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Toxoplasmosis is an important zoonotic disease that affects a wide range of warm-blooded host species. Neotropical primates (New World Primates; NWP) are highly susceptible, developing a lethal acute systemic disease. Toxoplasmosis in free-ranging NWP is poorly described, with only a few studies based on serosurveys. Herein we performed a retrospective study focusing on the epidemiology and pathology of toxoplasmosis among 1,001 free-ranging marmoset ( Callithrix spp.) deaths from the Brazilian Atlantic Forest. This study included marmosets necropsied at the Instituto Municipal de Medicina Veterinária Jorge Vaitsman (IJV) from January 2017 to July 2019, which were found dead from all regions in the State of Rio de Janeiro. Histopathology, immunohistochemistry, and transmission electron microscopy were performed to better characterize toxoplasmosis in this free-ranging population. All samples were also tested for Yellow Fever Virus (YFV) RT-qPCR by the official diagnostic service. A total of 1,001 free-ranging marmosets were included in this study, with 16 (1.6%) cases of lethal Toxoplasma gondii infections identified both as individual cases and in outbreaks. Presence of infection was not associated with sex, age, geographical distribution, or year of death, and no co-infection with YFV was observed. The main pathological feature in these cases was random necrotizing hepatitis with detection of intralesional T . gondii zoites in all infected cases. Interstitial pneumonia rich in alveolar foamy macrophages and fibrin deposition, necrotizing myocarditis and necrotizing splenitis were also pathological features in affected marmosets. Therefore, toxoplasmosis was considered the cause of death in 1.6% of free-ranging marmosets in this retrospective series, including some cases associated with outbreaks. Necrotizing random hepatitis was a consistent pathological finding in affected cases and sampling of liver should be ensured from Callitrichid post mortem cases.

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.

This study investigated the occurrence of selected pathogens of bovine respiratory disease in fetal pulmonary tissue of cattle and associated these with patterns of disease. Fetal pulmonary (n = 37) tissues were evaluated by histopathology; immunohistochemical assays identified intralesional antigens of bovine alphaherpesvirus 1 (BoAHV1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and Mycoplasma bovis. Molecular assays were performed to amplify reproductive disease pathogens and bovine gammaherpesvirus 6 (BoGHV6) from 12 lungs. The 2 patterns of pulmonary diseases were interstitial pneumonia (12/37) and suppurative bronchopneumonia (1/37). The frequency of the intralesional antigens identified was BRSV (16.2%; 6/37), BVDV (13.5%; 5/37), BoAHV1 (8.1%; 3/37), M. bovis (5.4%; 2/37), and BPIV-3 (2.7%; 1/37). Interstitial pneumonia was associated with BRSV (n = 3), BoAHV1 (n = 3), and BVDV (n = 2); suppurative bronchopneumonia contained a Gram-positive bacterium and BVDV and BRSV. Reproductive pathogens detected included Leptospira spp., (n = 3), BVDV, Neospora caninum, and Brucella abortus (n = 2). BoGHV6 DNA was identified in the lungs of two fetuses with interstitial pneumonia. These findings suggest that these fetuses were infected transplacentally by several pathogens. The role of some of these pathogens herein identified must be further elucidated in the possible participation of fetal disease.

Salmonella is a relevant pathogen under a clinical and public health perspective. Therefore, there has been a significant scientific effort to learn about pathogenic determinants of this pathogen. The clinical relevance of the disease, associated with the molecular tools available to study Salmonella as well as suitable animal models for salmonellosis, have provided optimal conditions to drive the scientific community to generate a large expansion of our knowledge about the pathogenesis of Salmonella-induced enterocolitis that took place during the past two decades. This research effort has also generated a wealth of information on the host immune mechanisms that complements gaps in the fundamental research in this area. This review focus on how the interaction between Salmonella, the microbiota and intestinal innate immunity leads to disease manifestation. As a highly successful enteropathogen, Salmonella actively elicits a robust acute intestinal inflammatory response from the host, which could theoretically lead to the pathogen demise. However, Salmonella has evolved redundant molecular machineries that renders this pathogen highly adapted to the inflamed intestinal environment, in which Salmonella is capable of outcompete resident commensal organisms. The adaptation of Salmonella to the inflamed intestinal lumen associated with the massive inflammatory response that leads to diarrhea, generate perfect conditions for transmission of the pathogen. These conditions illustrate the complexity of the co-evolution and ecology of the pathogen, commensals, and the host.

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and animals. Currently available live attenuated vaccines against brucellosis still have drawbacks. Therefore, subunit vaccines, produced using epitope-based antigens, have the advantage of being safe, cost-effective and efficacious. Here, we identified B. abortus small RNAs expressed during early infection with bone marrow-derived macrophages (BMDMs) and an apolipoprotein N-acyltransferase (Int) was identified as the putative target of the greatest expressed small RNA. Decreased expression of Int was observed during BMDM infection and the protein sequence was evaluated to rationally select a putative immunogenic epitope by immunoinformatic, which was explored as a vaccinal candidate. C57BL/6 mice were immunized and challenged with B. abortus , showing lower recovery in the number of viable bacteria in the liver, spleen, and axillary lymph node and greater production of IgG and fractions when compared to non-vaccinated mice. The vaccinated and infected mice showed the increased expression of TNF-α , IFN-γ , and IL-6 following expression of the anti-inflammatory genes IL-10 and TGF-β in the liver, justifying the reduction in the number and size of the observed granulomas. BMDMs stimulated with splenocyte supernatants from vaccinated and infected mice increase the CD86+ marker, as well as expressing greater amounts of iNOS and the consequent increase in NO production, suggesting an increase in the phagocytic and microbicidal capacity of these cells to eliminate the bacteria.

Pseudoplacentational endometrial hyperplasia (PEH) is a common uterine lesion in dogs. A high frequency of pyometra has been associated with PEH in dogs, suggesting that PEH might be related to the pathogenesis of pyometra. This study aimed to assess transcription levels and expression of Toll like receptors (TLR) 1, 2 and 4; alpha estrogen receptors (ESR1), progesterone receptors (PR) and prolactin receptors (PRLR) in uteri with PEH. Furthermore, the inflammatory response of dog endometrium with PEH against ex vivo bacterial stimulus was also investigated. Uteri were classified as controls or with PEH. Uterine explants were cultured for 6 and 12 hours after in vitro stimulus with inactivated Escherichia coli . Transcription of receptors and proinflammatory cytokines, namely interleukin-6 ( IL-6 ) and CXCL8 were evaluated. Expression of receptors was also evaluated in uterine explants and uteri from biopsy archives. CXCL8 concentration was measured in supernatants from all cultured explants. Transcription levels and expression of both PR and ESR1 were lower in uteri explants with PEH not stimulated and cultured for 6 hours. Expression of PRLR was higher in uteri with PEH from biopsy archives. Proinflammatory response by transcription levels of interleukin 6 demonstrated downregulation in uteri with PEH at 6 hours of stimulation followed by upregulation at 12 hours. However, no differences between groups were observed. Both control and uteri with PEH secreted similar concentrations of CXCL8 at 6 hours of bacterial stimulation. At 12 hours, no response to stimulation was observed in the PEH group and supernatant concentrations of CXCL8 were higher in the control group. The inflammatory response to bacterial stimulus in uteri with PEH had different pattern than the control group, with an inversion in IL-6 transcription levels between 6–12 hours of culture. Additionally, CXCL8 production ceased earlier in explants with PEH than in control.

Serologic diagnosis of canine brucellosis caused by Brucella canis remains quite challenging since the currently available methods have considerable limitations, although the relevance and awareness about this zoonotic pathogen is increasing over the past few years. Therefore, the development of novel strategies is highly desirable. In this study, in silico analyses resulted in prediction of B. canis specific B cell epitopes, which were validated by synthesizing the corresponding peptides on a membrane followed by immunobloting with sera from dogs naturally infected with B. canis or uninfected controls as well as by excluding epitopes that cross reacted with sera from cattle and sheep infected with B. abortus and B. ovis , respectively. This approach resulted in the identification of 26 epitopes that reacted exclusively with sera from dogs infected with B. canis , from which the 15 with strongest signal in the immunoblot were selected and synthesized in soluble form for further analyses. The best 10 synthetic peptides (considering the noise to signal ratio) were used in combination as antigens for development of a B. canis specific indirect enzyme-linked immunosorbent assay (iELISA) protocol, which yielded improved specificity to differentiate from other common canine pathogens ( Leishmania sp. and Babesia sp.) and an improved performance (100% specificity and 80% sensitivity) when compared to crude bacterial protein extracts used as antigen for iELISA. These selected epitopes were also incorporated in a multi- B. canis epitope protein that was expressed in E. coli and employed as antigen for detection of anti- B. canis IgG and IgM from naturally infected dogs, resulting in analytical performance similar to the iELISA protocol in which synthetic peptides were used as antigens (100% specificity and 75% sensitivity). The results clearly indicate that combination of detection of IgM and IgG may result in higher sensitivity. Therefore, this study provides novel tools for improving the accuracy of serologic diagnosis of canine brucellosis caused by B. canis .

Serological methods are valuable diagnostic tools for bovine brucellosis, a zoonotic infectious disease with a worldwide distribution. Currently employed diagnostic methods utilize crude bacterial extract or regions of LPS, a molecule that can exhibit cross-reactions with other infectious agents. The goal of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISAi) using synthetic peptides or a multi-epitope protein based on in silico-predicted B cell epitopes. Peptides were synthesized on a cellulose membrane using spot synthesis, and immunoblots were performed to evaluate reactivity by densitometry against bovine sera of interest. Peptides reacting with serum from positive cattle and non-reactive to the serum from negative controls were selected, synthesized in a soluble form, and used as antigens for the development of the ELISAi. Two peptides (P1 and P2) were selected and, after standardization of the ELISAi, positive (25) and negative (175) samples were tested, resulting in a sensitivity of 84% (21/25) and specificity of 83.43% (146/175). The sequence of the two peptides in replicates with spacers were inserted into a multi-epitope protein, which was also used as antigen in an ELISAi resulting in a sensitivity of 72% (18/25) and a specificity of 61.71% (108/175). This study provided a preliminary analytical assessment of in silico-predicted epitopes for developing novel serologic diagnostic tests.

Aerococcus viridans is an emerging pathogen for humans and livestock animals, mainly associated with genitourinary infections cases. Its occurrence in wild mammals has never been reported. The aim of this study was to determine the etiological agent associated with clinical a case of a genital infection in a female African elephant (Loxodonta africana). Phylogenetic analysis and antimicrobial susceptibility profile of the isolate were also addressed. The animal presented frequent cases of genital infection with intermittent white secretion. Purulent secretion was sampled and submitted to bacteriological exam. The isolate obtained was thus identified by phenotypic and molecular methods as A. viridans and was found to be similar to human pathogenic isolates in BLASTn and phylogenetic analysis. The isolate was sensitive to almost all antimicrobials evaluated, presenting resistance to ciprofloxacin and norfloxacin. This is the first report of occurrence of A. viridans infection in the genital tract of an African elephant. RESUMO: Aerococcus viridans é um patógeno emergente para seres humanos e animais de produção, principalmente associado a casos de infecções geniturinárias. Sua ocorrência em mamíferos selvagens nunca foi relatada. O objetivo deste estudo foi determinar o agente etiológico associado a um caso clínico de infecção genital em uma fêmea de elefante africano (Loxodonta africana). Análises filogenéticas e perfil de susceptibilidade antimicrobiana do isolado também foram avaliados. O animal apresentou casos frequentes de infecção genital com eliminação de secreção branca intermitente. A secreção purulenta foi coletada e submetida a exame bacteriológico. O isolado obtido foi identificado por métodos fenotípicos e moleculares como A. viridans e apresentou alta similaridade a isolados humanos patogênicos nas análises de BLASTn e filogenética. O isolado foi sensível a quase todos os antimicrobianos avaliados, apresentando resistência à ciprofloxacina e norfloxacina. Este é o primeiro relato de ocorrência de infecção por A. viridans no trato genital de elefante africano.

The giant anteater ( Myrmecophaga tridactyla ) is a vulnerable species in South America and is considered endangered or near extinction in Central America. Therefore, studies describing the reproductive characteristics of this species are pivotal for its conservation. Thus, this study aimed to provide a morphological description of the female reproductive tissues of this species. We collected tissue samples from six female giant anteaters and performed gross, morphological, and histochemical analyses. Five adult subjects and one juvenile were included in the study. In the ovary, classifications were made according to the follicle and oocyte sizes: primordial, primary, secondary, early antral, or antral. Typical follicles with a single oocyte surrounded by a simple or stratified layer of cubic epithelium, atretic follicles, corpora lutea, corpora albicans, and ovarian cysts were also observed. No ovarian lesions were observed. By contrast, endometritis, metritis, mucometra, and endometrial cysts were identified in the uterus. Uterine alterations in these subjects were frequent and could affect reproduction.