Limited exposure of captive Australian marsupials to plastics.

The global concern regarding the ubiquitous presence of plastics in the environment has led to intensified research on the impact of these materials on wildlife. In the Australian context, marsupials represent a unique and diverse group of mammals, yet little is known about their exposures to plastics. This study aimed to assess the contamination levels of seven common plastics (i.e., polystyrene (PS), polycarbonate (PC), poly-(methyl methacrylate) (PMMA), polypropylene (PP), polyethylene terephthalate (PET), polyethylene (PE), and polyvinyl chloride (PVC)) in both the diet and faeces of kangaroos, wallabies and koalas sampled from a sanctuary in Northeastern Australia. Quantitative analysis was performed by pressurized liquid extraction followed by double-shot microfurnace pyrolysis coupled to gas chromatography mass spectrometry. Interestingly, the analysis of the food and faeces samples revealed the absence of detectable plastic particles; with this preliminary finding suggesting a relatively limited exposure of captive Australian marsupials to plastics. This study contributes valuable insights into the current state of plastic contamination in Australian marsupials, shedding light on the limited exposures and potential risks, and highlighting the need for continued monitoring and conservation efforts. The results underscore the importance of proactive measures to mitigate plastic pollution and protect vulnerable wildlife populations in Australia's unique ecosystems.

Exploring the interplay between small and large scales movements in a neotropical small mammal.

We record and analyze the movement patterns of the marsupial Didelphis aurita at different temporal scales. Animals trajectories are collected at a daily scale by using spool-and-line techniques and, with the help of radio-tracking devices, animals traveled distances are estimated at intervals of weeks. Small-scale movements are well described by truncated Lévy flight, while large-scale movements produce a distribution of distances which is compatible with a Brownian motion. A model of the movement behavior of these animals, based on a truncated Lévy flight calibrated on the small scale data, converges towards a Brownian behavior after a short time interval of the order of 1 week. These results show that whether Lévy flight or Brownian motion behaviors apply, will depend on the scale of aggregation of the animals paths. In this specific case, as the effect of the rude truncation present in the daily data generates a fast convergence towards Brownian behaviors, Lévy flights become of scarce interest for describing the local dispersion properties of these animals, which result well approximated by a normal diffusion process and not a fast, anomalous one. Interestingly, we are able to describe two movement phases as the consequence of a statistical effect generated by aggregation, without the necessity of introducing ecological constraints or mechanisms operating at different spatio-temporal scales. This result is of general interest, as it can be a key element for describing movement phenomenology at distinct spatio-temporal scales across different taxa and in a variety of systems.

A family of olfactory receptors uniquely expanded in marsupial and monotreme genomes are expressed by a T cell subset also unique to marsupials and monotremes.

Olfactory receptors (OR), expressed on olfactory neurons, mediate the sense of smell. Recently, OR have also been shown to be expressed in non-olfactory tissues, including cells of the immune system. An analysis of single-cell transcriptomes of splenocytes of the grey short-tailed opossum (Monodelphis domestica) found OR are expressed on a subset of T cells, the γµ T cells, that are unique to marsupials and monotremes. A majority of opossum γµ T cells transcriptomes contain OR family 14 transcripts, specifically, from the OR14C subfamily. Amongst the mammals, the OR14 gene family is expanded in the genomes of marsupials and monotremes, and rarer or absent in placental mammals. In summary, here we demonstrate the intriguing correlation that a family of OR genes, abundant in the genomes of marsupials and monotremes, are ectopically expressed in a particular subset of T cells unique to the marsupials and monotremes.

Expanding the evo-devo toolkit: generation of 3D mammary tissue from diverse mammals.

The varying pathways of mammary gland development across species and evolutionary history are underexplored, largely due to a lack of model systems. Recent progress in organoid technology holds the promise of enabling in-depth studies of the developmental adaptations that have occurred throughout the evolution of different species, fostering beneficial phenotypes. The practical application of this technology for mammary glands has been mostly confined to rodents and humans. In the current study, we have successfully created next-generation 3D mammary gland organoids from eight eutherian mammals and the first branched organoid of a marsupial mammary gland. Using mammary organoids, we identified a role for ROCK protein in regulating branching morphogenesis, a role that manifests differently in organoids from different mammals. This finding demonstrates the utility of the 3D organoid model for understanding the evolution and adaptations of signaling pathways. These achievements highlight the potential for organoid models to expand our understanding of mammary gland biology and evolution, and their potential utility in studies of lactation or breast cancer.

Zoonotic Cryptosporidium and Giardia in marsupials-an update.

Marsupials, inhabiting diverse ecosystems, including urban and peri-urban regions in Australasia and the Americas, intersect with human activities, leading to zoonotic spill-over and anthroponotic spill-back of pathogens, including Cryptosporidium and Giardia. This review assesses the current knowledge on the diversity of Cryptosporidium and Giardia species in marsupials, focusing on the potential zoonotic risks. Cryptosporidium fayeri and C. macropodum are the dominant species in marsupials, while in possums, the host-specific possum genotype dominates. Of these three species/genotypes, only C. fayeri has been identified in two humans and the zoonotic risk is considered low. Generally, oocyst shedding in marsupials is low, further supporting a low transmission risk. However, there is some evidence of spill-back of C. hominis into kangaroo populations, which requires continued monitoring. Although C. hominis does not appear to be established in small marsupials like possums, comprehensive screening and analysis are essential for a better understanding of the prevalence and potential establishment of zoonotic Cryptosporidium species in small marsupials. Both host-specific and zoonotic Giardia species have been identified in marsupials. The dominance of zoonotic G. duodenalis assemblages A and B in marsupials may result from spill-back from livestock and humans and it is not yet understood if these are transient or established infections. Future studies using multilocus typing tools and whole-genome sequencing are required for a better understanding of the zoonotic risk from Giardia infections in marsupials. Moreover, much more extensive screening of a wider range of marsupial species, particularly in peri-urban areas, is required to provide a clearer understanding of the zoonotic risk of Cryptosporidium and Giardia in marsupials.

Marked genetic diversity within Blastocystis in Australian wildlife revealed using a next generation sequencing-phylogenetic approach.

Blastocystis is a genus of intestinal stramenopiles that infect vertebrates, and may cause disease of the alimentary tract. Currently, at least 40 genotypes ("subtypes") of Blastocystis are recognised worldwide based on sequence data for the small subunit of the nuclear ribosomal RNA (SSU-rRNA) gene. Despite the numerous studies of Blastocystis worldwide, very few studies have explored Blastocystis in wild animals, particularly in Australia. Here, we used a PCR-based next generation sequencing (NGS)-phylogenetic approach to genetically characterise and classify Blastocystis variants from selected wildlife in the Australian state of Victoria. In total, 1658 faecal samples were collected from nine host species, including eastern grey kangaroo, swamp wallaby, common wombat, deer, European rabbit, canines and emu. Genomic DNA was extracted from these samples, a 500 bp region of the SSU-rRNA gene amplified by polymerase chain reaction (PCR) and, then, a subset of samples sequenced using Illumina technology. Primary PCR detected Blastocystis in 482 of the 1658 samples (29%), with the highest percentage in fallow deer (63%). Subsequent, Illumina-based sequencing of a subset of 356 samples revealed 55 distinct amplicon sequence variants (ASVs) representing seven currently-recognised subtypes (STs) [ST13 (prominent in marsupials), ST10, ST14, ST21, ST23, ST24 and ST25 (prominent in deer)] and two novel STs (ST45 and ST46) in marsupials. Mixed infections of different STs were observed in macropods, deer, emu and canids (fox, feral dog or dingo), but no infection was detected in rabbits or wombats. This study reveals marked genetic diversity within Blastocystis in a small number of species of wild animals in Australia, suggesting complexity in the genetic composition and transmission patterns of members of the genus Blastocystis in this country.

Molecular detection and characterization of Bartonella spp. in small mammals in the Amazonia and Cerrado biomes, midwestern Brazil.

Although Bartonella spp. have been worldwide described in rodents and bats, few studies have reported these agents in marsupials. The present work aimed to investigate the occurrence and genetic diversity of Bartonella in small mammals (rodents, marsupials, and bats) and associated ectoparasites in two ecoregions (Amazonia and Cerrado biomes) in midwestern Brazil. For this purpose, DNA samples from 378 specimens of small mammals (128 rodents, 111 marsupials, and 139 bats) and 41 fleas (Siphonaptera) were screened for the Bartonella genus employing a quantitative real-time PCR assay (qPCR) based on the nuoG (nicotinamide adenine dinucleotide dehydrogenase gamma subunit) gene. Then, positive samples in qPCR were submitted to conventional PCR (cPCR) assays targeting the gltA, ftsZ, and rpoB genes. One (0.78 %) rodent, 23 (16.54 %) bats, and 3 (7.31 %) fleas showed positive results in the qPCR for Bartonella sp. After cPCR amplification and sequencing, 13 partial Bartonella DNA sequences of the following genes were obtained only from bats´ blood samples: 9 gltA (citrate synthase), 3 ftsZ (cell division protein), and 1 rpoB (RNA polymerase beta subunit). The maximum likelihood inference based on the gltA gene positioned the obtained sequences in three different clades, closely related to Bartonella genotypes previously detected in other bat species and bat flies sampled in Brazil and other countries from Latin America. Similarly, the ftsZ sequences clustered in two different clades with sequences described in bats from Brazil, other countries from Latin America, and Georgia (eastern Europe). Finally, the Bartonella rpoB from a specimen of Lophostoma silvicolum clustered with a Bartonella sp. sequence obtained from a Noctilio albiventris (KP715475) from French Guiana. The present study provided valuable insights into the diversity of Bartonella genotypes infecting bats from two ecoregions (Amazonia and Cerrado) in midwestern Brazil and emphasized that further studies should be conducted regarding the description and evaluation of different lineages of Bartonella in wild small mammals and their ectoparasites in different Brazilian biomes.

Anaerobic gut fungal communities in marsupial hosts.

The anaerobic gut fungi (AGF) inhabit the alimentary tracts of herbivores. In contrast to placental mammals, information regarding the identity, diversity, and community structure of AGF in marsupials is extremely sparse. Here, we characterized AGF communities in 61 fecal samples from 10 marsupial species belonging to four families in the order Diprotodontia: Vombatidae (wombats), Phascolarctidae (koalas), Phalangeridae (possums), and Macropodidae (kangaroos, wallabies, and pademelons). An amplicon-based diversity survey using the D2 region of the large ribosomal subunit as a phylogenetic marker indicated that marsupial AGF communities were dominated by eight genera commonly encountered in placental herbivores (Neocallimastix, Caecomyces, Cyllamyces, Anaeromyces, Orpinomyces, Piromyces, Pecoramyces, and Khoyollomyces). Community structure analysis revealed a high level of stochasticity, and ordination approaches did not reveal a significant role for the animal host, gut type, dietary preferences, or lifestyle in structuring marsupial AGF communities. Marsupial foregut and hindgut communities displayed diversity and community structure patterns comparable to AGF communities typically encountered in placental foregut hosts while exhibiting a higher level of diversity and a distinct community structure compared to placental hindgut communities. Quantification of AGF load using quantitative PCR indicated a significantly smaller load in marsupial hosts compared to their placental counterparts. Isolation efforts were only successful from a single red kangaroo fecal sample and yielded a Khoyollomyces ramosus isolate closely related to strains previously isolated from placental hosts. Our results suggest that AGF communities in marsupials are in low abundance and show little signs of selection based on ecological and evolutionary factors.IMPORTANCEThe AGF are integral part of the microbiome of herbivores. They play a crucial role in breaking down plant biomass in hindgut and foregut fermenters. The majority of research has been conducted on the AGF community in placental mammalian hosts. However, it is important to note that many marsupial mammals are also herbivores and employ a hindgut or foregut fermentation strategy for breaking down plant biomass. So far, very little is known regarding the AGF diversity and community structure in marsupial mammals. To fill this knowledge gap, we conducted an amplicon-based diversity survey targeting AGF in 61 fecal samples from 10 marsupial species. We hypothesize that, given the distinct evolutionary history and alimentary tract architecture, novel and unique AGF communities would be encountered in marsupials. Our results indicate that marsupial AGF communities are highly stochastic, present in relatively low loads, and display community structure patterns comparable to AGF communities typically encountered in placental foregut hosts. Our results indicate that marsupial hosts harbor AGF communities; however, in contrast to the strong pattern of phylosymbiosis typically observed between AGF and placental herbivores, the identity and gut architecture appear to play a minor role in structuring AGF communities in marsupials.

Toxoplasma gondii Does Not Inhibit the Assisted Colonization of Eastern Barred Bandicoots (Perameles gunnii) to Phillip Island, Victoria, Australia.

Eastern barred bandicoots (Perameles gunnii) are thought to be highly susceptible to disease caused by infection with the protozoan parasite Toxoplasma gondii. This study followed a population of 67 P. gunnii introduced onto the Summerland Peninsula, Phillip Island, Australia, where the prevalence of T. gondii infection in the feral cat population was known to be very high. Prior to release, bandicoots were tested for serologic exposure to T. gondii using the modified agglutination test. A subset of bandicoots was tested on four occasions after release onto the peninsula. No seroconversion was detected at any time point. A subset of bandicoots was radiotracked after release and at two additional trapping sessions to help monitor survival. Toxoplasma gondii DNA was not detected by PCR in eight carcasses recovered for necropsy. Fourteen founder bandicoots (21% of founders) were known to be alive at 500 d post-release. A total of 29 unmarked bandicoots were trapped over the study period, confirming that the bandicoots were successfully reproducing on the island. Body weight, packed cell volume, and total plasma protein were used as measures of individual animal health; population health was inferred from these data. Body weight was significantly associated with trip number, with a general trend of increasing weight after release onto the island. This study showed that eastern barred bandicoots were able to establish a new population despite a probably high environmental load of T. gondii.

Bacterial and Viral Diversity of Didelphid Opossums from Brazil.

Marsupials belonging to the Didelphis genus are widely distributed in the American Continent, and Didelphis albiventris and Didelphis aurita, are common in all of their areas of distribution in Brazil. Here we describe the bacterial and viral diversity of samples from opossums captured in three forest fragments in the State of São Paulo, Brazil. Microbiomes from the same body site were more similar across species and sampling sites while oral swabs presented higher bacterial diversity than rectal swabs. We also identified sequences related to bacterial species involved in zoonotic diseases. The detection of pathogens in such abundant mammal species warns for the possibility of emergence in other species.

Naturally Acquired Rabies in White-Eared Opossum, Brazil.

Opossums are considered resistant to rabies. Nonhematophagous bats are reservoirs of rabies in urban areas of South America. We analyzed bats and opossums tested for rabies during 2021 in a highly urbanized city in Brazil to understand spillover in an urban setting. Wildlife surveillance is necessary to prevent rabies in humans and domestic animals.

Reconstructing Pleistocene Australian herbivore megafauna diet using calcium and strontium isotopes.

Isotopes in fossil tooth enamel provide robust tools for reconstructing food webs, which have been understudied in Australian megafauna. To delineate the isotopic composition of primary consumers and understand dietary behaviour at the base of the food web, we investigate calcium (Ca) and strontium (Sr) isotope compositions of Pleistocene marsupial herbivores from Wellington Caves and Bingara (New South Wales, Australia). Sr isotopes suggest small home ranges across giant and smaller marsupial herbivores. Ca isotopes in Pleistocene marsupial herbivores cover the same range as those in modern wombats and placental herbivores. Early forming teeth are depleted in heavy Ca isotopes compared to late-forming teeth of a given individual, suggesting a weaning signal. Distinct Ca compositions between taxa can be interpreted as dietary niches. Some niches conform to previous dietary reconstructions of taxa, while others provide new insights into niche differentiation across Australian herbivores. Combined with the small roaming ranges suggested by Sr isotopes, the Ca isotope niche diversity suggests rich ecosystems, supporting a diversity of taxa with various diets in a small area.

A novel Babesia sp. of the "Western Babesia group", detected in opossums from Guatemala.

Babesia spp. are tick-borne protozoans that involve birds and mammals in their transmission cycles and cause babesiosis, a severe hemolytic malaria-like disease. Opossums of the genus Didelphis are recognized hosts of tick-borne pathogens. Therefore, exploring tick-borne agents in Didelphis species is important to understand the circulation of pathogens in areas where opossums occur. In this study, we targeted Anaplasmataceae, Babesia, Borrelia and Hepatozoon DNA in ticks, blood and organ samples collected from three hunted Didelphis marsupialis specimens in eastern Guatemala. While the samples were negative for Hepatozoon and bacterial DNA, sequences of Babesia 18S rDNA, cox1 and cytb genes were retrieved from two opossums. Ticks collected on the animals included Amblyomma parvum and an undetermined Ornithodoros sp. The Babesia sp. detected in this study (Babesia sp. THB1-2) clusters phylogenetically within the "Western Babesia group", which includes pathogenic species such as Babesia conradae, Babesia duncani, and Babesia negevi. Our results represent the first record of a Babesia sp. in Guatemala and highlight the importance of D. marsupialis as potential spreaders of ticks and pathogens in Central America.

Characterization of extracellular spike waveforms recorded in wallaby primary visual cortex.

Extracellular recordings were made from 642 units in the primary visual cortex (V1) of a highly visual marsupial, the Tammar wallaby. The receptive field (RF) characteristics of the cells were objectively estimated using the non-linear input model (NIM), and these were correlated with spike shapes. We found that wallaby cortical units had 68% regular spiking (RS), 12% fast spiking (FS), 4% triphasic spiking (TS), 5% compound spiking (CS) and 11% positive spiking (PS). RS waveforms are most often associated with recordings from pyramidal or spiny stellate cell bodies, suggesting that recordings from these cell types dominate in the wallaby cortex. In wallaby, 70-80% of FS and RS cells had orientation selective RFs and had evenly distributed linear and nonlinear RFs. We found that 47% of wallaby PS units were non-orientation selective and they were dominated by linear RFs. Previous studies suggest that the PS units represent recordings from the axon terminals of non-orientation selective cells originating in the lateral geniculate nucleus (LGN). If this is also true in wallaby, as strongly suggested by their low response latencies and bursty spiking properties, the results suggest that significantly more neurons in wallaby LGN are already orientation selective. In wallaby, less than 10% of recorded spikes had triphasic (TS) or sluggish compound spiking (CS) waveforms. These units had a mixture of orientation selective and non-oriented properties, and their cellular origins remain difficult to classify.

Comparative genomics of the T cell receptor µ locus in marsupials and monotremes.

T cells are a primary component of the vertebrate adaptive immune system. There are three mammalian T cell lineages based on their T cell receptors (TCR). The αß T cells and γδ T cells are ancient and found broadly in vertebrates. The more recently discovered γµ T cells are uniquely mammalian and only found in marsupials and monotremes. In this study, we compare the TCRµ locus (TRM) across the genomes of two marsupials, the gray short-tailed opossum and Tasmanian devil, and one monotreme, the platypus. These analyses revealed lineage-specific duplications, common to all non-eutherian mammals described. There is conserved synteny in the TRM loci of both marsupials but not in the monotreme. Our results are consistent with an ancestral cluster organization which was present in the last common mammalian ancestor which underwent lineage-specific duplications and divergence among the non-eutherian mammals.

Clade-specific forebrain cytoarchitectures of the extinct Tasmanian tiger.

The thylacine, or Tasmanian tiger, is the largest of modern-day carnivorous marsupials and was hunted to extinction by European settlers in Australia. Its physical resemblance to eutherian wolves is a striking example of evolutionary convergence to similar ecological niches. However, whether the neuroanatomical organization of the thylacine brain resembles that of canids and how it compares with other mammals remain unknown due to the scarcity of available samples. Here, we gained access to a century-old hematoxylin-stained histological series of a thylacine brain, digitalized it at high resolution, and compared its forebrain cellular architecture with 34 extant species of monotremes, marsupials, and eutherians. Phylogenetically informed comparisons of cortical folding, regional volumes, and cell sizes and densities across cortical areas and layers provide evidence against brain convergences with canids, instead demonstrating features typical of marsupials, and more specifically Dasyuridae, along with traits that scale similarly with brain size across mammals. Enlarged olfactory, limbic, and neocortical areas suggest a small-prey predator and/or scavenging lifestyle, similar to extant quolls and Tasmanian devils. These findings are consistent with a nonuniformity of trait convergences, with brain traits clustering more with phylogeny and head/body traits with lifestyle. By making this resource publicly available as rapid web-accessible, hierarchically organized, multiresolution images for perpetuity, we anticipate that additional comparative insights might arise from detailed studies of the thylacine brain and encourage researchers and curators to share, annotate, and preserve understudied material of outstanding biological relevance.

Influence of the vestibular system on the neonatal motor behaviors in the gray short-tailed opossum (Monodelphis domestica).

Marsupials are born very immature yet must be sufficiently autonomous to crawl on the mother's belly, find a teat and attach to it to pursue their development. Sensory inputs are necessary to guide the newborn to a teat and induce attachment. The vestibular system, which perceives gravity and head movements, is one of the senses proposed to guide newborns towards the teats but there are conflicting observations about its functionality at birth (postnatal day (P) 0). To test if the vestibular system of opossum newborns is functional and can influence locomotion, we used two approaches. First, we stimulated the vestibular apparatus in in vitro preparations from opossums aged from P1 to P12 and recorded motor responses: at all ages studied, mechanical pressures applied on the vestibular organs induced spinal roots activity whereas head tilts did not induce forelimb muscle contractions. Second, using immunofluorescence, we assessed the presence of Piezo2, a protein involved in mechanotransduction in vestibular hair cells. Piezo2 labeling was scant in the utricular macula at birth, but observed in all vestibular organs at P7, its intensity increasing up to P14; it seemed to stay the same at P21. Our results indicate that neural pathways from the labyrinth to the spinal cord are already in place around birth but that the vestibular organs are too immature to influence motor activity before the end of the second postnatal week in the opossum. It may be the rule in marsupial species that the vestibular system becomes functional only after birth.

Helminth community structure of the white-bellied woolly mouse opossum Marmosa constantiae Thomas, 1904 in Central-West Brazil.

Marmosa constantiae is a species of marsupial restricted to the central portion of South America. In Brazil, it occurs in the northwestern region including five states of the Amazon, Cerrado, and Pantanal biomes. However, there is no study of the helminth fauna or helminth community structure for this marsupial. The aims of this study were to describe the species composition and to analyse the structure of the helminthic community of M. constantiae in an area of the Amazon Arc in Sinop, north of the state of Mato Grosso, Brazil. Parasites were searched in 53 specimens of this marsupial, among which 44 were infected with at least one helminth species. Parasitic helminths were counted and identified. Nine species were collected: seven nematodes, one cestode, and one acanthocephalan. The most abundant species were Gracilioxyuris agilisis, Travassostrongylus scheibelorum, Pterygodermatites sinopiensis, and Subulura eliseae. These species were the only dominant ones in the component community. No significant differences were observed in the abundance and prevalence of helminths between male and female hosts. Host body size significantly influenced helminth abundance in males. The pattern of community structure considering the infracommunities in this locality indicated more species replacement than species loss along the environmental gradient. This is the first study to report the helminth fauna and the helminth community structure of M. constantiae.

Description of the Pliocene marsupial Ambulator keanei gen. nov. (Marsupialia: Diprotodontidae) from inland Australia and its locomotory adaptations.

Diprotodontids were the largest marsupials to exist and an integral part of Australian terrestrial ecosystems until the last members of the group became extinct approximately 40 000 years ago. Despite the frequency with which diprotodontid remains are encountered, key aspects of their morphology, systematics, ecology and evolutionary history remain poorly understood. Here we describe new skeletal remains of the Pliocene taxon Zygomaturus keanei from northern South Australia. This is only the third partial skeleton of a late Cenozoic diprotodontid described in the last century, and the first displaying soft tissue structures associated with footpad impressions. Whereas it is difficult to distinguish Z. keanei and the type species of the genus, Z. trilobus, on dental grounds, the marked cranial and postcranial differences suggest that Z. keanei warrants genus-level distinction. Accordingly, we place it in the monotypic Ambulator gen. nov. We, also recognize the late Miocene Z. gilli as a nomen dubium. Features of the forelimb, manus and pes reveal that Ambulator keanei was more graviportal with greater adaptation to quadrupedal walking than earlier diprotodontids. These adaptations may have been driven by a need to travel longer distances to obtain resources as open habitats expanded in the late Pliocene of inland Australia.

Prevalence of Trypanosoma lainsoni and its effects of parasitism on the health of non-volant small mammals from the Brazilian Cerrado.

Small mammals are important hosts and/or reservoirs of Trypanosoma spp. This study aimed to verify the prevalence of Trypanosoma spp. in non-volant small mammals from the Brazilian Cerrado and to test the effects of T. lainsoni on the neutrophil/lymphocyte ratio (N/L) and body condition in rodent and marsupial populations. For this, we collected blood samples of 293 individuals captured in five forest fragments between 2019 and 2020. Blood was used to prepare the blood smears and packed on filter paper for DNA extraction. Generalized linear models were performed to test the effects of T. lainsoni on host health. The DNA was submitted to nested PCR targeting the Trypanosoma spp. 18S rRNA gene. From blood smears analyzed by microscopy, we obtained a positivity rate of 7.2% for Trypanosoma spp. About 31.1% of Gracilinanus agilis, Didelphis albiventris, and Rhipidomys macrurus samples were positive in nested PCR. From the obtained sequences, 83.3% were genetically identical to T. lainsoni and about 11% to T. cruzi TcI. In addition, we reported the infection of T. lainsoni in Hylaeamys megacephalus. We suggest that T. lainsoni does not influence the body condition and N/L ratio for either G. agilis or R. macrurus. Overall, our results expand the host list of T. lainsoni and demonstrate the infection of small mammals by T. cruzi TcI in peri-urban areas.