and the detection of Chlamydiaceae in 72% of vaginal swabs of the animals suggest a pathogenic role by Chlamydiaceae in riverine buffalo and cows. The main Chlamydiaceae found in the genital tract of cattle in Egypt were Cp. psittaci and Cp. abortus.

CLINICAL RELEVANCE: Chlamydophila spp. should be included in diagnostic algorithms for reproductive disorders, in order to assess the real burden of Chlamydophila associated disease in buffalo and cattle and to evaluate the potential value of vaccines.     

THE EFFECT OF CHALLENGE WITH VIRULENT BRUCELLA ABORTUS ON BEEF CATTLE VACCINATED AS CALVES OR ADULTS WITH EITHER BRUCELLA ABORTUS STRAIN 19 OR 45/20

SUMMARY Groups of female calves were vaccinated subcutaneously with the standard dose of Brucella abortus strain 19 (S19) or with B. abortus 45/20 (S45/20). These calves and non-vaccinated control calves were mated at 15 months of age and challenged by way of the conjunctival sac with B. abortus strain 544 (S544). The incidence of abortion, stillbirths, weakling calves and healthy calves was observed after challenge and specimens were collected for culture at parturition and slaughter. Fifteen healthy calves were born to 18 animals vaccinated with S19, 12 were born to 18 animals vaccinated with S45/20 and 2 were born to 8 animals that were not vaccinated. B. abortus was isolated from 5 of the animals vaccinated with S19, 13 of the animals vaccinated with S45/20 and 9 of the 12 animals that were not vaccinated. Only one of the 5 infected animals vaccinated with S19 was vaccinated as an adult.     

Molecular identification of chlamydial cause of abortion in small ruminants in Jordan

Chlamydophila abortus (Ch. abortus) is the etiological agent of ovine enzootic abortion (OEA) and one of the most common infectious agents of abortion in small ruminants worldwide. RFLP-PCR analysis of the outer membrane protein gene (OMP2 gene) was used for diagnosis and characterization of chlamydial causes of abortion in small ruminants in Jordan. Sixty-six placental tissues and 15 vaginal swabs were collected from aborted ewes and does to identify cause of abortion in Jordan. Thirty-eight placental samples (58 %) and 13 vaginal swabs (87 %) were positive for chlamydial DNA. Shedding of bacteria in vaginal swabs was detected within 7 days after abortion. The results of this study showed that chlamydiosis is one of the important causes of abortion in small ruminants in Jordan. In addition, vaginal swab is an excellent sample for molecular diagnosis of chlamydiosis. DNA sequencing and RFLP analysis of the OMP2 reveal that all chlamydial cause of abortion in small ruminants in Jordan are due to Ch. abortus. While, Ch. pecorum was not detected in any sample. OMP2 gene of the isolated Jordanian strain was identical (100 %) to Ch. abortus FAS strain. In conclusion, Ch. abortus is an important cause of abortion in Jordan; vaginal swab within 7 days of abortion can be used for molecular diagnosis of chlamydiosis in small ruminants.     

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiolog y of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep.

METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp.

RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected.

CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks.     

Outbreak of Brucellosis in Domestic Elk in Korea

Seven of 18 elk on a deer farm were found by the official Rose‐Bengal agglutination test (RBT) and tube agglutination test to be brucellosis reactors/suspects. Evaluation with the competitive ELISA (C‐ELISA) and the fluorescence polarization assay (FPA) tests revealed that six and five sera were positive respectively. The seven reactors/ suspects were slaughtered and their blood and tissues were collected. Brucella species could be isolated from three of the slaughtered animals, with nine isolates being obtained from the popliteal, supramammary and submandibular lymph nodes, vaginal discharge, mammary tissue and spleen. Brucella genus‐specific PCR based on 16S rRNA and AMOS‐PCR, which is specific for differential Brucella species, revealed that all nine isolates were Brucella abortus. These nine were further confirmed to be B. abortus biovar 1 by classical biotyping scheme assays. This is the first report of an outbreak of brucellosis in domestic elk in Korea. Our observations suggest that deer should be included in the routine Brucella surveillance programme for the effective control and prevention of brucellosis in Korea.     

Monitoring clinical outcomes,pathological changes and shedding of Chlamydophila abortus following experimental challenge of periparturient ewes utilizing the natural route of infection

Enzootic abortion of ewes (EAE) caused by Chlamydophila abortus is an important disease resulting in significant lamb loss in most sheep producing countries. Ewes are considered to be naturally infected with C. abortus via the oral–nasal route and may become persistent carriers, shedding during subsequent oestrous cycles and at lambing. The aim of this study was to monitor the clinical outcomes, pathological changes and shedding of C. abortus in 18 periparturient orally infected sheep for two breeding seasons. In the first season, C. abortus was detected by real-time PCR (rt-PCR) in 13/18 conjunctival swabs at oestrus. Three out of the 15 pregnant ewes gave birth to 1 live and 1 dead lamb, and 2 of them aborted. Following parturition/abortion, C. abortus was detected in 12/15 vaginal swabs and in all the collected foetal membranes. However, only those membranes containing high copy numbers of the bacterium displayed the EAE typical lesions. In the second season, none of the 13 pregnant ewes aborted, and 5 of them gave birth to dead or weak lambs. C. abortus was not detected in conjunctival or vaginal swabs at oestrus or parturition. The bacterium was detected at low levels in 36% of the foetal membranes, but with no evidence of histopathological lesions. These results indicate that C. abortus can be detected in a large proportion of animals during the first pregnancy after oral infection. However, this proportion is reduced at the subsequent breeding season, confirming the occurrence of a chronic low level persistent infection in post-abortion/lambing ewes.     

A mixed methods study of ruminant brucellosis in central-eastern Tunisia

In this study, we conducted an investigation to determine the true prevalence of bovine and ovine brucellosis in central-eastern Tunisia. A total of 1134 veterinary samples taken from 130 ruminant herds were screened for brucellosis using IS711-based real-time PCR assay. Sera collected from the ruminants were tested using the Rose Bengal test and indirect enzyme-linked immunosorbent assay. Based on serological and molecular results, the true adjusted animal population level prevalence was 23.5 % in cattle, against 13.5 % in sheep. In addition, the true adjusted herd level prevalence of brucellosis was 55.6 % in cattle and 21.8 % in sheep. A statistically significant association was found between vaginal and milk shedding for ruminants. In addition, our results showed that Brucella abortus could be responsible for bovine and ovine brucellosis. Multivariable logistic regression analysis at the animal population level indicated that age and origin variables were important risk factors for cattle. However, age and abortion variables were found to be associated with ovine brucellosis. At the herd level, risk factors for Brucella positivity were as follows: abortion and herd composition for cattle against herd composition, mortality rates, and hygiene for sheep. Animal hygiene, food quality, and sanitary practices on the farm should be applied as strategies to control brucellosis in herds.