Orthohantaviruses in Misiones Province, Northeastern Argentina.

Few cases of hantavirus pulmonary syndrome have been reported in northeastern Argentina. However, neighboring areas show a higher incidence, suggesting underreporting. We evaluated the presence of antibodies against orthohantavirus in small rodents throughout Misiones province. Infected Akodon affinis montensis and Oligoryzomys nigripes native rodents were found in protected areas of Misiones.

Experimental manipulation of food availability and macroparasite prevalence reveal differential effects on space use in wild rodents.

Research Highlight: Mistrick, J., Veitch, J. S. M., Kitchen, S. M., Clague, S., Newman, B. C., Hall, R. J., Budischak, S. A., Forbes, K. M., & Craft, M. E. (2024). Effects of food supplementation and helminth removal on space use and spatial overlap in wild rodent populations. Journal of Animal Ecology. http://doi.org/10.1111/1365-2656.14067. The spread of pathogens has been of long-standing interest, even before dramatic outbreaks of avian influenza and the coronavirus pandemic spiked broad public interest. However, the dynamics of pathogen spread in wild populations are complex, with multiple effects shaping where animals go (their space use), population density and, more fundamentally, the resultant patterns of contacts (direct or indirect) among individuals. Thus, experimental studies exploring the dynamics of contact under different sets of conditions are needed. In the current field study, Mistrick et al. (2024) used a multifactorial experimental design, manipulating food availability and individual pathogen infection state in wild bank voles (Clethrionomys glareolus). They found that while food availability, individual traits and seasonality can affect how far individual voles moved, the degree of overlap between individual voles remained largely the same despite a high variation in population density-which itself was affected by food availability. These results highlight how biotic and abiotic factors can shape patterns of space use and balance the level of spatial overlap through multiple pathways.

Immune interactions and heterogeneity in transmission drives the pathogen-mediated invasion of grey squirrels in the UK.

Mathematical models highlighted the importance of pathogen-mediated invasion, with the replacement of red squirrels by squirrelpox virus (SQPV) carrying grey squirrels in the UK, a well-known example. In this study, we combine new epidemiological models, with a range of infection characteristics, with recent longitudinal field and experimental studies on the SQPV dynamics in red and grey squirrel populations to better infer the mechanistic basis of the disease interaction. A key finding is that a model with either partial immunity or waning immunity and reinfection, where individuals become seropositive on the second exposure to infection, that up to now has been shown in experimental data only, can capture the key aspects of the field study observations. By fitting to SQPV epidemic observations in isolated red squirrel populations, we can infer that SQPV transmission between red squirrels is significantly (4×) higher than the transmission between grey squirrels and as a result our model shows that disease-mediated replacement of red squirrels by greys is considerably more rapid than replacement in the absence of SQPV. Our findings recover the key results of the previous model studies, which highlights the value of simple strategic models that are appropriate when there are limited data, but also emphasise the likely complexity of immune interactions in wildlife disease and how models can help infer disease processes from field data.

Gut protozoa of wild rodents - a meta-analysis.

Protozoa are well-known inhabitants of the mammalian gut and so of the gut microbiome. While there has been extensive study of a number of species of gut protozoa in laboratory animals, particularly rodents, the biology of the gut protozoa of wild rodents is much less well-known. Here we have systematically searched the published literature to describe the gut protozoa of wild rodents, in total finding records of 44 genera of protozoa infecting 228 rodent host species. We then undertook meta-analyses that estimated the overall prevalence of gut protozoa in wild rodents to be 24%, with significant variation in prevalence among some host species. We investigated how host traits may affect protozoa prevalence, finding that for some host lifestyles some protozoa differed in their prevalence. This synthesis of existing data on wild rodent gut protozoa provides a better understanding of the biology of these common gut inhabitants and suggests directions for their future study.

First molecular characterisation of Hydatigera taeniaeformis (Cestoda: Taeniidae) from Rattus rattus in urban market centres from Tamale, Ghana.

BACKGROUND: The common cat tapeworm, Hydatigera taeniaeformis, is widely distributed across many regions of the world but its global genetic diversity, ecology, and other biological features are not fully understood. The predator-prey relationship of felids and rodents sustains the transmission in both sylvatic and synanthropic or urban settings. This study was carried out in three urban market centres in Tamale, Ghana to determine the prevalence of H. taeniaeformis infection in urban rats and characterise the local population genetics using the mitochondrial nad1 gene sequences. METHODS: From January to May 2023, rats were captured and euthanized to identity and isolate the H. taeniaeformis metacestodes. The full mitochondrial nad1 gene was amplified, sequenced and used to characterise the cestode population. RESULTS: In total 64/80 rats from the three market centres were found naturally infected with metacestodes of H. taeniaeformis. Averagely, infected rats harboured two cysts with one exceptional case of 25 cysts in one liver. Each cyst harboured one metacestode. The DNA sequences revealed four haplotypes in a star-like distribution network across the three market centres. All four haplotypes were found to be unique, clustered with H. taeniaeformis sensu stricto (s.s.) global isolates and had only marginal variation among themselves. CONCLUSION: This is the first account of the genetic variability within H. taeniaeformis in Ghana and would contribute to ongoing and future studies on African regional biogeographic distribution of the common cat tapeworm.

Histopathological and morphological characterization of Cysticercus fasciolaris isolated from domestic and wild rodents in Morocco.

Cysticercus fasciolaris is a parasitic helminth that usually infects feline and canine mammal hosts. The intermediate hosts (rodents, occasionally lagomorphs, and humans) get infected by the consumption of feed or water contaminated with eggs. Rodents are vectors of disease and reservoirs of various zoonotic parasites. The current survey was aimed at determining endoparasitic helminth infections in rodents in central Morocco. Sampled rodents after specific identification were sacrificed and examined to identify parasitic helminths following ethical guidelines. Parasites were identified using morphological characteristics. A total of 197 specimens of rodents were collected and examined in this study. Ten rodent species were identified morphologically as Rattus rattus, R. norvegicus, Apodemus sylvaticus, Mus musculus, M. spretus, Mastomys erythroleucus, Meriones shawi, M. libycus, Gerbillus campestris, and Lemniscomys barbarus. The parasitological results showed that metacestode of tapeworms was found encysted in the liver, the larval stage of Taenia taeniaeformis develops large multinodular fibrosarcomas which envelope the tapeworm cysts in the liver of the R. rattus and R. norvegicus. Based on morphological data, the metacestode was identified as C. fasciolaris in 23 (23/80) R. rattus 2 (2/8) and R. norvegicus with a prevalence of 11.7 % and 1.0 %, respectively. Rodents are major vectors of human and domestic animal diseases worldwide, and therefore, important parasitic zoonotic agents (C. fasciolaris), which are transmitted by black rats (R. rattus) and brown rats (R. norvegicus), must be considered to prevent the infectivity of humans, domestic animals, and livestock such as cattle, sheep, and rabbits.

Gastrointestinal parasites of Wolffsohn's viscacha (Chinchillidae: Lagidium wolffsohni), an endemic rodent species from the wild Patagonia.

Parasites are ubiquitous in wildlife populations and have a profound impact on population dynamics. Interest in parasites of wildlife has increased significantly in recent years, particularly in those with relevant conservation status. Patagonia is one of the wildest and remote areas of the world. The Wolffsohn's viscacha lives in a small mountainous area of Patagonia. Until now, little is known about the biology and ecology of this species. The aim of this research was to study the gastrointestinal parasite diversity in this rodent from a coprological survey. A total of 125 fecal samples from 25 colonies were examined. Each sample was rehydrated, homogenized, and analyzed using three parasitological techniques: spontaneous sedimentation, Mini-FLOTAC, and centrifugation-flotation in sucrose-saturated solution, followed by examination under optical microscopy. The samples, eggs, and oocysts of parasites were described, measured, and photographed. All colonies were positive for at least one parasite species. A total of 10 parasitic species were identified: Viscachataenia sp., possibly V. quadrata, Monoecocestus sp., an unidentified anoplocephalid, Heteroxynema sp., possibly H. (Cavioxyura) viscaciae, Helminthoxys sp., possibly H. effilatus, an unidentified strongylid-type egg, Trichuris sp., two morphologies of unidentified coccidians and Eimeria sp. This is the first exhaustive study of gastrointestinal parasites in L. wolffsohni and a large number of eggs and oocysts of parasites were found. Our results highlight the use of noninvasive techniques for the study of parasites of wildlife hosts; as in the case of this rodent with a remote habitat, which makes sampling difficult. The results of our study provide baseline information on gastrointestinal parasite infections in this species.

Phylogenetic analysis of a novel Hepatozoon species (Hepatozoon sp. SK3) and an additional yet unknown Hepatozoon species (Hepatozoon sp. BV2) besides H. erhardovae in small rodents from Central Europe.

Hepatozoon spp. are tick-borne apicomplexan parasites of terrestrial vertebrates that occur worldwide. Tissue samples from small rodents and their parasitizing fleas were sampled for molecular detection and phylogenetic analysis of Hepatozoon-specific 18S rRNA gene region. After alignment and tree inference the Hepatozoon-sequences retrieved from a yellow-necked mouse (Apodemus flavicollis) placed into a strongly supported single clade demonstrating the presence of a novel species, designated Hepatozoon sp. SK3. The mode of transmission of Hepatozoon sp. SK3 is yet unknown. It is important to note that this isolate may be identical with the previously morphologically described Hepatozoon sylvatici infecting Apodemus spp.; however, no sequences are available for comparison. Furthermore, the previously reported variants Hepatozoon sp. BV1/SK1 and BV2/SK2 were detected in bank voles (Clethrionomys glareolus). It has been suggested that these variants should be identified as Hepatozoon erhardovae leading to the assumption that BV1 and BV2 are paralogous 18S rRNA gene loci of this species. Evidence has also been presented that fleas are vectors of H. erhardovae. In this study, we show with high significance that only the Hepatozoon sp. BV1 variant, but not BV2, infects the studied flea species Ctenophthalmus agyrtes, Ctenophthalmus assimilis, and Megabothris turbidus (p < 0.001). This finding suggests that Hepatozoon sp. BV2 represents an additional species besides H. erhardovae (= Hepatozoon sp. BV1), for which alternative arthropod vectors or non-vectorial modes of transmission remain to be identified. Future studies using alternative molecular markers or genome sequencing are required to demonstrate that BV1/SK1 and BV2/SK2 are different Hepatozoon species.

Molecular characterizations of Cryptosporidium spp. in brown rat (Rattus norvegicus) from an animal feedlot in Xinjiang, China.

Cryptosporidium infection is a common occurrence in rodents worldwide. In this study, 435 wild brown rats were captured from an animal feedlot in Xinjiang, China, with a fecal sample obtained directly from the rectal contents of each rat. The DNA extracted from these fecal samples was analyzed for Cryptosporidium spp. using PCR targeting the SSU rRNA gene. The prevalence of Cryptosporidium infection in brown rats was found to be 5.5% (24 out of 435). Interestingly, the infection rates varied among different animal enclosures, with rates of 0% in the chicken coop (0/51), cowshed (0/3), and varying rates in other areas including the sheepfold (6.1%, 6/98), the pigsty (7.6%, 10/132), the dovecote (7.0%, 5/71), and outdoor environments (3.8%, 3/80). The study identified three species and one genotype of Cryptosporidium, namely C. occultus (n = 10), C. parvum (n = 4), C. ditrichi (n = 1), and Cryptosporidium rat genotype IV (n = 9). Additionally, two of the C. parvum isolates were successfully subtyped as IIdA19G1 (n = 2) at the gp60 gene. These results offer valuable insights into the prevalence and genetic diversity of Cryptosporidium in brown rats within the region.

Potentially zoonotic pathogens and parasites in opportunistically sourced urban brown rats (Rattus norvegicus) in and around Helsinki, Finland, 2018 to 2023.

BackgroundBrown rats (Rattus norvegicus) are synanthropic rodents with worldwide distribution, which are known to harbour many zoonotic pathogens and parasites. No systematic zoonotic surveys targeting multiple pathogens and parasites have previously been conducted in urban rats in Finland.AimIn Helsinki, Finland, we explored the presence and prevalence in brown rats of certain pathogens and parasites (including helminths, viruses and bacteria) across potentially zoonotic taxa.MethodsWe opportunistically received rat carcasses from pest management operators and citizens from 2018 to 2023. We searched for heart- or lungworms, performed rat diaphragm digestion to check for Trichinella and morphologically identified intestinal helminths. We assessed virus exposure by immunofluorescence assay or PCR, and detected bacteria by PCR (Leptospira) or culture (Campylobacter).ResultsAmong the rats investigated for helminths, no heart- or lungworms or Trichinella species were detected and the most common finding was the cestode Hymenolepis nana (in 9.7% of individuals sampled, 28/288). For some of the surveyed virus taxa, several rats were seropositive (orthopoxviruses, 5.2%, 11/211; arenaviruses, 2.8%, 6/211; hantaviruses 5.2%, 11/211) or tested positive by PCR (rat hepatitis E virus, 1.8%, 4/216). Campylobacter jejuni (6.6%, 17/259) and Leptospira interrogans (1.2%, 2/163) bacteria were also present in the rat population examined.ConclusionsPrevalences of potentially zoonotic pathogens and parasites in brown rats in Helsinki appeared low. This may explain low or non-existent diagnosis levels of rat-borne pathogen and parasite infections reported in people there. Nevertheless, further assessment of under-diagnosis, which cannot be excluded, would enhance understanding the risks of zoonoses.

Leptospira infection and carrier survey on rats from wet market areas in Kuala Lumpur, Malaysia.

BACKGROUND OBJECTIVES: Leptospirosis is an important zoonotic infection that has caused significant mortality and morbidity worldwide. This disease is endemic in Malaysia and as a developing tropical country, leptospirosis is concerning as it threatens Malaysian public health and the country's economic sectors. However, there is limited information on leptospirosis in Malaysia, especially regarding leptospiral seroepidemiology among carriers in Malaysia. Therefore, more epidemiological information on the source of the disease and reservoir are needed for better disease control and source intervention. The objectives of this study are to gather information on Leptospira infection and the carrier status of rats captured from selected wet markets of Kuala Lumpur metropolitan city in Malaysia. METHODS: Live rat trappings were performed in four major wet markets in Kuala Lumpur, namely, Pudu, Chow Kit, Datuk Keramat, and Petaling Street. Animal samplings were performed for 12 months in 2017, where blood and kidney samples were collected and tested for anti-leptospiral antibodies via Microscopic Agglutination Test (MAT) and pathogenic Leptospira screening via Polymerase Chain Reaction (PCR) amplification offlaB gene. RESULTS: MAT showed that 34.7% (n = 50/144) of the captured rats were positive for anti-leptospiral antibody of which the most prominent serovar was Malaya followed by a local strain, IMR LEP 175. In parallel, 50 rats were also positive for pathogenic Leptospira DNA. INTERPRETATION CONCLUSION: This study showed that there are persistent Leptospira infections among rats in Kuala Lumpur wet markets and these rats are important reservoir hosts for the bacteria.

Parasite infestation patterns differ between ticks and chigger mites on two rodent host species in Taiwan.

Parasites are typically concentrated on a few host individuals, and identifying the mechanisms underlying aggregated distribution can facilitate a more targeted control of parasites. We investigated the infestation patterns of hard ticks and chigger mites on two rodent species, the striped field mouse, Apodemus agrarius, and the lesser ricefield rat, Rattus losea, in Taiwan. We also explored abiotic and biotic factors that were important in explaining variation in the abundance of ticks and chiggers on rodent hosts. Ticks were more aggregated than chiggers on both rodent species. Factors important for the variation in parasitic loads, especially biotic factors, largely differed between ticks and chiggers. Variation partitioning analyses revealed that a larger proportion of variation in chiggers than in ticks can be explained, especially by abiotic factors. If, as proposed, the higher number of parasites in males is due to a larger range area or immunity being suppressed by testosterone, when A. agrarius males host more ticks, they are expected to also host more chiggers, given that chiggers adopt a similar host finding approach to that of ticks. Instead, the similar abundance of chiggers in male and female A. agrarius implies that a large home range or suppressed immunity does not predispose males to inevitably host more parasites. More variations were explained by abiotic than biotic factors, suggesting that controlling practices are more likely to be successful by focusing on factors related to the environment instead of host traits. Our study indicated that the extent of parasitism is rarely determined by a sole factor, but is an outcome of complex interactions among animal physiology, animal behavior, characteristics of parasites, and the environments.

Pathogens detected in ticks (Ixodes ricinus) feeding on red squirrels (Sciurus vulgaris) from city parks in Warsaw.

The European red squirrel (Sciurus vulgaris) is a common host for Ixodes ricinus ticks in urban and rural habitats, however, studies on ticks and tick-borne pathogens (TBPs) of squirrels have not been conducted in Poland yet. Thus, the aims of the current study were to assess and compare the prevalence and abundance of ticks on red squirrels trapped at two sites in the Warsaw area (in an urban forest reserve and an urban park) and using molecular tools, to assess the genetic diversity of three pathogens (Borrelia burgdorferi sensu lato, Rickettsia and Babesia spp.) in I. ricinus ticks collected from squirrels. For the detection of Rickettsia spp. a 750 bp long fragment of the citrate synthase gltA gene was amplified; for B. burgdorferi s.l. 132f/905r and 220f/824r primers were used to amplify the bacterial flaB gene fragments (774 and 605 bp, respectively) and for Babesia spp., a 550 bpfragment of 18S rRNA gene was amplified. In total, 91 red squirrels were examined for ticks. There were differences in tick prevalence and mean abundance of infestation in squirrels from the urban forest reserve and urban park. Three species of B. burgdorferi s.l., Rickettsia spp., and Babesia microti were detected in ticks removed from the squirrels. Our results broaden knowledge of S. vulgaris as an important host for immature I. ricinus stages and support the hypothesis that red squirrels act as a reservoir of B. burgdorferi. Moreover, we conclude that red squirrels may also play a role in facilitating the circulation of other pathogens causing serious risk of tick-borne diseases in natural and urban areas.

Toxoplasma gondii in small mammals in Romania: the influence of host, season and sampling location.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including humans. Small rodents and insectivores play an important role in the epidemiology of T. gondii and may serve as a source of infection for both, domestic and wild definitive felid hosts. Factors influencing the occurrence of T. gondii in wild small mammals are unknown, despite the fact that many intermediate host species are identified. We have used small mammals (Rodentia and Lipotyphla) captured over two years in various habitats, both in urbanised and in natural landscapes. We assessed the importance of land-use, season and host ecology on T. gondii infection. RESULTS: We examined 471 individuals belonging to 20 small mammal species, collected at 63 locations spread over wide altitude, habitat and land-use ranges from Romania. Heart tissue samples were individually analysed by PCR targeting the 529 bp repetitive DNA fragment of T. gondii. The overall prevalence of infection was 7.3%, with nine species of rodents and two species of shrews being found to carry T. gondii DNA. Five species showed high frequency of infection, with the highest prevalence found in Myodes glareolus (35.5%), followed by Spermophilus citellus (33.3%), Sorex minutus (23.1%), S. araneus (21.7%) and Micromys minutus (11.1%). Adults seemed more often infected than young, however when controlling for season, the difference was not significant, as in spring both adults and young showed higher infection rates, but more adults were sampled. Contrary to our expectations, urban/rural areas (with their implicit high density of domestic feline presence) had no effect on infection prevalence. In addition, neither habitat, nor land-use at sampling sites was important as only geographical location and host species were contributing factors to the infection risk. CONCLUSIONS: High prevalence of T. gondii infection showed a highly localised, patchy occurrence, with long living and higher mobility host species being the most common carriers, especially during autumn.

Experimental Infection of Peromyscus Species Rodents with Sin Nombre Virus.

We demonstrate that 6 distinct Peromyscus rodent species are permissive to experimental infection with Sin Nombre orthohantavirus (SNV). Viral RNA and SNV antibodies were detected in members of all 6 species. P. leucopus mice demonstrated markedly higher viral and antibody titers than P. maniculatus mice, the established primary hosts for SNV.

Culling of Urban Norway Rats and Carriage of Bartonella spp. Bacteria, Vancouver, British Columbia, Canada.

We investigated the effects of culling on Bartonella spp. bacteria carriage among urban rats in Canada. We found that the odds of Bartonella spp. carriage increased across city blocks except those in which culling occurred. Removing rats may have prevented an increase in Bartonella spp. prevalence, potentially lowering human health risks.

Viral Zoonoses in Small Wild Mammals and Detection of Hantavirus, Spain.

We screened 526 wild small mammals for zoonotic viruses in northwest Spain and found hantavirus in common voles (Microtus arvalis) (1.5%) and high prevalence (48%) of orthopoxvirus among western Mediterranean mice (Mus spretus). We also detected arenavirus among small mammals. These findings suggest novel risks for viral transmission in the region.

Tracing Transmission of Sin Nombre Virus and Discovery of Infection in Multiple Rodent Species.

Sin Nombre orthohantavirus (SNV), a negative-sense, single-stranded RNA virus that is carried and transmitted by the North American deer mouse Peromyscus maniculatus, can cause infection in humans through inhalation of aerosolized excreta from infected rodents. This infection can lead to hantavirus cardiopulmonary syndrome (HCPS), which has an ∼36% case-fatality rate. We used reverse transcriptase quantitative PCR (RT-qPCR) to confirm SNV infection in a patient and identified SNV in lung tissues in wild-caught rodents from potential sites of exposure. Using viral whole-genome sequencing (WGS), we identified the likely site of transmission and discovered SNV in multiple rodent species not previously known to carry the virus. Here, we report, for the first time, the use of SNV WGS to pinpoint a likely site of human infection and identify SNV simultaneously in multiple rodent species in an area of known host-to-human transmission. These results will impact epidemiology and infection control for hantaviruses by tracing zoonotic transmission and investigating possible novel host reservoirs. IMPORTANCE Orthohantaviruses cause severe disease in humans and can be lethal in up to 40% of cases. Sin Nombre orthohantavirus (SNV) is the main cause of hantavirus disease in North America. In this study, we sequenced SNV from an infected patient and wild-caught rodents to trace the location of infection. We also discovered SNV in rodent species not previously known to carry SNV. These studies demonstrate for the first time the use of virus sequencing to trace the transmission of SNV and describe infection in novel rodent species.

Secondary contact between diverged host lineages entails ecological speciation in a European hantavirus.

The diversity of viruses probably exceeds biodiversity of eukaryotes, but little is known about the origin and emergence of novel virus species. Experimentation and disease outbreak investigations have allowed the characterization of rapid molecular virus adaptation. However, the processes leading to the establishment of functionally distinct virus taxa in nature remain obscure. Here, we demonstrate that incipient speciation in a natural host species has generated distinct ecological niches leading to adaptive isolation in an RNA virus. We found a very strong association between the distributions of two major phylogenetic clades in Tula orthohantavirus (TULV) and the rodent host lineages in a natural hybrid zone of the European common vole (Microtus arvalis). The spatial transition between the virus clades in replicated geographic clines is at least eight times narrower than between the hybridizing host lineages. This suggests a strong barrier for effective virus transmission despite frequent dispersal and gene flow among local host populations, and translates to a complete turnover of the adaptive background of TULV within a few hundred meters in the open, unobstructed landscape. Genetic differences between TULV clades are homogenously distributed in the genomes and mostly synonymous (93.1%), except for a cluster of nonsynonymous changes in the 5' region of the viral envelope glycoprotein gene, potentially involved in host-driven isolation. Evolutionary relationships between TULV clades indicate an emergence of these viruses through rapid differential adaptation to the previously diverged host lineages that resulted in levels of ecological isolation exceeding the progress of speciation in their vertebrate hosts.

Virus isolation data improve host predictions for New World rodent orthohantaviruses.

Identifying reservoir host species is crucial for understanding the ecology of multi-host pathogens and predicting risks of pathogen spillover from wildlife to people. Predictive models are increasingly used for identifying ecological traits and prioritizing surveillance of likely zoonotic reservoirs, but these often employ different types of evidence for establishing host associations. Comparisons between models with different infection evidence are necessary to guide inferences about the trait profiles of likely hosts and identify which hosts and geographical regions are likely sources of spillover. Here, we use New World rodent-orthohantavirus associations to explore differences in the performance and predictions of models trained on two types of evidence for infection and onward transmission: RT-PCR and live virus isolation data, representing active infections versus host competence, respectively. Orthohantaviruses are primarily carried by muroid rodents and cause the diseases haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) in humans. We show that although boosted regression tree (BRT) models trained on RT-PCR and live virus isolation data both performed well and capture generally similar trait profiles, rodent phylogeny influenced previously collected RT-PCR data, and BRTs using virus isolation data displayed a narrower list of predicted reservoirs than those using RT-PCR data. BRT models trained on RT-PCR data identified 138 undiscovered hosts and virus isolation models identified 92 undiscovered hosts, with 27 undiscovered hosts identified by both models. Distributions of predicted hosts were concentrated in several different regions for each model, with large discrepancies between evidence types. As a form of validation, virus isolation models independently predicted several orthohantavirus-rodent host associations that had been previously identified through empirical research using RT-PCR. Our model predictions provide a priority list of species and locations for future orthohantavirus sampling. More broadly, these results demonstrate the value of multiple data types for predicting zoonotic pathogen hosts. These methods can be applied across a range of systems to improve our understanding of pathogen maintenance and increase efficiency of pathogen surveillance.