The aims of this study were to address the protective immune response induced by S19 vaccination (n=10) and RB51 revaccination, in pregnant (n=9) and non-pregnant (n=10) S19 calfhood-vaccinated cattle as follows: evaluate the in vitro CD4(+) and CD8(+) T-lymphocytes specific proliferation, and in vitro expression of IFN-? by CD4(+) and CD8(+) T-cells and IL-4 by CD4(+), CD8(+) and CD21(+) lymphocytes subset. Upon in vitro stimulation with ?-irradiated Brucella abortus 2308, blood mononuclear cells from S19 vaccinated and RB51 revaccinated cows exhibited significantly higher proliferation of CD4(+) and CD8(+) T-lymphocytes and CD4(+)IFN-?(+) T-cells compared to non-vaccinated animals. RB51 revaccination, regardless of the pregnancy status, did not enhance the proliferation of CD4(+) or CD8(+) T-cells nor IFN-? or IL-4 production. Data from the present study suggest that cattle's cellular immune response induced after brucellosis vaccination and revaccination is due to CD4(+) and CD8(+) T-lymphocytes, being CD4(+) T-cells the main source of IFN-?.
This prospective longitudinal study investigated the epidemiology of enteric disease associated with infections in calves aging up to 70 days. A total of 850 fecal samples were collected from 67 calves. Seventeen isolates of Salmonella spp. were recovered from feces of 11 calves (16.4 %), and statistical analysis revealed no association between the presence of Salmonella spp. and clinical signs of diarrhea or age. Virulence factors of Escherichia coli were identified in 103 strains: eae (7), K99/STa (7), Stx1 (7), Stx1/eae (36), Stx1/Stx2/eae (2), Stx2 (43), and Stx2/eae (1). There was statistical association between diarrheic animals carrying E. coli Stx1/eae (+) in their feces at 2 and 4 weeks of age (P?=?0.003) and E. coli Stx2 (+) at 5 weeks of age (P?=?0.03). Rotavirus was detected in 49 (5.76 %) fecal samples collected from 33 calves (49.2 %). The presence of rotavirus was correlated with diarrheic feces (P?0.0001) rather than feces with normal consistency. There was a significant relationship between age group and diarrhea (P?=?0.001). Bovine coronavirus (BCoV) was detected in 93 fecal samples collected from 46 calves (68.6 %). There was an association (P?0.0001) between diarrheic animals positive for BCoV and age groups. The results demonstrate the importance of the pathogens studied in the etiology of diarrhea in calves.
Mycobacterium tuberculosis complex (MTC) comprises a group of bacteria that have a high degree of genetic similarity. Two species in this group, Mycobacterium tuberculosis and Mycobacterium bovis, are the main cause of human and bovine tuberculosis, respectively. M. bovis has a broader host range that includes humans; thus, the differentiation of mycobacterium is of great importance for epidemiological and public health considerations and to optimize treatment. The current study aimed to evaluate primers and molecular markers described in the literature to differentiate M. bovis and M. tuberculosis by PCR. Primers JB21/22, frequently cited in scientific literature, presented in our study the highest number of errors to identify M. bovis or M. tuberculosis (73%) and primers Mb.400, designed to flank region of difference 4 (RD4), were considered the most efficient (detected all M. bovis tested and did not detect any M. tuberculosis tested). Although also designed to flank RD4, primers Mb.115 misidentified eight samples due to primer design problems. The results showed that RD4 is the ideal region to differentiate M. bovis from other bacteria classified in MTC, but primer design should be considered carefully.
Ovine epididymitis is predominantly associated with Brucella ovis infection. Molecular characterization of Brucella spp. achieved by multi-locus variable number of tandem repeats (VNTR) analyses (MLVA) have proved to be a powerful tool for epidemiological trace-back studies. Thus, the aim of this study was to evaluate the genetic diversity of Brucella ovis isolates from Rio Grande do Sul State, Brazil, by MLVA16.
Brucellosis caused by Brucella abortus is one of the most important zoonoses in the world. Multiple-locus variable-number tandem repeat analysis (MLVA16) has been shown be a useful tool to epidemiological traceback studies in B. abortus infection. Thus, the present study aimed (i) to evaluate the genetic diversity of B. abortus isolates from a brucellosis outbreak, and (ii) to investigate the in vivo stability of the MLVA16 markers.
The aim of this study was to evaluate the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis isolates from eight different hosts in twelve countries. Ninety-nine C. pseudotuberculosis field strains, one type strain (ATCC 19410T) and one vaccine strain (1002) were fingerprinted using the ERIC-1R and ERIC-2 primers, and the ERIC-1R+ERIC-2 primer pair. Twenty-nine different genotypes were generated by ERIC 1-PCR, 28 by ERIC 2-PCR and 35 by ERIC 1+2-PCR. The discriminatory index calculated for ERIC 1, ERIC 2, and ERIC 1+2-PCR was 0.89, 0.86, and 0.92, respectively. Epidemiological concordance was established for all ERIC-PCR assays. ERIC 1+2-PCR was defined as the best method based on suitability of the amplification patterns and discriminatory index. Minimal spanning tree for ERIC 1+2-PCR revealed three major clonal complexes and clustering around nitrate-positive (biovar Equi) and nitrate-negative (biovar Ovis) strains. Therefore, ERIC 1+2-PCR proved to be the best technique evaluated in this study for genotyping C. pseudotuberculosis strains, due to its usefulness for molecular epidemiology investigations.
The aims of the present study were (i) to assess the in vitro genetic stability of S19 and RB51 Brucella abortus vaccines strains and (ii) to evaluate the ability of multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) as a tool to be used in the quality control of live vaccines against brucellosis. Sixty-three batches of commercial S19 (n=53) and RB51 (n=10) vaccines, produced between 2006 and 2009, were used in this study. S19 and RB51 vaccines were obtained from, respectively, seven and two different manufacturers. Ten in vitro serial passages were performed on reference strains and on selected batches of commercial vaccines. All batches, reference strains and strains of serial passages were typed by the MLVA16. The results demonstrated that B. abortus S19 and RB51 vaccine strains are genetically stable and very homogeneous in their respective groups. Anyway, batches of S19 from one manufacturer and batches of RB51 from another presented genotypes distincts from the reference vaccine strains. In both cases, differences were found on locus Bruce07, which had addition of one repeat unit in the case of S19 batches and the deletion of one repeat unit in the case of RB51 batches. In summary, MLVA16 proved to be a molecular tool capable of discriminating small genomic variations and should be included in in vitro official tests.
Live attenuated Brucella abortus S19 is the most effective vaccine against brucellosis in cattle. The assessment of the immunological parameters is essential to guarantee the biological quality of live anti-bacteria vaccines. The evaluation of genetic stability of live bacterial vaccines is also important in quality control. The aims of the present study were to compare (i) the immunogenicity and residual virulence, and (ii) the genotypic profile (MLVA15) of the eight S19 vaccines commercialized in Brazil to the USDA S19 reference strain. Two batches of each of the eight S19 commercial vaccines used in Brazil (A-H) were tested. They were submitted to the potency and residual virulence in vivo tests recommended by OIE and typed by the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) described for Brucella spp. Our results demonstrated that all S19 vaccines commercialized in Brazil would be approved by Brazilian and OIE recommendations for potency and residual virulence. Furthermore, the S19 vaccine is genetically very homogeneous, as all but two batches (from the same manufacturer) tested showed identical MLVA15 profile. The two batches with different profiles presented six repeat units in locus Bruce07, instead of the five found in all other strains, including the USDA S19 reference strain. Although presenting a slightly different profile, this vaccine was also protective, as demonstrated by the immunogenicity and residual virulence assays performed. Therefore, the commercial Brazilian S19 vaccines were in accordance to Brazilian and international standards for immunogenicity and residual virulence tests. Moreover, our results also show that MLVA could be a useful inclusion to the list of in vitro tests required by the official control authorities to be applied to the commercial S19 vaccines, as an efficient assay to guarantee the quality and stability of the vaccine strains.
Brucellosis is a worldwide distributed zoonosis that causes important economic losses to animal production. In Brazil, information on the distribution of biovars and genotypes of Brucella spp. is scarce or unavailable. This study aimed (i) to biotype and genotype 137 Brazilian cattle isolates (from 1977 to 2008) of B. abortus and (ii) to analyze their distribution. B. abortus biovars 1, 2 and 3 (subgroup 3b) were confirmed and biovars 4 and 6 were first described in Brazil. Genotyping by the panel 1 revealed two groups, one clustering around genotype 40 and another around genotype 28. Panels 2A and 2B disclosed a high diversity among Brazilian B. abortus strains. Eighty-nine genotypes were found by MLVA16. MLVA16 panel 1 and 2 showed geographic clustering of some genotypes. Biotyping and MLVA16 genotyping of Brazilian B. abortus isolates were useful to better understand the epidemiology of bovine brucellosis in the region.
Campylobacter fetus subsp. venerealis is the etiologic agent of bovine genital campylobacteriosis, a sexually transmitted disease of cattle that is of worldwide importance. The complete sequencing and annotation of the genome of the type strain C. fetus subsp. venerealis NCTC 10354(T) are reported.
Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.
Brucella ovis infection is a major cause of epididymitis and infertility in rams, resulting in reproductive failure and significant economic losses worldwide. The goal of this study was to develop a PCR test targeting specific B. ovis genomic sequences. Specific primer pairs were designed targeting 12 of those ORFs. Samples of blood, serum, semen, urine, and preputial wash were collected from experimentally infected rams (n=9) every other week up to 180 days post infection (dpi), when tissue samples were obtained. Blood, serum, semen, urine, and preputial wash samples were obtained, in weekly intervals for 1 month, from eight rams belonging to a B. ovis-free flock. Semen samples were also obtained from rams belonging to naturally infected flocks (n=40). The limit of detection of this PCR protocol was 100, 10, and 1 CFU/mL for semen, urine and prepucial wash samples, respectively. Sensitivity and specificity values obtained with this PCR method were similar to that of bacteriology when evaluating biological samples. Agreement between PCR and bacteriology results was greater than 90%. These results clearly indicate that this species-specific PCR method is highly efficient for the diagnosis of B. ovis infection in semen, urine, preputial wash and tissue samples from infected rams.
Among 63 Haemophilus parasuis isolates from 17 Brazilian pig herds, 33 genotypes were identified by enterobacterial repetitive intergenic consensus (ERIC)-PCR, with a diversity index of 0.96. Eight serovars were detected, with serovar 4 (15.9%) being most frequent; 60.3% of isolates were non-typeable. There was no strong association between site of isolation and genotype or serovar.
Bovine brucellosis is one of the most important zoonotic diseases worldwide, and is of particular significance in developing countries. The disease, which results in serious economic losses due to late term abortion, stillborn and weakly calves, is caused by Gram negative coccobacilli bacteria of the genus Brucella. Lesions consist of necrotic placentitis and interstitial mastitis in pregnant cows, and fibrinous pleuritis with interstitial pneumonia in aborted fetuses and newborn calves. This article considers the pathogenesis of Brucella abortus and reviews the ability of the pathogen to invade phagocytic and non-phagocytic host cells, resist the acidified intraphagosomal environment, and inhibit phagosome-lysosome fusion. Significant aspects of innate and adaptive immunity against brucellosis are also discussed.
The genus Campylobacter contains pathogens causing a wide range of diseases, targeting both humans and animals. Among them, the Campylobacter fetus subspecies fetus and venerealis deserve special attention, as they are the etiological agents of human bacterial gastroenteritis and bovine genital campylobacteriosis, respectively. We compare the whole genomes of both subspecies to get insights into genomic architecture, phylogenetic relationships, genome conservation and core virulence factors. Pan-genomic approach was applied to identify the core- and pan-genome for both C. fetus subspecies and members of the genus. The C. fetus subspecies conserved (76%) proteome were then analyzed for their subcellular localization and protein functions in biological processes. Furthermore, with pathogenomic strategies, unique candidate regions in the genomes and several potential core-virulence factors were identified. The potential candidate factors identified for attenuation and/or subunit vaccine development against C. fetus subspecies contain: nucleoside diphosphate kinase (Ndk), type IV secretion systems (T4SS), outer membrane proteins (OMP), substrate binding proteins CjaA and CjaC, surface array proteins, sap gene, and cytolethal distending toxin (CDT). Significantly, many of those genes were found in genomic regions with signals of horizontal gene transfer and, therefore, predicted as putative pathogenicity islands. We found CRISPR loci and dam genes in an island specific for C. fetus subsp. fetus, and T4SS and sap genes in an island specific for C. fetus subsp. venerealis. The genomic variations and potential core and unique virulence factors characterized in this study would lead to better insight into the species virulence and to more efficient use of the candidates for antibiotic, drug and vaccine development.
In this study, multiplex PCR was employed to investigate the virulence factors of Escherichia coli strains isolated from 60-day-old calves. Faecal samples were collected from 54 calves at 12 dairy farms in the state of Minas Gerais, Brazil. A total of 156 isolates were obtained after culture and microbiological isolation and were tested by multiplex PCR for the presence of genes encoding toxins (Stx1, Stx2 and STa) and adherence factors (intimin, F41 and F5). Seventy of 156 isolates were positive for at least one virulence factor: ten (14.3 %) from diarrhoeic animals and 60 (85.7 %) from healthy calves. The virulence markers identified were: Stx1 (82.8 %), eae (24.3 %), F41 (11.4 %), F5 (10 %), STa (4.28 %) and Stx2 (4 %). In diarrhoeic animals, Stx1 (70 %) and F41 (30 %) were identified, while Stx1 (83.3 %), eae (28.3 %), F41 (8.3 %), F5 (11.6 %), STa (5 %) and Stx2 (1.6 %) were detected in isolates from healthy calves. Mixed infections with pathotypes Shiga toxin-producing E. coli (STEC)/enteropathogenic E. coli, STEC/enterohaemorrhagic E. coli and STEC/other (eae/F5, Stx1/STa) were detected in five healthy calves. Pathogenic E. coli were identified in 59.26 % of all calves and on 75 % of the dairy farms studied, not only in diarrhoeic (five of six) but also in healthy calves (27 of 48), which demonstrates the importance of this agent in the aetiology of diarrhoea in calves in the state of Minas Gerais.
We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.
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