Ezio Ferroglio

Diseases shared between wildlife and livestock: a European perspective

Wildlife diseases are in fashion. This is creating an explosion of related knowledge. Despite this, the dynamics of both wildlife and diseases and the changes in livestock and wildlife management make it increasingly difficult to overview the current situation of wildlife diseases in Europe. This paper aims to discuss the available management possibilities and to highlight current research priorities. One area that causes severe concern to authorities is diseases largely under control in domestic populations but still existing as a reservoir in wildlife. Multihost situations are also of concern for wildlife management and conservation, as diseases can affect the productivity and density of wildlife populations with an economic or recreational value. Concern about emerging diseases is rising in recent years, and these may well occur at the fertile livestock–wildlife interface. Wildlife-related zoonoses are a diverse and complex issue that requires a close collaboration between wildlife ecologists, veterinarians and public health professionals. A few risk factors can be identified in most of the relevant wildlife diseases. Among them are (1) the introduction of diseases through movements or translocations of wild or domestic animals, (2) the consequences of wildlife overabundance, (3) the risks of open air livestock breeding, (4) vector expansion and (5) the expansion or introduction of hosts. Wildlife disease control requires the integration of veterinary, ecology and wildlife management expertise. In addition to surveillance, attempts to control wildlife diseases or to avoid disease transmission between wildlife and livestock have been based on setting up barriers, culling, hygienic measures, habitat management, vector control, treatments and vaccination. Surveillance and descriptive studies are still valuable in regions, species or diseases that have received less attention or are (at least apparently) emerging. Nonetheless, limiting the research effort to the mere reporting of wildlife disease outbreaks is of limited value if management recommendations are not given at the same time. Thus, more experimental approaches are needed to produce substantial knowledge that enables authorities to make targeted management recommendations. This requires policy makers to be more aware of the value of science and to provide extra-funding for the establishment of multidisciplinary scientific teams.

The northward spread of leishmaniasis in Italy: evidence from retrospective and ongoing studies on the canine reservoir and phlebotomine vectors

Visceral leishmaniasis (VL) incidence has been increased in Italy in humans and dogs since the 1990s, with new foci being detected within traditional boundaries of endemic transmission but also in northern regions previously regarded as non‐endemic. To monitor the putative VL spreading, surveillance was implemented in northern continental Italy comprising: analysis of human cases recorded from 1990 through 2005; retrospective literature analysis of canine leishmaniasis (CanL) and phlebotomine sandfly records through 2002; prospective investigations in dogs from 2003 through 2005 and surveys on sandflies in 2003 and 2004. Two‐hundred‐thirty human cases (11% of Italian cases) were recorded. Their stratification by age and HIV status disclosed a sharp decrease of HIV/VL co‐infections paralleled by concomitant increase of paediatric and HIV‐negative adult patients during the study period. Four patients had no travel history. Seven leishmaniasis foci were retrospectively identified since 1990, whereas prospective investigations in dogs disclosed 47 autochthonous clinical cases and 106 autochthonous seropositives among 5442 dogs (2.1%) from 16 foci of six regions. Parasites were typed as Leishmania infantum MON‐1. Four vector species were identified among 1696 Phlebotomus (Larroussius) collected specimens. Comparisons with historical data showed that P. perniciosus and P. neglectus have increased in density and expanded their geographic range in the study area. Northern continental Italy is now focally endemic for VL and a moderate risk for human disease does exist, although the intensity of transmission seems to be lower than in traditional settings of Mediterranean VL.

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.

Asymptomatic Leishmania infantum Infection in an Area of Northwestern Italy (Piedmont Region) Where Such Infections Are Traditionally Nonendemic

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.

Canine Leishmaniasis, Italy

We report the results of a survey to determine the prevalence of canine leishmaniasis and the presence of sand flies in northwestern Italy, where autochthonous foci of canine leishmaniasis have not been reported. Active foci of canine leishmaniasis were identified, which suggests that the disease is now also endemic in continental climate areas.

Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes of Leishmania infantum for Serodiagnosis of Visceral Leishmaniasis

ABSTRACT Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3′-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts.

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 (χ2 = 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.

Role of wildlife in the epidemiology of Leishmania infantum infection in Europe

Although dogs are considered the main reservoir of Leishmania infantum infection in endemic areas in Europe, the existence of other wild vertebrate reservoirs has been proposed as a possible cause of the lack of success of control measures. Evidence of L. infantum infection in European wildlife has been reported in carnivores, lagomorphs, and rodents. The red fox (Vulpes vulpes) received most attention, probably due to its taxonomic relationship with the dog and because it is the most abundant wild carnivore in Europe. Foxes and other wild carnivores often displayed high prevalences of infection but their infectiveness to the sandfly vector has never been demonstrated. However, xenodiagnosis demonstrated that black rats (Rattus rattus), are infectious to sandflies. This, together with their relative abundance, high rates of infection, and the fact that infected rats have been found on a Mediterranean island where dogs are not present, makes rats good candidate to be reservoirs of L. infantum. Recently, the Iberian hare (Lepus granatensis) has been recognized as the origin of a leishmaniosis outbreak in humans in Spain and xenodiagnosis showed that this species is also able to infect sandflies. In contrast, a recent survey in cave bats failed to detect infected individuals. In the future, the comparison of parasite isolates from humans, dogs and wildlife, xenodiagnosis studies in wild carnivores, and the study of other vertebrate taxonomic groups will help determine the current role of European wildlife in the epidemiology of leishmaniosis.

Piroplasmosis in wildlife: Babesia and Theileria affecting free-ranging ungulates and carnivores in the Italian Alps

BackgroundPiroplasmosis are among the most relevant diseases of domestic animals. Babesia is emerging as cause of tick-borne zoonosis worldwide and free-living animals are reservoir hosts of several zoonotic Babesia species. We investigated the epidemiology of Babesia spp. and Theileria spp. in wild ungulates and carnivores from Northern Italy to determine which of these apicomplexan species circulate in wildlife and their prevalence of infection.MethodsPCR amplification of the V4 hyper-variable region of the 18S rDNA of Babesia sp./Theileria sp was carried out on spleen samples of 1036 wild animals: Roe deer Capreolus capreolus (n = 462), Red deer Cervus elaphus (n = 52), Alpine Chamois Rupicapra rupicapra (n = 36), Fallow deer Dama dama (n = 17), Wild boar Sus scrofa (n = 257), Red fox Vulpes vulpes (n = 205) and Wolf Canis lupus (n = 7). Selected positive samples were sequenced to determine the species of amplified Babesia/Theileria DNA.ResultsBabesia/Theileria DNA was found with a mean prevalence of 9.94% (IC95% 8.27-11.91). The only piroplasms found in carnivores was Theileria annae, which was detected in two foxes (0.98%; IC95% 0.27-3.49). Red deer showed the highest prevalence of infection (44.23%; IC95% 31.6-57.66), followed by Alpine chamois (22.22%; IC95% 11.71-38.08), Roe deer (12.55%; IC95% 9.84-15.89), and Wild boar (4.67%; IC95% 2.69-7.98). Genetic analysis identified Babesia capreoli as the most prevalent piroplasmid found in Alpine chamois, Roe deer and Red deer, followed by Babesia bigemina (found in Roe deer, Red deer and Wild boar), and the zoonotic Babesia venatorum (formerly Babesia sp. EU1) isolated from 2 Roe deer. Piroplasmids of the genus Theileria were identified in Wild boar and Red deer.ConclusionsThe present study offers novel insights into the role of wildlife in Babesia/Theileria epidemiology, as well as relevant information on genetic variability of piroplasmids infecting wild ungulates and carnivores.

Technical Note : A Rapid Diagnostic Test Detects Plague in Ancient Human Remains: An Example of the Interaction Between Archeological and Biological Approaches (Southeastern France, 16th-18th Centuries)

A rapid diagnostic test (RDT) that detects Yersinia pestis F1 antigen was applied to 28 putative plague victims exhumed from seven burial sites in southeastern France dating to the 16th-18th centuries. Yersinia pestis F1 antigen was detected in 19 of the 28 (67.9%) samples. The 27 samples used as negative controls yielded negative results. Soil samples taken from archeological sites related to both positive and negative samples tested negative for F1 antigen. The detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a preliminary retrospective diagnosis of Y. pestis infection in human remains. The high specificity and sensitivity of the assay were confirmed. For two sites positive to F1 antigen (Lambesc and Marseille), Y. pestis-specific DNA (pla gene) had been identified previously by PCR-sequence based analyses. Specifically, the positive results for two samples, from the Lambesc cemetery and the Marseille pit burial, matched those previously reported using PCR. Independent analyses in Italy and France of different samples taken from the same burial sites (Draguignan and Martigues) led to the identification of both Y. pestis F1 antigen and Y. pestis pla and gplD genes. These data are clear evidence of the presence of Y. pestis in the ancient human remains examined in this study.