Arboviruses in Mammals in the Neotropics: A Systematic Review to Strengthen Epidemiological Monitoring Strategies and Conservation Medicine.

Arthropod-borne viruses (arboviruses) are a diverse group of ribonucleic acid (RNA) viruses, with the exception of African swine fever virus, that are transmitted by hematophagous arthropods to a vertebrate host. They are the important cause of many diseases due to their ability to spread in different environments and their diversity of vectors. Currently, there is no information on the geographical distribution of the diseases because the routes of transmission and the mammals (wild or domestic) that act as potential hosts are poorly documented or unknown. We conducted a systematic review from 1967 to 2021 to identify the diversity of arboviruses, the areas, and taxonomic groups that have been monitored, the prevalence of positive records, and the associated risk factors. We identified forty-three arboviruses in nine mammalian orders distributed in eleven countries. In Brazil, the order primates harbor the highest number of arbovirus records. The three most recorded arboviruses were Venezuelan equine encephalitis, Saint Louis encephalitis and West Nile virus. Serum is the most used sample to obtain arbovirus records. Deforestation is identified as the main risk factor for arbovirus transmission between different species and environments (an odds ratio of 1.46 with a 95% confidence interval: 1.34-1.59). The results show an increase in the sampling effort over the years in the neotropical region. Despite the importance of arboviruses for public health, little is known about the interaction of arboviruses, their hosts, and vectors, as some countries and mammalian orders have not yet been monitored. Long-term and constant monitoring allows focusing research on the analysis of the interrelationships and characteristics of each component animal, human, and their environment to understand the dynamics of the diseases and guide epidemiological surveillance and vector control programs. The biodiversity of the Neotropics should be considered to support epidemiological monitoring strategies.

From roads to biobanks: Roadkill animals as a valuable source of genetic data.

To protect biodiversity we must understand its structure and composition including the bacteria and microparasites associated with wildlife, which may pose risks to human health. However, acquiring this knowledge often presents challenges, particularly in areas of high biodiversity where there are many undescribed and poorly studied species and funding resources can be limited. A solution to fill this knowledge gap is sampling roadkill (animals that die on roads as a result of collisions with circulating vehicles). These specimens can help characterize local wildlife and their associated parasites with fewer ethical and logistical challenges compared to traditional specimen collection. Here we test this approach by analyzing 817 tissue samples obtained from 590 roadkill vertebrate specimens (Amphibia, Reptilia, Aves and Mammalia) collected in roads within the Tropical Andes of Ecuador. First, we tested if the quantity and quality of recovered DNA varied across roadkill specimens collected at different times since death, exploring if decomposition affected the potential to identify vertebrate species and associated microorganisms. Second, we compared DNA stability across taxa and tissues to identify potential limitations and offer recommendations for future work. Finally, we illustrate how these samples can aid in taxonomic identification and parasite detection. Our study shows that sampling roadkill can help study biodiversity. DNA was recovered and amplified (allowing species identification and parasite detection) from roadkill even 120 hours after death, although risk of degradation increased overtime. DNA was extracted from all vertebrate classes but in smaller quantities and with lower quality from amphibians. We recommend sampling liver if possible as it produced the highest amounts of DNA (muscle produced the lowest). Additional testing of this approach in areas with different environmental and traffic conditions is needed, but our results show that sampling roadkill specimens can help detect and potentially monitor biodiversity and could be a valuable approach to create biobanks and preserve genetic data.

First Molecular Identification of Trypanosomes and Absence of Babesia sp. DNA in Faeces of Non-Human Primates in the Ecuadorian Amazon.

Trypanosomes are a group of pathogens distributed in the continents of Africa, America, Asia and Europe, and they affect all vertebrates including the neotropical primate group. Information about the trypanosome's diversity, phylogeny, ecology and pathology in non-human primates (NHPs) from the neotropical region is scarce. The objective of the study was to identify Trypanosoma and Babesia molecularly in NHPs under the phylogenetic species concept. We extracted DNA from a total of 76 faecal samples collected between 2019 and 2021, from a total of 11 non-human primate species of which 46 are from captive NHPs and 30 are free-living NHPs in the Western Amazon region of Ecuador. We did not detect DNA of Babesia sp. by polymerase chain reaction test in any of the faecal samples. However, the nested-PCR-based method revealed Trypanosoma parasites by ITS gene amplification in two faecal samples; one for the species Leontocebus lagonotus (from the captive population) and a second one for Cebus albifrons (from the free-ranging population). Maximum parsimony and likelihood methods with the Kimura2+G+I model inferred the evolutionary history of the two records, which showed an evolutionary relationship with the genus Trypanosoma. Two sequences are monophyletic with Trypanosoma. However, the number of sequences available in GenBank for their species identification is limited. The two samples present different molecular identifications and evolutionary origins in the tree topology. We are most likely referring to two different species, and two different localities of infection. We suggest that health management protocols should be implemented to prevent the transmission of blood-borne pathogens such as Trypanosoma sp. among captive populations. In addition, these protocols also protect the personnel of wildlife rehabilitation centers working in close proximity to NHPs and vice versa.

Pollen preferences of stingless bees in the Amazon region and southern highlands of Ecuador by scanning electron microscopy and morphometry.

Stingless bees are effective pollinators of native tropical flora. Their environmental service maintains flow of pollen through pollination, increase reproductive success and influence genetic structure in plants. The management of stingless bees "meliponiculture", is an activity limited to the countryside in Ecuador. The lack of knowledge of their managers about pollen resources can affect the correct maintenance/production of nests. The objective is to identify botanical families and genera of pollen grains collected by stingless bees by morphological features and differentiate potential species using geometric morphometry. Thirty-six pot pollen samples were collected from three Ecuadorian provinces located in two climatically different zones. Pollen type identification was based on the Number, Position, Character system. Using morphological features, the families and genera were established. Morphometry landmarks were used to show variation for species differentiation. Abundance, diversity, similarity and dominance indices were established by counting pollen grains, as well as spatial distribution relationships by means of Poisson regression. Forty-six pollen types were determined in two study areas, classified into 27 families and 18 genera. In addition, it was possible to identify more than one species, classified within the same family and genus, thanks to morphometric analysis. 1148 ± 799 (max 4211; min 29) pollen grains were counting in average. The diversity showed a high richness, low dominance and similarity between pollen resources. Families Melastomataceae and Asteraceae, genera Miconia and Bidens, were found as the main pollen resources. The stingless bee of this study are mostly generalist as shown the interaction network. The results of the present survey showed that stingless bees do not collect pollen from a single species, although there is evidence of a predilection for certain plant families. The diversity indexes showed high richness but low uniformity in the abundance of each family identified. The results of the study are also meaningful to the meliponiculture sector as there is a need to improve management practices to preserve the biodiversity and the environment.

Zoonotic Blood-Borne Pathogens in Non-Human Primates in the Neotropical Region: A Systematic Review.

Background: Understanding which non-human primates (NHPs) act as a wild reservoir for blood-borne pathogens will allow us to better understand the ecology of diseases and the role of NHPs in the emergence of human diseases in Ecuador, a small country in South America that lacks information on most of these pathogens. Methods and principal findings: A systematic review was carried out using PRISMA guidelines from 1927 until 2019 about blood-borne pathogens present in NHPs of the Neotropical region (i.e., South America and Middle America). Results: A total of 127 publications were found in several databases. We found in 25 genera (132 species) of NHPs a total of 56 blood-borne pathogens in 197 records where Protozoa has the highest number of records in neotropical NHPs (n = 128) compared to bacteria (n = 12) and viruses (n = 57). Plasmodium brasilianum and Trypanosoma cruzi are the most recorded protozoa in NHP. The neotropical primate genus with the highest number of blood-borne pathogens recorded is Alouatta sp. (n = 32). The use of non-invasive samples for neotropical NHPs remains poor in a group where several species are endangered or threatened. A combination of serological and molecular techniques is common when detecting blood-borne pathogens. Socioecological and ecological risk factors facilitate the transmission of these parasites. Finally, a large number of countries remain unsurveyed, such as Ecuador, which can be of public health importance. Conclusions and significance: NHPs are potential reservoirs of a large number of blood-borne pathogens. In Ecuador, research activities should be focused on bacteria and viruses, where there is a gap of information for neotropical NHPs, in order to implement surveillance programs with regular and effective monitoring protocols adapted to NHPs.

Molecular Identification of Plasmodium falciparum from Captive Non-Human Primates in the Western Amazon Ecuador.

Background: Malaria is a disease caused by hemoparasites of the Plasmodium genus. Non-human primates (NHP) are hosts of Plasmodium sp. around the world. Several studies have demonstrated that Plasmodium sp. emerged from Africa. However, little information is currently available about Plasmodium falciparum in the neotropical NHP and even less in Ecuador. Indeed, the objective of our study was to identify by molecular phylogenetic analyses the Plasmodium species associated with NHP from the Western Amazon region of Ecuador, and to design a molecular taxonomy protocol to use in the NHP disease ecology. Methods: We extracted DNA from faecal samples (n = 26) from nine species of captive (n = 19) and free-ranging (n = 7) NHP, collected from 2011 to 2019 in the Western Amazon region of Ecuador. Results: Using a pan-Plasmodium PCR, we obtained one positive sample from an adult female Leontocebus lagonotus. A maximum likelihood phylogenetic analysis showed that this sequence unequivocally clustered with Plasmodium falciparum. Conclusions: The identification of Plasmodium sp. in NHP of the Ecuadorian Amazon would be essential to identify their role as potential zoonotic reservoirs, and it is also important to identify their origin in wildlife and their transmission in captive NHP.

Detection of Babesia spp. in High Altitude Cattle in Ecuador, Possible Evidence of the Adaptation of Vectors and Diseases to New Climatic Conditions.

BACKGROUND: Babesia species are intraerythrocytic protozoa, distributed in tropical and subtropical areas of the world, causing anemic diseases in many animals, including cattle. This disease, called babesisosis, is transmitted from one animal to another through ticks (Tick Borne-Disease or TBD). On the other hand, Ecuador has a tropical climate that allows the development of the vector Rhipicephalus microplus, and therefore favors the transmission of Babesia spp. in cattle. METHODS AND PRINCIPAL FINDINGS: We determined the presence of Babesia spp. by PCR using 18s ribosomal gene as target (18s PCR) in 20 farms in the area of El Carmen (zone below 300 m above sea level) and 1 farm in Quito (2469 m.a.s.l.). In addition, we analyzed parameters such as age, sex, and packed cell volume (PCV) as explanatory variable associated with the disease. RESULTS: The 18s PCR test showed that 18.94% (14.77% Babesia bovis and 4.17% Babesia bigemina) and 20.28% (14.69% B. bovis and 5.59% B. bigemina) of the cattle were positive for Babesia spp in farms sampled in El Carmen and in Quito, respectively. Age influenced the presence of animals positive for Babesia spp., but sex and PCV did not. The phylogenetic analysis of sequences showed 4 isolates of B. bovis and 3 isolates of B. bigemina in the 2 study zones, with similarities between 99.73 and 100% with other sequences. One B. bovis isolate was similar in the zone of El Carmen and Quito. CONCLUSION AND SIGNIFICANCE: This work is the first molecular characterization of B. bigemina and B. bovis in Ecuador, and it is also the first evidence of Babesia spp. in cattle in the area of Quito at an altitude of 2469 m.a.s.l., being the highest altitude reported for animals with babesiosis and for the tick R. microplus. Climatic factors as well as mobility of tick-carrying animals without any control allow the presence of Babesiosis outbreaks in new geographical areas.

Gastrointestinal parasites in captive and free-ranging Cebus albifrons in the Western Amazon, Ecuador.

Currently, there is a lack of surveys that report the occurrence of gastrointestinal parasites in the white-headed capuchin monkey (Cebus albifrons). We therefore assessed the presence and richness (= number of different parasite genera) of parasites in C. albifrons in wildlife refuges (n = 11) and in a free-ranging group near a human village (n = 15) in the Ecuadorian Amazon. In the 78 samples collected (median of 3 samples per animal), we identified a total of 6 genera of gastrointestinal parasites, representing protozoa, nematodes, acanthocephalans and cestodes. We observed a high prevalence (84%) across the 26 individuals, with the most prevalent parasite being Strongyloides sp. (76.9%), followed by Hymenolepis sp. (38.5%) and Prosthenorchis elegans (11.5%). We found Entamoeba histolytica/dispar/moskovskii/nuttalli and Capillaria sp. in only a minority of the animals (3.8%). In addition, we observed unidentified strongyles in approximately one-third of the animals (34.6%). We found a total of 6 parasite genera for the adult age group, which showed higher parasite richness than the subadult age group (5) and the juvenile age group (3). Faecal egg/cyst counts were not significantly different between captive and free-ranging individuals or between sexes or age groups. The free-ranging group had a higher prevalence than the captive group; however, this difference was not significant. The only genus common to captive and free-ranging individuals was Strongyloides sp. The high prevalence of gastrointestinal parasites and the presence of Strongyloides in both populations support results from previous studies in Cebus species. This high prevalence could be related to the high degree of humidity in the region. For the free-ranging group, additional studies are required to gain insights into the differences in parasite prevalence and intensity between age and sex groups. Additionally, our study demonstrated that a serial sampling of each individual increases the test sensitivity.