From molecules to ecosystems: Insights into a network of interactions for a Chagas disease outbreak using Triatoma brasiliensis as natural samplers.

Exploring the dynamics of disease transmission involves an understanding of complex interactions within the eco-epidemiologic framework. In the context of Chagas disease (CD), elements are mainly represented by the interactions among the pathogen, insect vector, host, humans and the environment. We performed quantitative and qualitative analyses on a dataset derived from 98 Triatoma brasiliensis infected by trypanosomatids, which were linked to a CD outbreak in the semi-arid region of northeastern Brazil. We extracted invertebrate-derived DNA (iDNA) from these insects, comprising 18 populations around the outbreak area, each indicative of various strata of anthropogenic influence. Food source (FS) diversity, representing potential parasite reservoirs, was determined through mitochondrial gene (cyt b) sequencing of vertebrates, and parasite genotyping was accessed using fluorescent amplified fragment barcodes (FFLB) of trypanosomatids. We also assessed the residents' awareness of breeding sites for CD vectors in the inspected houses. The quantification of Trypanosoma cruzi was estimated via real-time PCR and is denominated here as the average parasite load (PL) per insect (T. cruzi/intestinal unit). We aimed to address vector-parasite-host-environment interactions that were discussed based on their significance among the components. Notably, among the significant interactions, we observed that the PL in the insects was significantly influenced by FS. Infected insects that fed on the classic reservoir, Didelphis albiventris, and Galea spixii exhibited higher PLs, compared to those that fed on Kerodon rupestris (p < 0.04)-a primary host. While D. albiventris is already recognized as a synanthropic species, we propose that G. spixii may also be undergoing a synanthropic process. Conversely, domestic cats are frequently identified as FS in infected insects from the sylvatic environment, suggesting a possible change in their behavior towards a wild state. Therefore, we propose that neglected anthropogenic actions have facilitated the reciprocal (sylvatic-peridomestic) circulation of T. cruzi-especially noted for TcI because it was predominant in insects found in peridomestic environments. Residents are often unaware of the existence of insect breeding grounds near their homes, particularly when it involves the storage of materials without planning for use, such as piles of tiles, bricks and wood. Although indirect inferences about the interaction among vector-parasite-host-environment are still incipient, we highlight the potential use of vectors as natural samplers of biological and ecological components in transmitting the disease.

RACCOONS (PROCYON LOTOR) SHOW HIGHER TRYPANOSOMA CRUZI DETECTION RATES THAN VIRGINIA OPOSSUMS (DIDELPHIS VIRGINIANA) IN SOUTH CAROLINA, USA.

Chagas disease, a significant public health concern in the Americas, is caused by a protozoan parasite, Trypanosoma cruzi. The life cycle of T. cruzi involves kissing bugs (Triatoma spp.) functioning as vectors and mammalian species serving as hosts. Raccoons (Procyon lotor) and opossums (Didelphis virginiana) have been identified as important reservoir species in the life cycle of T. cruzi, but prevalence in both species in the southeastern US is currently understudied. We quantified T. cruzi prevalence in these two key reservoir species across our study area in South Carolina, US, and identified factors that may influence parasite detection. We collected whole blood from 183 raccoons and 126 opossums and used PCR to detect the presence of T. cruzi. We then used generalized linear models with parasite detection status as a binary response variable and predictor variables of land cover, distance to water, sex, season, and species. Our analysis indicated that raccoons experienced significantly higher parasite detection rates than Virginia opossums, with T. cruzi prevalence found to be 26.5% (95% confidence interval [CI], 20.0-33.8) in raccoons and 10.5% (95% CI, 5.51-17.5) in opossums. Overall, our results concur with previous studies, in that T. cruzi is established in reservoir host populations in natural areas of the southeastern US.

Space-environment relationship in the identification of potential areas of expansion of Trypanosoma cruzi infection in Didelphis aurita in the Atlantic Rainforest.

Ecological Niche Modeling is widely used for animals, but rarely for understanding the parasite ecology. Trypanosoma cruzi is a heterogeneous and widely dispersed multi-host parasite. Didelphis aurita is a generalist species, both in terms of diet and environments. We modeled the D. aurita niche and T. cruzi infection in the Brazilian Atlantic Rainforest, using the models of two common vector species (Triatoma vitticeps and Panstrongylus megistus) as biotic variables, predicting their occurrence. Records of T. cruzi infected and non-infected D. aurita were analyzed through climate and landscape approaches by the Ecoland method. Models for each triatomine species and infected and noninfected D. aurita were produced considering climate and landscape: resolution of ~1km2 selected by Pearson's correlation [-0.7≤α≤0.7]. For modeling, seven algorithms available in ModleR package were used. True Skill Statistic was used to evaluate the models' performance (≥ 0.7). T. vitticeps indicates that there is a spatial dependence with warm areas in the southeastern region while P. megistus presented a distribution with high environmental suitability concentrated in the Southeast. High values of climatic suitability, landscape and potential presence of T. vitticeps and P. megistus were considered necessary, but not sufficient for the presence of D. aurita infected by T. cruzi. Climate models showed an ecological niche with suitability variations homogeneous, and landscape models showed a distribution of habitat conditions along the biome, with a fragmented profile and heterogeneous between locations. Ecoland demonstrated that D. aurita has different degrees of impact on its role in the enzootic cycle in different locations of the Atlantic Rainforest. Associating the models with the Ecoland method allowed the recognition of areas where D. aurita are important T. cruzi reservoirs. Areas of high suitability for the presence of marsupials are a necessary, but not sufficient for D. aurita to act as a reservoir for T. cruzi.

Molecular detection of the natural infection by trypanosomatid parasites in Didelphis marsupialis from a rural area in northern Colombia. / Detección molecular de infección natural por parásitos tripanosomátidos en Didelphis marsupialis de una zona rural del norte de Colombia.

We studied the prevalence of infection by trypanosomatid parasites in Didelphis marsupialis and its relationship with morphological/age aspects in a rural area of El Carmen de Bolivar, Colombia. Five visits were made to the Vereda El Alférez; each of which lasted three consecutive nights. During these visits, Tomahawk® traps were installed in the peridomestic and wild ecotopes of the Vereda El Alférez. Body measurements, sex and age were determined from the collected animals. Blood was extracted by cardiopuncture, after sedation, in order to obtain total deoxyribonucleic acid (DNA) and amplify the conserved region of the kinetoplast minicircle DNA (kDNA) of parasitic trypanosomatids. The association between morphological parameters of didelphids and their frequency of infection by parasitic trypanosomatids was determined by binomial regression. Thirty D. marsupialis specimens (60.0% females and 40.0% males/66.7% adults and 33.3% juveniles) were collected. Molecular diagnosis revealed a frequency of trypanosomatid parasite infection of 46.7%. Stage (p=0.024) was a determinant for infection. We discuss the role of D. marsupialis as a potential reservoir of parasitic trypanosomatids in the Vereda El Alférez.

Se evaluó la prevalencia de infección por parásitos tripanosomátidos en Didelphis marsupialis y su relación con los aspectos morfológicos/etarios en una zona rural de El Carmen de Bolívar, Colombia. En cinco visitas (2018-2019) se instalaron trampas Tomahawk® en los ecótopos peridoméstico y silvestre en la Vereda El Alférez, durante tres noches consecutivas/visita. A los animales recolectados, se les determinaron medidas corporales, sexo y edad; y se les extrajo sangre por cardiopuntura, previa sedación, para extracción del ácido desoxirribonucleico (ADN) total y amplificación de la región conservada del ADN de minicírculos de kinetoplasto (ADNk) de parásitos tripanosomátidos. La asociación entre parámetros morfológicos de los didélfidos y su frecuencia de infección por parásitos tripanosomátidos fue determinada mediante una regresión binomial. Se recolectaron 30 individuos de D. marsupialis (60,0% hembras y 40,0% machos/66,7% adultos y 33,3% juveniles). El diagnóstico molecular reveló una frecuencia de infección por parásitos tripanosomátidos del 46,7%. El estadio (p=0,024) fue determinante para la infección. Se discute el papel de D. marsupialis como potencial reservorio de parásitos tripanosomátidos en la zona evaluada.

MORPHOLOGICAL AND GENETIC CHARACTERIZATION OF DIDELPHONEMA LONGISPICULATA (NEMATODA: SPIRUROIDEA) IN THE BLACK-EARED OPOSSUM DIDELPHIS MARSUPIALIS.

Didelphonema longispiculata (Hill, 1939), a gastric nematode parasite of the black-eared opossum, Didelphis marsupialis Linnaeus, 1758, collected from 2 municipalities of Mato Grosso state, Brazil, in the ecotone region of the Amazon and Cerrado biomes was analyzed with integrative taxonomy using light and scanning electron microscopy (SEM) for morphological studies and sequencing of the 18S small subunit ribosomal RNA for phylogenetic inference through maximum likelihood and Bayesian phylogenetic inference. Here details of the helminth surface, oral aperture with octagonal border, pseudo- and inter-labia, amphids, external cephalic papillae, 2 dorsal and ventral internal plates distally indented, and stoma with strongly chitinized wall are presented. Caudal male papillae, spicules, female vulva, anus, and caudal tip were detailed using SEM. Morphological characteristics and phylogenetic data corroborated the taxonomic placement of the genus Didelphonema within the subfamily Ascaropsinae.

Population genetic structure and morphological diversity of Cruzia tentaculata (Nematoda: Ascaridida), a parasite of marsupials (Didelphinae), along the Atlantic Forest on the eastern coast of South America.

Cruzia tentaculata is a helminth parasite of marsupials and has a wide geographic distribution from Mexico to Argentina. The aim of this study was to analyse the genetic population structure of this nematode along the Atlantic Forest biome. Cruzia tentaculata specimens were recovered from Didelphis aurita, Didelphis albiventris and Philander quica in 9 localities. Morphological and morphometric data were investigated for phenotypic diversity among localities and hosts using multivariate discriminant analysis of principal components. Phylogenetic relationships of C. tentaculata were determined using maximum likelihood and Bayesian inference. The population structure was analysed by fixation indices, molecular variance analysis, Tajima's D and Fu's Fs neutrality tests, Mantel tests and Bayesian clustering analysis. A higher significant morphometric difference for males was observed between localities. In the haplogroup networks, 2 groups were recovered, separating locations from the north and from the south/southeast. The morphometric variation in C. tentaculata between different localities was compatible with this north and southeast/south pattern, suggesting adaptation to different ecological conditions. Population genetic analyses suggested a pattern of evolutionary processes driven by Pleistocene glacial refugia in the northeast and southeast of the Atlantic Forest based on the distribution of genetic diversity.

Population genetic structure and phenotypic diversity of Aspidodera raillieti (Nematoda: Heterakoidea), a parasite of Didelphini marsupials in Brazil's South and Southeast Atlantic Forest.

BACKGROUND: The population genetics of parasites may be influenced by host specificity, life cycle, host geographical range, evolutionary history, and host population structure. The nematode Aspidodera raillieti infects different marsupial and rodent hosts in the Nearctic and Neotropical regions, implying a gene flow among populations. However, niche diversification of the main hosts of A. raillieti in superimposed areas may provide conditions for population genetic structuring within this parasite species. We examined the genetic structuring of A. raillieti infecting three marsupial species co-occurring along the South and Southeast Brazilian Atlantic Forest, a hotspot of biodiversity. METHODS: We employed morphometric analyses and partial mitochondrial cytochrome c oxidase I gene sequences (MT-CO1) to characterize populations via phylogenetic and phylogeographic analyses. RESULTS: Among 175 A. raillieti specimens recovered from the marsupial hosts Didelphis aurita, D. albiventris, and Philander quica, we identified 99 MT-CO1 haplotypes forming four haplogroups and four clades in networks and phylogenetic trees, respectively. Clades I and II encompassed parasites of D. albiventris from the South region, clade III comprised parasites of D. aurita from the South and Southeast regions, and clade IV encompassed parasites of D. aurita and D. albiventris from the South and Southeast regions and parasites of P. quica from the South region. High genetic differentiation between clades, with a high fixation index and greater genetic variation in the analysis of molecular variance (AMOVA), indicated low gene flow between clades. Haplotypes shared among host species revealed a lack of host specificity. A significant correlation in the Mantel test suggested parasite isolation by distance, while there was no evidence of geographical structure between populations. Negative neutrality test values for clades III and IV suggested recent population expansion. Morphometric differentiation between A. raillieti specimens recovered from different host species, as well as from different localities, was more evident in males. CONCLUSION: The genetic structure of A. raillieti populations in the South and Southeast Atlantic Forest resulted from historical events rather than from current geographical distribution or host specificity. We also demonstrate morphometric variation associated with host species and localities, suggesting phenotypic plasticity to host attributes and to spatial variables.

Helminth community structure of Didelphis marsupialis (Didelphimorphia, Didelphidae) in a transition area between the Brazilian Amazon and the Cerrado.

Although the common opossum, Didelphis marsupialis (Didelphimorphia: Didelphidae) is a species widely distributed in South America, knowledge about their helminth parasites and helminth community structure is scarce. The aims of this study were to describe the species composition and analyze the structure of the helminth community of the common opossum in an area of the Amazonian Arc in northern Mato Grosso. The helminths were recovered, counted, and identified in 32 individuals. Overall, 10,198 specimens were categorized into 9 helminths taxa (seven nematodes, one cestode, and one acanthocephalan). The most abundant species were Aspidodera raillieti, Viannaia hamata, and Travassostrongylus orloffi. No statistically significant differences in helminth abundance and prevalence were observed between host sexes. However, young hosts had higher abundance and prevalence of Didelphonema longispiculata, whereas Oligacanthorhynchus microcephalus had higher abundance and prevalence in adult hosts. This was the first study to analyze the helminth fauna and helminth community structure of D. marsupialis in the Amazonian Arc. This is the first report of the presence of A. raillieti, D. longispiculata, T. orloffi, T. minuta, V. hamata, and O. microcephalus in the state of Mato Grosso, Brazil.

Over-dispersed Trypanosoma cruzi parasite load in sylvatic and domestic mammals and humans from northeastern Argentina.

BACKGROUND: The distribution of parasite load across hosts may modify the transmission dynamics of infectious diseases. Chagas disease is caused by a multi-host protozoan, Trypanosoma cruzi, but the association between host parasitemia and infectiousness to the vector has not been studied in sylvatic mammalian hosts. We quantified T. cruzi parasite load in sylvatic mammals, modeled the association of the parasite load with infectiousness to the vector and compared these results with previous ones for local domestic hosts. METHODS: The bloodstream parasite load in each of 28 naturally infected sylvatic mammals from six species captured in northern Argentina was assessed by quantitative PCR, and its association with infectiousness to the triatomine Triatoma infestans was evaluated, as determined by natural or artificial xenodiagnosis. These results were compared with our previous results for 88 humans, 70 dogs and 13 cats, and the degree of parasite over-dispersion was quantified and non-linear models fitted to data on host infectiousness and bloodstream parasite load. RESULTS: The parasite loads of Didelphis albiventris (white-eared opossum) and Dasypus novemcinctus (nine-banded armadillo) were directly and significantly associated with infectiousness of the host and were up to 190-fold higher than those in domestic hosts. Parasite load was aggregated across host species, as measured by the negative binomial parameter, k, and found to be substantially higher in white-eared opossums, cats, dogs and nine-banded armadillos (range: k = 0.3-0.5) than in humans (k = 5.1). The distribution of bloodstream parasite load closely followed the "80-20 rule" in every host species examined. However, the 20% of human hosts, domestic mammals or sylvatic mammals exhibiting the highest parasite load accounted for 49, 25 and 33% of the infected triatomines, respectively. CONCLUSIONS: Our results support the use of bloodstream parasite load as a proxy of reservoir host competence and individual transmissibility. The over-dispersed distribution of T. cruzi bloodstream load implies the existence of a fraction of highly infectious hosts that could be targeted to improve vector-borne transmission control efforts toward interruption transmission. Combined strategies that decrease the parasitemia and/or host-vector contact with these hosts would disproportionally contribute to T. cruzi transmission control.

Are Virginia opossums really ecological traps for ticks? Groundtruthing laboratory observations.

Virginia opossums (Didelphis virginiana) are a common synanthrope in North America, and serve as host to many species of ectoparasites. Research on captive Virginia opossums estimated that opossums eat, on average, 5500 larval ticks (Acari: Ixodidae) per week. To investigate this apparent preference exhibited by opossums for ingesting ticks, we comprehensively analyzed stomach contents of 32 Virginia opossums from central Illinois. Using a dissecting microscope, we searched the contents exhaustively for ticks and tick body parts, without sieving or pre-rinsing the stomach contents. We did not locate any ticks or tick parts in the stomach contents of Virginia opossums. We also performed a vigorous literature search for corroborating evidence of tick ingestion. Our search revealed 23 manuscripts that describe diet analyses of Virginia opossums, 19 of which were conducted on stomach or digestive tract contents and four of which were scat-based analyses. None of the studies identified ticks in their analyses of diet items. We conclude that ticks are not a preferred diet item for Virginia opossums. Considering that wildlife unconditioned to laboratory conditions may exhibit non-typical behaviors, we recommend that lab-based studies of wildlife behavior be groundtruthed with studies based in natural conditions.

Functional traits shape small mammal-helminth network: patterns and processes in species interactions.

Understanding the role of species traits in mediating ecological interactions and shaping community structure is a key question in ecology. In this sense, parasite population parameters allow us to estimate the functional importance of traits in shaping the strength of interactions among hosts and parasites in a network. The aim of this study was to survey and analyse the small mammal-helminth network in a forest reserve of the Brazilian Atlantic Forest in order to understand (i) how functional traits (type of parasite life cycle, site of infection in their host, host and parasite body length, host diet, host locomotor habit and host activity period) and abundance influence host­parasite interactions, (ii) whether these traits explain species roles, and (iii) if this relationship is consistent across different parasite population parameters (presence and absence, mean abundance and prevalence). Networks were modular and their structural patterns did not vary among the population parameters. Functional traits and abundance shaped the interactions observed between parasites and hosts. Host species abundance, host diet and locomotor habit affected their centrality and/or vulnerability to parasites. For helminths, infection niche was the main trait determining their central roles in the networks.

Chagas disease in urban and peri­urban environment in the Amazon: Sentinel hosts, vectors, and the environment.

Chagas disease is an anthropozoonosis, caused by a flagellated protozoan, Trypanosoma cruzi, in which the enzootic cycle occurs between mammals and triatomines. Two dogs with a history of sudden death were necropsied at the Federal University of Pará (UFPA). One dog had a pale area in the myocardium, which on histopathological examination showed a T. cruzi amastigote nest; immunohistochemistry (IHC) analysis characterized it as acute Chagas disease (ACD). The second dog showed no macroscopic changes. Microscopically, a few cardiomyocytes were replaced by adipocytes, and IHC result was negative for T. cruzi. However, results of polymerase chain reaction (PCR) of the cardiac tissue of both dogs was positive for T. cruzi DNA. After that, an epidemiological study was conducted in the region. For this study, we selected four areas in Castanhal. One of the four areas (Area 1) is where one of the dogs lived. The other three areas were chosen because they were recently deforested for housing. Blood samples were collected from dogs, cats, wild small mammals (marsupials and rodents), and the digestive tract of triatomines. Nested PCR was performed on all the blood samples and the triatomine digestive tracts. In Area 1, T. cruzi DNA was detected in 50% (12/24) of the tested dogs, in the only tested cat (1/1), 50% (1/2) of the tested marsupials (Didelphis marsupials), and 100% of the captured triatomines (Rhodnius pictipes) (2/2). In Area 2, T. cruzi DNA was not detected in any of the 11 (0/11) dogs and two marsupials tested (0/2), and no triatomines were found in this area. In Area 3, T. cruzi DNA was detected in 42.25% (30/71) of the dogs, in 66,6% (2/3) of the cats, the only captured marsupial (D. marsupialis) (1/1), and all three triatomines (3/3) (R. pictipes) tested. In Area 4, the two dogs tested were negative (0/2), 25% (1/4) of the captured marsupials (D. marsupialis) was positive, and no triatomine was captured in this area. The data demonstrate the importance of detecting T. cruzi in dogs, cats, small rodents, and marsupials in the Amazon metropolitan areas, where ecotopes carry reservoirs and vectors capable of participating in the Chagas disease cycle. The proximity between humans and T. cruzi vectors in these places might contribute to increased disease transmission risk and maintenance of agents. It was noted that high-standard condominiums, previously thought to reduce the risk for this disease, presented a new epidemiological risk. The presence of T. cruzi DNA in a dog who, a year earlier had tested negative, when another dog in the same house died of ACD, shows that the transmission cycle is present and active, with a high possibility of disease transmission to animals and humans.

Lesions caused by Sarcocystis spp., parasites of opossums (Didelphis aurita), in acute and chronic infections in birds (Melopsittacus undulatus).

Protozoa of the genus Sarcocystis are obligatory heterogenous parasites with both definitive and intermediate hosts. Opossums (Didelphis aurita) can shed multiple species of Sarcocystis with birds as the intermediate host. The pathologies of Sarcocystis species in birds have not been thoroughly elucidated. Therefore, the aim of the present study to determine the main lesions that can occur in acute and chronic infections in intermediate hosts, when they ingest infective sporocysts that are shed in the opossum's feces, using budgerigars as a model. To this end, 12 budgerigars, Melopsittacus undulatus, were divided into two groups that received an inoculum with 60 and 120 sporocysts. Birds that died or were euthanized were necropsied, and the lung, tongue, liver, brain, heart, and skeletal striated muscles were collected and fixed in 10% formalin for histopathological analysis. The infectivity varied according to the sample and infective dose. Acute histopathological lesions were characterized by evidence of slightly degenerated hepatocyte cords that permeated the region of the blood vessel and hepatic sinusoids. Pulmonary tissue lesions were also observed in the parabronchial region with the presence of inflammatory infiltrates associated with areas of edema and atelectasis. In chronic infections, few mature cysts were observed in the chest, and many mature cysts in the thigh and tongue muscles. Thus, it was possible to conclude that lesions are highly characteristic in acute infection and, in chronic infections, cysts were present but without major lesions. In this case, the preferred organs of parasitism were the thigh and the tongue.

Association of the invasive Haemaphysalis longicornis tick with vertebrate hosts, other native tick vectors, and tick-borne pathogens in New York City, USA.

Haemaphysalis longicornis, the Asian longhorned tick, is an invasive ixodid tick that has rapidly spread across the northeastern and southeastern regions of the United States since first reported in 2017. The emergence of H. longicornis presents a potential threat for livestock, wildlife, and human health as the host associations and vector potential of this invasive pest in the United States are poorly understood. Previous field data from the United States has shown that H. longicornis was not associated with natural populations of small mammals or birds, but they show a preference for medium sized mammals in laboratory experiments. Therefore, medium and large sized mammals were sampled on Staten Island, New York, United States, to determine H. longicornis host associations and vector potential for a range of human and veterinary pathogens. A total of 97 hosts were sampled and five species of tick (Amblyomma americanum, Dermacentor variabilis, H. longicornis, Ixodes scapularis, Ixodes cookei) were found feeding concurrently on these hosts. Haemaphysalis longicornis was found in the highest proportions compared with other native tick species on raccoons (55.4%), Virginia opossums (28.9%), and white-tailed deer (11.5%). Tissue, blood, and engorged larvae were tested for 17 different pathogens using a nanoscale PCR platform. Infection with five pathogens (Borrelia burgdorferi, Anaplasma phagocytophilum, Rickettsia spp., Mycoplasma haemocanis, and Bartonella spp.) was detected in host samples, but no pathogens were found in any larval samples. These results suggest that although large and medium sized mammals feed large numbers of H. longicornis ticks in the environment, there is presently a low potential for H. longicornis to acquire pathogens from these wildlife hosts.

Trypanosomatid species in Didelphis albiventris from urban forest fragments.

Urbanization results in loss of natural habitats and, consequently, reduction of richness and abundance of specialist to the detriment of generalist species. We hypothesized that a greater richness of trypanosomatid in Didelphis albiventris would be found in fragments of urban forests in Campo Grande, Mato Grosso do Sul, Brazil, that presented a larger richness of small mammals. We used parasitological, molecular, and serological methods to detect Trypanosoma spp. infection in D. albiventris (n = 43) from forest fragments. PCR was performed with primers specific for 18S rDNA, 24Sα rDNA, mini-chromosome satellites, and mini-exon genes. IFAT was used to detect anti-Trypanosoma cruzi IgG. All hemoculture was negative. We detected trypanosomatid DNA in blood of 35% of opossum. Two opossums were seropositive for T. cruzi. The trypanosomatid species number infecting D. albiventris was higher in the areas with greater abundance, rather than richness of small mammals. We found D. albiventris parasitized by T. cruzi in single and co-infections with Leishmania spp., recently described molecular operational taxonomic unit (MOTU) named DID, and Trypanosoma lainsoni. We concluded that (i) trypanosome richness may be determined by small mammal abundance, (ii) D. albiventris confirmed to be bio-accumulators of trypanosomatids, and (iii) T. lainsoni demonstrated a higher host range than described up to the present.

Detection of Trypanosoma cruzi DNA in false negative samples of collected triatomines, xenodiagnosis material, and biopsies of experimentally infected animals.

Direct test over the gut material from triatomine vectors and xenodiagnosis over mammalian hosts are classical techniques for Trypanosoma cruzi parasitological diagnosis. Nevertheless, negative results can be a source of uncertainty. Experimental models have allowed evaluating the tissue invasion of different strains of T. cruzi, but conventional techniques for tissue biopsies involve time-consuming and elaborated procedures and have low sensitivity. Gut material of collected triatomines (microscopically negative) (n = 114), material of mammal xenodiagnoses (microscopically negative) (n = 138), and biopsy material (microscopically negative) from experimentally infected animals (n = 34) with isolates from endemic areas of Chagas' disease from Venezuela were used for DNA extraction and PCR for the amplification of kinetoplast DNA (kDNA) and satellite DNA (sDNA) of T. cruzi. Positive PCR was observed in 53.6% of collected triatomine material, 15.8% of parasitological negative xenodiagnosis material, and 70.6% in biopsies, revealing underestimation by the parasitological tests and the valour of this analysis with preserved material. Anzoátegui was the state with the highest percentage of infection, and the triatomine species Rhodnius prolixus and Panstrongylus geniculatus had the highest percentages of infection. Didelphis marsupialis and Canis familiaris were the most infected by T. cruzi revealed by PCR of xenodiagnosis material. In addition, the PCR technique allowed demonstrating the invasion of T. cruzi in all tissues analyzed, constituting a molecular marker of tissue invasion.

Urban foci of murine typhus involving cat fleas (Ctenocephalides felis felis) collected from opossums in Mexico City.

Murine typhus, a neglected rickettsiosis caused by Rickettsia typhi, is a common disease in several Latin-American countries. The sylvatic life cycle of R. typhi encompasses the presence of several wild mammals, particularly opossums of the genus Didelphis and their associated fleas. Due to the colonization of wild environments by human populations, the increase in contact with opossum fleas has generated the presence of urban outbreaks of typhus. For this reason, the aim of our study was to identify the presence and diversity of Rickettsia sp. in fleas collected from opossums of an urban reserve in Mexico City. Opossums were captured from February to September 2017. For the detection of Rickettsia DNA, fragments of 800 bp of the citrate synthase (gltA) and the outer membrane protein B (ompB) were amplified. A total of 141 fleas (111 ♀, 30 ♂) of a single species (Ctenocephalides felis felis) were recovered from 31 Didelphis virginiana. Rickettsia DNA was detected in 17.7% (25/141) of the analysed fleas, recovered from seven infested opossums. The Maximum likelihood of sequences exhibited an identity of 99%-100% with sequences of R. typhi from southern United States. This work represents the first record of R. typhi in fleas from opossums in Mexico.

Didelphis albiventris as a carrier of Leptospira sp. in the central nervous tissue in the semiarid region of Northeast, Brazil.

Leptospirosis has been investigated in several species of wild animals. The white-eared opossum (Didelphis albiventris) is a mammal common in the brazilian semi-arid, so, this study aimed to investigate its role in the occurrence of the leptospirosis in the region Northeast of Brazil. 12 animals were used, from which samples were collected for the attempt of isolation, molecular detection and serological examination. There was no microbial growth, nor were any anti-Leptospira sp. antibodies found in the serological samples. The PCR detected leptospiric DNA in the central nervous system (CNS) of five animals (41.7 %). The gene in one of the samples was sequenced and showed identity with Leptospira interrogans. The presence of Leptospira sp. in the CNS of Didelphis albiventris does not allow the characterization of the studied animals as reservoirs with potential for transmission of the pathogen in the region, however it represents a site that needs to be further investigated.

High prevalence of Ancylostoma caninum infection in black-eared opossums (Didelphis aurita) in an urban environment.

Marsupials of the genus Didelphis, such as black-eared opossums (Didelphis aurita), are common synanthropic animals in urban areas of Brazil. These marsupials are frequently parasitized by numerous helminth species, including ancylostomatid nematodes. This study aimed to report the occurrence of Ancylostoma caninum in black-eared opossums captured in an urban environment of Southeastern Brazil and discuss the potential impact of these findings for public health. From January to June 2019, we collected fecal samples from 49 restrained opossums and evaluated by a simple flotation method; Helminth eggs were observed at different magnifications and identified according to morphological and morphometric features. Genomic DNA was extracted from Ancylostomatidae eggs and screened by duplex PCR for Ancylostoma spp. and Necator americanus using primers that amplify a region of internal transcribed spacer 2 and the 28S ribosomal RNA (ITS2-28S rRNA). Ancylostoma spp. eggs were detected in 65.3% (32/49) of the animals. Sequence analysis revealed 100% homology with A. caninum sequences from GenBank. Our results demonstrate a new host-parasite interaction for A. caninum, suggesting that black-eared opossums may participate in the zoonotic cycle of this parasite in urban areas of Brazil.

Ticks, fleas and endosymbionts in the ectoparasite fauna of the black-eared opossum Dipelphis aurita in Brazil.

Ticks and fleas are essential vectors of pathogens that affect humans and animals, and among their hosts, synanthropic animals such as the black-eared opossum, Didelphis aurita, play a role in public health due to their ability to move between urban centers and forested areas in Brazil. This study aimed to assess the ectoparasite fauna of D. aurita, as well as the presence of pathogens and endosymbionts in ticks and fleas. Opossums (n = 58) captured in Tomahawk livetraps were examined for ectoparasites, and their blood sampled for further analysis. Additionally, spleen samples were collected in individuals found dead. Samples were PCR screened for Rickettsia spp., Borrelia spp., Anaplasmataceae, and Babesia spp. Two tick species were morphologically identified as Ixodes loricatus 24/58 (41.4%) and Amblyomma sculptum 1/58 (1.7%). For fleas, Ctenocephalides felis was detected in 60.3% (35/58) of the animals, and Xenopsylla cheopis in 5.2% (3/58). PCR analysis detected Anaplasmataceae DNA in 34% (16/47) of pooled samples of C. felis, and in 66.7% (2/3) pooled samples of X. cheopis. Sequence analysis revealed Wolbachia pipientis symbiont in all positive samples. Tick, blood and spleen samples were all negative for the microorganisms assessed. These findings suggest that these arthropods circulate among wildlife and urban environments, which may implicate in their participation in the cycle of zoonotic pathogens among opossums, humans and companion animals.