M. C. Prati

Natural Vectors of Dirofilariasis in Rural and Urban Areas of the Tuscan Region, Central Italy

Abstract Entomological investigations by means of dog- and human-baited traps were carried out in summers 2000–2002 in urban and rural areas of the Tuscan region in central Italy. The aim of the study was to define the mosquito species involved in the transmission of Dirofilaria nematodes and to assess the risk that their presence might represent for animal and human health. Nocturnal fieldwork on host-seeking activity and feeding preferences was followed by microscopic identification of the mosquito species attracted and by molecular identification of Dirofilaria parasites in mosquitoes. In total, 3,611 mosquito females belonging to 12 species, largely represented by Culex pipiens L. and Aedes caspius (Pallas), were caught. Some females of each species collected fed on the dogs, indicating their possible role as an intermediate host, but filarial DNA was found only in Cx. pipiens, Anopheles maculipennis s.l. (Meigen), and Coquillettidia richiardii (Ficalbi). In rural environments, the DNA evidence indicated the presence of infective larvae of Dirofilaria immitis, whereas in urban areas, infective larvae of Dirofilaria repens were present. The role of Cx. pipiens as a vector for heartworm disease and subcutaneous infections in natural and artificial environments was confirmed, whereas Ae. caspius seemed refractory to the infection. The different role of the collected species is discussed. The vector competence of An. maculipennis and Cq. richiardii needs further investigation, because the importance of these species poorly represented, and the role of species such as Aedes albopictus (Skuse), characterized by a dominant diurnal activity pattern, has to be evaluated.

Endoparasites of red fox (Vulpes vulpes) in central italy

A parasitologic study on 129 red foxes (Vulpes vulpes) from Tuscany (central Italy) was carried out in 2004–2006. Five intestinal species were found at necropsy: Dipylidium caninum (prevalence 57.3%), Mesocestoides lineatus (45.4%), Uncinaria stenocephala (39.1%), Toxocara canis (9.1%), and Toxascaris leonina (5.4%). Other parasites not associated with the intestine included Crenosoma vulpis (14.7%), Capillaria aerophila (7.0%), Angiostrongylus vasorum (7.0%), and filarial parasites (17.8%). Coprologic tests were less sensitive and less specific in identifying parasites than direct examinations at necropsy. Trichinella larvae were not found in muscles submitted to artificial digestion. By immunologic assay, antigens of Echinococcus spp. were detected in fecal samples of 20 foxes, but results could not be confirmed by fecal examination or molecular tests.

Extraintestinal nematodes of the red fox Vulpes vulpes in north-west Italy.

Extraintestinal nematodes of the red fox (Vulpes vulpes) are a wide group of parasites that infect wild and domestic carnivores and occasionally humans. Nematodes in the cardiopulmonary system, stomach, urinary apparatus and muscle tissue of 165 red foxes (Vulpes vulpes) from north-west Italy (Liguria and Piedmont) were investigated between 2009 and 2012. Of the cardiopulmonary nematodes, a high prevalence of Angiostrongylus vasorum and Eucoleus aerophilus (syn. Capillaria aerophila) was found, 78.2% and 41.8% respectively; Crenosoma vulpis (15.8%) and Filaroides spp. (4.8%) were also found. Spirocerca lupi (23.5%), Aonchotheca putorii (syn. Capillaria putorii) (8.6%) and Physaloptera spp. (2.5%) were detected in the stomach and Pearsonema plica (syn. Capillaria plica) (56.8%) in the bladder. Eucoleus boehmi (syn. Capillaria boehmi) was also detected in the nasal cavities of one of the two foxes examined. A coprological examination revealed eggs of E. aerophilus, A. putorii, S. lupi, Physaloptera spp. and eggs of intestinal parasites. Filarial worms were absent in all the 165 animals examined, nor was there evidence of Trichinella spp. in any of the foxes. The foxes were found to host a high prevalence of many species of extraintestinal nematodes. The prevalence of A. vasorum in foxes found in the present study is among the highest in Europe. In addition, to the best of our knowledge, E. boehmi and Filaroides spp. have never been reported before in this host in Italy.

Feline leukaemia virus (FeLV) and feline immunodeficiency virus infections in cats in the Pisa district of Tuscany, and attempts to control FeLV infection in a colony of domestic cats by vaccination

The seroprevalence of feline immunodeficiency virus (FIV) in 203 apparently healthy domestic cats living in the district of Pisa, central Italy, was 11·3 per cent, and the prevalence of feline leukaemia virus (FeLV) was 8·4 per cent. The prevalence of FIV depended significantly on the lifestyle and age of the cats; cats living outdoors were more likely to be FIV-positive than cats living indoors, and the proportion of FIV-positive cats increased with age. In contrast, there was no significant relationship between these variables and the prevalence of FeLV. There was no significant relationship between the cats’ seropositivity for FIV and FeLV. The results of a five-year field study to control FeLV infection by vaccination in a colony of 30 domestic adult cats naturally exposed to the infection suggest that the vaccination was effective in FIV-negative cats, but failed to protect FIV-positive cats against FeLV.

First report of Eucoleus boehmi (syn. Capillaria boehmi) in dogs in north-western Italy, with scanning electron microscopy of the eggs.

Dogs can be infected by several nematodes of the Trichuridae family. Trichuridae eggs are all similar, barrel shaped with polar plugs, and misdiagnosis among different species can occur. The most common species is Trichuris vulpis, while the respiratory parasites Eucoleus boehmi (syn. Capillaria boehmi) and Eucoleus aerophilus (syn. Capillaria aerophila) are rarely observed in pets. E. boehmi is reported for the first time in this study in north-western Italy with other Trichuridae. Dog faecal samples (270) were examined by flotation. E. boehmi (2.2%), E. aerophilus (4.4%) and T. vulpis (12.2%) were found; identification was done with measurements and through observation of morphological characters already known. The specific identification of E. boehmi was confirmed using scanning electron microscopy: its egg shell shows a dense network with a fine mesh, surrounding small pits, on the contrary E. aerophilus eggs present a thick mesh with wide depressions, while T. vulpis eggs surface is smooth.

Canine filarial infections in Tuscany, central Italy.

This study was conducted in order to investigate the epidemiology of filarial species in a region of central Italy (Tuscany) in dogs that did not undergo prophylaxis for filariasis. From 2007 to 2009, 630 blood samples were collected from 40 kennels throughout the regional territory. Samples were analysed with Knotts modified test and with an enzyme-linked immunosorbent assay (ELISA) for the detection of Dirofilaria immitis antigens, those positive for microfilariae were also subjected to histochemical staining for acid phosphatase activity to validate the identification of the species. An overall elevated prevalence of filariasis (23.2%) was found. Dirofilaria immitis and D. repens were found to be the dominant species, with similar prevalences (12.5% and 12.1% respectively), while Acanthocheilonema reconditum was less common (1.9%). Results of this study indicate that monitoring for filariasis should be kept at a high level. A constant monitoring of the canine sanitary status should also be carried out for the protection of human health, considering the zoonotic potential of filarial worms.

Seroprevalence of feline heartworm disease in Tuscany

M. Magi, DVM, B. Sebastiani, DVM, MSc, P. Bandecchi, Prof, Dipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti, Universita degli Studi di Pisa, Italy M. C. Prati, Scuola Normale Superiore di Pisa, INFN Sezione di Pisa, Italy V. Guberti, DVM, Istituto Nazionale per la Fauna Selvatica, Ozzano Emilia (BO), Italy FELINE heartworm disease caused by Dirofilaria immitis is a clinical problem of which veterinary surgeons are increasingly becoming aware. It may cause acute signs, often followed by death, or it may be chronic and asymptomatic (Atkins and others 2000). The disease is present mainly in cats living in zones where it is endemic for dogs, which are the reservoir of the disease. The cat is not an effective reservoir because microfilariaemia is transient (one to two months) or absent. Female mosquitoes propagate the disease by ingesting microfilariae in the blood of an infected dog. In Italy, the main vector for the cat is Culexpipiens (Di Sacco and others 1992, Genchi and others 1992b), but many other species of mosquito can act as vectors of the infection (Cancrini and Kramer 2000). Canine heartworm disease is present in all European countries in the Mediterranean area, the largest endemic area being the Po river valley in Italy where the prevalence among untreated dogs is estimated to be between 50 and 80 per cent (Genchi and others 1998b). The disease spreads due to several factors such as changing climatic conditions, caused by global warming and pollution, favourable for the vectors, the increased number of pets and the increased mobility of people and, as a consequence, their pets. Several cases of canine heartworm disease have been recognised in northern European countries such as Austria, Germany, the Netherlands and northern France and even the UK is at risk, due to the presence of potential vectors (Byrne and Nichols 1999). Recently, various diagnostic tests specific for feline heartworm disease have been developed. Antibody tests, which can detect patent, prepatent and occult infections, are useful in estimating the percentage of cats exposed to the infection (Genchi and others 1998a, Watkins and others 1998). The antibody seroprevalence in cats is important as it allows an estimation of the risk of infection for the cats living in the area of interest. It is not directly correlated to a high prevalence of the disease in dogs because the most important factor for the diffusion of the infection is the abundance of vectors (Dye 1990). In Italy, feline heartworm disease was first recognised in the district of Pisa in 1989 (Sassetti 1989, Sassetti and others 1998) and in northern Italy in 1990 (Di Sacco and others 1990). In 1996, the disease had been recognised only in four districts; however, by 2001, 21 districts were affected by the disease. The regions involved were Tuscany and Emilia Romagna in central Italy, Piedmont, Lombardy, Veneto and Friuli in northern Italy (Genchi and others 1992a, 1998b, 2001, Sassetti and others 1998, Falconelli 1999). The aim of the present study was to evaluate the antibody seroprevalence of feline heartworm disease in Tuscany, which is an endemic zone for canine heartworm disease. Some preliminary results were given by Magi and Sebastiani (1999). It is very difficult to estimate the actual prevalence ofcanine heartworm disease in Tuscany. Almost all dogs going to veterinary practices with their owners are under preventive therapy, so that among them the prevalence should be low. There are, however, some large kennels where stray dogs are collected and no prevention is carried out; the prevalence there should be very high. The possible association between feline heartworm disease seropositivity and the breed, sex, age, living place, lifestyle, feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) status of the cat was also investigated. From November 1998 to April 2000, 349 serum samples from cats living in Tuscany were tested for antibodies against feline heartworm disease using the test ASSURE/FH (Synbiotics), according to the manufacturers instructions. The test can detect antibodies as early as six to eight weeks after infection with the L4 larval stage of the parasite. The sensitivity and specificity of the test are 98 per cent and 99 per cent, respectively (Bestul and others 1998). The sample of cats was chosen randomly from among the animals taken for normal checks or routine operations to veterinary clinics. For every cat examined the veterinary surgeon filled out a standard form with the following data: name of the owner; living place; age, sex and breed of the animal; its lifestyle; and the presence of a dog in the same house. The living places of the animals in each district were classified into three zones according to climatic and environmental factors as follows: coast, flat hinterland, hinterland hills. Canine filariasis is endemic in all three zones (Magi and others 1989) and mosquitoes are abundant throughout, due to the mildness and humidity of the climate. The cats were divided into three groups based on their lifestyle: living always inside the home, living always outside the home, living partly inside and partly outside the home. One hundred and ninety-eight serum samples were also tested for antibodies against FIV using ViraCHEK/FIV (Synbiotics) and 180 samples were tested for antibodies against FeLV using ViraCHEK/FeLV (Synbiotics). In order to verify any association between feline heartworm disease and the breed, sex, age, living place, lifestyle, FIV and FeLV status of the cat, chi-squared tests were performed. A logistic regression analysis was also performed (stepwise method) using SPSS 7.5 (spss), with feline heartworm disease seropositivity as the dependent variable. Positivity for FIV and FeLV were considered as interactive terms. From the output of the logistic regression the odds ratio (OR) of being positive to feline heartworm disease for cats with different lifestyles was calculated as stated in Hosmer and Lemeshow (2000). The P value for significance was 0*05. The 349 serum samples came from cats living in the dis-

Canine filarial infections in Liguria, north-west Italy

This paper reports the findings of a study on the presence of various species of filarial nematodes in dogs in Liguria, north-west Italy, a region traditionally considered free from the disease. Between 2009 and 2012 blood samples were taken from 365 dogs in rural areas in Liguria. The blood samples were then submitted to Knotts test, histochemical staining, polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) for Dirofilaria immitis antigens. Overall, 35 of the 365 dogs were positive using Knotts test for microfilariae (prevalence 9.6%; 95% confidence interval (CI): 6.6-12.6%). Acanthocheilonema reconditum was the most prevalent species (8.0%), while Dirofilaria repens (1.4%) and Dirofilaria immitis (0.6%) were less common. One co-infection by D. repens and A. reconditum was observed. All morphological identifications were confirmed by histochemical staining and PCR. In addition, a retrospective analysis of data on D. immitis antigens in 11,363 samples of canine sera was carried out. Sera were collected and analysed for D. immitis antigens by the Istituto Zooprofilattico Sperimentale (IZS) of Piedmont, Liguria and Aosta Valley (Imperia section) between 2004 and 2013 during annual tests for leishmaniasis on autochthonous dogs throughout Liguria. Serological data from IZS showed an overall seroprevalence of 0.65% (95% CI: 0.50-0.80%) for D. immitis throughout the region. The present study updates the epidemiological map of canine filarial infections in Italy and suggests the need for surveillance and prophylaxis in Liguria.

Eucoleus aerophilus (syn. Capillaria aerophila) and Other Trichinelloid Nematodes in Dogs from Liguria (Northwest Italy)

As a part of a wider study that examined endoparasites of carnivores in northwest Italy, 270 canine fecal samples were analyzed with flotation techniques. Overall prevalence was 7.4 % for Capillaria spp. and 12.2 % for Trichuris vulpis. This article stresses the importance of an accurate morphological and morphometrical analysis to distinguish eggs of T. vulpis from eggs of the genus Capillaria. We also suggest that different Capillaria spp. are present in the Italian dog population.

Cardiorespiratory and gastrointestinal parasites of dogs in north-west Italy

Summary The present study was conducted to evaluate the prevalence of gastrointestinal and cardio-respiratory parasites in dogs in Liguria (northwest Italy) which is a region that has scarcely been studied. A total of 450 dogs (260 males and 190 females) were enrolled in the study from 2009 and 2013. All dogs lived in a rural or semi-rural environment in the provinces of Imperia (n = 352) or Savona (n = 98), Liguria. Coprological examinations showed that 197 dogs (43.8 %, CI 38.7-48.9 %) were infected by parasites. Specifically, 3.3 % of the dogs presented only cardio-respiratory species, 32.4 % only intestinal species, and 8.0 % presented a mixed infection. The most frequent intestinal parasites were Toxocara canis (20 %), Trichuris vulpis (17.8 %), Ancylostomatidae (12 %), Coccidia (2.7 %), Aonchotheca putorii (1.8 %) and Toxascaris leonina (1.8 %). The cardio-respiratory species found were Eucoleus aerophilus (9.6 %), Eucoleus boehmi (1.6 %), Angiostrongylus vasorum (0.7 %), and Crenosoma vulpis (0.2 %). A total of 116 dogs (25.8 %) were parasitized by a single species, multiple infections were observed in 81 dogs (18.0 %) up to a six-order infection. This preliminary study highlighted that the prevalence of intestinal parasites in investigated area is high. Cardio respiratory parasites were detected in an area that has not been investigated before. The creation of a more extensive sampling programme of the area, on a provincial basis in order to build a more detailed map of prevalences for different species of dog parasites throughout Liguria, a more extensive sampling programme of the area needs to be created - ideally for each province.