Ecological and molecular associations between neotropical wild felids and Taenia (Cestoda: Taeniidae) in the Atlantic Forest: a new report for Taenia omissa.

Ecological associations between wild felids and parasites from the Taeniidae family are related to predator-prey interactions, where felids act as definitive hosts while their prey, herbivores and/or omnivores, act as intermediate hosts. In the Atlantic Forest, six neotropical felid species coexist in sympatry, but the ecological parasite-host interactions remain poorly studied. Taenia omissa is a tapeworm that parasitizes cougars (Puma concolor) as its only definitive host and their ungulate prey as intermediate hosts. The aim of this study was to identify tapeworms present in road-killed fauna using both molecular and morphological characteristics and their predator-prey relationship. Adult tapeworms found in a cougar, a jaguarundi (Herpailurus yagouaroundi), and two ocelots (Leopardus pardalis); and metacestodes found in a red brocket deer (Mazama americana) and a wild guinea pig (Cavia aperea) were analyzed. Through morphological analysis of rostellar hooks and molecular analysis of the mitochondrial genetic marker cox1, Taenia omissa adult individuals were identified in the cougar, and metacestodes in the red brocket deer, proving the existence of a full host-parasite life cycle in the Atlantic Forest region. This new report reveals the southernmost record of T. omissa and broadens its geographic distribution. In addition, isolates of the Taenia genus divergent from those described so far in molecular databases were reported and suggested a wild cycle that involves the jaguarundi and agouti (Dasyprocta asarae) as definitive and intermediate hosts, respectively. These results highlight the complexity of the tapeworm population in the region and the need to study it with both morphological and molecular approaches.

A putative new Besnoitia species in the southern black-eared opossum Didelphis aurita.

Besnoitia spp. are cyst-forming coccidian parasites with a broad host range, infecting various wild and domestic animal species. Northamerican opossums (Didelphis virginiana) are severely affected by the infection with B. darlingi. This study presents a case of infection with Besnoitia in a road-killed female southern black-eared opossum (Didelphis aurita) in Misiones, Argentina. Many 0.5-1 mm cysts were observed in several muscles and visceral organs and were microscopically identified in skeletal muscles, tongue, and heart. Histological analysis disclosed multiple spherical cysts with a myriad of bradyzoites like-cells and a well-defined cyst wall. A small number of degenerate and ruptured cysts, surrounded by mild to moderate inflammation were observed. Genomic DNA from an individual cyst and muscle was extracted and ITS1 marker and 18S rRNA gene fragments from sarcocystid protozoa were successfully amplified by PCR and sequenced. The 18S sequence exhibited 100% identity with sequences of B. darlingi and B. oryctofelisi. Comparison of the complete ITS1 sequence (259 bp) revealed an identity of 99.2% with B. oryctofelisi and 97.7% with B. darlingi. This result together with the phylogeny positioning, suggest that the Besnoitia sp. in the present case differ from B. darlingi, being closely related with B. oryctofelisi.

Detection of Mycobacterium bovis in Free-Ranging Sapajus nigritus, Argentina.

Mycobacterium bovis and Mycobacterium tuberculosis are the most relevant among pathogenic mycobacteria, both belonging to the M. tuberculosis complex (MTC). Samples of blood, liver, spleen, kidneys, lungs and caseous tubercles were collected from a free-ranging juvenile black capuchin monkey (Sapajus nigritus) showing non-specific signs of illness. Macroscopic findings included emaciation, a caseous lesion in a tooth and gingiva, disseminated nodules in both lungs and left kidney parenchyma and caseous nodules on the pleura and mesentery. The lesions suggested MTC infection, a diagnosis subsequently supported in the lung by bacilloscopy, immunochromatography and PCR. A multiplex PCR further validated the presence of M. bovis genes. Cases of tuberculosis in platyrrhine primates have only been reported in animals maintained in captivity. We describe for the first time the pathological and molecular findings of M. bovis infection in a free-ranging platyrrhine monkey within an area of intense human-wildlife interaction, which has important implications from a One Health perspective.

First identification and molecular phylogeny of Sparganum proliferum from endangered felid (Panthera onca) and other wild definitive hosts in one of the regions with highest worldwide biodiversity.

After decades of being neglected, broad tapeworms now attract growing attention thanks to the increasing number of reports from humans but also thanks to many advancements achieved by application of molecular methods in diagnosis and epidemiological studies. Regarding sparganosis, unfortunately general uniformity of most species, their high intraspecific variability and lack of agreement among researchers has led to confusion about the classification of Spirometra/Sparganum species. For the first time we determined adult, eggs and plerocercoid life cycle stages and the molecular phylogeny of Sparganum proliferum obtained from endangered wild felids (Panthera onca, Leopardus pardalis, Leopardus guttulus and Herpailurus yagoauroundi) in one of the largest continuous remnants of worldwide biodiversity, the Atlantic Forest from South America. Our results showed that at least 57% of total species of wild felids in this natural area could act as definitive hosts of Sparganum proliferum. We conclude that the availability of more morphological characteristics are needed in order to secure reliable characterization and diagnosis of sparganosis. The integration of these data with molecular analysis of mitochondrial DNA sequences will be useful for species discrimination.

Revisiting the Phylogenetic History of Helminths Through Genomics, the Case of the New Echinococcus oligarthrus Genome.

The first parasitic helminth genome sequence was published in 2007; since then, only ∼200 genomes have become available, most of them being draft assemblies. Nevertheless, despite the medical and economical global impact of helminthic infections, parasite genomes in public databases are underrepresented. Recently, through an integrative approach involving morphological, genetic, and ecological aspects, we have demonstrated that the complete life cycle of Echinococcus oligarthrus (Cestoda: Taeniidae) is present in South America. The neotropical E. oligarthrus parasite is capable of developing in any felid species and producing human infections. Neotropical echinococcosis is poorly understood yet and requires a complex medical examination to provide the appropriate intervention. Only a few cases of echinococcosis have been unequivocally identified and reported as a consequence of E. oligarthrus infections. Regarding phylogenetics, the analyses of mitogenomes and nuclear datasets have resulted in discordant topologies, and there is no unequivocal taxonomic classification of Echinococcus species so far. In this work, we sequenced and assembled the genome of E. oligarthrus that was isolated from agoutis (Dasyprocta azarae) naturally infected and performed the first comparative genomic study of a neotropical Echinococcus species. The E. oligarthrus genome assembly consisted of 86.22 Mb which showed ∼90% identity and 76.3% coverage with Echinococcus multilocularis and contained the 85.0% of the total expected genes. Genetic variants analysis of whole genome revealed a higher rate of intraspecific genetic variability (23,301 SNPs; 0.22 SNPs/kb) rather than for the genomes of E. multilocularis and Echinococcus canadensis G7 but lower with respect to Echinococcus granulosus G1. Comparative genomics against E. multilocularis, E. granulosus G1, and E. canadensis G7 revealed 38,762, 125,147, and 170,049 homozygous polymorphic sites, respectively, indicating a higher genetic distance between E. oligarthrus and E. granulosus sensu lato species. The SNP distribution in chromosomes revealed a higher SNP density in the longest chromosomes. Phylogenetic analysis using whole-genome SNPs demonstrated that E. oligarthrus is one of the basal species of the genus Echinococcus and is phylogenetically closer to E. multilocularis. This work sheds light on the Echinococcus phylogeny and settles the basis to study sylvatic Echinococcus species and their developmental evolutionary features.

Twice upon a time: The progression of canine visceral leishmaniasis in an Argentinean city.

Canine Visceral Leishmaniasis (CVL) prevalence, spatial distribution and associated factors were assessed in four locations in Iguazú department in 2014 and in Puerto Iguazú city again in 2018. The city areas were divided into a grid of 400x400m cells. All cells were sampled in 2014 and a random subsampling was developed in 2018. In each cell, five dogs clustered in a 'critical scenario' (prone to have vectors) were sampled. A rapid immunochromatographic dipstick was used to detect antibodies against Leishmania infantum, confirming by lymph node smears observation and PCR. For Puerto Iguazú, Generalized Linear Models (GLMs) were constructed considering environmental, dog and clinical variables. Pearson's Chi square and Fisher's exact tests were employed to evaluate the association between CVL, dog clinical signs and infestation with other parasites. Cartographic outputs were made and Moran's I indices were calculated as spatial autocorrelation indicators. CVL prevalence rates were 26.18% in 2014 and 17.50% in 2018. No associations were established in environmental models, but dog age and repellent use were significant when running 2014 dog models. Clinical models showed significant associations between seropositive dogs and ophthalmological, dermal signs and onychogryphosis in 2014. In 2018, only adenomegaly was associated. The results of global Moran´s I were not significant but regarding local analysis, six sites in 2014 and one in 2018 presented autocorrelation with neighboring sites. The decrease in CVL prevalence may be associated to transmission stabilization, which could explain the lack of associations with dog-related variables. Further, spatial distribution of CVL is a poor evidence for design of transmission control measures but could be important in case of intensive parasite circulation or when the first autochthonous cases appear. For control success, sensitivity of diagnostic methods, political will and adequate material resources remain critical. Modeling of multiple variables will be required to identify factors that drive disease stabilization/destabilization.

Factors associated with hard tick (Acari: Ixodidae) parasitism in medium-sized mammals in the Atlantic Rainforest region of Argentina.

Associations with environmental and host parameters were assessed to describe tick parasitism patterns in two medium-sized mammals of the Atlantic rainforest region of Argentina. Ticks found on 93 specimens of Nasua nasua and 26 specimens of Didelphis aurita captured at six sites in the Iguazú National Park were collected. Generalized linear models were constructed to explain the presence and abundance of ticks and the most appropriate ones were selected after stepwise simplification. The season, site and host body mass variables were important to explain the abundance of Amblyomma coelebs nymphs, while site was important to describe larval abundance of this species. Season was the most important variable for larvae and nymphs of Haemaphysalis juxtakochi, while Amblyomma brasiliense nymphs were associated with host age. Amblyomma ovale larvae were related to host age, and adult ticks with host sex. The results indicate that these medium-sized mammals are important to support the immature tick stages and that both host and environmental factors may be associated with parasite loads.

Echinococcus oligarthrus in the subtropical region of Argentina: First integration of morphological and molecular analyses determines two distinct populations.

Echinococcosis is a parasitic zoonosis that is considered as a neglected disease by the World Health Organization. The species Echinococcus oligarthrus is one of the causative agents of Neotropical echinococcosis, which is a poorly understood disease that requires a complex medical examination, may threaten human life, and is frequently associated with a low socioeconomic status. Morphological and genetic diversity in E. oligarthrus remains unknown. The aim of this work is to identify and characterize E. oligarthrus infections in sylvatic animals from the Upper Paraná Atlantic Forest in the province of Misiones, Argentina, by following an integrative approach that links morphological, genetic and ecological aspects. This study demonstrates, for the first time, one of the complete life cycles of E. oligarthrus in an important ecoregion. The Upper Paraná Atlantic Forest constitutes the largest remnant continuous forest of the Atlantic Forest, representing 7% of the world's biodiversity. This is the first molecular determination of E. oligarthrus in Argentina. In addition, the agouti (Dasyprocta azarae), the ocelot (Leopardus pardalis) and the puma (Puma concolor) were identified as sylvatic hosts of Neotropical echinococcosis caused by E. oligarthrus. Mitochondrial and nuclear molecular marker analyses showed a high genetic diversity in E. oligarthrus. Moreover, the genetic distance found among E. oligarthrus isolates is higher than the one observed among Echinococcus granulosus genotypes, which clearly indicates that there are at least two different E. oligarthrus populations in Argentina. This study provides valuable information to understand the underlying conditions that favour the maintenance of E. oligarthrus in sylvatic cycles and to evaluate its zoonotic significance for devising preventive measures for human and animal wellbeing.