Brucella ovis mutant in ABC transporter protects against Brucella canis infection in mice and it is safe for dogs.

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.

Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of Brucella melitensis.

Brucella spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR) sensor IRE1α (inositol-requiring enzyme 1) and ER-associated autophagy confer susceptibility to Brucella melitensis and Brucella abortus intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from Ern1 (encoding IRE1) conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to Brucella intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of Brucella intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of Brucella.

Comparative experimental infection of Listeria monocytogenes and Listeria ivanovii in bovine trophoblasts.

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.

Iron acquisition pathways and colonization of the inflamed intestine by Salmonella enterica serovar Typhimurium.

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.

The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages.

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.

Encapsulated Brucella ovis Lacking a Putative ATP-Binding Cassette Transporter (ΔabcBA) Protects against Wild Type Brucella ovis in Rams.

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.

Salmonella enterica Serovar Typhi conceals the invasion-associated type three secretion system from the innate immune system by gene regulation.

Delivery of microbial products into the mammalian cell cytosol by bacterial secretion systems is a strong stimulus for triggering pro-inflammatory host responses. Here we show that Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, tightly regulates expression of the invasion-associated type III secretion system (T3SS-1) and thus fails to activate these innate immune signaling pathways. The S. Typhi regulatory protein TviA rapidly repressed T3SS-1 expression, thereby preventing RAC1-dependent, RIP2-dependent activation of NF-κB in epithelial cells. Heterologous expression of TviA in S. enterica serovar Typhimurium (S. Typhimurium) suppressed T3SS-1-dependent inflammatory responses generated early after infection in animal models of gastroenteritis. These results suggest that S. Typhi reduces intestinal inflammation by limiting the induction of pathogen-induced processes through regulation of virulence gene expression.

Clinical and pathological changes in rams experimentally infected with Actinobacillus seminis and Histophilus somni.

Infectious epididymitis is considered a major cause of economic losses for the sheep industry worldwide. This study aimed to investigate clinical and pathological changes associated with experimental infections with A. seminis and H. somni in rams. Twenty rams of age 18 to 24 months were infected by intraepididymal inoculation of A. seminis (n = 10) and H. somni (n = 10). Rams were weekly examined and biological samples were collected during six weeks. All rams inoculated with A. seminis and 80% inoculated with H. somni became infected. The recovery of bacteria was possible in semen and urine samples and tissues in both experimental groups. Clinically, there were a decrease in testicular consistency and an increase in measures of the left epididymis tails in both experimental groups. The main gross changes were observed in the reproductive tract. Microscopically, the main lesions were inflammatory changes in the genitourinary tract and testicular degeneration. A. seminis and H. somni were able to colonize several organs of the genitourinary tract in rams, being indistinguishable by clinical exam, necropsy or histopathology. For differential diagnosis, it is important to use diagnostic techniques for direct confirmation of the etiologic agent.

Hemorrhagic colitis associated with Salmonella enterica serotype Infantis infection in a captive western lowland gorilla (Gorilla gorilla gorilla) in Brazil.

BACKGROUND: Enteric diseases are among the most common causes of morbidity and mortality in gorillas, and it is often caused by bacteria. METHODS: A thirteen-year-old captive female western lowland gorilla (Gorilla gorilla gorilla) developed hemorrhagic diarrhea. Despite the treatment, the animal died 7 days after the onset of clinical signs. The animal was submitted to a thorough pathological and microbiological evaluation. RESULTS: Pathologic examination revealed a severe acute hemorrhagic colitis, neutrophilic splenitis, glomerulitis, and interstitial pneumonia. Salmonella enterica serotype Infantis was isolated from a mesenteric lymph node. CONCLUSION: A diagnosis of hemorrhagic colitis associated with Salmonella enterica serotype Infantis was established.

Brucella ovis lacking a species-specific putative ATP-binding cassette transporter is attenuated but immunogenic in rams.

Ovine brucellosis caused by Brucella ovis is considered one of the most important reproductive diseases of rams worldwide. This study aimed to characterize the kinetics of infection of a ΔabcAB B. ovis mutant strain in rams. Twelve 1-year-old crossbred rams were used. Six rams were challenged with 2 mL of a suspension containing 1.2×10(9) CFU/mL of B. ovis strain ATCC25840 (wild type) by intraprepucial inoculation and additional 50 µL in each conjunctival sac of a suspension containing 1.2×10(10) CFU/mL of the same strain. The other six rams were challenged with an equivalent number of CFU of the mutant strain ΔabcAB B. ovis through the same routes. Serum samples for serology and semen and urine samples for bacteriologic culture and PCR were collected weekly during 24 weeks. At 24 weeks post infection, tissue samples were collected for bacteriologic culture and PCR. All rams inoculated with wild type or the ΔabcAB strain seroconverted at the fourth week post infection, remaining positive up to the 16th week post infection. PCR and bacteriology demonstrated that only rams inoculated with the wild type strain shed the organism in semen and urine. Lymphocytes from rams inoculated with wild type or ΔabcAB B. ovis had significantly higher proliferation in response to B. ovis antigens when compared with unstimulated controls. Tissue bacteriology and PCR detected B. ovis in all rams challenged with the wild type strain, whereas only one ΔabcAB-infected ram had a positive iliac lymph node sample by PCR.

Distribution of caprine arthritis encephalitis virus provirus, RNA, and antigen in the reproductive tract of one naturally and seven experimentally infected bucks.

Caprine arthritis encephalitis is a worldwide, multisystemic disease caused by a small ruminant lentivirus. Although the main route of transmission is oral, detection of proviral DNA of the caprine arthritis encephalitis virus (CAEV) in caprine semen has been previously described. However, the presence of viral antigens in the male reproductive tract has apparently never been reported. The objective was to study lesions in the buck reproductive system and to detect, in these tissues, the presence of proviral DNA, viral RNA and CAEV antigens. Tissues from eight CAEV-infected bucks (one naturally and seven experimentally infected) were analyzed by histopathology, nested polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and immunohistochemistry. Interstitial pneumonia, synovitis, and lesions in the male reproductive tract were detected in some of the bucks. Proviral DNA was detected in the lungs and joints as well as in the reproductive systems of all animals, whereas viral RNA was detected only in the genital tract of the naturally infected buck. Viral antigens were immunostained in most of the organs of the male reproductive tract. This report was apparently the first to clearly demonstrate CAEV antigen expression in the male reproductive tract, which indicates the possibility of venereal transmission of CAEV.

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

BACKGROUND: Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. RESULTS: The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. CONCLUSIONS: The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

Salmonellosis in cattle: advantages of being an experimental model.

Salmonellosis is an important disease of cattle caused predominantly by Salmonella enterica serotypes Typhimurium (S. typhimurium) and Dublin (S. dublin). S. typhimurium causes acute enteritis and exudative diarrhea in calves. In addition to enteric disease, S. dublin can cause systemic infections, and may cause abortion in pregnant cows. Calves are considered a relevant model for non-typhoidal salmonellosis in humans. Experimental oral infections or inoculation of ligated ileal loops in calves have been extensively studied recently. This article reviews relevant published results regarding bovine salmonellosis as a natural disease or as an animal model.

Tissue distribution of Leishmania chagasi and lesions in transplacentally infected fetuses from symptomatic and asymptomatic naturally infected bitches.

Visceral leishmaniasis (VL) is primarily transmitted by an invertebrate vector, but transmission in the absence of the vector has been reported. Vertical transmission of VL has been described in man and dogs. The aim of this study was to evaluate the distribution of Leishmania amastigotes in fetal organs and histopathologic changes associated with parasitism and to determinate the frequency of transplacental transmission and potential of vertical transmission by symptomatic and asymptomatic pregnant bitches. Symptomatic (n=4) and asymptomatic (n=4) pregnant bitches, serologically and parasitologically positive for Leishmania sp., carrying a total of 53 fetuses (26 from symptomatic and 27 from asymptomatic bitches) were selected at the Veterinary Hospital of the National University of Asuncion, Paraguay. Samples of placenta and fetal organs such as liver, spleen, lymph nodes, bone marrow, kidney and heart were histologically evaluated and processed for immunodetection of amastigotes and PCR. There were no lesions compatible with VL in fetal tissues in spite of the presence of amastigotes, particularly in lymphoreticular tissues. However, fetal hepatocytes had marked degenerative changes that were independent of the presence of amastigotes in liver. Twenty-six out of 53 placentas (13 symptomatic and 13 asymptomatic) and a total of 17 fetuses out of 53 (nine symptomatic and eight asymptomatic) were PCR positive. Together these findings indicate a high frequency of transplacental transmission and no differences in the potential of transmission when symptomatic were compared to asymptomatic pregnant bitches.