Detection of Rickettsia felis in ectoparasites collected from domestic animals.

Ticks and fleas are arthropods widely distributed around the world involved in the transmission of various vector-borne diseases (VBDs), including Brazilian Spotted Fever (BSF), Baggio-Yoshinari Syndrome and the plague, with outstanding consequences for the public health. The aim of this study was to investigate the presence of Rickettsia spp., Borrelia spp. and Yersinia pestis in arthropods collected from dogs, cats and horses living in the state of Pernambuco, Northeastern Brazil. From January 2017 to April 2019, ectoparasites were collected, morphologically identified and molecularly analysed through PCR and sequencing. In total 401 specimens were collected from 86 animals, being 68% (n = 273) and 32% (n = 128) from rural and urban areas, respectively. The most commonly detected species were the ticks Dermacentor nitens, Amblyomma sculptum, Rhipicephalus sanguineus sensu lato (s.l.), Rhipicephalus microplus, and Amblyomma ovale, and the fleas Ctenocephalides felis and Ctenocephalides canis. DNA of Rickettsia felis was detected in D. nitens collected from horses, and C. felis, and R. sanguineus s.l. collected from dogs. All samples scored negative for Borrelia spp. and Y. pestis DNA. This study provides valuable data on ectoparasite fauna from domestic animals and identifies the circulation of a zoonotic pathogen (i.e., R. felis) in the population of the arthropods assessed. Therefore, preventive measures should be adopted in order to reduce the risk of occurrence of neglected VBD caused by this pathogen in animal and human hosts.

Rhipicephalus turanicus, a new vector of Hepatozoon canis.

The distribution of Hepatozoon canis mainly encompasses areas where its main tick vector, Rhipicephalus sanguineus sensu lato, is present. However, the detection of this pathogen in dogs, foxes and golden jackals well outside the areas inhabited by this tick species reinforced the hypothesis that additional ixodids are involved in the life cycle and transmission of this protozoon. The present study provides, for the first time, data supporting the sporogonic development of H. canis in specimens of Rhipicephalus turanicus collected from a naturally infected fox from southern Italy. The epidemiological role of R. turanicus as a vector of H. canis is discussed, along with information on the potential use of cell cultures for the experimental infection with H. canis sporozoites. The in vitro infection of canine leucocytes by sporozoites from ticks is proposed as a potential tool for future in-depth studies on the biology of H. canis.

Filarial infection caused by Onchocerca boehmi (Supperer, 1953) in a horse from Italy.

Equids can be infected by a range of skin-dwelling filarial nematodes, including four species of the genus Onchocerca. Current literature on equine onchocercosis is fragmentary and often limited to isolated case reports. The present study aimed to describe a clinical case of equine onchocercosis caused by Onchocerca boehmi (Supperer, 1953) (syn. Elaeophora boehmi) in an 8-year-old gelding Belgian show jumper from northern Italy. The horse was presented with a firm and painless mass on the proximal third of the right metacarpal region. Ultrasound examination showed a peritendinous enlargement around the palmaro-lateral area of the tendons, characterized by an elongated hypoechoic and well-defined structure, embedding a coiled hyperechoic line. The metacarpal nodule was resected and histologically examined. Fragments of a parasitic nematode were detected, isolated and examined. The morphological analysis allowed identifying the nematode as O. boehmi. In addition, total genomic DNA was extracted from individual fragments using a commercial kit for the nematode identification and a comparative sequence analysis of the nematode cytochrome oxidase subunit 1 (cox1) sequence with data available in the GenBankTM database revealed the closest identity (i.e. 91 %) with that of Onchocerca lupi. Thus far, O. boehmi has only been reported in Austria and Iran, and information about its life-cycle and vectors is lacking. The systematic position of this species within the genus Onchocerca, not in Elaeophora where it was originally described, is in concordance with the morphological and molecular analysis. In this article, we describe the first autochthonous case of equine onchocercosis in Italy caused by O. boehmi and discuss novel parasitological, clinical, and pathological data on these pathogens of horses.

Ixodes ventalloi: morphological and molecular support for species integrity.

Despite their medical and veterinary importance, some tick species are so poorly studied, that their role within pathogen vector transmission cycles is difficult to assess. The tick Ixodes ventalloi is one such species, and its biology and phylogenetic status remain an issue of debate. In the present study, specimens of adult I. ventalloi (n = 65 females; n = 31 males) infesting cats in the Lipari Island (Aeolian archipelago, Sicily, southern Italy) were characterized morphologically and molecularly, the latter based on mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes. The genetic data and phylogenetic analyses for both mitochondrial genes suggest the existence of two distinct genogroups. The ecological and epidemiological significance of the genetic structure within the I. ventalloi endemic population remains to be determined. The results highlight the need for further analysis of this tick species, including whole mitochondrial genome sequencing and crossbreeding studies, which will be pivotal to complement features of its status as a vector of pathogens.

Canine Infections with Onchocerca lupi Nematodes, United States, 2011-2014.

Infections with Onchocerca lupi nematodes are diagnosed sporadically in the United States. We report 8 cases of canine onchocercosis in Minnesota, New Mexico, Colorado, and Florida. Identification of 1 cytochrome c oxidase subunit 1 gene haplotype identical to 1 of 5 from Europe suggests recent introduction of this nematode into the United States.

Crenosoma vulpis in wild and domestic carnivores from Italy: a morphological and molecular study.

Crenosoma vulpis is a metastrongyloid nematode primarily associated with respiratory tract infections of red foxes in North America and Europe. Sporadic cases have also been reported in domestic dogs. The present study aimed to provide morphological, molecular, and epidemiological data on the geographical distribution of this nematode throughout Italy. From 2012 to 2014, 12 of the 138 foxes examined, three dogs and one badger scored positive for C. vulpis. Forty adults were isolated from foxes and the badger, whereas first-stage larvae were detected in the three dogs. All specimens were morphologically identified as C. vulpis, and 28 nematodes were also molecularly characterized by sequencing mitochondrial (12S ribosomal DNA (rDNA)) and nuclear (18S rDNA) ribosomal genes. Four haplotypes were identified based on the 12S rDNA target gene, with the most representative (78.5%) designated as haplotype I. No genetic variability was detected for the 18S rDNA gene. The molecular identification was consistent with the distinct separation of species-specific clades inferred by the phylogenetic analyses of both mitochondrial and ribosomal genes. Data herein reported indicates that C. vulpis has a wide distribution in foxes from southern Italy, and it also occurs in dogs from southern and northern regions of the country. Practitioners should consider the occurrence of this nematode in the differential diagnosis of canine respiratory disease, particularly in dogs living close to rural areas where foxes are present.

Development of the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior in Helix aspersa snails.

Aelurostrongylus abstrusus (Strongylida, Angiostrongylidae) and Troglostrongylus brevior (Strongylida, Crenosomatidae) are regarded as important lungworm species of domestic felids, with the latter considered an emerging threat in the Mediterranean region. The present study aimed to assess their concurrent development in the mollusc Helix aspersa (Pulmonata, Helicidae). Thirty snails were infested with 100 first-stage larvae (L1) of A. abstrusus and T. brevior, isolated from a naturally infested kitten. Larval development was checked by digesting five specimens at 2, 6 and 11 days post infestation. Larvae retrieved were morphologically described and their identification was confirmed by specific PCR and sequencing. All H. aspersa snails were positive for A. abstrusus and T. brevior, whose larval stages were simultaneously detected at each time point. In addition, snails were exposed to outdoor conditions and examined after overwintering, testing positive up to 120 days post infestation. Data herein presented suggest that A. abstrusus and T. brevior develop in H. aspersa snails and may eventually co-infest cats. Data on the morphology of both parasitic species in H. aspersa provide additional information on their development and identification, to better understand the population dynamics of these lungworms in receptive snails and paratenic hosts.

A preliminary investigation of serological tools for the detection of Onchocerca lupi infection in dogs.

Onchocerca lupi is a neglected filarioid causing nodular lesions associated with acute or chronic ocular disease in dogs. Despite the recent appraisal of its zoonotic potential, human cases are increasingly reported in the Old and New Worlds. Therefore, the development of accurate tools for the rapid diagnosis of O. lupi infections in dogs is becoming a priority. In this study, we conducted a preliminary investigation aimed at evaluating the usefulness of a commercially available ELISA test for the detection of O. lupi antigens in canine sera. The potential use of this tool for larger epidemiological studies of canine onchocerciasis is discussed.

Feline lungworm Oslerus rostratus (Strongylida: Filaridae) in Italy: first case report and histopathological findings.

Oslerus rostratus syn. Anafilaroides rostratus (Strongylida: Filaroididae) is a metastrongyloid transmitted by snails, which localizes in peri-bronchial tissues and in the lung parenchyma of wild as well as domestic cats. In Europe, this nematode has been reported only on two occasions, being diagnosed in cats from Majorca Island and in northern Spain. Here, we describe a case of O. rostratus infection in a necropsied 4-year-old cat in Sicily (southern Italy). At the inspection of lungs, slender and greyish nematodes (four females and two males) were found embedded in the peri-bronchial tissues and in the bronchial walls. Parasites were morphological and molecularly identified as O. rostratus, with their 18S sequences being identical among them and showing a high homology (99%) with those available in public databases. At the histology, nematodes were encapsulated in a pseudo-cystic formation surrounded by an interstitial inflammatory process and fibrous tissue. Lung lesions were mainly represented by peri-luminal fibrosis, hyperplasia and hypertrophy of the bronchial mucosa and glands, respectively. This first record of O. rostratus infection from Italy indicates that this parasite should be included in the differential diagnosis of feline of lungworm infection.

Spirocerca lupi infection in a dog from southern Italy: an "old fashioned" disease?

Canine spirocercosis caused by Spirocerca lupi is a life-threatening helminthic disease featured by severe clinical signs and potential development of oesophageal neoplasia. This infection is considered fairly frequent in Europe but almost unknown in Italy, from where only few reports have been published in local journals at the beginning of the XXI century. In the present study, an autochthonous case of canine spirocercosis in a 2-year-old dog from southern Italy is described. The animal was admitted to a private veterinary clinic in the municipality of Potenza (Basilicata region) due to persistent dyspnoea, vomiting, and regurgitation. At the abdominal ultrasound, a mass (1.3 × 2 cm) was observed and, thereafter, surgically removed from the apical part of the stomach. A female specimen of S. lupi was morphologically identified during the histological examination of the nodule and its eggs were detected in the faeces. In addition, the morphological identification was confirmed by molecular amplification and sequencing of partial cox2 gene sequence of S. lupi. Veterinarians should be aware of the potential risks derived from infection with this spirurid, which requires specific diagnostic and preventive measures.

Quantifying the neglected: Initial estimation of the global burden and economic impact of human toxocariasis.

Toxocariasis is a parasitic zoonotic infection caused by Toxocara spp., ascarid nematodes of companion animals (dogs and cats) affecting people in both high-income and low/middle-income countries. Toxocariasis can manifest as several distinct syndromes. The most frequent, often termed common toxocariasis, is a self-limiting and mild febrile illness. Ocular and visceral larva migrans are severe disease manifestations affecting the eye and other internal organs, respectively, but their reported occurrence is rare. The vast majority of symptomatic cases are thought due to common toxocariasis, which has also been associated with cognitive impairment in children. Few studies to date have sought to quantity the health burden of toxocariasis in humans. In this study we provide a preliminary estimation using the Disability-Adjusted Life Year (DALY) approach. We estimate a total of 23,084 DALYs are lost annually in 28 selected countries due to common toxocariasis. Extrapolating based on a global average seroprevalence rate of 19%, we estimate 91,714 DALYs per year are lost across all countries due to toxocariasis, of which 40,912 are attributable to less severe forms, i.e. common toxocariasis, and 50,731 to cognitive impairment in children. Clinically diagnosed and reported ocular and visceral larva migrans represent a small proportion of estimated total health burden. We also found a positive correlation at national level between prevalence in cats or dogs and seroprevalence in humans, but no correlation between estimated soil contamination and seroprevalence in humans. Finally, we estimate the potential economic impact of toxocariasis in selected countries at 2.5 billion USD per annum, from costs of medical treatment and lost income. These preliminary estimates should serve as a call to action for further research and evidence-based measures to tackle toxocariasis.

Zoonotic Onchocerca lupi infection in dogs, Greece and Portugal, 2011-2012.

Onchocerca lupi infection is reported primarily in symptomatic dogs. We aimed to determine the infection in dogs from areas of Greece and Portugal with reported cases. Of 107 dogs, 9 (8%) were skin snip-positive for the parasite. DNA sequences of parasites in specimens from distinct dog populations differed genetically from those in GenBank.

Evidence for direct transmission of the cat lungworm Troglostrongylus brevior (Strongylida: Crenosomatidae).

Metastrongyloids of cats are emerging pathogens that may cause fatal broncho-pulmonary disease. Infestation of definitive hosts occurs after ingestion of intermediate or paratenic hosts. Among metastrongyloids of cats, Troglostrongylus brevior and Troglostrongylus subcrenatus (Strongylida: Crenosomatidae) have recently been described as agents of severe broncho-pulmonary disease. Here, we provide, for the first time, observational evidence suggesting the direct transmission of T. brevior from queen cat to suckling kittens. This new knowledge will have a significant impact on current scientific information of this parasite and shed new light into the biology and epidemiology of metastrongyloid nematodes.

Survival of first-stage larvae of the cat lungworm Troglostrongylus brevior (Strongylida: Crenosomatidae) under different conditions.

Troglostrongylus brevior has recently been recognised as a neglected causative agent of broncho-pulmonary infestation in domestic cats. Nonetheless, information on the biology and ecology of this nematode is still scarce. In this study, the survival of T. brevior first-stage larvae (L1) was evaluated in water and in faecal samples at different controlled temperatures in the laboratory (i.e., 4 and 26°C) or in the outdoor environment (mean temperature 14±3.1°C). Vitality of larvae was microscopically assessed every 7days, until their death. Larval survival ranged from 7days in both water and faeces at 26°C up to 142days at 4°C, respectively. Larvae maintained in the environment (14°C) survived from 35 up to 63days in faeces and water, respectively. Data herein presented clearly indicate that the survival of T. brevior L1s is negatively correlated with temperature (P<0.001). According to the results of this study, the infection in mollusc intermediate hosts in the Mediterranean area may easily occur during winter/spring, while during hot and dry seasons a reduction of transmission should be expected.

Comments on potential efficacy of monthly administrations of spot-on moxidectin 2.5%/imidacloprid 10% in the simultaneous prevention of major canine filarioses.

Information on the efficacy of pharmaceutical protocols for the prevention of the major canine filarioses (i.e., Dirofilaria immitis, Dirofilaria repens, and Acanthocheilonema reconditum) under natural conditions is scant. Chemoprophylaxis for canine filarioses under field conditions deserves to be studied more fully and information about vector biology, ecology, and seasonality has to be well appreciated to correctly set control protocols. It is advisable that researchers planning field trials to assess the efficacy of any product for the prevention of canine vector-borne diseases should consider different eco-epidemiological aspects of diseases, including their dynamics of transmission, which are driven by complex interactions between animals, pathogens, and vectors.

Effect of egg clustering on the fitness of Rhipicephalus sanguineus larvae.

Several biological aspects of Rhipicephalus sanguineus have been studied, but scant information is available on the effect of egg clustering on incubation period and larval survival. Herein, R. sanguineus eggs were separated in groups composed by 1 (GI), 10 (GII), 50 (GIII), 100 (GIV), 500 (GV) and 1,000 (GVI) eggs (10 replicates), and incubation and larval survival period were recorded. No correlation was observed between the amount of egg batches and hatching percentage. Larvae from GI hatched in a longer time (mean 17 days), than those of other groups (mean 14.9 days). This difference was significant in eggs from group GI vs. GV and GI vs. GVI (P<0.05). Conversely, GI displayed a shorter survival period (mean 28.7 days) compared with other groups (P<0.01). The maximum survival time was recorded in GV (mean 49.8 days). Data suggest that R. sanguineus egg clustering affects incubation period and larval survival.

Tick vectors of Cercopithifilaria bainae in dogs: Rhipicephalus sanguineus sensu lato versus Ixodes ricinus.

Recently, dermal microfilariae of a Cercopithifilaria species (Spirurida, Onchocercidae), namely Cercopithifilaria bainae , were detected in dogs from several geographical areas of the Mediterranean basin. Evidence from both laboratory and field studies support the role of the brown dog tick, Rhipicephalus sanguineus sensu lato, as an intermediate host of this nematode. In the present study, we investigated the competence of Ixodes ricinus nymphs as vectors of C. bainae. On November 2012, fully engorged nymphs of I. ricinus (n = 174) and R. sanguineus s.l. (n = 10) were collected from a dog infected by C. bainae. The presence of C. bainae in I. ricinus was assessed by both microscopic dissection of specimens and detection of nematode DNA (PCR), at days 3, 10, 20 and 30 (T1-T4) post-collection; due to the small number of specimens available, R. sanguineus s.l. were examined using the same methods at T4 only. No developing larva of C. bainae was detected in I. ricinus specimens at different time points (T1-T4), even if four of these specimens were PCR-positive at T1. Seven out of ten R. sanguineus s.l. were positive for C. bainae third-stage larvae (L3) at both microscopical and molecular analysis at T4. This study indicates that C. bainae does not develop in I. ricinus nymphs, which preclude the role of this tick as an intermediate host of this parasite. Data presented herein provide new insights into the biology of this filarioid species and will lead to a better understanding of the role of different tick species as vectors of nematodes.

Nodular lesions due to infestation by Dirofilaria repens in dogs from Italy.

BACKGROUND: Canine subcutaneous dirofilariosis caused by Dirofilaria repens is an important cause of human dirofilariosis in the Old World. OBJECTIVES: To describe the clinical and pathological features of infestation by D. repens in dogs with cutaneous nodules and to discuss key diagnostic procedures. ANIMALS: Sixteen privately owned dogs of different breeds, ages and genders with subcutaneous nodules were positive for D. repens on at least one diagnostic test. METHODS: Diagnostic tests included cytological examination of fine-needle aspiration biopsies, detection of circulating larvae by Knott's test, morphological and molecular identification of adult parasites and microfilariae, and histopathological examination of excised nodules. RESULTS: Twelve animals were positive for D. repens microfilariae, using cytology and at least one other diagnostic test. Four of twelve dogs screened by Knott's test had circulating D. repens microfilariae, and three of them were co-infected with Dirofilaria immitis. In eight dogs, adult nematodes were morphologically identified as D. repens. Nodular panniculitis was observed in all cases, with nodules containing either one or several cross-sections of nematodes and microfilariae. The nematodes extracted were identified as D. repens by an RT-PCR on the second internal transcribed spacer (ITS-2). CONCLUSIONS AND CLINICAL IMPORTANCE: Adults and larvae of D. repens can induce skin nodules in dogs, and these lesions should be carefully evaluated in endemic areas. Clinicians should be aware that correct tissue sampling (including the storage of part of a sample in ethanol instead of formalin) is pivotal to achievement of a specific diagnosis. Cytology of fine-needle tissue aspirates was shown to be useful.

Effective immuno-therapeutic treatment of Canine Leishmaniasis.

BACKGROUND: Canine Leishmaniasis (CanL) caused by the L. infantum species is one of the biggest threats to the health of the South American canine population. Chemotherapeutics currently used for the treatment of CanL fail to induce a total parasite clearance while inducing numerous side effects. As CanL is an immunomodulated disease, the use of immuno-treatments should strengthen the deficient immune response of infected dogs. In this study, we evaluated a nasally administered immunotherapy in dogs naturally infected with L. infantum (stage 2), with both visceral and cutaneous manifestations. Noteworthy, some of them were also infected by other parasites (E. canis, D. immitis, A. platys), what worsen their chance of survival. METHODOLOGY/PRINCIPAL FINDINGS: The treatment was based on 2 intranasal (IN.) administrations of a killed L. infantum parasite loaded into maltodextrin nanoparticles, which treatment was compared with the classical oral administration of Miltefosine (2 mg/kg) for 28 days, as well as a combination of these 2 treatments. The results showed that two IN administrations significantly reduced the serology, and were at least as efficient as the chemotherapy to reduce the skin and bone marrow parasite burden, as well as clinical scores, and that unlike Miltefosine treatments, this nasally administered nanoparticle vaccine was without side effects. CONCLUSIONS: These results confirm the feasibility of a simple therapeutic immuno-treatment against L. infantum infected dogs, which is a promising tool for future developments.

Leishmaniases in a tropical area of Brazil: a spatio-temporal analysis.

Leishmaniases are a group of tropical and neglected diseases caused by the protozoa of the genus Leishmania which are transmitted by insect sandflies. Despite of the major efforts undertaken at global level for the control of these diseases, a recent spreading to non-endemic areas been observed. In this study we analyzed the spatio-temporal distribution of Visceral Leishmaniasis (VL) and Tegumentary Leishmaniasis (TL) cases notified in a tropical region of Brazil. A retrospective evaluation was carried out using secondary data (e.g., demographics, origin, age and area of occurrence) of human patients obtained from 2009 to 2019. Choropleth maps with levels of color intensity represented the spatial distribution of VL and TL cases. Overall, 116 (10.5 ± 8.5) cases of leishmaniases were registered in the study area during the whole period. Of those, 61 (5.5 ± 5.5) and 55 (5 ± 4.3) were diagnosed as VL and TL, respectively. VL cases were recorded in 61.9% (13/21) and TL cases in 42.8% (9/21) of the municipalities. Most of the cases were predominantly notified in men from rural areas, and specifically for TL with an age range of 30-59 years old. These findings will be useful to drive strategies of control (e.g., vector control, dog surveillance and notification of suspected cases) against these neglect diseases, preventing the spreading to non-endemic areas.