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In recent decades, the number of autochthonous cases and foci of Dirofilaria immitis in dogs from southern regions has increased considerably, suggesting that the distribution of the species is not limited to northern Italian regions. This epidemiological picture emerges from case reports or studies in specific locations where outbreaks of heartworm disease have occasionally been reported together with the presence of mosquito vectors. To obtain a more comprehensive picture of the current distribution of D. immitis in southern Italy, a multicenter cross-sectional survey of canine filariasis was conducted. Owned and sheltered dogs (n = 1,987) were included in the survey regardless their breed, attitude and/or sex. All included dogs were older than 1 year and had no history of chemoprophylactic treatment against filarioses. A blood sample was collected from enrolled dogs and screened by modified Knott’s test and, when positive, tested using D. immitis specific ELISA rapid test (SNAP 4DX, IDEXX). The overall microfilaremia prevalence was 17% (n = 338) being single-species infection (92.6%) more common that mixed (7.4%). Remarkably, D. immitis was the most frequent species detected with an overall prevalence of 11.4% (n = 227), followed by Dirofilaria repens (n = 74; 3.7%), and Acanthocheilonema reconditum (n = 12; 0.6%). Sheltered dogs were significantly more infected by D. immitis, as well as mongrel dogs and animals housed in rural areas. Data here reported indicate that D. immitis is largely present in southern Italy, raising awareness about the necessity of proper screening and chemoprophylactic treatments in exposed animals.

Hedgehogs are synanthropic mammals, reservoirs of several vector-borne pathogens and hosts of ectoparasites. Arthropod-borne pathogens (i.e., Rickettsia spp., Borrelia spp., and Anaplasmataceae) were molecularly investigated in ectoparasites collected on hedgehogs (n = 213) from Iran (161 Hemiechinus auritus, 5 Erinaceus concolor) and Italy (47 Erinaceus europaeus). In Iran, most animals examined (n = 153; 92.2%) were infested by ticks (Rhipicephalus turanicus, Hyalomma dromedarii), and 7 (4.2%) by fleas (Archeopsylla erinacei, Ctenocephalides felis). Of the hedgehogs infested by arthropods in Italy (i.e., 44.7%), 18 (38.3%) were infested by fleas (Ar. erinacei), 7 (14.9%) by ticks (Haemaphysalis erinacei, Rh. turanicus, Rhipicephalus sanguineus sensu lato), and 6 (12.8%) by mites (Caparinia tripilis, Acarus nidicolous, Ornithonyssus spp.). Phoretic behavior of C. tripilis on Ar. erinacei was detected in two flea specimens from Italy. At the molecular analysis Rickettsia spp. was detected in 93.3% of the fleas of Italy. In Iran, Rickettsia spp. was detected in 8.0% out of 212 Rh. turanicus ticks, and in 85.7% of the Ar. erinacei fleas examined. The 16S rRNA gene for Ehrlichia/Anaplasma spp. was amplified in 4.2% of the 212 Rh. turanicus ticks. All sequences of Rickettsia spp. from fleas presented 100% nucleotide identity with Rickettsia asembonensis, whereas Rickettsia spp. from Rh. turanicus presented 99.84%–100% nucleotide identity with Rickettsia slovaca, except for one sequence, identical to Rickettsia massiliae. The sequences of the 16S rRNA gene revealed 99.57%–100% nucleotide identity with Anaplasma spp., except for one, identical to Ehrlichia spp. A new phoretic association between C. tripilis mites and Ar. erinacei fleas has been herein reported, which could be an important route for the spreading of this mite through hedgehog populations. Additionally, spotted fever group rickettsiae were herein detected in ticks and fleas, and Anaplasma/Ehrlichia spp. in ticks, suggesting that hedgehogs play a role as reservoirs for these vector-borne pathogens.

The proper diagnosis of gastrointestinal parasites of small ruminants requires the development of multivalent techniques characterized by high sensitivity, specificity, precision, reproducibility, and the ability to quickly detect and monitor infections that pose risks to animal health. The aim of this study was to evaluate the performance of Mini-FLOTAC technique and McMaster egg counting (gold standard) in the detection of gastrointestinal parasites of goats and sheep. A total of 789 fecal samples were analyzed (401 of sheep and 388 of goats). From those, nematode eggs were detected in 80.86% (638/789), being 72.57% (291/401) from sheep and 89.43% (347/388) from goats. The Mini-FLOTAC technique presented a better performance to detect helminth eggs in both goats (88.40%; 343/388) and sheep (71.57%; 287/401) (?2 = 10.358; p < 0.0001). Kappa analysis revealed a weak concordance between techniques for goats (k = 0.342; p < 0.001) and sheep (k = 0.281; p < 0.001). Data herein reported suggests that the Mini-FLOTAC is a technique more sensitive than the McMaster egg counting, therefore its use might be adopted for the diagnosis of gastrointestinal infection in small ruminants.

Leishmania major and Leishmania tropica cause cutaneous leishmaniasis in humans and dogs in several parts of the world, with a large number of cases recorded in the Middle East. However, when they occur in sympatry, the role of each species of Leishmania in the epidemiology of cutaneous leishmaniasis (CL) is not clear. To assess the frequency and to identify the species of Leishmania that infect humans and stray dogs in Riyadh and Al-Qaseem (Saudi Arabia), 311 stray dogs and 27 human patients who were suspected for Leishmania infection were examined for CL by a nested polymerase chain reaction (nPCR). Seven (25.9%) out of 27 human patients scored positive for Leishmania spp. (i.e., L. major in five patients from Riyadh and L. tropica in two patients from Al-Qaseem). Out of 311 dogs, five (1.6%) were infected by L. tropica. Data herein presented demonstrate the occurrence of L. tropica in dogs and humans in Saudi Arabia, as well as the occurrence of L. major in humans.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne filarioids of zoonotic concern, which primarily cause canine heartworm disease (HWD) and subcutaneous dirofilariosis, respectively. The geographical distribution of these nematodes is constantly changing, due to many factors such as the colonization by new invasive mosquito species, the increased movement of animals and the absence of chemoprophylactic strategies in non-endemic regions. Although HWD has always been considered endemic in northern Italy, an increase in the prevalence of this disease has been recorded in the last decades in central and southern regions. We describe the southernmost hyperendemic European focus of heartworm disease in the Pelagie archipelagos. From June to November 2020, 157 dogs and 46 cats were sampled in Linosa and Lampedusa islands for the detection of Dirofilaria immitis and Dirofilaria repens by modified Knott's, rapid antigen and molecular tests. Mosquitoes were collected using CDC light and BG sentinel-2 traps and aspirators, and tested for host blood meal and Dirofilaria spp. DNA. Out of 56 dogs in Linosa and 101 in Lampedusa, 33 (i.e. 58.9%) and 8 (i.e. 7.9%) were positive to D. immitis and D. repens, respectively. Three cats scored positive to D. immitis (i.e. 17.6%) in Linosa. Six mosquito species were identified, and the abdomen of a non-engorged Aedes albopictus was positive for D. immitis and human DNA. The results suggest that D. immitis infection could spread to new previously non-endemic territories in southern Europe, representing a real threat to animal and human health.

Medium sized opossums (Didelphisspp.) are among the most fascinating mammals of the Americas, playing important ecological roles (e.g., dispersal of seeds and control of insect populations) in the environment they inhabit. Nevertheless, as synanthropic animals, they are well adapted to human dwellings, occupying shelters within the cities, peripheral areas, and rural settings. These marsupials can harbor numerous pathogens, which may affect people, pets, and livestock. Among those, some protozoa (e.g.,Leishmania infantum,Trypanosoma cruzi,Toxoplasma gondii), helminths (e.g.,Ancylostoma caninum,Trichinella spiralis,Alaria marcianae,Paragonimusspp.) and arthropods (e.g., ticks, fleas) present substantial public health and veterinary importance, due to their capacity to cause disease in humans, domestic animals, and wildlife. Here, we reviewed the role played by opossums on the spreading of zoonotic parasites, vectors, and vector-borne pathogens, highlighting the risks of pathogens transmission due to the direct and indirect interaction of humans and domestic animals withDidelphisspp. in the Americas.

Bartonellae are emerging vector‐borne pathogens infecting humans, domestic mammals and wildlife. Ninety‐seven red foxes (Vulpes vulpes), 8 European badgers (Meles meles), 6 Eurasian wolves (Canis lupus), 6 European hedgehogs (Erinaceus europaeus), 3 beech martens (Martes foina) and 2 roe deer (Capreolus capreolus) from Italian Nature Conservatory Parks were investigated for Bartonella infection. Several Bartonella species (9.84%; 95% CI: 4.55–15.12), including zoonotic ones, were molecularly detected among wolves (83.3%; 95% CI: 51–100.00), foxes (4.12%; 95% CI: 0.17–8.08), hedgehogs (33.33%; 95% CI: 0.00–71.05) and a roe deer. Bartonella rochalimae was the most common Bartonella species (i.e. in 4 foxes and 2 wolves) detected. Candidatus B. merieuxii and B. vinsonii subsp. berkhoffii were identified for the first time in wolves. Furthermore, Bartonella schoenbuchensis was identified in a roe deer and a new clone with phylogenetic proximity to B. clarridgeiae was detected in European hedgehogs. Zoonotic and other Bartonella species were significantly more frequent in Eurasian wolves (p

Toxoplasmosis is a worldwide-distributed zoonotic disease of great relevance to public health. Many wildlife species, including marsupials of the genus Didelphis, are considered hosts for Toxoplasma gondii, which makes them to have a possible role in the dispersion and maintenance of this parasite in nature. This study provides data on the molecular detection of T. gondii in D. aurita opossums from an urbanized area of Southeastern Brazil. Animals were captured and blood and/or spleen samples were collected. Real Time PCR was performed for the detection of T. gondii. From the opossums captured, 26.3% (n = 15/57) scored positive, with a frequency of 21.6% (n = 11/51) in blood, and 66.7% (n = 6/9) in spleen samples. BLAST analysis demonstrated 100% identity and 100% cover query with sequences of T. gondii available in GenBank database. Data herein reported present great public health importance, since Didelphis spp. are usually observed inhabiting close to human dwellings, which facilitates their contact with people and domestic animals, and consequently, the transmission of zoonotic agents. However, further studies are needed to elucidate whether these opossums play an important role in the zoonotic cycle of T. gondii in urban areas of Brazil.

Eimeria spp. infections lead to acute or chronic intestinal disorders in small ruminants, being responsible for important economic losses worldwide. The aim of this study was to assess the risk factors associated with Eimeria spp. infection in small ruminants of the microregion of Garanhuns, state of Pernambuco, Northeastern Brazil. Fecal samples (n = 822) were obtained from goats (n = 414) and sheep (n = 408) and evaluated by the modified Gordon and Whitlock technique. Risk factors were assessed through univariate analysis and logistical regression. Oocysts of Eimeria species were detected in 62.9% (517/822) of the animals, with 77.8% (322/414) in goats and 47.8% (195/408) in sheep. For goats, the herd size (OR = 5.52), rearing system (OR = 1.57), feeding place (OR = 2.60), absence of mineral salt in the diet (OR = 2.54), flooring type (OR = 2.83) and periodicity of cleaning (OR = 5.39) were considered risk factors. Conversely, for sheep only the herd size (OR = 3.16) and rearing system (OR = 2.45) were important factors associated with infection by Eimeria spp. Data herein obtained brings meaningful information on the epidemiology of coccidiosis in small ruminants in Northeastern Brazil. The knowledge of these risk factors is useful to contribute to the development of preventive measures, thereby, reducing the economic impact caused by these protozoa in small ruminant production.

Currently, a great proportion of the emerging infectious human diseases are zoonotic, with most of the pathogens originated from wildlife. In this sense, synanthropic animals such as marsupials play important role in the dissemination of pathogens due to their proximity to human dwellings. These hosts are affected by many gastrointestinal parasites, including species with zoonotic potential. The aim of this study was to assess the diversity of gastrointestinal parasites infecting the black-eared opossum D. aurita captured in urban areas of Southeastern, Brazil. In addition, the potential risk for the human population based on the One Health perspective has been discussed. Forty-nine marsupial specimens were captured with Tomahawk live traps and fecal samples were collected. The samples were evaluated by parasitological procedures. Eggs and oocysts were analyzed at different magnifications (400 × and 1000 ×), and their identification, together with adult nematodes, was established on morphological and morphometric data. Forty-three hosts (87.76%) scored positive for at least one gastrointestinal parasite, being 83.67% (41/49) for helminths, and 65.30% (32/49) for protozoa. For Cryptosporidium sp., only 13 samples were evaluated due to insufficient amount of feces obtained of some animals. A prevalence of 23.08% (3/13) was reported for this parasite. PCR analysis revealed Ancylostomatidae eggs to belong to the genus Ancylostoma. Our results demonstrated that multiparasitism is frequently found in these animals and a high percentage of potentially zoonotic parasites are observed, implying that D. aurita may be involved in zoonotic cycles in urban environments.

Ticks and fleas are essential vectors of pathogens that affect humans and animals, and among their hosts, synanthropic animals such as the black-eared opossum, Didelphis aurita, play a role in public health due to their ability to move between urban centers and forested areas in Brazil. This study aimed to assess the ectoparasite fauna of D. aurita, as well as the presence of pathogens and endosymbionts in ticks and fleas. Opossums (n = 58) captured in Tomahawk livetraps were examined for ectoparasites, and their blood sampled for further analysis. Additionally, spleen samples were collected in individuals found dead. Samples were PCR screened for Rickettsia spp., Borrelia spp., Anaplasmataceae, and Babesia spp. Two tick species were morphologically identified as Ixodes loricatus 24/58 (41.4%) and Amblyomma sculptum 1/58 (1.7%). For fleas, Ctenocephalides felis was detected in 60.3% (35/58) of the animals, and Xenopsylla cheopis in 5.2% (3/58). PCR analysis detected Anaplasmataceae DNA in 34% (16/47) of pooled samples of C. felis, and in 66.7% (2/3) pooled samples of X. cheopis. Sequence analysis revealed Wolbachia pipientis symbiont in all positive samples. Tick, blood and spleen samples were all negative for the microorganisms assessed. These findings suggest that these arthropods circulate among wildlife and urban environments, which may implicate in their participation in the cycle of zoonotic pathogens among opossums, humans and companion animals.

Wolbachia, the endosymbiont of arthropods and onchocercid nematodes is present in many medically important insect species, being also considered for the indirect control of parasitic ones. Archaeopsylla erinacei is a flea species infesting hedgehogs acting as vector of Rickettsia felis, Bartonella henselae, and Rickettsia helvetica, thus having public health relevance. The Wolbachia surface protein (wsp) and 16S rRNA genes were used to determine the presence, prevalence and molecular typing of Wolbachia in this flea species collected in two regions of southern Italy. Of the 45 fleas tested (n = 16 males, 35.6%; n = 29 females, 64.4%), 43 (95.6%; 95% CI: 84.8–99.2) scored positive for Wolbachia, of which 15 (33.3%) and 28 (62.2%) were males and females, respectively. The sex-wise prevalence of this endosymbiont was almost equal in both sexes (males 93.8%; 95% CI: 69.5–99.7; females 96.7%; 95% CI: 83.1–99.8). Single locus sequence analysis (SLST) of Wolbachia revealed two sequence types for 16S rRNA gene, named as wAr_15227 and wAr_15234, which came from two different areas, equally distributed in male and female fleas, whilst only one sequence type was identified for wsp gene. The phylogenetic analysis placed the two 16S rRNA sequence types in paraphyletic clades belonging to the supergroup A and B, respectively. Whilst, the tree of wsp gene clustered the corresponding sequence in the same clade including those of Wolbachia supergroup A. In MLST analyses, both Wolbachia sequence types clustered in a monophyletic clade with Drosophila nikananu (wNik) and Drosophila sturtevanti (wStv) from supergroup A. ClonalFrame analysis revealed a recombination event in the wAr_15234 strain which came from Apulia region. Scientific knowledge of the presence/prevalence of Wolbachia among medically important fleas, may contribute to develop an alternative biological method for the vector control.

Boars are exotic animals in Brazil, which have bred with domestic pigs resulting in a hybrid known as “javaporco”. Both cause problems related to invasion of forests and farms. These animals are ho...

Parasites of the genus Eimeria induce relevant economic losses in small ruminants worldwide. The aim of this study was to investigate the epidemiology of goats and sheep coccidiosis in a semi-arid region of North-eastern Brazil and to assess whether the rearing system represents a risk factor for the distribution of the infection. A total of 822 fresh faecal samples were collected from the rectum of goats (n = 414) and sheep (n = 408) All samples were individually processed using the technique described by Gordon and Whitlock, and after sporulation in 2.5% Potassium dichromate (K2Cr2O7), an accurate morphometric analysis was performed, with oocysts morphologically identified at species level. The analysis of the risk factors associated with Eimeria infection and system of rearing was performed through univariate analysis and logistic regression. Out of all animals sampled, 70.07% (576/822) scored positive for coccidia oocysts, with an overall prevalence of the infection of 73.91% (306/414) in goats and 66.18% (270/408) in sheep (χ2 = 5.50; p = 0.01). Eight Eimeria species were identified either in goats (i.e., E. arloingi, E. ninakohlyakimovae, E. alijevi, E. jolchijevi, E. caprina, E. chirstenseni, E. caprovina and E. hirci) and in sheep (i.e., E. ovinoidalis, E. parva, E. crandallis, E. granulosa, E. bakuensis, E. ashata, E. faurei, E. pallida). The analysis of risk factors revealed that the semi-intensive system (OR = 1.57) was a risk factor for goats, and, incontrast, intensive system for sheep (OR = 2.45) was safer, relative to Eimeria species transmission. Data reported in this study indicate that a wide species diversity and frequency of coccidia affect small ruminants in the study area. Finally, these findings are pivotal to better understand the dynamics of infection by this coccidian in these herds, as well as sound as an alert for practice veterinarians of the region, regarding the most common pathogenic species.

Background Onchocerca lupi and Cercopithifilaria spp. are vector-borne filarioids of dogs, which harbour skin microfilariae (mfs), the former being of zoonotic concern. Proper treatment studies using compounds with microfilaricidal activity have not been performed. Therefore, this study aimed to assess the efficacy of a commercially available spot-on formulation containing moxidectin 2.5%/imidacloprid 10% for the treatment of O. lupi or Cercopithifilaria spp. skin-dwelling mfs in naturally infected dogs. Methods Privately owned dogs (n = 393) from southern Portugal were sampled via skin biopsies to identify and count mfs in 20 µl of skin sediment. A total of 22 mfs-positive dogs were allocated to treatment group (n = 11; G1) or left untreated as a control (n = 11; G2). As a pilot investigation to test the treatment efficacy, five dogs assigned to G1 were treated four times at monthly intervals with moxidectin 2.5%/imidacloprid 10% spot-on formulation on SDs 0, 28 (± 2), 56 (± 2), and 84 (± 2). Based on the negative results for both O. lupi and/or Cercopithifilaria spp. mfs of dogs in the pilot study from SD28 onwards, the remaining six dogs in G1 were treated at SD0 and assessed only at SD28. Results Of the 393 animals sampled, 78 (19.8%) scored positive for skin-dwelling mfs. At the pilot investigation, a mean number of 19.6 mfs for O. lupi was recorded among five infected dogs whereas no mfs were detected at SD28. At SD0, the mean number of Cercopithifilaria spp. larvae was 12.6 for G1 and 8.7 for G2. The mean number of mfs for G2 was 20.09. Conclusions Results herein obtained suggest that a single treatment with moxidectin 2.5%/imidacloprid 10% spot-on formulation is efficacious against skin-dwelling mfs in dogs. The microfilaricidal effect of moxidectin could also be useful in reducing the risk of O. lupi infection for humans. Graphical Abstract

Background Canine vector-borne diseases (CVBDs) associated to ticks are among the most important health issues affecting dogs. In Italy, Ehrlichia canis, Anaplasma spp., Rickettsia conorii and Borrelia burgdorferi (s.l.) have been studied in both healthy canine populations and those clinically ill with suspected CVBDs. However, little information is currently available on the overall prevalence and distribution of these pathogens in the country. The aim of this study was to assess the prevalence and distribution of tick-borne pathogens (TBPs) in clinically suspect dogs from three Italian macro areas during a 15-year period (2006–2020). Methods A large dataset (n = 21,992) of serological test results for selected TBPs in three macro areas in Italy was analysed using a Chi-square test to evaluate the associations between the categorical factors (i.e. macro area, region, year, sex and age) and a standard logistic regression model (significance set at P = 0.05). Serological data were presented as annual and cumulative prevalence, and distribution maps of cumulative positive cases for TBPs were generated. Results Of the tested serum samples, 86.9% originated from northern (43.9%) and central (43%) Italy. The majority of the tests was requested for the diagnosis of E. canis (47%; n = 10,334), followed by Rickettsia spp. (35.1%; n = 7725), B. burgdorferi (s.l.) (11.6%; n = 2560) and Anaplasma spp. (6.2%; n = 1373). The highest serological exposure was recorded for B. burgdorferi (s.l.) (83.5%), followed by Rickettsia spp. (64.9%), Anaplasma spp. (39.8%) and E. canis (28.7%). The highest number of cumulative cases of Borrelia burgdorferi (s.l.) was recorded in samples from Tuscany, central Italy. Rickettsia spp. was more prevalent in the south and on the islands, particularly in dogs on Sicily older than 6 years, whereas Anaplasma spp. was more prevalent in the north and E. canis more prevalent in the south and on the islands. Conclusions The results of this study highlight the high seroprevalence and wide distribution of the four TBPs in dogs with clinically suspected CVBDs from the studied regions of Italy. The very high seroprevalence of B. burgdorferi (s.l.) exemplifies a limitation of this study, given the use of clinically suspect dogs and the possibility of cross-reactions when using serological tests. The present research provides updated and illustrative information on the seroprevalence and distribution of four key TBPs, and advocates for integrative control strategies for their prevention. Graphical Abstract

Citizen science may be described as a research involving communities and individuals, other than scientists. Following this approach, along with the evidence of a high prevalence of Rickettsia spp. in Dermacentor marginatus from wild boars in hunting areas of southern Italy, this study aimed to assess the occurrence of tick-borne pathogens (TBPs) in ticks collected from hunters and their hunting dogs. From October 2020 to May 2021, ticks were collected from wild boar hunters (n = 347) and their dogs (n = 422) in regions of southern Italy (i.e., Apulia, Basilicata, Calabria, Campania and Sicily). All ticks were morphologically identified, classified according to gender, feeding status, host, geographic origin, and molecularly screened for zoonotic bacteria. Adult ticks (n = 411) were collected from hunters (i.e., n = 29; 8.4%; mean of 1.6 ticks for person) and dogs (i.e., n = 200; 47.4%; mean of 1.8 ticks for animal) and identified at species level as D. marginatus (n = 240, 58.4%), Rhipicephalus sanguineus sensu lato (n = 135, 32.8%), Rhipicephalus turanicus (n = 27, 6.6%) and Ixodes ricinus (n = 9, 2.2%). Overall, 45 ticks (i.e., 10.9%, 95% CI: 8.3-14.3) tested positive for at least one tick-borne agent, being Rickettsia slovaca the most frequent species (n = 37, 9.0%), followed by Rickettsia raoultii, Rickettsia aeschlimannii, Rickettsia monacensis, Coxiella burnetii, Borrelia lusitaniae and Candidatus Midichloria mitochondrii (n = 2, 0.5% each). Data herein presented demonstrate a relevant risk of exposure to TBPs for hunters and hunting dogs during the hunting activities. Therefore, the role of hunters to monitor the circulation of ticks in rural areas may be considered an effective example of the citizen science approach, supporting the cooperation toward private and public health stakeholders.

Didelphis aurita Wied-Neuwied, 1826 is a marsupial well adapted to anthropogenic activity and commonly found in urban areas of Brazil. Among the gastrointestinal parasites found in this opossum, protozoa of the genus Eimeria are frequently detected. This study investigated the biodiversity of Eimeria species infecting D. aurita in Southeastern Brazil, and provides morphological data on a newly discovered species of Eimeria. From January to June 2019, 43 D. aurita were captured, and their fecal samples were collected and evaluated by salt flotation; positive samples were allowed to sporulate in 2.5% potassium dichromate (K2Cr2O7), and detailed morphological measurements were performed to determine the species present. Thirty-two of forty-three (74.4%) opossums were infected with from one to five Eimeria spp. Four of the eimerians were described and named previously by others: Eimeria auritanensis, Eimeria caluromydis, Eimeria gambai, and Eimeria philanderi. Additionally, sporulated oocysts of a species new to science were detected. Oocysts of this new Eimeria species are spheroidal to subspheroidal, 21.7 × 20.7 (20-23 × 19-23), length/width (L/W) ratio 1.05, with a highly refractile polar granule, but lacking a micropyle and oocyst residuum. Sporocysts are ovoidal, 10.6 × 8.0 (9-12 × 7-9), L/W ratio 1.3, with a small, Stieda body and a sporocyst residuum of diffuse granules. Of the infected opossums, 5/32 (16%) were infected with only one Eimeria sp., 6/32 (19%) with two, 15/32 (47%) with three, 5/32 (16%) with four and 1/32 (3%) with five Eimerians.

The illegal wildlife trade (IWT) is a criminal practice bringing several ecological and public health consequences, such as the spreading of zoonotic pathogens and/or the introduction of exotic species of animals into new geographical areas. Here, we discuss potential risks of IWT on the spreading and emergence of zoonotic pathogens.

Red foxes (Vulpes vulpes) have been recognised to harbour and transmit a wide range of tick-borne pathogens (TBPs) including those of zoonotic concern. To investigate the prevalence and the distribution of TBPs and of Leishmania infantum in foxes (n = 244), spleen samples were collected within the frame of a multi-regional wildlife health surveillance program in Italy. A combined PCR/sequencing approach was performed for the detection of Anaplasma spp., Babesia spp., Borrelia spp., Ehrlichia spp., Hepatozoon spp. and L. infantum DNA. Overall, 146 foxes (59.8 %, 95 % CI: 53.6-65.8) tested positive for at least one pathogen with Hepatozoon canis being the most prevalent (i.e., n = 124; 50.8 %, 95 % CI: 44.6-57.0), followed by Babesia vulpes (n = 20; 8.2 %, 95 % CI: 5.4-12.3), different spirochete species from Borrelia burgdorferi sensu lato complex (n = 9; 3.7 %, 95 % CI: 1.9-6.9), Ehrlichia canis and L. infantum (n = 7; 2.9 % each, 95 % CI: 1.4-5.8), Anaplasma platys (n = 4; 1.6 %, 95 % CI: 0.6-4.1), Anaplasma phagocytophilum ecotype I and Candidatus Neoehrlichia sp. (n = 3; 1.2 % each, 95 % CI: 0.4-3.5). All samples scored negative for Babesia canis and Borrelia miyamotoi. This study revealed the presence of spirochetes from B. burgdorferi s.l. complex, Ca. Neoehrlichia sp., A. platys and A. phagocytophilum ecotype I in red fox population from Italy, underling the necessity to monitoring these carnivores, mainly because they live in contact with dogs and humans. Data on the tick fauna circulating on wildlife species will complement information herein obtained, instrumentally to establish preventive strategies for minimizing the risk of infection for animals and humans.

Graphical abstract, Highlights • Transmammary transmission of Troglostrongylus brevior in domestic cats is suggested. Snails, rats and lizards play a role in the epidemiology of this feline lungworm. Observational parasitology is important in studying events that may occur in small ecological niches around us., Feline lungworms such as Aerulostrongylus abstrusus and Troglostrongylus brevior are snail-borne pathogens causing respiratory disease in domestic cats. Paratenic hosts such as rodents and reptiles have also been implicated in the epidemiology of these parasites. Although A. abstrusus has been recognized for a long time as the most prevalent lungworm among cats worldwide, T. brevior is of major concern in kittens. Bearing in mind that disease due to T. brevior occurs mainly in pediatric patients younger than 6 months of age, the diagnosis of this parasite in two kittens presenting severe respiratory disease from the garden of one of the authors inspired us to investigate the potential routes of transmission for T. brevior in domestic cats. Of the three queens (A, B and C) that delivered kittens (n = 8), only cat A was positive for T. brevior, presenting her two kittens severe respiratory clinical signs, which lead to the exitus in one of them, 18 days of age. In addition, three kittens, the offspring of queen B, turned to be positive at the coprological examination after suckling from queen A, whereas those from queen C (that suckled only on their own mother) remained negative. A series of coprological, histological and molecular tests were conducted to confirm the presence of T. brevior in the patients as well as in the other cats cohabiting the same garden. Adult nematodes were retrieved from the trachea and bronchi of the dead kitten (kitten 1A), and larvae at the histology of the lung and liver parenchyma associated with bronco pneumonitis and lymphocytic pericholangitis, respectively. Cornu aspersum (n = 60), Eobania vermiculata (n = 30) snails (intermediate hosts) as well as lizards and rats (potential paratenic hosts) were collected from the same garden and processed through tissue digestion and molecular detection. Troglostrongylus brevior larvae were recovered through tissue digestion from two C. aspersum (3.33%) and it was confirmed by PCR-sequencing approach, which also detected T. brevior DNA in the liver and lungs of one rat and one gecko lizard. During the COVID-19 lockdown, when scientists spent more time at home, we grasp the opportunity to decipher T. brevior biology and ecology starting in a small ecological niche, such as the garden of our house. Data herein presented led us to suggest: i) the transmammary transmission of T. brevior in domestic cats; ii) the role of intermediate and paratenic hosts (including reptiles) in the epidemiology of the infection which they transmit; as well as iii) the importance of observational parasitology in studying any event that certainly occurs in small ecological niches, as it could be in our home gardens.

Toxoplasmosis is a worldwide-distributed zoonotic disease of great relevance to public health. Many wildlife species, including marsupials of the genus Didelphis, are considered hosts for Toxoplasma gondii, which makes them to have a possible role in the dispersion and maintenance of this parasite in nature. This study provides data on the molecular detection of T. gondii in D. aurita opossums from an urbanized area of Southeastern Brazil. Animals were captured and blood and/or spleen samples were collected. Real Time PCR was performed for the detection of T. gondii. From the opossums captured, 26.3% (n = 15/57) scored positive, with a frequency of 21.6% (n = 11/51) in blood, and 66.7% (n = 6/9) in spleen samples. BLAST analysis demonstrated 100% identity and 100% cover query with sequences of T. gondii available in GenBank database. Data herein reported present great public health importance, since Didelphis spp. are usually observed inhabiting close to human dwellings, which facilitates their contact with people and domestic animals, and consequently, the transmission of zoonotic agents. However, further studies are needed to elucidate whether these opossums play an important role in the zoonotic cycle of T. gondii in urban areas of Brazil.

Eimeria spp. infections lead to acute or chronic intestinal disorders in small ruminants, being responsible for important economic losses worldwide. The aim of this study was to assess the risk factors associated with Eimeria spp. infection in small ruminants of the microregion of Garanhuns, state of Pernambuco, Northeastern Brazil. Fecal samples (n = 822) were obtained from goats (n = 414) and sheep (n = 408) and evaluated by the modified Gordon and Whitlock technique. Risk factors were assessed through univariate analysis and logistical regression. Oocysts of Eimeria species were detected in 62.9% (517/822) of the animals, with 77.8% (322/414) in goats and 47.8% (195/408) in sheep. For goats, the herd size (OR = 5.52), rearing system (OR = 1.57), feeding place (OR = 2.60), absence of mineral salt in the diet (OR = 2.54), flooring type (OR = 2.83) and periodicity of cleaning (OR = 5.39) were considered risk factors. Conversely, for sheep only the herd size (OR = 3.16) and rearing system (OR = 2.45) were important factors associated with infection by Eimeria spp. Data herein obtained brings meaningful information on the epidemiology of coccidiosis in small ruminants in Northeastern Brazil. The knowledge of these risk factors is useful to contribute to the development of preventive measures, thereby, reducing the economic impact caused by these protozoa in small ruminant production.

Marsupials of the genus Didelphis, such as black-eared opossums (Didelphis aurita), are common synanthropic animals in urban areas of Brazil. These marsupials are frequently parasitized by numerous helminth species, including ancylostomatid nematodes. This study aimed to report the occurrence of Ancylostoma caninum in black-eared opossums captured in an urban environment of Southeastern Brazil and discuss the potential impact of these findings for public health. From January to June 2019, we collected fecal samples from 49 restrained opossums and evaluated by a simple flotation method; Helminth eggs were observed at different magnifications and identified according to morphological and morphometric features. Genomic DNA was extracted from Ancylostomatidae eggs and screened by duplex PCR for Ancylostoma spp. and Necator americanus using primers that amplify a region of internal transcribed spacer 2 and the 28S ribosomal RNA (ITS2-28S rRNA). Ancylostoma spp. eggs were detected in 65.3% (32/49) of the animals. Sequence analysis revealed 100% homology with A. caninum sequences from GenBank. Our results demonstrate a new host-parasite interaction for A. caninum, suggesting that black-eared opossums may participate in the zoonotic cycle of this parasite in urban areas of Brazil.

Rhipicephalus sanguineus sensu lato (s.l.) is one of the most widespread ixodid ticks and is a competent vector of several vector-borne pathogens of veterinary and medical concern. For instance, this tick species transmits nematodes of the genus Cercopithifilaria and protozoa of the genus Hepatozoon to carnivores, including dogs. Here we investigated the occurrence of Cercopithifilaria spp. and Hepatozoon spp. in a population of ticks collected from naturally infested dogs living in rural areas of Northeastern Brazil. From August 2016 to June 2017, 758 tick specimens (mean ticks per month = 68.9 ± 71.4) were sampled from 75 dogs (mean ticks per dog = 10.11 ± 5.2) and dissected under a stereomicroscope in order to visualize Cercopithifilaria spp. larvae and Hepatozoon spp. oocysts and sporocysts. R. sanguineus s.l. was the only species collected, peaking in September (n = 273) and decreasing in February 2017 (n = 39). Different larval stages of Cercopithifilaria bainae were identified in 7 out of 758 (0.93%) ticks. In addition, 4 specimens (0.53%) were positive for oocysts and free sporocysts of Hepatozoon canis. The identity of both species of parasites was molecularly confirmed. These results account for the predominance of R. sanguineus (s.l.) in domestic dogs from rural locations of the study area, as well as for the presence C. bainae and H. canis in these tick populations.

Currently, a great proportion of the emerging infectious human diseases are zoonotic, with most of the pathogens originated from wildlife. In this sense, synanthropic animals such as marsupials play important role in the dissemination of pathogens due to their proximity to human dwellings. These hosts are affected by many gastrointestinal parasites, including species with zoonotic potential. The aim of this study was to assess the diversity of gastrointestinal parasites infecting the black-eared opossum D. aurita captured in urban areas of Southeastern, Brazil. In addition, the potential risk for the human population based on the One Health perspective has been discussed. Forty-nine marsupial specimens were captured with Tomahawk live traps and fecal samples were collected. The samples were evaluated by parasitological procedures. Eggs and oocysts were analyzed at different magnifications (400 × and 1000 ×), and their identification, together with adult nematodes, was established on morphological and morphometric data. Forty-three hosts (87.76%) scored positive for at least one gastrointestinal parasite, being 83.67% (41/49) for helminths, and 65.30% (32/49) for protozoa. For Cryptosporidium sp., only 13 samples were evaluated due to insufficient amount of feces obtained of some animals. A prevalence of 23.08% (3/13) was reported for this parasite. PCR analysis revealed Ancylostomatidae eggs to belong to the genus Ancylostoma. Our results demonstrated that multiparasitism is frequently found in these animals and a high percentage of potentially zoonotic parasites are observed, implying that D. aurita may be involved in zoonotic cycles in urban environments.

Dirofilariosis by Dirofilaria immitis and Dirofilaria repens is endemic in dogs from countries of the Mediterranean basin. Both species may infect humans, with most of the infected patients remaining asymptomatic. Based on the recent description of the southernmost hyperendemic European focus of heartworm disease in dogs from the Pelagie archipelagos, we performed a serological and molecular survey in human population of that area. Human blood samples were collected in the islands of Linosa (n=101) and Lampedusa (n=296) and tested by ELISA and molecular test for the detection of D. immitis and D. repens. Samples were also screened for filarioid-associated endosymbionts, Wolbachia sp. The seroprevalence of D. immitis and D. repens was, respectively, 7.9% and 3.96% in Linosa, and 7.77% and 19.93% in Lampedusa. Out of 397 human blood samples tested molecularly, 4 scored positive (1%) for Dirofilaria spp. by qPCR (i.e., three for D. immitis and one for D. repens) and 6 (1.5%) for Wolbachia. Of the qPCR positive for Dirofilaria spp., only D. repens was amplified by cPCR and was positive for Wolbachia. In the phylogenetic analysis, the sequence of Wolbachia detected in D. repens positive samples clustered along with other C supergroup filarioids. Our results overlap with the recent prevalence data collected on dogs from the same area, where D. immitis is prevalent in Linosa and D. repens prevails in Lampedusa. Molecular detection of D. immitis in human blood is quite unusual considering that humans are dead-end hosts for dirofilarial infection and most of the human cases described so far in Europe were ascribed to D. repens. An integrative diagnostic approach using serum analysis and Wolbachia detection is also presented. In endemic areas for canine dirofilarioses humans are exposed to the infection, suggesting the importance of One Health approach in diagnosing, treating and controlling this zoonotic parasitosis.

Trypanosoma evansi, the causative agent of surra, is a hemoflagellate protozoan mechanically transmitted by hematophagous flies, mainly in tropical and subtropical regions. This protozoan affects several mammalian hosts, including dogs, which are highly susceptible to the infection. To investigate the occurrence of T. evansi in dogs, a total of 672 DNA samples from India (n = 228), Indonesia (n = 57), Malaysia (n = 45), the Philippines (n = 103), Thailand (n = 120), and Vietnam (n = 119) were screened by using species-specific conventional PCR. Of the tested dogs, 10 (1.5%) scored positive to T. evansi. In particular, positive samples were detected in canine blood samples collected from India (n = 4; 1.8%), Indonesia (n = 4; 7%), and Malaysia (n = 2; 4.4%). All tested samples from the Philippines, Thailand and Vietnam were negative. Nucleotide sequence analysis revealed a high variation (i.e. from 0.4% to 6.2%) among the RoTat 1.2 variant surface glycoprotein (vsg) gene. Although the number of sequences included in this analysis is relatively small, this nucleotide variation may indicate the divergence of T. evansi RoTat 1.2 vsg gene among different strains. The high incidence of T. evansi previously reported in cattle and buffaloes in India and Southeast Asia suggests that these animals are the main source of infection to dogs.

Ehrlichia canis is among the most prevalent tick-borne pathogens infecting dogs worldwide, being primarily vectored by brown dog ticks, Rhipicephalus sanguineus sensu lato (s.l.). The genetic variability of E. canis has been assessed by analysis of different genes (e.g., disulfide bond formation protein gene, glycoprotein 19, tandem repeat protein 36 - TRP36) in the Americas, Africa, Asia, and in a single dog sample from Europe (i.e., Spain). This study was aimed to assess the variations in the TRP36 gene of E. canis detected in naturally infected canids and R. sanguineus s.l. ticks from different countries in Asia and Europe. DNA samples from dogs (n = 644), foxes (n = 146), and R. sanguineus s.l. ticks (n = 658) from Austria, Italy, Iran, Pakistan, India, Indonesia, Malaysia, the Philippines, Singapore, Thailand, Vietnam, and Taiwan were included in this study. Ehrlichia canis 16S rRNA positive samples (n = 115 from the previous studies; n = 14 from Austria in this study) were selected for molecular examination by analyses of TRP36 gene. Out of 129 E. canis 16S rRNA positive samples from dogs (n = 88), foxes (n = 7), and R. sanguineus s.l. ticks (n = 34), the TRP36 gene was successfully amplified from 52. The phylogenetic analysis of the TRP36 pre-repeat, tandem repeat, and post repeat regions showed that most samples were genetically close to the United States genogroup, whereas two samples from Austria and one from Pakistan clustered within the Taiwan genogroup. TRP36 sequences from all samples presented a high conserved nucleotide sequence in the tandem repeat region (from 6 to 20 copies), encoding for nine amino acids (i.e., TEDSVSAPA). Our results confirm the US genogroup as the most frequent group in dogs and ticks tested herein, whereas the Taiwan genogroup was present in a lower frequency. Besides, this study described for the first time the US genogroup in red foxes, thus revealing that these canids share identical strains with domestic dogs and R. sanguineus s.l. ticks.

INTRODUCTION: Areas at risk of transmission of arboviruses have been monitored using ovitraps. This study aimed to evaluate the spatial distribution of Aedes aegypti in vulnerable areas for the transmission of arboviruses and assess the influence of climatic conditions on the infestation of these culicids. METHODS: Ovitraps were installed in Agrestina, Pernambuco, Northeastern Brazil. RESULTS: Overall, 44,936 eggs were collected, and the indexes of infestation varied. Relative humidity was significantly associated with the infestations. CONCLUSIONS: Using ovitraps, entomologic indexes and analysis of climatic factors might be good strategies for monitoring vulnerable areas for the transmission of arboviruses.

Hippoboscid flies (Diptera: Hippoboscidae), commonly known as keds or louse flies, have been for long time overlooked by the scientific community, and their vector role of infectious agents to humans and domestic animals has been scantly investigated. This is partly due to the fact that the host range for most species is primarily restricted to wildlife, being rarely reported on domestic animals and humans. This led to a scarce scientific knowledge about their biology, ecology, behaviour, epidemiology as well as vector competence. However, the life history of some hippoboscid species, e.g., Melophagus ovinus, Lipoptena cervi and Hippobosca equina, suggests that these ectoparasites are important candidates to vector infectious disease agents (e.g., Rickettsia spp., Borrelia spp., Bartonella spp., Anaplasma phagocytophilum, Theileria ovis). Indeed, the peculiar biological and behavioural traits (i.e., obligatory blood sucking and reproductive physiology) of many ked species make them a suitable pabulum for pathogen's multiplication and for their transmission to receptive hosts. Therefore, studies focusing on the ked bio-ecological aspects as well as on their vector role are advocated along with the control of keds affecting different animal species. This review discusses current information on keds, highlighting their importance as vectors of pathogens of medical and veterinary concern to all animal species, with a special focus on mammals.

Phlebotomines have worldwide distribution with many species present in Brazil, including the northeastern region, where the fauna is very diverse. The aim of this study was to identify the sandfly fauna in an area endemic for American cutaneous leishmaniasis (ACL) in the state of Pernambuco. Sandflies were caught on three consecutive nights every month from October 2015 to September 2016, from 5 pm to 5 am, using seven light traps of Centers for Disease Control (CDC) type. Females were identified and used for molecular Leishmania detection. A total of 2,174 specimens belonging to ten species were collected: Lutzomyia choti (88.2%; 1,917/2,174) was the most abundant species, followed by Lutzomyia whitmani (8.1%; 176/2,174) and Lutzomyia sordellii (1.5%; 33/2,174). The majority of the specimens were collected in peridomestic areas (64.1%; 1,394/2,174) and during the rainy period. All the samples examined were negative for Leishmania spp. The presence of Lutzomyia whitmani indoors and in peridomestic areas indicates that the inhabitants of this area are exposed to the risk of infection by the parasites responsible for ACL. Resumo Os flebotomíneos apresentam uma ampla distribuição mundial com muitas espécies presentes no Brasil, inclusive na região Nordeste, onde a fauna é bastante rica. O objetivo desse estudo foi identificar a fauna de flebotomíneos em uma área endêmica para Leishmaniose Tegumentar Americana (LTA), no estado de Pernambuco. As capturas foram realizadas mensalmente, durante três noites consecutivas das 17h às 5h, utilizando sete armadilhas luminosas tipo CDC, no período de outubro de 2015 a setembro de 2016. As fêmeas identificadas foram utilizadas para análise molecular para detecção de Leishmania. Um total de 2.174 espécimes pertencentes a dez espécies foram coletadas: Lutzomyia choti (88,2%; 1.917/2.174) a espécie mais abundante, seguida por Lutzomyia whitmani (8,1%; 176/2.174) e Lutzomyia sordellii (1,5%; 33/2.174). A maioria dos espécimes foi coletada no peridomicílio (64,1%; 1.394/2.174) e no período chuvoso. Todas as amostras avaliadas foram negativas para Leishmania spp. A presença de Lutzomyia whitmani no intradomicílio e peridomicílio indica que a população residente nesta área está exposta ao risco de infecção por parasitos causadores de LTA.

The biological control of ticks represents an alternative method to the chemical control, given its ecological-friendly approach. Amongst the alternatives, the use of parasitoids of the genus Ixodiphagus (Hymenoptera: Encyrtidae) has been largely investigated. The aim of this study was to document and molecularly characterize Ixodiphagus wasps in ticks from a tropical region of Brazil. From October 2015 to March 2016, Rhipicephalus sanguineus sensu lato ticks (n = 1814) were collected from naturally infested dogs and Ixodiphagus larvae were detected by microscopic examination. In addition, adult wasps were obtained in the laboratory. Larvae and adults were molecularly identified as Ixodiphagus hookeri . These findings suggest that this type of parasitism deserves to be studied in local tick populations, in order to elucidate the role of these wasps as a potential alternative to chemical tick control.

Rhipicephalus sanguineus sensu lato (s.l.) ticks act as intermediate host for a range of canine vector-borne pathogens, including nematodes ranked in the genus Cercopithifilaria. Though being the object of several studies in the last years, information on the distribution of these parasites is still lacking. In this study, the occurrence of Cercopithifilaria spp. was investigated in on-host population of R. sanguineus s.l. collected from naturally infested dogs. Ticks (n=1906, including one larva, 294 nymphs and 1611 adults) were sampled on domestic dogs (n=155) living in the municipality of Garanhuns (northeastern Brazil). Tick collections (n=36) were performed every 8 days, from October 2015 to June 2016. Filarioid larvae detected at tick dissection were morphologically and morphometrically identified at species level. At the end of the study, only R. sanguineus s.l. ticks were collected, with the highest number in January 2016 (n=254) and the lowest in June 2016 (n=26). Out of 1906 dissected ticks, 2.68% (51/1906) harboured Cercopithifilaria bainae larvae, whose identification was molecularly confirmed, with a nucleotide identity of 99% with C. bainae. Data here reported indicate that, in the study area, R. sanguineus s.l. is the predominant tick infesting domestic dogs. Accordingly, these animals are at a high risk of C. bainae infection.