Hepatozoon spp. in stray cats from the metropolitan area of Rio de Janeiro, Brazil.

In the last few years, the number of studies on feline hepatozoonosis has increased, but our knowledge on the actual species of Hepatozoon and/or different genotypes affecting felines is still incipient. At least three species, namely Hepatozoon felis, H. canis, and H. silvestris, have been isolated from domestic cats in various countries. Additionally, there are indications that other species and genotypes may affect felines in given geographic areas. This study was carried out to investigate the occurrence of Hepatozoon spp. in cats from Niterói, a municipality within the metropolitan area of Rio de Janeiro, Brazil. Individual blood samples were collected from 28 cats enrolled in a spaying/castration program. DNA was extracted from all samples and subjected to sequencing specific for Hepatozoon spp. DNA of H. felis was found in 21/28 cats (75%), and four genetic polymorphisms never described thus far were detected. This is the first report of H. felis in cats living in the State of Rio de Janeiro, and the present data confirm that H. felis is a species complex encompassing different genotypes circulating within cat populations. Further studies are warranted to investigate whether different genotypes have different biology or pathogenicity for felids.

Title: Hepatozoon spp. chez les chats errants de la zone métropolitaine de Rio de Janeiro, Brésil. Abstract: Au cours des dernières années, le nombre d'études sur l'hépatozoonose féline a augmenté, mais nos connaissances sur les espèces d'Hepatozoon et/ou différents génotypes affectant les félins sont encore naissantes. Au moins trois espèces, à savoir Hepatozoon felis, H. canis et H. silvestris, ont été isolées chez des chats domestiques dans divers pays. De plus, il semble que d'autres espèces et génotypes puissent affecter les félins dans des zones géographiques données. Cette étude a été réalisée pour étudier la présence d'Hepatozoon spp. chez des chats de Niterói, une municipalité de la zone métropolitaine de Rio de Janeiro, au Brésil. Des échantillons de sang ont été prélevés individuellement sur 28 chats d'un programme de castration. L'ADN a été extrait de tous les échantillons et soumis à un séquençage spécifique de Hepatozoon spp. L'ADN de H. felis a été trouvé chez 21 chats sur 28 (75%) et quatre polymorphismes génétiques, jamais décrits jusqu'à présent, ont été détectés. Il s'agit du premier signalement de H. felis chez des chats vivant dans l'État de Rio de Janeiro et les données actuelles confirment que H. felis est un complexe d'espèces englobant différents génotypes circulant au sein des populations de chats. Des études supplémentaires sont nécessaires pour déterminer si les différents génotypes ont une biologie ou une pathogénicité différente pour les félidés.

Survey of ectoparasites affecting dog and cat populations living in sympatry in Gamo Zone, Southern Ethiopia.

BACKGROUND: Ectoparasites of dogs and cats are implicated to be responsible for life-threatening anaemia, allergic dermatitis and pruritic and non-pruritic skin disorders. In Ethiopia, few studies have been conducted on the arthropods of dogs and cats. OBJECTIVES: In order to shed light on some of these aspects, a survey was conducted to investigate the presence of ectoparasites in dogs and cats living in sympatry in the urban and rural areas of Gamo Zone, Ethiopia. METHODS: A total of 297 dogs and 110 cats were examined for ectoparasites, and questionnaires were employed to obtain information concerning owner's knowledge about arthropods and vector-borne diseases (VBDs). RESULTS: The overall prevalence of ticks, fleas and lice in dogs was 36.7%, 69.7% and 4.7%, respectively. Similarly, on cats, an overall prevalence of 2.7% ticks and 21.8% fleas was recorded. On dogs, fleas (Ctenocephalides felis 69.4%, Echidnophaga gallinacea 1.3%, Ctenocephalides canis 1.0% and Pulex irritans 0.3%), ticks (Amblyomma variegatum 22.9%, Rhipicephalus sanguineus 14.1%, Haemaphysalis leachi 8.8%, Rhipicephalus praetextatus 4.0% and Rhipicephalus pulchellus 3.4%) and lice (Heterodoxus spiniger 4.0% and Trichodectes canis 0.7%) were identified. Likewise, on cats, fleas (C. felis [15.5%] and E. gallinacea [7.3%]) and ticks (H. leachi [2.7%]) were identified. The abundance of C. felis was significantly higher (p < 0.001) on dogs, whereas in cats, the abundance of E. gallinacea was significantly higher (p = 0.002) than the other ectoparasites. On dogs, a significantly higher prevalence of Rh. sanguineus was recorded in urban areas (<0.001) and on dogs which live in indoor environments (p = 0.003) than on dogs which live in other environments. On the other hand, the prevalence of A. variegatum in rural areas and midland agroecology was significantly higher (p < 0.001). The prevalence of H. leachi was significantly higher in midland (p < 0.001) and on adult dogs (p = 0.001). Overall, fleas were more prevalent in rural (p = 0.029) than in urban areas, and female dogs were with higher infestation than the male (p = 0.047) dogs; C. felis was prevalent in female (p = 0.038) dogs than males. Overall, 88.3% owners in the study area had no knowledge about ectoparasites and VBDs of dogs and cats. Majority of the owners (64.8%) attest that they had never visited veterinary clinics. CONCLUSIONS: In conclusion, the data presented in the present study provide additional knowledge on the importance of ectoparasites of dogs and cats and are believed to contribute in awareness creation and strengthening of veterinary services of dogs and cats of the study area.

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.

Effective Methods of Estimation of Pathogen Prevalence in Pooled Ticks.

Since tick-borne diseases (TBDs) incidence, both in human and animal populations, is increasing worldwide, there is the need to assess the presence, distribution and prevalence of tick-borne pathogens. Reliable estimates on tick-borne pathogens (TBPs) prevalence represent the public health foundation to create risk maps and take effective prevention and control actions against TBDs. Tick surveillance consists of collecting and testing (usually in pools) thousands of specimens. Construction and analysis of tick pools represent a challenge due to the complexity of tick-borne pathogens and tick-borne diseases ecology. The aim of this study is to provide a practical guideline on appropriate pooling strategies and statistical analysis of infection prevalence through: (i) reporting the different pooling strategies and statistical methodologies commonly used to calculate pathogen prevalence in tick populations and (ii) practical comparison between statistical methods utilising a real dataset of infection prevalence in ticks collected in Northern Italy. Reporting detailed information on tick pool composition and size is as important as the correct TBPs prevalence estimation. Among the prevalence indexes, we suggest using maximum-likelihood estimates of pooled prevalence instead of minimum infection rate or pool positivity rate given the merits of the method and availability of software.

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.

Ticks, Fleas, and Harboured Pathogens from Dogs and Cats in Cyprus.

Ticks and fleas are blood-sucking ectoparasites that cause irritation and anaemia to their hosts and act as vectors of pathogens (vector-borne pathogens, VBPs) of relevance for animal and human health. In the present study, tick and flea species in dogs and cats from Cyprus were recorded and VBPs were detected in the collected specimens. Ectoparasites were collected from 220 animals (161 dogs and 59 cats), and a questionnaire including demographic, clinical, and other information was filled out for each animal. The ectoparasites were morphologically identified and the detection of VBPs was performed by PCR-coupled sequencing. Rhipicephalus sanguineus sensu lato was found on 108 dogs and 13 cats, and Ixodes gibbosus on 2 dogs. Ctenocephalides felis was the predominant flea species (on 62 dogs and 45 cats), while one dog and one cat were infested by Ctenocephalides canis and Echidnophaga gallinacea, respectively. The VBPs in ticks were Anaplasma platys, Rickettsia massiliae, Rickettsia conorii, Rickettsia felis, Hepatozoon felis and Hepatozoon canis, while Rickettsia felis, Rickettsia sp., Bartonella koehlerae, Bartonella clarridgeiae, and Bartonella henselae were recorded in fleas. Statistical analysis (chi-square test and multiple univariate generalized linear model) showed that animals up to 6 months of age were less likely to be infested with ticks than older animals, but more likely to be infested with fleas. Ticks were more prevalent in sheltered than in owned animals, while the odds ratio of flea presence was higher in owned animals than those living in shelters. The present study is the first investigation on the occurrence of ticks and fleas in dogs and cats from Cyprus, showing the presence of different VBPs in these important ectoparasites. The results point out the importance of systematic ectoparasite control in dogs and cats.

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.

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.

High Prevalence of Tick-Borne Zoonotic Rickettsia slovaca in Ticks from Wild Boars, Northeastern Italy.

Tick-borne rickettsiae are emerging pathogens that are becoming widespread in Europe. Rickettsiae are endemic in Italy, but epidemiological data are currently scarce. This study aimed to improve our knowledge about rickettsial infections in tick and wild boar populations. Blood and ticks were collected from 102 wild boars in 2010 and 2018. Ticks were also collected from the vegetation in the area. All of the samples were examined using real-time PCR targeting the gltA gene to detect Rickettsia DNA. Positivity was confirmed by PCR amplifying the gltA and/or ompB genes. A total of 254 ticks and 89 blood samples were analyzed. Zoonotic rickettsiae were detected in the ticks but not in the blood samples. Rickettsia slovaca (R. slovaca) was the most prevalent in ticks and was found in 23.7% of Dermacentor marginatus (D. marginatus) and in 3.4% of Ixodes ricinus (I. ricinus). Other zoonotic species were identified, such as Rickettsia monacensis, which was detected in 12% of I. ricinus ticks, and Rickettsia helvetica which was found in 3.4% of questing I. ricinus ticks and in 1.1% of D. marginatus collected from wild boars. This study highlights a high prevalence of zoonotic rickettsiae, particularly that of R. slovaca, in northeastern Italy. As rickettsioses are underreported and underdiagnosed in human medicine, both clinicians and researchers should pay more attention to this topic.

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.

Potential Role of Hepatozoon canis in a Fatal Systemic Disease in a Puppy.

Canine hepatozoonosis caused by Hepatozoon canis is an emerging disease in Europe. Clinical pictures vary from subclinical to life-threatening and non-specific clinical signs are predominantly reported. A 2-month-old female puppy originating from Southern Italy was adopted and moved to Northern Italy. Then, the dog was brought to a local veterinary practice for gastrointestinal signs, migrating lameness and pruritic dermatitis, and then tested positive for Hepatozoon spp. gamonts at the blood smear. After treatment with imidocarb dipropionate and doxycycline, the dog showed an initial clinical improvement. However, gastrointestinal signs recurred, and diffuse superficial pyoderma appeared on the thoracolumbar region, along with fever, lethargy, and weight loss. Eight months from the first onset of clinical signs, the dog was referred to a veterinary clinic and subjected to complete blood count, urine and fecal analysis, along with abdominal ultrasonography, whole-body CT and gastroduodenal endoscopy. Skin biopsies and blood samples were subjected to a PCR-coupled sequencing protocol, which scored both positive for H. canis. Alterations were consistent with a pre-existing cholangiohepatitis and multiple acquired extrahepatic shunts secondary to portal hypertension. The dog was euthanatized due to a clinical worsening two months later. The potential role of H. canis in the systemic disease observed, clinic-pathological findings and epizootiological implications are discussed.

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.

The Influence of Temperature on the Larval Development of Aelurostrongylus abstrusus in the Land Snail Cornu aspersum.

The metastrongyloid Aelurostrongylus abstrusus has an indirect lifecycle involving gastropod intermediate hosts. The widespread snail Cornu aspersum is an efficient intermediate host of A. abstrusus. As the temperature may influence the developmental rate of metastrongyloids from first (L1) to the third infective larval stage (L3) inside molluscs, this study evaluated the effect of two controlled temperatures on the development of A. abstrusus in C. aspersum. Overall, 300 snails were infected with 500 L1 of A. abstrusus and kept at ∼25 °C. Fifteen days post infection (D15), the overall developmental rate to L3 (0.8%) was assessed in a subset of 20 snails. The remaining gastropods were divided in 2 groups, i.e., 180 still kept at ∼25 °C (G1) and 100 hibernated at ∼4 °C (G2). On D30, the larval development was evaluated in 20 snails from each group, while another batch of 80 snails was selected random from G1 and hibernated at ∼4 °C (G3). The larval developmental rate was determined digesting 20 snails from each of the three groups on D45, D60, and D75. The higher mean developmental rate was registered in G1 (3.8%) compared to G2 (1.9%) and G3 (2.3%), indicating that the development to L3 of A. abstrusus in C. aspersum is positively influenced by the increase of temperature.

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.

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.