Toxoplasma gondii infection and food consumption: A systematic review and meta-analysis of case-controlled studies.

BACKGROUND: Toxoplasmosis is a zoonotic disease causing severe symptoms in pregnant women and immunocompromised individuals. On average, worldwide, around 30% of people are seropositive. The oral transmission route is of great significance and food, particularly meat, is an important transmission vehicle for T. gondii. However, the role of different food matrices is debated. OBJECTIVES: The aim of this review was to assess the risk of humans developing acute T. gondii infection via the foodborne route. STUDY ELIGIBILITY CRITERIA: Case-control studies including acute cases of T. gondii infection were included after literature searches, without time limits, in several databases. All studies estimating the risk of acquiring T. gondii infection after consumption of specific food categories were included. RESULTS: Three risk factors proved to be significantly associated with acute T. gondii infection in humans: consumption of raw/undercooked meat, Odds Ratio (OR) 3.44 (1.29-9.16), consumption of raw/undercooked beef, OR 2.22 (1.57-3.12), and consumption of raw/undercooked sheep meat, OR 3.85 (1.85-8.00). Consumption of raw/undercooked pork, raw eggs, and unpasteurized milk proved to be non-significant risk factors. LIMITATIONS: Limitations in the present review and meta-analysis are due to the low number of case-control studies available for analysis and the lack of a search strategy targeting gray literature. CONCLUSION: Consumption of raw/undercooked beef and sheep meat are important risk factors for T. gondii infection. Their consumption should be avoided in order to prevent toxoplasmosis, particularly by those in at-risk categories, including pregnant women. The review protocol is registered in PROSPERO database (CRD42016043295).

Mitochondrial haplotypes of Aelurostrongylus abstrusus and Troglostrongylus brevior (Nematoda, Metastrongyloidea) from domestic and wild felids.

The lungworm Aelurostrongylus abstrusus is the most important respiratory parasite of domestic cats. Pulmonary aelurostrongylosis has been reported in wild felids, though unequivocally evidence of wildlife infection by A. abstrusus is scant. Recently, Troglostrongylus brevior, a lungworm usually infecting wild felids, has been described in domestic cats from Mediterranean areas. The present work evaluates the sequence variation of an informative region within the gene encoding the mitochondrial cytochrome c oxidase subunit 1 of A. abstrusus and T. brevior, in order to provide novel information on the genetic make-up of these lungworms. Parasitic stages of A. abstrusus and T. brevior were collected from domestic and wild hosts (i.e., domestic cat, European wildcat, caracal, serval, and lion) from Italy, Greece, and South Africa. Five (HI-HV) and four (HI-HIV) haplotypes were recorded for A. abstrusus and T. brevior, respectively, mostly shared between domestic and wild felids in different geographical areas. The phylogenetic analysis showed that all haplotypes of A. abstrusus and T. brevior clustered as monophyletic groups with a strong nodal support, indicating that all haplotypes identified were distinct from each other. All sequence types represent two distinct species, A. abstrusus and T. brevior, and these genetic convergences are also detected within and among populations of these nematodes, irrespective of their hosts and geographical origin. The occurrence of A. abstrusus and T. brevior haplotypes in different hosts from the same regions and between different countries indicates that the same lungworm populations circulate in domestic and wild hosts under the same routes of transmission.

Molecular detection of Giardia duodenalis and Cryptosporidium spp. in canine faecal samples contaminating public areas in Northern Italy.

Giardia duodenalis and Cryptosporidium spp. are common intestinal pathogens of humans and animals. Dogs may be infected by zoonotic isolates of G. duodenalis and Cryptosporidium spp. that, consequently, have high interest under public health perspective. This study estimated the occurrence of these protozoa in canine faeces polluting public areas of Padua municipality (Northern Italy), towards a potential evaluation of health risks for dogs and humans. A total of 705 canine stools was collected in green (n = 270) and urban (n = 435) areas and processed by duplex real-time PCR and real-time PCR SYBR® Green I for the detection of both protozoa. Positive samples were submitted to specific nested PCRs (i.e. ß-giardin/SSU-rRNA genes for Giardia; SSU-rRNA gene for Cryptosporidium) to obtain detailed information on the isolates retrieved.Giardia and Cryptosporidium prevalence were 28.9% and 1.7%, respectively. Twenty-one Giardia-positive samples were successfully identified as dog-specific assemblages C and D, and 1 as the human-specific assemblage B. One isolate was identified as Cryptosporidium canis, while the other 11 were confirmed to belong to the Cryptosporidium parvum species complex. Contrariwise to the Cryptosporidium low prevalence, the wide distribution of Giardia suggests a high risk of infection for dogs attending public areas. Although data indicate a limited risk for human health, it is necessary to improve general education to reduce canine faecal pollution towards a widespread awareness of health risks.

Copromicroscopic and molecular investigations on intestinal parasites in kenneled dogs.

Intestinal parasites are common in dogs worldwide, and their importance has recently increased for a renewed awareness on the public health relevance that some of them have. In this study, the prevalence of helminths and protozoa was evaluated by microscopy in 318 canine faecal samples collected from eight rescue shelters in the North-eastern Italy; 285 of them were also submitted to the molecular characterization of Giardia duodenalis and Cryptosporidium spp. isolates. An analysis was performed to evaluate the prevalence rates in relation to canine individual data, shelter provenance and anthelmintic treatments. Overall, 52.5% (167/318) of faecal samples were positive for at least one parasite. Trichuris vulpis showed the highest overall prevalence rate (29.2%), followed by G. duodenalis (15.1%), Toxocara canis (9.7%), ancylostomatids (8.2%) and Cystoisospora (5.7%). The prevalence of G. duodenalis, evaluated by real-time PCR, was 57.9% (165/285), and 79 isolates were characterized by nested PCR on the ß-giardin gene. The assemblages found were mainly the host-specific genotypes C and D, while only one assemblage was identified as the human-specific genotype B1. Isolates of Cryptosporidium spp., recorded in 3/285 (1.1%) stool samples, were Cryptosporidium parvum based on the characterization of the Cryptosporidium oocyst wall protein (COWP) gene. Although the results describe a relatively limited risk of dog-originating zoonoses, there is the need to improve the quality of shelter practices towards better health managements for safe pet-adoption campaigns and a minimization of the environmental faecal pollution with canine intestinal parasites.

Respiratory nematodes in cat populations of Italy.

The occurrence of common respiratory parasites of domestic cats (the metastrongyloid "cat lungworm" Aelurostrongylus abstrusus and the trichuroid Capillaria aerophila) and of neglected respiratory nematodes of felids (Troglostrongylus brevior, Angiostrongylus chabaudi and Oslerus rostratus) was here evaluated in two and three geographical sites of Northern and Central Italy, respectively. In 2014-2015, individual fecal samples of 868 domestic cats were examined microscopically and genetically, and epidemiological data related to parasitic infections were evaluated as possible risk factors by binary logistic regression models. The most common parasite was A. abstrusus in both mono- and poli-specific infections, followed by T. brevior and C. aerophila, while cats scored negative for other parasites. Cats positive for A. abstrusus (1.9-17 % infection rate) and C. aerophila (0.9-4.8 % infection rate) were found in all examined sites, while cats scored positive for T. brevior (1-14.3 % infection rate) in four sites. Also, T. brevior was here found for the first time in a domestic cat from a mountainous area of Northern Italy. The occurrence of lungworms was statistically related to the presence of respiratory signs and more significant in cats with mixed infection by other lungworms and/or intestinal parasites. Cats living in site C of Central Italy resulted statistically more at risk of infection for lungworms than cats living in the other study sites, while animals ageing less than 1 year were at more risk for troglostrongylosis. Finally, the presence of lungworms was more significant in cats with mixed infection by other lungworms and/or intestinal parasites. These results are discussed under epidemiological and clinical points of views.

Mixed infection by Aelurostrongylus abstrusus and Troglostrongylus brevior in kittens from the same litter in Italy.

Parasitic nematodes affecting the respiratory system of felids are spreading in endemic regions and emerging in previously free areas and/or hosts. This is particularly the case of the cat lungworm Aelurostrongylus abstrusus, which can cause respiratory signs in cats all over the world. Additionally, Troglostrongylus brevior has been recently found in domestic cats from Ibiza Island in Spain and Southern Italy. The present paper describes the first mixed infection by these lungworms in kittens belonging to the same litter. Two ∼10-11 weeks old kittens were found infected by A. abstrusus and T. brevior at a copromicroscopical examination. The identity of larvae shed by faeces were confirmed with an already validated PCR specific for A. abstrusus and a novel DNA-based assay specific for T. brevior. One kitten showed severe respiratory signs and died despite an anthelmintic treatment, while the other had a subclinical infection and recovered after a parasiticide administration with milbemycin oxime. New insights into epidemiology, biology, clinical aspects and control of these parasitoses are discussed.

Microfilarial periodicity of Dirofilaria repens in naturally infested dogs.

In the past years, canine and human cases of infestation by Dirofilaria repens (Spirurida, Onchocercidae) have been increasingly reported in several European countries. Subcutaneous dirofilariosis by D. repens may either be asymptomatic in dogs or may be characterized by subcutaneous nodules and other symptoms. Information on the periodicity of D. repens microfilariae in naturally infested animals is scant, and this might impair the accurate diagnosis of subcutaneous dirofilariosis and appropriate control plans. In the present study, eight dogs infested with D. repens were sampled twice daily at 12-h intervals for ten consecutive days, and the dog with the highest mean value of microfilariaemia was further sampled every 4 h for four consecutive days. The blood was microscopically and molecularly examined for microfilariae, and, additionally, negative samples were also subjected to a real-time PCR to evaluate the level of circulating DNA. The results demonstrated significant variations in circadian rhythms of D. repens larvae, with higher values of microfilariae per milliliter in the evening samples. A significant variation was also found at the individual level for the dogs with the highest values of microfilariaemia. All samples which were negative at the light microscopy and positive at the real-time PCR displayed levels of circulating parasite DNA <1 microfilaria per milliliter. Biological and clinical implications have been here discussed.

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.

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.

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.

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.

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.

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.

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.

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.