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Our preliminary data using bone marrow-derived macrophages (BMDMs) collected from ICR mice treated with anti-sirtuin (anti-SIRT) 1 antibody showed that Brucella uptake was significantly attenuated. We then further investigated the effect of an inhibitor of SIRT1/2, cambinol, in the progression of Brucella. The in vitro results using RAW264.7 cells revealed that cambinol treatment had no effect on adhesion, uptake, intracellular survival and nitric oxide (NO) production during B. abortus infection, nor did it directly affect bacterial growth for up to 72 h. Finally, intraperitoneal treatment of 8-week-old female ICR mice infected with Brucella showed no differences in the total average weights of spleens and livers; however, the treated mice displayed higher Brucella colony-forming units (CFUs) from the spleens. Furthermore, the interleukin (IL)-10 serum level was observed to be lower in treated mice at 7 d post-infection, and none of the cytokines tested showed a change at 14 d post-infection. The overall findings showed that cambinol treatment had no effect on the proliferation of Brucella in RAW264.7 macrophages but exacerbated the splenic proliferation of the bacteria in mice and displayed reduced anti-inflammatory cytokine IL-10 at the first week of infection, suggesting that cambinol as an inhibitory of SIRT1/2 could be beneficial in the context of Brucella dissemination in animal hosts and that exploration of activating SIRTs could be an alternative treatment against Brucella infection. [ABSTRACT FROM AUTHOR]

Brucellosis is an infectious bacterial zoonotic disease of domestic and wild animals frequently caused by Brucella abortus, B. ovis, B. melitensis, B. suis, and B. canis species. The present study was conducted on 100 serum samples from swine with a history of abortion and reproductive failure in the southern states of India. The samples were screened by Rose Bengal plate agglutination test (RBPT) and standard tube agglutination test (STAT) for detection and quantification of brucella antibody respectively. The samples were also subjected to 16S rRNA gene and Brucella cell surface salt-extractable (BCSP 31) based PCR assays for Brucella genome screening and confirmation. Brucella genome-positive samples were also subjected to AMOS multiplex PCR for B. abortus, B. ovis, B. melitensis, and B. suis species differential diagnosis. The serological screening revealed 3% (n=3) seropositivity for Brucellosis with antibody tires of 1: 160, 1:160, 1:320 for TL7, TL12 and KA-9 samples respectively. Molecular screening evidenced 5% (n=5) positivity for the Brucella genome including three seropositive samples yielded a specific amplicon of 800 and 223 bp amplicons by 16S rRNA and BCSP-31 PCR respectively. Further species identification by AMOS PCR yielded an amplicon of 498 bp specific to B. abortus genome in all three seropositive samples (TL7, TL12, and KA-9) and two PCR-positive samples (TN17 and KA-21) yielded 285bp amplicon specific to B. suis. Molecular screening of Brucella-positive samples for viral co-infections such as PCV2, PPV, CSFV, and PRRSV evidenced non-involvement of above screened 4 viral aetiologies in all 5 Brucella-positive samples indicating the primary infection of Brucella species.

Abstract Background Brucellosis, a zoonotic disease in Türkiye, which has significant direct and indirect impacts on the healthcare system and livestock. This study, which aimed to investigate the differences among Brucella spp. isolates originating from different regions of Türkiye, for implications for public health and veterinary medicine. Method Twenty-one isolates from ruminants and two isolates from humans obtained from various regions of Türkiye were utilized in the study. The isolates were identified and biotyped using traditional microbiological procedures, and whole-genome sequencing (WGS) was performed. This was followed by single nucleotide polymorphism (SNP)--based phylogenetic analysis and WGS-based analysis of virulence and resistance genes. Additionally, phenotypic antimicrobial resistance and phage susceptibilities were determined. The obtained data were then compared for concordance, ensuring the validity and reliability of the results. Results Our study, employing culture methods, polymerase chain reaction (PCR), and WGS analyses, identified 11 Brucella melitensis (bv 3 (n = 9), one each bv 1 and bv 2) and 12 B. abortus (bv 3 (n = 11), bv 9 (n = 1)) isolates All B. abortus isolates were of bovine origin, while the B. melitensis isolates were from sheep (n = 7), goat (n = 1), ram (n = 1), and humans (n = 2). In the whole-genome SNP-based phylogenetic tree, all B. melitensis strains were found to be of the IIb subtype of genotype II associated with the Eastern Mediterranean lineage. Ten different genotypes were identified in the SNP analysis of the isolates, with a maximum SNP difference of 278 and a minimum SNP difference of 4 among these genotypes. According to the WGS-SNP-based phylogenetic tree of B. abortus isolates, they were grouped in clade C1. In the SNP analysis, where ten different genotypes were identified, the SNP difference among these genotypes was a maximum of 316 and a minimum of 6. In the in silico MLST analysis performed with WGS data, B. melitensis isolates were identified as ST8 and ST102 genotypes, while B. abortus isolates were identified as ST2 and ST3 genotypes. The dominant genotypes were ST8 for B. melitensis and ST2 for B. abortus, respectively. Virulence gene analysis conducted based on WGS data of the 23 B. abortus and B. melitensis isolates revealed 43 virulence gene-associated regions in all strains, irrespective of species, host, or isolation year. Although classical resistance-related genes were not detected by WGS-based antimicrobial resistance gene analysis, phenotypic resistance analysis revealed resistance to azithromycin, rifampin, and trimethoprim/sulfamethoxazole in B. abortus and B. melitensis isolates. Conclusion Both B. melitensis and B. abortus were circulating species in animals and human. The dominant genotypes were ST8 for B. melitensis and ST2 for B. abortus, respectively. All B. melitensis strains were found to be of the IIb subtype of genotype II associated with the Eastern Mediterranean lineage, while B. abortus isolates, they were grouped in clade C1. Further, a comprehensive study with a sufficient number of isolates covering all regions of Türkiye would provide more accurate information about the current epidemiological situation in the country.

Background: Brucellosis, a zoonotic disease in Türkiye, which has significant direct and indirect impacts on the healthcare system and livestock. This study, which aimed to investigate the differences among Brucella spp. isolates originating from different regions of Türkiye, for implications for public health and veterinary medicine. Method: Twenty-one isolates from ruminants and two isolates from humans obtained from various regions of Türkiye were utilized in the study. The isolates were identified and biotyped using traditional microbiological procedures, and whole-genome sequencing (WGS) was performed. This was followed by single nucleotide polymorphism (SNP)--based phylogenetic analysis and WGS-based analysis of virulence and resistance genes. Additionally, phenotypic antimicrobial resistance and phage susceptibilities were determined. The obtained data were then compared for concordance, ensuring the validity and reliability of the results. Results: Our study, employing culture methods, polymerase chain reaction (PCR), and WGS analyses, identified 11 Brucella melitensis (bv 3 (n = 9), one each bv 1 and bv 2) and 12 B. abortus (bv 3 (n = 11), bv 9 (n = 1)) isolates All B. abortus isolates were of bovine origin, while the B. melitensis isolates were from sheep (n = 7), goat (n = 1), ram (n = 1), and humans (n = 2). In the whole-genome SNP-based phylogenetic tree, all B. melitensis strains were found to be of the IIb subtype of genotype II associated with the Eastern Mediterranean lineage. Ten different genotypes were identified in the SNP analysis of the isolates, with a maximum SNP difference of 278 and a minimum SNP difference of 4 among these genotypes. According to the WGS-SNP-based phylogenetic tree of B. abortus isolates, they were grouped in clade C1. In the SNP analysis, where ten different genotypes were identified, the SNP difference among these genotypes was a maximum of 316 and a minimum of 6. In the in silico MLST analysis performed with WGS data, B. melitensis isolates were identified as ST8 and ST102 genotypes, while B. abortus isolates were identified as ST2 and ST3 genotypes. The dominant genotypes were ST8 for B. melitensis and ST2 for B. abortus, respectively. Virulence gene analysis conducted based on WGS data of the 23 B. abortus and B. melitensis isolates revealed 43 virulence gene-associated regions in all strains, irrespective of species, host, or isolation year. Although classical resistance-related genes were not detected by WGS-based antimicrobial resistance gene analysis, phenotypic resistance analysis revealed resistance to azithromycin, rifampin, and trimethoprim/sulfamethoxazole in B. abortus and B. melitensis isolates. Conclusion: Both B. melitensis and B. abortus were circulating species in animals and human. The dominant genotypes were ST8 for B. melitensis and ST2 for B. abortus, respectively. All B. melitensis strains were found to be of the IIb subtype of genotype II associated with the Eastern Mediterranean lineage, while B. abortus isolates, they were grouped in clade C1. Further, a comprehensive study with a sufficient number of isolates covering all regions of Türkiye would provide more accurate information about the current epidemiological situation in the country. [ABSTRACT FROM AUTHOR]

Brucellosis is an economically important zoonotic disease affecting humans, livestock, and wildlife health globally and especially in Africa. Brucella abortus and B. melitensis have been isolated from human, livestock (cattle and goat), and wildlife (sable) in South Africa (SA) but with little knowledge of the population genomic structure of this pathogen in SA. As whole genome sequencing can assist to differentiate and trace the origin of outbreaks of Brucella spp. strains, the whole genomes of retrospective isolates (n = 19) from previous studies were sequenced. Sequences were analysed using average nucleotide identity (ANI), pangenomics, and whole genome single nucleotide polymorphism (wgSNP) to trace the geographical origin of cases of brucellosis circulating in human, cattle, goats, and sable from different provinces in SA. Pangenomics analysis of B. melitensis (n = 69) and B. abortus (n = 56) was conducted with 19 strains that included B. abortus from cattle (n = 3) and B. melitensis from a human (n = 1), cattle (n = 1), goat (n = 1), Rev1 vaccine strain (n = 1), and sable (n = 12). Pangenomics analysis of B. melitensis genomes, highlighted shared genes, that include 10 hypothetical proteins and genes that encodes for acetyl-coenzyme A synthetase (acs), and acylamidase (aam) amongst the sable genomes. The wgSNP analysis confirmed the B. melitensis isolated from human was more closely related to the goat from the Western Cape Province from the same outbreak than the B. melitensis cattle sample from different cases in the Gauteng Province. The B. melitensis sable strains could be distinguished from the African lineage, constituting their own African sub-clade. The sequenced B. abortus strains clustered in the C2 lineage that is closely related to the isolates from Mozambique and Zimbabwe. This study identified genetically diverse Brucella spp. among various hosts in SA. This study expands the limited known knowledge regarding the presence of B. melitensis in livestock and humans in SA, further building a foundation for future research on the distribution of the Brucella spp. worldwide and its evolutionary background. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Our preliminary data using bone marrow-derived macrophages (BMDMs) collected from ICR mice treated with anti-sirtuin (anti-SIRT) 1 antibody showed that Brucella uptake was significantly attenuated. We then further investigated the effect of an inhibitor of SIRT1/2, cambinol, in the progression of Brucella. The in vitro results using RAW264.7 cells revealed that cambinol treatment had no effect on adhesion, uptake, intracellular survival and nitric oxide (NO) production during B. abortus infection, nor did it directly affect bacterial growth for up to 72 h. Finally, intraperitoneal treatment of 8-week-old female ICR mice infected with Brucella showed no differences in the total average weights of spleens and livers; however, the treated mice displayed higher Brucella colony-forming units (CFUs) from the spleens. Furthermore, the interleukin (IL)-10 serum level was observed to be lower in treated mice at 7 d post-infection, and none of the cytokines tested showed a change at 14 d post-infection. The overall findings showed that cambinol treatment had no effect on the proliferation of Brucella in RAW264.7 macrophages but exacerbated the splenic proliferation of the bacteria in mice and displayed reduced anti-inflammatory cytokine IL-10 at the first week of infection, suggesting that cambinol as an inhibitory of SIRT1/2 could be beneficial in the context of Brucella dissemination in animal hosts and that exploration of activating SIRTs could be an alternative treatment against Brucella infection.

The aim of the present study was to evaluate the effectiveness of analysis of the high resolution melting curves obtained after amplification of VNTR loci for the identification and differentiation of Brucella strains.Materials and methods. 16 strains of Brucella species – B. canis (n=1), B. abortus (n=9), B. melitensis (n=2), B. suis (n=4) – of different geographical origin were used as objects of the research. The MLVA-typing was performed using conventional PCR followed by separation of amplicons in agarose gel and real-time PCR with post-amplification analysis of the curves of VNTR loci melting in the presence of intercalating dye SybrGreen. Bioinformatics analysis was conducted with the help of Vector NTI 9.1, Mega 11 software (MUSCLE algorithm). Phylogenetic analysis was carried out applying UPGMA method using the Mega 11 program.Results and discussion. MLVA approach based on the analysis of the melting point curves of the obtained after amplification of VNTR-loci PCR fragments has shown that each of the 16 strains of Brucella is characterized by a unique melting temperature profile. PCR followed by electrophoresis has demonstrated that despite the high variability of the used VNTR sequences (h=0.48…0.74), only post-amplification melting curves of the Bru7, Bru9, Bru18, Bru21 loci had sufficient information content to determine the genetic polymorphism of the studied Brucella strains. Based on the results of phylogenetic analysis of the Bru7, Bru9, Bru18, Bru21 sequences, it has been found that the majority of the studied Brucella strains are distributed in the dendrogram in accordance with their taxonomic and geographical position. Thus, HRM analysis of melting curves obtained after amplification of the Bru7, Bru9, Bru18, Bru21 loci has the potential to be used for differentiating Brucella 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.

Abstract 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.

To investigate the incidence of the virulence genes among predominant Brucella strains in infected cattle, buffaloes, sheep, goats, and camels as well as in humans in Egypt, a total of 263 samples (85 milk, 167 tissue, 11 whole blood samples), yielded 140 (53.2%) Brucella isolates. Confirmation of Brucella isolates was carried out by conventional biotyping and by PCR using IR1/IR2 primers tar-geting Brucella genus showing an amplicon of 839 bp in all Brucella isolates. Conventional bioty¬ping, as well as duplex PCR of the isolated non-repetitive Brucella strains, identified 107 (76.4 %) as B. melitensis with an amplicon of 731 bp and 33 (23.6%) as B. abortus with an amplicon of 498 bp. Out of 87 Brucella strains isolated from cattle, 63 (72.4%) were B. melitensis. No Brucella isolates were obtained from 7 lymph nodes of camels or 11 human blood samples; however, DNA extraction from 7 human sera and 3 camel lymph nodes gave positive PCR yield. All these samples gave PCR products indicating infection with B. melitensis. The distribution of the virulence genes among 33 B. abortus isolates revealed that virB recorded the highest incidence (97%), then followed bvfA, ure, and omp25 (93.9%), wbkA (90.9%), manB (87.9%) and amiC (84.8%). All 107 B. melitensis isolates had the bvfA, virB, and omp25 genes, while the prevalence of ure was 99.1%, that of wbkA: 96.3%, manB: 95.3% and amiC: 94.4%. The obtained results indicated the high incidence of virulence genes among field Brucella strains among farm animals in Egypt.

Brucellosis is an important bacterial zoonosis responsible for considerable economic losses in livestock and health-related burden worldwide. The objective of this study was to determine the seroprevalence of brucellosis in communal and smallholder cattle farming in four districts of the North West province of South Africa (Dr Ruth Segomotsi Mompati, Ngaka Modiri Molema, Bojanala platinum and Dr Kenneth Kaunda districts). Seven hundred and seventy blood samples from farmed animals (n = 378) and abattoir-slaughtered animals (n = 392) were collected. In addition, milk samples (n = 22) were collected from lactating farmed cows. Rose Bengal test (RBT), complement fixation test (CFT) and milk ring test (MRT) were used to detect antibodies against Brucella species. The RBT showed a seroprevalence of 2% at 95% confidence interval (CI: 1.35–3.35), CFT confirmed an overall seroprevalence of 1.95% (95% CI: 1.14–3.12) for all four districts sampled. Although the seroprevalence of brucellosis was found to be low, the possibility of undetected cases of the disease could not be ruled out. Overall, the findings of this study confirmed that brucellosis is endemic in the surveyed areas of the North West province of South Africa. Contribution: The outcome of this study will contribute to the National Brucellosis Project organised by the Department of Agriculture, Land Reform and Rural Development (2016–2026) to assist in the effective implementation of the disease control measures with a view to prevent further outbreaks in the country’s cattle population.

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. [ABSTRACT FROM AUTHOR]

Background: Brucellosis is one of the most important zoonotic diseases caused by Gram-negative bacteria belonging to the genus Brucella. Detection of Brucella species in different countries is of utmost importance. Aims: This study aimed to detect Brucella abortus and Brucella melitensis in domestic ruminant blood samples and their ticks in western Iran. Methods: Sampling was conducted on ruminants from August to September 2020 in four different counties of Kurdistan Province, including Divandareh, Marivan, Baneh, and Sanandaj. Totally, 250 blood samples were collected from 250 small ruminants. There were no ticks on the skin of six (2.4%) ruminants, and 244 ticks were isolated from 244 animals. After genomic DNA extraction from all the collected samples, quantitative polymerase chain reaction (qPCR) was performed to detect IS711 gene. Results: Based on qPCR results, Brucella genus was detected in two blood samples (0.8%) from female sheep and four ticks (1.6%) from male sheep, including three Dermacentor marginatus (1.22%) and one Rhipicephalus turanicus (0.4%). Although B. melitensis was not detected in any tick or blood sample, one tick sample (D. marginatus) was positive for B. abortus. Conclusion: Considering the positivity of ticks for brucellosis in this study, there is a possibility of Brucella transmission from infected ticks to humans and animals through tick bites, nevertheless, in order to identify the Brucella transmission relationship between ticks and animals, serological tests should be used in future studies. [ABSTRACT FROM AUTHOR]

La Brucelosis es una enfermedad zoonótica extendida a nivel mundial en el ganado bovino, ocasionada principalmente por Brucella abortus, previamente reportada en ciertas regiones del Ecuador. La caracterización de las cepas circulantes de Brucella spp. en ganaderías lecheras resulta importante para comprender la epidemiología de esta enfermedad. La tipificación bacteriológica de la Brucella spp. es un proceso lento, riesgoso y requiere de laboratorios especializados. El objetivo de este estudio fue tipificar las cepas de Brucella spp. que afectan al ganado bovino en la provincia del Azuay, mediante ensayos moleculares, a partir de muestras de sangre y leche de vacas seropositivas a brucelosis. En fincas seropositivas a ELISA-Indirecto en leche, se seleccionaron 70 vacas Holstein mestizas, reactoras individualmente a las pruebas Rosa de Bengala y confirmadas con ELISA competitivo. Se extrajo el ADN de esas muestras de sangre y leche confirmando en un inicio la viabilidad del material genético de bovino con oligonucleótidos específicos para el género Bos. La amplificación de ADN para Brucella spp. se realizó por PCR-AMOS con cebadores de genero para la región IS711 y de especie para Brucella abortus, Brucella mellitensis, Brucella suis y Brucella ovis. Se pudo identificar ADN bovino en 65 (92,8 %) muestras de leche y en 62 (88,5 %) muestras de sangre. Un total de 62 muestras de ADN extraído de leche resultaron positivas (95,4 %) al género Brucella spp. y todas las muestras de sangre resultaron negativas. El PCR-AMOS mostró bandas con un peso molecular de 498 pb en muestras de cuatro animales correspondiente a B. abortus. Este es el primer estudio de identificación molecular en la provincia del Azuay con evidencia científica de la especie de Brucella spp. circulante en las ganaderías bovinas de la zona, contribuyendo de base para la identificación futura de los biovares de B. abortus aún no reportados en esta zona del país.

Brucellosis is a widely prevalent zoonotic disease of major public health significance. A collection of Brucella melitensis and Brucella abortus field isolates of animal and human origin were subjected to MLVA-15 typing followed by phylogeography studies. The MLVA-15 analysis of B. melitensis (n = 65) field isolates resulted in 48 different profiles. The panel I marker bruce45 was found to be most conserved, while the rest of the panel I markers showed low to moderate length polymorphism. Among the panel II markers, bruce04, bruce16 and bruce30 showed a high discriminatory index. The MLVA-15 typing of 13 B. abortus field isolates revealed 13 different genotypes with panel II markers showing higher discriminatory ability vis-à-vis panel I. The minimum spanning tree analysis (MST) in comparison with isolates from the international database revealed that all B. melitensis and B. abortus isolates from this study belonged to the 'Eastern Mediterranean' and the 'abortus C' lineage, respectively. The MLVA-15 typing could differentiate field isolates of B. abortus and B. melitensis originating from different regions, reaffirming the technique's potential of high resolution and suitability for local epidemiological studies. The MLVA scheme also has the advantage of comparison of local isolates with a worldwide database, allowing for phylogeographical studies. [ABSTRACT FROM AUTHOR]

Brucella is the causative agent of brucellosis and can be transmitted to humans through aerosolized particles or contaminated food. Brucella abortus (B. abortus), Brucella melitensis (B. melitensis), and Brucella suis (B. suis) are the most virulent of the brucellae , but the traditional detection methods to distinguish them are time-consuming and require high instrumentation. To obtain epidemiological information on Brucella during livestock slaughter and food contamination, we developed a rapid and sensitive triplex recombinant polymerase amplification (triplex-RPA) assay that can simultaneously detect and differentiate between B. abortus, B. melitensis , and B. suis. Three pairs of primers (B1O7F/B1O7R, B192F/B192R, and B285F/B285R) were designed and screened for the establishment of the triplex-RPA assay. After optimization, the assay can be completed within 20 min at 39°C with good specificity and no cross-reactivity with five common pathogens. The triplex-RPA assay has a DNA sensitivity of 1–10 pg and a minimum detection limit of 2.14 × 104–2.14 × 105 CFU g−1 in B. suis spiked samples. It is a potential tool for the detection of Brucella and can effectively differentiate between B. abortus, B. melitensis , and B. suis S2, making it a useful tool for epidemiological investigations. [ABSTRACT FROM AUTHOR]

Brucellosis is an important bacterial zoonosis responsible for considerable economic losses in livestock and health-related burden worldwide. The objective of this study was to determine the seroprevalence of brucellosis in communal and smallholder cattle farming in four districts of the North West province of South Africa (Dr Ruth Segomotsi Mompati, Ngaka Modiri Molema, Bojanala platinum and Dr Kenneth Kaunda districts). Seven hundred and seventy blood samples from farmed animals (n = 378) and abattoir-slaughtered animals (n = 392) were collected. In addition, milk samples (n = 22) were collected from lactating farmed cows. Rose Bengal test (RBT), complement fixation test (CFT) and milk ring test (MRT) were used to detect antibodies against Brucella species. The RBT showed a seroprevalence of 2% at 95% confidence interval (CI: 1.35-3.35), CFT confirmed an overall seroprevalence of 1.95% (95% CI: 1.14-3.12) for all four districts sampled. Although the seroprevalence of brucellosis was found to be low, the possibility of undetected cases of the disease could not be ruled out. Overall, the findings of this study confirmed that brucellosis is endemic in the surveyed areas of the North West province of South Africa. Contribution: The outcome of this study will contribute to the National Brucellosis Project organised by the Department of Agriculture, Land Reform and Rural Development (2016-2026) to assist in the effective implementation of the disease control measures with a view to prevent further outbreaks in the country's cattle population. [ABSTRACT FROM AUTHOR]

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Brucellosis is a common zoonotic disease in Iran. Antimicrobial-resistant (AMR) Brucella isolates have been reported from different developing countries, posing an imminent health hazard. The objective of this study was to evaluate AMR and virulence-associated factors in Brucella isolates recovered from humans and animals in different regions of Iran using classical phenotyping and next generation sequencing (NGS) technology. Our findings revealed that B. melitensis is the most common species in bovines, small ruminants and camels. B. abortus was isolated only from one human case. Probable intermediate or resistant phenotype patterns for rifampicin, trimethoprim-sulfamethoxazole, ampicillin-sulbactam and colistin were found. Whole genome sequencing (WGS) identified mprF, bepG, bepF, bepC, bepE, and bepD in all isolates but failed to determine other classical AMR genes. Forty-three genes associated with five virulence factors were identified in the genomes of all Brucella isolates, and no difference in the distribution of virulence-associated genes was found. Of them, 27 genes were associated with lipopolysaccharide (LPS), 12 genes were related to a type IV secretion system (virB1-B12), two were associated with the toll-interleukin-1 receptor (TIR) domain-containing proteins (btpA, btpB), one gene encoded the Rab2 interacting conserved protein A (ricA) and one was associated with the production of cyclic β-1,2 glucans (cgs). This is the first investigation reporting the molecular-based AMR and virulence factors in brucellae isolated from different animal hosts and humans in Iran. Iranian B. abortus and B. melitensis isolates are still in vitro susceptible to the majority of antibiotics used for the treatment of human brucellosis. WGS failed to determine classical AMR genes and no difference was found in the distribution of virulence-associated genes in all isolates. Still, the absence of classical AMR genes in genomes of resistant strains is puzzling, and investigation of phenotypic resistance mechanisms at the proteomic and transcriptomic levels is needed. [ABSTRACT FROM AUTHOR]

Background and Aim: Brucellosis is a zoonotic disease which has become endemic in Iran. Contaminated milk with Brucella bacteria is the main way of transmission of this disease in humans. Therefore, the aim of this study was to investigate the prevalence of Brucella spp. and B. abortus in raw milk samples collected from farms belongs to six geographical areas of Lorestan province. Materials and Methods: In the present study, 100 raw milk samples of cows which were less than 4 years, 4 to 6 years and over 6 years old were randomly collected. The isolates were identified by PCR method using specific bcsp31 and IS711 primers for Brucella spp. and B. abortus, respectively. Results & Conclusion: The results showed that 26 of the samples were infected with Brucella bacteria, of which 19 samples (73%) were B. abortus. Most of the brucellosis infection in cows belonged to cows less than 4 years (31.4%) and 4 to 6 years (31.4%) categories, 11 samples in both groups, so that in cows older than 6 years this rate was 13% (4 samples). The east of the province with 12 samples and the northwest of the province with one sample showed the highest and lowest levels of Brucella infection, respectively. The findings demonstrated that there is a significant difference between the frequency of Brucella contamination in milk in the eastern regions of Lorestan province with other regions (p<0.05). It should be noted that the possibility of transmitting brucellosis to humans through consumption of contaminated raw milk and dairy products in Lorestan province is high. [ABSTRACT FROM AUTHOR]

A storm of abortions was reported in a Holstein dairy farm (150 heads) at Dakahlia governorate, Delta region, Egypt. The abortion rate was 25.9% among the pregnant cows between the 5th and 8th months of pregnancy. All animals inside the farm (n=150) have been sampled for a serological survey. Additionally, abomasal contents, retained placenta, and milk samples were sampled for bacteriological isolation and characterization of the causative pathogen of abortion. A total of 16.6 % of the aborted animals were seropositive with RBPT and BAPAT. Abortion materials and retained fetal membranes showed significant association with seropositivity. Moreover, B. abortus bv. 1 was bacteriologically isolated and then underwent confirmation by AMOS-PCR in samples of 20 animals. Increase awareness of occupational personnel on the farm, immediate slaughtering of the sero-positive animals, and vaccination of the sero-negative animals with B. abortus RB51 vaccine (2ml subcutaneous for each animal), are collectively recommended for a rapid control of brucellosis on the farm and for prevention of further abortions. [ABSTRACT FROM AUTHOR]

(1) Background: MALDI-TOF mass spectrometry (MS) is the gold standard for microbial fingerprinting, however, for phylogenetically closely related species, the resolution power drops down to the genus level. In this study, we analyzed MALDI-TOF spectra from 44 strains of B. melitensis, B. suis and B. abortus to identify the optimal classification method within popular supervised and unsupervised machine learning (ML) algorithms. (2) Methods: A consensus feature selection strategy was applied to pinpoint from among the 500 MS features those that yielded the best ML model and that may play a role in species differentiation. Unsupervised k-means and hierarchical agglomerative clustering were evaluated using the silhouette coefficient, while the supervised classifiers Random Forest, Support Vector Machine, Neural Network, and Multinomial Logistic Regression were explored in a fine-tuning manner using nested k-fold cross validation (CV) with a feature reduction step between the two CV loops. (3) Results: Sixteen differentially expressed peaks were identified and used to feed ML classifiers. Unsupervised and optimized supervised models displayed excellent predictive performances with 100% accuracy. The suitability of the consensus feature selection strategy for learning system accuracy was shown. (4) Conclusion: A meaningful ML approach is here introduced, to enhance Brucella spp. classification using MALDI-TOF MS data. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Brucellosis is recognized as a major public health concern leading to critical economic losses in livestock animals. The present study assessed Brucella spp. isolated from aborted ovine and caprine fetuses in different parts of Iran between 2016 and 2019. It used classic and molecular methods in order to determine the Brucella species carrying higher risks of abortion complications in these animals. A total of 189 samples from 35 cases/case series from milk (16 sheep, and 8 goats), 19 abomasum content (sheep), and 146 aborted fetuses (116 sheep, and 30 goats) were bacteriologically examined. Subsequently, the resultant Brucella isolates were further characterized by phenotypic and molecular approaches. The multiplex Polymerase chain reaction (PCR) (Bruce-ladder) and IS711-based PCR were performed on all the extracted DNA to evaluate the presence of Brucella spp. As suggested by the obtained results, all recovered isolates from ovine and caprine abortion samples were either B. melitensis or B. abortus. An issue of concern was the implication of B. melitensis vaccine strain Rev1 in a small portion of sheep and goat abortion cases. Despite the recent B. abortus burden in ovine, aborted cases were predominantly associated with B. melitensis infections in both ovine and caprine, and B. melitensis biovar 1 was responsible for the majority of studied cases. These data and the techniques implemented in the present study can shed light on the level of implication of different Brucella species in ovine and caprine abortion in Iran. The present study identified Brucella agents responsible for abortion in small ruminants at the biovar level. Therefore, it provides precious information for future control programs and vaccination strategies in Middle Eastern regions.

Brucellosis is a zoonotic infectious disease with a worldwide distribution. This cross-sectional study aimed to determine the occurrence of Brucella in milk and blood samples of ruminants and the importance factors associated with animal brucellosis in Eastern Iran. A total of 200 paired samples, including blood (100) and milk (100), were obtained from the goats, sheep, and cows in Eastern Iran. Serum agglutination (SAT) and 2-mercapto ethanol (2-ME) tests were performed on the sera. A multiplex-polymerase chain reaction (m-PCR) assay was performed to identify the following species of Brucella, including B. abortus biovar 1, 2, 4, 3b, 5, 6, and 9, B. abortus S19, B. melitensis, and B. melitensis Rev.1 vaccine strain. B. abortus RB51 vaccine strain was also investigated in a second PCR assay. Risk factors for infection with Brucella spp. including the effect of abortion, contact with the wild animals, herd type, age, and previous vaccination in predicting blood contamination with B. abortus biovar 1, 2, and 4 were modeled by use of the artificial neural network. A total of 23 samples were seropositive regarding SAT and 2-ME tests. In total, B. abortus was detected in 35% and 15% of blood and milk samples, respectively, by the m-PCR assay. One sample of each of milk and blood was detected to have B. melitensis. Some samples were simultaneously contaminated with two Brucella species or two biovars of B. abortus. B. abortus S19 and B. melitensis Rev.1 vaccine strains were also detected in milk and blood samples. The sensitivity and specificity of the ANN model were 81% and 62%, respectively. In conclusion, B. abortus had higher frequency than B. melitensis in blood and milk samples. ANN determined herd type, previous vaccination, and age of the animal as the largest predictors of blood contamination with B. abortus. [ABSTRACT FROM AUTHOR]

The RB51 vaccine strain of Brucella abortus, which confers safe and effective protection of cattle from B. abortus infection, was originally generated via serial passage of B. abortus 2308 to generate spontaneous, attenuating mutations. While some of these mutations have been previously characterized, such as an insertional mutation in the wboA gene that contributes to the rough phenotype of the strain, a comprehensive annotation of genetic differences between RB51 and B. abortus 2308 genomes has not yet been published. Here, the whole genome sequence of the RB51 vaccine strain is compared against two available 2308 parent sequences, with all observed single nucleotide polymorphisms, insertions, and deletions presented. Mutations of interest for future characterization in vaccine development, such as mutations in eipA and narJ genes in RB51, were identified. Additionally, protein homology modeling was utilized to provide in silico support for the hypothesis that the RB51 capD mutation is the second contributing mutation to the rough phenotype of RB51, likely explaining the inability of wboA-complemented strains of RB51 to revert to a smooth phenotype.

Brucellosis is a common zoonotic disease in Iran. Antimicrobial-resistant (AMR) Brucella isolates have been reported from different developing countries, posing an imminent health hazard. The objective of this study was to evaluate AMR and virulence-associated factors in Brucella isolates recovered from humans and animals in different regions of Iran using classical phenotyping and next generation sequencing (NGS) technology. Our findings revealed that B. melitensis is the most common species in bovines, small ruminants and camels. B. abortus was isolated only from one human case. Probable intermediate or resistant phenotype patterns for rifampicin, trimethoprim-sulfamethoxazole, ampicillin-sulbactam and colistin were found. Whole genome sequencing (WGS) identified mprF, bepG, bepF, bepC, bepE, and bepD in all isolates but failed to determine other classical AMR genes. Forty-three genes associated with five virulence factors were identified in the genomes of all Brucella isolates, and no difference in the distribution of virulence-associated genes was found. Of them, 27 genes were associated with lipopolysaccharide (LPS), 12 genes were related to a type IV secretion system (virB1-B12), two were associated with the toll-interleukin-1 receptor (TIR) domain-containing proteins (btpA, btpB), one gene encoded the Rab2 interacting conserved protein A (ricA) and one was associated with the production of cyclic β-1,2 glucans (cgs). This is the first investigation reporting the molecular-based AMR and virulence factors in brucellae isolated from different animal hosts and humans in Iran. Iranian B. abortus and B. melitensis isolates are still in vitro susceptible to the majority of antibiotics used for the treatment of human brucellosis. WGS failed to determine classical AMR genes and no difference was found in the distribution of virulence-associated genes in all isolates. Still, the absence of classical AMR genes in genomes of resistant strains is puzzling, and investigation of phenotypic resistance mechanisms at the proteomic and transcriptomic levels is needed.

ABSTRACTIn the present study, a total of 7793 samples from 5 different types of hosts were collected and tested, with a seroprevalence of 2.4% (184/7793). Although the seroprevalence of human and animal brucellosis is relatively low, numbers of human brucellosis cases reported have increased continuously from 2004 to 2018. A total of 118 Brucella strains containing 4 biotypes were obtained, including Brucella melitensis bv.1 (n = 8) and bv.3 (n = 106), Brucella abortus bv.3 (n = 3) and bv.7 (n = 1). Twenty-one shared MLVA-16 genotypes, each composed of 2 to 19 strains obtained from different hosts, suggest the occurrence of a brucellosis outbreak epidemic with multiple source points and laboratory infection events. Moreover, 30 shared MLVA-16 genotypes were observed among 59.6% (68/114) B. melitensis isolates from Zhejiang and strains from other 21 different provinces, especially northern provinces, China. The analysis highlighted the imported nature of the strains from all over the northern provinces with a dominant part from the developed areas of animal husbandry. These data revealed a potential transmission pattern of brucellosis in this region, due to introduced infected sheep leading to a brucellosis outbreak epidemic, and eventually causing multiple laboratory infection events. It is urgent to strengthen the inspection and quarantine of the introduced animals.

(1) Background: MALDI-TOF mass spectrometry (MS) is the gold standard for microbial fingerprinting, however, for phylogenetically closely related species, the resolution power drops down to the genus level. In this study, we analyzed MALDI-TOF spectra from 44 strains of B. melitensis, B. suis and B. abortus to identify the optimal classification method within popular supervised and unsupervised machine learning (ML) algorithms. (2) Methods: A consensus feature selection strategy was applied to pinpoint from among the 500 MS features those that yielded the best ML model and that may play a role in species differentiation. Unsupervised k-means and hierarchical agglomerative clustering were evaluated using the silhouette coefficient, while the supervised classifiers Random Forest, Support Vector Machine, Neural Network, and Multinomial Logistic Regression were explored in a fine-tuning manner using nested k-fold cross validation (CV) with a feature reduction step between the two CV loops. (3) Results: Sixteen differentially expressed peaks were identified and used to feed ML classifiers. Unsupervised and optimized supervised models displayed excellent predictive performances with 100% accuracy. The suitability of the consensus feature selection strategy for learning system accuracy was shown. (4) Conclusion: A meaningful ML approach is here introduced, to enhance Brucella spp. classification using MALDI-TOF MS data.

Brucella abortus is an important zoonotic pathogen that causes severe economic loss to husbandry and poses a threat to human health. The B. abortus A19 live vaccine has been extensively used to prevent bovine brucellosis in China. However, it is difficult to distinguish the serological response induced by A19 from that induced by natural infection. In this study, a novel genetically marked vaccine, A19ΔvirB12, was generated and evaluated. The results indicated that A19ΔvirB12 was able to provide effective protection against B. abortus 2308 (S2308) challenge in mice. Furthermore, the safety and protective efficacy of A19ΔvirB12 have been confirmed in natural host cattle. Additionally, the VirB12 protein allowed for serological differentiation between the S2308 challenge/natural infection and A19ΔvirB12 vaccination. However, previous studies have found that the accuracy of the serological detection based on VirB12 needs to be improved. Therefore, we attempted to identify potential supplementary antigens with differential diagnostic functions by combining label-free quantitative proteomics and protein chip technology. Twenty-six proteins identified only in S2308 were screened; among them, five proteins were considered as potential supplementary antigens. Thus, the accuracy of the differential diagnosis between A19ΔvirB12 immunization and field infection may be improved through multi-antigen detection. In addition, we explored the possible attenuation factors of Brucella vaccine strain. Nine virulence factors were downregulated in A19ΔvirB12. The downregulation pathways of A19ΔvirB12 were significantly enriched in quorum sensing, ATP-binding cassette transporter, and metabolism. Several proteins related to cell division were significantly downregulated, while some proteins involved in transcription were upregulated in S2308. In conclusion, our results contribute to the control and eradication of brucellosis and provide insights into the mechanisms underlying the attenuation of A19ΔvirB12.

Brucella abortus is an important zoonotic pathogen that causes severe economic loss to husbandry and poses a threat to human health. The B. abortus A19 live vaccine has been extensively used to prevent bovine brucellosis in China. However, it is difficult to distinguish the serological response induced by A19 from that induced by natural infection. In this study, a novel genetically marked vaccine, A19ΔvirB12, was generated and evaluated. The results indicated that A19ΔvirB12 was able to provide effective protection against B. abortus 2308 (S2308) challenge in mice. Furthermore, the safety and protective efficacy of A19ΔvirB12 have been confirmed in natural host cattle. Additionally, the VirB12 protein allowed for serological differentiation between the S2308 challenge/natural infection and A19ΔvirB12 vaccination. However, previous studies have found that the accuracy of the serological detection based on VirB12 needs to be improved. Therefore, we attempted to identify potential supplementary antigens with differential diagnostic functions by combining label-free quantitative proteomics and protein chip technology. Twenty-six proteins identified only in S2308 were screened; among them, five proteins were considered as potential supplementary antigens. Thus, the accuracy of the differential diagnosis between A19ΔvirB12 immunization and field infection may be improved through multi-antigen detection. In addition, we explored the possible attenuation factors of Brucella vaccine strain. Nine virulence factors were downregulated in A19ΔvirB12. The downregulation pathways of A19ΔvirB12 were significantly enriched in quorum sensing, ATP-binding cassette transporter, and metabolism. Several proteins related to cell division were significantly downregulated, while some proteins involved in transcription were upregulated in S2308. In conclusion, our results contribute to the control and eradication of brucellosis and provide insights into the mechanisms underlying the attenuation of A19ΔvirB12. [ABSTRACT FROM AUTHOR]

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.

Background: We previously elucidated the protective mechanism of Korean red ginseng oil (RGO) against Brucella abortus infection, and our phytochemical analysis revealed that palmitic acid (PA) was an abundant component of RGO. Consequently, we investigated the contribution of PA against B. abortus. Objectives: We aimed to investigate the efficacy of PA against B. abortus infection using a murine cell line and a murine model. Methods: Cell viability, bactericidal, internalization, and intracellular replication, western blot, nitric oxide (NO), and superoxide (O2 -) analyses and flow cytometry were performed to determine the effects of PA on the progression of B. abortus infection in macrophages. Flow cytometry for cytokine analysis of serum samples and bacterial counts from the spleens were performed to determine the effect of PA in a mouse model. Results: PA did not affect the growth of B. abortus. PA treatment in macrophages did not change B. abortus uptake but it did attenuate the intracellular survivability of B. abortus. Incubation of cells with PA resulted in a modest increase in sirtuin 1 (SIRT1) expression. Compared to control cells, reduced nitrite accumulation, augmented O2 -, and enhanced pro-inflammatory cytokine production were observed in PA-treated B. abortus-infected cells. Mice orally treated with PA displayed a decreased serum interleukin-10 level and enhanced bacterial resistance. Conclusions: Our results suggest that PA participates in the control of B. abortus within murine macrophages, and the in vivo study results confirm its efficacy against the infection. However, further investigations are encouraged to completely characterize the mechanisms involved in the inhibition of B. abortus infection by fatty acids. [ABSTRACT FROM AUTHOR]

Brucellosis is a highly contagious zoonosis caused by a species under the genus Brucella. A duplex recombinase polymerase amplification (Duplex RPA) assay for the specific detection of Brucella melitensis and Brucella abortus was developed in this study. Primers were designed targeting hypothetical protein genes and membrane transporter genes of B. melitensis and B. abortus, respectively. The newly developed assay was validated for its analytical sensitivity and specificity. Different samples were collected from the Qinghai, Inner Mongolia, and Xinjiang provinces. After DNA extraction, the samples were analyzed by Duplex RPA, real-time PCR, and multiplex AMOS PCR to estimate the prevalence of brucellosis in sheep and yak in West China. The analytical sensitivities of Duplex RPA were 9 × 102 plasmid copies of B. melitensis and 9 × 101 plasmid copies of B. abortus, but by mixing the reaction tubes after 4 min of incubation, the sensitivities were 4 × 100 and 5 × 100 copies of B. melitensis and B. abortus, respectively. There was no cross-reactivity with Brucella suis, Chlamydia abortus, Salmonella typhimurium, Escherichia coli, and Toxoplasma gondii. The screening of field samples by Duplex RPA revealed that the prevalence of B. melitensis in sheep and yak was 75.8% and the prevalence of B. abortus was 4.8%. Multiplex AMOS PCR showed that the prevalence of B. melitensis was 19.3%, and that of B. abortus was 4.8%. It was concluded that the developed Duplex RPA is sensitive and specific to the detection of and differentiation between B. melitensis and B. abortus which will be useful in epidemiological surveillance and in the clinical settings.

Abstract Background Brucella causes a chronic and debilitating infection that leads to great economic losses and a public health burden. In this study, we demonstrated the brucellacidal effect of heat shock mediated by the induction of pro-inflammatory cytokines, reactive oxygen species (ROS) accumulation and apoptosis in murine macrophages and in mice. Results RAW264.7 cells were incubated at 43 °C, and BALB/c mice were subjected to whole body hyperthermia. The data showed a reduction in bacterial survival in the mice after daily heat exposure. This was accompanied by increased levels of cytokines TNF, IL-6, IL-1β and IFN-γ in the sera of the mice. Gene expression of NF-κB and inducible nitric oxide production were also induced in the mouse splenic cells. In parallel with the bacterial reduction in the mouse model, an increased bactericidal effect was observed in RAW264.7 cells after exposure to heat stress. In addition, the heat stress increased both the nuclear translocation of NF-κB and the expression of the heat shock proteins HSP70 and HSP90 in murine macrophages. Furthermore, heat exposure induced the increase of pro-inflammatory cytokines, ROS accumulation and apoptosis but did not affect the production of nitric oxide (NO) in macrophages. Conclusion This study demonstrated the induction of innate immune responses by heat stress that significantly reduced the intracellular survival of B. abortus in vitro and in vivo. Transcriptional factor NF-κB, which is a master regulator, could be termed a key activator of heat-induced immunity against Brucella. The increase in the expression and activation of NF-κB in splenic cells and macrophages was followed by enhanced antimicrobial effectors, including cytokines, ROS and NO that may contribute to the reduction of bacterial survival.

Copyright of Archives of Razi Institute is the property of Institut Razi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Brucella infection is most evidently manifested in abortion. This investigation involved aborted samples of 190 animals and 170 human patients. Clinical, bacteriological, serological and molecular assays were performed on these samples for the identification of Brucella infection. Rose Bengal (RB) and Enzyme-Linked Immunosorbent Assay (ELISA) tests were adopted as serological investigations to screen infection(s). A comparative account of results of the RB and ELISA tests have been presented. Aborted fetuses, vaginal emissions and milk samples were used for identifying and collecting Brucella samples. About seven and three isolates were recovered from aborted tissues of cows, respectively. B4 and B5 primers were shown amplify a 223-bp fragment in isolates of Brucella. The presence of brucellosis was confirmed by using (ELISA) technique, a valuable serological test. To conclude, RB and ELISA combined tests are to be necessarily conducted to screen brucellosis, although other tests such as molecular byPolymerase chain reaction (PCR) investigations are efficient evaluator tests that may be performed for verification of brucellosis disease. [ABSTRACT FROM AUTHOR]

Brucellosis is a highly contagious disease that affects public health and wildlife species worldwide. The efficient determination is required to reduce the transmission of infection in human and animals. In this research, a new nano-biosensor was developed for diagnosis of Brucella abortus (B. abortus) bacteria based on graphene quantum nanoparticles (GQNPs), magnetic nanoparticles (MNPs) and polyclonal antibody. First, GQNPs and MNPs were synthesized and conjugated with B. abortus polyclonal antibody (named here IgG 1 and IgG 2 , respectively). The morphology and size of the synthesized nanoparticles were characterized using SEM, DLS and FTIR. Then, the GQNP-IgG 1 and MNP-IgG 2 conjugates were added to B. abortus to form a sandwich structure. After isolating the sandwiches by a magnet, the detection method was approved successfully using a fluorescence spectrophotometer as a quick and precise procedure for detection of B. abortus. [Display omitted] • A fluorescence nanobiosensor based on GQNPs and MNPs was developed for detection of Brucella abortus. • The detection limit was 37 CFU ml−1, Y= 9.178x + 10.714 and R2: 0.9988. • Sensitivity and specificity were determined by a fluorescence spectrophotometry with linearity 4.6 × 101 up to 4.6 × 108. • Detection by a fluorescence spectrophotometry utilizing the fluorescence properties of GQNPs. [ABSTRACT FROM AUTHOR]

Bovine brucellosis is a zoonotic disease of global public health and economic importance. South Africa has had a national bovine brucellosis eradication scheme since 1979; however, no published report on elimination progress from any province exists. We analysed laboratory test results of all cattle herds participating in the Gauteng Provincial Veterinary Services’ eradication scheme between 2013 and 2018. Herd reactor status and within-herd seroprevalence, modelled using mixed-effects logistic and negative binomial regression models, respectively, showed no significant change over the period. However, provincial State Vet Areas, Randfontein (OR = 1.6; 95% CI: 1.2–2.1; p < 0.001) and Germiston (OR = 1.9; 95% CI: 1.5–2.5, p = 0.008) had higher odds of reactor herds than the Pretoria Area and within-herd prevalence count ratios for these areas were 1.5-fold greater than the Pretoria State Vet Area (p < 0.001). Reactor herds were associated with increased herd size (p < 0.001) and larger herd sizes were associated with lower within-herd prevalence (p < 0.001). Despite no evidence of significant progress toward bovine brucellosis elimination in Gauteng province, variability in bovine brucellosis prevalence between State Vet Areas exists. A public health and farmer-supported strategy of ongoing district-based surveillance and cattle vaccination targeting small- to medium-sized herds combined with compulsory test and slaughter of reactors in larger herds is recommended for the province.

Brucellosis in humans is under-detected and underreported in sub-Saharan Africa. Risk factors associated with Brucella infection and health seeking behaviour in response to brucellosis-like symptoms, amongst cattle farm workers and veterinary officials in South Africa, are unknown. Farm workers and veterinary officials (N = 230) were screened for brucellosis using commercial Rose Bengal Test (RBT®), IgM Enzyme-linked Immunoassay (ELISA)®, IgG ELISA® and the BrucellaCapt® test. Knowledge of brucellosis and risk factors for exposure to Brucella were also investigated. Seroprevalence varied according to test used: 10.1% (RBT®), 20.9% (IgG ELISA®) and 6.5% (BrucellaCapt®). Only 22.2% (6/27) of veterinary officials opt to visit a clinic, doctor, or hospital in response to self-experienced brucellosis-like symptoms, compared to 74.9% (152/203) of farm workers (p < 0.001). Of the BrucellaCapt® seropositive participants, 53% (7/15) did not visit a clinic in response to brucellosis-like symptoms. Weak evidence of an association between the handling of afterbirth or placenta and infection of a short evolution (RBT®, IgM ELISA® and IgG ELISA® seropositive) was found (OR = 8.9, 95% CI: 1.0–81.1, p = 0.052), and strong evidence of an association between this outcome and the slaughter of cattle (OR = 5.3, 95% CI: 1.4–19.6, p = 0.013). There was strong evidence of a positive association between inactive/resolved infection and veterinary officials vs. farm workers exposed to seropositive herds (OR = 7.0, 95% CI: 2.4–20.2, p < 0.001), with a simultaneous negative association with the handling of afterbirth or placenta (OR = 3.9, 95% CI: 1.3–11.3, p = 0.012). Findings suggest a proportion of undetected clinical cases of brucellosis amongst workers on cattle farms in Gauteng.

The purposes of this study were to determine phenotypic and genotypic characteristics of Brucella isolates from the Republic of Kazakhstan and to determine their biotype. The focus was laid on culture-morphological, biochemical, and biological properties of 59 Brucella isolates from primary cultures. Material was isolated from blood and tissue of serum-positive killed, dead diseased, or aborted domestic cattle from different regions of Kazakhstan where brucellosis is a common problem. Multiple-locus variable number tandem repeat analysis (MLVA) of all strains, isolated in different regions, has shown that Brucella isolates from the epizootic form two clusters. Based on the comparison with strains available in the MLVA database, B. abortus 0015/B is alike the B. abortus strains isolated from Italy and Portugal. B. melitensis 0016/B isolated from the Almaty region fits the third cluster and is alike the B. melitensis strains isolated from humans in Turkey, China, and Portugal. More than 90% of the overall B. abortus samples were isolated from the northern regions of the East and West Kazakhstan, while B. melitensis strains were registered in the southeast Kazakhstan. The most frequently recorded B. abortus biovar is biovar 3. The most frequently recorded B. melitensis biovars are biovars 1 and 3. Significance and impact of study: These results contribute to a better understanding of the geographic pattern of Brucella infection in Kazakh cattle also important for developing the specific control measures. The results of current research can be used for creating a gene bank of Brucella strains circulating in Kazakhstan for producing diagnostic and therapeutic agents. The research material will be used to solve the problems of genetic characterization of Brucella species and to establish the phylogenetic relationships of strains. [ABSTRACT FROM AUTHOR]

• Interference of IL-1α augmented susceptibility of RAW 264.7 cells to Brucella. • IL-1α signaling was IL-1R/MyD88-dependent during infection. • IL-1α signaling was associated with increased lysosomal-mediated killing and NO. • IL-1β and TNF were dependent on several transcription factors. • Phagolysosomal genes were dependent on c-Jun and CEBPA. The interleukin-1 (IL-1) family of cytokines, particularly IL-1α and IL-1β, are potent regulators of innate immunity that play key roles in host defense against infection, hence we evaluated the role of these cytokines in the control of brucellosis within RAW 264.7 cells. Marked expression and secretion of IL-1α and IL-1β were observed during Brucella infection in macrophages. Blocking of IL-1α and IL-1β reduced induction of IL-10, IL-1β and TNF, and IL-6 and TNF, respectively. However, interference of IL-1α and not IL-1β signaling notably augmented susceptibility of macrophages to Brucella infection which indicates that IL-1α is required for a downstream signaling cascade of innate immunity for efficient clearance of Brucella. This protection requires binding to interleukin-1 receptor (IL-1R) mediated by myeloid differentiation factor 88 (MyD88) signaling and associated with increased lysosomal-mediated killing and nitric oxide (NO) production. Expression of pro-inflammatory cytokines was observed to be mediated via NF-κB-p50, HIF-1α and CEBPA, but negatively controlled by CEBPB while transcription of some important phagolysosomal genes was regulated via CEBPA and c-Jun which indicates the important role of these transcription factors in the control of Brucella infection in macrophages via IL-1α signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Background Brucellosis is a debilitating zoonotic disease affecting humans and animals. A comprehensive, evidence-based assessment of literature and officially available data on animal and human brucellosis for Kenya are missing. The aim of the current review is to provide frequency estimates of brucellosis in humans, animals and risk factors for human infection, and help to understand the current situation in Kenya. Methods A total of accessible 36 national and international publications on brucellosis from 1916 to 2016 were reviewed to estimate the frequency of brucellosis in humans and animals, and strength of associations between potential risk factors and seropositivity in humans in Kenya. Results The conducted studies revealed only few and fragmented evidence of the disease spatial and temporal distribution in an epidemiological context. Bacteriological evidence revealed the presence of Brucella (B.) abortus and B. melitensis in cattle and human patients, whilst B. suis was isolated from wild rodents only. Similar evidence for Brucella spp infection in small ruminants and other animal species is unavailable. The early and most recent serological studies revealed that animal brucellosis is widespread in all animal production systems. The animal infection pressure in these systems has remained strong due to mixing of large numbers of animals from different geographical regions, movement of livestock in search of pasture, communal sharing of grazing land, and the concentration of animals around water points. Human cases are more likely seen in groups occupationally or domestically exposed to livestock or practicing risky social-cultural activities such as consumption of raw blood and dairy products, and slaughtering of animals within the homesteads. Many brucellosis patients are misdiagnosed and probably mistreated due to lack of reliable laboratory diagnostic support resulting to adverse health outcomes of the patients and routine disease underreporting. We found no studies of disease incidence estimates or disease control efforts. Conclusion The risk for re-emergence and transmission of brucellosis is evident as a result of the co-existence of animal husbandry activities and social-cultural activities that promote brucellosis transmission. Well-designed countrywide, evidence-based, and multidisciplinary studies of brucellosis at the human/livestock/wildlife interface are needed. These could help to generate reliable frequency and potential impact estimates, to identify Brucella reservoirs, and to propose control strategies of proven efficacy.

Brucellosis is a common zoonotic disease in Egypt. However, there are limited data available on the genetic diversity of brucellae circulating in Egypt and other Mediterranean areas. One hundred and nine Brucella (B.) strains were isolated from different animal species in thirteen Egyptian governorates. Multi-locus variable number tandem repeats (VNTRs) analysis (MLVA-16) was employed to determine the geographical relatedness and the genetic diversity of a panel of selected Egyptian strains (n = 69), with strains originating from Italy (n = 49), Portugal (n = 52), Greece (n = 63), and Tunisia (n = 4). Egyptian B. melitensis strains clustered into two main clusters containing 21 genotypes. Egyptian B. abortus strains clustered into three main clusters containing nine genotypes. The genotypes were irregularly distributed over time and space in the study area. Egyptian strains of B. melitensis showed MLVA-16 patterns closer to that of Italian strains. Egyptian B. abortus strains isolated from cattle share the same genotype with strains from Portugal and similar to strains from Italy with low genetic diversity. Strains with similar MLVA patterns isolated from different governorates highlight the movement of the pathogen among governorates. Hence, it may also reflect the long endemicity of brucellosis in Egypt with earlier dispersal of types and great local genetic diversity. Open markets may contribute to cross-species transmission and dissemination of the new types nationwide. The presence of West Mediterranean lineages of B. melitensis and relatedness of B. abortus strains from the studied countries is a result of the socio-historical connections among the Mediterranean countries. Transnational eradication of brucellosis in the Mediterranean basin is highly demanded.

Brucella melitensis and Brucella abortus are of the most important causes of brucellosis, an infectious disease which is transmitted either directly or indirectly including consuming unpasteurized dairy products. Both strains are considered endemic in Iran. Common diagnostic methods such as bacteriologic cultures are difficult and time consuming regarding the bacteria. The aim of this study was to suggest a single-stage PCR method using a pair of primers to detect both B. melitensis and B. abortus. The primers were named UF1 and UR1 and the results showed that the final size of PCR products were 84 bp and 99 bp for B. melitensis and B. abortus, respectively. Therefore the method could be useful for rapid detection of B. melitensis and B. abortus simultaneously.

This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection.