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Canine brucellosis is an infectious and zoonotic disease caused byBrucella canis, which has been reported worldwide, and is a major public health concern due to close contact between dogs and humans. In dogs, canine brucellosis manifests with abortion outbreaks, reproductive failure, enlargement of lymph nodes, and occasionally affects the osteoarticular system, although the occurrence of asymptomatic infections in dogs are not uncommon. In humans, the disease is associated with a febrile syndrome, commonly with non-specific symptoms including splenomegaly, fatigue, and weakness. Infection of dogs occurs mostly by the oronasal route when in contact with contaminated tissues such as aborted fetuses, semen, urine, and vaginal secretions. In humans, contact with contaminated fluids from infected dogs is an important source of infection, and it is an occupational risk for veterinarians, breeders, laboratory workers, among other professionals who deal with infected animals or biological samples. The diagnosis in dogs is largely based on serologic methods. However, serologic diagnosis of canine brucellosis remains very challenging due to the low accuracy of available tests. Molecular diagnostic methods have been increasingly used in the past few years. Treatment of infected dogs is associated with a high frequency of relapse, and should be employed only in selected cases. Currently there are no commercially available vaccines for prevention of canine brucellosis. Therefore, development of novel and improved diagnostic methods as well as the development of efficacious and safe vaccination protocols are needed for an effective control of canine brucellosis and its associated zoonotic risk.

Macrophages are critical mediators of the inflammatory process, playing a relevant role in the pathogenesis of Salmonella Typhimurium. The protocols for isolation, culture, and differentiation of monocytes into macrophages and their interaction with Salmonella are well established in humans and murine models, but little information is available in swine. The aims of this study were to establish an efficient protocol for macrophage culture and to evaluate the interaction of the invA mutant strain and the wild type (WT) Salmonella Typhimurium with porcine macrophages. Peripheral blood monocyte-derived macrophages from pigs were obtained, separated by density-gradient centrifugation, and cultured in Teflon vials for 10 days. After the differentiation period, cultures consisted of 92.4% CD14(+) cells. In addition, these cells showed phagocytic ability, demonstrated by the presence of the same amount of WT and invA mutant Salmonella Typhimurium 1 h after interaction with macrophages. The early cytotoxic effect was Salmonella pathogenicity island (SPI)-[1]dependent, in which log-phase WT strains were more efficient (p

Bovine brucellosis poses a risk to human health and causes serious economic losses for the animal industry. This report describes the use of different diagnostic methods for the diagnosis of brucellosis in cattle affected by cervical bursitis from a slaughterhouse located in São Luís, Maranhão, Brazil. Serum samples from a total of 47 cattle with bursitis were collected and submitted to the Rose Bengal Test (RBT), and RBT-positive samples were further confirmed by the 2-mercaptoethanol (2-ME) assay. RBT indicated 85.1% (40/47) of positive samples, from which 78.7% (37/47) were confirmed by 2-ME. Immunohistochemistry detected Brucella spp. in 34.0% (16/47) of tissues with bursitis. PCR and/or bacterial isolation demonstrated that 63.8% (30/47) of samples were positive and morphologically compatible with Brucella sp. All colonies suggestive of Brucella sp. were confirmed by PCR. Isolates were further characterized by PCR Multiplex AMOS-ENHANCED, which indicated that the isolates corresponded to biovar 1, 2, 4 (43.33%). This study evidences an association between cervical bursitis and Brucella spp. infection in cattle, and that different biovars of Brucella circulate in bovine herds in Maranhão.

Brucella canis infection is an underdiagnosed zoonotic disease. Knowledge about perinatal brucellosis in dogs is extremely limited, although foetuses and neonates are under risk of infection due to vertical transmission. In this study, immunohistochemistry was used to determine tissue distribution and cell tropism of B. canis in canine foetuses and neonates. Diagnosis of B. canis in tissues of naturally infected pups was based on PCR and sequencing of amplicons, bacterial isolation, and immunohistochemistry, whose specificity was confirmed by laser capture microdissection. PCR positivity among 200 puppies was 21%, and nine isolates of B. canis were obtained. Tissues from 13 PCR-positive puppies (4 stillborn and 9 neonates) presented widespread immunolabeling. Stomach, intestines, kidney, nervous system, and umbilicus were positive in all animals tested. Other frequently infected organs included the liver (92%), lungs (85%), lymph nodes (69%), and spleen (62%). Immunolabeled coccobacilli occurred mostly in macrophages, but they were also observed in erythrocytes, epithelial cells of gastrointestinal mucosa, renal tubules, epidermis, adipocytes, choroid plexus, ependyma, neuroblasts, blood vessels endothelium, muscle cells, and in the intestinal lumen. These results largely expand our knowledge about perinatal brucellosis in the dog, clearly demonstrating a pantropic distribution of B. canis in naturally infected foetuses and neonates.

Background/Objectives Vaccination is the most important tool for controlling brucellosis, but currently there is no vaccine available for canine brucellosis, which is a zoonotic disease of worldwide distribution caused by Brucella canis. This study aimed to evaluate protection and immune response induced by Brucella ovis ΔabcBA (BoΔabcBA) encapsulated with alginate against the challenge with Brucella canis in mice and to assess the safety of this strain for dogs. Methods Intracellular growth of the vaccine strain BoΔabcBA was assessed in canine and ovine macrophages. Protection induced by BoΔabcBA against virulent Brucella canis was evaluated in the mouse model. Safety of the vaccine strain BoΔabcBA was assessed in experimentally inoculated dogs. Results Wild type B. ovis and B. canis had similar internalization and intracellular multiplication profiles in both canine and ovine macrophages. The BoΔabcBA strain had an attenuated phenotype in both canine and ovine macrophages. Immunization of BALB/c mice with alginate-encapsulated BoΔabcBA (108 CFU) induced lymphocyte proliferation, production of IL-10 and IFN-γ, and protected against experimental challenge with B. canis. Dogs immunized with alginate-encapsulated BoΔabcBA (109 CFU) seroconverted, and had no hematologic, biochemical or clinical changes. Furthermore, BoΔabcBA was not detected by isolation or PCR performed using blood, semen, urine samples or vaginal swabs at any time point over the course of this study. BoΔabcBA was isolated from lymph nodes near to the site of inoculation in two dogs at 22 weeks post immunization. Conclusion Encapsulated BoΔabcBA protected mice against experimental B. canis infection, and it is safe for dogs. Therefore, B. ovis ΔabcBA has potential as a vaccine candidate for canine brucellosis prevention.

A 2-year-old captive gorilla (Gorilla gorilla gorilla) was diagnosed with visceral leishmaniasis in Brazil, and treated with a single dose of liposomal amphotericin B, which resulted in clinical cure. This is the first report of visceral leishmaniasis in gorillas, and the first reported liposomal amphotericin B treatment in great apes.

Made available in DSpace on 2020-12-10T17:01:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-21 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Canine brucellosis is an infectious and contagious disease associated with reproductive losses in breeding kennels. As a zoonotic disease, it poses a risk to human health, especially for veterinarians and breeders who handle materials potentially contaminated with Brucella canis. However, canine brucellosis is a neglected and underestimated disease given the difficulties in establishing a definitive diagnosis. We evaluated the frequency of detection of B. canis in 5 breeding kennels by using various serologic methods and PCR. Circulation of B. canis in these kennels was confirmed by bacterial isolation. The frequency of positive serologic results varied from 6.3% by AGID to 16.5% by dot-ELISA. There was no positive serology for smooth Brucella. PCR testing was positive in 13.9% of samples. The only detection tests with reasonable agreement were PCR and 2ME-MAT. The diagnosis of canine brucellosis remains challenging. The use of a single laboratory method, or even the use of different laboratory methods, may not be sufficient to reach a definitive diagnosis. Univ Fed Minas Gerais, Dept Clin & Cirurgia Vet, Escola Vet, Av Presidente Antonio Carlos 6627, BR-30161970 Belo Horizonte, MG, Brazil Univ Fed Minas Gerais, Dept Med Vet Prevent, Escola Vet, Belo Horizonte, MG, Brazil Univ Fed Minas Gerais, Inst Ciencias Biol, Dept Patol Geral, Belo Horizonte, MG, Brazil Univ Vila Velha, Curso Med Vet, Vila Velha, ES, Brazil Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Med Vet Prevent & Reprod Anim, Sao Paulo, Brazil Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Med Vet Prevent & Reprod Anim, Sao Paulo, Brazil

The aim of the current study is to diagnose Brucella spp. infection using methods such as serology, bacterial isolation, and molecular analysis in buffaloes bred in Maranhão State. In order to do so, 390 samples of buffalo serum were subjected to serological tests, to Rose Bengal Plate Test (RBPT) and to 2-mercaptoethanol (2-ME) combined with slow agglutination test (SAT). Vaginal swabs were collected from seropositive animals and subjected to bacterial isolation and to generic PCR. According to the serological test, 16 animals had a positive reaction to the confirmatory test (2-ME/SAT). As for bacterial isolation, three samples resulted in the isolation of Brucella spp.-characteristic colonies, which were confirmed through PCR. These results confirmed Brucella spp. infection in the buffalo herd from Maranhão State.

Brucella ovis is an important cause of epididymitis in rams, which results in impaired fertility and economic losses. This study demonstrated the role of TLR during the acute phase of infection in the mouse model. C57BL/6 wild type and TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) mice were infected with B. ovis and bacteriology, histopathology, and pro-inflammatory gene expression were evaluated at 7days post-infection. MyD88(-/-) mice had higher bacterial loads in the spleen when compared to wild type mice. This enhanced susceptibility was associated with decreased inflammatory response in the liver. TLR9(-/-) mice also had higher bacterial loads when compared to wild type mice, but, surprisingly, they developed stronger inflammatory response. TLR2(-/-) and TLR4(-/-) mice were as susceptible as wild type mice to B. ovis infection. Therefore, MyD88 and TLR9 are required for controlling B. ovis multiplication during the early stages of infection.

© 2016 Elsevier GmbH Salmonella enterica serotype Typhimurium is able to expand in the lumen of the inflamed intestine through mechanisms that have not been fully resolved. Here we utilized streptomycin-pretreated mice and dextran sodium sulfate (DSS)-treated mice to investigate how pathways for S. Typhimurium iron acquisition contribute to pathogen expansion in the inflamed intestine. Competitive infection with an iron uptake-proficient S. Typhimurium strain and mutant strains lacking tonB feoB, feoB, tonB or iroN in streptomycin pretreated mice demonstrated that ferric iron uptake requiring IroN and TonB conferred a fitness advantage during growth in the inflamed intestine. However, the fitness advantage conferred by ferrous iron uptake mechanisms was independent of inflammation and was only apparent in models where the normal microbiota composition had been disrupted by antibiotic treatment.

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.

Leishmania (Leishmania) infantum is the cause of visceral leishmaniasis in the Americas. The disease is transmitted mostly through the bite of the invertebrate vector, the phlebotomine Lutzomyia longipalpis in the New World. Although the domestic dog is considered the most important reservoir of the disease, other mammalian, including wildlife, are susceptible to infection. The goal of this study was to perform xenodiagnosis to evaluate the capacity of naturally infected maned wolves (Chrysocyon brachyurus) and bush dogs (Speothos venaticus) to transmit Leishmania infantum to female sand flies (L. longipalpis). Xenodiagnoses were performed in February and August, 2013, when 77.7% (three maned wolves and four bush dogs) or 100% of the animals were positive, respectively. However, parasite loads in the engorged sand flies was low (

Transcription of non-classical major histocompatibility complex class I (MHC-I) was assessed in the bovine placenta throughout gestation. Additionally, the effect of Brucella abortus infection on expression of non-classical MHC-I was also evaluated using a chorioallantoic membrane explant model of infection. The non-classical MHC-I genes MICB and NC3 had higher levels of transcription in the intercotyledonary region when compared to the placentome, which had higher levels of transcription at the second trimester of gestation. NC1 and classical MHC-I had very low levels of transcription throughout gestation. Trophoblastic cells of B. abortus -infected chorioallantoic membrane explants had an increase in transcription of non-classical MHC-I at 4 h post infection. Therefore, this study provides an analysis of non-classical MHC-I transcription at different stages of gestation and different placental tissues, and during B. abortus infection. These findings provide additional knowledge on immune regulation in placental tissues, a known immune-privileged site.

Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment.

This study aimed to evaluate protection induced by the vaccine candidate B. ovis ΔabcBA against experimental challenge with wild type B. ovis in rams. Rams were subcutaneously immunized with B. ovis ΔabcBA encapsulated with sterile alginate or with the non encapsulated vaccine strain. Serum, urine, and semen samples were collected during two months after immunization. The rams were then challenged with wild type B. ovis (ATCC25840), and the results were compared to non immunized and experimentally challenged rams. Immunization, particularly with encapsulated B. ovis ΔabcBA, prevented infection, secretion of wild type B. ovis in the semen and urine, shedding of neutrophils in the semen, and the development of clinical changes, gross and microscopic lesions induced by the wild type B. ovis reference strain. Collectively, our data indicates that the B. ovis ΔabcBA strain is an exceptionally good vaccine strain for preventing brucellosis caused by B. ovis infection in rams.

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.

Listeria monocytogenes is a Gram-positive, facultative intracellular and invasive bacterium that has tropism to the placenta, and causes fetal morbidity and mortality in several mammalian species. While infection with L. monocytogenes and L. ivanovii are known as important causes of abortion and reproductive failure in cattle, the pathogenesis of maternal-fetal listeriosis in this species is poorly known. This study used the bovine chorioallantoic membrane explant model to investigate the kinetics of L. monocytogenes, L. ivanovii, and L. innocua infections in bovine trophoblastic cells for up to 8 h post infection. L. monocytogenes and L. ivanovii were able to invade and multiply in trophoblastic cells without causing cell death or inducing expression of pro-inflammatory genes. Although L. innocua was unable to multiply in bovine trophoblastic cells, it induced transcription of the pro-inflammatory mediator CXCL6. This study demonstrated for the first time the susceptibility of bovine trophoblastic cells to L. monocytogenes and L. ivanovii infection.

© 2014 Silva et al. Brucella ovis is a major cause of reproductive failure in rams and it is one of the few well-described Brucella species that is not zoonotic. Previous work showed that a B. ovis mutant lacking a species-specific ABC transporter (ΔabcBA) was attenuated in mice and was unable to survive in macrophages. The aim of this study was to evaluate the role of this ABC transporter during intracellular survival of B. ovis. In HeLa cells, B. ovis WT was able to survive and replicate at later time point (48 hpi), whereas an ΔabcBA mutant was attenuated at 24 hpi. The reduced survival of the ΔabcBA mutant was associated with a decreased ability to exclude the lysosomal marker LAMP1 from its vacuolar membrane, suggesting a failure to establish a replicative niche. The ΔabcBA mutant showed a reduced abundance of the Type IV secretion system (T4SS) proteins VirB8 and VirB11 in both rich and acid media, when compared to WT B. ovis. However, mRNA levels of virB1, virB8, hutC, and vjbR were similar in both strains. These results support the notion that the ABC transporter encoded by abcEDCBA or its transported substrate acts at a post-transcriptional level to promote the optimal expression of the B. ovis T4SS within infected host cells.

The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; P

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.

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation.

Brucella abortus é considerada o principal agente etiológico da brucelose bovina, uma zoonose que provoca grandes perdas econômicas em todo o mundo. O objetivo desse estudo foi determinar o perfil diferencial de expressão proteica de células trofoblásticas bovinas nas fases iniciais da infecção por B. abortus. Foi usado o modelo de explantes confeccionados a partir de membrana cório-alantóidea de fetos no último trimestre de gestação infectados por suspensão deB. abortus 2308 (1.0 x 108 bactérias - MOI 1:1000). No estudo da cinética da infecção foram avaliados os tempos de 0,5, 2, 4 e 8 h pós-infecção no qual foi obtida reprodutibilidade alta entre triplicatas dos géis comparados (Match > 84%; IC > 0,77). O perfil de expressão deproteínas foi similar entre os tempos avaliados (Match > 75%). Os tempos de 0,5 e 4 h pósinfecção apresentaram as maiores diferenças qualitativas entre os géis e foram escolhidos para análise diferencial em gel (DIGE). Nessa análise não foram identificados spots com diferençasquantitativas significativas entre os géis comparados. Por outro lado, 103 spots presentes apenas em um grupo experimental foram selecionados para identificação por espectrometria de massa. As proteínas BLVRA, LGALS7, RPLP1, SCAMP2, TOLLIP, GALM, AHCY, PSAPL1/PSAP, OAT, C1QBP, NDUFS8, PRCII, RAB11A, CALM1, MDH1, PRDX3, ABHD14B, KRT14, KRT7, HMGB1, HEXB, AKR1B1, PDIA3 e TPM4 foram identificadas apenas nos extratos proteicos de células trofoblásticas infectadas. As proteínas identificadas nesse estudo servirão como alvos para novas pesquisas relacionadas à interação patógeno-hospedeiro na infecção por B. abortus. Brucella abortus is considered the main etiological agent of bovine brucellosis, a zoonotic disease that causes great economic losses worldwide. The aim of this study was to determine the differential profile of proteins expression of bovine trophoblast cells in the early stages ofinfection by B. abortus. Was used the explants model prepared from chorio-alantóidea of fetuses in the last trimester of pregnancy infected by suspending the B. abortus 2308 containing 1.0 x 108 bacteria (MOI 1:1000). In the study of the kinetics of infection was evaluated the timesof 0.5, 2, 4 and 8 h post infection with B. abortus. Was obtained high reproducibility between triplicate of gels compared (Match> 84%; CI > 0.77) and the proteins expression profile was similar among the times evaluated (Match > 75%). Times of 0.5 and 4 h post infection showedthe highest qualitative differences between the gels and were used for the differential in gel electrophoresis (DIGE). In this analysis have not been identified spots with significant quantitative differences between the compared gels. Moreover, 103 spots are present only in an experimental group were selected for identification by mass spectrometry. The proteins BLVRA, LGALS7, RPLP1, SCAMP2, TOLLIP, GALM, AHCY, PSAPL1/PSAP, OAT, C1QBP, NDUFS8, PRCII, RAB11A, CALM1, MDH1, PRDX3, ABHD14B, KRT14, KRT7, HMGB1, HEXB, AKR1B1, PDIA3 and TPM4 were identified only in protein extracts of trophoblastic cells infected by B. abortus. The proteins identified in this study will serve as targets for further research related to host-pathogen interaction in infection by B. abortus.

Infectious abortion is a significant cause of reproductive failure and economic losses in cattle. The goal of this study was to detect nucleic acids of several infectious agents known to cause abortion including Arcanobacterium pyogenes, Bovine Herpesvirus 1, Brucella abortus, Campylobacter fetus subsp. venerealis, Chlamydophila abortus, Leptospira sp., Listeria monocytogenes, Salmonella sp., Mycoplasma bovis, Mycoplasma bovigenitalium, Neospora caninum, and Tritrichomonas foetus. Tissue homogenates from 42 fetuses and paraffin-embedded tissues from 28 fetuses and 14 placentas/endometrium were included in this study. Brucella abortus was detected in 14.2% (12/84) of the samples. Salmonella sp. DNA was amplified from 2 fetuses, and there was one positive for Neospora caninum, and another for Listeria monocytogenes. This PCR-based approach resulted in identification of the etiology in 19% of samples, or 20% if considered fetal tissues only.

Background 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. Results Three-hundred and seventy-five clinical samples, including 275 vaginal swabs and 100 milk samples, were cultured from a brucellosis outbreak in a cattle herd, which adopted RB51 vaccination and test-and-slaughter policies. Thirty-seven B. abortus isolates were obtained, eight from milk and twenty-nine from post-partum/abortion vaginal swabs, which were submitted to biotyping and genotyping by MLVA16. Twelve B. abortus isolates obtained from vaginal swabs were identified as RB51. Twenty four isolates, seven obtained from milk samples and seventeen from vaginal swabs, were identified as B. abortus biovar 3, while one isolate from vaginal swabs was identified as B. abortus biovar 1. Three distinct genotypes were observed during the brucellosis outbreak: RB observed in all isolates identified as RB51; W observed in all B. abortus biovar 3 isolates; and Z observed in the single B. abortus biovar 1 isolate. Epidemiological and molecular data show that the B. abortus biovar 1 genotype Z strain is not related to the B. abortus biovar 3 genotype W isolates, and represents a new introduction B. abortus during the outbreak. Conclusions The results of the present study on typing of multiple clinical B. abortus isolates from the same outbreak over a sixteen month period indicate the in vivo stability of MLVA16 markers, a low genetic diversity among B. abortus isolates and the usefulness of MLVA16 for epidemiological studies of bovine brucellosis.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Background: Brucella ovis infection is one of the leading causes of sub fertility and infertility in ovine, been characterized mainly by epididymitis, orchitis and testicular atrophy in rams. This study aimed to determine the frequency of B. ovis positivity in rams and goats flocks in the State of Minas Gerais, Brazil, by agarose gel immunodiffusion (AGID), ELISA, Rose Bengal, PCR and bacteriological isolation as diagnostic tools. Findings: Serum and urine samples were collected from properties with sheep or goat flocks, or from properties with mixed flock. Out of 50 sheep flocks, 6 % (3/50) were seropositive by AGID while 4 % (2/50) were positive by urine PCR for B. ovis. Out of five goat farms, 20 % (1/5) were seropositive for B. ovis by AGID. Mixed flock farms had 11.1 % (2/18) of positivity by AGID. By ELISA, 19.5 % (8/41) of sheep properties and 61.1 % (11/18) of the properties with mixed flocks were positive for B. ovis. No samples were positive in the test of Rose Bengal, ruling out exposure to smooth LPS Brucella species (particularly Brucella melitensis) and indicating that the positive in the ELISA was associated with Brucella spp. LPS rough (presumably B. ovis). No urine sample from sheep or goat was positive by bacteriological isolation. Conclusions: Our results demonstrate serologic or molecular evidence of B. ovis infection in several rams and billy goats from meso-regions of the State of Minas Gerais, Brazil. Also, this study report the indirect ELISA as an important tool for the diagnosis of B. ovis infection, as indirect ELISA in this study demonstrated to be the most sensitive diagnostic method adopted.

A mortalidade fetal e neonatal na espécie canina é reponsável por perdas significativas para os criadores e proprietários de cães. Diversos autores afirmam que as causas infecciosas são importantes, mas estudos epidemiológicos são muito escassos e restritos a algumas raças e canis, e ainda não foram realizados no Brasil. O objetivo deste trabalho foi investigar as doenças relacionadas a mortalidade fetal e neonatal canina com a associação de métodos anatomopatológicos, microbiológicos e moleculares. Foram necropsiados 203 filhotes de 89 ninhadas, provenientes de 32 canis localizados no estado do Espírito Santo, no sudeste do Brasil. As lesões inflamatórias predominaram e frequentemente estavam associadas à visualização de agentes bacterianos ao exame microscópico e/ou sua detecção pelos exames auxiliares. As lesões mais frequentes foram: pneumonia (85,4%), onfalite (56,3%), hepatite (47,2%), epicardite (38,5%), glossite (21,1%) e miocardite (17,4%). Malformações ocorreram em 7,5% dos animais examinados. Alterações cadavéricas impediram a visualização de lesões macro e microscópicas em 15 animais. O congelamento dos cadáveres antes da necropsia impediu a análise microscópica em 51 animais. Infecções bacterianas foram a principal causa de morte, tanto em infecções primárias, quanto em infecções secundárias. Os agentes bacterianos isolados com maior frequência foram Staphylococcus sp. (62,6%), Escherichia coli (36,0%), Klebsiella pneumoniae (17,8%) e Enterococcus sp. (9,8%). O isolamento de mais de um gênero bacteriano ocorreu em grande número de filhotes (55,8%). Por meio da reação em cadeia da polimerase, verificou-se positividade de 21% para Brucella sp., 1,5% para herpesvírus canino e 1,5% para Leishmania infantum. A brucelose canina mostrou-se disseminada em muitos filhotes e canis, contudo, a frequência de aborto em ninhadas infectadas foi baixa (1/31). Este estudo evidenciou que a brucelose perinatal canina estava relacionada principalmente a natimortalidade e mortalidade neonatal nos canis positivos estudados, enquanto o aborto foi raro nessa população. Os filhotes Brucella-positivos na PCR analisados por imuno-histoquímica apresentaram ampla distribuição do agente nos tecidos, frequentemente com infecção bacteriana oportunista, especialmente por Staphylococcus (54%) e Escherichia coli (40,5%). Dentre os agentes virais, a infecção por herpesvírus em sua forma generalizada foi observada em apenas um indivíduo. Contudo, uma infecção viral caracterizada por gastroenterite, glossite e dermatite ulcerativa associadas a inclusões intracelulares sugestivas de infecção viral ocorreu em 60/161 (37,3%) dos animais examinados histopatologicamente, sempre associada a infecção bacteriana oportunista. Não foi possível identificar o agente etiológico destas lesões neste trabalho. Um caso de babesiose neonatal devido a infecção por Babesia canis vogeli foi diagnosticado por métodos anatomopatológicos e moleculares. Antígenos de Toxocara canis foram demonstrados por meio de imuno-histoquímica em focos de hepatite necrótica granulomatosa, hepatite portal e pneumonia intersticial, indicando que tais lesões podem estar relacionadas a migração visceral de larvas. T. canis foi diagnosticado também na forma adulta intestinal em três neonatos com nove a 20 dias de idade. Coinfecções foram a principal causa de morte dos fetos e neonatos neste estudo, geralmente com agentes bacterianos oportunistas, agentes virais e Brucella sp. Infecções por agentes bacterianos oportunistas são facilmente diagnosticadas por métodos de rotina e podem ocultar a ocorrência da doença primária. As infecções diagnosticadas nos fetos e neonatos estudados, de forma geral, tiveram uma morfologia perinatal diferente daquela observada em outras faixas etárias na espécie canina e são pouco documentadas na literatura, o que pode levar ao subdiagnóstico em muitos casos. A caracterização anatomopatológica das doenças perinatais caninas é a principal contribuição deste trabalho Canine fetal and neonatal mortality is responsible for many losses to breeders and dog owners. Many researchers consider infectious diseases as important causes of death, but epidemiologic investigations are limited to some breeds or kennels and had not been performed in Brazil before. The goal of this study was to investigate diseases related to canine fetal and neonatal mortality through anatomopathological, microbiological, and molecular analysis. A total of 203 puppies were submitted to necropsy, from 89 litters and 32 kennels located in the State of Espirito Santo, southeastern Brazil. Inflammatory lesions predominated, frequently with visualization of bacterial agents in tissue lesions by microscopy and/or their detection by the complementary analysis. Lesions most frequently diagnosed were pneumonia (85,4%), omphalitis (56,3%), hepatitis (47,2%), epicarditis (38,5%), glossitis (21,1%), and myocarditis (17,4%). Malformations were diagnosed in 7,5% of puppies. Post mortem changes prevented morphologic diagnosis in 15 animals. Freezing artefacts jeopardized microscopy in 51 animals. Bacterial infections were the main cause of death, mainly as secondary but also as primary infections. Bacterial agents isolated more frequently were Staphylococcus sp. (62,6%), Escherichia coli (36,0%), Klebsiella pneumoniae (17,8%), and Enterococcus sp. (9,8%). Isolation of more than one bacterial genus occurred in many puppies (55,8%). Polymerase chain reaction detected positivity in puppies for Brucella (21%), canine herpesvirus (1,5%), and Leishmania infantum (1,5%). Canine brucellosis was spread in puppies and kennels, although the frequency of abortions of infected litters was low (1/31). This study brings evidence that canine perinatal brucellosis was mainly related to stillbirth and neonatal death in the positive kennels studied, while abortion was rare in this population. Brucella-PCR positive puppies submitted to immunohistochemistry presented wide tissue distribution of the agent, frequently associated with opportunistic bacterial infection, especially with Staphylococcus (54%) and Escherichia coli (40,5%). Among viral agents, herpesvirus generalized infection occurred in only one neonate. A viral infection characterized by gastroenteritis, glossitis, and ulcerative dermatitis with intranuclear inclusions suggestive of viral infection was detected in 60/161 (37,3%) of puppies submitted to histopathology, always in association with opportunistic bacterial infection. It was not possible to identify the etiologic agent of these lesions with the methods employed in this research. One case of neonatal babesiosis due to Babesia canis vogeli infection was diagnosed through anatomopathological and molecular methods. Toxocara canis antigens were detected through immunohistochemistry in foci of necrotizing and granulomatous hepatitis, portal hepatitis, and interstitial pneumonia, suggesting visceral larvae migration. Adult T. canis worms were also detected in intestinal lumen of three neonates, nine to 20 days old. Co-infections were the cause of death of the fetuses and neonates in this study, mainly due to association between opportunistic bacterial agents, viruses, and Brucella sp. Opportunistic bacterial infections are easily diagnosed by routine methods and may hide the primary disease if analysis are not accurate or directed towards specific agents as Brucella and viruses. The infections diagnosed in the fetuses and neonates in this study, in many cases, were morphologically distinct from that described for dogs in other age ranges and poorly documented in the literature, what may lead to underdiagnoses and misdiagnosis in many cases. Anatomopathological characterization of canine perinatal diseases is the main contribution of this study.

Patotipos de Escherichia coli se destacam como importante enteropatógenos envolvidos na síndrome diarreica. Amostras de E. coli patogênicas são classificadas em patotipos, de acordo com a presença de fatores de virulência e mecanismos pelos quais causam doença. Já foram identificados cinco patotipos de E. coli associados à diarreia em bezerros: E. coli enterotoxigênica (ETEC), E. coli enteropatogênica (EPEC), E. coli enterohemorrágica (EHEC), E. coli produtora de toxina Shiga (STEC) e E. coli necrotoxigênica (NTEC). No presente estudo, amostras de E. coli isoladas de fezes de bezerros bovinos e bubalinos e carcaças de frangos foram estudadas. A análise filogenética de 336 estirpes de E. coli isoladas de fezes de bezerros bovinos mostrou que 21 (6,25%) pertencem ao filogrupo A, 228 (67,85%) ao filogrupo B1, 2 (0,6%) ao filogrupo B2, 5 (1,49%) ao filogrupo C, 57 (16,96%) ao filogrupo E e 3 (0,9%) ao filogrupo F. O filogrupo D não foi identificado e vinte amostras (5,95%) foram designadas como filogrupo "desconhecido". Somente ETEC foi associada com a presença de diarreia (P = 0,002) e não houve associação entre o grupo filogenético de E. coli e a presença de diarreia (P = 0,164). A análise de correspondência mostrou que EHEC e STEC são classificados principalmente como filogrupo B1, EAEC (EAST-1) filogrupo A, ETEC e EPEC filogrupo E. Duzentos e dezessete amostras de E. coli foram isoladas de fezes de bezerros bubalinos. Os patotipos identificados em fezes diarreicas foram ETEC (2/84), NTEC (16/84), STEC (20/84), EPEC (3/84), EHEC (3/84), e EAEC (33/84). E em fezes não diarreicas foram NTEC (21/50), STEC (17/50), EHEC (1/50), e EAEC (7/50). Fezes de animais com 1 a 30 dias de idade tiveram maior frequência de E. coli positivas para os fatores de virulência (P = 0,094). Patotipo EAEC (EAST-1) (P = 0,008) e filogrupo C (P = 0,03) foram associados com a presença de diarreia. A análise dos filogrupos de E. coli mostrou que 58.95% pertenciam ao filogrupo B1, seguidos dos filogrupos E (9.70%), B2 (5.9%), C (5.9%), D (5.22%), A (2.24%) e F (1.50%). Amostras de E. coli isoladas de carcaças de frangos com aparência normal liberadas para consumo e carcaças condenadas por colibacilose em um matadouro com Serviço de Inspeção Federal no estado de Tocantins foram analisadas. Cento e trinta e sete amostras de E. coli foram obtidas após isolamento e identificação bioquímica. Oitenta e quatro amostras de E. coli foram isoladas de carcaças de frangos de corte condenados, dos quais 11 foram isoladas do coração, 7 do fígado e 66 do trato respiratório. Das 53 amostras de E. coli isoladas de carcaças de frangos normais, 5 foram isoladas de coração, 4 de fígado e 44 de trato respiratório. Os filogrupos A seguido por B1 foram os filogrupos mais comuns de E. coli isoladas de carcaças de frangos saudáveis, enquanto os filogrupos B1 seguido por A foram os mais comuns em carcaças de frangos condenadas. Os filogrupos B2, C, D, E e F foram os menos comuns. Foi estudada também a distribuição dos grupos filogenéticos de 529 amostras de Escherichia coli isoladas das três espécies animais. A frequência dos filogrupos foi: A = 15,31%, B1 = 60,49%, B2 = 2,46%, C = 4,35%, D = 2,46%, E = 12,29% e F = 2,64%. Os filogrupos A (P 0,001) e F (P = 0,002) foram associados com E. coli isoladas de aves, os filogrupos B1 (P 0,001) e E (P 0,001) com E. coli isoladas de bovinos, e os filogrupos B2 (P 0,001) e D (P = 0,007) com E. coli isoladas de búfalos. Os resultados demonstram que alguns filogrupos de E. coli estão associados com o hospedeiro estudado e permitem um melhor entendimento da composição filogenética de E. coli nos animais domésticos. Escherichia coli pathotypes are important enteropathogens involved in the diarrheal syndrome. Pathogenic E. coli are classified into pathotypes according to the production of virulence factors and mechanisms which they cause the disease. Five pathotypes have been identified associated E. coli diarrhea in calves: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC) and necrotoxigenic E. coli (NTEC). In this study, E. coli strains isolated from feces of cattle and buffalo calves and poultry carcasses were studied. Phylogenetic analysis of the 336 strains of E. coli isolated from bovine calves showed that 21 (6.25%) belonged to phylogroup A, 228 (67.85%) to phylogroup B1, 2 (0.6%) to phylogroup B2, 5 (1.49%) to phylogroup C, 57 (16.96%) to phylogroup E and 3 (0.9%) the filogrupo F. Phylogroup D was identified and twenty samples (5.95%) were designated as phylogroup "unknown". Only ETEC was associated with the presence of diarrhea (P = 0.002) and there was no association between the phylogenetic group of E. coli and the presence of diarrhea (P = 0.164). The correspondence analysis showed that EHEC and STEC are classified primarily as phylogroup B1.EAEC (EAST-1) phylogroup A, ETEC and EPEC phylogroup E. In total, 217 E. coli strains were isolated from feces of buffalo calves. Pathotypes identified in diarrheic feces were ETEC (2/84), NTEC (16/84), STEC (20/84), EPEC (3/84), EHEC (3/84) and EAEC (33/84). In non-diarrheic feces the pathotypes identified were NTEC (21/50), STEC (17/50), EHEC (1/50) and EAEC (7/50). Feces from animals aged 1 to 30 days were more frequently positive for E. coli virulence factors. Pathotype EAEC (EAST-1) (P = 0.008) and phylogroup C (P = 0.03) were associated with the presence of diarrhea. The analysis of E. coli phylogroup showed that 58.95% were phylogroup B1, followed by phylogroup s E (9.70%), B2 (5.9%), C (5.9%), D (5.22%), A (2.24%) and F (1.50%). E. coli strains isolated from poultry carcasses with normal appearance and carcasses condemned of colibacillosis in a slaughterhouse with Federal Inspection Service in the state of Tocantins were analyzed. One hundred and thirty seven samples of E. coli were obtained after isolation and biochemical identification. Eighty-four samples of E. coli were isolated from broiler carcasses condemned, of which 11 were isolated from the heart, 7 from the liver and 66 from the respiratory tract. Of the 53 strains isolated from normal poultry carcasses, 5 were isolated from heart, 4 from liver and 44 from the respiratory tract. The phylogroups A followed byB1 were the most common E.coli phylogroups isolated from healthy broiler carcasses, while phylogroups B1 followed by the A were more common in broiler carcasses condemned. The phylogroups B2, C, D, E and F were the least common phylogroups. The distribution of phylogenetic groups of 529 E. coli strains isolated from the three animal species were studied The frequency of phylogroups was: A = 15.31%, 60.49% = B1, B2 = 2.46%, C = 4.35%, D = 2.46%, 12.29% and E = F = 2.64%. The phylogroups A (P 0.001) and F (p = 0.002) were associated with E. coli isolated from poultry, phylogroups B1 (P 0.001) and E (P 0.001) with E. coli isolated from cattle and phylogroups B2 (P 0.001) and D (P = 0.007) with E. coli isolated from buffalo. The results demonstrate that some phylogroups ofE. coli are associated with the host studied and allow a better understanding of the phylogenetic composition of E. coli isolated fromin domestic animals

Infecção por Brucella ovis é associada à epididimite, orquite e infertilidade em carneiros. As informações disponíveis sobre B. ovis e sua interação com células hospedeiras foram geradas com macrófagos murinos ou linhagens de células epiteliais humanas, mas a interação entre B. ovis e macrófagos primários ovinos nunca foi estudada. O objetivo deste estudo foi avaliar o papel do transportador ABC codificado pelo gene abcEDCBA de B. ovis e o sistema de secreção do Tipo IV (SST4) codificado pelo operon virB durante a sobrevivência intracelular de B. ovis em macrófagos derivados de monócitos do sangue periférico de ovinos. As cepas mutantes abcBA e virB2 de B. ovis foram incapazes de sobreviver no ambiente intracelular quando comparado com a cepa selvagem (WT) de B. ovis às 48 horas pós-infecção (hpi). Além disso, estas cepas mutantes não foram capazes de excluir o marcador lisossomal LAMP-1 da membrana de seus vacúolos e seus vacúolos não adquiriram o marcador de retículo endoplasmático calreticulina, ao passo que esse marcador estava presente no vacúolo contendo WT B. ovis. Níveis mais altos de produção de óxido nítrico (NO) foram observados em macrófagos infectados com WT B. ovis no tempo de 48 hpi quando comparados com macrófagos infectados com as cepas mutantes abcBA ou virB2. Por outro lado, níveis mais elevados de espécies reativas de oxigênio (ROS) foram detectadas em macrófagos infectados com os mutantes abcBA ou virB2 48 hpi quando comparados aos macrófagos infectados com a cepa WT. Nossos resultados demonstram que B. ovis é capaz de persistir e de se multiplicar em macrófagos ovinos, enquanto que mutações que provocam a perda de funcionalidade do transportador ABC e do SST4 impedem a multiplicação intracelular, favorecem a fusão do fagolisossoma e prejudicam a maturação do vacúolo de B. ovis para um compartimento com características de retículo endoplasmático rugoso. Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Information available on B. ovis and host cell interaction have been generated using murine macrophages or human epithelial cell lines, whereas the interaction between B. ovis and primary ovine macrophages has never been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. B. ovis abcBA and virB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP-1 from its vacuolar membrane, and their vacuoles did not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the abcBA or virB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the abcBA or virB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while abcBA and virB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment

A salmonelose, uma das principais doenças de origem alimentar, é causada pela infecção por Salmonella enterica, bactéria Gram negativa pertencente à família Enterobacteriaceae. A infecção por S. enterica em seres humanos pode causar a enterite não-tifoide, que se caracteriza pelo quadro de enterite neutrofílica e é causada predominantemente por S. enterica sorotipos Typhimurium e Enteritidis, ou a febre tifoide, que se caracteriza por disseminação sistêmica de S. enterica sorotipos Typhi ou Paratyphi A, Paratyphi B ou Paratyphi C. Os objetivos deste trabalho foram avaliar mecanismos de patogenicidade de S. Typhimurium e de S. Typhi no intestino de animais usados como modelos experimentais de infecção. Buscou-se avaliar os mecanismos de absorção de ferro ferroso e férrico relacionados à sobrevivência intestinal de S. Typhimurium em condições de inflamação. Foram utilizados os modelos murino pré-tratado com estreptomicina, murino tratado com DSS a 3% e de ligadura cirúrgica de alças ilíacas de bezerro. Nos modelos murinos pré-tratado com estreptomicina e tratado com DSS a 3%, as coinfecções com a cepa de referência de S. Typhimurium e uma das mutantes tonB feoB, feoB, tonB ou iroN demonstraram que a absorção de ferro férrico mediada por IroN e TonB confere vantagem competitiva a S. Typhimurium em ambiente inflamado. Paralelamente, durante este trabalho foram estudados mecanismos que S. Typhi utiliza para suprimir a resposta inflamatória no ambiente intestinal. Foi demonstrado que a inserção do gene tviA, regulador da síntese da proteína Vi capsular de S. Typhi, no genoma de S. Typhimurium não altera a capacidade de invasão de S. Typhimurium mas causa diminuição do acúmulo de fluido e da inflamação no modelo de ligadura de alça ilíaca de bezerro. Os modelos de infecção animal demonstraram ser ferramentas importantes para avaliação dos mecanismos de patogenicidade tanto de S. Typhimurium quanto de S. Typhi no ambiente intestinal. Salmonellosis is one of the most important foodbourne diseases, and it is caused by Salmonella enterica, a Gram negative bacterium belonging to the Enterobacteriaceae family. S. enterica infection in humans may cause non-typhoidal salmonellosis, characterized by enteritis with severe neutrophilic infiltration, and it is caused mostly by S. enterica serotypes Typhimurium and Enteritidis, or typhoid fever, which is characterized by systemic dissemination of serotypes Typhi, Paratyphi and A, B, or C. The goals of this study were to assess distinct pathogenic mechanisms of S. Typhimurium and S. Typhi in the intestine of animal models of infection. Mechanisms by which S. Typhimurium uptakes ferrous and ferric iron in the inflamed intestinal environment were evaluated. There were utilized streptomycin pre-treated mice, mice treated with 3% DSS, and bovine ligated ileal loops models In mice pretreated with streptomycin and treated with 3% DSS, co-infections with reference strain and one of the mutants tonB feoB, feoB, tonB or iroN indicated that absorption of ferric iron mediated by IroN and TonB provides competitive advantage to S. Typhimurium in the inflamed intestinal environment. In addition, mechanisms by which S. typhi represses inflammation in the intestine were investigated. Insertion of tviA, regulatory Vi capsular gene of S. Typhi, into the genome of S. typhimurium did not alter the invasion rate of S. typhimurium but resulted in decreased fluid accumulation and inflammation in the bovine ligated ileal loop model. In these studies, animal models have proven to be important tools for assessment of pathogenic mechanisms of both S. Typhimurium and S. Typhi in the intestinal environment.

A esquistossomose mansoni é uma das principais doenças parasitárias no mundo, sendo provocada por trematódeo da espécie Schistosoma mansoni, o qual possui caramujos do gênero Biomphalaria como hospedeiros invertebrados. No Brasil, Biomphalaria tenagophila é o segundo mais importante hospedeiro, apresentando linhagens altamente suscetíveis ao parasito e outras absolutamente resistentes. A natureza da resistência e suscetibilidade de B. tenagophila não é completamente esclarecida, mas admite-se que os hemócitos, células efetoras do sistema interno de defesa dos moluscos, desempenham papel crucial na contenção da infecção. Assim, o estudo expressão proteica pelos hemócitos em resposta à infecção por S. mansoni pode gerar informações relevantes que contribuam para a melhor compreensão de aspectos da interação parasito-hospedeiro. Este estudo comparou os perfis proteômicos de hemócitos da hemolinfa de populações de B. tenagophila resistentes (Taim/RS e Anchieta/ES) e suscetível (Cabo Frio/RJ), desafiadas ou não por S. mansoni. Através do DIGE e eletroforese bidimensional os perfis proteicos das três populações antes e após o desafio foram avaliados e, posteriormente, as proteínas diferencialmente expressas foram identificadas por espectrometria de massa. Foi possível observar proteínas com expressão alterada entre populações resistentes e suscetíveis, principalmente após a exposição a S. mansoni. Na população de Taim desafiada foi observada expressão aumentada de um componente da família das proteínas que contem tioéster (TEP) que é relacionada à fagocitose ou encapsulação de patógenos, bem como da proteína hipotética LOTGIDRAFT_234865 envolvida com a ligação e transporte de um produto resultante da formação de espécies reativas de oxigênio. O aumento da expressão de HSP70 e de frutose-bisfosfato aldolase em Cabo Frio desafiada pode estar relacionada com o fenótipo suscetível desta linhagem. A identificação das proteínas e a análise nos bancos de dados foram afetadas pela ausência do genoma completo de B. glabrata. Ainda assim, os resultados encontrados foram significativos e confirmaram a já conhecida complexidade da interação molecular entre Biomphalaria e S. mansoni. Schistosomiasis mansoni remains one of the main parasitic diseases in the world and is caused by a trematode of the species Schistosoma mansoni, whose invertebrate hosts are molluscs of the genus Biomphalaria. Biomphalaria tenagophila is an important host in Brazil, with some strains being highly susceptible to the parasite, whereas others are absolutely resistant to infection. The nature of the resistance and susceptibility of B. tenagophila is not completely understood, but it is assumed that the hemocytes, effector cells of molluscs internal defense system, play a crucial role in controlling trematode infections. Thus, the study of proteins produced by hemocytes in response to infection by S. mansoni can generate relevant contributions to a better understanding of host-parasite interaction. This study compared the proteomic profiles of hemocytes from the hemolymph of resistant B. tenagophila populations (Taim / RS and Anchieta / ES) and a susceptible one (Cabo Frio / RJ), challenged or not with S. mansoni. Through the two-dimensional electrophoresis and DIGE the protein profiles of the three populations before and after challenge were evaluated and then differentially expressed proteins were identified by mass spectrometry. The findings showed proteins with altered expression between resistance and susceptibility phenotypes, especially after exposure to S. mansoni. In the Taim challenged population, it was observed an elevation of two relevant proteins: a member of the thioester-containing proteins (TEP) family, which is related to phagocytosis or encapsulation of pathogens; and a hypothetical protein LOTGIDRAFT_234865 involved in binding and transport of a product of reactive oxygen species formation. Increased HSP70 and fructose-bisphosphate aldolase expression in Cabo Frio challenged may be related to this strain susceptible phenotype. The absence of the complete B. glabrata genome affected proteins identification and database analysis. Nevertheless, results were significant and confirmed the already known complexity of the molecular interaction between Biomphalaria and S. mansoni.

Brucella ovis é a principal causa de falha reprodutiva em carneiros e é uma das poucas espécies de Brucella spp. que não é zoonótica. Os objetivos deste trabalho foram realizar análise proteômica comparativa do mutante sem um transportador ABC espécie-específico funcional (abcAB) com B. ovis selvagem (WT) e caracterizar seu tráfego intracelular, para elucidar o papel do sistema ABC na sua sobrevivência in vitro e intracelular. Na análise proteômica, foram identificados 55 spots de proteínas diferentemente expressas (p

FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais Campilobacteriose Genital Bovina é uma doença venérea que causa problemas reprodutivos em rebanhos, com grande importância em países onde há largo emprego da monta natural. Phage Display é uma técnica que aplica bibliotecas de bacteriófagos que expõem peptídeos fusionados ao capsídeo viral em seleções biológicas contra proteínas alvo. Com o objetivo de selecionar peptídeos miméticos à bactéria Campylobacter fetus subsp. venerealis e fragmentos de anticorpos recombinantes (scFv) foram empregadas biblioteca comercial de peptídeos conformacionais de sete aminoácidos e biblioteca de scFv construída contra células tumorais. Foram selecionados cinco peptídeos considerados miméticos a C. fetus subsp. venerealis NCTC 10354 – P3, com os quais foram produzidos soros hiperimune em camundongo e realizados imunoensaios com antígenos bacterianos e amostras de muco cérvico-vaginal de novilhas. Oito clones que expressam moléculas de scFv reativos contra C. fetus subsp. venerealis NCTC 10354 – P3 foram selecionados e triados por meio de ELISA Indireto com outras bactérias. Dentre os peptídeos miméticos, dois foram considerados promissores (ALASLPL e LSYLFPP) devido à reação do soro hiperimune contra a bactéria e dos peptídeos sintéticos com muco cérvico-vaginal. Os clones de scFv apresentaram reações cruzadas entre bactérias do gênero Campylobacter e dois (3H6 e 4G7) apresentaram baixa reatividade contra Escherichia coli e Staphylococcus aureus. Bovine genital Campylobacteriosis is a venereal disease that causes reproductive problems in herds, with great importance in countries where there is widespread use of natural service. Phage display is a technique that applies bacteriophage libraries that displays peptides fused to the viral capsid proteins in selections against biological target. The aim of the present study was to select mimetic peptides to Campylobacter fetus subsp. venerealis and recombinant monoclonal antibody fragments (scFv). Library of conformational seven aminoacids peptides and scFv library constructed from tumor cells were uses. Five peptides was considered mimetics to C. fetus subsp. venerealis NCTC 10354 - P3, and hyperimmune sera were produced in mice and performed immunoassays with bacterial antigens and samples of cervical-vaginal mucus of heifers. Eight clones expressing scFv antibodies reactive against C. fetus subsp. venerealis NCTC 10354 - P3 were selected and screened by ELISA. Among the mimetic peptides, two were considered promising (ALASLPL and LSYLFPP) due to the reaction of hyperimmune serum against bacteria and synthetic peptides with cervical-vaginal mucus. ScFv antibodies showed cross-reactivity between bacteria of the genus Campylobacter and two clones (3H6 and 4G7) showed low reactivity against Escherichia coli and Staphylococcus aureus.

A epididimite infecciosa, considerada uma das maiores causas de prejuízos econômicos na ovinocultura mundial, é causada principalmente pelas bactérias Brucella ovis, Actinobacillus seminis e Histophilus somni. Este trabalho teve por objetivo investigar as lesões genitais causadas pelas infecções por A. seminis e H. somni em ovinos e desenvolver um método de diagnóstico direto e diferencial para as três infecções. Foram realizadas infecções experimentais, por A. seminis e por H. somni, em ovinos de 18 a 24 meses de idade. Os carneiros foram examinados semanalmente e foram coletadas diferentes amostras biológicas para fins de diagnóstico. A. seminis e H. somni foram capazes de causar infecção, colonizando diferentes órgãos do trato genito-urinário, sendo indistinguíveis apenas por exame clínico, achados de necropsia ou histopatologia. Mesmo sem diferenciar o agente etiológico, o exame clínico continuou sendo imprescindível como teste de triagem, pois possibilitou a detecção de indícios de epididimite infecciosa, mesmo antes do teste laboratorial. Por outro lado, para o diagnóstico diferencial, é importante o uso de técnicas de diagnóstico direto, com confirmação do agente etiológico. O ensaio de PCR multiplex espécie-específico desenvolvido no presente estudo foi igualmente eficaz à bacteriologia (padrão-ouro) para o diagnóstico diferencial das três maiores causas de epididimite infecciosa ovina, podendo ser usado em substituição ao isolamento bacteriano. Além disso, urina e lavado prepucial também podem ser usados como amostras alternativas ao sêmen para extração de DNA e emprego da PCR multiplex descrita neste estudo. Infectious epididymitis is considered a major cause of economic losses for the sheep industry worldwide. The most common causative agents include Brucella ovis, Actinobacillus seminis and Histophilus somni. This study aimed to investigate changes associated with experimental infections with A. seminis and H. somni in rams, and to develop a direct method for differential diagnosis for these three infections. Experimental infections with A. seminis and H. somni were carried out in rams with 18 to 24 months of age. Rams were weekly examined and biological samples were collected for diagnostic purposes. A. seminis and H. somni werecapable of causing infection, colonizing several organs of the genitourinary tract, being indistinguishable only by clinical exam, necropsy or histopathology. Nevertheless, the clinical exam proved to be indispensable as a screening method. Conversely, the importance of laboratorial diagnostic techniques for direct confirmation of the etiologic agent was demonstrated in this study. Therefore, the species-specific multiplex PCR assay developed in this study can be successfully used for the diagnosis of the three most common causes of ovine infectious epididymitis. Furthermore, this technique is a practical alternative to bacteriology. Urine and preputial wash can be used as alternatives to semen samples for DNA extraction and application of the multiplex PCR described in this study.

A Brucella ovisé uma importante causa de epididimite em ovinos e consequentemente ocasiona perdas econômicas para a ovinocultura por afetar a reprodução dos animais. Considerando a escassez de estudos sobre como a B. ovis interage com componentes dos sistema imunológico inato, como os receptores semelhante a Toll (TLR), este trabalho caracterizou a importância da sinalização por TLR para o controle da infecção por B. ovis e a proteína TcpB, descrita anteriormente como mecanismo de evasão da sinalização TLR em B. abortus e B. melitensis. Camundongos C57/BL6 knockout para TLR2, TLR4, TLR9 e MyD88 foram infectados com B. ovise submetidos a eutanásia após sete dias de infecção (dpi) para avaliação bacteriológica, histopatológica e número de cópias de RNA. Observou-se que os camundongos MyD88-/- apresentaram maior colonização esplênica quando comparados com os camundongos C57/BL6 selvagens. Este aumento de susceptibilidade ocorreu devido ao menor desenvolvimento de resposta inflamatória destes animais, demonstrado por menor inflamação em baço e fígado e menor transcrição de citocinas pró-inflamatórias como TNF-e IL-6, necessárias para o desenvolvimento adequado de resposta contra Brucella. Os animais TLR9-/- também apresentaram maior colonização bacteriana esplênica quando comparados com camundongos selvagens mas, interessantemente desenvolveram uma resposta inflamatória maior que o grupo controle. Em comparação com camundongos selvagens, animais TLR2-/- e TLR4-/- não apresentaram diferenças de susceptibilidade à infecção por B. ovis. Portanto, MyD88 é necessário para o desenvolvimento de uma resposta inflamatória eficaz contra B. ovis no modelo murino. Adicionalmente, foi gerado uma amostra mutante de B. ovis construída a partir da deleção do gene tcpBe a cinética de infecção foi realizada em camundongos C57/BL6. A ausência deste geneacarretou em fenótipo atenuado in vivo caracterizado pela menor colonização bacteriana desde o primeiro dpi e incapacidade de persistir no hospedeiro até 30 dpi. Contudo, esta amostra mutante apresentou uma deficiência de crescimento in vitro. Portanto, a proteína TcpB funcional é necessária para a virulência e sobrevivência de B. ovisem camundongos, mas estudos bacteriológicos com o mutante desenvolvido neste estudo devemser realizados para determinar se a amostra apresenta um defeito metabólico ou de crescimento. Brucella ovisis a significant cause of epididymitis and thus causes economic loss to the sheep industry by affecting animal reproduction. Considering the scarcity of studies on the interaction of B. ovis with innate system components, such as Toll-like receptors (TLR), this work highlights the importance of TLR signalling to resistance to B. ovis and the protein TcpB, described as an evasion mechanism of TLR signaling in B. abortus and B. melitensis, as a virulence factor. C57/BL6 knockout forTLR2, TLR4, TLR9, and MyD88 mice were infected with B. ovisand euthanized after seven days of infection (dpi) for bacteriologic, histopathologic and gene expression evaluation. MyD88-/- mice showed higher spleen colonization when compared with wild-type C57/BL6 mice. This increase in susceptibility was due to smaller inflammatory response in these animals, demonstrated by smaller inflammation in spleen and liver and smaller transcription of proinflammatory citokines such as TNF-and IL-6, necessary for proper response against Brucella. TLR9-/- mice also presented larger spleen bacterial colonization in comparison with wild-type mice, having, however, developed larger inflammatory response than the control group. TLR2-/- e TLR4-/- mice showed no diferent susceptibility to B. ovisinfection in comparison with wild-type mice. Therefore, MyD88 is necessary for the developmentof efficient inflammatory response against B. ovis in the murine model. Additionally, a mutant strain of B. ovis was generated from the deletion of the tcpBgene. B. ovis tcpB was inoculated in C57/BL6 mice and the infection kinetic was carried out. The absence of this gene resulted in in vivo attenuated phenotype, characterized by smaller bacterial colonization from 1 dpi and lack of capacity to persist in host. However, this mutant strain showed a growth defect in vitro. Therefore, the functional protein TcpB is necessary for B. ovisvirulence and survival in mice, but further bacteriological studies of the mutation developed in this work should be carried out to determineif the sample shows a metabolic or growth defect.