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.

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.

Epidemiology of blood flukes (Digenea: Spirorchiidae) in sea turtles from Tyrrhenian and Adriatic Seas, off Italy.

BACKGROUND: The Spirorchiidae is a family of blood flukes parasitizing turtles. Spirorchiids may cause a wide range of inflammatory reactions in the vascular system of their host being frequently implicated with stranding and death of sea turtles worldwide. Recent studies revealed the presence of two spirorchiid species in the Mediterranean basin. Our study presents comparative epidemiological data of spirorchiid infections in loggerhead turtles (Caretta caretta) stranded during an eight-year period from Adriatic and Tyrrhenian Seas, and the first report of Neospirorchis Neogen-11 in a green turtle (Chelonia mydas). METHODS: We screened a total of 319 carcasses of loggerhead turtles stranded from January 2011 to December 2018 along the Tyrrhenian coast (n = 111) and the north-western Adriatic coast (n = 208) of Italy using traditional (copromicroscopy and histopathology) and molecular assays. Three green turtles from the Tyrrhenian coast were also included in the study. RESULTS: A total of 56 (17.5%) loggerhead turtles and one green turtle (33.3%) were found to be infected with spirorchiid flukes. Amplification, sequencing of the ITS2 region of the ribosomal RNA gene cluster and BLAST analysis confirmed the presence of Hapalotrema mistroides and Neospirorchis Neogen-11 in 51 (16.0%) and 24 (7.5%) loggerhead turtles, respectively, and Neospirorchis Neogen-11 in an infected green turtle. Differences in prevalence of infection between the two sampling areas were found. CONCLUSIONS: The risk of spirorchiid infection in the Tyrrhenian Sea is lower than in the Adriatic Sea and in general the risk of infection in the Mediterranean is lower than in other geographical locations. Differences in the prevalence of infection between the two sampling areas were related to the differences of regional habitats supporting different abundance of spirorchiid intermediate hosts. A systematic monitoring to evaluate the progress of the infection is recommended, as well as studies on the occurrence and distribution of spirorchiid species from other Mediterranean areas.

Contamination of Italian parks with canine helminth eggs and health risk perception of the public.

The contamination of public areas by dog faeces is a social behaviour and public health problem. In fact, the most frequently isolated intestinal helminths in dogs are distributed worldwide, and most of them have zoonotic potential (i.e., ascarids and ancylostomatids). The aims of this survey were to evaluate citizen awareness of health risks for animals and humans related to canine faecal pollution and to estimate the presence and prevalence of intestinal helminths in dog faeces collected in green public areas in three municipalities of Italy (Padua, Rome and Teramo). The awareness of citizens about the health risks related to faecal pollution was evaluated using questionnaires submitted to 313 dog owners and 159 non-dog owners in Padua (n = 341) and Rome (n = 131). Most dog owners (85.4%) declared they picked up their dog's faeces every time, and these data were confirmed by operators secretly observing dog owners. Moreover, 84.3% participants were aware of the existence of a municipal regulation concerning the correct management of animals in public areas with no significant differences between dog owners and non-dog owners, whereas Rome citizens were significantly more aware than Padua citizens. Nonetheless, only 10.9% (51/469) of responders knew the health risks related to canine faecal pollution, with no significant differences between dog and non-dog owners. A total of 677 dog stool samples were collected and copromicroscopically analysed. Forty-eight (7.1%) samples were positive for at least one parasite species, with significantly lower prevalence values in Padua (2.2%) than in Rome (11.9%) and Teramo (8.6%). The highest prevalence was detected for Trichuris vulpis (4.4%), followed by Toxocara canis (1.9%); T. vulpis presented significantly lower prevalence in Padua than in the other cities. Other helminths were found with values under 0.5% in the investigated cities. This survey shows that most citizens are unaware of the health risk related to abandoned canine faeces on public soils. Nevertheless, laboratory results suggest a limited risk for dog and human health, but the zoonotic risk due to the high vitality of infective helminths eggs in the soil should always be considered.

A new approach to outbreak management for bovine Cystic Echinococcosis cases in hypo-endemic areas.

Cystic Echinococcosis (CE) surveillance in Italy is based on detection of its larval stage (hydatic cysts) at the slaughterhouse. In northern Italy, a hypo-endemic area, local health authorities investigate each individual farm with positive animals to treat their dogs with cestocidal drugs, but this system is time-consuming and poorly effective for bovine farms. The study applied a new approach based on targeted epidemiological surveys in areas with aggregation of bovine CE cases, and compared the outcome with that of two control areas with farms individually investigated. The presence of territorial cluster of bovine farms with CE cases was investigated for 3 consecutive years (2013-2014-2015) in a high-risk area of Veneto Region (north-eastern Italy), using a spatial scan statistic. Epidemiological investigations, consisting of a questionnaire survey and canine faecal samples collection, were conducted in cluster and control areas. All faecal samples were analyzed for taeniids eggs and positive samples were sequenced and identified. In total, 99 farms were surveyed and 208 faecal samples were retrieved from dogs. Sixty-two farms (42 bovine and 20 sheep) were investigated in cluster areas and 37 farms (33 bovine and 4 sheep) in control areas. Based on the results of the cestode egg isolation procedure, 14 animals (6.7%) were positive to taeniid eggs. For molecular analysis, two dogs resulted positive to Echinococcus granulosus, and seven to Taenia hydatigena. Twelve positive dogs were found in targeted survey areas and ten of these dogs were shepherd dogs, belonging to transhumant sheep flocks known to pass in cluster areas. The new approach demonstrated to successfully identify the probable source of infection of CE positive bovines. Most of positive dogs belonged to transhumant flocks, underlining the importance to include shepherd dogs in the surveillance system for CE.

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).

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.

Microfilaricidal efficacy of a single administration of Advocate(®) (Bayer Animal Health) in dogs naturally infected with Dirofilaria immitis or Dirofilaria repens.

The present study evaluated the microfilaricidal efficacy of a single application of the spot-on containing imidacloprid 10%/moxidectin 2.5% (Advocate(®), Bayer Animal Health) in dogs naturally infected either by Dirofilaria immitis or Dirofilaria repens. Dogs living in north-eastern and central-southern Italy, endemic for D. immitis and D. repens respectively, were randomly screened. Sixteen animals, eight infected with D. immitis and eight with D. repens, and fulfilling inclusion criteria were enrolled. Dogs infected with D. immitis received an adulticide treatment prior to the study and Advocate(®) 3 weeks after. The animals were divided in blocks of two (1:1, T1:T2) animals each, where Day 0 (D0) had an interval of 15days to compare T2 vs. T1 dogs during the first fortnight of examination (i.e. T2 dogs acted as control animals at each examination). At baseline (Days -15 and 0 for T2 and T1 dogs, respectively) the animals had a range of microfilaraemia of 180-99.700mff/ml (D. immitis) and 60-750 mff/ml (D. repens). All animals received a topical administration of Advocate(®) at D0 and were examined for microfilariae with microscopic and molecular tests at D15, D30, D60 and D90. All animals scored negative for mff at the first control post-treatment and throughout the study, with the exception of two D. immitis- infected animals that had a 2 mff/ml count at D15, and then become negative from Day 30 onwards. No adverse events were observed. The present study demonstrates the safety and the high microfilaricidal efficacy (99.97% and 100% for D. immitis and D. repens, respectively) of a single dose of moxidectin contained in Advocate(®) in naturally infected dogs.

Fatal Angiostrongylus dujardini infection in callitrichid monkeys and suricates in an Italian zoological garden.

This paper reports four fatal cases of metastrongylid nematode Angiostrongylus dujardini infection observed in a Saguinus oedipus and a Callimico goeldii monkey and in two suricates (Suricata suricatta). All animals were kept in captivity in a zoo of central Italy. The two monkeys died with no premonitory signs, while the two-month-old suricates showed malaise, anorexia and tachypnea for a few days prior to death. Cardiomegaly and/or granulomatous pneumonia were the major anatomo-pathological findings. Inflammatory lesions were observed in the liver, heart and kidney of the suricates at histology. A. dujardini diagnosis was confirmed through both morphological identification of adult worms recovered at necropsy and molecular characterization of larvae in tissue samples. Callitrichidae and suricates are active predators and maintain their hunting behaviour in captivity and it is then likely that they were exposed to infection by preying on parasitized gastropods, intermediate hosts of A. dujardini, entering zoo enclosures from the surrounding environment. This is the first report of A. dujardini in Italy and in S. suricatta.

Investigating the Determinants of Toxoplasma gondii Prevalence in Meat: A Systematic Review and Meta-Regression.

BACKGROUND: Toxoplasma gondii is one of the most widespread parasites in humans and can cause severe illness in immunocompromised individuals. However, its role in healthy people is probably under-appreciated. The complex epidemiology of this protozoan recognizes several infection routes but consumption of contaminated food is likely to be the predominant one. Among food, consumption of raw and undercooked meat is a relevant route of transmission, but the role of different meat producing animal species and meats thereof is controversial. OBJECTIVES: The aim of the present work is to summarize and analyse literature data reporting prevalence estimates of T. gondii in meat animals/meats. DATA SOURCES: We searched Medline, Web of Science, Science Direct (last update 31/03/2015). ELIGIBILITY CRITERIA: Relevant papers should report data from primary studies dealing with the prevalence of T. gondii in meat from livestock species as obtained through direct detection methods. Meta-analysis and meta-regression were performed. RESULTS: Of 1915 papers screened, 69 papers were included, dealing mainly with cattle, pigs and sheep. Pooled prevalences, based on random-effect models, were 2.6% (CI95 [0.5-5.8]) for cattle, 12.3% (CI95 [7.6-17.8]) for pigs and 14.7% (CI95 [8.9-21.5]) for sheep. Due to the high heterogeneity observed, univariable and multivariable meta-regression models were fitted showing that the geographic area for cattle (p = 0.032), the farming type for pigs (p = 0.0004) and the sample composition for sheep (p = 0.03) had significant effects on the prevalences of Toxoplasma detected/estimated. Moreover, the role of different animal species was dependent on the geographic location of animals' origin. LIMITATIONS: Limitations were due mainly to a possible publication bias. CONCLUSIONS AND IMPLICATIONS: The present work confirms the role of meat, including beef, as T. gondii sources, and highlights the need for a control system for this parasite to be implemented along the meat production chain. Moreover, consumer knowledge should be strengthened in order to reduce the impact of disease.

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.

Ecological niche model of Phlebotomus perniciosus, the main vector of canine leishmaniasis in north-eastern Italy.

With respect to the epidemiology of leishmaniasis, it is crucial to take into account the ecoclimatic and environmental characteristics that influence the distribution patterns of the vector sand fly species. It is also important to consider the possible impact of on-going climate changes on the emergence of this disease. In order to map the potential distribution of Phlebotomus perniciosus, the main vector species of canine leishmaniasis in north-eastern Italy, geographical information systems tools, ecological niche models (ENM) and remotely sensed environmental data were applied for a retrospective analysis of an entomological survey conducted in north-eastern Italy over 12 years. Sand fly trapping was conducted from 2001 to 2012 in 175 sites in the provinces of Veneto, Friuli-Venezia Giulia and Trentino-Alto Adige. We developed a predictive model of potential distribution of P. perniciosus using the maximum entropy algorithm software, based on seasonal normalized difference vegetation index, day and night land surface temperature, the Corine land cover 2006, a digital elevation model (GTOPO30) and climate layers obtained from the WorldClim database. The MaxEnt prediction found the more suitable habitat for P. perniciosus to be hilly areas (100-300 m above the mean sea level) characterised by temperate climate during the winter and summer seasons, high winter vegetation cover and moderate rainfall during the activity season of vector sand fly. ENM provided a greater understanding of the geographical distribution and ecological requirements of P. perniciosus in the study area, which can be applied for the development of future surveillance strategies.

Cytauxzoon sp. infection in two free ranging young cats: clinicopathological findings, therapy and follow up.

Two young brother male free-ranging domestic shorthair cats were evaluated for diarrhea. They presented with intraerythrocytic piroplasms on blood smear evaluation. Only the first cat was anemic (mild non-regenerative anemia). A partial segment of the 18S rRNA was amplified and sequenced, revealing a homology of 99% with Cytauxzoon sp. and of 93% with Cytauxzoon felis. The first cat was treated with doxycycline and imidocarb dipropionate and monitored by serial laboratory exams, resulting negative for Cytauxzoon sp. infection after the end of the therapy (follow-up period of 175 days). The second cat received the same therapy, but doxycycline was discontinued by the owner after 1 week. He was monitored for 130 days, remaining erythroparasitemic and asymptomatic. We described cases of Cytauxzoon sp. infection in domestic cats with detailed clinical data, description of two therapeutic protocols, and follow-up after treatment with opposite parasitological responses (parasitological cure versus persistence of infection).

Retrospective and spatial analysis tools for integrated surveillance of cystic echinococcosis and bovine cysticercosis in hypo-endemic areas.

Cystic echinococcosis (CE) and bovine cysticercosis (BC) are two important parasitic zoonoses, whose prevalence varies among European countries. Few data are available on prevalence and geographic distribution of these two diseases in Veneto region in North-Eastern Italy, where they are generally perceived as minor public health problems. Available data from regional farms on cattle positive to CE and BC and slaughtered in the period 2006-2010 were analysed by spatial scan statistic using a Bernoulli probablility model. Out of 576 bovines testing positive to CE, 467 were found to be autochthonous cases. Three significant CE clusters were identified, the most likely one (P < 0.0001) located in the eastern part of the Veneto region. As for BC, two clusters were identified from 148 animals resting positive, 91 which were autochthonous. An epidemiological survey was conducted and the most likely CE cluster was centered, collecting faecal samples from 28 dogs living in the farms of the area. Out of five animals (all shepherd dogs) found positive for taenid eggs by copromicroscopy, one was confirmed positive for Echinoccus granulosus by means of polymerase chain reaction. The study demonstrates the usefulness of integration of slaughterhouse data and geographical coordinates of farms involved for effective surveillance of CE and BC. The reliability of the spatial analysis in the identification of clusters of EC cases was confirmed by the finding of one dog positive for E. granulosus.

Human exposure to piroplasms in Central and Northern Italy.

TA serosurvey has been conducted in Northern and Central Italy to investigate the presence in humans of antibodies against zoonotic Babesia and Theileria species. The study focused on a total of 432 volunteers, of which 290 were persistently exposed to tick bites because of their jobs (forester employees, livestock keepers, veterinary practitioners, farmers and hunters) and 142 resident in the same area less frequently exposed. An indirect fluorescent antibody test (IFAT) for humans was used to detect antibodies to Babesia microti, IFAT tests for veterinary use were modified to detect reactivity to Babesia bovis, Babesia canis and Theileria equi. A laboratory-derived ELISA was employed to detect antibodies to Babesia divergens. Both reactive and 10 negative sera were analysed against plasmodial antigens to evaluate possible aspecificity. A high reactivity to piroplasm antigens was found, showing significant difference between the sera of the two groups of volunteers (24% vs 7.%; p<0.001). No cross-reactivity was observed, while each professional group showed reactivity that would fit with the professional risk exposure. In particular, a high reactivity to B. microti and B. divergens antigens was observed in foresters and hunters (32% and 12%, respectively). This is the first report on the human seroreactivity to piroplasms in Italy; it also provides additional epidemiological information on these tick-borne zoonoses in Europe. Our findings suggest the possible occurrence of piroplasm infections in Italy and alert physicians to consider these otherwise neglected parasitic diseases when dealing with any febrile illness, especially in subjects exposed to tick bites.

Environmental contamination by canine geohelminths.

Intestinal nematodes affecting dogs, i.e. roundworms, hookworms and whipworms, have a relevant health-risk impact for animals and, for most of them, for human beings. Both dogs and humans are typically infected by ingesting infective stages, (i.e. larvated eggs or larvae) present in the environment. The existence of a high rate of soil and grass contamination with infective parasitic elements has been demonstrated worldwide in leisure, recreational, public and urban areas, i.e. parks, green areas, bicycle paths, city squares, playgrounds, sandpits, beaches. This review discusses the epidemiological and sanitary importance of faecal pollution with canine intestinal parasites in urban environments and the integrated approaches useful to minimize the risk of infection in different settings.

Preliminary study of the effects of preventive measures on the prevalence of Canine Leishmaniosis in a recently established focus in northern Italy.

Canine Leishmaniosis is endemic in Mediterranean areas, with a well-documented northward spread. The mass use of preventive measures against sandfly bites (collar and spot-on formulations) was tested in a small focus recently established in an isolated hilly area of north-eastern Italy (Colli Euganei). In 2006 and 2007, a total of 449 dogs living in the southern part of Colli Euganei were screened against Leishmania infantum using an immunofluorescence antibody test (IFAT), and 31 (6.9%) were seropositive. A risk factor analysis clearly described the focus as limited to a small village named Calaone. In 2010, 63 animals from Calaone were sampled and their owners interviewed to verify the effectiveness of the preventive measures. According to what reported by owners, dogs started to be protected in 2006 (66.7% dogs protected), and protection rate incremented (around 90%) during the subsequent years. The seroprevalence value (4.2%) of the youngest age class (<5 years) was significantly lower than other classes, demonstrating that animals born after 2006 had low probabilities of getting infected. Besides, seroprevalence value referred only to dogs living in Calaone was 32.4% (23/71) in 2006-2007 and 20.6% (13/63) in 2010, showing a decreasing trend. Although still preliminary, the results show high sensitization of dog owners and suggest that the mass use of collars and spot-on acts positively in reducing the circulation of L. infantum.

Risk of canine and human exposure to Dirofilaria immitis infected mosquitoes in endemic areas of Italy.

BACKGROUND: The occurrence of infections by Dirofilaria immitis in canine and human populations depends on several factors linked to both the definitive and intermediate hosts. Little data are available on the risk of human and dog exposure to D. immitis in endemic areas. Data collected on dog- and human-bait traps in endemic areas of north-eastern Italy were used to estimate the likelihood of a receptive host coming into contact with an infected vector. METHODS: From 1997 to 1999, mosquitoes were collected from three sampling sites of north-eastern Italy on D. immitis microfilaraemic dogs and on human baits. The bite/night/host rates were determined based on the number of feeding and probing mosquitoes on dogs and humans, respectively. The survival/mortality rates of different species of mosquitoes following the blood meal, and the rate of natural Dirofilaria infection in unfed specimens were estimated. The risk of exposure of dogs and humans to infected mosquito species was determined by combining the bite/host/night and the mosquito infection rates. RESULTS: A total of 1,165 mosquitoes were collected on human (n=815) and dog (n=350) baits with varying species composition (i.e., Culex pipiens, 87.3% and Ochlerotatus caspius, 11.6%). Overall, dogs were more attractive to Cx pipiens than humans (feeding rate 70.2% vs probing rate 25.9%). The highest bite/night/host rate was 84.0 for dogs and 26.5 for humans. Cx pipiens displayed a mortality rate of 76.3% within 13 days and Oc. caspius of 100% within two days following the infective blood meal. In addition, D. immitis DNA was detected in unfed Cx pipiens (infection rate of 0.26%-2.07%). The infection rate adjusted for mosquito mortality was 0.38%. Based on data collected, the contact between an infected mosquito and a host can occur as often as every four nights for D. immitis infected-mosquitoes in dogs and within two weeks for humans. CONCLUSIONS: Cx pipiens was confirmed as the most efficient natural vector of D. immitis in the studied area. In endemic areas, the risk of transmission can be very high for dogs and relevant for humans. Despite the increased awareness of veterinarians and owners on canine dirofilarioses, dogs from rural areas still maintain the natural life cycle of Dirofilaria spp., therefore acting as a source of infection to humans through vector bites.

Development of culture-based serological assays to diagnose Babesia divergens infections.

Babesioses are hematic tick-borne diseases that induce malaria-like disorders in domestic, wild animals, and humans. Although indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) commercial kits are available to test the presence of antibodies against most Babesia species, no kit exists to serologically diagnose the infections due to Babesia divergens, one of the most important zoonotic species. To fill this gap and to develop assays to detect animal and human infections, in vitro cultures (microaerophilous stationary phase system) of B. divergens were organized. Infected erythrocytes were adsorbed as corpuscular antigen (CA) on IFAT slides and ELISA microwells. The supernatant medium of the cultures (metabolic antigen, MA) was collected and employed in ELISA and western blot (WB) assays. B. divergens was also used to produce positive sera in Meriones unguiculatus and to infect a calf. Serological tests were set up with sera from experimentally/naturally infected animals, and possible cross-reactions were evaluated using heterologous sera from cattle positive to other piroplasms. Sera from clinically healthy people at risk of infection were also tested. As expected, assays based on the purified MAs from in vitro cultures proved more sensitive and specific than CA-IFAT and CA-ELISA. In fact, MA-ELISA provided satisfactory performances (even if 8.4%-15.7% cross-reactions were evidenced), and the WB developed proved totally sensitive and specific. WB indicated as immunodominant antigens two major protein bands at 33 and 37 kDa, which were also evidenced in 2.2% of the human sera tested, proving the parasite transmission to humans also in Italy.

Tick reservoirs for piroplasms in central and northern Italy.

Ticks, collected in central and northern Italy from pets, livestock, wild animals and the environment (n=2107), were identified by microscopy and processed by molecular diagnostics to determine the species that act as a reservoir for piroplasms. A total of 11 ixodid tick species were identified, with five of them proving to be piroplasm positive. Molecular diagnostics identified Theileria equi and eight Babesia species in 52 adult specimens, mostly (n=50) removed from piroplasm-free vertebrate hosts. Ixodes ricinus hosted the highest number of species, although the highest infection rate was recorded in Hyalomma marginatum (9.1%), followed by I. ricinus (5.1%), Dermacentor marginatus (5%), Rhipicephalus turanicus (3.1%) and R. sanguineus (1.2%). Novel tick/pathogen associations were detected, suggesting that certain tick species (such as Hy. marginatum, R. sanguineus and I. ricinus) are vector of more piroplasm species than previously thought. Trans-stadial maintenance of the piroplasms was observed in each positive tick species; vertical transmission of B. canis canis was demonstrated in R. sanguineus. Finally, the detection of Babesia sp., B. microti-like species and B. rodhaini, phylogenetically related to zoonotic species, suggests that the human population could be at risk of infection in the studied area.