A study on the epidemiology of brucellosis in bovine population of peri-urban and rural areas of district Multan, southern Punjab, Pakistan.

BACKGROUND: Brucellosis is a zoonotic disease caused by a bacterial pathogen belonging to the genus Brucella. It is one of the most frequent bacterial zoonoses globally but unfortunately, it is still considered as a neglected disease in the developing world. Keeping in view, this study was conducted to determine the prevalence and risk determinants of brucellosis in large ruminants of peri-urban and rural areas of district Multan-Pakistan. For this purpose, blood samples (n = 490) were collected from the cattle (n = 245) and buffalo (n = 245) population of the study area and subjected to preliminary screening of brucellosis using local and imported RBPT reagents. All the samples were further analyzed using commercially available multi-specie indirect ELISA kit followed by their confirmation by PCR using genus and species-specific primers. Data obtained from lab analysis and questionnaires were subjected to statistical analysis for Pearson Chi-square, Odds Ratio and Confidence intervals (95%). RESULTS: The results showed that the maximum seropositivity was recorded with local RBPT reagent (VRI, Pakistan; 12.45%; 95%CI = 9.72-15.65%) followed by RBPT-IDEXX (12.24%; 95%CI = 9.52-15.45%) and RBPT-ID.vet (11.84%; 95%CI = 9.18-14.95%) however statistical difference was non-significant (P = 0.956). The ELISA results showed an overall seroprevalence rate of 11.22% (95%CI = 8.59-14.33%) with comparatively higher rate in cattle (12.65%; 95%CI = 8.82-17.44%) as compared to buffaloes (9.80%; 95%CI = 6.49-14.15%). The PCR analysis confirmed the presence of genus Brucella in all seropositive samples whereas frequency of B. abortus and B. melitensis in seropositive samples was 80% and 20%, respectively. The co-existence of both species was also observed in 5.45% samples. The statistical analysis showed a significant association of bovine brucellosis with herd size, breed, reproductive disorders, mode of insemination, educational status and farmers' awareness about brucellosis (P < 0.05). Conversely, locality, age, weight, gender, pregnancy status, parity and puberty status had no associations with brucellosis (P > 0.05). CONCLUSION: In conclusion, brucellosis is prevalent in large ruminants of district Multan, Pakistan. It is suggested to devise and implement stringent policies for the effective control and prevention of brucellosis in the region. Further, the current situation also warrants the need to strengthen interdisciplinary coordination among veterinarians and physicians in one health perspective to ensure and strengthen the human and animal health care systems in the region.

Is it safe to use ovarian follicular fluid from cows seropositive for brucellosis in the production of in vitro embryos?

Animal reproduction biotechniques are important tools for the technological advancement of livestock, as they allow the selection of the reproductive potential of superior quality females and males; however, infectious diseases that have a predilection for the reproductive system can be a hindrance for the use of these technologies. Therefore, the present study aimed to detect Brucella spp. in the ovarian follicular fluid of brucellosis-positive bovine cows. A total of 47 bovine ovarian follicular fluid aspirates from cows, positive in tests for brucellosis and from Brucella-positive herd, were submitted to PCR. The primers used in the PCR were specific to the genus Brucella (bcsp31 gene). All 47 bovine aspirates were negative for Brucella spp. 0.00% (95% CI: 0.00-4.00%). Our results demonstrated that Brucella spp. was absent in the ovarian follicular fluid from seropositive cows, which indicates that Brucella spp.-infected cows could be used for reproductive biotechnologies carried out with follicular aspirates. Future studies are needed to more precisely evaluate the feasibility and safety of using these oocytes from brucellosis-seropositive cows to transfer embryos to heifers/cows not infected by Brucella, aiming to produce calves free of the infection.

Distribution, seroprevalence and risk factors for bovine brucellosis in Brazil: Official data, systematic review and meta-analysis.

Bovine brucellosis is an endemic disease in Brazil, and evidence-based assessments of the available literature on its seroprevalence and risk factors are limited. The aim of this study was to systematically review and summarize studies related to seroprevalence and risk factors of bovine brucellosis in the entire Brazil, in addition to comparing published data with the most recent official reports. Articles available in scientific databases and published between October 2006 and October 2021 were evaluated. Forty-five publications were included in the meta-analysis on the seroprevalence of brucellosis and 29 publications in the review on risk factors. The largest number of publications was found for the State of Mato Grosso do Sul (n=4), and the highest and lowest seroprevalences were observed in Acre (11%; 95% CI: 8.0-14.0%) and in the Federal District (0.4%; 95% CI: 0.2-0.7%). The main risk factors were the purchase of animals for breeding, vaccination, the number of heifers (female ≥2 years), the presence of calving paddocks and the occurrence of abortions. The need for new official studies has been suggested to determine the true prevalence of bovine brucellosis in Brazil, supported by the National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis.

Transcriptome and the gut microbiome analysis of the impacts of Brucella abortus oral infection in BALB/c mice.

Brucellosis is a zoonotic infectious disease caused by Brucella spp, which could cause serious economic losses to animal husbandry and threaten human public health. Ingestion of contaminated animal products is a common way to acquire Brucella infection in humans, while research on effect of oral Brucella infection on host gut microbiota and the gene expression in intestinal tissues is limited. In the present study, 16S rRNA sequencing and RNA sequencing were conducted to explore gut microbiota and expression profiles of mRNAs in the colon of BALB/c mice, which were infected by Brucella abortus 2308. The fecal samples were collected at 7 and 28 days post infection to observe changes in the gut microbiota during Brucella infection. In the alpha diversity analysis, significantly increased Chao 1 index was observed at 28 days after Brucella infection. The Bray-Curtis distancebased principal coordinate analysis indicated that the WT group showed a separation from the Brucella infection groups. In addition, analysis of composition of microbes revealed that Prevotellaceae_NK3B31_group were more abundant in 1 week and 4 week infection groups, while Turicibacter was only more abundant in 4 week infection group. Based on the RNA-seq assay, a total of 45 differentially expressed genes were detected between Brucella abortus infection group and control group. Furthermore, KEGG pathway enrichment analysis showed that protein processing in endoplasmic reticulum, Legionellosis, Spliceosome, Hippo signaling pathway and Influenza A were significantly enriched in response to Brucella abortus infection. Our finding will help to improve the knowledge of the mechanisms underlying Brucella infection and may provide novel targets for future treatment of this pathogen infection.

Economic analysis of bovine brucellosis control in the Rondônia state, Brazil.

Bovine brucellosis, mainly caused by Brucella abortus, is a worldwide distribution anthropozoonosis that causes great economic losses. In 2001, Brazil launched the National Program for the Control and Eradication of Brucellosis and Tuberculosis (PNCEBT). Contemporaneously, a great effort to characterize the epidemiology of the disease in Brazilian states was started. In the state of Rondônia, a first epidemiological study was carried out in 2004, revealing a prevalence of 35.2% of infected herds and 6.22% of seropositive females. In 2014, after a successful heifer vaccination program with strain 19 (S19), a second study detected a reduction in the prevalence of infected herds to 12.3% and of seropositive females to 1.9%. The present study aimed to quantify and compare the costs and benefits related to the control of bovine brucellosis in the state through an accounting analysis. Vaccinating heifers and performing serological tests to move animals were computed as private costs. The expenditures of the state official veterinary service for brucellosis control were considered public cost. The considered benefits of lowering prevalence were decreased cow replacement, decreased abortions, decreased perinatal and cow mortality, and increased milk production. Considering private and public costs, the net present value (NPV) was estimated at US$ 18.3 million, the internal rate of return (IRR) was calculated at 23%, and the benefit-cost ratio (BCR) was 1.7. When considering only the private costs, the NPV was US$34.9 million, the IRR was 49%, and the BCR was 3.0, meaning that the bovine producer had a return of 3 for each unit of currency invested. The results showed that the bovine brucellosis control measures implemented in the state of Rondônia, which had as its main strategy the vaccination of heifers with S19, produced highly advantageous economic results. The state should continue with its vaccination program, stimulating the use of the RB51 vaccine in addition to S19, to achieve further reductions in prevalence at low cost.

Evaluation of serological diagnostic tests for bovine brucellosis in dairy cattle herds in an endemic area: a multicenter study.

Brucellosis is known as one of the most common zoonotic diseases worldwide affecting both livestock and humans. It causes abortions, reduces milk production, and infertility in infected animals. The disease is routinely diagnosed through three serological techniques, such as rose bengal plate test (RBPT), standard agglutination test (SAT), and indirect enzyme-linked immunosorbent assay (I-ELISA). The aim of this study was to identify and compare the brucellosis seroprevalence among dairy cattle farms through these different serological tests. From 2112 sampled dairy cattle in different parts of Iran, RBPT, SAT, and I-ELISA led to 296 (14.02%), 215 (10.18%), and 297 (14.06%) positive results, respectively. Brucella abortus biovar 3 (62 cases) was identified as the most common cause of brucellosis in tested animals. Our results showed that the specificity and sensitivity of I-ELISA were higher than those obtained by RBPT and SAT. In this study, the overall agreement of RBPT and SAT with I-ELISA reached 95.21% and 94.12% in dairy cattle farms, respectively. Furthermore, Cohen's kappa statistical analysis revealed that the best degree of agreement was seen between RBPT and I-ELISA (0.80), followed by RBPT and SAT (0.78) and finally SAT and I-ELISA (0.72), thereby indicating a strong agreement between RBPT and I-ELISA methods and good agreement between SAT and I-ELISA methods. The McNemar analysis also showed that a significant difference exists between positive and negative results determined by SAT and I-ELISA methods (p < 0.0001). However, the positive and negative results determined by I-ELISA and RBPT did not show a significant difference (p = 0.9207). Therefore, I-ELISA was a more specific and sensitive serological test when compared to RBPT and SAT and could remarkably decrease non-specific reaction by improving the serological screening specificity for an accurate brucellosis diagnosis in endemic areas.

Tumor Necrosis Factor Alpha Contributes to Inflammatory Pathology in the Placenta during Brucella abortus Infection.

Research on Brucella pathogenesis has focused primarily on its ability to cause persistent intracellular infection of the mononuclear phagocyte system. At these sites, Brucella abortus evades innate immunity, which results in low-level inflammation and chronic infection of phagocytes. In contrast, the host response in the placenta during infection is characterized by severe inflammation and extensive extracellular replication of B. abortus. Despite the importance of reproductive disease caused by Brucella infection, our knowledge of the mechanisms involved in placental inflammation and abortion is limited. To understand the immune responses specifically driving placental pathology, we modeled placental B. abortus infection in pregnant mice. B. abortus infection caused an increase in the production of tumor necrosis factor alpha (TNF-α), specifically in the placenta. We found that placental expression levels of Tnfa and circulating TNF-α were dependent on the induction of endoplasmic reticulum stress and the B. abortus type IV secretion system (T4SS) effector protein VceC. Blockade of TNF-α reduced placental inflammation and improved fetal viability in mice. This work sheds light on a tissue-specific response of the placenta to B. abortus infection that may be important for bacterial transmission via abortion in the natural host species.

Development of a novel glycoprotein-based immunochromatographic test for the rapid serodiagnosis of bovine brucellosis.

AIMS: Bovine brucellosis is a worldwide zoonotic disease that causes important economic losses and public health concerns. Because control of the disease depends on vaccination, serodiagnosis and isolation of the infected animals, affordable, rapid and accurate point of care (POC) tests are needed. METHODS AND RESULTS: We developed and evaluated a novel glycoprotein-based immunochromatographic test for the detection of IgG antibodies against the O-polysaccharide of Brucella in bovine serum samples. Brucella GlycoStrip combines the power of immunochromatographic and bacterial glycoengineering technologies for the diagnosis of bovine brucellosis. The analysis of positive and negative reference samples indicated that the test has a diagnostic sensitivity and specificity of 96.9% (95% CI: 92.7%-100.0%) and 100%, respectively. CONCLUSIONS: Due to the recombinant glycoprotein-based antigen OAg-AcrA, which consists of the O-side chain of Brucella smooth lipopolysaccharide (sLPS) covalently linked to the carrier protein AcrA, the test is highly accurate, allows the differentiation of infected animals from those vaccinated with a rough strain or with a single dose of a smooth strain and fulfil the minimum diagnostic requirements established by the national and international regulations. SIGNIFICANCE AND IMPACT OF STUDY: This strip test could provide a rapid (10 min) and accurate diagnosis of bovine brucellosis in the field contributing to the control of the disease.

Intracellular Passage Triggers a Molecular Response in Brucella abortus That Increases Its Infectiousness.

Brucella abortus is a facultatively extracellular-intracellular pathogen that encounters a diversity of environments within the host cell. We report that bacteria extracted from infected cells at late stages (48 h postinfection) of the intracellular life cycle significantly increase their ability to multiply in new target cells. This increase depends on early interaction with the cell surface, since the bacteria become more adherent and penetrate more efficiently than in vitro-grown bacteria. At this late stage of infection, the bacterium locates within an autophagosome-like compartment, facing starvation and acidic conditions. At this point, the BvrR/BvrS two-component system becomes activated, and the expression of the transcriptional regulator VjbR and the type IV secretion system component VirB increases. Using bafilomycin to inhibit BvrR/BvrS activation and using specific inhibitors for VjbR and VirB, we showed that the BvrR/BvrS and VjbR systems correlate with increased interaction with new host cells, while the VirB system does not. Bacteria released from infected cells under natural conditions displayed the same phenotype as intracellular bacteria. We propose a model in which the B. abortus BvrR/BvrS system senses the transition from its replicative niche at the endoplasmic reticulum to the autophagosome-like exit compartment. This activation leads to the expression of VirB, which participates in the release of the bacterium from the cells, and an increase in VjbR expression that results in a more efficient interaction with new host cells.

Comparative analysis of the main outer membrane proteins of Brucella in the diagnosis of brucellosis.

Brucellosis has placed a heavy economic burden on numerous countries and has consumed considerable medical resources worldwide. To improve the specificity and sensitivity of serological methods for diagnosing brucellosis, it is important to develop new diagnostic antigens. Brucella outer membrane proteins(omps) possess good immunogenicity, but there is a scarcity of comparative studies of these proteins in the clinical diagnosis of brucellosis. In this study, six recombinant Brucella outer membrane proteins, omp10, omp16, omp19, omp25, omp31 and BP26, were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera of humans, bovines and goats with brucellosis were analyzed by indirect ELISA using these proteins, lipopolysaccharide(LPS) and Rose Bengale Ag, served as positive-control antigens. In diagnosing human and goat serum, BP26 exhibited the highest diagnostic accuracy of 96.45% and 95.00%, respectively, while omp31 exhibited the strongest ability to detect Brucella in bovine serum with an accuracy of 84.03%. Cross-reaction experiments also confirmed that the diagnostic specificities of omp31 and BP26 were higher than those of the LPS and Rose Bengale Ag antigens. The results of this study indicate that omp31 and BP26 are candidate antigens with high potential application value in the clinical diagnosis of brucellosis.

Epidemiological Investigation of Bovine Brucellosis in Indigenous Cattle Herds in Kasulu District of Tanzania.

Background: Livestock-wildlife interfaces create unique hotspots of many infectious diseases including brucellosis. Methods: A combination of epidemiological field studies utilizing Participatory Epidemiology tools and cross-sectional studies investigating Abortion Associated Syndrome (AAS) was conducted in livestock wildlife interface areas in Kasulu district, Tanzania from 23 to 28 July 2019. A total of 285 cattle from 27 herds were examined and sampled. Individual animal and herd-level data were collected using a structured questionnaire. Serum samples were screened for anti-Brucella antibodies using the Rose Bengal Plate Test (RBPT). Results: Ranking and proportional piling showed cattle to be the most important animal species kept but also significantly contribute to the livelihood of the livestock keepers. Matrix scoring results showed weak to moderate agreement between informant groups perception on the AAS and risk factors. The overall seroprevalence of anti-Brucella antibodies in individual animal was 30.8% (95% Confidence intervals (CI) = 25.5 - 36.2) and the corresponding herd prevalence was 77.7% (95% CI = 59.2 - 89.4). Fifty-one (37.0%) out of the 138 cows that had history of abortion over the previous 2 years (2018-2019) prior to the study was seropositive on RBPT. Univariate logistic regression analysis showed sex and age of the animals as a potential predictor for individual animal seroprevalence. Conclusions: The results showed that farmer knowledge and perception about diseases including AAS to be moderate and further confirm brucellosis to be prevalent and widely distributed locally. Heightened routine surveillance, further studies, and institution of preventive and control measures particularly among young female stock should be implemented. Creating disease awareness especially amongst livestock keepers and general public who are at high risk of contracting brucellosis is desirable.

Seroprevalence and risk factors for bovine brucellosis in the state of Paraná, Brazil: an analysis after 18 years of ongoing control measures.

Seroprevalence and risk factors of bovine brucellosis (Brucella abortus) in herds and cattle were estimated by a cross-sectional study in the state of Paraná, Brazil. The state was divided into seven regions and a random, two-stage sampling was performed on properties and cattle from each region between 2018 and 2019. Serum samples were collected from 11,592 cows over 24 months from 1,757 properties and a questionnaire was applied to identify potential risk factors. As recommended by the National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), serological testing for the detection of anti-Brucella antibodies included the buffered plate agglutination test (screening test) and the fluorescence polarization assay (confirmatory test). The seroprevalence of bovine brucellosis on properties and in cattle was 4.87% (95% confidence interval [CI]: 3.98-5.93%) and 2.24% (95% CI: 1.47-3.41%), respectively. Multiple logistic regression analysis identified larger herd size and failure to test for brucellosis as risk factors for the presence of anti-B. abortus antibodies. These results demonstrate no change in the prevalence when comparing initial studies conducted in 2002. Given our findings, it is recommended that policies for brucellosis control include a widespread vaccination program for higher prevalence areas and eradication approach to lower prevalence areas. All steps related to correct immunization of the herds should be verified and improved by training and education. Health education action must be carried out informing farmers about the risks of introducing animals not tested for brucellosis into their herds and the benefits of testing their herds regularly.

Epidemiology of bovine brucellosis in Hisar, India: identification of risk factors and assessment of knowledge, attitudes, and practices among livestock owners.

Brucellosis caused by facultative intracellular bacteria, Brucella, remains a global threat to both animal and human health. In this study we aimed to identify potential risk factors of bovine brucellosis and to assess the knowledge, attitudes, and practices (KAPs) of livestock keepers in Hisar, India. A standardized questionnaire was used to collate information regarding potential risk factors of bovine brucellosis and livestock owners' KAPs. A total of 127 livestock keepers were involved. Serum samples from their animals (n = 635) were tested for the presence of antibodies against Brucella by Rose Bengal Plate Test (RBPT) and indirect enzyme-linked immunosorbent assay (iELISA). Out of these, 78 (61.4%) of the herds had at least one seropositive animal, and 302 (47.6%) of the cattle were seropositive. Univariate and multivariate analysis revealed significant associations between intensive farm type (OR = 4.6; 95% CI, 1.6-16.7; P = 0.009), hygienic disposal of aborted fetuses (OR = 0.3; 95% CI, 0.08-0.9; P = 0.04) and herd seropositivity for brucellosis. The majority, 96 (75.6%) of the respondents, were males aged 18-50, and 82 (64.6%) owned a small-backyard farm. Only 51 (40.2%) of the participants knew about brucellosis; out of them, 54.9% (28/51) could not identify clinical signs of brucellosis. Six (11.8%) participants indicated abortion as the most noticeable clinical sign, and 45.1% indicated that consumption of raw milk is associated with high risk of contracting brucellosis. A large proportion of respondents confirmed that milk from their animals was regularly consumed (86.6%) and sold (59.8%) to other people. These results suggest that bovine brucellosis is endemic in Haryana, where Brucella-contaminated milk is likely being regularly sold. Brucellosis control efforts in Haryana should include education programs to raise awareness of the disease and means to control it in cattle and to prevent zoonotic transmission.

Brucella species circulating in rural and periurban dairy cattle farms: a comparative study in an endemic area.

Brucellosis is among the most important zoonotic infectious diseases worldwide affecting both humans and domestic animals. The present study aimed to determine and compare the seroprevalence of brucellosis among rural and periurban dairy cattle farms of four Iranian provinces from 2017 to 2019. We applied different serological tests, including RBT, SAT, and iELISA to evaluate the brucellosis prevalence among 2808 dairy cattle. Species-specific multiplex PCR and biotyping tests were also used to further identify the implicated Brucella species. Serological screening using RBT, SAT, and iELISA led to 157 (5.6%), 112 (3.9%), and 139 (4.9%) positive results among tested cattle, respectively. Brucella abortus biovars 1 (2 cases) and biovars 3 (42 cases) were identified by biotyping experiments and multiplex PCR in all 44 tested lymph node samples. Further, Cohen's kappa statistical analysis revealed that the best degree of agreement was seen between RBT and iELISA (99.4%), followed by SAT/iELISA (98.5%) and finally RBT/SAT (98.4%). Our results also showed a significantly lower seroprevalence of brucellosis in periurban dairy cattle when compared to rural dairy cattle population (p value= 0.01). These results reflect the need for better vaccine coverage using RB51 combined with an appropriate test-and-slaughter program in the rural dairy cattle population.

Effect of co-positivity for brucellosis and tuberculosis on milk yield and fertility of Holstein cows.

This study aimed to determine whether cows detected as tuberculosis (bTB) reactors and seropositive to brucellosis (bBR), as well as co-positive to bBR and bTB (bBR-bTB) and with a complete lactation before slaughter, were associated with reduced milk production and fertility. A total of 8068 productive and reproductive records of high-yielding Holstein cows from a single large dairy herd with a high prevalence of bTB and bBR were collected from 2012 to 2015. Lactation derived either from calving (n = 6019) or hormonally induced lactation (n = 2049), and all cows received growth hormone throughout lactation. For cows not induced into lactation, pregnancy rate to first service for healthy cows (C; 26.6%) was higher (P < 0.01) than bBR (15.2%), bTB (15.8%), and bBR-bTB (1.3%) cows. For induced cows, pregnancy rate to first service did not differ significantly among C, bBR, and bTB (14.5-17.3%) cows, but the percentage success of first service was extremely low (1.3%; P < 0.01) in bBR-bTB cows. Services per pregnancy (only pregnant cows) were lowest for C (3.3 ± 2.9; P < 0.01) and highest (6.4 ± 3.4) for bBR-bTB non-induced cows. This variable was lowest for C (2.9 ± 2.5; P < 0.01) and highest for bBR-bTB non-induced cows (6.3 ± 3.1). Pregnancy rate to all services did not differed for C (79.5%), bBR (76.7%), and bTB (75.9%) but was lower (58.9%; P < 0.01) for bBR-bTB non-induced cows. For induced cows this variable was highest for bBR (53.3%) and lowest for bBR-bTB (34.1%; P < 0.01) non-induced cows. 305-d milk production was increased by 4%, and total milk yield by 7% in TB-positive cows compared to that of the negative cows non-induced hormonally into lactation. This study showed the negative impact of the co-positivity for bTB and bBR on the reproductive efficiency of Holstein cows, although positive bTB and bBR tests enhanced milk yield.

Immunoproteomics of Brucella abortus reveals potential of recombinant antigens for discriminating vaccinated from naturally infected cattle.

Brucellosis serodiagnosis is still a challenge and vaccination is the main measure used to control bovine brucellosis, being S19 and RB51 the most currently used vaccines. So, in order to contribute to brucellosis control, a bidimensional (2D) immunoblot-based approach was used to find immunogenic proteins to be used in serodiagnosis, particularly with ability to be employed in DIVA (Differentiating Infected from Vaccinated Animals) strategy. Immunoproteomic profile of Brucella abortus 2308 was analyzed in 2D western blotting using pooled sera from S19 vaccinated animals, RB51 vaccinated animals, B. abortus naturally infected animals and non-vaccinated seronegative animals. Evaluation of the antigens differentially immunoreactive against the groups of sera showed three proteins of particular importance: MDH (malate dehydrogenase) immunoreactive for S19-vaccinated animals, SOD (superoxide dismutase) reactive for infected animals and ABC transporter (multispecies sugar ABC transporter) reactive against sera from vaccinated animals (S19 and RB51). These three proteins were produced in E. coli and tested in an indirect ELISA (I-ELISA). For MDH, comparison between the vaccinated animals (independent of the vaccine used) and the seropositive and seronegative animals in I-ELISA showed significant differences. Data on the I-ELISA using SOD showed that sera from non-vaccinated naturally infected animals exhibited significant difference in comparison with all other groups. Otherwise, sera from vaccinated animals (S19 and RB51) and from non-vaccinated naturally infected animals did not show significant difference in OD values, but they were all significant different from non-vaccinated seronegative animals using ABC transporter as antigen in I-ELISA. In conclusion, together the 2D western blot analysis and the preliminary I-ELISA results suggest that the combined use of MDH and SOD could be successful employed in a LPS-free protein based serodiagnosis approach to detect bovine brucellosis and to discriminate vaccinated from naturally infected animals, in early post-vaccination stages.

Brucella melitensis B115-based ELISA to unravel false positive serologic reactions in bovine brucellosis: a field study.

BACKGROUND: Brucellosis is a zoonosis whose incidence is not declining worldwide despite the global effort to control the disease. Accurate and precise diagnosis is a crucial step in any prophylaxis program but single tests to unequivocally detect animals infected with Brucella spp. are currently unavailable. In Italy, serological diagnosis of bovine brucellosis is performed with two official tests: a rapid agglutination test (i.e., Rose Bengal Plate test, RBPT) and a complement fixation test (CFT) that detect antibodies directed mainly to the smooth lipopolysaccharide (S-LPS). Neither of the two tests is able to avoid the detection of false positive serological reactions (FPSRs) caused by bacteria sharing S-LPS components with Brucella spp. and responsible for the single reactors (SR) phenomenon. A B. melitensis R strain-based ELISA showed a good diagnostic performance in unravelling FP animals; however, since a limited number of animals were analyzed in that study, a large field study was conducted here to discriminate between Brucella-infected from FP animals, with the final aim of reducing the unnecessary slaughter of the latter. An ELISA based on a R strain of Brucella, i.e., Brucella melitensis B115, was employed to measure specific IgG responses in a collection of bovine sera (n = 648). Sera were obtained from 180 farms (either officially brucellosis-free or not brucellosis-free) recruited during an extended period of time (2014-2018) and were preliminarily assayed with the official tests by the Italian Reference Centers and then subjected to the ELISA. RESULTS: Negative sera, when subjected to the ELISA, gave O.D. values below the cutoff; SR sera, i.e. RBPT positive and CFT negative, as well as double positive (DP) sera, i.e. RBPT and CFT positive, gave O.D. values that were below the cutoff. All positive sera, i.e. from Brucella-infected animals, were RBPT positive and CFT positive (ICFTU ranging from 20 to 1280) and gave ELISA O.D. values above the cutoff. CONCLUSIONS: The B. melitensis B115-based ELISA systematically unravelled all false positive (FP) sera while confirming the diagnosis in Brucella-infected animals. Thus, the test employed in the present study may complement the official assays to avoid the costly slaughter of FP animals.

Differential expression of cytokines in PBMC of Bos indicus and Bos taurus × Bos indicus cattle due to Brucella abortus S19 antigen.

Brucellosis is the most dreadful disease among bovines, although breed differences have been observed in prevalence of disease, worldwide. In present study, antibody response and relative expression of proinflammatory cytokines was compared in Bos indicus (zebu) and Bos taurus × Bos indicus (crossbred) cattle vaccinated by live attenuated Brucella abortus S19 antigen. Six female calves (4-6 months age) of both groups were vaccinated with B.abortus S19 strain. Blood samples were collected before vaccination (0d) and 7th (7d), 14th (14d) and 28th (28d) days after vaccination. Indirect ELISA showed high (p < .05) anti-Brucella antibody level after vaccination; with no significant difference between the groups. During Real-time expression, IFNγ, TNFα, IL6 and IL10 genes initially showed down regulation followed by upregulation in both the groups; however, the trend was much prominent in crossbreds. The expressions of IFNγ, TNFα and IL6, proinflammatory molecules important for initial containment of the Brucella were significantly (p < .01) higher in crossbred. The study showed that the Sahiwal cattle were less responsive to B.abortus S19 antigen than crossbreds, indicating its lower sensitivity to the Brucella, comparatively. In contrary, higher expression of the proinflammatory molecules in crossbreds could be important for containment of the organism during initial stage of infection.

A retrospective sero-epidemiological survey of bovine brucellosis on commercial and communal farming systems in Namibia from 2004 to 2018.

Cattle production is the major livestock production activity and the mainstay of Namibia's economy. Sustained beef exports are contingent on a sound sanitary environment where diseases such as brucellosis are under control. In this retrospective study, 49,718 bovine brucellosis testing results from 2004 to 2018 were analyzed to determine the proportion of sero-positive cattle and herds, and the spatial distribution of positive reactors from commercial and communal areas. In total, 244 positive reactors were identified based on the Rose Bengal Test (RBT) and the Complement Fixation Test (CFT) in series, giving an overall proportion of infected animals of 0.49% (244/49,718; 95% CI, 0.43-0.56%) and an overall proportion of infected herds of 9.26% (78/842; 95% CI, 7.49-11.41%). There was a higher proportion of sero-positive communal herds (33.09%) and cattle (10.27%) than commercial herds (4.67%) and cattle (0.24%; p < 0.05). Annually, the proportion of positive reactors was 0-1.37% in the commercial area and 0-52.38% in the communal areas, with a clear decline in positive reactors in the communal areas. Within the commercial sector, the proportion of positive reactor dairy, beef, and export cattle was 0.19% (51/27,067; 95% CI, 0.14-0.25%), 0.30% (48/16,098; 95% CI, 0.22-0.40%), and 0.33% (16/4811; 95% CI, 0.20-0.54%), respectively. Abortions were found to be the major reason for Brucella testing in the communal areas. About 12.65% (96/759) of abortion-linked sera tested positive in the communal areas, but none were positive in beef or dairy cattle. Widespread vaccination of cattle and robust planned surveillance is recommended to reduce the incidence of the disease, its associated production losses and public health risk.

Brucellosis in India: results of a collaborative workshop to define One Health priorities.

Brucellosis is an important zoonosis worldwide. In livestock, it frequently causes chronic disease with reproductive failures that contribute to production losses, and in humans, it causes an often-chronic febrile illness that is frequently underdiagnosed in many low- and middle-income countries, including India. India has one of the largest ruminant populations in the world, and brucellosis is endemic in the country in both humans and animals. In November 2017, the International Livestock Research Institute invited experts from government, national research institutes, universities, and different international organizations to a one-day meeting to set priorities towards a "One Health" control strategy for brucellosis in India. Using a risk prioritization exercise followed by discussions, the meeting agreed on the following priorities: collaboration (transboundary and transdisciplinary); collection of more epidemiological evidence in humans, cattle, and in small ruminants (which have been neglected in past research); Economic impact studies, including cost effectiveness of control programmes; livestock vaccination, including national facilities for securing vaccines for the cattle population; management of infected animals (with the ban on bovine slaughter, alternatives such as sanctuaries must be explored); laboratory capacities and diagnostics (quality must be assured and better rapid tests developed); and increased awareness, making farmers, health workers, and the general public more aware of risks of brucellosis and zoonoses in general. Overall, the meeting participants agreed that brucellosis control will be challenging in India, but with collaboration to address the priority areas listed here, it could be possible.