Genomic Diversity and Zoonotic Potential of Brucella neotomae.

After reports in 2017 of Brucella neotomae infections among humans in Costa Rica, we sequenced 12 strains isolated from rodents during 1955-1964 from Utah, USA. We observed an exact strain match between the human isolates and 1 Utah isolate. Independent confirmation is required to clarify B. neotomae zoonotic potential.

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.

The Reasons Behind Long-Term Endemicity of Brucellosis in Low and Middle-Income Countries: Challenges and Future Perspectives.

Brucellosis is a neglected worldwide zoonotic disease with more than 500,000 new human cases each year. Direct contact with infected animals and consumption of undercooked animal origin foods are the main routes of brucellosis transmission to humans. Although long endeavor has been applied to control and eliminate brucellosis from animal and human populations in developing countries especially in low- and middle-income countries (LMICs), the disease is still endemic in these regions. Many common or unique factors including raw milk consumption, unhygienic slaughter of livestock, extensive husbandry, budgetary limitations, misdiagnosis, and other conditions play a role in long-term endemicity of brucellosis in these locations. It has been shown that One Health is the only practical approach to control brucellosis; however, applying such methods is challenging in low-resource areas. In such conditions, brucellosis is continuously maintained in animals and repeatedly spread to human populations. In this article, factors playing a critical role in brucellosis endemicity, and the real conditions challenging the application of One Health approach in control of brucellosis are highlighted.

Quantification of Brucella abortus population structure in a natural host.

Cattle are natural hosts of the intracellular pathogen Brucella abortus, which inflicts a significant burden on the health and reproduction of these important livestock. The primary routes of infection in field settings have been described, but it is not known how the bovine host shapes the structure of B. abortus populations during infection. We utilized a library of uniquely barcoded B. abortus strains to temporally and spatially quantify population structure during colonization of cattle through a natural route of infection. Introducing 108 bacteria from this barcoded library to the conjunctival mucosa resulted in expected levels of local lymph node colonization at a 1-wk time point. We leveraged variance in strain abundance in the library to demonstrate that only 1 in 10,000 brucellae introduced at the site of infection reached a parotid lymph node. Thus, cattle restrict the overwhelming majority of B. abortus introduced via the ocular conjunctiva at this dose. Individual strains were spatially restricted within the host tissue, and the total B. abortus census was dominated by a small number of distinct strains in each lymph node. These results define a bottleneck that B. abortus must traverse to colonize local lymph nodes from the conjunctival mucosa. The data further support a model in which a small number of spatially isolated granulomas founded by unique strains are present at 1 wk postinfection. These experiments demonstrate the power of barcoded transposon tools to quantify infection bottlenecks and to define pathogen population structure in host tissues.

Comparative evaluation of RBPT, I-ELISA, and CFT for the diagnosis of brucellosis and PCR detection of Brucella species from Ethiopian sheep, goats, and cattle sera.

BACKGROUND: Brucellosis is an economically devastating animal disease and has public health concern. Serological methods such as Rose Bengal Plate Test (RBPT), Complement Fixation Test (CFT), and Indirect-Enzyme-Linked Immunosorbent Assay (I-ELISA) have been used to detect brucellosis. However, there is limited comparative evaluation studies and lack of molecular confirmation of the causative agents in the study areas. The study was aimed to compare RBPT, I-ELISA, CFT, and confirmation using Polymerase Chain Reaction (PCR). A total of 2317 sera samples were collected from brucellosis-affected areas of Ethiopia with no vaccination history. All sera were subjected to comparative serological assays. Post-cross tabulation, sensitivity, and specificity were determined using Receiver Operating Characteristics (ROC) curve analysis software. PCR was performed on 54 seropositive samples using genus- and species-specific primers. RESULTS: Among the 2317 sera tested for comparative serological assays, 189 (8.16%) were positive for RBPT, 191 (8.24%) for I-ELISA, and 48 (2.07%) for CFT. Sensitivity to RBPT was 100% (95%) in shoats and 74% (95%) in cattle. Specificity on RBPT was 98.69% (95%), 99.28% (95%), 100% (95%) in sheep, goats, and cattle, respectively. CFT sensitivity was 4 (95%) in sheep, 9.65 (95%) goats, and 72 (95%) cattle. Specificity on CFT was 100% (95%) for sheep, goats, and cattle. A 223bp Brucella genus-specific and 156bp B. abortus species-specific detected. However, B. melitensis not detected. CONCLUSION: In this study, I-ELISA was the most sensitive and specific test. RBPT detected all Brucellosis-infected sheep and goats; nevertheless, it showed false positive in sheep and goats and false negative in cattle. The presence of B. abortus in small and large ruminants was confirmed by PCR. This is the first report of B. abortus detection in small ruminant in Ethiopia. B.abortus detected in non-preferred hosts. The findings suggest further study on molecular epidemiology of Brucella species.

The canonical Brucella species-host dependency is changing, however, the antibiotic susceptibility profiles remain unchanged.

Brucellosis is a chronic disease caused by Brucella species with a wide range of hosts, from marine mammals to terrestrial species, but with strict host preferences. With the zoonotic character, the prevalence of human brucellosis cases is a reflection of animal infections. This study aimed to identify 192 Brucella isolates obtained from various sources by Bruce-ladder PCR and to determine their antibiotic susceptibilities by gradient diffusion method (E-test). As a result of the PCR, all human isolates (n = 57) were identified as B. melitensis. While 58 (82.9%) of the cattle isolates were identified as B. abortus, 59 (90.8%) of the sheep isolates were identified as B. melitensis. In addition, 12 (17.1%) of the cattle isolates and 6 (9.2%) of the sheep isolates were determined as B. melitensis and B. abortus, respectively. The primary host change behavior of B. melitensis was 1.9 times higher than that of B. abortus. While gentamicin and ciprofloxacin susceptibilities of Brucella isolates were 100%, tetracycline, doxycycline, streptomycin, trimethoprim/sulfamethoxazole and rifampicin susceptibilities were 99%, 99%, 97.4%, 91.7% and 83.9%, respectively. The lowest sensitivity of the isolates was determined against to cefoperazone as 26%. A triple-drug resistance was detected in 1 B. abortus isolate that included simultaneous resistance to cefoperazone, rifampicin, and trimethoprim/sulfamethoxazole. The high susceptibility profiles we found against to antibiotics such as tetracycline, doxycycline gentamicin and ciprofloxacin, used widely in treatment, are encouraging. However, the change in the canonical Brucella species-primary host preference suggests the need to reconsider eradication program, including updating vaccine formulations.

Clearance of bacteria from lymph nodes in sheep immunized with Brucella suis S2 vaccine is associated with M1 macrophage activation.

Ovine brucellosis is a global zoonotic disease of sheep caused by Brucella melitensis, which inflicts a significant burden on human and animal health. Brucella suis strain S2 (B. suis S2) is a smooth live attenuated vaccine for the prevention of ovine brucellosis in China. However, no previous studies have assessed the immunogenicity of B. suis S2 vaccine after oral immunization in sheep. Here, we attempted to evaluate the ovine immune response over the course of B. suis S2 immunization and to identify in vivo predictors for vaccine development. Body temperature, serum Brucella antibodies, serum cytokines (IL-12p70 and interferon [IFN]-γ), and bacterial load in the mandibular lymph nodes (LN), superficial cervical LN, superficial inguinal LN, and spleen were investigated to determine the safety and efficacy of the vaccine. The abnormal body temperature of sheep occurred within 8 days post-infection (dpi). Brucella suis S2 persisted for a short time (< 21 dpi) in the mandibular LN. The highest level of IL-12p70 was observed at 9 dpi, whereas serum IFN-γ levels peaked at 12 dpi. Transcriptome analysis and quantitative reverse transcription PCR were performed to determine gene expression profiles in the mandibular LN of sheep. Antigen processing and presentation pathway was the dominant pathway related to the dataset. Our studies suggest that the immune response in ovine LN resembled type 1 immunity with the secretion of IL-12p70 and IFN-γ after B.suis S2 immunization and the vaccine may eliminate Brucella via stimulation of M1 macrophages through the course of Th cells.

Genotype diversity of brucellosis agents isolated from humans and animals in Greece based on whole-genome sequencing.

BACKGROUND: Brucellosis is a zoonotic disease whose causative agent, Brucella spp., is endemic in many countries of the Mediterranean basin, including Greece. Although the occurrence of brucellosis must be reported to the authorities, it is believed that the disease is under-reported in Greece, and knowledge about the genomic diversity of brucellae is lacking. METHODS: Thus, 44 Brucella isolates, primarily B. melitensis, collected between 1999 and 2009 from humans and small ruminants in Greece were subjected to whole genome sequencing using short-read technology. The raw reads and assembled genomes were used for in silico genotyping based on single nucleotide substitutions and alleles. Further, specific genomic regions encoding putative virulence genes were screened for characteristic nucleotide changes, which arose in different genotype lineages. RESULTS: In silico genotyping revealed that the isolates belonged to three of the known sublineages of the East Mediterranean genotype. In addition, a novel subgenotype was identified that was basal to the other East Mediterranean sublineages, comprising two Greek strains. The majority of the isolates can be assumed to be of endemic origin, as they were clustered with strains from the Western Balkans or Turkey, whereas one strain of human origin could be associated with travel to another endemic region, e.g. Portugal. Further, nucleotide substitutions in the housekeeping gene rpoB and virulence-associated genes were detected, which were characteristic of the different subgenotypes. One of the isolates originating from an aborted bovine foetus was identified as B. abortus vaccine strain RB51. CONCLUSION: The results demonstrate the existence of several distinct persistent Brucella sp. foci in Greece. To detect these and for tracing infection chains, extensive sampling initiatives are required.

Scent detection of Brucella abortus by African giant pouched rats (Cricetomys ansorgei).

BACKGROUND: Brucellosis is a contagious zoonosis caused by bacteria of the genus Brucella. While the disease has been eradicated in most developed countries, it remains endemic in sub-Saharan Africa where access to reliable diagnostics is limited. African giant pouched rats (Cricetomys ansorgei) have been trained to detect the scent of Mycobacterium tuberculosis to increase case detection in sub-Saharan Africa. Given the similar diagnostic challenges facing brucellosis and tuberculosis, we explored the feasibility of training African giant pouched rats to detect Brucella. RESULTS: After 3 months of training, rats reliably identified cultured Brucella, achieving an average sensitivity of 93.56% (SD = 0.650) and specificity of 97.65% (SD = 0.016). Rats readily generalized to novel, younger Brucella cultures that presumably generated a weaker volatile signal and correctly identified at least one out of three fecal samples spiked with Brucella culture during a final test of feasibility. DISCUSSION: To our knowledge, these experiments are the first to demonstrate Brucella emits a unique odor profile that scent detection animals can be trained to identify. Importantly, cultured E. coli samples were included throughout training and test to ensure the rats learned to specifically identify Brucella bacteria rather than any bacteria in comparison to bacteria-free culture medium. E. coli controls therefore served a crucial function in determining to what extent Brucella abortus emits a unique odor signature. Further research is needed to determine if a Brucella-specific volatile signature is present within clinical samples. If confirmed, the present results suggest trained rats could serve as a valuable, novel method for the detection of Brucella infection.

A review of three decades of use of the cattle brucellosis rough vaccine Brucella abortus RB51: myths and facts.

Cattle brucellosis is a severe zoonosis of worldwide distribution caused by Brucella abortus and B. melitensis. In some countries with appropriate infrastructure, animal tagging and movement control, eradication was possible through efficient diagnosis and vaccination with B. abortus S19, usually combined with test-and-slaughter (T/S). Although S19 elicits anti-smooth lipopolysaccharide antibodies that may interfere in the differentiation of infected and vaccinated animals (DIVA), this issue is minimized using appropriate S19 vaccination protocols and irrelevant when high-prevalence makes mass vaccination necessary or when eradication requisites are not met. However, S19 has been broadly replaced by vaccine RB51 (a rifampin-resistant rough mutant) as it is widely accepted that is DIVA, safe and as protective as S19. These RB51 properties are critically reviewed here using the evidence accumulated in the last 35 years. Controlled experiments and field evidence shows that RB51 interferes in immunosorbent assays (iELISA, cELISA and others) and in complement fixation, issues accentuated by revaccinating animals previously immunized with RB51 or S19. Moreover, contacts with virulent brucellae elicit anti-smooth lipopolysaccharide antibodies in RB51 vaccinated animals. Thus, accepting that RB51 is truly DIVA results in extended diagnostic confusions and, when combined with T/S, unnecessary over-culling. Studies supporting the safety of RB51 are flawed and, on the contrary, there is solid evidence that RB51 is excreted in milk and abortifacient in pregnant animals, thus being released in abortions and vaginal fluids. These problems are accentuated by the RB51 virulence in humans, lack diagnostic serological tests detecting these infections and RB51 rifampicin resistance. In controlled experiments, protection by RB51 compares unfavorably with S19 and lasts less than four years with no evidence that RB51-revaccination bolsters immunity, and field studies reporting its usefulness are flawed. There is no evidence that RB51 protects cattle against B. melitensis, infection common when raised together with small ruminants. Finally, data acumulated during cattle brucellosis eradication in Spain shows that S19-T/S is far more efficacious than RB51-T/S, which does not differ from T/S alone. We conclude that the assumption that RB51 is DIVA, safe, and efficaceous results from the uncritical repetition of imperfectly examined evidence, and advise against its use.

CRISPR-Cas12a test strip (CRISPR/CAST) package: In-situ detection of Brucella from infected livestock.

BACKGROUND: Brucellosis is a common zoonotic disease caused by Brucella, which causes enormous economic losses and public burden to epidemic areas. Early and precise diagnosis and timely culling of infected animals are crucial to prevent the infection and spread of Brucella. In recent years, RNA-guided CRISPR/Cas12a(Clustered Regularly Interspaced Short Palindromic Repeats and its associated protein 12a) nucleases have shown great promise in nucleic acid detection. This research aims to develop a CRISPR/CAST (CRISPR/Cas12a Test strip) package that can rapidly detect Brucella nucleic acid during on-site screening, especially on remote family pastures. The CRISPR/Cas12a system combined with recombinase polymerase amplification (RPA), and lateral flow read-out. RESULTS: We selected the conserved gene bp26, which commonly used in Brucella infection detection and compared on Genbank with other Brucella species. The genomes of Brucella abortus 2308, Brucella suis S2, Brucella melitansis 16 M, and Brucella suis 1330, et al. were aligned, and the sequences were found to be consistent. Therefore, the experiments were only performed on B. melitensis. With the CRISPR/CAST package, the assay of Brucella nucleic acid can be completed within 30 min under isothermal temperature conditions, with a sensitivity of 10 copies/µl. Additionally, no antigen cross-reaction was observed against Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis. The serum samples of 398 sheep and 100 cattle were tested by the CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR results and higher than that of the Rose Bengal Test (RBT, 19 sheep and 5 cattle were serum positive). CONCLUSIONS: The CRISPR/CAST package can accurately detect Brucella DNA in infected livestock within 30 min and exhibits several advantages, including simplicity, speed, high sensitivity, and strong specificity with no window period. In addition, no expensive equipment, standard laboratory, or professional operators are needed for the package. It is an effective tool for screening in the field and obtaining early, rapid diagnoses of Brucella infection. The package is an efficient tool for preventing and controlling epidemics.

Influence of landscape structure on previous exposure to Leptospira spp. and Brucella abortus in free-living neotropical primates from southern Brazil.

The environments in which neotropical primates live have been undergoing an intense fragmentation process, constituting a major threat to the species' survival and causing resource scarcity, social isolation, and difficulty in dispersal, leaving populations increasingly vulnerable. Moreover, the proximity of wild environments to anthropized landscapes can change the dynamics of pathogens and the parasite-host-environment relationship, creating conditions that favor exposure to different pathogens. To investigate the previous exposure of free-living primates in Rio Grande do Sul State (RS), southern Brazil, to the bacterial agents Leptospira spp. and Brucella abortus, we investigated agglutinating antibodies against 23 serovars of Leptospira spp. using the microscopic agglutination test and B. abortus acidified antigen test in primate serum samples; 101 samples from primates captured between 2002 and 2016 in different forest fragments were used: 63 Alouatta caraya, 36 Alouatta guariba clamitans, and 02 Sapajus nigritus cucullatus. In addition, the forest remnants where the primates were sampled were characterized in a multiscale approach in radii ranging from 200 to 1400 m to investigate the potential relationship of previous exposure to the agent with the elements that make up the landscape structure. The serological investigation indicated the presence of antibodies for at least one of the 23 serovars of Leptospira spp. in 36.6% (37/101) of the samples analyzed, with titers ranging from 100 to 1600. The most observed serovars were Panama (17.8%), Ballum (5.9%), Butembo (5.9%), Canicola (5.9%), Hardjo (4.9%), and Tarassovi (3.9%); no samples were seropositive for Brucella abortus. Decreased forest cover and edge density were the landscape factors that had a significant relationship with Leptospira spp. exposure, indicating that habitat fragmentation may influence contact with the pathogen. The data generated in this study demonstrate the importance of understanding how changes in landscape structure affect exposure to pathogenic microorganisms of zoonotic relevance. Hence, improving epidemiological research and understanding primates' ecological role in these settings can help improve environmental surveillance and conservation strategies for primate populations in different landscapes.

Investigation of Brucella canis and Brucella abortus Seropositivity by In-House Rapid Slide Agglutination Test and In-House ELISA in Northern Cyprus.

The incidence of Brucella canis (B. canis) in humans is unknown in Northern Cyprus. In this study, we investigated the prevalence of B. canis and Brucella abortus (B. abortus) infection in human sera and evaluated the results obtained by agglutination-based techniques using standardized antigens made from B. canis comparatively. All of the subjects were negative in terms of Rose-Bengal plate test. Undiluted serum samples were initially screened by rapid slide agglutination test (RSAT), and those which were found positive were retested in the dilution of 1/25-1/200. Confirmation of the positive results was performed by using 2-mercaptoethanol standard agglutination test (SAT). The test antigen was prepared from the less mucoid M (-) variant of B. canis, and 1/1,048 titered dog antiserum was used as positive control. In 225 serum samples, 3.6% (8/225) was positive by B. canis M (-) RSAT, 4.4 % (10/225) was positive by B. canis M (-) indirect enzyme-linked immunosorbent assay (iELISA). 5.3% (12/225) was positive by B. abortus S99 RSAT and 9.8% (22/225) was positive by B. abortus S99 iELISA. Nine samples were positive by both B. abortus S99 RSAT and B. abortus S99 iELISA. Seven samples were positive by both B. canis M (-) RSAT and B. canis M (-) iELISA. One patient was positive by all methods. It is important to evaluate patient samples with RSAT and iELISA. Until the notification system gives better results to the Ministry of Health, in order to reach the real data for Northern Cyprus, multicenter prevalence determination studies should be done for future.

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.

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.

First Case of Human Brucella canis Infection in the Netherlands.

A patient was diagnosed with Brucella canis following exposure to infected dogs in her breeding facility. Transboundary spread of B. canis through (illegal) import of infected dogs to non-endemic countries in Europe suggest that B. canis infection should be considered in European patients with occupational exposure to dogs.

BtpB inhibits innate inflammatory responses in goat alveolar macrophages through the TLR/NF-κB pathway and NLRP3 inflammasome during Brucella infection.

Brucella species are infectious facultative intracellular pathogens. They have evolved multiple strategies to thwart immune responses and replicate in macrophages for chronic persistence in the host. As a Brucella effector, BtpB is transferred into target cells through the type IV secretion system. BtpB, a Toll/interleukin-1 receptor domain-containing protein, blocks host innate immune responses by interfering with Toll-like receptor signaling. However, the intracellular targets and their activated downstream pathways remain unclear. In this study, we constructed a strain of Brucella suis S2 with a deletion in the gene for BtpB, ΔbtpB, and the complemented strain, C-ΔbtpB with a restored copy of the btpB gene. The bacterial growth curves and stress resistance results showed that BtpB did not affect B. suis S2 growth. Infection of alveolar macrophages with WT and ΔbtpB strains showed that BtpB inhibited TLR2 and TLR4 expression and attenuated NLRP3 inflammasome activation. BtpB also attenuated secretion of the Brucella-induced proinflammatory cytokines, IL-1ß, IL-6, and TNF-α, in alveolar macrophages while up-regulating IL-10 expression. In general, the results confirmed that BtpB specifically inhibits TLR2/TLR4 and disrupts NLRP3 signaling pathways to inhibit host immune responses in early Brucella infections.

A Brucella melitensis H38ΔwbkF rough mutant protects against Brucella ovis in rams.

Brucella melitensis and Brucella ovis are gram-negative pathogens of sheep that cause severe economic losses and, although B. ovis is non-zoonotic, B. melitensis is the main cause of human brucellosis. B. melitensis carries a smooth (S) lipopolysaccharide (LPS) with an N-formyl-perosamine O-polysaccharide (O-PS) that is absent in the rough LPS of B. ovis. Their control and eradication require vaccination, but B. melitensis Rev 1, the only vaccine available, triggers anti-O-PS antibodies that interfere in the S-brucellae serodiagnosis. Since eradication and serological surveillance of the zoonotic species are priorities, Rev 1 is banned once B. melitensis is eradicated or where it never existed, hampering B. ovis control and eradication. To develop a B. ovis specific vaccine, we investigated three Brucella live vaccine candidates lacking N-formyl-perosamine O-PS: Bov::CAΔwadB (CO2-independent B. ovis with truncated LPS core oligosaccharide); Rev1::wbdRΔwbkC (carrying N-acetylated O-PS); and H38ΔwbkF (B. melitensis rough mutant with intact LPS core). After confirming their attenuation and protection against B. ovis in mice, were tested in rams for efficacy. H38ΔwbkF yielded similar protection to Rev 1 against B. ovis but Bov::CAΔwadB and Rev1::wbdRΔwbkC conferred no or poor protection, respectively. All H38ΔwbkF vaccinated rams developed a protracted antibody response in ELISA and immunoprecipitation B. ovis diagnostic tests. In contrast, all remained negative in Rose Bengal and complement fixation tests used routinely for B. melitensis diagnosis, though some became positive in S-LPS ELISA owing to LPS core epitope reactivity. Thus, H38ΔwbkF is an interesting candidate for the immunoprophylaxis of B. ovis in B. melitensis-free areas.

Trans-species transmission of Brucellae among ruminants hampering brucellosis control efforts in Egypt.

AIMS: This study aimed to identify the genotypic fingerprinting of Brucella melitensis biovar 3 isolates from ruminants in Kafr El-Sheikh, Egypt, to compare with other peers globally and to highlight the epidemiology and potential causes of brucellosis control failure. METHODS AND RESULTS: A multilocus variable-number tandem-repeat analysis (MLVA 16) was carried out on 41 B. melitensis bv3 isolates, 31 from the preferential hosts (28 sheep and three goats) and 10 from atypical hosts (nine cattle and one buffalo), identified by bacteriological and molecular techniques. MLVA-16 analysis revealed 19 genotypes with nine as singletons. The most prevalent genotypes were M3_K.E (3,5,3,13,1,1,3,3,7,43,8,7,6,7,5,3), M13_K.E (3,5,3,13,1,1,3,3,7,43,8,5,8,7,7,3) and M5_K.E (3,5,3,13,1,1,3,3,7,43,8,4,8,7,11,3) circulating between different animal species. The B. melitensis isolation from aborted cows in farms that had never reared small ruminants indicates the likelihood of cow to cow B. melitensis transmission. Different genotypes of B. melitensis could be isolated from the same animal. The local geographic distribution of genotypes showed a very close genetic relatedness with genotypes reported outside the study area. Worldwide, our genotypes were mostly related to the Western Mediterranean lineage and less likely to the America's clonal lineage. CONCLUSION: There is a high genetic similarity of B. melitensis bv3 genotypes among different ruminant species, and the same animal could be infected with different genotypes. There is a high probability of spreading of B. melitensis among atypical hosts in the absence of the original hosts. The genetic relatedness of B. melitensis bv3 genotypes in the study area with other different geographic areas highlighted the national and international ruminants movement role as a potential factor for maintaining B. melitensis infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Further investigations are required to understand the impact of the presence of more than one genotype of B. melitensis in the same animal on the efficacy of brucellosis control strategies.

Brucella spp. distribution, hosting ruminants from Greece, applying various molecular identification techniques.

BACKGROUND: Brucellosis still remains an endemic disease for both livestock and human in Greece, influencing the primary sector and national economy in general. Although farm animals and particularly ruminants constitute the natural hosts of the disease, transmission to humans is not uncommon, thus representing a serious occupational disease as well. Under this prism, knowledge concerning Brucella species distribution in ruminants is considered a high priority. There are various molecular methodologies for Brucella detection with however differential discriminant capacity. Hence, the aim of this survey was to achieve nationally Brucella epidemiology baseline genotyping data at species and subtype level, as well as to evaluate the pros and cons of different molecular techniques utilized for detection of Brucella species. Thirty-nine tissue samples from 30 domestic ruminants, which were found positive applying a screening PCR, were tested by four different molecular techniques i.e. sequencing of the 16S rRNA, the BP26 and the OMP31 regions, and the MLVA typing panel 1 assay of minisatellite markers. RESULTS: Only one haplotype was revealed from the 16S rRNA sequencing analysis, indicating that molecular identification of Brucella bacteria based on this marker might be feasible solely up to genus level. BP26 sequencing analysis and MLVA were in complete agreement detecting both B. melitensis and B. abortus. An interesting exception was observed in 11 samples, of lower quality extracted DNA, in which not all expected MLVA amplicons were produced and identification was based on the remaining ones as well as on BP26. On the contrary OMP31 failed to provide a clear band in any of the examined samples. CONCLUSIONS: The present study reveals the constant circulation of Brucella bacteria in ruminants throughout Greece. Further, according to our results, BP26 gene represents a very good alternative to MLVA minisatellite assay, particularly in lower quality DNA samples.