Teane M. A. Silva

Venereal transmission of canine visceral leishmaniasis

Leishmania chagasi, the agent of visceral leishmaniasis in dogs in the Americas has a tropism to the male genital system, particularly the epididymis, prepuce, and glans penis, resulting in shedding of Leishmania in the semen. The goal of this study was to verify the possibility of venereal transmission of L. chagasi. Twelve Leishmania-free bitches, housed in the absence of the insect vector, copulated with multiple naturally infected dogs that were shedding Leishmania in the semen. PCR analysis of serially collected ejaculates indicated that shedding of Leishmania in the semen is intermittent. Three bitches seroconverted, and six were PCR positive by the end of the experimental period (165 days after the last copulation). These data support the notion that L. chagasi may be sexually transmitted from naturally infected dogs to susceptible bitches in the absence of the biological insect vector.

Laboratory animal models for brucellosis research.

Brucellosis is a chronic infectious disease caused by Brucella spp., a Gram-negative facultative intracellular pathogen that affects humans and animals, leading to significant impact on public health and animal industry. Human brucellosis is considered the most prevalent bacterial zoonosis in the world and is characterized by fever, weight loss, depression, hepato/splenomegaly, osteoarticular, and genital infections. Relevant aspects of Brucella pathogenesis have been intensively investigated in culture cells and animal models. The mouse is the animal model more commonly used to study chronic infection caused by Brucella. This model is most frequently used to investigate specific pathogenic factors of Brucella spp., to characterize the host immune response, and to evaluate therapeutics and vaccines. Other animal species have been used as models for brucellosis including rats, guinea pigs, and monkeys. This paper discusses the murine and other laboratory animal models for human and animal brucellosis.

Visceral leishmaniasis in captive wild canids in Brazil

Visceral leishmaniasis (VL) is endemic in Belo Horizonte (State of Minas Gerais, Brazil). Leishmania sp. can naturally infect several species of mammals, and the domestic dog is the most important reservoir of the disease in South America. This report describes five cases of visceral leishmaniasis in Brazilian canids. Among 15 animals kept in captivity in a zoo in Belo Horizonte (State of Minas Gerais, Brazil), two animals, a bush dog (Spheotos venaticos) and a hoary zorro (Lycalopex vetulus) were serologically positive and developed clinical signs of VL, whereas three other canids, including a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), and a hoary zorro (Lycalopex vetulus) had positive serological results without clinical signs.

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.

CD4+ T cell-derived IL-10 promotes Brucella abortus persistence via modulation of macrophage function.

Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4+CD25+ T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25+CD4+ T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection.

Lack of Endogenous IL-10 Enhances Production of Proinflammatory Cytokines and Leads to Brucella abortus Clearance in Mice

IL-10 is a cytokine that regulates the balance between pathogen clearance and immunopathology. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals. Here we evaluated the contribution of IL-10 in host immune response and pathology during B. abortus infection. To assess the role of IL-10 in vivo, IL-10 knockout (KO) or 129 Sv/Ev (wild-type) mice were infected with B. abortus and the number of viable bacteria from the spleen was determined at 1, 2, 3, 6 and 14-weeks postinfection. IL-10 KO mice showed reduced bacterial loads in the spleen when compared to wild-type mice during all time points studied. Additionally, at 14-weeks postinfection IL-10 KO mice had totally cleared the infection. This clearance was preceded by an enhanced IFN-γ, TNF-α and IL-17 responses in both the serum and the spleen of IL-10 KO mice. Additionally, dendritic cells from infected IL-10 KO mice produced elevated levels of IL-12 and TNF-α compared to wild-type animals. Histopathology analysis was performed and both KO and wild-type mice developed multifocal granulomas and necrosis in the liver. However, at six-weeks postinfection reduced numbers of granulomas was detected in IL-10 KO mice compared to wild-type animals. This reduced liver pathology at later stage of infection was accompanied by increased numbers of CD4+CD25+foxp3+ T cells and expression of TGF-β in IL-10 KO splenocytes. Taken together, our findings demonstrate that IL-10 modulates the proinflammatory immune response to B. abortus infection and the lack of IL-10 increases resistance to Brucella infection.

Development and evaluation of a species-specific PCR assay for the detection of Brucella ovis infection in rams

Brucella ovis infection is a major cause of epididymitis and infertility in rams, resulting in reproductive failure and significant economic losses worldwide. The goal of this study was to develop a PCR test targeting specific B. ovis genomic sequences. Specific primer pairs were designed targeting 12 of those ORFs. Samples of blood, serum, semen, urine, and preputial wash were collected from experimentally infected rams (n=9) every other week up to 180 days post infection (dpi), when tissue samples were obtained. Blood, serum, semen, urine, and preputial wash samples were obtained, in weekly intervals for 1 month, from eight rams belonging to a B. ovis-free flock. Semen samples were also obtained from rams belonging to naturally infected flocks (n=40). The limit of detection of this PCR protocol was 100, 10, and 1 CFU/mL for semen, urine and prepucial wash samples, respectively. Sensitivity and specificity values obtained with this PCR method were similar to that of bacteriology when evaluating biological samples. Agreement between PCR and bacteriology results was greater than 90%. These results clearly indicate that this species-specific PCR method is highly efficient for the diagnosis of B. ovis infection in semen, urine, preputial wash and tissue samples from infected rams.

Putative ATP-binding cassette transporter is essential for Brucella ovis pathogenesis in mice.

ABSTRACT Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic. Due to the scarcity of studies on B. ovis infection, a murine model of infection was developed. The roles of B. ovis genes encoding a putative hemagglutinin and an ABC transporter were investigated in the mouse model. The kinetics of B. ovis infection were similar in BALB/c and C57BL/6 mice, and both strains of mice developed multifocal microgranulomas in the liver and spleen, but only minimal colonization and histopathological changes were observed in the genital tract. Therefore, the mouse was considered a suitable infection model for B. ovis but not for B. ovis-induced genital disease. Two mutant strains were generated in this study (the ΔabcAB and Δhmg strains). The B. ovis ΔabcAB strain was attenuated in the spleens and livers of BALB/c mice compared to the wild-type (WT) strain (P < 0.001). Conversely, the Δhmg strain infected mice at the same level as WT B. ovis, suggesting that a putative hemagglutinin is not required for B. ovis pathogenesis. Additionally, the ΔabcAB strain did not survive in peritoneal macrophages, extracellularly in the peritoneal cavity, or in RAW 264.7 macrophages. Moreover, infection with the ΔabcAB strain was not lethal for male regulatory factor 1-knockout mice, whereas infection with the B. ovis WT strain was 100% lethal within 14 days postinfection. These results confirm that the predicted ABC transporter is required for the full virulence and survival of B. ovis in vivo.

The virB-encoded type IV secretion system is critical for establishment of infection and persistence of Brucella ovis infection in mice

Brucella spp. are gram-negative intracellular bacterial pathogens that cause chronic infections. Brucella virulence factors include a type IV secretion system (T4SS) and its lipopolysaccharide (LPS), which are essential for persistence. However, the role of the virB-encoded T4SS has not been investigated in naturally rough Brucella species such as Brucella ovis. In this study, male 6-week old BALBc mice were infected with B. ovis, Brucella abortus, and their respective ΔvirB2 mutant strains. During early infection, B. ovis and B. abortus wild type strains were similarly recovered from spleen. Interestingly, in contrast to ΔvirB2 B. abortus that was recovered at similar levels when compared to the wild type strain, the ΔvirB2 B. ovis was markedly attenuated as early as 24h post infection (hpi). The ΔvirB2 B. ovis was unable to survive and multiply in murine peritoneal macrophages and extracellularly within the peritoneal cavity at 12 and 24 hpi with lower splenic colonization than the parental strain at 6, 12 and 24 hpi. In contrast, wild type B. abortus and ΔvirB2 B. abortus had a similar kinetics of infection in this model. As expected, the T4SS was essential for intracellular replication of smooth and rough strains in RAW macrophages at 48 hpi. These results suggest that T4SS is important for survival of B. ovis in murine model, and that a T4SS deficient B. ovis strain is cleared at earlier stages of infection when compared to a similar B. abortus mutant.

The Predicted ABC Transporter AbcEDCBA Is Required for Type IV Secretion System Expression and Lysosomal Evasion by Brucella ovis

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.