The most effective systemic treatment in dogs with sarcoptic mange: a critically appraised topic.

BACKGROUND: Sarcoptic mange is a common, pruritic parasitic skin disease of dogs. Due to its highly contagious character, it represents a potential veterinary and public health risk. Because of clinical similarity with other diseases, cross-antigenicity, and low sensitivity of available diagnostic methods, therapeutical trial is frequently used to confirm the disease. Considering the variety of available acaricidal molecules as well as the need to use the most effective treatment, the present paper reviews evidence comparing different types of systemic treatment of canine scabies. RESULTS: Analysis of the results showed that afoxolaner, fluralaner and sarolaner as well as several macrocyclic lactones such as selamectin, moxidectin and milbemycin oxime can lead to parasitological and clinical cure. CONCLUSION: The similarity in the clinical and parasitological efficacy of these substances enhances the need for comparative studies, which could allow the identification of the most efficacious product.

Case report: Identification of the tropical rat mite (Ornithonyssus bacoti) on a domestic donkey in France.

A 25-year-old donkey was referred for a generalized, pruritic and severe exfoliative dermatitis that had been evolving for several years, with deterioration in the last few months. Close examination of the skin surface revealed numerous small, dark, mobile elements identified as Ornithonyssus bacoti confirmed by DNA sequencing. The severity, type and topography of the lesions called for complementary examinations, leading to a second diagnosis of cutaneous epitheliotropic T-cell lymphoma. The lack of clinical improvement after antiparasitic therapy despite parasite clearance, suggests opportunistic behavior of Ornithonyssus bacoti. To the best of our knowledge, this is the first report of the presence of a tropical rat mite on a donkey, thus expanding the known host spectrum of this zoonotic parasite. Further potential questions concern the implication of this new host as a possible source of human contamination.

Epidemiological Survey of the Main Tick-Borne Pathogens Infecting Dogs from the Republic of Moldova.

Despite the significant burden of tick-borne diseases (TBDs), epidemiologic studies are missing, and TBD awareness is low in the Republic of Moldova. Our study is the first to assess the prevalence of the main tick-borne pathogens (TBPs) infecting dogs in this country and associated risk factors. In this cross-sectional, multi-centre study (June 2018-July 2019), blood samples were collected from dogs presenting in veterinary clinics (Chisinau: N = 30) and hosted in public dog shelters (Cahul: N = 42; Chisinau: N = 48). TBPs were assessed by molecular techniques and risk factors by the logistic regression model. Hepatozoon canis was the most prevalent TBP (15.8% [19/120]), followed by Babesia canis (11.7% [14/120]), Anaplasma phagocytophilum (5.8% [7/120]), and Bartonella spp. (0.8% [1/120]). Blood samples tested negative for Borrelia spp., Rickettsia spp., Francisella tularensis, Anaplasma platys, and Ehrlichia canis. Dogs originating from the veterinary clinics had a higher prevalence of A. phagocytophilum infection than those from the shelters (16.6% versus 2.2%, respectively, p = 0.0292; OR: 27.0 [95%CI: 1.4-521.9]). Dogs from Chișinau had a higher prevalence of Hepatozoon canis infection versus those from Cahul (19.2% versus 9.5%, respectively, p = 0.0295; OR: 3.9 [95%CI: 1.1-13.4]). We recommend routine use of acaricides and deworming of dogs to prevent or/and limit TBD spread. Further TBD surveillance studies are needed.

Case Report: Notoedric Mange and Aelurostrongylidosis in Two Domestic Cats From Rural Environment in Romania.

This article describes two cases of notoedric mange concurrent with aelurostrongylidosis in two domestic-owned cats from a rural environment in Romania. Two European shorthair cats originating from the same litter were referred to our clinic, at 2 months apart, with a history of skin lesions, pruritus, weight loss, and respiratory signs. Cats lived mainly outdoor together with the queen and a third littermate. The latter two expressed the same clinical signs and had died before the presentation of the first cat. None of the four cats was vaccinated, dewormed, or treated for external parasites. Coat brushing, skin scrapings, skin cytology, earwax direct microscopic examination, and coproparasitological techniques (flotation and Baermann methods) were used as laboratory procedures. A blood sample was also collected for hematology, blood biochemistry, and feline immunodeficiency virus/feline leukemia virus (FIV/FeLV) test. Notoedres cati mites in high numbers were identified by all complementary tests, alongside fleas and Aelurostrongylus abstrusus first-stage larvae. The blood analysis revealed neutrophilia, and the FIV/FeLV fast test was negative. The cats were successfully treated off-label with selamectin spot-on formulation (Stronghold®, Zoetis) three times at 1- or 4-week intervals. Furthermore, they were treated with amoxicillin trihydrate/clavulanic acid, housed indoor, and fed with a commercial diet. Before presentation to the clinic, the female cat was unsuccessfully treated with a combination of fipronil, S-methoprene, eprinomectin, and praziquantel. During this period, the female cat remained outdoor and fed with home wastes. The cats become negative for A. abstrusus L1 larvae after 2-4 months of treatment. The owners developed pruritic skin lesions 1 month after introducing the first cat in the house. In conclusion, notoedric mange and aelurostrongylidosis can be treated successfully with selamectin as a spot-on formulation and the treatment must be continued until no parasite will be detected through specific techniques. The success of treatment depends on improving the quality of animal life (nutrition and hygiene) and treatment of secondary complications.

Clinical and Microbiological Performances and Effects on Lipid and Cytokine Production of a Ceruminolytic Ear Cleaner in Canine Erythemato-Ceruminous Otitis Externa.

Erythemato-ceruminous otitis externa (ECOE) is the most common type of otitis in dogs and is generally associated with bacterial and/or yeast infections. The performance of an ear cleaner was assessed over two weeks in canine ECOE, associated with a mild or moderate secondary infection, in a prospective open-label study. Forty ear canals with ECOE that did not receive any type of aural treatment and were not cleaned for 7 days were included. Pruritus (PS), 0−3 Otitis Index Score (OTIS-3) and 0−4 scale cytology (CYTO) scores were assessed on Day (D) 0, D7 and D14. Concentrations of a panel of 13 cytokines on the ear canal surface and the lipid profile of the exudate were measured on D0 and D14. From D0 to D12 or D13, the dogs' ears were cleaned daily if the secretion score (SEC) was 3/3, every second day if the score was 2/3 and every third day if the score was 1/3. PS, OTIS-3, SEC and CYTO were significantly lower on D7 compared to baseline (−40%, −31%, −36%, −34%, respectively; p < 0.0001). The same parameters decreased further on D14 (−60%, −53%, −61%, −73%, respectively; p < 0.0001) and amounts of interleukin 8 and chemokine KC-like were also reduced compared to baseline (−45%, p < 0.01; −36%, p = 0.3, respectively). The lipid profile was also modified, with a decrease in free lipids and an increase in bound lipids.

The Effects of Electron Beam Irradiation on the Morphological and Physicochemical Properties of Magnesium-Doped Hydroxyapatite/Chitosan Composite Coatings.

This work reports on the influence of 5 MeV electron beam radiations on the morphological features and chemical structure of magnesium-doped hydroxyapatite/chitosan composite coatings generated by the magnetron sputtering technique. The exposure to ionizing radiation in a linear electron accelerator dedicated to medical use has been performed in a controllable manner by delivering up to 50 Gy radiation dose in fractions of 2 Gy radiation dose per 40 s. After the irradiation with electron beams, the surface of layers became nano-size structured. The partial detachment of irradiated layers from the substrates has been revealed only after visualizing their cross sections by scanning electron microscopy. The energy dispersive X-ray spectral analysis of layer cross-sections indicated that the distribution of chemical elements in the samples depends on the radiation dose. The X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction analysis have shown that the physicochemical processes induced by the ionizing radiation in the magnesium doped hydroxyapatite/chitosan composite coatings do not alter the apatite structure, and Mg remains bonded with the phosphate groups.

An epidemiological survey of Dirofilaria spp. and Acanthocheilonema spp. in dogs from the Republic of Moldova.

BACKGROUND: During the last decades, filarial infections caused by Dirofilaria spp. have spread rapidly within dog populations of several European countries. Increasing scientific interest in filariasis, and the availability of new diagnostic tools, has led to improved knowledge of the biology, morphology, and epidemiology of different species of filarial worms. However, data are still scarce for a number of countries, including the Republic of Moldova. Thus, we assessed the epidemiological status of canine filariasis in the Republic of Moldova to address part of this knowledge gap. METHODS: A total of 120 blood samples were collected between June 2018 and July 2019 from dogs originating from the cities of Cahul and Chisinau. The samples were examined microscopically, and multiplex polymerase chain reaction was performed to evaluate filarioid species diversity. RESULTS: Microscopic examination revealed that 12 dogs (10.0%) were positive for circulating microfilariae. The molecular test showed that one dog was positive for Acanthocheilonema reconditum (0.8%), one for Dirofilaria immitis (0.8%), six for Dirofilaria repens (5.0%), and four (3.3%) harboured a co-infection with D. immitis and D. repens. Prevalence was significantly higher in dogs aged ≥ 2 years. CONCLUSIONS: The epidemiological survey presented here for the Republic of Moldova confirmed the presence D. immitis, D. repens and A. reconditum in dogs that had not received any heartworm preventive.

Toxocara cati and Other Parasitic Enteropathogens: More Commonly Found in Owned Cats with Gastrointestinal Signs Than in Clinically Healthy Ones.

Intestinal parasites are involved in the health and wellbeing of cats and some of them, due to their zoonotic potential, represent a problem for public health. This study aimed to assess the prevalence of parasitic infections in cats with gastrointestinal signs. Fecal samples collected from 137 cats were analyzed by the flotation method using a sodium chloride saturated solution. The overall prevalence of intestinal parasites was 50.4%. Intestinal parasites were significantly (p < 0.0001) more common in cats with digestive clinical signs (66.7%) than in clinically healthy ones (19.2%). Toxocara cati (40.2%) was the most frequently identified intestinal parasite, followed by Cystoisospora spp. (10.2%), hookworms (3.7%), Taeniidae (2.2%), Giardia duodenalis (2.2%), and Toxoplasma gondii (0.7%). Toxocara cati (53.3%, p < 0.0001) and Cystoisospora spp. (15.6%, p < 0.001) were significantly more frequently diagnosed in cats with clinical signs. A lack of deworming in the last three months (OR: 15.9), outdoor access (OR: 13.8), the presence of digestive symptoms (OR: 5.4), and young age (OR: 4.2) were identified as risk factors for T. cati infection by logistic regression analysis. Regardless of age, gastrointestinal signs like vomiting, diarrhea, and inappetence were positively associated with T. cati.

First case report of dermatitis associated with Leporacarus gibbus in cat.

BACKGROUND: Leporacarus gibbus is a highly specific acarian parasitizing in rabbits, with a proven zoonotic potential. While the majority of cases of L. gibbus infestation are asymptomatic, several cases of pruritic cutaneous condition in both laboratory and pet rabbits were reported. Up to date, L. gibbus has not been linked with clinical signs in any other species than rabbits and humans. CASE PRESENTATION: This case report described the clinical case of a 14-month-old cat with a dermatitis linked to L. gibbus. Mites specimens were collected by brushing, followed by light microscopy examination and species identification. To the best of our knowledge, this is the first report of L. gibbus-related dermatitis in cat. CONCLUSIONS: L. gibbus infestation should be considered as a possible differential diagnosis of pruritic skin conditions in cat.

Detection of Leishmania infantum DNA and antibodies against Anaplasma spp., Borrelia burgdorferi s.l. and Ehrlichia canis in a dog kennel in South-Central Romania.

Canine vector-borne diseases are caused by pathogens transmitted by arthropods including ticks, mosquitoes and sand flies. Many canine vector-borne diseases are of zoonotic importance. This study aimed to assess the prevalence of vector-borne infections caused by Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in a dog kennel in Argeș County, Romania. Dog kennels are shelters for stray dogs with no officially registered owners that are gathered to be neutered and/or boarded for national/international adoptions by various public or private organizations. The international dog adoptions might represent a risk in the transmission of pathogens into new regions. In this context, a total number of 149 blood samples and 149 conjunctival swabs from asymptomatic kennel dogs were assessed using serology and quantitative real-time polymerase chain reaction. Antibodies against B. burgdorferi s.l. were detected in one dog (0.6%), anti-Anaplasma antibodies were found in five dogs (3.3%), while ten dogs (6.7%) tested positive for D. immitis antigen. Overall, 20.1% (30/149) of dogs were positive for L. infantum DNA. All samples were seronegative for anti-Leishmania antibodies. When adopting dogs from this region of Romania, owners should be aware of possible infection with especially L. infantum. The travel of infected dogs may introduce the infection to areas where leishmaniasis is not present.

Multiple Tick-Borne Pathogens in Ixodes ricinus Ticks Collected from Humans in Romania.

Ticks are medically important vectors of infectious diseases that are able to transmit pathogens to humans and animals. Tick-borne diseases represent a major health concern, posing an increasing risk to the public health during the last century and affecting millions of people. The aim of the current study was to provide epidemiological data regarding the presence of certain tick-borne pathogens in ticks feeding on humans in Romania. Overall, 522 Ixodes ricinus ticks collected from humans were screened for six pathogens: Borrelia spp., Neoehrlichia mikurensis, Babesia spp., Coxiella spp., Bartonella spp., and Francisella tularensis. Ticks attached to humans were collected between 2013-2015 in Cluj County, Romania. Conventional, nested and quantitative PCR were used to detect specific genetic sequences of each pathogen. For identifying the infectious agents, positive samples were sequenced. The infection prevalence was 21.07% from which 8.18% were mixed infections. The detected agents were Borrelia spp., N. mikurensis and Babesia spp. The present data reveal the endemic occurrence of potentially zoonotic pathogens in Romania. Revealing the current distribution of tick-borne pathogens in ticks collected from humans may provide new insights in understanding the complex ecology of tick-borne diseases and enlightens current knowledge about the infection prevalence at local, regional and national levels.

Molecular Survey of Metastrongyloid Lungworms in Domestic Cats (Felis silvestris catus) from Romania: A Retrospective Study (2008-2011).

: Background: Lungworms are recognized as important agents in the pathology of the respiratory system in domestic cats. While Aelurostrongylus abstrusus is worldwide known and studied, Troglostrongylus brevior has gained the attention of the scientific community only in the last decade. The pathogenicity of this species seems to be higher than A. abstrusus, causing more severe clinical presentations and being potentially fatal, especially in young animals. METHODS: In this study, 371 DNA isolates of faecal samples were tested by multiplex polymerase chain reaction for the presence of A. abstrusus, T. brevior, and Angiostrongylus chabaudi. RESULTS: The results showed that 30.2% and 6.7% of the investigated domestic cats were positive for A. abstrusus and T. brevior respectively, stressing out the importance of these parasites as agents of respiratory conditions in domestic cats from Romania. None of the samples were positive for A. chabaudi. The age, the outdoor access, and the lack of deworming were identified as significant risk factors for infection with A. abstrusus. CONCLUSIONS: This paper represents the first report of T. brevior in domestic cats from Romania. Moreover, it presents the most northern localization in Europe of T. brevior.

First report of canine ocular thelaziosis in the Republic of Moldova.

BACKGROUND: Countries of eastern Europe are considered, due to several risk factors, more vulnerable to infections with newly (re)emerging pathogens. During the last decade, in several European countries, reports of autochthonous cases of ocular thelaziosis due to Thelazia callipaeda have been published, posing a great concern from both veterinary and public health perspective. However, in the Republic of Moldova only limited epidemiological data are available regarding zoonotic vector-borne pathogens and, until now, no data exist on the zoonotic nematode T. callipaeda. METHODS: In September 2018, an 11-year-old dog, mixed-breed, intact male was referred to a private veterinary clinic from Chișinau, Republic of Moldova, with a history of 2 weeks of an ocular condition affecting the right eye. The ophthalmological exam revealed the presence of nematode parasites in the conjunctival sac and under the third eyelid. The collected parasites were identified by morphological techniques and molecular analysis. RESULTS: A total of 7 nematodes were collected, and 5 females and 2 males of T. callipaeda were identified morphologically. The BLAST analysis confirmed the low genetic variability of this parasite in Europe. The travel history of the patient allowed us to confirm the autochthonous character of the case. CONCLUSIONS: To the best of our knowledge, this is the first report of thelaziosis in dogs from the Republic of Moldova, which confirms the spreading trend of T. callipaeda and the existence of an autochthonous transmission cycle of this zoonotic parasite in the country.

Efficacy of a commercial herbal formula in chicken experimental coccidiosis.

BACKGROUND: Coccidiosis represents a serious threat to the poultry industry, affecting production and causing high morbidity, mortality and significant costs resulting from treatment and prophylaxis. In-feed anticoccidials have been used for decades for managing avian coccidiosis and were very effective until drug resistance emerged. The use of natural remedies has become a promising alternative in combating coccidiosis in chickens. Therefore, the purpose of the present study was to assess the efficiency of a commercial herbal formula (H), as oral liquid preparations, in experimental chicken coccidiosis. METHODS: Two independent controlled battery experiments (BE1 and BE2) were designed and the product was tested in 3 different formulas (H1, H2 and H3): H1 contained a propylene glycol extract of Allium sativum and Thymus serpyllum; H2 contained Origanum vulgare, Satureja hortensis and Chelidonium majus; and H3 contained Allium sativum, Urtica dioica, Inula helenium, Glycyrrhiza glabra, Rosmarinus officinalis, Chelidonium majus, Thymus serpyllum, Tanacetum vulgare and Coriandrum sativum. Chickens were divided into five groups for each BE as follows: (i) uninfected untreated control (UU1, UU2); (ii) infected untreated control (IU1, IU2); (iii) infected treated with amprolium (ITA1, ITA2); and (iv, v) two experimental groups infected treated with H1 (ITH1) and H2 (ITH2) formulas in the BE1 and with H3 (ITH3-5 and ITH3-10) formula in the BE2. The chickens from infected groups were challenged with 5000 (BE1) and 50,000 (BE2) sporulated oocysts of Eimeria spp. (E. acervulina, E. tenella and E. maxima), respectively. The anticoccidial efficacy was assessed by recording the following: oocysts output (OPG), lesion score (LS), weight gain (WG), feed conversion ratio (FCR) and anticoccidial index (ACI). Additionally, polyphenolics and flavonoids (caffeic-chlorogenic acid, apigenin, kaempferol, luteolin, quercitin, quercitrin) from herb extracts found in H3 formula were determined by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. RESULTS: H1 and H2 reduced the WG, and increased the FCR and OPG compared with controls. H1 reduced the duodenal lesions, whilst H2 reduced the caecal lesions, compared with control. H3 decreased the OPG of Eimeria spp., reduced the total lesion score and improved the zootechnical performance (weight gain and feed conversion ratio). According to ACI value, H1 and H2 had no efficacy on Eimeria spp. infection, but H3 had good to marked anticoccidial effect, the ACI being slightly greater in the group ITH3-5. According to the results of LC-MS/MS, the concentration of polyphenols in H3 formula was the highest, the sum of chlorogenic acid and caffeic acid being 914.9 µg/ml. CONCLUSIONS: H3 formula is a promising natural anticoccidial and field trials are recommended in order to validate the obtained data.

Updates on the distribution and diversity of sand flies (Diptera: Psychodidae) in Romania.

BACKGROUND: Phlebotomine sand flies (Diptera: Psychodidae) are haematophagous insects that transmit the protozoan parasite Leishmania infantum (Kinetoplastida: Trypanosomatidae), the main causative agent of both zoonotic visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in the Mediterranean basin. Eight species of sand flies have been previously recorded in Romania: Phlebotomus papatasi, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus perfiliewi, Phlebotomus neglectus, Phlebotomus longiductus, Phlebotomus balcanicus and Sergentomyia minuta. Three of them (P. perfiliewi, P. neglectus and P. balcanicus) were incriminated as vectors of L. infantum. Recent reports of autochthonous CanL in Romania require updates on sand fly distribution and diversity in this country. METHODS: Between 2013-2014 and 2016-2018, CDC light traps and mouth aspirators were used to collect sand flies in 132 locations from Romania, indoors and around various animal species shelters. Species identification of collected specimens was done using morphological keys, genetic tools and MALDI-TOF protein profiling. RESULTS: Sand flies were present in seven localities (5.3%): Eibenthal, Baia Noua, Gura Vaii (south-western Romania, Mehedinti County); Fundatura, Pâhnesti, Epureni (eastern Romania, Vaslui County); and Schitu (southern Romania, Giurgiu County). Of the total number of collected sand flies (n = 251), 209 (83.27%) were Phlebotomus neglectus, 39 (15.53%) P. perfiliewi, 1 (0.40%) P. papatasi, 1 (0.40%) P. balcanicus and 1 (0.40%) P. sergenti (sensu lato). CONCLUSIONS: We confirmed the presence of five sand fly species previously recorded in Romania. However, their updated distribution differs from historical data. The diversity of sand fly species in Romania and their presence in areas with Mediterranean climatic influences constitutes a threat for the reemergence of vector-borne diseases. In the context of CanL and VL reemergence in Romania, but also due to imported cases of the diseases in both humans and dogs, updates on vector distribution are imperative.

Ocular thelaziosis due Thelazia callipaeda (Spirurida: Thelaziidae) in Romania: first report in domestic cat and new geographical records of canine cases.

Thelazia callipaeda has been regarded in Europe in the last few decades as a "new" agent of an ocular disease in dogs, cats, several wild species, and not least in humans. After the first European description in Italy, in 1989, the parasite has been reported in many other countries, showing a widespread distribution of this parasite. The first case of canine thelaziosis in Romania has been reported in 2015, and it has been followed by other studies confirming the presence of the nematode in dogs or broadening the host spectrum with some wild carnivore species that have been found positive. Despite being considered suitable primary hosts for T. callipaeda, infection with T. callipaeda in domestic cat is rarely reported in Europe, probably due to some behavioral and physical particularities of this host. This study describes the first autochthonous report of T. callipaeda infection in a domestic cat from Romania. Additionally, to these findings, new geographical records of canine thelaziosis are being revealed, confirming the increased risk of this emerging vector-borne zoonosis.

Prevalence and risk factors associated with endoparasitic infection in dogs from Transylvania (Romania): A retrospective study.

During six years (April 2010-April 2016) we examined individual feces samples collected from 1314 dogs located in Center and Northwest Romania (Transylvania). Stool samples were analyzed by saturated salt flotation, sedimentation technique and modified Ziehl-Nielsen staining method. The overall prevalence of endoparasitic infections was 66.6% (n=875). Sixteen species/genera of endoparasites were identified. The most prevalent species were Ancylostoma caninum/Uncinaria stenocephala (33.0%) (p=0.0001) followed by Trichocephalus vulpis (25.0%). Mixed infections, were significantly more frequent (p=0.0001) than single species infections. The age and the living condition/service of dogs were identified as the main risk factors for infection with endoparasites.

Mesocarnivores and macroparasites: altitude and land use predict the ticks occurring on red foxes (Vulpes vulpes).

BACKGROUND: The red fox Vulpes vulpes is the most common mesocarnivore in Europe and with a wide geographical distribution and a high density in most terrestrial habitats of the continent. It is fast urbanising species, which can harbor high numbers of different tick species, depending on the region. Here we present the results of a large-scale study, trying to disentangle the intricate relationship between environmental factors and the species composition of ectoparasites in red foxes. The samples were collected in Transylvania (Romania), a region with a diverse geography and high biodiversity. The dead foxes (collected primarily through the National Surveillance Rabies Program) were examined carefully for the presence of ticks. RESULTS: Ticks (n = 4578) were found on 158 foxes (out of 293 examined; 53.9%). Four species were identified: Dermacentor marginatus, Ixodes canisuga, I. hexagonus and I. ricinus. The most common tick species was I. hexagonus (mean prevalence 37.5%, mean intensity 32.2), followed by I. ricinus (15.0%; 4.86), I. canisuga (4.8%; 7.71) and D. marginatus (3.7%; 3.45). Co-occurrence of two or more tick species on the same host was relatively common (12.6%), the most common co-occurrence being I. hexagonus - I. ricinus. For D. marginatus and I. canisuga the highest prevalence was recorded in lowlands, for I. hexagonus in hilly areas, while for I. ricinus in mountains. CONCLUSIONS: Altitude influenced the intensity of parasitism, with highest intensity observed for all Ixodes species in hilly areas. Dermacentor marginatus occurred only in lowlands, I. canisuga in lowlands and hilly areas while the other two species occurred in all of the regions studied. Foxes from lower altitudes had the most tick species associated, with most incidences of co-parasitism also recorded here. Land use affected tick-species composition, with the presence of D. marginatus strongly associated with the extension of arable areas and lack of forests. The presence of I. hexagonus was determined only by the extent of arable lands. As foxes are frontrunners of wildlife urbanization process, with a continuous increase of their numbers in urban areas, the knowledge of their ticks' ecology (and the pathogens vectored by these) is of utmost importance.

Dirofilaria immitis and D. repens show circadian co-periodicity in naturally co-infected dogs.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively. The females are larviparous, releasing the larvae (microfilariae) into the bloodstream, which further develop in mosquito vectors. However, microfilaremia greatly fluctuates during a 24-h period. As the sampling time can greatly influence the accuracy of diagnosis, the aim of the present study was to assess the circadian periodicity of D. immitis and D. repens in naturally co-infected dogs in an endemic area of Romania and to investigate possible differences of periodicity between these two species. METHODS: Overall, four dogs harbouring natural co-infection with D. immitis and D. repens were selected and sampled every two hours for two consecutive days: two dogs in July 2014 and two in July 2015. At each sampling time, a 0.7 ml blood sample was taken. Modified Knott's test was performed on 0.5 ml, and the remaining 0.2 ml were used for DNA extraction and molecular amplification, both in single and duplex PCR reactions. Microfilariae of both species were morphologically identified and counted in each collected sample, microfilaremia was calculated, and fluctuation was charted. RESULTS: The dynamics of microfilaremia showed similar patterns for both Dirofilaria species. In all four dogs, D. immitis was present at all sampling times, with several peak values of microfilaremia, of which one was common for all dogs (1 am), while minimum counts occurred between 5 and 9 am. Similarly, for D. repens, one of the peak values was recorded in all dogs at 1 am, while minimum counts (including zero) occurred at 9 and 11 am. Single species-specific PCR reactions were positive for both D. immitis and D. repens in all collected samples, while duplex PCR failed to amplify D. repens DNA in many cases. CONCLUSIONS: Both Dirofilaria immitis and D. repens microfilariae are subperiodic, following a similar variation pattern, with peak values of microfilaremia registered during the night in Romania. Duplex PCR fails to identify the infection with D. repens in co-infected dogs when the ratio of microfilaremia is in favour of D. immitis.

Clinical and pathological effects of Dirofilaria repens and Dirofilaria immitis in a dog with a natural co-infection.

Canine dirofilarioses are mosquito-borne zoonotic diseases with a continuous expansion of their geographical distribution, as a consequence of different climatic and ecological factors. Dirofilaria immitis, the aetiological agent of heartworm disease, has gained a major veterinary interest, mainly due to its severe clinical implication. In the last decades, D. repens, despite of being regarded as a less pathogenic species, regained attention due to its recognized zoonotic potential. Romania has been traditionally regarded as a non-endemic country, but recent epidemiological surveys are highlighting the presence of both D. repens and D. immitis. The present case report describes the clinical and pathological features of a natural co-infection with D. repens and D. immitis in 5year old male Boxer from north-western Romania. Based on clinical and laboratory investigations, a diagnosis of kidney failure and chronical cystitis was established. The parasites were identified by morphology and confirmed by PCR and sequencing. To the best of our knowledge this is the first worldwide report of the ectopic presence of D. repens adults in the pelvic cavity and mesentery by natural infection and first report of the ocular localization of this parasite for Romania. The clinical and paraclinical findings suggest the implication of D. repens microfilariae in the aetiology of histopathological lesions. Further investigations are needed in order to establish the actual pathogenic potential of D. repens, a frequently neglected parasite.