Ana Paula Reinato Stynen

Modulation of the Bovine Trophoblastic Innate Immune Response by Brucella abortus

ABSTRACT Brucellosis is still a widespread zoonotic disease. Very little is known about the interaction between Brucella abortus and trophoblastic cells, which is essential for better understanding the pathogenesis of the Brucella-induced placentitis and abortion, a key event for transmission of the disease. The goal of this study was to evaluate the profile of gene expression by bovine trophoblastic cells during infection with B. abortus. Explants of chorioallantoic membranes were inoculated with B. abortus strain 2308. Microarray analysis was performed at 4 h after infection, and expression of cytokines and chemokines by trophoblastic cells was assessed by real-time reverse transcription-PCR at 6 and 12 h after inoculation. In addition, cytokine and chemokine expression in placentomes from experimentally infected cows was evaluated. Expression of proinflammatory genes by trophoblastic cells was suppressed at 4 h after inoculation, whereas a significant upregulation of CXC chemokines, namely, CXCL6 (GCP-2) and CXCL8 (interleukin 8), was observed at 12 but not at 6 h after inoculation. Placentomes of experimentally infected cows had a similar profile of chemokine expression, with upregulation of CXCL6 and CXCL8. Our data indicate that B. abortus modulates the innate immune response by trophoblastic cells, suppressing the expression of proinflammatory mediators during the early stages of infection that is followed by a delayed and mild expression of proinflammatory chemokines, which is similar to the profile of chemokine expression in the placentomes of experimentally infected cows. This trophoblastic response is likely to contribute to the pathogenesis of B. abortus-induced placentitis.

Evaluation of Brucella abortus S19 vaccines commercialized in Brazil: immunogenicity, residual virulence and MLVA15 genotyping.

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.

Campilobacteriose genital bovina e tricomonose genital bovina: epidemiologia, diagnóstico e controle

The present update deals with two of the most important sexually transmitted diseases of cattle: bovine genital campylobacteriosis and bovine genital trichomonosis. Epidemiological aspects, mainly their distribution in Brazil, alongside with their diagnosis in cattle are presented and commented. The main points in their diagnoses, including the description of the techniques and the interpretation of the results are also reviewed. Finally the control and prevention of both diseases are discussed.

Complete Genome Sequence of Type Strain Campylobacter fetus subsp. venerealis NCTC 10354T

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.

Brucellosis in working equines of cattle farms from Minas Gerais State, Brazil

The present survey aimed at estimating the seroprevalence of brucellosis in working equines of cattle farms from Minas Gerais State, Brazil, and investigating risk factors associated with the infection. Serum samples from 6439 animals, including 5292 horses, 1037 mules and 110 donkeys, were collected from 1936 herds, between September 2003 and March 2004, in 848 municipalities from the state of Minas Gerais, Brazil. The prevalence of antibodies against smooth Brucella spp. found in equines from Minas Gerais State was 1.37% (95% CI: 0.97-1.78), resulting in a prevalence of herds with infected animals of 4.28% (95% CI: 4.21-4.36). There were differences between regions but these were not of major epidemiological relevance nor were most of them statistically significant, given the considerable overlap of confidence intervals. Nevertheless, the point estimates suggest that the three northeastern regions have slightly higher prevalence than the rest of the state, both at the herd and animal levels. No association of Brucella spp. seropositivity with sex, age or host was observed. In conclusion, the present study showed a low but widespread prevalence of antibodies against smooth Brucella in equines kept in cattle farms in Minas Gerais, a state where bovine brucellosis is also widespread albeit with low prevalence.

Reduced Susceptibility to Rifampicin and Resistance to Multiple Antimicrobial Agents among Brucella abortus Isolates from Cattle in Brazil

This study aimed to determine the susceptibility profile of Brazilian Brucella abortus isolates from cattle to eight antimicrobial agents that are recommended for the treatment of human brucellosis and to correlate the susceptibility patterns with origin, biotype and MLVA16-genotype of the strains. Screening of 147 B. abortus strains showed 100% sensitivity to doxycycline and ofloxacin, one (0.68%) strain resistant to ciprofloxacin, two strains (1.36%) resistant to streptomycin, two strains (1.36%) resistant to trimethoprim-sulfamethoxazole and five strains (3.40%) resistant to gentamicin. For rifampicin, three strains (2.04%) were resistant and 54 strains (36.73%) showed reduced sensitivity. Two strains were considered multidrug resistant. In conclusion, the majority of B. abortus strains isolated from cattle in Brazil were sensitive to the antimicrobials commonly used for the treatment of human brucellosis; however, a considerable proportion of strains showed reduced susceptibility to rifampicin and two strains were considered multidrug resistant. Moreover, there was no correlation among the drug susceptibility pattern, origin, biotype and MLVA16-genotypes of these strains.

Complete Genome Sequence of Type Strain Campylobacter fetus subsp. fetus ATCC 27374

ABSTRACT Campylobacter fetus subsp. fetus is a zoonotic bacterium important for animal and public health. The complete sequencing and annotation of the genome of the type strain C. fetus subsp. fetus ATCC 27374 are reported here.

Parameter estimation and use of gamma interferon assay for the diagnosis of bovine tuberculosis in Brazil

This study aimed to evaluate the interference of tuberculin test on the gamma-interferon (INFg) assay, to estimate the sensitivity and specificity of the INFg assay in Brazilian conditions, and to simulate multiple testing using the comparative tuberculin test and the INFg assay. Three hundred-fifty cattle from two TB-free and two TB-infected herds were submitted to the comparative tuberculin test and the INFg assay. The comparative tuberculin test was performed using avian and bovine PPD. The INFg assay was performed by the BovigamTM kit (CSL Veterinary, Australia), according to the manufacturers specifications. Sensitivity and specificity of the INFg assay were assessed by a Bayesian latent class model. These diagnostic parameters were also estimate for multiple testing. The results of INFg assay on D0 and D3 after the comparative tuberculin test were compared by the McNemars test and kappa statistics. Results of mean optical density from INFg assay on both days were similar. Sensitivity and specificity of the INFg assay showed results varying (95% confidence intervals) from 72 to 100% and 74 to 100% respectively. Sensitivity of parallel testing was over 97.5%, while specificity of serial testing was over 99.7%. The INFg assay proved to be a very useful diagnostic method.