Trypanosoma cruzi in domestic and wild mammals in the northeast region of Colombia.

Trypanosoma cruzi hosts can serve as a source of infection for animals, vectors, and humans, contributing to the establishment of Chagas disease (CD) in a given area. Traditionally, the Department of Córdoba has not been considered a transmission area for CD; however, the report of several acute cases of Chagas disease highlights the importance of studying the dynamics of disease transmission in this region. This study aimed to detect T. cruzi in domestic and wild mammals in the department of Córdoba. In 2017, a cross-sectional descriptive study was conducted in six villages in two municipalities in the department of Córdoba. Blood samples from dogs living in the zones were collected in EDTA vacutainer tubes for domestic mammals. Wild mammals were collected using Sherman and Tomahawk traps and mist nets in crops and peridomiciles. T. cruzi DNA was detected using the kinetoplast DNA (kDNA) variable region and the tandem repeat satellite region of T. cruzi as molecular targets. We sampled 168 dogs and 146 wild mammals. The detected prevalence of T. cruzi was 6.37%; the TcI lineage was found in D. marsupialis, H. anomalus, and one canine. A specimen of D. marsupialis with TcI and TcII lineages was also identified. T. cruzi DNA was detected in domestic and wild animals in the study area, indicating the circulation of the parasite in peridomestic environments. D. marsupialis may represent an important host in maintaining this region's wild and domestic cycle.

Occurrence of Clostridium perfringens in Wild Mammals in the Amazon Biome.

The objective of this study was to evaluate the occurrence of Clostridium perfringens in stool samples and swabs collected from wild mammals in the Amazon biome. Sixty-five faecal and swab samples were collected in situ and ex situ from 16 species and three genera of wild mammals, some of which were in good health and some of which had diarrhoea. After pre-enrichment, the samples were plated on selective agar for C. perfringens. Characteristic colonies were subjected to multiplex PCR for the detection of genes encoding the main C. perfringens toxins (alpha, beta, epsilon, and iota toxin and enterotoxin). Among the 65 samples, 40 (61.5%) were positive for the gene encoding the alpha toxin and were classified as type A, 36 of which were asymptomatic animals and four were diarrheal. No other toxinotypes were found. The findings of this study suggest that C. perfringens type A is commonly found in mammal species of the Amazon biome. This seems to be the first study to identify C. perfringens type A in species such as B. variegatus (common ground sloth), C. didactylus (two-toed sloth), P. flavus (Jupará), T. tetradactyla (anteater), S. collinsi (squirrel monkey), S. niger (black marmoset), and S. apella (Guyana capuchin) and in the genus Didelphis sp. (opossum).

Meat-Borne-Parasite: A Nanopore-Based Meta-Barcoding Work-Flow for Parasitic Microbiodiversity Assessment in the Wild Fauna of French Guiana.

French Guiana, located in the Guiana Shield, is a natural reservoir for many zoonotic pathogens that are of considerable medical or veterinary importance. Until now, there has been limited data available on the description of parasites circulating in this area, especially on protozoan belonging to the phylum Apicomplexa; conversely, the neighbouring countries describe a high parasitic prevalence in animals and humans. Epidemiological surveillance is necessary, as new potentially virulent strains may emerge from these forest ecosystems, such as Amazonian toxoplasmosis. However, there is no standard tool for detecting protozoa in wildlife. In this study, we developed Meat-Borne-Parasite, a high-throughput meta-barcoding workflow for detecting Apicomplexa based on the Oxford Nanopore Technologies sequencing platform using the 18S gene of 14 Apicomplexa positive samples collected in French Guiana. Sequencing reads were then analysed with MetONTIIME pipeline. Thanks to a scoring rule, we were able to classify 10 samples out of 14 as Apicomplexa positive and reveal the presence of co-carriages. The same samples were also sequenced with the Illumina platform for validation purposes. For samples identified as Apicomplexa positive by both platforms, a strong positive correlation at up to the genus level was reported. Overall, the presented workflow represents a reliable method for Apicomplexa detection, which may pave the way for more comprehensive biomonitoring of zoonotic pathogens.

"Mi Casa, Tu Casa": the coati nest as a hub of Trypanosoma cruzi transmission in the southern Pantanal biome revealed by molecular blood meal source identification in triatomines.

BACKGROUND: The study of the ecology of Trypanosoma cruzi is challenging due to its extreme adaptive plasticity, resulting in the parasitism of hundreds of mammal species and dozens of triatomine species. The genetic analysis of blood meal sources (BMS) from the triatomine vector is an accurate and practical approach for gathering information on which wild mammal species participate in a local transmission network. South American coatis, Nasua nasua, act as important reservoir host species of T. cruzi in the Pantanal biome because of their high rate of infection and elevated parasitemia, with the main discrete typing unit (DTU) lineages (TcI and TcII). Moreover, the carnivore coati is the only mammal species to build high arboreal nests for breeding and resting that can be shared by various vertebrate and invertebrate species. Herein, we applied the sensitive and specific methodology of DNA barcoding and molecular cloning to study triatomines found in a coati nest to access the diversity of mammal species that explore this structure, and therefore, may be involved in the parasite transmission network. METHODS: Twenty-three Triatoma sordida were collected in one coati's nest in the subregion of Nhecolândia, Pantanal. The DNA isolated from the gut of insects was subjected to BMS detection by PCR using universal primers that flank variable regions of the cytochrome b (cytb) and 12S rDNA mitochondrial genes from vertebrates. The Trypanosoma spp. diagnosis and DTU genotyping were based on an 18S rDNA molecular marker and also using new cytb gene primers designed in this study. Phylogenetic analyses and chord diagrams were constructed to visualize BMS haplotypes, DTU lineages detected on vectors, and their interconnections. RESULTS: Twenty of 23 triatomines analyzed were PCR-positive (86.95%) showing lineages T. cruzi DTU TcI (n = 2), TcII (n = 6), and a predominance of TcI/TcII (n = 12) mixed infection. Intra-DTU diversity was observed mainly from different TcI haplotypes. Genetic analyses revealed that the southern anteater, Tamandua tetradactyla, was the unique species detected as the BMS of triatomines collected from the coati's nest. At least three different individuals of T. tetradactyla served as BMS of 21/23 bugs studied, as indicated by the cytb and 12S rDNA haplotypes identified. CONCLUSIONS: The identification of multiple BMS, and importantly, different individuals of the same species, was achieved by the methodology applied. The study demonstrated that the southern anteaters can occupy the South American coati's nest, serving as the BMS of T. sordida specimens. Since anteaters have an individualist nonsocial behavior, the three individuals detected as BMS stayed at the coati's nest at different times, which added a temporal character to BMS detection. The TcI and TcII infection, and significantly, a predominance of TcI/TcII mixed infection profile with different TcI and TcII haplotypes was observed, due to the discriminatory capacity of the methodology applied. Tamandua tetradactyla, a host which has been little studied, may have an important role in the T. cruzi transmission in that Pantanal subregion. The data from the present study indicate the sharing of coatis' nests by other mammal species, expanding the possibilities for T. cruzi transmission in the canopy strata. We propose that coatis' nests can act as the true hubs of the T. cruzi transmission web in Pantanal, instead of the coatis themselves, as previously suggested.

Investigation of Leishmania (Viannia) braziliensis Infection in Wild Mammals in Brazil.

PURPOSE: Leishmaniasis are infectious and zoonotic diseases and present in cutaneous and visceral forms. Cutaneous leishmaniasis is endemic and widely distributed throughout the state of Espírito Santo, Brazil. Several cases of cutaneous leishmaniasis in humans and dogs associated with Leishmania (Viannia) braziliensis have been reported in the state. This study aimed to investigate the occurrence of natural infection by Leishmania(Viannia) braziliensis in wild mammals found dead (by trampling or natural death) in the Sooretama Biological Reserve, Espírito Santo State. METHODS: From January 2018 to December 2019, 60 animals were collected. Of these, 47 animals from 12 different species were analyzed. The results were demonstrated using descriptive analysis of the observations to calculate the absolute and relative frequencies of the data. RESULTS: In the PCR, using specific primers for the genus Leishmania (D1, D2, and D3) and the species Leishmania (Viannia) braziliensis (ISVB/ISVC), 4 positive animals (8.5%) were detected: 1 Cuniculus paca (paca) (25%) and 3 Callithrix geoffroyi (white-faced marmoset) (25%). In the histopathological analysis, the parasitic amastigote form was not observed. CONCLUSIONS: The natural infection, detected by PCR, by Leishmania (Viannia) braziliensis in Cuniculus paca (paca) and Callithrix geoffroyi (white-faced marmoset) constitutes the first report of infection of this rodent and primate species in the literature. Despite the confirmation of Leishmania (Viannia) braziliensis infection in rodents and primates, the role of these species in the transmission of this zoonosis still needs further observational studies to identify their seasonal variation, transmissibility, infection stability, and the effects of a given parasite on the population and/or individual.

'Candidatus Mycoplasma haematonasua' and tick-borne pathogens in ring-tailed coatis (Nasua nasua Linnaeus, 1976) from the Iguaçu National Park, Paraná State, southern Brazil.

The Iguaçu National Park (INP) is the largest remnant of Atlantic Forest in southern Brazil, representing an ecological continuum with Argentina. The INP harbours a diverse fauna, with ring-tailed coatis (Nasua nasua Linnaeus, 1976, Carnivora: Procyonidae) in close contact with tourists either begging and/or snatching food from visitors. A potentially novel haemotropic Mycoplasma sp. has been previously detected in the ring-tailed coatis from central-western and southern Brazil. Therefore, the aims of this study were to investigate the occurrence of haemotropic Mycoplasma sp. and tick-borne pathogens in wild ring-tailed coatis from the INP, Foz do Iguaçu municipality, Paraná State, southern Brazil. Blood samples were collected from 18 wild ring-tailed coatis and evaluated by conventional PCR (cPCR) assays for haemotropic Mycoplasma spp. (16S and 23S rRNA), Theileria/Babesia spp. (18S rRNA) and Ehrlichia/Anaplasma spp. (16S rRNA, sodB, dsb and groEL). Eight out of 18 (44.44%; 95% CI: 24.56%-66.28%) animals were positive for haemotropic Mycoplasma spp. All ring-tailed coatis tested negative for Theileria/Babesia spp. and only one out of 18 (5.56%; 95% CI: 0.99%-25.76%) animals tested positive for Ehrlichia/Anaplasma spp. by the 16S rRNA cPCR. Unfortunately, multiple attempts to sequence the 16S rRNA gene of the Ehrlichia/Anaplasma-positive sample have failed. Phylogenetic and network analysis of the hemoplasma 16S and 23S rRNA gene fragments confirmed that animals were infected by a potentially novel haemotropic Mycoplasma sp. previously reported in ring-tailed coatis from Brazil. The name 'Candidatus Mycoplasma haematonasua' is proposed for this novel organism.

Relationships between vector-borne parasites and free-living mammals at the Brazilian Pantanal.

In nature, parasitic infections must be addressed as complex systems involving parasite-host relationships on a temporal and spatial scale. Since the parasites cover a great biological diversity, we can expect that wildlife are exposed simultaneously to different parasites. In this sense, the objective of this work was to determine the relationships between free-living mammals and their associated hemoparasites in the Brazilian Pantanal. We used the data published during 2017 and 2018 by de Sousa et al. regarding the detection of vector-borne pathogens (VBP), namely Anaplasma, Babesia, Bartonella, Cytauxzoon, Ehrlichia, Hepatozoon, Mycoplasma, and Theileria, in nine species of free-living mammals belonging to orders Carnivora, Rodentia, and Didelphimorphia. We assume as infected an individual positive on any of parasitological, molecular, and/or serological tests. We observed a strong association between the wild felid Leopardus pardalis with Cytauxzoon, the wild canid Cerdocyon thous with Hepatozoon, the small rodent Thrichomys fosteri with Bartonella, and the procyonid Nasua nasua with Mycoplasma and Theileria. Therefore, N. nasua, C. thous, T. fosteri, and the small rodent Oecomys mamorae can be considered key species for the maintenance of selected VBP in the Pantanal region, because they showed a high number of single and coinfections. Together, our results highlighted the importance of coinfection as a common phenomenon in nature.

Ovarian gene expression in collared peccary (Pecari tajacu Linnaeus, 1758) subjected to gonadotropin stimulation protocols.

Ovarian response of collared peccaries (Pecari tajacu), after hormonal stimulation with gonadotropin association (eCG/hCG), was accessed by both gene expression and follicular development. Thus, collared peccaries (n = 8) were treated with the dose used for sows (swine dose, SWD) or with dose adjusted for peccary's weight (allometric dose, ALD). The gene expression of receptors was evaluated for both gonadotropins (FSHR and LHCGR) and growth factors (proteins codified by TGFßR-1, BMPR1-A and BMPR2 genes) in antral follicles, cortex and corpora haemorrhagica (CH). Five days after gonadotropin injection, all females presented CH. The ovulation rate was similar (p > .05) between SWD (4.00 ± 1.17) and ALD (2.50 ± 0.43) group. The total number of follicles per animal and amounts of small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles was similar among groups. However, SWD produced large follicles heavier than ALD group, as accessed by weight of follicular wall biopsies. Ovarian follicles expressed both gonadotropin and growth factor receptors at levels which are independent from gonadotropin dose. In conclusion, the two gonadotropin doses (SWD and ALD) can be used for ovarian stimulation of collared peccary. Additionally, FSH and growth factors (TGFßR-1, BMPR1-A and BMPR2) receptors are more expressed in the early follicle development, while LH receptor seems to be more important in the final of follicular growth.

Study of Arboviruses in Philander opossum, Didelphis marsupialis and Nectomys rattus captured from forest fragments in the municipality of Belém, Pará, Brazil / Estudo de Arbovírus em Philander opossum, Didelphis marsupialis e Nectomys rattus capturados em fragmentos florestais no município de Belém, Pará

ABSTRACT: Arboviruses are viruses that maintain their life cycle in the wild and are transmitted to vertebrate hosts by hematophagous diptera. They are zoonotic and can establish an enzootic cycle in the urban areas; in humans, the infection can manifest from being encephalitogenic to hemorrhagic. This study aimed to report the occurrence of arboviruses in mammals of the order Didelphimorphia and Rodentia, captured from the Amazon. Serum samples were subjected to hemagglutination inhibition test using a viral panel of 19 species of arboviruses that are known to occur in the Amazon. Altogether, 14 wild mammals, 12 of Philander opossum, 1 of Didelphis marsupialis, and 1 of Nectomys rattus were captured. Eight of these were reported to be seropositive for arboviruses (57.14%) with monotypic seroprevalence for the Eastern Equine Encephalitis Virus (n=1), the Ilheus Virus (n=2), and the Catu virus (n=4); 4 heterotypic responses were observed for Flavivirus and Orthobunyavirus. In conclusion, several arbovirus species are in active circulation and maintenance, exhibiting enzootic characteristics in the wild mammals of the Amazon region; these animals prove to be potential hosts in the transmission of diseases to humans.

RESUMO: Os arbovírus são vírus que mantêm o seu ciclo de vida em ambiente silvestre. Eles são transmitidos aos hospedeiros vertebrados por dípteros hematófagos, tem caráter zoonótico podendo estabelecer um ciclo enzoótico no meio urbano, sendo que em humanos a infecção pode ter caráter encefalomiogênico a hemorrágico. Este estudo teve como objetivo relatar a ocorrência de arbovírus em mamíferos da ordem Didelphimorphia e Rodentia capturados na Amazônia. Os soros dos animais foram submetidos a testes de Inibição da Hemaglutinação utilizando um painel viral com as 19 principais espécies de arbovírus que ocorrem na Amazônia. Foram capturados 14 espécimes de mamíferos silvestres, 12 Philander opossum, 1 Didelphis marsupialis e 1 Nectomys rattus. A soropositividade para arbovírus foi observada em 57,14% (8/14) dos espécimes estudados, com soroprevalência monotípica para o vírus da Encefalite Equina do Leste (n = 1), o vírus Ilheus (n = 2) e o vírus Catu (n = 4) e quatro reações heterotípicos para Flavivírus e Orthobyavírus. Conclui-se que há manutenção e circulação de espécies de arbovírus com características enzoóticas em mamíferos silvestres da região amazônica, podendo ser hospedeiros em potenciais na transmissão da doença para humanos.

Bancos de recursos somáticos: Como eles contribuem para a conservação de grandes felídeos? / Somatic resource bank: How they contribute to the conservation of large felines?

Distintas estratégias de conservação têm sido adotadas visando a manutenção e recuperação da biodiversidade, especialmente de espécies que se encontram em diferentes níveis de ameaça à extinção. Neste grupo de espécies, encontram-se os grandes felídeos, os quais em virtude das ações antrópicas, como a destruição e a fragmentação de habitat, necessitam de esforços voltados para a conservação de seu material genético. Uma estratégia empregada para essa finalidade consiste nos bancos de recursos somáticos, os quais são definidos como a criopreservação de tecidos e células somáticas oriundas de diferentes populações. Essas amostras biológicas, quando adequadamente conservadas, são elementoschave para a multiplicação das espécies por meio de seu emprego na clonagem por transferência nuclear e indução de células à pluripotência. Assim, o objetivo desta revisão é abordar os aspectos relevantes envolvidos na formação de bancos de recursos somáticos em grandes felídeos, destacando os estudos desenvolvidos pelo Laboratório de Biotecnologia Animal (LBA), da Universidade Federal Rural do Semi-Árido (UFERSA) em onças-pintadas e onças-pardas.

Different conservation strategies have been adopted to maintain and recover biodiversity, especially for species that are at different levels of threat to extinction. In this group of species are the large felids, which, due to anthropic actions, such as habitat destruction and fragmentation, require efforts aimed at the conservation of their genetic material. A strategy employed for this purpose consists of somatic resource banks, which are defined as the cryopreservation of tissues and somatic cells from different populations. These biological samples, when properly preserved, are key elements for the multiplication of species through their use in cloning by nuclear transfer and induction of cells to pluripotency. Thus, the aim of this review is to address the relevant aspects involved in the formation of somatic resource banks in large felids, highlighting the studies carried out by the Laboratory of Animal Biotechnology (LBA) of the Federal Rural University of the Semi-Arid (UFERSA) in jaguars and pumas.

Study of Arboviruses in Philander opossum, Didelphis marsupialis and Nectomys rattus captured from forest fragments in the municipality of Belém, Pará, Brazil / Estudo de Arbovírus em Philander opossum, Didelphis marsupialis e Nectomys rattus capturados em fragmentos florestais no município de Belém, Pará

Arboviruses are viruses that maintain their life cycle in the wild and are transmitted to vertebrate hosts by hematophagous diptera. They are zoonotic and can establish an enzootic cycle in the urban areas; in humans, the infection can manifest from being encephalitogenic to hemorrhagic. This study aimed to report the occurrence of arboviruses in mammals of the order Didelphimorphia and Rodentia, captured from the Amazon. Serum samples were subjected to hemagglutination inhibition test using a viral panel of 19 species of arboviruses that are known to occur in the Amazon. Altogether, 14 wild mammals, 12 of Philander opossum, 1 of Didelphis marsupialis, and 1 of Nectomys rattus were captured. Eight of these were reported to be seropositive for arboviruses (57.14%) with monotypic seroprevalence for the Eastern Equine Encephalitis Virus (n=1), the Ilheus Virus (n=2), and the Catu virus (n=4); 4 heterotypic responses were observed for Flavivirus and Orthobunyavirus. In conclusion, several arbovirus species are in active circulation and maintenance, exhibiting enzootic characteristics in the wild mammals of the Amazon region; these animals prove to be potential hosts in the transmission of diseases to humans.(AU)

Os arbovírus são vírus que mantêm o seu ciclo de vida em ambiente silvestre. Eles são transmitidos aos hospedeiros vertebrados por dípteros hematófagos, tem caráter zoonótico podendo estabelecer um ciclo enzoótico no meio urbano, sendo que em humanos a infecção pode ter caráter encefalomiogênico a hemorrágico. Este estudo teve como objetivo relatar a ocorrência de arbovírus em mamíferos da ordem Didelphimorphia e Rodentia capturados na Amazônia. Os soros dos animais foram submetidos a testes de Inibição da Hemaglutinação utilizando um painel viral com as 19 principais espécies de arbovírus que ocorrem na Amazônia. Foram capturados 14 espécimes de mamíferos silvestres, 12 Philander opossum, 1 Didelphis marsupialis e 1 Nectomys rattus. A soropositividade para arbovírus foi observada em 57,14% (8/14) dos espécimes estudados, com soroprevalência monotípica para o vírus da Encefalite Equina do Leste (n = 1), o vírus Ilheus (n = 2) e o vírus Catu (n = 4) e quatro reações heterotípicos para Flavivírus e Orthobyavírus. Conclui-se que há manutenção e circulação de espécies de arbovírus com características enzoóticas em mamíferos silvestres da região amazônica, podendo ser hospedeiros em potenciais na transmissão da doença para humanos.(AU)

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines.

BACKGROUND: Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecosystem. We identified collared peccary fibroblasts by immunofluorescence and evaluated their morphology, growth and adherence capacity. Further, we monitored the viability and metabolic activity of the fibroblasts to determine the effects of passage number and cryopreservation on establishment of cell lines. METHODS: Skin biopsies were collected from the peripheral ear region from five adult animals in captivity. Initially, cells were isolated from fragments and cultured in the Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 2% antibiotic-antimycotic solution under a controlled atmosphere (38.5 °C, 5% CO2). We evaluated the maintenance of primary cells for morphology, adherence capacity of explants, explants in subconfluence, cell growth and absence of contamination. Moreover, we identified the fibroblast cells by immunofluorescence. Additionally, to evaluate the influence of the number of passages (first, third and tenth passage) and cryopreservation on establishment of cell lines, fibroblasts were analysed for the viability, metabolic activity, population doubling time (PDT), levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). RESULTS: All explants (20/20) adhered to the dish in 2.4 days ± 0.5 with growth around the explants in 4.6 days ± 0.7, and subconfluence was observed within 7.8 days ± 1.0. Moreover, by morphology and immunocytochemistry analyses, cells were identified as fibroblasts which presented oval nuclei, a fusiform shape and positive vimentin staining. No contamination was observed after culture without antibiotics and antifungals for 30 days. While there was no difference observed for cell viability after the passages (first vs. third: P = 0.98; first vs. tenth: P = 0.76; third vs. tenth: P = 0.85), metabolic activity was found to be reduced in the tenth passage (23.2 ± 12.1%) when compared to that in the first and third passage (100.0 ± 24.4%, P = 0.006). Moreover, the cryopreservation did not influence the viability (P = 0.11), metabolic activity (P = 0.77), or PDT (P = 0.11). Nevertheless, a greater ΔΨm (P = 0.0001) was observed for the cryopreserved cells (2.12 ± 0.14) when compared to that in the non-cryopreserved cells (1.00 ± 0.05). Additionally, the cryopreserved cells showed greater levels of intracellular ROS after thawing (1.69 ± 0.38 vs. 1.00 ± 0.22, P = 0.04). CONCLUSIONS: This study is the first report on isolation, characterization and cryopreservation of fibroblasts from collared peccaries. We showed that adherent cultures were efficient for obtaining fibroblasts, which can be used as donor cells for nuclei for species cloning and other applications.

BMP-15 activity on in vitro development of collared peccary (Pecari tajacu Linnaeus, 1758) preantral follicles.

This study investigated the effects of BMP-15 on the in vitro development of preantral follicles of collared peccaries. Ovarian fragments were cultured for 1 or 6 days in Tissue Culture Medium 199 (TCM199+ ) supplemented with BMP-15 at rates of 0, 1, 25 or 50 ng/ml. The fragments were analysed histologically by evaluating follicular morphology, activation and growth as well as the potential for proliferation of granulosa cells. Our results show the addition of 25 ng/ml BMP-15 in the medium provided the greatest percentage of normal follicles (79.67% ± 0.69) when compared to other treatments (p < .05); however, this result is similar to 1 ng/ml BMP-15 (74.00% ± 1.90, p > .05). Moreover, 25 and 50 ng/ml of BMP-15 promoted follicular activation. BMP-15 supplements did not affect oocyte and follicular growth. All concentrations of BMP-15 increased the number of nucleolus organizer regions (NORs) after 1 day of culture when compared to fresh fragments or the control samples (p < .05). However, at the end of the experiment, the number of NORs in follicles cultured in all treatments was higher than that observed in the fresh control (sample taken prior to culturing) (p > .05). In summary, the addition of 25 ng/ml BMP-15 to the culture medium of collared peccary preantral follicles maintained a high number of morphologically healthy follicles and stimulated the activation of primordial follicles after 6 days in culture.

Landmarks of the Knowledge and Trypanosoma cruzi Biology in the Wild Environment.

Trypanosomatids are ancient parasitic eukaryotes that still maintain prokaryotic characteristics. Trypanosoma cruzi, a primarily wild mammal parasite, infected humans already long before European colonization of the Americas. T. cruzi heterogeneity remains an unsolved question, and until now, it has still not been possible to associate T. cruzi genotypes with any biological or epidemiological feature. One of the first biochemical attempts to cluster the T. cruzi subpopulations recognized three main subpopulations (zymodemes) that have been associated with the transmission cycles in the wild (Z1; Z3) and in the domestic environment (Z2). The description of wild mammal species harboring Z2 two decades later challenged this assemblage attempt. Currently, the genotypes of T. cruzi are assembled in seven discrete typing units (DTUs). The biology of T. cruzi still shows novelties such as the description of epimastigotes multiplying and differentiating to metacyclic trypomastigotes in the lumen of the scent glands of Didelphis spp. and the capacity of the true meiosis in parallel to clonal reproduction. The study of the transmission cycle among wild animals has broken paradigms and raised new questions: (i) the interaction of the T. cruzi DTUs with each of its mammalian host species displays peculiarities; (ii) the impact of mixed genotypes and species on the transmissibility of one or another species or on pathogenesis is still unknown; (iii) independent T. cruzi transmission cycles may occur in the same forest fragment; (iv) the capacity to act as a reservoir depends on the peculiarities of the host species and the parasite genotype; and (v) faunistic composition is a defining trait of the T. cruzi transmission cycle profile. The development of models of environmental variables that determine the spatial distribution of the elements that make up T. cruzi transmission by spatial analysis, followed by map algebra and networking, are the next steps toward interpreting and dealing with the new profile of Chagas disease with its many peculiarities. There is no way to solve this neglected disease once and for all if not through a multidisciplinary look that takes into account all kinds of human and animal activities in parallel to environmental variations.

What do studies in wild mammals tell us about human emerging viral diseases in Mexico?

Multiple species of viruses circulate in wild mammals, some of them potentially causing zoonosis. Most of the suspected viral zoonotic diseases affecting human patients remain unidentified with regard to their aetiological agent. The aim of this study is to summarize the state of knowledge of the viral richness associated with wild mammals in Mexico throughout 1900-2018 and their relationship with human cases. We compiled two databases, one of them containing all available published studies on potentially zoonotic viruses in wild mammals and another with human cases related to zoonotic viruses. The database on wild mammals covers the period of 1900-2018; the human case database spans 2000-2013. We calculated the richness of viral potential zoonotic agents and evaluated their geographical distribution. We found 262 records of 42 potential zoonotic viral species associated with 92 wild mammal species in 28 states across Mexico. Records of human viral cases were only found in 29 states, which did not overlap with the reports in wild mammals. We detected 25.6% (42/164) of viral zoonotic agents reported worldwide. This analysis opens a relevant topic of discussion for public health attention.

Uncovering Trypanosoma spp. diversity of wild mammals by the use of DNA from blood clots.

Trypanosoma spp. infection in wild mammals is detected mainly through parasitological tests that usually display low sensitivity. We propose the use of DNA extracted directly from blood clots (BC), which are neglected sources of DNA for diagnosis and identification of Trypanosoma spp. This approach followed by nested PCR targeting the 18S SSU rDNA demonstrated to be sensitive and suitable to evaluate the diversity of trypanosomes infecting sylvatic mammals, including subpatent and mixed infections. Infection was detected in 95/120 (79.2%) samples from bats, carnivores and marsupials that included negative serological and hemoculture testing mammals. Thirteen Trypanosoma spp. or Molecular Operational Taxonomic Units (MOTUs) were identified, including two new MOTUs. The high diversity of trypanosomes species and MOTUs infecting bats and marsupials showed that these hosts can be considered as bio-accumulators of Trypanosoma spp., with specimens of Didelphis spp. displaying the highest trypanosome diversity. The use of blood clots allowed direct access to non-culturable parasites, mixed infections, besides bypassing the selective pressure on the parasites inherent to cultivation procedures. Trypanosoma cruzi was the species found infecting the highest number of individuals, followed by T. lainsoni. Positive PCR for T. cruzi was observed in 16 seronegative individuals and 30 individuals with negative hemocultures. Also, T. lainsoni, previously found only in rodents, showed to be capable of infecting bats and marsupials. This finding makes it clear that some species of Trypanosoma are more generalist than previously thought. Molecular diagnosis using nested PCR from DNA extracted from BC allowed the increase of the knowledge about host-spectrum and distribution of Trypanosoma spp. and allowed the identification of new MOTUs.

Campylobacter jejuni, Campylobacter coli, Salmonella spp. e Yersinia enterocolitica isoladas de animais silvestres em um centro de reabilitação / Campylobacter jejuni, Campylobacter coli, Salmonella spp. and Yersinia enterocolitica isolated from wildlife animals of a reabilitation center

Muitas espécies de animais silvestres de vida livre servem como reservatório de bactérias patogênicas que ameaçam a saúde humana e dos animais domésticos. Algumas bactérias, como Campylobacter jejuni, Campylobacter coli, Yersinia enterocolitica e Salmonella enterica, causam enfermidades em humanos e podem contaminar os animais domésticos e silvestres. O Núcleo de Reabilitação da Fauna Silvestre da Universidade Federal de Pelotas (NURFS-UFPel) soluciona uma demanda regional específica de atenção à fauna silvestre brasileira. O objetivo desse trabalho foi identificar a presença de Campylobacter jejuni, Campylobacter coli, Salmonella spp. e Yersinia enterocolitica em animais silvestres que se encontravam em processo de reabilitação. Foram coletadas amostras de fezes, com uso de zaragatoas estéreis, de 34 aves, 16 mamíferos e 23 répteis. Dos 73 animais amostrados, quatro (5,48%) albergavam Y. enterocolitica, sendo duas aves, um mamífero e um réptil. Salmonella e Campylobacter não foram isolados. Os perfis de bandas dos isolados de Y. enterocolitica analisados pela rep-PCR foram diferentes entre si. Esses resultados indicam que as cepas isoladas não estão relacionadas entre si, não possuindo uma origem comum recente. Vanellus chilensis, Turdus rufiventris, Didelphis albiventris e Pantherophis guttatus podem albergar Y. enterocolitica e eliminá-la nas fezes, oferecendo risco de disseminação desse micro-organismo no ambiente, além de constituírem possíveis fontes de contaminação para humanos e outros animais.(AU)

Wild animals can transmit pathogenic bacteria to human and domestic animal's health. Some bacteria, such as Campylobacter jejuni, Campylobacter coli, Yersinia enterocolitica and Salmonella enterica, cause diseases in humans and can contaminate domestic and wild animais. The Núcleo de Reabilitação da Fauna Silvestre of Universidade Federal de Pelotas (Nurfs-UFPel) attend a specific regional demand of wildlife in Brazil. The aim of this paper was to identify the presence of these pathogenic bacteria in wild animals in rehabilitation. Stool samples were collected using sterile swabs from 34 birds, 16 mammals and 23 reptilian that were housed at Nurfs. Of the 73 collections, Y. enterocolitica was isolated from four (5.48%) of two birds, one mammal and one reptile. Salmonella and Campylobacter were not isolated. The molecular profile of bands of Y. enterocolitica identified in rep-PCR had differences. These results indicated that the isolates did not have a recent common origin. Pantherophis guttatus, Didelphis albiventris, Turdus rufiventris and Vanellus chilensis could shelt Y. enterocolitica and eliminate the bacteria in stool, offering risk of dissemination of these microorganisms in the environment with possible contamination of humans and other animals.(AU)

In vitro maturation of collared peccary (Pecari tajacu) oocytes after different incubation times / Maturação in vitro de oócitos de cateto (Pecari tajacu) após diferentes períodos de incubação

Oocyte in vitro maturation (IVM) is the first step of the in vitro reproductive technologies that enables mature oocytes to be generated ex vivo and after used for embryo production. In this sense, the establishment of culture environment, as oocyte incubation time, is essential for the success of the IVM. Therefore, the study was carried out to investigate the relationship between the meiotic potential and the IVM times of collared peccary oocytes, wild mammals of great commercial and ecological interest. Thus, ovaries were collected of females derived from captivity and transported to the laboratory within 1 hour of slaughtering. The oocytes derived from follicles (3-6mm in diameter) were recovered by aspirated and sliced. Good quality oocytes (evenly granulated cytoplasm with a least one layer of surrounding cumulus cells) were selected and subjected to culture in TCM 199 supplemented with 10µg/mL FSH, 10% FBS and 100µM cysteamine at 38.5°C, 5% CO2 and maximum humidity for 24 or 48 hours. After the incubation period, the nuclear status, the presence of first polar body and the expansion of cumulus cells of oocytes were assessed. The data obtained were analyzed by Fisher exact test (P<0.05). A total of four sessions (2-3 females per session) were performed, resulting in eighteen aspirated and sliced ovaries with normal morphological characteristics. An oocyte recovery rate of about 83.1% (59/71) was obtained with 3.3 oocytes/ovary and 2.3 viable oocytes/ovary. After different incubation times, differences (P<0.05) were observed in 24 and 48 hours for expansion of the cumulus cells (38.1% vs. 100%), presence of first polar body (52.4% vs. 90.5%) and nuclear status in second metaphase (19.0% vs. 76.2%), respectively. In conclusion, 48 hours is suitable time for the in vitro maturation of oocytes derived from collared peccaries when compared to the time of 24 hours, according to the meiotic potential observed. Additional studies should be conducted to improve the quality of the oocyte culture environment, as medium composition, aiming to obtain viable mature oocytes for other in vitro biotechnologies.(AU)

A maturação in vitro (MIV) oocitária é a primeira etapa das tecnologias reprodutivas in vitro que permite que oócitos maturados sejam gerados ex vivo e depois usados para a produção de embriões. Nesse sentido, o estabelecimento do ambiente de cultivo, como o período de incubação de oócitos, é essencial para o sucesso da MIV. Portanto, o estudo foi realizado para investigar a relação entre o potencial meiótico e os períodos de MIV de oócitos derivados de catetos, mamíferos silvestres de grande interesse comercial e ecológico. Para tanto, os ovários foram coletados de fêmeas derivadas de cativeiro e transportados ao laboratório dentro de 1 h após o abate. Os oócitos derivados de folículos (3-6mm de diâmetro) foram recuperados por aspiração e fatiados. Oócitos de boa qualidade (citoplasma uniformemente granulado com pelo menos uma camada circundante de células cumulus) foram selecionados e submetidos ao cultivo em TCM 199 suplementado com 10µg/mL de FSH, 10% de SFB e 100μM de cisteamina a 38,5°C, 5% de CO2 e umidade máxima por 24 e 48 h. Após o período de incubação, o estado nuclear, a presença do primeiro corpúsculo polar e a expansão das células do cumulus dos oócitos foi avaliada. Os dados obtidos foram analisados pelo teste exato de Fisher (P<0,05). Um total de quatro sessões (2-3 fêmeas por sessão) foi realizado, resultando em dezoito ovários aspirados e fatiados com características morfológicas normais. Uma taxa de recuperação oocitária de aproximadamente 83,1% (59/71) foi obtida com 3,3 oócitos/ovário e 2,3 oócitos viáveis/ovário. Após diferentes períodos de incubação, diferenças (P<0,05) foram observadas entre 24 e 48 h para a expansão das células cumulus (38,1% vs. 100%), presença de primeiro corpúsculo polar (52,4% vs. 90,5%) e estado nuclear na segunda metáfase (19,0% vs. 76,2%), respectivamente. Em conclusão, 48 h é o período adequado para a maturação in vitro de oócitos derivados de catetos quando comparado ao tempo de 24 h, de acordo com o potencial meiótico observado. Estudos adicionais devem ser conduzidos para melhorar a qualidade do ambiente de cultivo oocitário, como a composição de meio, objetivando obter oócitos maturados viáveis para outras biotecnologias in vitro.(AU)

Campylobacter jejuni, Campylobacter coli, Salmonella spp. e Yersinia enterocolitica isoladas de animais silvestres em um centro de reabilitação / Campylobacter jejuni, Campylobacter coli, Salmonella spp. and Yersinia enterocolitica isolated from wildlife animals of a reabilitation center

Muitas espécies de animais silvestres de vida livre servem como reservatório de bactérias patogênicas que ameaçam a saúde humana e dos animais domésticos. Algumas bactérias, como Campylobacter jejuni, Campylobacter coli, Yersinia enterocolitica e Salmonella enterica, causam enfermidades em humanos e podem contaminar os animais domésticos e silvestres. O Núcleo de Reabilitação da Fauna Silvestre da Universidade Federal de Pelotas (NURFS-UFPel) soluciona uma demanda regional específica de atenção à fauna silvestre brasileira. O objetivo desse trabalho foi identificar a presença de Campylobacter jejuni, Campylobacter coli, Salmonella spp. e Yersinia enterocolitica em animais silvestres que se encontravam em processo de reabilitação. Foram coletadas amostras de fezes, com uso de zaragatoas estéreis, de 34 aves, 16 mamíferos e 23 répteis. Dos 73 animais amostrados, quatro (5,48%) albergavam Y. enterocolitica, sendo duas aves, um mamífero e um réptil. Salmonella e Campylobacter não foram isolados. Os perfis de bandas dos isolados de Y. enterocolitica analisados pela rep-PCR foram diferentes entre si. Esses resultados indicam que as cepas isoladas não estão relacionadas entre si, não possuindo uma origem comum recente. Vanellus chilensis, Turdus rufiventris, Didelphis albiventris e Pantherophis guttatus podem albergar Y. enterocolitica e eliminá-la nas fezes, oferecendo risco de disseminação desse micro-organismo no ambiente, além de constituírem possíveis fontes de contaminação para humanos e outros animais.(AU)

Wild animals can transmit pathogenic bacteria to human and domestic animal's health. Some bacteria, such as Campylobacter jejuni, Campylobacter coli, Yersinia enterocolitica and Salmonella enterica, cause diseases in humans and can contaminate domestic and wild animais. The Núcleo de Reabilitação da Fauna Silvestre of Universidade Federal de Pelotas (Nurfs-UFPel) attend a specific regional demand of wildlife in Brazil. The aim of this paper was to identify the presence of these pathogenic bacteria in wild animals in rehabilitation. Stool samples were collected using sterile swabs from 34 birds, 16 mammals and 23 reptilian that were housed at Nurfs. Of the 73 collections, Y. enterocolitica was isolated from four (5.48%) of two birds, one mammal and one reptile. Salmonella and Campylobacter were not isolated. The molecular profile of bands of Y. enterocolitica identified in rep-PCR had differences. These results indicated that the isolates did not have a recent common origin. Pantherophis guttatus, Didelphis albiventris, Turdus rufiventris and Vanellus chilensis could shelt Y. enterocolitica and eliminate the bacteria in stool, offering risk of dissemination of these microorganisms in the environment with possible contamination of humans and other animals.(AU)

In vitro maturation of collared peccary (Pecari tajacu) oocytes after different incubation times / Maturação in vitro de oócitos de cateto (Pecari tajacu) após diferentes períodos de incubação

Oocyte in vitro maturation (IVM) is the first step of the in vitro reproductive technologies that enables mature oocytes to be generated ex vivo and after used for embryo production. In this sense, the establishment of culture environment, as oocyte incubation time, is essential for the success of the IVM. Therefore, the study was carried out to investigate the relationship between the meiotic potential and the IVM times of collared peccary oocytes, wild mammals of great commercial and ecological interest. Thus, ovaries were collected of females derived from captivity and transported to the laboratory within 1 hour of slaughtering. The oocytes derived from follicles (3-6mm in diameter) were recovered by aspirated and sliced. Good quality oocytes (evenly granulated cytoplasm with a least one layer of surrounding cumulus cells) were selected and subjected to culture in TCM 199 supplemented with 10µg/mL FSH, 10% FBS and 100µM cysteamine at 38.5°C, 5% CO2 and maximum humidity for 24 or 48 hours. After the incubation period, the nuclear status, the presence of first polar body and the expansion of cumulus cells of oocytes were assessed. The data obtained were analyzed by Fisher exact test (P<0.05). A total of four sessions (2-3 females per session) were performed, resulting in eighteen aspirated and sliced ovaries with normal morphological characteristics. An oocyte recovery rate of about 83.1% (59/71) was obtained with 3.3 oocytes/ovary and 2.3 viable oocytes/ovary. After different incubation times, differences (P<0.05) were observed in 24 and 48 hours for expansion of the cumulus cells (38.1% vs. 100%), presence of first polar body (52.4% vs. 90.5%) and nuclear status in second metaphase (19.0% vs. 76.2%), respectively. In conclusion, 48 hours is suitable time for the in vitro maturation of oocytes derived from collared peccaries when compared to the time of 24 hours, according to the meiotic potential observed. Additional studies should be conducted to improve the quality of the oocyte culture environment, as medium composition, aiming to obtain viable mature oocytes for other in vitro biotechnologies.(AU)

A maturação in vitro (MIV) oocitária é a primeira etapa das tecnologias reprodutivas in vitro que permite que oócitos maturados sejam gerados ex vivo e depois usados para a produção de embriões. Nesse sentido, o estabelecimento do ambiente de cultivo, como o período de incubação de oócitos, é essencial para o sucesso da MIV. Portanto, o estudo foi realizado para investigar a relação entre o potencial meiótico e os períodos de MIV de oócitos derivados de catetos, mamíferos silvestres de grande interesse comercial e ecológico. Para tanto, os ovários foram coletados de fêmeas derivadas de cativeiro e transportados ao laboratório dentro de 1 h após o abate. Os oócitos derivados de folículos (3-6mm de diâmetro) foram recuperados por aspiração e fatiados. Oócitos de boa qualidade (citoplasma uniformemente granulado com pelo menos uma camada circundante de células cumulus) foram selecionados e submetidos ao cultivo em TCM 199 suplementado com 10µg/mL de FSH, 10% de SFB e 100μM de cisteamina a 38,5°C, 5% de CO2 e umidade máxima por 24 e 48 h. Após o período de incubação, o estado nuclear, a presença do primeiro corpúsculo polar e a expansão das células do cumulus dos oócitos foi avaliada. Os dados obtidos foram analisados pelo teste exato de Fisher (P<0,05). Um total de quatro sessões (2-3 fêmeas por sessão) foi realizado, resultando em dezoito ovários aspirados e fatiados com características morfológicas normais. Uma taxa de recuperação oocitária de aproximadamente 83,1% (59/71) foi obtida com 3,3 oócitos/ovário e 2,3 oócitos viáveis/ovário. Após diferentes períodos de incubação, diferenças (P<0,05) foram observadas entre 24 e 48 h para a expansão das células cumulus (38,1% vs. 100%), presença de primeiro corpúsculo polar (52,4% vs. 90,5%) e estado nuclear na segunda metáfase (19,0% vs. 76,2%), respectivamente. Em conclusão, 48 h é o período adequado para a maturação in vitro de oócitos derivados de catetos quando comparado ao tempo de 24 h, de acordo com o potencial meiótico observado. Estudos adicionais devem ser conduzidos para melhorar a qualidade do ambiente de cultivo oocitário, como a composição de meio, objetivando obter oócitos maturados viáveis para outras biotecnologias in vitro.(AU)