Evaluation of parasitic infections with histological and molecular detection in Anoles from St. Kitts.

Anoles are a widely distributed group of reptiles that are also increasing in popularity as a pet animal. These species have been described as paratenic and intermediate hosts for many parasites of veterinary and medical importance. However, while human and animals such as cats and dogs may often interact with anoles, little research has been conducted in terms of assessing the full potential of anoles to harbor parasites of public and veterinary health significance. The current study analyzed the parasites of anoles captured and dissected in St. Kitts, West Indies, to describe the parasitic population within these reptiles and evaluate the potential role of anoles as intermediate/paratenic hosts. From March until May of 2019, samples from 11 anoles were collected following humane euthanasia and evaluated grossly, histologically, and molecularly for the presence of parasites. Histologic evaluation revealed adult ascarids, oxyurids, and spirurids within the stomach, as well as intraluminal adult ascarids within the intestine, and encysted larval trematodes within the intestinal wall. Molecular analysis based on 18S rRNA and ITS2 DNA markers for nematodes and trematodes, respectively, identified Physaloptera sp. (99.4-99.8% identical with Physaloptera turgida, Physaloptera mirandai, Physaloptera retusa, Physaloptera rara) and Platynosomum illiciens (99.7% identical). Phylogenetic analysis of ITS 2 sequences of P. illiciens from this study cladded monophyletically with the same species from different geographic areas and hosts. Our study highlights the importance of understanding the role anoles play in the life cycles of less well-studied parasites, and the potential of these animals to act as intermediate or paratenic hosts.

In vitro maintenance of the endosymbiont Wolbachia of Dirofilaria immitis.

Wolbachia has an obligatory mutualistic relationship with many onchocercid nematodes of the subfamilies Dirofilariinae and Onchocercinae. Till date, no attempts have been made for the in vitro cultivation of this intracellular bacterium from the filarioid host. Hence, the current study attempted cell co-culture method using embryonic Drosophila S2 and the LD cell lines to cultivate Wolbachia from Dirofilaria immitis microfilariae (mfs) harvested from infected dogs. Microfilariae (mfs = 1500) were inoculated in shell vials supplemented with Schneider medium using both cell lines. The establishment and multiplication of the bacterium were observed during the initial inoculation, at day 0 and before every medium change (from days 14 to 115). An aliquot (50 µl) from each time point was tested by quantitative real-time PCR (qPCR). Comparing the average of Ct values, obtained by the tested parameters (i.e., LD/S2 cell lines and mfs with/without treatment), the S2 cell line without mechanical disruption of mfs provided the highest Wolbachia cell count by qPCR. Despite the maintenance of Wolbachia within both S2 and LD-based cell co-culture models for up to 115 days, a definitive conclusion is still far. Further trials using fluorescent microscopy and viable staining will help to demonstrate the cell line infection and viability of Wolbachia. Use of considerable amount of untreated mfs to inoculate the Drosophilia S2 cell lines, as well as the supplementation of the culture media with growth stimulants or pre-treated cells to increase their susceptibility for the infection and development of a filarioid-based cell line system are recommended for the future trials.

Molecular detection and characterization of the endosymbiont Wolbachia in the European hedgehog flea, Archaeopsylla erinacei.

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.

Molecular detection of zoonotic blood pathogens in ticks from illegally imported turtles in Italy.

International trade of animals may represent a gateway for the spreading of zoonotic pathogens and their vectors. Amongst animals, reptiles are commonly illegally imported worldwide, being Italy in the fifth position of importation of these animals. Thus, the current study analysed the pathogens associated with Hyalomma aegyptium ticks, which were collected from illegally imported tortoises from North Africa to Italy. All tick DNA samples were tested by conventional PCR for the presence of Anaplasma spp., Babesia spp., Borrelia spp., Coxiella burnetti, Ehrlichia spp., Hepatozoon spp., Rickettsia spp. and microfilariae of filarioids. Out of 22% (n=161) of ticks screened, 78.9% (n=127) were males and 21.1% (n=34) females. Among them, three male specimens collected from two different turtles (1.9%; 95% CI; 0.5-5.5) scored positive for Anaplasma spp./Ehrlichia spp., whereas all females were negative. BLAST analysis of the sequences obtained from positive samples revealed 99-99.3% nucleotide identity with the sequence of Ehrlichia ewingii available in GenBank. The finding of E. ewingii in ticks from imported reptiles warrants the need for imposing strict rules in the international trade of reptiles to effectively reduce the introduction of exotic pathogens and their vectors in new geographic areas.

Molecular detection of Wolbachia endosymbiont in reptiles and their ectoparasites.

Wolbachia, a maternally transmitted Gram-negative endosymbiont of onchocercid nematodes and arthropods, has a role in the biology of their host; thus it has been exploited for the filariasis treatment in humans. To assess the presence and prevalence of this endosymbiont in reptiles and their ectoparasites, blood and tail tissue as well as ticks and mites collected from them were molecularly screened for Wolbachia DNA using two sets of primers targeting partial 16S rRNA and Wolbachia surface protein (wsp) genes. Positive samples were screened for the partial 12S rRNA and cytochrome c oxidase subunit 1 (cox1) genes for filarioids. Of the different species of lizards (Podarcis siculus, Podarcis muralis and Lacerta bilineata) and snakes (Elaphe quatuorlineata and Boa constrictor constrictor) screened from three collection sites, only P. siculus scored positive for Wolbachia 16S rRNA. Among ectoparasites collected from reptiles (Ixodes ricinus ticks and Neotrombicula autumnalis, Ophionyssus sauracum and Ophionyssus natricis mites), I. ricinus (n = 4; 2.8%; 95% CI, 0.9-7) from P. siculus, N. autumnalis (n = 2 each; 2.8%; 95% CI, 0.9-6.5) from P. siculus and P. muralis and O. natricis (n = 1; 14.3%; 95% CI, 0.7-55.4) from Boa constrictor constrictor scored positive for Wolbachia DNA. None of the positive Wolbachia samples scored positive for filarioids. This represents the first report of Wolbachia in reptilian hosts and their ectoparasites, which follows a single identification in the intestinal cells of a filarioid associated with a gecko. This data could contribute to better understand the reptile filarioid-Wolbachia association and to unveil the evolutionary pattern of Wolbachia in its filarial host.

Wolbachia: endosymbiont of onchocercid nematodes and their vectors.

BACKGROUND: Wolbachia is an obligate intracellular maternally transmitted, gram-negative bacterium which forms a spectrum of endosymbiotic relationships from parasitism to obligatory mutualism in a wide range of arthropods and onchocercid nematodes, respectively. In arthropods Wolbachia produces reproductive manipulations such as male killing, feminization, parthenogenesis and cytoplasmic incompatibility for its propagation and provides an additional fitness benefit for the host to protect against pathogens, whilst in onchocercid nematodes, apart from the mutual metabolic dependence, this bacterium is involved in moulting, embryogenesis, growth and survival of the host. METHODS: This review details the molecular data of Wolbachia and its effect on host biology, immunity, ecology and evolution, reproduction, endosymbiont-based treatment and control strategies exploited for filariasis. Relevant peer-reviewed scientic papers available in various authenticated scientific data bases were considered while writing the review. CONCLUSIONS: The information presented provides an overview on Wolbachia biology and its use in the control and/or treatment of vectors, onchocercid nematodes and viral diseases of medical and veterinary importance. This offers the development of new approaches for the control of a variety of vector-borne diseases.

Molecular detection of Trypanosoma evansi in dogs from India and Southeast Asia.

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.

Genetic variability of Ehrlichia canis TRP36 in ticks, dogs, and red foxes from Eurasia.

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.

Spotted fever group rickettsiae in Dermacentor marginatus from wild boars in Italy.

Following the increase in wild boar population recorded in urban and peri-urban areas throughout Europe, the present survey aimed to assess the occurrence of zoonotic tick-borne pathogens (TBPs) in wild boars living in southern Italy and in their ticks for evaluating the potential risk of infection for animals and humans. From October to December 2019, a total of 176 ticks collected from 93 wild boars andtheir spleen samples were molecularly screened for selected TBPs. Overall, all the wild boars were infested by ticks (mean intensity, 1.9) with Dermacentor marginatus and Ixodes ricinus being identified in 99.4% and 0.6%, respectively. Out of 93 wild boars, 17 (18.3%) were infested by ticks which scored positive to spotted fever group (SFG) Rickettsia species. Rickettsia slovaca and Rickettsia raoultii were identified in 16 (9%) and 1 (0.6%) D. marginatus, respectively, whereas a single I. ricinus (0.6%) was infected by R. slovaca. A single wild boar (1.1%) tested positive to R. slovaca. All ticks and wild boars scored negative to Babesia/Theileria spp., Ehrlichia spp., Anaplasma spp., Candidatus Neoehrlichia spp., Coxiella burnetii and Borrelia burgdorferi sensu lato complex. Data herein obtained suggest that wild boars are involved in the maintenance of D. marginatus in the environment as in peri-urban areas herein investigated. An integrated management approach is advocated for wild boar population control and in preventing the potential risk of TBPs infection in animals and humans.

Canine vector-borne pathogens from dogs and ticks from Tamil Nadu, India.

Canine vector-borne diseases (CVBDs) pose a major health problem in dogs globally, with the potential to cause zoonoses, in particular in developing countries where scientific knowledge on the topic is minimal. Blood samples and ticks were collected from stray dogs in Tamil Nadu, South India to assess the prevalence of CVBD-causing pathogens (Anaplasma spp., Babesia spp., Ehrlichia spp., Hepatozoon spp., filarioids and Leishmania spp.). Of the 230 dogs examined, 229 (99.6%) were infested by ticks (mean intensity, 5.65) with Rhipicephalus sanguineus sensu lato and Rhipicephalus haemaphysaloides being morphologically identified in the 98.3% and 1.7% of the infested dogs, respectively. Overall, the 67.8% (n = 156) of dogs was positive for at least one pathogen with Hepatozoon canis being the most prevalent (37.8%) followed by Anaplasma platys (22.6%), Ehrlichia canis (16.1%) Babesia vogeli (10%), Anaplasma phagocytophilum (0.4%) and Babesia gibsoni (0.4%). Two filarioids (Dirofilaria sp. "hongkongensis" and Brugia malayi, 0.4%) were diagnosed in sampled animals. Co-infection with H. canis and A. platys was the most prevalent (8.3%, P = 0.00001), whilst all animals scored negative for Leishmania spp.. Out of 295 ticks analysed, 215 R. sanguineus s.l. (75.4%) and 8 R. haemaphysaloides (88.9%) were positive for at least one pathogen with H. canis as the predominant species (42.5%), followed by A. platys (33.8%), E. canis (16.9%), B. vogeli (3.8%) and A. phagocytophilum (0.3%). Fifty-six dogs (35.9%) harboured the same pathogen as the respective tick specimens, while 29 dogs (18.6%) had a different pathogen. Thirteen sequence types (STs) were identified for H. canis, with ST2 (49.4%) as the most representative in dogs and ST1 (73.5%) in ticks. Similarly, seven STs were found for Anaplasma spp. (i.e., five for A. platys, one for A. phagocytophilum and one for Anaplasma sp.), with ST2 as the most representative in dogs (70.6%) and ST3 (52.5%) in ticks for A. platys. Only one ST was identified for B. vogeli, B. gibsoni, E. canis, D. sp. "hongkongensis" and B. malayi. Regular surveillance and adoption of adequate treatment and control measures are needed to reduce the risk of disease-causing pathogens in stray dogs and of pathogens with zoonotic potential.