PIANTEDOSI D.1, VENEZIANO V.1, DI PRISCO F.2, ZOTTOLA T.3, D’ALESSIO N.2, SANTORO M.2, MARIANI U.2, FAGIOLO A.3, DI MUCCIO T.4, FOGLIA MANZILLO V.1, FIORENTINO E.4, SCALONE A.4,
NEOLA B.1, GRADONI L.4, OLIVA G.1, GRAMICCIA M.4 1
Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Via F. Delpino 1, 80137 Naples, Italy; neola_benedetto@libero.it
2
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via della Salute 2, 80055 Portici, Italy 3
Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, Sezione Provinciale di Latina, Strada Congiunte Destre, 04100 Latina, Italy
4
Unit of Vector-borne Diseases and International Health, MIPI, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
ABSTRACT
The aim of this study was to investigate on the prevalence of L. infantum infection in foxes living in two distinct areas of southern and central Italy. Sixty eight carcasses of foxes were subjected to PCR analysis. At external examination no clinical signs of leishmaniasis were recorded. Ten foxes were found to be positive by Leishmania nested-PCR, of which 4 in spleen, 2 in lymph nodes and 4 in both organs. Our results show that in southern and central Italy foxes may me subclinically infected by Leishmania, but their importance as reservoirs for the maintenance of the infection remains to be determined.
INTRODUCTION
Leishmania infantum is endemic in the Mediterranean basin. Domestic dogs act as the main reservoir hosts and may suffer severe disease, characterized by chronic evolution of viscero-cutaneous signs. Campania region, and some areas of central Italy, are historically considered territories of elevated endemicity for both human and canine leishmaniasis (CL); however limited information is available as regards potential wild reservoirs of L. infantum. The red fox (Vulpes vulpes) has been suspected to have an epidemiological role as a source of CL cases occurring in rural or peri-urban areas (Millán et al., 2014). Therefore, the aim of this study was to investigate on the prevalence of L. infantum infection in free-ranging foxes living in two areas in southern and central Italy.
MATERIALS AND METHODS
The first area of study was represented by the Picentini hills, a mountain range in Campania region of southern Italy, part of the Apennines. The area is located in the Salerno and Avellino provinces. The second area of study was represented by the southern part of Lazio region and sampling has been carried out in the provinces of Latina and Frosinone. Hunters were invited to bring carcasses of foxes, either shot or found dead because of vehicular accidents, to the Avellino and Latina sections of the Istituto Zooprofilattico.
Carcasses were weighted, examined externally and submitted to necropsy. The body condition was scored from 1 to 5 during necropsy. Whenever possible, spleen, lymph node and liver samples were obtained from each animal and stored at -20°C pending Leishmania examination. Frozen vulpine samples were thawed and total genomic DNA was extracted from approximately 20 mg fragments of liver or spleen tissue, and from whole lymph nodes using Easy-DNA kit (Invitrogen) following the manufacturer’s instruction. DNA was subjected to two consecutive PCR amplifications using the kinetoplastid-specific primers R221 and R332 in the first run, and the Leishmania genus specific primers R223 and R333 in the second run. Positive Leishmania nested-PCR samples were afterwards identified at species level using ribosomal internal transcribed spacer-1 (ITS-1) PCR-RFLP analysis by HaeIII enzyme.
RESULTS
Overall, sixty eight foxes were examined; 53 animals were shot between October 2012 – January 2013, while 15 vehicle-crashed foxes were found in a period between June and September 2013. From an examination of teeth, animals were classified as puppy (3), young (12), adult (49) and old (4). Thirty nine were males and 29 females, with a mean weight of 4.4 kg (range 0.9 – 7.0 kg). As regards geographical distribution, 48 animals were from Campania region and 20 were from Lazio region. At external examination no clinical signs of CL were recorded, except for frequent weight loss that could be attributed to other causes (e.g. intestinal worms). Likewise, no classical CL lesions such as spleen, liver or lymph node enlargement, nor renal macroscopic alterations were detected at necropsy. All target tissues were available for the examination in 43 foxes; spleen and lymph nodes were available in 2 cases, spleen and liver in other 2 cases, and spleen in 21 cases. Ten foxes (14.7 %) were found to be positive by Leishmania nested-PCR, of which 4 in spleen, 2 in lymph nodes and 4 in both organs. All liver samples were found negative at the analysis. Leishmania typing by ITS1 PCR- RFLP analysis of all positive samples confirmed L. infantum as the aetiological agent. As regards the estimated age of positive animals, 2 were puppies, 7 adults and 1 an old adult. Seven infected animals were recorded as presenting a poor body condition score (=1).
DISCUSSION
In our study, that confirmed indisputably the identification of the agent as L. infantum, 4/10 infected foxes where detected in January, indicating the persistence of the infection after the sand fly season, that in southern peninsular Italy occurs from the end of May through the end of October. The two positive puppies showing an apparent age of less than 2 months were detected early in June and in mid August, respectively; as regards the former case, a maternal transmission could also be hypothesized because sand fly infectious bites are estimated to increase in incidence starting from July. Our results showed a rate of Leishmania infection in foxes lower than previous data reported in Italy (range 40 – 52%) (Dipineto et al., 2007; Verin et al., 2010). Different rates of Leishmania infection have been reported in foxes from other Mediterranean regions endemic for CL, from very high in central Spain (74.6%) (Criado-Fornelio et al., 2000) to moderate (8.7%) in southern France (Davoust et al., 2014). In our study the low infection rate and the lack of the clinical disease support the hypothesis that foxes do not play a major role as reservoirs in endemic areas (Millàn et al., 2014). This latter conclusion was strongly suggested by Courtenay and colleagues (2002) as regards a different fox species in Brazil. A population of crab-eating foxes (Cerdocyon thous) was studied longitudinally by means of xenodiagnosis and quantitative PCR of skin samples. Results showed that L.infantum-infected foxes, which typically presented asymptomatic infections, were non-infectious for the vector (Lutzomyia longipalpis) and exhibited parasite loads similar to those detected in non- infectious subclinically infected dogs.
CONCLUSION
In conclusion, in southern and central Italy foxes may me subclinically infected by Leishmania, but their importance as effective reservoirs for the maintenance of CL remains to be determined.
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AKNOWLEDGMENTS
This study was partially funded by EU grant FP7-261504 EDENext, catalogued by the EDENext Steering Committee as EDENext000 (http://www.edenext.eu), and by the Management Committee of the Hunting District of Salerno (ATC SA 1).
NON-INVASIVE METHODOLOGIES TO EVALUATE STRESS ON HARE