Abstract: The prevalence of Leishmania infantum-specific antibodies and asymptomatic infection was assessed in a randomized sample of 526 healthy adults from a continental area of northwestern Italy where L. infantum is not endemic and where autochthonous cases of visceral leishmaniasis (VL) were recently reported. L. infantum-specific antibodies were detected by Western blotting (WB) in 39 subjects (7.41%), while L. infantum kinetoplast DNA was amplified from buffy coat in 21 out of 39 WB-positive subjects, confirming asymptomatic infection in 53.8% of seropositives. Risk factors significantly associated with WB positivity were uninterrupted residence since childhood in a local rural environment (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.7 to 7.3), daily contact with animals though not exclusively with dogs (OR, 3.7; 95% CI, 1.3 to 10.7), older age (OR, 2.31; 95% CI, 1.2 to 4.5), and agricultural/other outdoor activities (OR, 3.8; 95% CI, 0.99 to 3.7.) Logistic regression analysis showed that uninterrupted residence in a local rural environment and an age of >65 years were the only independent predictors of seropositivity assessed by WB. Follow-up at 24 months did not show evidence of VL in either seropositive or PCR-positive subjects. The detection of a high seroprevalence rate, confirmed as asymptomatic infection by PCR in more than half of the cases, among healthy residents in a continental area of northwestern Italy makes local L. infantum transmission very likely. In a region where VL is considered nonendemic, these findings warrant further epidemiological investigations as well as interventions with respect to both the canine reservoir and vectors, given the possible risks for immunosuppressed patients.
Abstract: 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
Abstract: 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.
Abstract: 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
Abstract: 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.
Abstract: 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
