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Background Dirofilaria sp. “hongkongensis” is a putative Dirofilaria species, initially identified in subcutaneous nodules in humans in Hong Kong and in other South and Southeast Asian regions. While it differs genetically from the better-known zoonotic species, Dirofilaria repens and Dirofilaria immitis , information on the lesions caused by Dirofilaria sp. “hongkongensis” in the hosts as well as on its biology is scarce. This study documents for the first time the presence of this filarioid nematode in subcutaneous nodules in dogs and cats in Hong Kong, where it was originally described in human patients, therefore providing evidence for the zoonotic nature of this parasite. Methods Records of Veterinary Diagnostic Laboratory of City University of Hong Kong were searched between 2019 and 2024 for histological reports of possible filarioid-associated lesions. Tissue samples were collected by excisional surgical biopsy and processed with routine paraffin techniques. Selected slides were stained using various staining techniques [i.e., hematoxylin and eosin, periodic acid–Schiff (PAS), Grocott methenamine silver (GMS) or Ziehl–Neelsen (ZN) and Gram stain]. DNA from formalin-fixed paraffin-embedded tissue were extracted, submitted to conventional polymerase chain reaction (cPCR) and sequencing (i.e., cox 1 and 12S rRNA genes) and phylogenetic analyzed. Results A total of five subcutaneous nodules from four cats and one from a dog with histopathology suggestive of filariosis were selected. The presence of Dirofilaria sp. “hongkongensis” was morphologically and molecularly confirmed in one dog and one cat. Both histopathological presentation and phylogenetic analysis enabled classification of this species close to D. repens and within the subgenus Nochtiella. In the remaining three cases, one showed histological evidence of aberrant nematode migration, while non-parasitic causes were identified in the other two. Conclusions This study provides the first evidence of Dirofilaria sp. “hongkongensis” in subcutaneous nodules in cats and dogs. The histology of clinical lesions of this filarioid species herein described is closely related to those caused by D. repens . Overall, this species should be considered in differential diagnoses of subcutaneous lesions in both animals and humans in the region. Graphical Abstract

While in much of the Western world snakes are feared, in the small, rural, mountainous town of Cocullo, in the middle of central Italy, snakes are annually collected and celebrated in a sacro-profane ritual. Every 1 st of May, Serpari (snake catchers) capture and showcase dozens of non-venomous snakes to celebrate the ritual of San Domenico . In order to detect potential zoonotic pathogens within this unique epidemiological context, parasites and microorganisms of snakes harvested for the “ festa dei serpari” ritual were investigated. Snakes ( n = 112) were examined and ectoparasites collected, as well as blood and feces sampled. Ectoparasites were identified morpho-molecularly, and coprological examination conducted through direct smear and flotation. Molecular screenings were performed to identify parasites and microorganisms in collected samples (i.e., Mesostigmata mites, Anaplasma / Ehrlichia spp., Rickettsia spp., Borrelia burgdorferi sensu lato, Coxiella burnetii , Babesia/Theileria spp., Cryptosporidium spp., Giardia spp., Leishmania spp. and helminths). Overall, 28.5% (32/112) of snakes were molecularly positive for at least one parasite and/or microorganism. Endosymbiont Wolbachia bacteria were identified from Macronyssidae mites and zoonotic vector-borne pathogens (e.g., Rickettsia , Leishmania ), as well as orally transmitted pathogens (i.e., Cryptosporidium , Giardia , Proteus vulgaris , Pseudomonas ), were detected from blood and feces. Thus, given the central role of the snakes in the tradition of Cocullo, surveys of their parasitic fauna and associated zoonotic pathogens may aid to generate conservation policies to benefit the human-snake interactions, whilst preserving the cultural patrimony of this event.

Background Ophionyssus natricis is the main species of mite that infests captive reptiles. High infestations may result in the host experiencing general discomfort and deleterious effects, even death. Moreover, O. natricis is an important vector of reptile vector-borne diseases and is considered to be the putative vector of the Reptarenavirus, the causal agent of the inclusion body disease. Despite the cosmopolitan distribution of O. natricis in captive reptiles, treatment options are limited. The aim of the present study was to assess the efficacy of afoxolaner (NexGard®; Boehringer Ingelheim, Ingelheim, Germany) in heavily infested, privately owned snakes, evaluate the prevalence of mites and drug availability in the plasma of treated snakes (pharmacokinetics) and perform a clinical examination of animals. Methods The study was conducted in two snake breeding facilities, where many snakes were infested with mites. Each animal was clinically examined and weighed, and mite infestations were assessed on the animals and in their enclosures (environment). Animals were treated with a dose of 2.5 mg afoxolaner per kilogram body weight (2.5 mg/kg) administered orally. All animals were examined pre-treatment (T0) and at various time points post-treatment (T1, 6 h; T2, 24 h; T3, 14 days; T4, 28 days). The collected mites were morphologically identified at the species level and the species identity also confirmed molecularly. Results Overall, 81 snakes from the two participating facilities (i.e. 70 from site 1 and 11 from site 2) were screened, and 31 (38.3%) snakes were found to have at least one mite. All mites were identified morphologically and molecularly as O. natricis. Lampropeltis was the genus of snakes with highest number of infested individuals. Mites were found to be alive on snakes at T1, but at T2 only dead mites were observed, and at T3 and T4 mites were no longer present on the animals or in their environment. No side effects were observed in the treated snakes. Conclusions A single oral administration of afoxolaner at 2.5 mg/kg was a safe treatment for snakes and 100% effective for the eradication of natural O. natricis infestation without the need to treat the environment of the snake. Graphical Abstract

Background Feline vector-borne pathogens (FeVBPs) are common in tropical and subtropical countries, mainly due to favorable climate conditions for arthropod perpetuation coupled with limited preventive measures. However, data regarding the actual burden of these infections among cats are still scarce compared with dogs. The present study aimed to provide an overview of the prevalence of FeVBPs infections in Iran. Methods From December 2018 to February 2023, a total of 848 cats of both sexes, different ages, and with outdoor lifestyle living in 7 provinces of Iran were blood sampled and molecularly screened for Hepatozoon spp., Babesia spp., Cytauxzoon spp., Dirofilaria spp., and Leishmania spp. Results Overall, 5.4% of cats scored positive for at least one VBP, with Hepatozoon spp. being the most common (3.8%), followed by Leishmania spp. (2.5%) and Dirofilaria immitis (0.7%). The Hepatozoon -positive cats lived in localities from the eastern, western, and central-northern regions; most of them ( n  = 25) were infected by Hepatozoon felis, and the remaining ( n  = 3) by Hepatozoon canis . Leishmania spp.-infected cats were detected from the east, center, and west of the country, while D. immitis -positive animals lived in central-north areas. Conclusions To our knowledge, this is the first large-scale molecular epidemiology study of vector-borne pathogens in cats in Iran. The circulation of several VBPs, including those with zoonotic potential (i.e., D .  immitis and Leishmania spp.) highlights the importance of endo- and ectoparasite control measures in owned cats and suggests that controlling the population of feral animals (e.g., through spaying and neutering campaigns) would contribute to reducing the risk of transmission of VBPs. Graphical abstract

Among vector-borne helminths, filarioids of the genus Dipetalonema (Spirurida: Onchocercidae) localize in several tissues and body cavities of several animal species, causing mild to moderate lesions. The pathological findings associated with Dipetalonema spp. infection in Neotropical monkeys from southern Brazil are herein described, along with a fatal case due to filarial polyserositis and entrapment of an intestinal segment. At necropsy, nematodes were observed in abdominal and thoracic cavities, or in the pericardium of 37 (31.3%) out of the 118 individuals examined (i.e., 35 Alouatta guariba clamitans and two Sapajus nigritus). In addition, at histology, 27.0% of positive animals presented microfilarie (inside blood vessels of lung, spleen, liver, and brain) and 8.1% presented adult nematodes in the heart, lung, and liver. In two cases, cross-sections of filarioids were associated with areas of epicardial thickening with intense fibrosis and pyogranulomatous inflammation in the brain, heart, liver, lungs, or spleen. The DNA fragment was amplify using the cox1 gene, sequenced and analyzed to identify the nematode species collected; presence of Wolbachia was assessed in the filarioids using the 16S rRNA gene. At BLAST analysis of the cox1 gene, 10 sequences showed 91.7% nucleotide identity with Dipetalonema gracile, and two with D. gracile (98.5%) and Dipetalonema graciliformis (98.3%). Phylogenetic analyses clustered sequences of the cox1 obtained in this study in two clades corresponding with the host species. Wolbachia sp. endosymbiont was detected in four samples. Data herein reported provide a description of pathological lesions associated with the infection by Dipetalonema spp., suggesting that they may cause disease in Neotropical monkeys. In addition, a better understanding of diversity and biology of Dipetalonema spp. in South America is needed to assess the impact they may cause in native non-human primates from Brazil.

The Anaplasmataceae family encompasses obligate intracellular α-proteobacteria of human and veterinary medicine importance. This study performed multi-locus sequencing to characterize Ehrlichia and Anaplasma in coati’s blood samples in Midwestern Brazil. Twenty-five samples (25/165—15.1%) were positive in the screening PCR based on the dsb gene of Ehrlichia spp. and were characterized using 16S rRNA, sodB, groEL, and gltA genes and the 23S-5S intergenic space region (ITS). Phylogenetic analyses based on all six molecular markers positioned the sequences into a new clade, with a common origin of Ehrlichia ruminantium. Haplotype analyses of 16S RNA sequences revealed the presence of two distinct Ehrlichia genotypes. Six samples (6/165, 3.6%) were positive in the screening nPCR for the 16S rRNA gene of Anaplasma spp. and were submitted to an additional PCR targeting the ITS for molecular characterization. Phylogenetic analyses based on both 16S rRNA gene and ITS positioned the Anaplasma sp. detected in the present study in a large clade with other Anaplasma sp. previously detected in ticks and wild animals and in a clade with ‘Candidatus Anaplasma brasiliensis’, respectively. Based on distinct molecular markers, the present work described a putative novel Anaplasmataceae agent, namely ‘Candidatus Ehrlichia dumleri’, and Anaplasma sp. closely related to the previously described ‘Candidatus Anaplasma brasiliensis’.

The low-land tapir (Tapirus terrestris) is the largest wild terrestrial mammal found in Brazil. Although T. terrestris has been already reported as a host of hemoparasites, the occurrence and genetic identity of Piroplasmida agents in this species is still cloudy. Although it is reported that Theileria equi, an endemic equid-infective agent in Brazil, is occurring in lowland tapirs, these reports are probably misconceived diagnoses since they are solely based on small fragments of 18S rRNA that may not achieve accurate topologies on phylogenetic analyses. The present study aimed to detect and investigate the identity of Theileria spp. in tapirs from Pantanal and Cerrado biomes. Blood-DNA samples from tapirs were screened for a partial (~800 bp) 18S rRNA gene fragment from Piroplasmida and 64 (64/122; 52.46% CI: 43.66–61.11%) presented bands of expected size. Samples were submitted to different protocols for molecular characterization, including near-full length 18S rRNA gene (~1500 bp), and the ema-1 gene from T. equi. Eight sequences were obtained for extended fragments (1182–1473 bp) from the 18S rRNA gene. Moreover, three sequences from partial cox-1 and five from partial hsp70 gene were obtained. None of the samples presented amplifications for the ema-1 gene. Phylogenetic and distance analyses from the 18S rRNA sequences obtained demonstrated a clear separation from tapirs’ Theileria spp. and T. equi. Phylogenetic analyses of cox-1 and hsp70 sequences obtained herein also showed a unique clade formed by tapir’s Theileria spp. Theileria terrestris sp. nov. is positioned apart from all other Theileria species in 18S rRNA, cox-1, and hps70 phylogenetic analyses. This novel proposed species represents a new Piroplasmida clade, yet to be characterized regarding biological features, vectors involved in the transmission cycles, additional vertebrate hosts, and pathogenicity.

ParSCo (Parasitology Summer Course) is an intense, 1-week-long summer course organized by the Parasitology Unit of the Department of Veterinary Medicine, University of Bari, Italy, with the support of the World Association for the Advancement of Veterinary Parasitology (WAAVP), the European Veterinary Parasitology College (EVPC) and Parasites and Vectors . The course, which is conducted in southern Italy, is planned for parasitologists and post-graduate students working in the field of parasitology. The course consists of theoretical and practical lessons, which include the collection, identification and diagnosis of parasites of pets, livestock and wildlife. The participants in ParSCo are afforded the opportunity to be involved in clinical examination and sample collection for the diagnosis of parasitic diseases (e.g. leishmaniosis, thelaziosis and many tick-borne diseases) present in the Mediterranean Basin. The course is conducted at Casa di Caccia , a hunting lodge situated in the Gallipoli Cognato Forest near the Basento River in the Basilicata region in southern Italy. In addition to the training purpose, ParSCo is a great opportunity for sharing knowledge and expertise while becoming part of the parasitology community in a pleasant environment. In this editorial, we share some information and celebrate 10 years of ParSCo, looking forward to forthcoming sessions of this unique parasitology summer course. Graphical Abstract

Understanding the diversity and ecology of ectoparasites in wild animals is essential for surveillance of vector-borne diseases. Coatis (Nasua nasua) easily adapt to anthropized areas, favoring close contact with domestic animals and humans, with the possibility of exchange of ectoparasites and pathogens. The present study aimed to identify the diversity of ticks parasitizing coatis from forest urban areas of midwestern Brazil, to evaluate the seasonal dynamics of ticks during the seasons of the year, and to assess the correlation between tick species and gender and age of the sampled coatis. For this purpose, 103 coatis were captured in two Conservation areas, both located in Campo Grande city, Mato Grosso do Sul state, Midwestern Brazil. The animals’ entire body was inspected for the presence of ectoparasites, and ticks were removed for taxonomic identification. In total, 168 captures were performed in both areas during the observational study considering the first capture and recaptures. In total, 2242 ticks were collected: 838 Amblyomma larvae, 1241 A. sculptum nymphs, and 150 A. dubitatum nymphs. Thirteen adult ticks were identified as three males and five females of A. sculptum and two males and three females of A. ovale. While a quantity of Amblyomma larvae was observed in the first months of the year (January, April and May), Amblyomma nymphs showed a higher quantity during the months of July, August, October and November. No statistical difference was observed when comparing mean intensity and prevalence of Amblyomma larvae, nymphs of A. sculptum and A. dubitatum between the two sampled areas, males vs. females and immature vs. mature animals. In conclusion, three tick species were identified parasitizing coatis from forested urban fragments in midwestern Brazil, namely A. dubitatum nymphs, A. sculptum adults and nymphs, and A. ovale adults. Coatis from anthropized areas seem to present tick species diversity lower than those from natural areas. The lack of statistical difference regarding mean intensity and prevalence of Amblyomma larvae and nymphs between males vs. females and immature vs. mature animals might have reflected the gregarious behavior of coatis, since adult males live together with females and offspring outside and inside the mating season, forming large groups of individuals.

Background Dirofilaria immitis and D. repens are mosquito-borne filaroids that primarily infect dogs but also cats. Diagnosing feline dirofilariosis is challenging because of the low parasitic burdens and transient or absent microfilaremia. To improve detection of antibodies against Dirofilaria spp. in cats, an indirect enzyme-linked immunosorbent assay (ELISA) using somatic antigens of D. immitis was standardized. Methods Serum samples from cats positive for D. immitis , D. repens , bronchopulmonary metastrongylids and gastrointestinal helminths as well as negative sera were tested to evaluate the sensitivity (Se) and specificity (Sp). Three different antigen concentrations (2, 3 and 4 μg/ml) and two concentrations of horseradish peroxidase (HRP) IgG anti-cat conjugate (1:10,000 and 1:20,000) were used to screen the optimal titration of the test. Once the best conditions were established, Se and Sp were assessed by testing 151 serum samples positive for D. immitis , D. repens and other parasites (i.e. feline lungworms, gastrointestinal helminths) or negative. Results The optimized ELISA showed 89% Se and 98% Sp in detecting D. immitis infections, presenting a cross-reactivity with D. repens . A cut-off point of 1.150 was established to classify positive and negative samples. Conclusions The developed ELISA may improve the detection of antibodies against Dirofilaria spp. in cats, also in epidemiological contexts characterized by multiple helminth infections. Future efforts will focus on reducing the cross-reactivity with D. repens , therefore increasing ELISA Sp. Graphical Abstract