Epidemiological evaluation of Leishmania infantum zoonotic transmission risk in the recently established endemic area of Northwestern Italy.

Leishmania infantum infection had been expanding into new areas due to changes in vector and host biology. Zoonotic visceral leishmaniasis has become endemic in previously unsuitable areas as vectors find favourable climatic conditions and an increasing number of reservoir dogs are moved between traditionally and new endemic areas. Monitoring vector and disease expansion in areas of recent colonization is needed to understand transmission mechanisms and patterns of disease establishment. Here, we studied the infection status of 815 human blood donors and of 803 sympatric dogs from five, newly endemic, areas in Northwestern Italy. In autochthonous dogs, the seroprevalence of anti-L. infantum antibodies, recorded by Western blot, reached 42.22%, while in humans, the seroprevalence was of 16.81%. No significant correlation between the infection status of dogs and that of their human owners was found, but L. infantum infection was recorded in the different study areas with significant levels of diversity. Restriction fragment length polymorphism showed a high genetic variability of the circulating strains and gave useful insights on patterns of disease establishment into a naïve area.

Epidemiology of Leishmania infantum, Toxoplasma gondii, and Neospora caninum in Rattus rattus in absence of domestic reservoir and definitive hosts.

Isolated environments are privileged settings to study transmission of infection. Montecristo is a small island where no wild or domestic carnivores are present. Invasive Black rats Rattus rattus (n=78) were captured and tested by PCR for Leishmania infantum, Toxoplasma gondii and Neospora caninum. We wanted to test, for these parasites, the existence of a sylvatic cycle independent of reservoir or definitive hosts. None of the rats tested positive by PCR for either T. gondii or N. caninum. We recorded a 15.5% prevalence (CI95% 8-26%) of L. infantum in the rats and Phlebotomus mascittii was captured in Montecristo, leading us to identify it as possible vector of the parasite.

Encephalitozoon cuniculi, Toxoplasma gondii and Neospora caninum infection in invasive Eastern Cottontail Rabbits Sylvilagus floridanus in Northwestern Italy.

Sylvilagus floridanus is a lagomorph introduced for hunting purposes from North America to Europe where, in certain areas like Northern Italy, its population reached high densities. Brain, kidney and skeletal muscle of 144 Eastern Cottontail Rabbits S. floridanus were examined by PCR for Encephalitozoon cuniculi, Toxoplasma gondii and Neospora caninum. DNA of E. cuniculi was found with a prevalence of 9.72% (CI 95% 0.058-0.156). T. gondii and N. caninum DNA was detected in 2.08% (CI 95% 0.0071-0.0595) and 2.78% (CI 95% 0.0109-0.0692) of the samples examined, respectively. This is the first report of E. cuniculi infection in a lagomorph species other than in its natural host Oryctolagus cuniculus, and this is also the first time N. caninum is found to naturally infect S. floridanus. E. cuniculi, T. gondii and N. caninum infect S. floridanus at low but relevant prevalences, considered the important role that these pathogens could play in both animal and human health.

Evaluation of a rapid device for serological diagnosis of Leishmania infantum infection in dogs as an alternative to immunofluorescence assay and Western blotting.

In this study, we compared a rapid immunochromatographic test (Speed Leish K; BVT Groupe Virbac, La Seyne sur Mer, France) with an indirect immunofluorescence assay (IFAT) and Western blotting (WB) for the detection of Leishmania infantum antibodies in dogs. A total of 250 serum samples were collected from 125 L. infantum-positive and 125 L. infantum-negative dogs. Among the positive samples, 81 were strongly positive at low IFAT dilutions, while 44 were low-reactivity sera (IFAT titers, 1:40 to 1:80). The sensitivity and specificity of the Speed Leish K were 96.3% and 100%, respectively, compared with those of the IFAT. When IFAT low-reactivity sera (titers, 1:40 or 1:80) were tested with the Speed Leish K, using WB results as a reference, the sensitivities were 93.75% for sera with a 1:80 titer and 73.33% for sera with a 1:40 titer, and the specificity was 100%. The Speed Leish K is easy to use and performs well, so it can be considered a quick and reliable tool for the diagnosis of L. infantum infection in dogs.

Canine leishmaniosis: in vitro efficacy of miltefosine and marbofloxacin alone or in combination with allopurinol against clinical strains of Leishmania infantum.

Despite the availability of different therapeutic options, canine visceral leishmaniosis (CVL) remains a challenging disease to treat. Recently miltefosine has been registered for use in dogs, and different studies have demonstrated its leishmanicidal effect. Moreover, it has been suggested that fluoroquinolones, compared to standard chemotherapeutic agents, could be an effective and pragmatic alternative to treat CVL. The efficacy of miltefosine and marbofloxacin alone or in combination with allopurinol against clinical strains of Leishmania infantum was assessed in vitro by incubating increasing concentrations of the drugs with a standard parasite inoculum. Miltefosine was significantly more efficacious than marbofloxacin (P < 0.05) against the two strains of L. infantum either alone or in combination with allopurinol. Both drugs were significantly (P < 0.05) more efficacious when associated with allopurinol than alone.

An investigation into alternative reservoirs of canine leishmaniasis on the endemic island of Mallorca (Spain).

The role of wild and free-roaming domestic carnivores as a reservoir of Leishmania infantum was investigated on the Mediterranean island of Mallorca (Balearic Islands, Spain), an endemic area for this disease. Serum, blood and/or spleen samples from 169 animals [48 dogs from a kennel, 86 wild-caught feral cats, 23 pine martens (Martes martes), 10 common genets (Genetta genetta) and two weasels (Mustela nivalis)] were analysed. Seroprevalence determined by Western blotting was 38% in dogs and 16% in feral cats, while the prevalence of infection determined by PCR was 44% in dogs, 26% in cats, 39% in pine martens and 10% in genets. This is the first report of infection by L. infantum in the pine marten or any other member of the Mustelidae family. Restriction fragment length polymorphism (RFLP) analysis found 33 different patterns in 23 dogs, 14 cats and three martens. Two patterns were shared by dogs and cats, two by different cats, and one by different dogs. Patterns were different to those previously reported in carnivores from peninsular Spain. No external lesions compatible with leishmaniasis were observed in any species other than the dogs. Although the dog is probably the primary reservoir of leishmaniasis in endemic areas, the prevalence and the absence of apparent signs of this disease within the island's abundant feral cat and pine marten populations could make these species potential primary or secondary hosts of L. infantum in Mallorca.

Distribution of Phlebotomus perniciosus in North-Italy: a study on 18S rDNA of phlebotomine sand flies.

Leishmaniasis has recently spread, and is now endemic, in many parts of North Italy, even if it is not clear how sand flies vectors have reached this area. In order to clarify the origin of the Phlebotomus perniciosus, the main sand flies specie found in these areas, we analyzed and compared the 18S rDNA sequence from 33 out of 122 P. perniciosus collected in new endemic areas, from neighbor (

Physaloptera sibirica in foxes and badgers from the Western Alps (Italy).

We investigated the presence of Physaloptera sibirica and its distribution as well as the association among the parasite, host (i.e. mange due to Sarcoptes scabiei) and environmental factors (i.e. altitudes) in foxes (Vulpes vulpes) and badgers (Meles meles) from the North-West of Italy. From 1996 to 2008 a total of 608 foxes, culled by hunters or road killed, and 39 road killed badgers were examined in order to investigate the presence of nematodes in the stomach. P. sibirica was found in 16 foxes (2.63%) and one badger (2.56%). As regards foxes' habitat type, prevalence was significantly higher (chi(2) = 16.36, p < or = 0.05) in mountain foxes (6.43%; 95%CI 3.25-11.22%) than those from hills (2.22%; 95%CI 0.46-6.36%) or irrigated plains (0.34%; 95%CI 0.01-1.90%). There were no significant differences between sex, age, months and years of the sampling. P. sibirica presence is significantly (chi(2) = 241.63, p > 0.000001) higher (73.33%; 95%CI 44.83-91.09%) in foxes with sarcoptic mange than foxes without mange (0.84%; 95%CI 0.21-1.84%). According to previous studies, in Southern Europe this parasite is associated with mountain areas, probably due to its intermediate hosts which require cold climate. Further studies are needed to evaluate the pathogenic role of P. sibirica in wildlife and its effects on host life history.

Comparison of two PCR protocols for the detection of Neospora caninum DNA in rodents.

The objective of the present study was, due to increasing interest in the epidemiological role of small mammals as potential reservoir of Neospora caninum, to compare two different PCR protocols for the diagnosis of N. caninum in rodents. We tested tissue samples from 50 house mice (Mus musculus), 50 rats (Rattus norvegicus) and 35 field mice (Apodemus sylvaticus). Two different PCR protocols based on primer pairs, Np4-Np7 and Np6plus-Np21plus, were used for diagnosis on these samples. While there were not mismatches between the results of both PCR from rats or field mice, 49 out of 50 samples from house mice gave positive results with Np4-Np7 primer set. However after cloning and sequencing the PCR products, only six of these were confirmed to be N. caninum, while all the other 43 amplicons matched with house mice DNA sequence from clone RP23-14F5 on chromosome 11 sequence. Our results evidence that Np4-Np7 PCR could not be reliable in diagnosis of N. caninum in rodents.

Characterization of widespread canine leishmaniasis among wild carnivores from Spain.

Visceral Leishmaniasis (VL) is an emerging zoonotic parasitic disease caused by Leishmania infantum in Mediterranean countries, with sand flies (Phlebotomus spp.) as vectors and dogs as the main domestic reservoir. The role of wild carnivores in the epidemiology of leishmaniasis is still controversial. In order to determine the prevalence of natural infection with L. infantum in wild carnivores from Spain, we analyzed 217 samples by PCR and western blotting and used restriction fragment length polymorphism (RFLP) to compare the patterns present in wild carnivores with those of domestic dogs from the same areas. DNA of the parasite was detected in spleen or blood samples from 35 (16.12%) analyzed wild carnivores, including 8 of 39 (20.5%) wolves (Canis lupus), 23 of 162 (14.1%) foxes (Vulpes vulpes), 2 of 7 (28.6%) Egyptian mongooses (Herpestes ichneumon), 1 of 4 genets (Geneta geneta), and 1 of 4 Iberian lynxes (Lynx pardinus). No significant sex or age differences in prevalence were observed in wolves and foxes (P>0.05), but there was a significant difference among regions in foxes (P<0.05). A total of 12 PCR-RFLP patterns were found in foxes, 6 in wolves, 4 in dogs, 2 in Egyptian mongooses and 1 in lynx and genet. RFLP patterns differed between dogs and foxes in the two areas where they could be compared. This is the first study of canine leishmaniasis in wild canids and other carnivores from different regions of Spain by PCR. The prevalence of infection indicates the existence of natural infection in apparently healthy wild carnivore populations, and our results are suggestive of a sylvatic cycle independent of dogs.

Evaluation of 65% permethrin spot-on and deltamethrin-impregnated collars for canine Leishmania infantum infection prevention.

During the 2004 and 2005 sand fly seasons, we evaluated the efficacy of a 65% spot-on solution of permethrin (Exspot, Schering & Plough) and deltamethrin-impregnated collar (Scalibor, Intervet) in reducing Leishmania infantum infection, in a canine leishmaniasis (CanL) endemic region (Liguria) in Italy. Immunofluorescent assay (IFA) revealed that three of 120 dogs (2.5%) treated with a 65% spot-on solution of permethrin, as three of 119 dogs (2.5%) treated with deltamethrin-impregnated collar have shown seroconversion after sand fly season. On the contrary, seroconversion was 15% in 188 untreated control dogs. Treatment reduced the risk of infection by 84%. The difference in treated dogs and control ones is highly significant (chi2 = 12.4; P = 0.0004). Our results show that treatment with 65% spot-on solution of permethrin and the deltamethrin-impregnated collar are effective in reducing the risk of acquiring L. infantum infection.

Evidence of Neospora caninum DNA in wild rodents.

Seventy-five house mice (Mus musculus), 103 rats (Rattus norvegicus) and 55 field mice (Apodemus sylvaticus) from North-West Italy were PCR analysed for Neospora caninum infection. Brain, kidney and muscle tissues collected from the above mentioned animals were tested by PCR using Np6 and Np21 primers. The brain tissue from 2 house mice and 2 rats, the kidney from 4 rats, 1 house mouse and 1 field mouse and muscle from 10 rats, 8 house mice and 1 field mouse were tested positive for N. caninum. Sequencing showed a 96-97% identity of PCR products with N. caninum NC1 sequence. Our findings support previous report on house mouse and rat, and for the first time, provides the evidence of the infection also in field mice. Based on our data, it could be hypothesized that mice can act as a reservoir of N. caninum, and they can play a role in maintaining/spreading N. caninum infection also in the sylvatic cycle. The possibility that dogs could be infected by eating infected house mice suggests new opportunities for N. caninum prophylaxis and control.

Seroprevalence of antibodies to Neospora caninum in urban and rural dogs in north-west Italy.

Sera were collected from 490 dogs from north-west Italy. One hundred and eighty-eight dogs were urban, while 302 dogs were rural. Among the latter, 190 were shepherd dogs and 112 were cattle farm dogs. Sera were tested for the presence of antibodies against Neospora caninum using the Neospora agglutination test. Seroprevalence at 1/40, 1/80, 1/160 dilution titres was significantly higher in rural (36.4%, 19.5%, 9.9% respectively) than in urban dogs (20.2%, 10.6%, 4.8% respectively). Seroprevalence did not differ significantly in males and females. In shepherd dogs, prevalence increased according to dogs' age, thus suggesting a post-natal exposure by horizontal transmission. The observed higher seroprevalence in rural dogs suggests the importance of lifestyle and alimentary habits (i.e. aborted foetuses, placentas and small mammals) in the acquisition of N. caninum infection. Our results confirm that dogs are exposed to N. caninum and play an important role in the epidemiology of N. caninum.

Evaluation of an ELISA rapid device for the serological diagnosis of Leishmania infantum infection in dog as compared with immunofluorescence assay and Western blot.

In this study we compared a commercial enzyme linked immunosorbent assay (ELISA) rapid test (Snap CLATK Canine Leishmania Antibody Test Kit, IDEXX-Snap) with indirect immunofluorescence assay (IFA) and Western blot (WB) for the detection of Leishmania infantum antibodies in dogs. In total sera from 234 dogs were collected: 59 positives and 51 doubtful sera (IFA 1:40-1:80) from an L. infantum endemic area and 124 negative sera from a non-endemic area were tested. To evaluate the Snap CLATK's performances on whole blood, blood in EDTA and sera from 37 dogs were tested in parallel with Snap CLATK. Snap CLATK sensitivity and specificity compared to IFA were 91.1% and 99.2%, while compared to WB were 93.4% and 98.3%, respectively. When IFA doubtful sera (titers of 1:40 or 1:80) were tested Snap CLATK, using WB as reference, sensitivity and specificity were 90.9% and 100%, respectively. Moreover, a complete concordance was observed when Snap CLATK rapid assay was carried out on whole blood or sera from 37 dogs. The Snap CLATK has demonstrated simplicity and performance and can be considered a quick and reliable alternative for the diagnosis of L. infantum infection in dogs.

Dogs' parasite and zoonotic risk: from old to new "emergencies" in the North-West of Italy.

Toxocariasis due to soil contamination from dog and cat faeces has been long described and represents one of the zoonotic risk linked with pets presence in human settlements. Soil samples were collected from private backyards and school playgrounds in Turin and tested for the presence of Toxocara spp. eggs. Samples from dogs and cats living in the same area were also analysed and our results seem to indicate a decrease in soil contamination respect to a survey carried out in 1985. Considering that recently new foci of Canine Leishmaniosis and the presence of competent sand fly vectors have also been reported in the North-West of Italy, a survey was carried out on dogs and humans living in Asti province. To assess the risk of local Leishmania infantum transmission between dog and humans, samples were also analysed by Restriction Fragment Length Polymorphism (PCR-RFLP). Our results have shown that more than 10% of autochthonous dogs and human being living in this previously non-endemic area have been infected by L. infantum. The identity of PCR-RFLP patterns from 3 human clinical cases and from the dogs of one of them allows us to confirm the autochthonous origin of these cases.

Equine Culicoides hypersensitivity: evaluation of a skin test and of humoral response.

Intradermal tests were carried out on 18 horses with clinical signs of Culicoides hypersensitivity (CHS) and 23 horses without clinical signs of CHS, and sera from these horses were analysed by SDS-PAGE and Western blotting (W-B). Intradermal injections of 0.1 ml of 25 microg/microl sterile Culicoides extract, 0.1 ml of 1:10,000 histamine (positive control) and 0.1 ml of physiological saline (negative control) were made in the dermis of the middle region of the neck. Analysis of reactions indicated that a 1 cm wheal and a skinfold thickness >10% at 24 h represented a valid cut-off between horses with and without CHS. In these conditions the test, even in winter when clinical signs were absent, had 100% sensitivity and specificity. The W-B was performed after running Culicoides extract on a 12% polyacrylamide gel. The test revealed the presence of several bands with molecular weight ranging from 6 to 200 kDa. In particular, a band of 65 kDa was predominantly found in hypersensitive horses by using an anti-IgE antibody while in normal horses the same band was mainly detected by using an anti-IgG antibody. Our results demonstrated that the skin test is a valid diagnostic test, with high sensitivity and specificity and that the band of about 65 kDa probably corresponds to the allergen involved in the pathogenesis of CHS.

Characterization of Leishmania infantum strains in blood samples from infected dogs and humans by PCR-RFLP.

Characterization of Leishmania infantum is based on zymodeme analysis, which requires parasite isolation and therefore is not routinely employed. Moreover, the majority of strains in the Mediterranean Basin belong to zymodeme MON-1, and this is a major limitation for this technique in epidemiological studies in this region. We developed a PCR-RFLP method based on kDNA amplification, which was able to discriminate L. infantum strains directly from peripheral blood. Twenty-eight samples were tested with this technique: four obtained from promastigote cultures, and 24 collected from dogs (18) and human donors (six) from traditionally endemic and newly endemic areas of northwestern Italy. Extracted DNAs were amplified using RV1-RV2 primers and PCR products were digested using two restriction enzymes separately: BsiY I and Mlun NI. Some patterns were specific to certain areas. In particular, the identity of PCR-RFLP patterns from a human patient from a newly endemic area and three dogs allow the confirmation of the autochthonous origin of this case. This approach could be applied to epidemiological studies in order to trace the diffusion of L. infantum within dog populations, as well as its transmission to humans.

Antibodies to Neospora caninum in stray cats from north Italy.

Antibodies to Neospora caninum were determined in serum samples from 282 stray cats coming from four colonies near Turin (north-west Italy). Sera were tested using a Neospora Agglutination Test (NAT). Seroprevalence was 24.8% at 1:80, 12.8% at 1:160 and 5.3% at 1:320 dilution. Seroprevalence in females and males, in different colonies and in different age classes, did not differ significantly. Our results confirm that domestic cats are exposed to N. caninum and the observed seroprevalences suggest that risk of exposure is high and N. caninum should be considered in differential diagnosis in cats with neurological signs.

Antibodies to Neospora caninum in wild animals from Kenya, East Africa.

The prevalence of antibodies to Neospora caninum was examined in six wild Artiodactyla species, and in five wild Carnivora species from Kenya. Blood sera (104 wild ungulates from Marula Estates (MEs), and 31 wild carnivores from Masai-Mara reserve and from other wildlife areas in northern and Southern Kenya), were screened using a Neospora agglutination test (NAT), with a twofold dilution (1:40-1:320 titres). Presence of NAT antibodies to N. caninun is reported here for the first time in zebra (Equus burchelli), eland (Taurotragus oryx), African buffalo (Syncerus caffer), Thompson gazelle (Gazella thompsoni), impala (Aepyceros melampus), warthog (Phacochoerus aethiopicus), spotted hyena (Crocuta crocuta) and in free-ranging cheetah (Acinonyx jubatus). At 1:80 dilution, prevalence was 61.5% in eland, 58.5% in zebra, 19.2% in Thompson gazelle, 33.3% in warthog, 50% in African buffalo, 30% in lion (Panthera leo), 20% in cheetah, and 33.3% in spotted hyena. Antibodies up to 1:320 titre were detected in eland (38.4%), zebra (19.5%), Thompson gazelle (3.8%) and lion (5%). Amongst herbivores, sero-prevalence was significantly (P<0.05) higher, at all dilutions, in "grazer/digger" species (e.g. eland and zebra) than in non-"grazer/digger" species (e.g. impala and Thompson gazelle). No antibodies to N. caninum were found in two leopards (Panthera pardus) and one serval (Felis serval). Our results indicates a steady presence of N. caninum in wild mammals from Kenya. The hypothesis of a sylvatic cycle of N. caninum could be suggested, but more data are needed to verify the hypothesis, as to evaluate the role of N. caninum infection on the dynamics of wild animals population in the study area.