Molecular survey of Cytauxzoon spp. and Hepatozoon spp. in felids using a novel real-time PCR approach.

Tick-transmitted apicomplexans of the genera Cytauxzoon and Hepatozoon affect a wide range of felids worldwide, but little is known about them. Recently, several studies addressed the species circulating in Europe, their distribution, and their hosts. Molecular assays are the method of choice for their detection. Unfortunately, conventional PCRs already described are time- and cost-consuming and specific for either Hepatozoon or Cytauxzoon detection. This study was developed to evaluate (i) the occurrence of Cytauxzoon and Hepatozoon in felids using a fast and cost-saving real-time PCR capable of detecting both protozoa simultaneously, (ii) the distribution of Cytauxzoon and Hepatozoon species in north-eastern Italy, and (iii) the involvement of other susceptible felid hosts in the same area. An SYBR® Green-based real-time PCR with primers targeting the 18S-rRNA was validated and applied to 237 felid samples, i.e., whole blood from 206 domestic cats and 12 captive exotic felids, and tissues from 19 wildcats. Positive results were obtained by melting temperature curve analysis due to the specific melting peak (i.e., 81°C Cytauxzoon spp.; 78-78.5°C Hepatozoon spp.). Positive samples were subjected to conventional PCR, followed by sequencing for species identification. Phylogenetic analyses were performed to assess relatedness among European isolates. Data on domestic cats (age class, sex, origin, management, and lifestyle) were recorded, and statistical analyses were performed to identify potential risk factors. A total of 31 (15%) domestic cats were positive for Hepatozoon spp. (i.e., 12 for H. felis, 19 for H. silvestris), while six (2.9%) for C. europaeus. The prevalence of Hepatozoon felis was significantly (p < 0.05) higher in domestic cats, while H. silvestris was higher in strays and animals from the Eastern region (i.e., Friuli-Venezia Giulia). Cytauxzoon europaeus was detected only in stray cats from Friuli-Venezia Giulia (province of Trieste). Among captive felids, one tiger was infected with H. felis and another with H. silvestris; eight out of 19 (42%) wildcats were positive for Hepatozoon spp. (i.e., six with H. felis, two with H. silvestris) and four out of 19 (21%) for Cytauxzoon europaeus. Outdoor lifestyle and origin (i.e., Friuli-Venezia Giulia region) were the most relevant risk factors for H. silvestris and C. europeus infections. Conversely, H. felis was most frequently isolated from domestic cats, suggesting different modes of transmission.

Use of In-Clinic Diagnostic Kits for the Detection of Seropositivity to Leishmania infantum and Other Major Vector-Borne Pathogens in Healthy Dogs.

Canine Vector-Borne Diseases (CVBDs) are widespread in Europe and enzootic in many other countries. Though severe illnesses may occur, dogs living in enzootic areas often show vague or no clinical signs of CVBDs. Undiagnosed infections/co-infections in subclinically infected animals favor the spread of CVBDs and increase the risk of transmission to other animals and, in some cases, humans. This study has evaluated the exposure of dogs living in key enzootic countries, i.e., Italy and Greece, to major CVBDs via the use of in-clinic diagnostic kits. Overall, 300 privately owned dogs without/with single mild clinical signs living in different regions of Italy (n. 150) and Greece (n. 150) were included in the study. As part of a clinical examination, a blood sample was collected from each dog and subjected to two serological rapid tests, i.e., the SNAP® 4Dx®Plus (IDEXX Laboratories Inc.) for the detection of antibodies against Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi s.l. and Dirofilaria immitis antigen and the SNAP®Leishmania (IDEXX Laboratories Inc.) for the detection of antibodies against Leishmania infantum. In all, 51 dogs (17%; 95% CI 12.9-21.7) were seropositive to at least 1 pathogen, i.e., 4 in Italy (2.7%; 95% CI 1.4-13.1) and 47 in Greece (31.3%; 95% CI 24-39.4). Dirofilaria immitis antigens were found in 39 dogs (13%; 95% CI 9.4-17.3), while antibodies against Ehrlichia, Anaplasma and Leishmania were detected in 25 (8.3%; 95% CI 5.5-12.1), 8 (2.7%; 95% CI 1.2-5.2) and 5 (1.7%; 95% CI 0.5-3.8) dogs, respectively. None of the dogs tested seropositive for B. burgdorferi s.l. Statistical analyses were performed to evaluate associations between exposure to CVBDs and possible risk factors. The present results indicate that dogs living in enzootic areas may be seropositive for one or more CVBDs in absence of clinical signs. Rapid kits are among first line tools for the detection of CVBDs in clinical settings, as they are cost-effective, straightforward and quick to use. Also, in-clinic tests used herein allowed detection of co-exposure to CVBDs investigated.

Seroexposure to Zoonotic Anaplasma and Borrelia in Dogs and Horses That Are in Contact with Vulnerable People in Italy.

Equine and canine anaplasmosis and borreliosis are major tick-borne zoonotic diseases caused by Anaplasma phagocytophilum and various species of Borrelia (the most important being Borrelia burgdorferi s.l.), respectively. This study evaluated the seroexposure to Anaplasma and Borrelia in dogs and horses used in Animal-Assisted Interventions or living in contact with children, elderly people or immunocompromised persons. A total of 150 horses and 150 dogs living in Italy were equally divided into clinically healthy animals and animals with at least one clinical sign compatible with borreliosis and/or anaplasmosis (present at clinical examination or reported in the medical history). Serum samples were tested with ELISA and immunoblot for the presence of antibodies against A. phagocytophilum and B. burgdorferi s.l., and the association between seropositivity and possible risk factors was analyzed using multivariate and univariate tests. Overall, 13 dogs (8.7%) and 19 horses (12.7%) were positive for at least one of the two pathogens. In addition, 1 dog (0.7%) and 12 horses (8%) were positive for antibodies against A. phagocytophilum, while 12 dogs (8.0%) and 10 horses (6.7%) had antibodies against B. burgdorferi s.l. Tick infestation in the medical history of the dogs was significantly associated with seropositivity to at least one pathogen (p = 0.027; OR 7.398). These results indicate that, in Italy, ticks infected with A. phagocytophilum and/or B. burgdorferi circulate in places where horses and dogs are in contact with people at risk of developing severe diseases. Awareness should be increased, and adequate control plans need to be developed to protect human and animal health, especially where vulnerable, at-risk individuals are concerned.

Epidemiological Survey on Tick-Borne Pathogens with Zoonotic Potential in Dog Populations of Southern Ethiopia.

Dogs are known to host several tick-borne pathogens with zoonotic potential; however, scant information is available on the epidemiology of these pathogens in low-income tropical countries and in particular in sub-Saharan Africa. With the aim of investigating a wide range of tick-borne pathogens (i.e., Rickettsia spp., Anaplasma spp., Erhlichia spp., Borrelia spp., Hepatozoon spp. and Babesia spp.), 273 blood samples were collected from dogs in selected districts of Ethiopia and analyzed by real-time and/or end-point PCR. The results of the study showed that Hepatozoon canis was the most prevalent pathogen (53.8%), followed by Anaplasma phagocythophilum (7.0%), Babesia canis rossi (3.3%), Ehrlichia canis (2.6%) and Anaplasma platys (2.2%). Furthermore, five samples tested positive for Borrelia spp., identified as Borrelia afzelii (n = 3) and Borrelia burgdorferi (n = 2), and two samples for Rickettsia spp., identified as Rickettsia conorii (n = 1) and Rickettsia monacensis (n = 1). The finding of Anaplasma phagocythophilum and different species of the genera Borrelia and Rickettsia with zoonotic potential was unexpected and alarming, and calls for further investigation on the roles of dogs and on the tick, species acting as vector in this specific context. Other pathogens (Hepatozoon canis, Babaesia canis rossi, Anaplasma platys, Ehrlichia canis) are already known to have an important impact on the dogs' health but have minor zoonotic potential as they were rarely or never reported in humans. Dogs from rural areas were found to be at higher risk for different pathogens, probably due to the presence of other wild canids in the same environment. The findings of the present study contribute to a better knowledge of the epidemiology of tick-borne pathogens, which is relevant to human and animal health.

First autochthonous clinical case of Hepatozoon silvestris in a domestic cat in Italy with unusual presentation.

Hepatozoon spp. is the causative agent of a vector-borne parasitic disease in many animal species. In felids, Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris have been molecularly isolated. Hepatozoonosis usually causes asymptomatic infections in domestic cats, but clinical cases have recently been reported in Europe. We describe the first Italian case of hepatozoonosis in a cat with an unusual presentation. An 11-year-old neutered European shorthair cat was urgently hospitalized for intestinal intussusception. Hematology, biochemistry, FIV-FeLV tests, blood smears and molecular investigation targeting the 18S rRNA gene of Hepatozoon spp. were performed on blood samples; in addition, histological and molecular investigations were performed to analyze surgical samples to identify Hepatozoon infection. Hepatozoon gamonts were detected in granulocytes in the blood smear, and Hepatozoon spp. DNA was confirmed by PCR on blood. The intussusception was caused by a sessile endoluminal nodule that was surgically removed. Histologically, many elements referring to parasitic tissue forms were identified in the intestinal cells, and then the specimens were molecularly confirmed to harbor H. silvestris. This is the first description of symptomatic hepatozoonosis in a domestic cat in Italy. Hepatozoon silvestris has been described in wild felids, which are usually resilient to the infection, whereas the domestic cat seems to be more susceptible. Indeed, H. silvestris in cats usually presents tropism for skeletal muscle and myocardium with subsequent clinical manifestations. This is the first description of a domestic cat with H. silvestris localized in the intestinal epithelium and associated with intussusception.

Comparison of Different Copromicroscopic Techniques in the Diagnosis of Intestinal and Respiratory Parasites of Naturally Infected Dogs and Cats.

Several copromicroscopic techniques, including tools belonging to the FLOTAC group, are available for the qualitative and/or quantitative diagnosis of canine and feline parasitoses. The present study was carried out to compare the diagnostic performance of different copromicroscopic methods for detecting common intestinal and extra-intestinal parasites of dogs and cats. Fecal samples of 100 dogs and 105 cats were randomly selected from different regions of Italy. All samples were subjected to conventional flotation, McMaster, Mini-FLOTAC, and Baermann. Fifty-six dogs and twenty-five cats were found positive to at least one technique, and, among them, flotation (55% and 20.9% of the dogs and cats, respectively) and Mini-FLOTAC (52% and 20.9% of the dogs and cats, respectively) detected the highest number of positive samples. Larvae of the feline metastrongyloids Aelurostrongylus abstrusus and Troglostrongylus brevior were identified only using the Baermann test in two (1.9%) and one (0.9%) cat respectively. No larvae were found with the Baermann examination of dog feces or any of the other methods. The present results show that the Mini-FLOTAC represents a possible alternative to conventional flotation in clinical settings for the detection of intestinal and respiratory parasites e.g., Toxocara spp., Toxascaris leonina, Ancylostomatidae, Cystoisospora spp., Trichuris vulpis and Capillaria spp., although Baermann's test remains the most recommended technique for the diagnosis of infections caused by metastrongyloid lungworms.

Evidence of Dirofilaria immitis in Felids in North-Eastern Italy.

Dirofilaria immitis is a mosquito-borne nematode, causing heartworm (HW) disease in wild and domestic canids. HW can also affect felids with different clinical patterns from asymptomatic pictures to sudden death, making the monitoring and diagnosis complicated. Canine HW is endemic in North-eastern Italy; however, very little information has been recorded for felids. This study aims to provide new information on HW in felids in North-eastern Italy. Two hundred and six domestic cats from Veneto, Friuli-Venezia Giulia, Trentino Alto-Adige regions (North-eastern Italy), nine captive felids from zoological parks from Veneto, and nineteen European wildcats from Friuli Venezia Giulia were recruited. Sera/plasma was analysed for the detection of anti-HW antibodies (Ab) and HW antigens (Ag); positive blood samples were molecularly analysed, targeting the HW DNA (5S-rRNA gene). Twelve out of two hundred and six (5.8%) cats presented with Ab, and three out of two hundred and six (1.5%) presented with Ag, mainly those from the Veneto region, already known as a canine HW-endemic area. Among Ab-positive cats, two were from Belluno, a mountain province previously considered free, suggesting the expansion of HW into the northern areas. No cats were positive for both Ab and Ag. Three out of nineteen (15.8%) wildcats were Ag-positive, constituting the first HW report in Italy. No captive felids were positive. Dirofilaria immitis DNA was not amplified in positive samples, suggesting the low sensitivity of PCR on blood. This study provides new data on the occurrence of HW in domestic cats and wildcats in North-eastern Italy.

A Comparison of Copromicroscopic and Molecular Methods for the Diagnosis of Cat Aelurostrongylosis.

The gold standard method for the diagnosis of cat aelurostrongylosis is the detection of Aelurostrongylus abstrusus first stage larvae with the Baermann's examination. Nevertheless, molecular assays have shown higher diagnostic performances compared to copromicroscopy. This study evaluated the usefulness of an A. abstrusus species-specific PCR on different biological samples collected in clinical settings from 100 privately-owned cats in Italy (n. 60) and Greece (n. 40). A fecal sample was collected from each animal and a pharyngeal swab was also obtained for cats from Italy. All stool samples were subjected to flotation and Baermann's test. The cats were categorized in three groups based on the results of copromicroscopy, i.e., Group A (n. 50 cats with A. abstrusus infection regardless of positivity for other helminths), Group B (n. 25 cats negative for A. abstrusus but positive for at least one of any other helminth), Group C (n. 25 cats negative for any helminth). DNA was extracted from individual aliquots of feces, flotation supernatant, Baermann's sediment and the pharyngeal swab and then subjected to a PCR specific for A. abstrusus. At least one fecal aliquot or the pharyngeal swab scored positive by the A. abstrusus-specific PCR for 48/50 (96%) cats enrolled in Group A; in particular, 38/50 (76%), 35/50 (70%), 41/50 (82%) and 21/25 (84%) DNA extracts from feces, flotation supernatant, Baermann's sediment and pharyngeal swabs were positive by PCR. These results confirm that molecular tools are highly sensitive and specific and indicate that pharyngeal swabs are the most suitable sample for molecular analysis in clinical settings.

Monitoring and detection of new endemic foci of canine leishmaniosis in northern continental Italy: An update from a study involving five regions (2018-2019).

Canine leishmaniosis (CanL) is an emerging zoonosis caused by Leishmania infantum and transmitted in southern Europe by phlebotomine sand flies of the subgenus Phlebotomus (Larroussius). Endemic foci of CanL have been recorded in northern continental Italy since early 1990s and attributed to the northward expansion of vector populations due to climatic changes in association with travelling/relocated infected dogs from the southern Mediterranean littoral. In this study, further spread of endemic Leishmania foci was monitored during 2018-2019 in five regions (Aosta Valley, Piedmont, Lombardy, Veneto and Friuli-Venezia Giulia), with focus to territories where investigations were not performed, or they have been inconclusive. Clinical cases of CanL identified by local veterinary practitioners and confirmed by reference diagnosis centers were regarded as autochthonous if their origin from, or travel to, areas endemic for CanL were excluded in the previous ≥2 years. Around these index cases, i) serosurveys for L infantum were carried out where indicated, ii) sampling from potential autochthonous cases in healthy or clinically-suspected resident dogs was intensified by collaborating veterinary practitioners, and iii) suitable sites were investigated for the presence of competent phlebotomine vectors. Fifty-seven municipalities whose enzootic status of CanL was unreported before 2018, were identified as endemic. The stability of 27 foci recorded over the past decade, was also confirmed. Competent phlebotomine vectors, mainly Phlebotomus perniciosus, were collected for the first time in 23 municipalities. The newly recorded endemic municipalities appear to be distributed over a west-to-east decreasing gradient: 30 in Piedmont, 21 in Lombardy, 4 in Veneto and 2 in Friuli-Venezia Giulia. As regards Veneto, it should be noted that a relatively restricted territory was investigated as several municipalities of the region had already been surveyed and detected as endemic for CanL in the past. Cold climate conditions of the easternmost region of Friuli-Venezia Giulia bordering non-endemic territories of Slovenia, are probably less favorable to L infantum transmission.

Simultaneous Exposure to Angiostrongylus vasorum and Vector-Borne Pathogens in Dogs from Italy.

Several drivers have recently fostered the expansion of Angiostrongylus vasorum throughout Europe, where Vector-Borne Pathogens (VBPs) are also spreading. However, the level of simultaneous risk of infection is still unknown in canine populations. This study evaluated the simultaneous exposure to A. vasorum and major canine VBPs in dogs of Italy. Sera of 294 dogs were subjected to two ELISAs, detecting A. vasorum circulating antigens and antibodies against the parasite, and to the following assays: (i) SNAP® 4DX (IDEXX Laboratories Inc.) detecting Dirofilaria immitis antigens, and antibodies vs. Borrelia burgdorferi, Anaplasma spp. and Ehrlichia spp. and (ii) IFAT for the detection of antibodies vs. Leishmania infantum, Babesia canis and Rickettsia conorii. Twenty-two (7.5%, CI: 4.8-11.1%) and six (2%, CI: 0.7-4.4%) dogs scored positive for circulating A. vasorum antibodies and antigens, respectively. Seventeen dogs (5.8%, CI: 3.4-9.1%) were positive for A. vasorum antibodies + at least one VBP, three (1%, CI: 0.2-3%) for A. vasorum antigen + at least one VBP, while one dog (0.3%, CI: 0.01-1.88%) was positive for A. vasorum antigen + A. vasorum antibodies + B. canis antibodies. These results show that dogs living in different regions of Italy are at risk of simultaneous infections with both A. vasorum and VBPs. Despite the same scenario being likely in other countries of Europe, the current knowledge is scant. Therefore, further studies are warranted to amplify current epizootiological information and to understand whether control programs should be improved.

Cytauxzoon sp. and Hepatozoon spp. in Domestic Cats: A Preliminary Study in North-Eastern Italy.

Knowledge on the presence of Cytauxzoon sp. and Hepatozoon spp. in Italy is scant and mostly limited to a few areas of Northern and Southern regions, respectively. The present study updated the current epidemiological scenario by investigating the occurrence of these protozoa in domestic cats from three broad regions of North-Eastern Italy. Blood samples from cats at risk of vector-borne diseases were processed by PCR to detect Cytauxzoon and Hepatozoon DNA. Blood smears were observed for haemoparasite inclusions. The influence of cat individual data (e.g., provenance, management, indoor/outdoor lifestyle) on the prevalence of haemoprotozoan infections was statistically evaluated. Among 158 cats, Cytauxzoon and Hepatozoon DNA were detected in 6 (3.8%) and 26 (16.5%) animals, respectively. No Hepatozoon gamonts were detected in blood smears, whereas all Cytauxzoon PCR-positive samples were microscopically positive, though with low levels of parasitaemia. Two species of Hepatozoon were identified, Hepatozoon felis (n = 10) and Hepatozoon silvestris (n = 16). Hepatozoon silvestris prevalence values were significantly (p < 0.05) higher in the region Friuli Venezia Giulia and in stray cats. Cytauxzoon sp. was detected in 6/39 (15.4%) stray cats from Friuli Venezia Giulia (Trieste province). These data add new information on the occurrence of these neglected protozoa in domestic cats' populations.

Efficacy of fipronil/(S)-methoprene/eprinomectin/praziquantel (Broadline®) against Thelazia callipaeda in naturally infected cats.

BACKGROUND: The present clinical field trial was conducted to assess the efficacy of a broad-spectrum parasiticide spot-on formulation containing eprinomectin (Broadline®) against Thelazia callipaeda eyeworm in naturally infected cats. METHODS: Fifteen privately owned cats harboring at least one live adult T. callipaeda were included in the study. Cats were randomly allocated to an untreated control group of seven cats or to a Broadline®-treated group of eight cats. Cats were treated on Day 0; ocular examinations were performed at inclusion and on Days 7 and 14; eyeworms were recovered and counted on Day 14. The primary efficacy assessment was based on group comparison of number of T. callipaeda on Day 14. RESULTS: Seven days after treatment, six of eight treated cats were negative for eyeworm infection per visual examination, and on Day 14 no eyeworms were found in the treated cats while the seven untreated cats were still infected (geometric mean: 1.97). All cats had inflammatory ocular signs at inclusion; on Day 14, five of eight treated cats had recovered while all untreated control cats were still symptomatic. All collected parasites were confirmed to be T. callipaeda by morphology and molecular characterization. CONCLUSIONS: A single treatment with Broadline® provided 100% efficacy against feline thelaziosis and improved related ocular inflammation signs.

Tritrichomonas foetus in cats from Central Italy: Clinical signs and risk factors.

Tritrichomonas foetus (Trichomonadida, Tritrichomonadidae) is a parasitic protozoan which causes a diarrheic disease in domestic cats worldwide. The epizootiological and pathogenic impact of this parasite in many geographic locations is still to be unraveled; thus, the present study has investigated the occurrence of T. foetus in cats from central Italy, along with the analysis of risk factors and clinical signs. One hundred and five cats referred to the University Veterinary Teaching Hospital of the University of Teramo were tested with copromicroscopic techniques for common intestinal and extra-intestinal parasites and a specific PCR for T. foetus. A statistical analysis evaluating associations between T. foetus positivity and possible risk factors was performed. One animal scored positive for Giardia (0.95%), 2 for Toxocara cati (1.90%), 1 for Eucoleus aerophilus (syn. Capillaria aerophila) (0.95%), 9 for Aelurostrongylus abstrusus (8.57%) and 1 for Dipylididum caninum (0.95%), while T. foetus was molecularly recorded in 18/105 (17.14%) of the study cats. In two cats, T. foetus was found in association respectively with Giardia and A. abstrusus. Animals with chronic gastrointestinal signs were more significantly infected by T. foetus (40.90% vs 10.84%; p < 0.01; 95% CI = 1.90-17.04; OR = 5.69) in comparison to cats with no gastrointestinal distresses. No other statistically significant associations were found. This study confirms that T. foetus is enzootic in cats in Italy and that it should be included in the differential diagnosis of feline chronic gastrointestinal diseases.

Exposure to Major Vector-Borne Diseases in Dogs Subjected to Different Preventative Regimens in Endemic Areas of Italy.

Vector-borne diseases (VBDs) are globally widespread arthropod-transmitted diseases with a significant impact on animal and human health. Many drivers have recently spurred the geographic spread of VBDs in dogs. This study has evaluated the exposure to most important VBDs in dogs under different preventative treatments in different regions of Italy, i.e., Veneto, Friuli Venezia-Giulia, Umbria, Giglio Island (Tuscany), Abruzzo and Latium. Serological analyses were performed to detect antibodies against Leishmania infantum, Babesia canis, Anaplasma phagocytophilum/Anaplasma platys, Ehrlichia canis/Ehrlichia ewingii, Borrelia burgdorferi, Rickettsia conorii and the circulating antigen of Dirofilaria immitis. Dogs were categorized according to the treatment schedule usually received, and the association between seropositivity and possible risk factors was statistically evaluated. Overall, 124/242 (51.2%) dogs tested positive for at least one pathogen, while 34 (14.0%) were exposed to two or more pathogens. The most detected seropositivity was against R. conorii, followed by Anaplasma spp., L. infantum, B. canis, and the other pathogens under study. Significant statistical associations were found according to geographical provenance, history of tick infestation, lifestyle and inadequate prophylactic treatments. Random/irregular treatments have been identified as a clear risk factor. These results show that adequate prophylactic treatment protocols are overlooked by dog owners, despite the availability of several effective products, with possible implications in veterinary medicine and on public health.

Occurrence of Phlebotomine sand flies (Diptera: Psychodidae) in the northeastern plain of Italy.

BACKGROUND: Recent climate and environmental changes have resulted in the geographical expansion of Mediterranean Leishmania infantum vectors towards northern latitudes and higher altitudes in different European countries, including Italy, where new foci of canine leishmaniasis have been observed in the northern part of the country. Northern Italy is also an endemic area for mosquito-borne diseases. During entomological surveillance for West Nile virus, mosquitoes and other hematophagous insects were collected, including Phlebotomine sand flies. In this study, we report the results of Phlebotomine sand fly identification during the entomological surveillance conducted from 2017 to 2019. METHODS: The northeastern plain of Italy was divided by a grid with a length of 15 km, and a CO2-CDC trap was placed in each geographical unit. The traps were placed ~ 15 km apart. For each sampling site, geographical coordinates were recorded. The traps were operated every two weeks, from May to November. Sand flies collected by CO2-CDC traps were identified by morphological and molecular analysis. RESULTS: From 2017 to 2019, a total of 303 sand flies belonging to the species Phlebotomus perniciosus (n = 273), Sergentomyia minuta (n = 5), P. mascittii (n = 2) and P. perfiliewi (n = 2) were collected, along with 21 unidentified specimens. The trend for P. perniciosus collected during the entomological surveillance showed two peaks, one in July and a smaller one in September. Sand flies were collected at different altitudes, from -2 m above sea level (a.s.l.) to 145 m a.s.l. No correlation was observed between altitude and sand fly abundance. CONCLUSIONS: Four Phlebotomine sand fly species are reported for the first time from the northeastern plain of Italy. Except for S. minuta, the sand fly species are competent vectors of Leishmania parasites and other arboviruses in the Mediterranean Basin. These findings demonstrate the ability of sand flies to colonize new environments previously considered unsuitable for these insects. Even though the density of the Phlebotomine sand fly population in the plain areas is consistently lower than that observed in hilly and low mountainous areas, the presence of these vectors could herald the onset of epidemic outbreaks of leishmaniasis and other arthropod-borne diseases in areas previously considered non-endemic.

Efficacy of milbemycin oxime/afoxolaner chewable tablets (NEXGARD SPECTRA®) against Capillaria aerophila and Capillaria boehmi in naturally infected dogs.

BACKGROUND: Capillaria aerophila and Capillaria boehmi parasitize the respiratory system of wild and domestic carnivores. Capillaria aerophila inhabits the trachea and bronchi of dogs and cats, while C. boehmi affects the nasal cavities and sinuses of dogs. In dogs the infection may be subclinical or characterized by varying respiratory distress. METHODS: The present study evaluated the efficacy of an oral formulation containing milbemycin oxime and afoxolaner (NEXGARD SPECTRA®) in dogs naturally infected with C. aerophila and/or C. boehmi from three enzootic areas of Italy. Dogs were enrolled pending fecal examination and molecular confirmation of respiratory capillarioses. Dogs were allocated in two groups: Group 1 (G1, 25 dogs), treated with a negative control product with no anthelmintic activity (afoxolaner, NEXGARD®), and Group 2 (G2, 26 dogs), treated with NEXGARD SPECTRA®. At the day of treatment administration (Day 0), all dogs were clinically examined. Dogs were again subjected to clinical and fecal examinations at Days 28 (± 4) and 56 (± 2). The primary criterion for treatment efficacy was the reduction of fecal Capillaria egg counts in G2 compared with G1. The regression of/recovery from baseline clinical signs was considered as a further efficacy criterion. RESULTS: Percentage reduction of fecal Capillaria egg counts in the NEXGARD SPECTRA® group compared to the control group was > 97% on Day 28 and 100% on Day 56, respectively (p < 0.05 for both time points). Twelve of the 13 dogs in the NEXGARD SPECTRA® group with respiratory signs prior to treatment were free of clinical signs at the end of the study. Conversely, the six control group dogs with respiratory signs prior to treatment remained symptomatic. CONCLUSIONS: Results of the present study showed that NEXGARD SPECTRA® was safe and highly efficacious in the reduction of C. aerophila and C. boehmi eggs after one treatment with a complete reduction of the egg output after the second administration associated with a recovery from respiratory signs.

First record of Hepatozoon spp. in domestic cats in Greece.

Feline hepatozoonosis is an emerging disease of domestic and wild felids though there is limited knowledge of this infection, e.g. regarding geographical distribution and parasite species involved. The present study evaluated microscopically and molecularly the occurrence of Hepatozoon spp. in domestic cats from insular (Crete, Mykonos and Skopelos) and continental (prefectures of Attica and Thessaloniki) Greece. Out of 282 cats examined, 72 (25.5 %) scored positive by PCR for Hepatozoon spp. and of them, 9 (12.5 %) showed gamonts on the blood smear microscopic examination. Sequences obtained from 35 of the amplicons proved the presence of two haplotypes of Hepatozoon felis. One, herein called H1 (34/35 amplicons) resulted 100 % identical with H. felis from Italy and isolates from other continents, and ∼98 % similar with a H. felis isolate causing severe clinical signs in Austria. The haplotype H2, found in a cat in Skopelos, had ∼94 % identity with H1, with H. felis isolates from Italy, Israel, Spain, a ∼92 % identity with the isolate from Austria, and ∼94-98 % with isolates from South Africa. These are the first records of H. felis in cat populations from Greece and indicate that the infection may be present at high prevalences in different regions of the country. Furthermore, the results of the molecular and phylogenetic analysis support a recent hypothesis indicating the existence of a species-complex classification for H. felis. Further studies aiming at elucidating the genetic make-up of Hepatozoon populations and possible variations in terms of geographic distribution and clinical relevance are necessary. The importance of a continuous epizootiological monitoring is crucial for the establishment of preventative and control measures protecting the health of cats living in or travelling to enzootic areas.

Surveillance of Zoonotic Parasites in Animals Involved in Animal-Assisted Interventions (AAIs).

Animal-assisted interventions (AAIs) are based on the establishment of a therapeutic relationship between animals and beneficiaries that is certain to provide positive effects, while currently, it reads as if AAIs aim at exposing stakeholders to potential risk of infection. The surveillance of zoonotic pathogens is necessary for guaranteeing common health. This study investigated the presence of potentially zoonotic parasites, including dermatophytes, in animals involved in AAIs. Between 2015 and 2017, 190 animals (equids, dogs, cats, birds, rabbits, rodents, and goats) were investigated. Anamnestic and management data were recorded. Individual faecal samples were analysed using a copromicroscopic procedure. Fur and skin were examined for ectoparasites during clinical examinations, and samples for mycological investigation were collected by brushing. Parasites were described in 60 (31.6%) investigated animals. Thirteen out of the 60 (21.7%) animals harboured potentially zoonotic parasites, mainly recovered in dogs (Ancylostomatidae, Eucoleusaerophilus, Toxocaracanis, and Giardiaduodenalis) and a cat (G.duodenalis). Nannizziagypsea and Paraphytonmirabile, potential agents of cutaneous mycosis, were isolated in a dog and a horse, respectively. No ectoparasites were found. AAIs might represent a source of infections either directly or via environmental contamination. Thus, active surveillance is necessary and animal screenings should be planned and scheduled according to the risk of exposure.

Parasitological survey to address major risk factors threatening alpacas in Andean extensive farms (Arequipa, Peru).

Extensive alpaca farming in the Colca Valley (Arequipa, Peru) is the most important resource for farmers who live in this remote area of the country. Grazing is the major available source of food, whereas forage production and supply are limited. Food availability is low during the dry season predisposing animals to parasitic diseases and reproduction failure. In this study, we looked into gaining an overview about flock health management and nutritional and parasitological status of different age groups in medium-large alpaca farms at the beginning of the dry season. A total of 20 herds were included in the survey and 288 fecal samples were collected and analyzed to determine prevalence and oocysts/eggs output level of the most common gastro-intestinal parasites that affect alpacas. Body condition scoring (BCS), was used to determine the nutritional status of sampled animals, belonging to different physiological classes (i.e. crias, tuis, lactating females, non-lactating females, males). Coccidiosis was the parasitic disease with the highest prevalence and output level. It was the most relevant parasitic disease in all classes and especially in young pre-breeding animals. Higher burdens were found in subjects with lower BCS. More specifically, the farms with higher cria mortality rates showed also a tendency to have higher prevalence values of Eimeria macusaniensis. An improved management of nutritional aspects and parasite control, in particular regarding the weaning phase, is expected to enhance the farming reproductive and productive performances of the alpacas in this region.

Canine Leishmaniosis Control through the Promotion of Preventive Measures Appropriately Adopted by Citizens.

Canine leishmaniosis (CanL) is a disease caused by the protist Leishmania infantum and transmitted to dogs by sand fly (Diptera: Phlebotominae) bites. In 2005, a new autochthonous focus of CanL was recognised in the southern part of Euganei hills (northeastern Italy). In subsequent years, this outbreak was monitored, testing dogs and evaluating sand fly population. Moreover, dog owners were sensitized on the adoption of preventive measures, thanks to the collaboration of local administration, health authorities, and private veterinarians. This study includes serological tests on dogs, questionnaires submitted to dog owners regarding the use of preventive measures on their animals, and the evaluation of sand fly abundance. Data collected were statistically compared with those of previous years. The canine seroprevalence was significantly lower than that recorded at the beginning of the outbreak, despite the fact that sand fly abundance did not significantly decrease. In addition, most of the dog owners declared using regularly the topical insecticides on their dogs during the sand fly season. This experience demonstrated that a collaborative approach among scientific researchers, local authorities, and private veterinarians can achieve excellent results in the management of a leishmaniosis outbreak.