Swine Brucellosis Research Articles

Swine Brucellosis in the Kadiogo Province of Burkina Faso: Seroprevalence, Knowledge and Zoonotic Transmission Risk Behaviours among Farmers

Swine Brucellosis in the Kadiogo Province of Burkina Faso: Seroprevalence, Knowledge and Zoonotic Transmission Risk Behaviours among Farmers

Tracking sero-molecular trends of swine brucellosis in Hawai'i and the central Pacific.

Brucellosis is a zoonotic disease of mammals caused by bacterial species of the Brucella genus. The reservoir for disease is typically mammals, with species of Brucella found infecting amphibians, bats, and marine mammals. Brucella spp. can pass directly to humans through contact with infected animals or their products. Brucella spp. can cause chronic debilitating infections in mammals, including humans, and is associated with spontaneous abortions in infected animals, causing reduced fecundity. In Hawai'i, terrestrial species that could harbor Brucella spp. include swine, cattle, horses, and axis deer among others. The numerous feral swine in Hawai'i are known to carry Brucella suis, with evidence supporting infections in cattle. Brucella suis also poses infection risk to humans, dogs, and potentially horses across the state. In this study, 3,274 feral swine serum samples collected from 5 of the 8 main islands over a 15-year span were analyzed for exposure to B. suis. Of the 558 watersheds in the state, 77 were sampled as part of this effort. Spatial analysis was used to identify watersheds of concern. MLVA and whole genome SNP analysis was used for molecular epidemiological analysis. Statewide seropositivity rates were triple that of feral swine found in the conterminous United States. Smoothed positivity rates were highest on Maui, followed by O'ahu, and the island of Hawai'i. Island-by-island analysis found high brucellosis positivity levels associated with specific watersheds and agricultural areas. Local spatial autocorrelation identified hot spots on O'ahu and Hawai'i. MLVA analysis of available B. suis from Hawai'i found molecular epidemiological connections with B. suis found in French Polynesia and the mainland US while differing from those in Tonga, Western Polynesia. Strains from Hawai'i are phylogenetically closest to strains from the United States. MLVA and SNP analysis found B. suis strains from Hawai'i fell into the genetic group that contains biovar 1 B. suis. This work identified islands and watersheds of high brucellosis seropositivity in feral swine of Hawai'i, highlighting the magnitude of the zoonotic risk. Introduction of strains in recent history is unlikely due to modern animal trade and disease control practices. Genomic analysis of strains in Hawai'i and the Pacific area can provide hidden historical and local clues to brucellosis epidemiology in the state.

Serological, bacteriological, and molecular detection of brucellosis in pigs of Tamil Nadu, India

Brucellosis caused by the facultative intracellular bacteria of the genus Brucella is associated with huge economic and zoonotic implications worldwide. The disease is among the top priority zoonosis for integrated “one health” approach and has gained wide attention in developed countries. Epidemiological data on brucellosis in ruminants is readily available; however, reports on swine brucellosis remain scanty. Hence, the present study has been undertaken to assess the endemicity of brucellosis in the swine population of Tamil Nadu, India and to identify the infecting Brucella species using conventional bacteriological and molecular techniques. The apparent seroprevalence of brucellosis in pig farms were assessed by RBPT and indirect ELISA (i-ELISA) as 5.2 per cent and 10.1 per cent respectively. There was a moderate agreement between the RBPT and i-ELISA results (Cohen’s kappa coefficient (κ) = 0.512, percent agreement = 93 per cent) Six Brucella isolates could be obtained from various samples; biovar typing by biochemical and molecular methods revealed B. suis biovar 1 as the most prevalent type among swine population in Tamil Nadu. Thus, the determination of Brucella biovar in swine is an important step for epidemiologic characterization of the disease to design the control and eradication strategies.

Seroprevalence and risk factors for brucellosis and leptospirosis in swine from rural communities of Argentina

Objectives. To determine the seroprevalence of brucellosis and leptospirosis in pigs reared on small-scale farms in rural areas of the district of Tandil, Buenos Aires Province, Argentina; to analyse the associated risk factors; to describe the prevalent Leptospira spp. serogroups; and to determine the spatial distribution of both diseases. Materials and methods. A total of 340 serum samples were obtained. Buffer Plate Agglutination Test, Rose Bengal Test, and Fluorescence Polarisation Assay were used to detect antibodies against Brucella spp. Microscopic Agglutination Test was applied for serological diagnosis of leptospirosis. Statistical methods were used to evaluate the factors associated with the infections. Results. Seroprevalence of 0% and 22.6% was observed for brucellosis and leptospirosis, respectively. The most prevalent Leptospira serogroups identified were Canicola, Ballum, Icterohaemorrhagiae and Pomona. The main risk factors associated with leptospirosis infection were farms with a higher number of pigs and the presence of horses and wild boars near or within the farms. The use of milling by-products of cereal grains as animal feed and the presence of weaning cages were also associated. A relevant spatial cluster of seropositivity to Leptospira spp. was identified in a low-altitude area. Conclusions. Our results suggest that brucellosis is probably controlled, but Leptospira spp. are present in the farming systems under study. Knowledge of brucellosis and leptospirosis seroprevalence, spatial distribution and associated risk factors can be useful for the prevention and control of endemic zoonotic diseases in the region.

Disease Progression and Serological Assay Performance in Heritage Breed Pigs following Brucella suis Experimental Challenge as a Model for Naturally Infected Feral Swine.

Invasive feral swine (Sus scrofa) are one of the most important wildlife species for disease surveillance in the United States, serving as a reservoir for various diseases of concern for the health of humans and domestic animals. Brucella suis, the causative agent of swine brucellosis, is one such pathogen carried and transmitted by feral swine. Serology assays are the preferred field diagnostic for B. suis infection, as whole blood can be readily collected and antibodies are highly stable. However, serological assays frequently have lower sensitivity and specificity, and few studies have validated serological assays for B. suis in feral swine. We conducted an experimental infection of Ossabaw Island Hogs (a breed re-domesticated from feral animals) as a disease-free proxy for feral swine to (1) improve understanding of bacterial dissemination and antibody response following B. suis infection and (2) evaluate potential changes in the performance of serological diagnostic assays over the course of infection. Animals were inoculated with B. suis and serially euthanized across a 16-week period, with samples collected at the time of euthanasia. The 8% card agglutination test performed best, whereas the fluorescence polarization assay demonstrated no capacity to differentiate true positive from true negative animals. From a disease surveillance perspective, using the 8% card agglutination test in parallel with either the buffered acidified plate antigen test or the Brucella abortus/suis complement fixation test provided the best performance with the highest probability of a positive assay result. Application of these combinations of diagnostic assays for B. suis surveillance among feral swine would improve understanding of spillover risks at the national level.

Seroprevalence of Brucella Infection in Wild Boars (Sus scrofa) of Bavaria, Germany, 2019 to 2021 and Associated Genome Analysis of Five B. suis Biovar 2 Isolates

Brucella species are highly pathogenic zoonotic agents and are found in vertebrates all over the world. To date, Germany is officially declared free from brucellosis and continuous surveillance is currently limited to farm ruminants. However, porcine brucellosis, mostly caused by B. suis biovar 2, is still found in wild boars and hares. In the present study, a three-year monitoring program was conducted focusing on the wild boar population in the state of Bavaria. Serologic screening of 11,956 animals and a direct pathogen detection approach, including a subset of 681 tissue samples, was carried out. The serologic incidence was 17.9%, which is in approximate accordance with previously published results from various European countries. Applying comparative whole genome analysis, five isolated B. suis biovar 2 strains from Bavaria could be assigned to three known European genetic lineages. One isolate was closely related to another strain recovered in Germany in 2006. Concluding, porcine brucellosis is endemic in Bavaria and the wild boar population represents a reservoir for genetically distinct B. suis biovar 2 strains. However, the transmission risk of swine brucellosis to humans and farm animals is still regarded as minor due to low zoonotic potential, awareness, and biosafety measures.

Monoclonal Antibody based Blocking ELISA for Diagnosis of Brucellosis

Background: Porcine brucellosis is a contagious and emerging zoonosis but neglected in most of the endemic countries including India. The disease in pigs is rarely reported due to non-availability of diagnostics or major focus is on bovine brucellosis. Hence, the necessity was felt to diagnose porcine brucellosis by Monoclonal antibody based ELISA, RBPT and STATS for the detection of anti-Brucella antibodies and to record spatial seroprevalence of porcine brucellosis in the country. Methods: The Rose Bengal Plate Test (RBPT), Standard tube agglutination test (STAT) and Bru Alert monoclonal antibody based c-ELISA was employed to screen 148 sera samples for the presence of Brucella antibody. Result: A total 148 samples were collected and tested, the overall prevalence of Brucella infection in pig was 43.2% per cent (64/148) by C-ELISA followed by RBPT 40% (60/148) and STAT 25% (40/148) in the study area comparatively high seropositivity was found in female animals. The agreement between the two test was excellent (Kappa = 0.167) and also, chisquare test indicated an evidence of strong diagnostic weapon for detection of swine brucellosis.

Swine brucellosis in the state of Maranhão: Prevalence in slaughterhouses without sanitary inspection service

Raising subsistence pigs is common in the state of Maranhão. Most of the time, this creation is mixed with cattle, canines and birds, being represented by loose animals, fed with leftovers of human food, with precarious slaughter, without sanitary inspection, exposed to the risk of contamination with pathogenic agents. Among the diseases that can affect the swine herd, brucellosis, an infectious disease caused by bacteria of the genus Brucella, which produces chronic infection in animals, can also infect humans, one of the most important and widespread zoonoses worldwide. Given this scenario, the objective of the study was to estimate the prevalence of anti-Brucella spp. in pigs slaughtered in slaughterhouses without inspection service. 224 swine serum samples were evaluated by the Buffered Acidified Antigen (AAT) screening test and confirmed by means of 2-Mercaptoethanol (2-ME) combined with Slow Seroagglutination (SAL). In the AAT test, 30/224 (13.39%) samples presented agglutination reaction in a total of 9/12 (75%) municipalities. In the 2-ME test, 3/224 (1.34%) samples were considered inconclusive and 12/224 (5.36%) positive samples in a total of 6/12 (50%) municipalities. These results demonstrate the presence of Brucella spp. in the swine species in the state of Maranhão which, in addition to being an imminent risk to public health, could harm the main objective of the National Program for the Control and Eradication of Brucellosis and Tuberculosis in cattle and buffaloes: the eradication of the disease.

Countrywide cross-sectional study of swine brucellosis sero-prevalence in Indian subcontinent during 2018-2019.

Brucellosis in swine is a contagious disease with greater zoonotic potential caused by Brucella suis. The study describes PAN India swine brucellosis sero-prevalence in 5431 stratified random serum samples collected during 2018-2019 from 26 out of 29 states and two out of seven union territories. The serum samples were tested for anti-Brucella antibodies by indirect ELISA and overall, 4.33% apparent prevalence (AP) was recorded. The AP is ≥ 10% in five states among 26 states, P ≥ 50% in four districts out of 117 districts screened and cent percent prevalence in two epi units out of 264 sampled. Significantly high seropositivity (p < 0.05) in male (6.08%) than female pigs (3.46%) and in ≥ 24-month-old pigs indicated older and male pigs as potential carriers of the disease. The study recorded endemicity of the swine brucellosis in few regions of India requiring periodical surveillance for control of the disease. Brucella testing of boars before breeding and awareness among farmers and veterinarians will aid in reduction of disease burden in the absence of vaccination policy.

Study of Swine Brucellosis Infection Rate in the Avan Community of Aragatsotn Region

Investigations on the swine brucellosis has been conducted for the first time within 2021-2022 throughout the recent 60 years. Probably, the infection carrier small and large cattle have become the cause of brucellosis infection. Joint housing and zoohygienic conditions, as well as the remnants of infected dairy products have served as predisposing factors formorbidity. Rose-Bengal test and agglutination reaction were used to detect antibodies in blood serum. As a result of epidemiological analysis, the indicators of morbidity and insecurity rates have amounted to 0.0025 (0.25 %), 0.0075 (0.75 %), respectively, while the economic damage has made AMD 1 mln 310 thousand.

Loci Associated With Antibody Response in Feral Swine (Sus scrofa) Infected With Brucella suis.

Feral swine (Sus scrofa) are a destructive invasive species widespread throughout the United States that disrupt ecosystems, damage crops, and carry pathogens of concern for the health of domestic stock and humans including Brucella suis—the causative organism for swine brucellosis. In domestic swine, brucellosis results in reproductive failure due to abortions and infertility. Contact with infected feral swine poses spillover risks to domestic pigs as well as humans, companion animals, wildlife, and other livestock. Genetic factors influence the outcome of infectious diseases; therefore, genome wide association studies (GWAS) of differential immune responses among feral swine can provide an understanding of disease dynamics and inform management to prevent the spillover of brucellosis from feral swine to domestic pigs. We sought to identify loci associated with differential antibody responses among feral swine naturally infected with B. suis using a case-control GWAS. Tissue, serum, and genotype data (68,516 bi-allelic single nucleotide polymorphisms) collected from 47 feral swine were analyzed in this study. The 47 feral swine were culture positive for Brucella spp. Of these 47, 16 were antibody positive (cases) whereas 31 were antibody negative (controls). Single-locus GWAS were performed using efficient mixed-model association eXpedited (EMMAX) methodology with three genetic models: additive, dominant, and recessive. Eight loci associated with seroconversion were identified on chromosome 4, 8, 9, 10, 12, and 18. Subsequent bioinformatic analyses revealed nine putative candidate genes related to immune function, most notably phagocytosis and induction of an inflammatory response. Identified loci and putative candidate genes may play an important role in host immune responses to B. suis infection, characterized by a detectable bacterial presence yet a differential antibody response. Given that antibody tests are used to evaluate brucellosis infection in domestic pigs and for disease surveillance in invasive feral swine, additional studies are needed to fully understand the genetic component of the response to B. suis infection and to more effectively translate estimates of Brucella spp. antibody prevalence among feral swine to disease control management action.

Occurrence of brucellosis in pigs kept in confinement and free ranging systems in the Democratic Republic of the Congo

Swine brucellosis is a zoonosis disease affecting pigs widely. It is caused by Brucella suis whose distribution is not well documented in various areas including the Democratic Republic of the Congo (DRC). This study was initiated to determine the extent of swine brucellosis in pigs kept in confinement or in divagation in peri-urban and rural areas of Kinshasa and Kongo Central provinces of the DRC. A total of 814 samples were collected in both provinces. ELISA and Rose Bengal Test (RBT) were performed to screen all the collected samples. A prevalence of 4.42% and 3.19% of cumulative IgG seroprevalence of B. suis was obtained in both provinces sampled using ELISA and RBT, respectively. At the province level, no difference was obtained in the seroprevalence obtained in Kinshasa and Kongo Central when ELISA (3.53% and 5.38%, RR: 0.6570 p=0.20; χ2 =1.23) or RBT (2.59% and 3.85%, p=0.41; χ2 =0.66) were used, respectively. Similarly, the seroprevalence obtained in animals aged between 7-12 months and those aged more than 12 months did not show any significant difference using ELISA (3.53% and 5.54%, p=0.166; χ2 =1.9167) or RBT (3.09% and 3.32%, p=0.85; χ2 =0, 04), respectively. When animals reared in confinement or divagation systems were compared, no differences were also obtained using ELISA (6.76% versus 3.62%, p=0.058; χ2 =3.5980) or RBT (3.86% and 2.97%, p=0.525; χ2 =0.4038), respectively. However higher seroprevalence was recorded in males compared to females using ELISA (8.06% versus 3.15%, p=0.0028; χ2 =8.90) or RBT (5.68% versus 2.32%, p=0.017; χ2 =5.7256), respectively. Similarly, when animals were compared based on their breeds, higher seroprevalences were recorded in indigenous pig breed compared to improved breed ones using ELISA (15.18% and 2.7%, p=0.001; χ2 =32.65) or RBT (9.82% and 2.1%, p=0.001; χ2 =16, 04), respectively. As far as it can be ascertained, this study reports the first investigation of swine brucellosis infecting indigenous and improved pig breeds in Kinshasa and Kongo Central provinces in the DRC. The presence of brucellosis antibodies in animals in these provinces highlights a high public health concern for this major zoonotic disease not well documented in health care structures in RDC.

Development of attenuated live vaccine candidates against swine brucellosis in a non-zoonotic B. suis biovar 2 background

Brucella is a genus of gram-negative bacteria that cause brucellosis. B. abortus and B. melitensis infect domestic ruminants while B. suis (biovars 1–3) infect swine, and all these bacteria but B. suis biovar 2 are zoonotic. Live attenuated B. abortus S19 and B. melitensis Rev1 are effective vaccines in domestic ruminants, though both can infect humans. However, there is no swine brucellosis vaccine. Here, we investigated the potential use as vaccines of B. suis biovar 2 rough (R) lipopolysaccharide (LPS) mutants totally lacking O-chain (Bs2ΔwbkF) or only producing internal O-chain precursors (Bs2Δwzm) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2ΔwadB and Bs2ΔwadD). We also investigated mutants in the pyruvate phosphate dikinase (Bs2ΔppdK) and phosphoenolpyruvate carboxykinase (Bs2ΔpckA) genes encoding enzymes bridging phosphoenolpyruvate and the tricarboxylic acid cycle. When tested in the OIE mouse model at the recommended R or S vaccine doses (108 and 105 CFU, respectively), CFU/spleen of all LPS mutants were reduced with respect to the wild type and decreased faster for the R than for the S mutants. At those doses, protection against B. suis was similar for Bs2ΔwbkF, Bs2Δwzm, Bs2ΔwadB and the Rev1 control (105 CFU). As described before for B. abortus, B. suis biovar 2 carried a disabled pckA so that a double mutant Bs2ΔppdKΔpckA had the same metabolic phenotype as Bs2ΔppdK and ppdK mutation was enough to generate attenuation. At 105 CFU, Bs2ΔppdK also conferred the same protection as Rev1. As compared to other B. suis vaccine candidates described before, the mutants described here simultaneously carry irreversible deletions easy to identify as vaccine markers, lack antibiotic-resistance markers and were obtained in a non-zoonotic background. Since R vaccines should not elicit antibodies to the S-LPS and wzm mutants carry immunogenic O-chain precursors and did not improve Bs2ΔwbkF, the latter seems a better R vaccine candidate than Bs2Δwzm. However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2ΔwbkF is advantageous over Bs2ΔwadB or Bs2ΔppdK requires experiments in the natural host.

Comparison of four serological tests for the diagnosis of swine brucellosis

The purpose of this article is to present results of a research in which the results of four serological tests were compared for the diagnosis of swine brucellosis in pigs from Brazilian herds in which the outbreak occurred in relation to other brucellosis-free. Rose Bengal (RBT), complement fixation (CFT), agglutination plus 2-mercaptoethanol test (TAT+ME), and commercial fluorescence polarization assay (FPA) were used to test 333 sera (271 sows and 62 finishing pigs) from a Brucella suis infected swine herd and 1,100 swine sera from brucellosis free pigs taken at a slaughterhouse. Considering infected all the 271 sows from the outbreak, and interpreting results of the FPA according to the manufacturer directions, sensitivities observed were 95.94% for FPA, 94.83% for RBT, 93.73% for CFT, and 92.25% for TAT+ME. Considering infected all the pigs from the infected herd with a positive result in at least one test, sensitivities observed were 98.92% for RBT, 97.13% for CFT, 96.06% for FPA, and 94.98% for TAT+ME. Specificities of the tests were CFT and TAT+ME, 100%; FPA, 99.55%; and RBT 99.27%. Results indicated a good performance of all the tests, and the agreement among them was almost perfect.

Diagnosis of Yersinia enterocolitica serovar O:9 in a commercial 2400-sow farm with false-positive Brucella suis serology using western blot, competitive ELISA, bacterial isolation, and whole genome sequencing

Despite eradication of swine brucellosis from US commercial swine, Brucella suis still exists in feral swine. Therefore, brucellosis surveillance occurs to detect and eliminate any disease introduction from feral swine to domestic swine. As serology for swine brucellosis has imperfect specificity, false-positive serological reactions (FPSRs) occur and true brucellosis infection must be ruled out. In this case report, we detail a process to rule out B suis infection in a commercial sow herd using additional diagnostics including bacterial culture, whole genome sequencing, western blot, and competitive enzyme-linked immunosorbent assay. It was determined Yersinia enterocolitica serovar O:9 caused the FPSRs.

A Serological Survey of Brucellosis in Eurasian Wild Boar (Sus scrofa) in Campania Region, Italy

Swine brucellosis is a zoonotic disease that affects both domestic pigs (Sus scrofa) and wild pigs such as wild boar (Sus scrofa). We evaluated the presence of antibodies against Brucella spp. in wild boar hunted in the Campania region, Italy, during the hunting season of 2016-17. Serum samples were collected from 434 wild boars and tested for antibodies against Brucella spp. For detection of antibodies to swine brucellosis, the rose Bengal test (RBT) and a blocking enzyme-linked immunosorbent assay (ELISA) were performed. Of 434 serum samples, 22 (5.1%) tested by RBT and 58 (13.5%) tested using the blocking ELISA were positive for brucella. The seroprevalences of Brucella spp. did not differ between the sexes or age classes, while the prevalence of infected animals was positively correlated with the Avellino province. Our data showed that wild boar in the Campania region are exposed to brucella infection, and that the prevalence of the disease has increased in recent years.

Vaccine safety studies of Brucella abortus S19 and S19ΔvjbR in pregnant swine

Brucellosis in swine is caused by Brucella suis, a bacterial infection of nearly worldwide distribution. Brucella suis is also transmissible to humans, dogs and cattle and is considered a reemerging disease of public health concern. To date, there is no effective vaccine for swine. This prompted us to investigate the potential use of the commercially available vaccine for cattle or the live attenuated vaccine candidate S19ΔvjbR. As the first step, we sought to study the safety of the vaccine candidates when administered in pregnant sows, since one of the major drawbacks associated with vaccination using Live Attenuated Vaccines (LAV) is the induction of abortions when administered in pregnant animals. Fifteen pregnant gilts at mid-gestation were divided into four groups and subsequently vaccinated subcutaneously using different formulations containing 2.0 ± 0.508 × 109 CFU of either S19 or S19ΔvjbR. Vaccination in pregnant animals with the vaccine candidates did not induce abortion, stillbirths or a reduction in litter size. Multiple tissues in the gilts and piglets were examined at the time of delivery to assess bacterial colonization and histopathological changes. There was no evidence of vaccine persistence in the gilts or bacterial colonization in the fetuses. Altogether, these data suggest that both vaccine candidates are safe for use in pregnant swine. Analysis of the humoral responses, specifically anti-Brucella IgG levels measured in serum, demonstrated a robust response induced by either vaccine, but of shorter duration (4–6 weeks post-inoculation) compared to that observed in cattle or experimentally infected mice. Such a transient humoral response may prove to be beneficial in cases where the vaccine is used in eradication campaigns and in the differentiation of vaccinated from infected animals. This study provides evidence to support future efficacy studies of both vaccine candidates in swine.

Estudo epidemiológico de brucelose em suínos em criatórios de subsistência do Distrito Federal, Brasil

Brucellosis is caused by 10 species of Brucella sp. and is responsible for huge economic losses. There are no specific prophylactic measures present for household pigs, which can be also reservoirs, in the brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis. Therefore, we conducted a cross-sectional study of blood serum samples from 1793 household pigs from 823 farms in Federal District of Brazil. Serum samples were collected between August and October in 2011, 2012, and 2014 by the veterinarians under the Secretary of Agriculture. The Rose Bengal plate test (RBPT) was used for screening for antibodies against smooth Brucella sp. Positive results were confirmed by the slow agglutination test (SAT) and 2-mercaptoethanol test (2-ME). Among the 1793 samples screened with RBPT, 125 were positive for antibodies against smooth Brucella sp.; 46 (32%) of these samples showed agglutination in SAT, but only one (0.8%) sample was considered positive by 2-ME. The animal with the positive blood serum was traced back in a household farm whose owner had supplemented feeds with cow milk whey. For preventing swine brucellosis, it is necessary to prevent the cross-infection either by the consumption of contaminated cow milk or by direct contact with infected animals. The government policy should enhance the sanitary education for encouraging household farmers to employ prophylactic measures and ensure biosecurity. The present study results indicate that swine brucellosis risk in household farms occurs &nbsp;in a low prevalence rate in pigs from the Federal District of Brazil.

Phylogeography and epidemiology of Brucella suis biovar 2 in wildlife and domestic swine

Swine brucellosis due to Brucella suis biovar 2 (bv2) is enzootic in wild boar and hare in continental Europe and may cause major economic losses to the pig industry, mainly in free-ranged pig farms. The high nucleotide identity found among the B. suis biovar 2 isolates has long hindered the full understanding of the epidemiology and the phylogeography of the disease. Here, we used multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) and whole-genome analysis to identify single-nucleotide polymorphisms (SNPs) in order to gain insights from the largest B. suis bv2 dataset analyzed so far composed of domestic pigs and wildlife isolates collected throughout Europe since the 1970s. We found four major clades with a specific phylogeographic pattern. The Iberian clade contains isolates exclusively from the Iberian Peninsula. The Central European clade includes most isolates from France, Northern Italy, Switzerland and an important proportion of those of Northern Spain. The Eastern European clade clustered isolates from Croatia and Hungary mainly but also from areas of France, Germany, Italy and Poland. Finally, a separated Sardinian clade grouped three isolates from this island. At fine scale, MLVA demonstrated an endemic status of the infection in Europe and it allowed tracking a large outbreak formed by different farms from Spain linked to the same infection source. The whole genome SNP analysis showed that the strains form genetically distinct clades, shared between wild boar and pigs, in agreement with the MLVA clades. Interestingly, all hare isolates clustered together within two groups composed exclusively of wildlife isolates. Our results support the hypothesis that maintenance and spread of B. suis bv2 in Europe is a dynamic process linked to the natural expansion of wild boar as the main wild reservoir of the infection, while spread over long distances is found largely dependent on anthropogenic activities.

Patho-epidemiological and molecular diagnosis of swine brucellosis in Punjab

Brucellosis is one of the major reproductive tract diseases of swine and causes a huge economic loss to pig farmers. In this study, serological, gross pathological, histopathological, immunohistochemical and Polymerase Chain Reaction technique(s) have been applied for diagnosis of brucellosis in pig. Seroprevalence studies showed prevalence of brucellosis to be 16.7% by Rose Bengal Plate Test (RBPT) and 12.5% by competitive ELISA(c-ELISA). In this study, comparison was done between RBPT and c-ELISA for serological diagnosis of brucellosis which revealed substantial degree of agreement (Kappa= 0.676, 95% CI= 0.372-0.826) between two tests. Histopathological examination revealed focal to diffuse mixed infiltration of neutrophils or lymphocytesin the fetal organs like lung especially in bronchi and interstitial septa. Lesions in the placenta were characterized by diffuse lympho-mononuclear cell infiltration. Histopathological changes in female reproductive tracts were characterized by cellular infiltration of the endometrial stroma, endometrial glands and hyperplasia of periglandular connective tissue. Immunohistochemical (IHC) staining revealed intracytoplasmic immunostaining of Brucella in bronchial epithelial cells of lung, endothelial cells in spleen, hepatocytes of liver, cells within the lymph node, kidney of fetus and placenta. In addition, positive immunostaining to Brucella was also seen in lamina propria cells of uterus. By PCR, eight out of 34 samples (2 from stomach contents, 2 from placental cotyledons and 4 from pooled tissue(s) produced 193 bpsized ampliconspecific for Brucella genus. In the present study, PCR and IHC provide a reliable test for the diagnosis of brucellosis in abortion cases on aborted fetal tissue and placental cotyledons. However, serology is important for detection of Brucella positive animal in a farm .