Molecular epidemiology of brucellosis in Asia: insights from genotyping analyses.

Brucellosis infects humans and animals worldwide but is particularly prevalent in Asia. In many Asian countries, molecular diagnostic tools for accurate molecular diagnostics and molecular epidemiology are lacking. Nonetheless, some countries have conducted in-depth molecular epidemiological studies. The objective of this study was to reveal the genetic relationships, geographic origins, and distributions of Brucella strains across Asia for two primary species, B. abortus and B. melitensis. For this, we systematically searched genotyping data from published studies on the molecular epidemiology of Brucella species for both humans and livestock in Asia. We used data from multilocus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing analysis of Brucella strains. We also analyzed the MLVA genotypes of 129 B. abortus isolates and 242 B. melitensis isolates with known origins in Asia from an online MLVA database using MLVA-11 data in minimum spanning trees and MLVA-16 data in neighbor-joining trees. We found that the B. melitensis East Mediterranean lineage is predominant across the continent, with only a small number of samples from the Africa and Americas lineages, and none from the West Mediterranean lineage. The "abortus C" genotype was the most common group of B. abortus in Asia, with limited genetic variation for this species. Several studies also reported that Near Eastern countries frequently encounter human brucellosis cases of B. abortus from genotypes 42 and 43. Our study highlights the inconsistent collection of genetic data for Brucella species across Asia and a need for more extensive sampling in most countries. Finally, a consistent nomenclature is necessary to define various groupings of strains within a lineage (i.e., clade) so uniform terminology should denote particular genetic groups that are understood by all researchers.

Epidemiological Investigation of Animal Brucellosis in Domestic Ruminants in Greece from 2015 to 2022 and Genetic Characterization of Prevalent Strains.

Brucellosis is one of the most important zoonotic diseases in Greece, causing a significant burden on both human and animal vitality as well as economic loss. The present study was conducted from 2015 to 2022 on 711,415 serum samples by determining the seroepidemiology of Brucellosis among livestock in 24 geographical areas in Greece using the Rose Bengal Test (RBT) and the complement fixation test (CFT) and further performing genetic analysis of Brucella spp. by species-specific real-time PCR and MLVA Brucella analysis. A total of 3086 serum samples from goats, sheep, and cattle showed positive results using the RBT and CFT, and only strongly positive samples (n = 800) were preserved in the Βlood Bank of the Veterinary Laboratory of Brucellosis. From these, 212 sera samples were randomly selected for molecular and genetic analysis. The results indicated that the incidence rate of Brucellosis is higher in cattle herds in comparison with other animal species. Overall, 48 samples tested positive by real-time PCR, of which forty-seven of them were B. abortus and one was B. melitensis. Genetic analysis of two B. abortus samples revealed a common pattern, indicating two Bruce04, two Bruce18, four Bruce07, two Bruce09, three Bruce16, and four Bruce30 for both samples, which, interestingly, were not identical with the known genotypes in the public MLVA Brucella database. Our findings substantiate that animal Brucellosis remains a health issue in Greece, with a stable but apparent incidence rate, and further investigation is needed to fully characterize the newly identified Brucella strains in Greece.

Molecular Investigation of Small Ruminant Abortions Using a 10-Plex HRM-qPCR Technique: A Novel Approach in Routine Diagnostics.

The objective of this study was to apply and preliminarily evaluate a High-Resolution Melting (HRM) analysis technique coupled with qPCR, that allows the simultaneous detection of 10 different ruminant abortogenic pathogens, for investigating abortions in sheep and goats throughout Greece. A total of 264 ovine and caprine vaginal swabs were obtained the week following the abortion from aborted females and analyzed using a commercially available kit (ID Gene™ Ruminant Abortion Multiplex HRM, Innovative Diagnostics). Results indicated a high prevalence of Coxiella burnetii and Chlamydophila spp., which were detected in 48.9% and 42.4% of the vaginal swabs, respectively. Results for these most commonly detected pathogens were compared with those of a well-established commercial qPCR kit, with near-perfect agreement. Toxoplasma gondii, Salmonella spp., Brucella spp., Anaplasma phagocytophilum, Campylobacter fetus, and Neospora caninum were also identified, the two latter reported for the first time in the country in small ruminants. Mixed infections occurred in 35.6% of the animals examined. This technique allows for the simultaneous detection of many abortogenic pathogens in an accurate and cost-effective assay. Detection of uncommon or not previously reported pathogens in various cases indicates that their role in ovine and caprine abortions may be underestimated.

The Prevalence of Brucella Spp. in Dairy Products in Iran: A Systematic Review and Meta-Analysis.

Brucellosis, a significant zoonotic disease, threatens food safety substantially, particularly in developing nations such as the Middle East. This study aimed to comprehensively assess the prevalence of Brucella spp. in Iranian milk and dairy products through a systematic review and meta-analysis. A thorough search of international and domestic databases from January 2008 to October 2023 identified 38 relevant studies encompassing 11,130 samples for meta-analysis. The overall prevalence of Brucella spp. in Iranian dairy products was 22% (95% CI: 16-28%). The highest and lowest overall prevalence of Brucella spp. in milk were found in raw goat milk 27% (95% CI: 11-42%) and raw camel milk 15% (95% CI: -0.42 to 72%), respectively. The overall prevalence of traditional cheese, ice cream, and cream is estimated to be 9% (95% CI: -16 to 35%), 2% (95% CI: -2.78 to 2.82%), and 9% (95% CI: -0.94 to 1.12%). Geographical disparities were evident, with Zanjan province reporting the highest contamination rate, 53%, while Razavi Khorasan province had the lowest, 1%. However, the prevalence of Brucella spp. in Iranian dairy products has fluctuated over time, with a significant association between the study year and sample size. Comprehensive planning, robust policy implementation, and rigorous monitoring are imperative to mitigate and ultimately eliminate Brucella contamination in dairy products effectively. Further research is essential to refine prevalence estimates and develop targeted prevention strategies to safeguard public health.

Serologic Survey of Brucella spp. in Culled Invasive Alien Mammals from El Palmar National Park, Argentina, and in Exposed Consumers.

Brucellosis is a worldwide zoonotic disease caused by Brucella spp. and transmitted from domestic and wild animals to humans. Brucellosis causes important economic losses in livestock, has a significant impact on public health, and may affect the health of wildlife. Hunting and consumption of meat from culled wildlife constitute a risk for Brucella spp. infection in humans and hunting dogs. In El Palmar National Park (EPNP), Argentina, the invasive alien mammals wild boar (Sus scrofa) and axis deer (Axis axis) are controlled, slaughtered in situ, and consumed by hunters, with meat trimmings and offal often fed to dogs. In this study, we evaluated but did not detect anti-Brucella antibodies in wild boar (n=95) and axis deer (n=238) from EPNP or in game consumers, dogs (n=39) and humans (n=61). These results suggest a lack of exposure to Brucella spp. at this site during the study period. Despite negative findings in the sampled location, One Health surveillance across multiple species contributes to our understanding of pathogen dynamics and enables targeted interventions to minimize health risks.

Multiple pathogens co-exposure and associated risk factors among cattle reared in a wildlife-livestock interface area in Kenya.

Introduction: Understanding multi-pathogen infections/exposures in livestock is critical to inform prevention and control measures against infectious diseases. We investigated the co-exposure of foot-and-mouth disease virus (FMDV), Brucella spp., Leptospira spp., and Coxiella burnetii in cattle in three zones stratified by land use change and with different wildlife-livestock interactions in Narok county, Kenya. We also assessed potential risk factors associated with the transmission of these pathogens in cattle. Methods: We identified five villages purposively, two each for areas with intensive (zone 1) and moderate wildlife-livestock interactions (zone 2) and one for locations with low wildlife-livestock interactions (zone 3). We sampled 1,170 cattle from 390 herds through a cross-sectional study and tested the serum samples for antibodies against the focal pathogens using enzyme-linked immunosorbent assay (ELISA) kits. A questionnaire was administered to gather epidemiological data on the putative risk factors associated with cattle's exposure to the investigated pathogens. Data were analyzed using the Bayesian hierarchical models with herd number as a random effect to adjust for the within-herd clustering of the various co-exposures among cattle. Results: Overall, 88.0% (95% CI: 85.0-90.5) of the cattle tested positive for at least one of the targeted pathogens, while 41.7% (95% CI: 37.7-45.8) were seropositive to at least two pathogens. FMDV and Brucella spp. had the highest co-exposure at 33.7% (95% CI: 30.9-36.5), followed by FMDV and Leptospira spp. (21.8%, 95% CI: 19.5-24.4), Leptospira spp. and Brucella spp. (8.8%, 95% CI: 7.2-10.6), FMDV and C. burnetii (1.5%, 95% CI: 0.7-2.8), Brucella spp. and C. burnetii (1.0%, 95% CI: 0.3-2.2), and lowest for Leptospira spp. and C. burnetii (0.3%, 95% CI: 0.0-1.2). Cattle with FMDV and Brucella spp., and Brucella spp. and Leptospira spp. co-exposures and those simultaneously exposed to FMDV, Brucella spp. and Leptospira spp. were significantly higher in zone 1 than in zones 2 and 3. However, FMDV and Leptospira spp. co-exposure was higher in zones 1 and 2 than zone 3. Discussion/conclusion: We recommend the establishment of a One Health surveillance system in the study area to reduce the morbidity of the targeted zoonotic pathogens in cattle and the risks of transmission to humans.

Hunting for Answers: Assessing Brucella spp. Seroprevalence and Risks in Red Deer and Wild Boar in Central Portugal.

Between 2016 and 2023, a cross-sectional study was conducted in the central region of Portugal in order to better understand the epidemiology and public health risks resulting from the handling and consumption of game animals infected with Brucella spp. The seroprevalence and risk factors for Brucella spp. seropositivity were evaluated. Antibodies against Brucella spp. were determined using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Results showed that in the 650 serum samples collected from red deer (n = 298) and wild boars (n = 352) in Portugal, 21.7% (n = 141; 95% CI: 18.6-25.1%) tested positive. Wild boar had a significantly higher prevalence (35.5%; 95% CI: 30.5-40.8%) than red deer (5.4%, 95% CI: 3.1-8.6%; p ≤ 0.001). Risk factors for seropositivity were investigated using multivariable logistic regression models. The odds of being seropositive was 8.39 (95% CI: 4.75-14.84; p ≤ 0.001) times higher in wild boar than in red deer. Correlations between sex, age, body condition, and seropositivity could not be observed. The higher seroprevalence in wild boar suggests that this species may primarily contribute to the Brucella spp. ecology in central Portugal.

Seroprevalence study of brucellosis in wild boar hunted for private consumption in northeast Portugal.

Brucellosis is an important infectious disease caused by bacteria of the genus Brucella. In the northeast region of Portugal, infection with Brucella melitensis is endemic in small ruminants, and there are also humans' cases. However, the epidemiological role of the wild boar in the dynamics of this disease in this region is unknown. In this study, a total of 332 blood samples were collected from wild boar hunted in thirty-six hunting areas during the 2022/2023 hunting season. All were taken by the hunters for private consumption, with no evisceration or examination in the field. Serum samples were tested by indirect ELISA (i-ELISA). It was observed that 88 wild boars were exposed to Brucella spp., pointing to a seroprevalence of 26.5% (95% CI: 21.8 - 31.3%). This high prevalence underlines the importance that wild boar may have in the dynamics of this disease in the region and its potential transmission to other animals, and to humans (for example, during the handling of carcasses). Increased awareness and knowledge of brucellosis in wild boar is essential for the implementation of effective practices and habits and, consequently, for the control and prevention of this important zoonosis.

Seroprevalence of Coxiella burnetii, Leptospira interrogans serovar hardjo, and Brucella species and associated reproductive disorders in cattle in southwest Ethiopia.

In Ethiopia, Coxiella burnetii, Leptospira Hardjo, and Brucella spp are recognized as the primary factors contributing to cattle reproductive issues. A cross-sectional study was conducted in southwest Ethiopia from October 2020 to October 2021 to assess the risk of reproductive disorders associated with L. Hardjo, Coxiella burnetii, and Brucella spp. Moreover, the study aimed to identify the factors associated with reproductive disorders. Using an indirect ELISA, antibodies against these pathogens were observed in serum samples collected from 461 cattle. We employed multivariable random effect logistic regression analysis to identify potential risk factors associated with reproductive disorders in cattle. The study areas showed a prevalence of 25.16 % (95 % CI: 21.20-29.12) for cattle reproductive disorders. The presence of Leptospira Hardjo (OR = 2.9, 95 % CI: 1.17-4.02) and Coxiella burnetii (OR = 3.0, 1.49-5.94) antibodies was associated to the occurrence of cattle reproductive disorders. Seropositivity to pathogens B. abortus, C. burnetii, and L. Hardjo, along with co-infection of all three, showed association with cattle abortion. The presence of L. Hardjo seropositivity and co-infection with C. burnetii were related to dystocia in cattle. Cattle with retained fetal membranes were associated with co-infection seropositivity to these pathogens. Additionally, B. abortus seropositivity was linked to cases of repeated breeding in cattle. Age, breeding practices, and dog access to cattle showed associations with reproductive disorders, with odds ratios of 2.3 (95 % CI: 2.03-4.69), 2.9 (95 % CI: 1.83-4.82), and 6.5 (95 % CI: 1.04-2.53) respectively. This research indicates that Brucella abortus, Coxiella burnetii, and Leptospira Hardjo, which are responsible for severe zoonotic diseases, have a substantial negative impact on cattle production by causing reproductive disorders. To address the transmission of these diseases, it is essential to implement effective mitigation strategies and enhance public awareness. Additional investigation is necessary to identify and understand the factors contributing to cattle reproductive disorders in the specified area.

Antibodies against the Mycobacterium tuberculosis complex and Brucella spp. in captive and free-living European bison (Bison bonasus) in Poland.

BACKGROUND: The European bison (Bison bonasus), a symbol of Polish nature, is a protected species that requires active health monitoring. However, conservation efforts are made difficult by the zoonotic diseases such as brucellosis and tuberculosis. OBJECTIVE: The aim of this study was to screen the Polish European bison population for exposure to the Mycobacterium tuberculosis complex (MTC) and Brucella spp. METHODS: A total of 323 free-living and captive European bison from 13 localities were tested serologically for antibodies against the M. bovis P22 multi-protein complex (in-house ELISA) and against Brucella spp. (commercial ELISA). RESULTS: Antibodies against the MTC (P22) were detected in 7% (22/323) of the tested European bison. Anti-MTC antibody positivity was not significantly different by sex, age, and captive/free range status. Anti-MTC antibodies were found in six of 13 populations sampled, always in populations with larger sample sizes including the four free-living ones. Antibodies against Brucella spp. were detected in 36% (116/323) of the tested bison. While Brucella spp. antibody prevalence was not different by sex, it was significantly different by age (lower in adults) and captive/free-living status. Brucella spp. seroprevalence decreased with sample size and seropositive bison were found in 12 of 13 sampling populations. CONCLUSIONS: Our findings identify potential emerging threats to the European bison population and confirm the first serological response to P22 in European bison. As Poland is currently officially free of brucellosis and bovine tuberculosis, our results require careful interpretation. Further studies are needed to establish the presence of cross-reactions with atypical mycobacteria in the case of MTC and other bacteria (e.g. Yersinia enterocolitica O:9) in the case of Brucella spp.

Seroprevalence of Chlamydia abortus and Brucella spp. and risk factors for Chlamydia abortus in pigs from China.

Chlamydiosis and brucellosis induced abortions have resulted in significant economic losses in the global livestock industry. Although there have been numerous reports on these two diseases in ruminants in China, limited information is available regarding the prevalence of Chlamydia abortus (C. abortus) and Brucella spp. infection in pigs. This study aimed to investigate the prevalence of C. abortus and Brucella spp. infections in pig serum using serology and to identify potential risk factors. In total, 2816 serum samples were collected from 12 provinces in China. The presence of C. abortus antibodies was determined using an enzyme-linked immunosorbent assay (ELISA), while the presence of Brucella spp. antibodies was examined using the Rose Bengal Plate Test (RBPT) and the Standard Agglutination Test (SAT). The seroprevalences of C. abortus and Brucella spp. were 8.38 % (236/2816) and 0.11 % (3/2816), respectively. Geographical location, season, and age were found to be risk factors associated with C. abortus infection in pig herds in China (p<0.01), and the seropositive rate for C. abortus in sow herds was strongly associated with the occurrence of abortion (p<0.01). Overall, in China, pigs exhibit a higher seroprevalence of C. abortus, whereas the prevalence of Brucella is limited. This study represents the first comprehensive survey of C. abortus and Brucella spp. in pig herds in China that established potential risk factors and provided data for the prevention and control of intraspecies and interspecies transmission of C. abortus to humans.

Seroprevalence and Risk Factors Related to Bovine Brucellosis in Continental Ecuador.

Bovine brucellosis is a worldwide zoonotic contagious disease. According to World Animal Health Information System reports Ecuador has presented an increasing number of bovine brucellosis outbreaks in the continental territory over the past years (756 in 2018 versus 964 in 2021), generating economic losses for producers and causing a risk to public health. A cross-sectional study was conducted to investigate the seroprevalence of bovine brucellosis and associated risk or protective factors between May and June 2018. This stratified random study was implemented in 290 cattle herds located in the 23 provinces of continental Ecuador, which represents a total of 3737 cows aged 24 months or older. A competitive ELISA was used to detect Brucella antibodies. Simultaneously, an epidemiological survey was implemented to assess the brucellosis risk or protective factors. The apparent prevalence of bovine brucellosis at the herd level was 21.3% (95% CI: 16.8-26.6) and 6.2% (95% CI: 5.5-7) at the animal level. Univariate and multivariate logistic regression analyses were performed to determine the relationship between the potential factors associated with the presence of bovine brucellosis. The risk factors identified after multivariate analysis were a surface in ha per herd > 70 ha (OR = 2.73; 95% CI: 1.18-6.32) and the number of parturitions per animal (two or more with OR ≥ 1.8 and p-value ≤ 0.047). On the contrary, the protective factors were the region (farms located in the eastern region) and the absence of reported clinical signs. In addition, in herds where extensive production predominates, farmers have a low level of knowledge, and the farm biosecurity level is low. These results can guide the authorities in managing the risk factors identified, understanding the current epidemiological situation in Ecuador, improving the bovine brucellosis control program and food safety, as well as increase the one-health approach.

Modelling the Unidentified Abortion Burden from Four Infectious Pathogenic Microorganisms (Leptospira interrogans, Brucella abortus, Brucella ovis, and Chlamydia abortus) in Ewes Based on Artificial Neural Networks Approach: The Epidemiological Basis for a Control Policy.

Unidentified abortion, of which leptospirosis, brucellosis, and ovine enzootic abortion are important factors, is the main cause of disease spread between animals and humans in all agricultural systems in most developing countries. Although there are well-defined risk factors for these diseases, these characteristics do not represent the prevalence of the disease in different regions. This study predicts the unidentified abortion burden from multi-microorganisms in ewes based on an artificial neural networks approach and the GLM. METHODS: A two-stage cluster survey design was conducted to estimate the seroprevalence of abortifacient microorganisms and to identify putative factors of infectious abortion. RESULTS: The overall seroprevalence of Brucella was 70.7%, while Leptospira spp. was 55.2%, C. abortus was 21.9%, and B. ovis was 7.4%. Serological detection with four abortion-causing microorganisms was determined only in 0.87% of sheep sampled. The best GLM is integrated via serological detection of serovar Hardjo and Brucella ovis in animals of the slopes with elevation between 2600 and 2800 meters above sea level from the municipality of Xalatlaco. Other covariates included in the GLM, such as the sheep pen built with materials of metal grids and untreated wood, dirt and concrete floors, bed of straw, and the well water supply were also remained independently associated with infectious abortion. Approximately 80% of those respondents did not wear gloves or masks to prevent the transmission of the abortifacient zoonotic microorganisms. CONCLUSIONS: Sensitizing stakeholders on good agricultural practices could improve public health surveillance. Further studies on the effect of animal-human transmission in such a setting is worthwhile to further support the One Health initiative.

White-Toothed Shrews (Genus Crocidura): Potential Reservoirs for Zoonotic Leptospira spp. and Arthropod-Borne Pathogens?

Three species of white-toothed shrews of the order Eulipotyphla are present in central Europe: the bicolored (Crocidura leucodon), greater (Crocidura russula) and lesser (Crocidura suaveolens) white-toothed shrews. Their precise distribution in Germany is ill-defined and little is known about them as reservoirs for zoonotic pathogens (Leptospira spp., Coxiella burnetii, Brucella spp., Anaplasma phagocytophilum, Babesia spp., Neoehrlichia mikurensis and Bartonella spp.). We investigated 372 Crocidura spp. from Germany (n = 341), Austria (n = 18), Luxembourg (n = 2) and Slovakia (n = 11). West European hedgehogs (Erinaceus europaeus) were added to compare the presence of pathogens in co-occurring insectivores. Crocidura russula were distributed mainly in western and C. suaveolens mainly in north-eastern Germany. Crocidura leucodon occurred in overlapping ranges with the other shrews. Leptospira spp. DNA was detected in 28/227 C. russula and 2/78 C. leucodon samples. Further characterization revealed that Leptospira kirschneri had a sequence type (ST) 100. Neoehrlichia mikurensis DNA was detected in spleen tissue from 2/213 C. russula samples. Hedgehogs carried DNA from L. kirschneri (ST 100), L. interrogans (ST 24), A. phagocytophilum and two Bartonella species. This study improves the knowledge of the current distribution of Crocidura shrews and identifies C. russula as carrier of Leptospira kirschneri. However, shrews seem to play little-to-no role in the circulation of the arthropod-borne pathogens investigated.

False Positives in Brucellosis Serology: Wrong Bait and Wrong Pond?

This review summarizes the status of resolving the problem of false positive serologic results (FPSR) in Brucella serology, compiles our knowledge on the molecular background of the problem, and highlights some prospects for its resolution. The molecular basis of the FPSRs is reviewed through analyzing the components of the cell wall of Gram-negative bacteria, especially the surface lipopolysaccharide (LPS) with details related to brucellae. After evaluating the efforts that have been made to solve target specificity problems of serologic tests, the following conclusions can be drawn: (i) resolving the FPSR problem requires a deeper understanding than we currently possess, both of Brucella immunology and of the current serology tests; (ii) the practical solutions will be as expensive as the related research; and (iii) the root cause of FPSRs is the application of the same type of antigen (S-type LPS) in the currently approved tests. Thus, new approaches are necessary to resolve the problems stemming from FPSR. Such approaches suggested by this paper are: (i) the application of antigens from R-type bacteria; or (ii) the further development of specific brucellin-based skin tests; or (iii) the application of microbial cell-free DNA as analyte, whose approach is detailed in this paper.

Seroprevalence of zoonotic abortive diseases and their associated risk factors in Tunisian sheep.

BACKGROUND: Abortion is a serious problem for sheep flocks and it is responsible for considerable economic losses. The epidemiological situation of abortion causing agents in sheep is poorly documented in Tunisia. This study aims to investigate the status of three abortion causing agents (Brucella spp, Toxoplasma gondii, and Coxiella burnetii) among organized flocks in Tunisia. RESULTS: A total of 793 sample blood collected from twenty-six flocks in seven governorates in Tunisia, were tested by indirect enzyme-linked immunosorbent assay (i-ELISA) for antibodies against three abortion causing agents (Brucella spp, Toxoplasma gondii, and Coxiella burnetii). Risk factors for individual-level seroprevalence were analyzed using a logistic regression model. Results revealed that 19.7%, 17.2%, and 16.1% of the tested sera were positive for toxoplasmosis, Q fever, and brucellosis, respectively. Mixed infection was found in all the flocks with 3 to 5 responsible abortive agents simultaneously. Logistic regression showed that the management practices (control of new introduction, common grazing and watering point, workers exchange, presence of lambing box on the farm) and the history of infertility and the presence of abortion in neighboring flocks were likely to increase the probability of being infected by the three abortive agents. CONCLUSIONS: Evidence of the positive relationship between seroprevalence of abortion causing agents and several risk factors, suggests further investigations to better understand the etiology of infectious abortions in flocks to develop an applicable preventive and control program.

In-depth immunophenotyping reveals significant alteration of lymphocytes in buffalo with brucellosis.

Water buffalo (Bubalus bubalis) has a prominent position in the livestock industry worldwide but still suffers from limited knowledge on the mechanisms regulating the immune against infections, including brucellosis (BRC), one of the most significant neglected zoonotic diseases of livestock. Seventy-three buffalo were recruited for the study. Thirty-five were naturally infected with Brucella spp. The aims of the study were to (i) verify the cross-reactivity of 16 monoclonal antibodies (mAbs) developed against human, bovine, and ovine antigens; (ii) evaluate lymphocyte subset alterations in BRC positive buffalo; (iii) evaluate the use of the canonical discriminant analysis (CDA), with flow cytometric data, to discriminate BRC positive from negative animals. A new set of eight mAbs (anti CD3e, CD16, CD18, CD45R0, CD79a; CD172a) were shown to cross-react with water buffalo orthologous molecules. BRC positive animals presented a significant (p < 0.0001) decrease in the percentage of PBMC (29.5 vs. 40.3), total, T and B lymphocytes (23.0 vs. 35.5, 19.2 vs. 28.9, 2.6 vs. 5.7, respectively). In contrast, they showed an increase in percentage of granulocytes (65.2 vs. 55.1; p < 0.0001) and B lymphocytes CD21neg (22.9 vs. 16.1; p = 0.0067), a higher T/B lymphocyte ratio (10.3 vs. 6.4; p = 0.0011) and CD3+ /CD21+ (14.7 vs. 8.3; p = 0.0005) ratio. The CDA, applied to 33 different flow cytometric traits, allowed the discrimination of all BRC positive from negative buffalo. Although this is a preliminary study, our results show that flow cytometry can be used in a wide range of applications in livestock diseases, including in support of uncertain BRC diagnoses.

Detection of Brucella S2 vaccine strain by a loop-mediated isothermal amplification (LAMP) method.

Introduction: Brucellosis is a highly prevalent zoonotic disease caused by Brucella spp. Brucella suis S2 vaccination is an effective strategy to prevent animal brucellosis. However, S2 induces antibodies against the smooth lipopolysaccharide,making it challenging to distinguish field infected from vaccinated livestock. Early and accurate diagnosis is essential for infection control and prevention. In this study, we aimed to develop a quick and accurate assay to distinguish the BrucellaS2 vaccine strain from closely related B. abortus and B. melitensis. Methods: Whole-genome sequencing of B. suis S2 was performed, and the sequence was compared with that of the genomes of B. abortus and B. melitensis. One specific gene, GL_0002189, was selected as a marker to differentiate the BrucellaS2vaccine strain from B. abortus and B. melitensis. A loop-mediated isothermal amplification (LAMP) assay was developed, based on the GL_0002189 gene, and then assessed for target specificity, lower limit of detection, and repeatability. Results: Our results revealed that there was no cross-reaction with other strains, and the LAMP assay displayed high sensitivity for detecting S2 with a minimum detection limit of 18.9×103 copies/µL DNA input, it is nearly 100 times higher than conventional PCR technology. Concordance between the LAMP assay and a conventional polymerase chain reaction method was assessed using 54 blood samples collected from sheep with suspected brucellosis. Total concordance between the two assays was 92.6%, without a significant difference (p > 0.05) in the test results. Conclusion: This is the first report of a LAMP assay for the detection of the B. suis S2vaccine strain. Our approach can be helpful for the control and eradication of brucellosis, and its simplicity in requiring no specialized equipment or personnel makes it useful for implementation in resource-limited settings as well as for field use.

Molecular characterization of Brucella spp. from seropositive herds of cattle farmed at the wildlife-livestock-human interface in Rwanda.

Seroprevalence studies showed that brucellosis is prevalent in cattle in Rwanda with no recent study on the characterization of Brucella spp. Therefore, this study aimed to characterize Brucella spp. in seropositive herds of cattle farmed at the wildlife-livestock-human interface. Whole blood samples (n = 118), milk (n = 41), and vaginal swabs (n = 51) were collected from 64 seropositive herds. All samples (n = 210) were inoculated onto modified Centro de Investigacion y Tecnologia Agroalimentaria (CITA) selective medium. Cultures were analyzed to detect Brucella spp. using 16S-23S ribosomal DNA interspacer region (ITS) PCR, the Brucella cultures were speciated using AMOS and Bruce-ladder PCR assays. Brucella spp. were detected in 16.7% (35/210) of the samples established from the samples using ITS-PCR. The AMOS PCR assay identified mixed Brucella abortus and B. melitensis (n = 6), B. abortus (n = 7), and B. melitensis (n = 1) from cultures from blood samples; mixed B. abortus and B. melitensis (n = 1) and B. abortus (n = 4) from cultures from milk samples; mixed B. abortus and B. melitensis (n = 6), B. abortus (n = 8), and B. melitensis (n = 1) from cultures from vaginal swabs. Bruce-ladder PCR assay confirmed B. abortus and B. melitensis cultures. The isolation of Brucella spp. was significantly associated with districts, with the Nyagatare district having more isolates than other districts (p = 0.01). This study identified single or mixed B. abortus and B. melitensis infections in cattle samples in Rwanda, which emphasizes the need to improve brucellosis control at the wildlife-livestock-human interface and raise the awareness of cattle keepers, abattoir workers, laboratory personnel, and consumers of cattle products.

Brucella spp. Contamination in Artisanal Unpasteurized Dairy Products: An Emerging Foodborne Threat in Tunisia.

Brucellosis is a worldwide zoonotic disease transmitted to humans, predominantly by the consumption of contaminated raw milk and dairy products. This study aimed to investigate the occurrence of Brucella spp. in 200 raw milk, ricotta, and artisan fresh cheese samples, collected from individual marketing points in four districts in Tunisia. Samples were analyzed for the presence of Brucella spp. by IS711-based real-time PCR assay. Positive samples were further analyzed by qPCR for B. melitensis and B. abortus species differentiation. The DNA of Brucella spp. was detected in 75% of the samples, B. abortus was detected in 31.3%, and B. melitensis was detected in 5.3% of positive samples. A percentage of 49.3% of samples co-harbored both species, while 14% of the Brucella spp. positive samples were not identified either as B. abortus or B. melitensis. High contamination rates were found in ricotta (86.2%), cheese (69.6%), and raw milk (72.5%) samples. The study is the first in Tunisia to assess the occurrence of Brucella spp. contamination in artisanal unpasteurized dairy products and showed high contamination rates. The detection of both B. abortus and B. melitensis highlights that zoonotic high-pathogen agent control remains a challenge for food safety and consumer health protection and could represent a serious emerging foodborne disease in Tunisia.