Sero-epidemiological investigation on Toxoplasma gondii infection in Apennine wolf (Canis lupus italicus) and wild boar (Sus scrofa) in Italy.

BACKGROUND: The wild boar (Sus scrofa) and the Apennine wolf (Canis lupus italicus) are two wild species that have both increased their presence in the Italian territory, albeit in varying numbers. They can be occasionally found in peri-urban areas as well. Both of these species can serve as intermediate hosts for Toxoplasma gondii, as they can become infected either through the consumption of oocysts found in water, soil, or on vegetables, or through the ingestion of meat containing bradyzoites. Consequently, these animals can be regarded as key indicators of Toxoplasma presence in the wild or peri-urban environment. In our study, we examined a total of 174 wild boar meat juice and 128 wolf sera from Italy for the detection of T. gondii IgG using the indirect fluorescent antibody test (IFAT). RESULTS: The results showed that 40 (22.6%) of the wild boar meat juice and 34 (26.6%) of the wolf serum samples tested positive. Interestingly, there were no significant differences in seropositivity with respect to gender, age group, or the region of origin in both species. CONCLUSIONS: Overall the results indicate a moderate exposure in both the species under investigation, highlighting the spread of T. gondii in sylvatic and periurban environments. The prevalence of T. gondii in wild boar is consistent with findings from other studies conducted in Europe. Our study, with a considerably larger sample size compared to the available research in European context, provides valuable data on the seroprevalence of T. gondii in wolves.

Observational longitudinal study on Toxoplasma gondii infection in fattening beef cattle: serology and associated haematological findings.

Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is a globally distributed zoonotic infection with significant implications for human and animal health. This study investigated the prevalence of T. gondii infection in a population of beef cattle at three different stages of their productive lifespan and examined the impact of T. gondii serological status on blood parameters. A commercial beef fattening unit in Italy was the setting for this research, which involved a biosecurity assessment upon cattle arrival, blood sampling at three time points and Toxoplasma-specific serological testing using indirect fluorescent antibody tests (IFAT). Results revealed a dynamic pattern of T. gondii seropositivity in cattle, with an initial prevalence of 30.6% at arrival (T0) that increased to 44.6% at 14 days (T1) and then decreased slightly to 39.3% at slaughter after 5 months (T2). Interestingly, seroconversion was observed during the study, indicating ongoing infections, and antibody waning occurred in some animals. In terms of blood parameters, seropositive cattle exhibited significantly lower mean corpuscular volume (MCV) and a higher neutrophil-lymphocyte (N/L) ratio, suggesting an activation of the innate immune response. Furthermore, cattle with higher antibody titres displayed higher neutrophil counts. However, all blood parameters with a statistical significance were within the reference range. This study provides for the first time a longitudinal investigation on the serological status for T. gondii in naturally exposed beef cattle. These findings provide valuable insights into the clinico-pathological aspects of natural T. gondii exposure in cattle and underscore the importance of monitoring and managing T. gondii infection in livestock production systems.

Detection of Lotmaria passim in honeybees from Emilia Romagna (Italy) based on a culture method.

Lotmaria passim is considered an emerging field of study in honeybee pathology, since it can threaten the health of the colony leading to a higher mortality rate. However, there is a lack of knowledge regarding the diffusion of this trypanosomatid in Italy. In this study, we highlight the presence of L. passim in the province of Bologna through its culture isolation from honeybee guts and microscopic observation.

Detection of Leishmania sp. kDNA in questing Ixodes ricinus (Acari, Ixodidae) from the Emilia-Romagna Region in northeastern Italy.

To date, sand flies (Phlebotominae) are the only recognized biological vectors of Leishmania infantum, the causative agent of human visceral leishmaniasis, which is endemic in the Mediterranean basin and also widespread in Central and South America, the Middle East, and Central Asia. Dogs are the main domestic reservoir of zoonotic visceral leishmaniasis, and the role of secondary vectors such as ticks and fleas and particularly Rhipicephalus sanguineus (the brown dog tick) in transmitting L. infantum has been investigated. In the present paper, the presence of Leishmania DNA was investigated in questing Ixodes ricinus ticks collected from 4 rural areas included in three parks of the Emilia-Romagna Region (north-eastern Italy), where active foci of human visceral leishmaniasis have been identified. The analyses were performed on 236 DNA extracts from 7 females, 6 males, 72 nymph pools, and 151 larvae pools. Four samples (1.7%) (i.e., one larva pool, 2 nymph pools, and one adult male) tested positive for Leishmania kDNA. To the best of our knowledge, this is the first report of the presence of Leishmania kDNA in questing I. ricinus ticks collected from a rural environment. This finding in unfed larvae, nymphs, and adult male ticks supports the hypothesis that L. infantum can have both transstadial and transovarial passage in I. ricinus ticks. The potential role of I. ricinus ticks in the sylvatic cycle of leishmaniasis should be further investigated.

Analysis of honey environmental DNA indicates that the honey bee (Apis mellifera L.) trypanosome parasite Lotmaria passim is widespread in the apiaries of the North of Italy.

Lotmaria passim is a trypanosomatid that infects honey bees. In this study, we established an axenic culture of L. passim from Italian isolates and then used its DNA as a control in subsequent analyses that investigated environmental DNA (eDNA) to detect this trypasonosomatid. The source of eDNA was honey, which has been already demonstrated to be useful to detect honey bee parasites. DNA from a total of 164 honey samples collected in the North of Italy was amplified with three L. passim specific PCR primers and 78% of the analysed samples gave positive results. These results indicated a high prevalence rate of this trypanosomatid in the North of Italy, where it might be considered another threat to honey bee health.

A case of a dog refractory to different treatments for pulmonary capillariasis.

Pulmonary capillariasis is a parasitic disease caused by the nematode Eucoleus aerophilus which affects wild and domestic carnivores. Currently, there are no anthelmintics approved for use in the treatment of dogs infected with E. aerophilus. The use of several anthelmintics has been reported in a few case reports and field efficacy studies in cats; much less is known on the treatment of dogs infected with E. aerophilus. The paper describes a case of a 4-month-old, mixed breed intact male referred to the Veterinary Teaching Hospital (VTH) of the Department of Veterinary Medical Science of the University of Bologna for a routine vaccination and tested positive for E. aerophilus. The dog has not been responding to three different administered treatments, such as moxidectin, fenbendazole, and milbemycin oxime. Eighteen months after the first fecal examination, owner has brought in the dog for a routine visit; a coprological examination was requested and performed resulting negative for parasites. Veterinary practitioners, parasitologists, diagnostic laboratories, and dog owners need to be aware of the increased danger of possible treatment failure when attempting to control parasitic infections for which there are no approved anthelmintics with established efficacies available for use.

In vitro activity of chemicals and commercial products against Saprolegnia parasitica and Saprolegnia delica strains.

Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments pushes towards the selection of more safe and environment-friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100 ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250 ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon™ S was effective in inhibiting the growth of the mycelium (MIC = MLC = 1,000 ppm). Actidrox® and Detarox® AP showed MIC = 5,000 and 1,000 ppm, respectively, while MLCs were 10-fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.

Crenosoma vulpis in red foxes (Vulpes vulpes) in Northern Italy.

Crenosoma vulpis, the fox lungworm, is a nematode parasite of wild and domestic canids belonging to the super-family Metastrongyloidea. A survey of infection was carried out examining 88 red foxes (Vulpes vulpes) obtained during the regular hunting season (2014-2015) from the Emilia-Romagna region of Italy. Carcasses were stored frozen (- 21 °C) prior to necropsy. Lungs were examined for the presence of adult worms by dissection of the trachea, bronchi, and bronchioles, and then the lung tissue was examined for first-stage larvae (L1) by the Baermann method. No adult stages were detected, but L1, identified based on morphology as Crenosoma vulpis, were recovered from 28.4% (25/88) of the fox lungs. No significant differences in infection were found based on sex or geographical distribution. A brief review on C. vulpis report in red foxes in Italy and other European countries was also carried out.

Risk factors for Toxoplasma gondii infection in dogs: a serological survey.

BACKGROUND: Dogs, as well as a wide variety of other warm-blooded animals, act as intermediate host of Toxoplasma gondii. In dogs, most cases of toxoplasmosis are subclinical, although clinical disease has been sporadically reported. Beyond its role in diagnostic pathways, seropositivity also functions as a reflection of the parasite's spread within the dog's living environment. The aim of the present study was to evaluate the possible risk factor associated with seropositivity to T. gondii in dogs in Central-Northern Italy, analysing 120 dogs sera for the presence of IgG antibodies by indirect fluorescence antibody test (IFAT). RESULTS: The population examined was composed of 54.2% hunting dogs, 24.2% companion dogs, 14.2% truffle dogs and 7.5% watchdogs. Thirty-four (29.2%) dogs tested positive for T. gondii IgG, with titres ranging from 1:40 to 1:1280. Seroprevalence and antibodies titres were not related to dog gender, age or function. The logistic regression and ordered logistic regression results indicated that seroprevalence, and antibody titres were significantly higher in dogs cohabiting with cats, exhibiting coprophagy habits, and living constantly outdoors. Notably, the lifestyle factor showed the highest odds-ratios in the study: dogs living constantly outdoors were found to be at approximately 5 times greater risk of testing positive and having higher antibody titres compared to dogs living both indoors and outdoors. CONCLUSION: Both logistic and ordered logistic regression results support the key role of living with cats, engaging in coprophagy behaviours, and maintaining an outdoor lifestyle in increasing the risk of T. gondii infection in dogs. These identified risk factors collectively suggest that both ingesting oocysts, as observed through cat cohabitation and coprophagy, and engaging in predatory behaviours, as possible for outdoor living dogs, are indicating likely sources of T. gondii infection in this host species.

Sentinels in the shadows: Exploring Toxoplasma gondii and other Sarcocystidae parasites in synanthropic rodents and their public health implications.

Synanthropic rodents play a crucial role in maintaining the life cycle of Toxoplasma gondii in anthropized regions and can serve as indicators of environmental oocyst contamination. This investigation aimed to explore the occurrence of T. gondii infection within synanthropic rodent populations using a molecular diagnostic technique targeting the 18S rDNA gene, which is generic for Coccidia, with subsequent specific PCR confirmation. We examined 97 brown rats (Rattus norvegicus), 67 black rats (R. rattus), 47 house mice (Mus musculus), and 1 common shrew (Sorex araneus). PCR tests were conducted on the brain, heart, and tongue tissues. PCR tested positive in at least one of the examined tissues in 26 R. norvegicus (26.8%), 13 R. rattus (19.4%), and 13 M. musculus (27.6%). Sequencing comparisons by BLAST allowed us to identify four different species of cyst-forming Apicomplexa. In particular, T. gondii DNA was detected in 13 (6.1%) rodents, Hammondia hammondi (including H. hammondi-like organisms) in 36 (17%) subjects, Besnoitia sp. (in two cases identified as B. besnoiti) in 8 (3.7%), and Sarcocystis gigantea in two (0.94%). Rodents from peri-urban and urban environments can act as indicators of environmental contamination by oocysts of apicomplexan parasites with cats as definitive hosts, such as T. gondii, H. hammondi, and S. gigantea, the latter of which has never been previously recorded in rodents. Moreover, the presence of B. besnoiti, a parasite with an unidentified definitive host in Europe, sheds light on the potential role of these hosts as infection sentinels.

Survey on the presence of Leishmania sp. in peridomestic rodents from the Emilia-Romagna Region (North-Eastern Italy).

Leishmaniasis is a neglected vector-borne parasitic disease caused in Italy only by the species Leishmania infantum of the Leishmania donovani complex, which is the causative agent of the zoonotic visceral leishmaniasis (VL) and the sporadic cutaneous leishmaniasis (CL) in humans, and of the canine leishmaniasis (CanL). The disease is considered endemic in southern, central, and insular Italian regions and recognizes phlebotomine sand flies as vector and dogs as main reservoir. Among northern Italian region, Emilia-Romagna shows peculiar epidemiological situation and recent studies are questioning the role of dog as main reservoir of L. infantum. Due to their synanthropic relationship with humans, rodents have been tested for Leishmania spp. in several European countries. The aim of this study was to assess the presence of Leishmania spp. in peridomestic rodents in the Emilia-Romagna. The study was carried out on 136 peridomestic rodents collected by professional pest control services: 47 brown rats (Rattus norvegicus), 39 black rats (Rattus rattus) and 50 mice (Mus musculus). Specimens of earlobe skin, spleen, liver and prescapular lymph nodes were tested with a real-time PCR. Fifteen (11%) rodents, tested positive for Leishmania spp. in particular five brown rats (10.6%), five black rats (12.8%) and five mice (10%). Positivity was obtained from different target organs. These findings revealed the presence of Leishmania spp. in peridomestic rodents of Emilia-Romagna Region, also in two species never tested before in Italy, namely R. norvegicus and M. musculus.

Migratory Wild Birds as Potential Long-Distance Transmitters of Toxoplasma gondii Infection.

Toxoplasma gondii is a worldwide distributed zoonotic protozoan capable of infecting a wide range of mammals (including humans) and birds as intermediate hosts. Migratory wild birds, through interconnecting countries along their flyways, can play a role in the spatial spread of T. gondii and could contribute to its sylvatic cycle. Additionally, hunted wild birds used for meat consumption could represent a further source of human infection. To determine the presence of T. gondii in wild birds, a total of 50 individuals belonging to the Anseriformes and Charadriiformes orders were sampled during the 2021-2022 hunting season in Northern Italy. Cardiac muscle samples of three Northern shovelers (Anas clypeata), two wild mallards (A. platyrhynchos), one Eurasian teal (A. crecca), and one Northern lapwing (Vanellus vanellus) were positive for the molecular detection of T. gondii based on a targeted amplification of the B1 gene. A 14% (7/50) overall positivity was observed in the sampled population. Results from this study suggest a moderate exposure of wild aquatic birds to T. gondii, highlighting the importance of a further characterization of T. gondii in its wildlife hosts.

A Case of Bovine Eosinophilic Myositis (BEM) Associated with Co-Infection by Sarcocystis hominis and Toxoplasma gondii.

Bovine eosinophilic myositis (BEM) is a specific inflammatory myopathy, often associated with Sarcocystis spp., with multifocal gray-green lesions leading to carcass condemnation with considerable economic losses. Here is described a peculiar case of BEM that occurred in an adult (16 month) cattle, born in France, bred, and slaughtered in Italy at the end of 2021. On inspection, muscles showed the typical multifocal gray-green lesions that were sampled for, cytological, histological, and molecular investigations, while meat juice was subjected to IFAT for Toxoplasma IgG. Genomic DNA was extracted from lesions, portions of healthy muscle and from meat juice pellet and analyzed by PCR targeting 18S rDNA, COI mtDNA and B1 genes, and sequenced. The cytology showed inflammatory cells mostly referable to eosinophils; at histology, protozoan cysts and severe granulomatous myositis were observed. A BEM lesion and meat juice pellet subjected to PCR showed, concurrently, sequences referable both to S. hominis and T. gondii. Meat juice IFAT resulted negative for T. gondii IgG. Our findings highlight the first detection of T. gondii DNA in association with S. hominis in a BEM case, suggesting a multiple parasite infection associated with this pathology, although the actual role of T. gondii infection in the pathophysiology of the diseases should be clarified.

Unveiling a novel parasitosis: Trichostrongylus colubriformis infection in captive ring-tailed lemurs (Lemur catta).

Ring-tailed Lemur (Lemur catta) is the only surviving semi-terrestrial diurnal lemur in Madagascar. Despite being the most intensively studied of lemur taxa, only a few helminths have been described in this species. In this study we describe a persistent infection due to Trichostrongylus colubriformis in a captive population of L. catta hosted in a zoological park of northern Italy. In the context of a parasitological survey on zoo animals, we investigated parasites in a captive colony of ring-tailed lemurs within a zoological park. Parasitological analysis included necropsy of a deceased lemur in 2019, subsequent fecal sample collections in 2021-2022, followed by coprological examination and coprocolture. Morphological and molecular analyses were conducted on adult helminths, larvae and eggs, involving microscopy, scanning electron microscopy (SEM), and sequencing of the ITS rDNA region. Trichostrongylidae parasites were primarily found after necropsy in the intestine of the lemur. Morphological and molecular investigations on adults and eggs/larvae recovered from feces collected at different times from lemurs of the same captive population, allowed to properly identify the parasite as T. colubriformis. To the best of our knowledge this is the first description of T. colubriformis in L. catta. Although its presence in wild populations is not necessarily implied by our finding, this parasitosis represent a cause of concern in captive lemurs, considering the possibility of interspecies transmission and the zoonotic implications.

Endoparasites in dogs diagnosed at the Veterinary Teaching Hospital (VTH)-University of Bologna, combined with clinicopathological results. A long-term retrospective secondary data study.

Humans and dogs commonly share the same domestic environment. Europe, and Italy specifically, have a substantial and growing dog population. Potentially zoonotic parasites may be harbored even by dogs receiving regular veterinary care. Thus, transmission of zoonotic or potentially zoonotic parasites to owners and their families should not be underestimated. Frequently, endoparasite infections occur as a subclinical infection and clinicopathological alterations have been documented including anemia, hypoalbuminemia, and eosinophilia. The aim of this large retrospective secondary data study was to analyze coprological endoparasite results and putative risk factors obtained from owned dogs, through a 9-year-period (2011-2019). Possible associations between diagnosed endoparasites and sex, age, seasonality, and year of examination were evaluated. Additionally, parasitological diagnoses were combined to complete blood count parameters and biochemical profiles, when available, to check for any possible hematological alteration from parasitism. A total of 1,972 dogs were evaluated for endoparasites using common fecal diagnostic tests over a 9-year period. The overall proportion of endoparasite-positive animals was 10%. The most common endoparasites detected were Cystoisospora spp. (3%), Toxocara canis (2.8%), Giardia duodenalis (1.6%), and Trichuris vulpis (1.2%). Of these parasites detected, Toxocara poses the greatest zoonotic risk, while Giardia species are considered to have a low potential to be zoonotic. There was no significant diagnostic trend across the years through the study period. Dogs were more frequently diagnosed endoparasite-positive when young and during cold seasons compared to the baselines of mature dogs and warm seasons. The clinicopathological profiles indicated that parasitized dogs had mild hematological alterations. The frequency of detected potentially zoonotic endoparasites in this study highlights that the risk should not be underestimated. Parasitic infection was found to be mostly dependent on age and season. Having this information may help clinicians to develop anthelmintic protocols to reduce the risk of transmission.

Microfluidic manufacturing of tioconazole loaded keratin nanocarriers: Development and optimization by design of experiments.

Fungal infections of the skin, nails, and hair are a common health concern affecting a significant proportion of the population worldwide. The current treatment options include topical and systematic agents which have low permeability and prolonged treatment period, respectively. Consequently, there is a growing need for a permeable, effective, and safe treatment. Keratin nanoparticles are a promising nanoformulation that can improve antifungal agent penetration, providing sustainable targeted drug delivery. In this study, keratin nanoparticles were prepared using a custom-made 3D-printed microfluidic chip and the manufacturing process was optimized using the design of experiments (DoE) approach. The total flow rate (TFR), flow rate ratio (FRR), and keratin concentration were found to be the most influential factors of the size and polydispersity index (PDI) of the nanoparticles. The crosslinking procedure by means of tannic acid as safe and biocompatible compound was also optimized. Keratin nanoparticles loaded with a different amount of tioconazole showed a size lower than 200 nm, a PDI lower than 0.2 and an encapsulation efficiency of 91 ± 1.9 %. Due to their sustained drug release, the formulations showed acceptable in vitro biocompatibility. Furthermore, a significant inhibitory effect compared to the free drug against Microsporum canis.

Evaluation of the Acaricidal Effectiveness of Fipronil and Phoxim in Field Populations of Dermanyssus gallinae (De Geer, 1778) from Ornamental Poultry Farms in Italy.

The poultry red mite Dermanyssus gallinae is the most important blood-sucking ectoparasite in egg-laying hen facilities. The aim of this study was to evaluate the in vitro acaricidal activity of different concentration of authorized (phoxim, ByeMite®, 500 mg/mL) and unauthorized (fipronil, Frontline® 250 mg/100 mL spray) molecules on 14 field isolates of D. gallinae collected from different ornamental poultry farms from different Italian regions. The sensitivity test was performed by contact exposure to four concentrations of each insecticide diluted at 1:5 (10,000-2000-400-80 ppm for phoxim, 500-100-20-4 ppm for fipronil) on a filter paper. The effectiveness of the treatment was significantly (p < 0.0001) associated with the dose of the pesticide used. Considering the mean lethality, phoxim has greater efficacy compared to fipronil (p < 0.001). A great variability of lethality rate was observed with the increase in fipronil dilution; conversely, for phoxim, some outliers were observed, particularly in one farm, suggesting the hypothesis that a certain degree of resistance in the mite population could occur possibly as a consequence of the continual contact with the molecule. This underlines the importance of the use of licensed products administered at correct dosages and the need for alternative molecules to avoid the onset of drug resistance phenomena.

Laboratory analysis as support to slaughterhouse inspection in Songea cattle abattoir (Tanzania): A public health perspective.

Livestock breeding is one of the main agricultural activities in Tanzania, recognized as one of the African countries with the greatest livestock resources. Cattle is the most widely bred animal species, especially with traditional farming methods such as pastoralism and agro-pastoralism. Slaughtering takes place mainly in rural slaughter slabs and municipal slaughterhouses, and according to local legislation it must be supervised by Official Veterinarians, who report any organs to be discarded and, after stamping the carcass, authorize its free consumption. Since the Ruvuma region lacks data on cattle diseases, the aim of this study was to collect information at Songea slaughterhouse, with particular attention to conditions of parasitic aetiology and potential zoonotic interest, deepening inspective diagnoses by means of parasitological and histological investigations on tissue samples. Overall, the slaughter data of 614 animals are reported. In most cases slaughtered cattle belonged to local breeds and came from the Mbeya region. Fasciolosis (diagnosed in 44.6% of animals) was the most frequent condition, followed by respiratory diseases (24.4%), aortic onchocerciasis (6.5%), hydatidosis (5.5%) and bovine cysticercosis (3.4%). Laboratory analysis identified Fasciola gigantica as the species involved in hepatic distomatosis in this area. Cases macroscopically ascribed to cysticercosis turned out to be a muscular form of onchocerciasis (by Onchocerca dukei), a neglected and unrecognised parasitic disease in Tanzania, questioning the real frequence of a zoonosis that causes primarily major economic losses. Pulmonary cysts, referred as hydatidosis at visual inspection, were histologically identified as haemal nodes, lymphoid organs common in ruminants, but described in cattle lungs for the first time in the present paper. Slaughterhouse is an important epidemiological observatory, especially for neglected parasitoses. The possibility of having basic laboratory diagnostics as an aid to visual inspection can ensure greater efficiency of veterinary services in the control of important livestock diseases and zoonoses, in the frame of a One Health perspective.

Roe deer (Capreolus capreolus) are a novel potential reservoir for human visceral leishmaniasis in the Emilia-Romagna region of northeastern Italy.

Leishmaniasis is a complex human disease caused by intracellular parasites of the genus Leishmania, predominantly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused exclusively by Leishmania infantum, responsible for the human and canine visceral leishmaniases (HVL and CVL, respectively). Within the Emilia-Romagna region, two different foci are active in the municipalities of Pianoro and Valsamoggia (both in the province of Bologna). Recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different, suggesting that there is an animal reservoir other than dogs for human visceral leishmaniasis in the Emilia-Romagna region. In this work, we analyzed specimens from wild animals collected during hunts or surveillance of regional parks near active foci of human visceral leishmaniasis for L. infantum infection in the province of Bologna. Out of 70 individuals analyzed, 17 (24%) were positive for L. infantum. The infection prevalence in hedgehogs (Erinaceus europaeus), roe deer (Capreolus capreolus), badgers (Meles meles), and bank voles (Myodes glareolus) was 80, 33, 25, and 11%, respectively. To distinguish the two strains of L. infantum we have developed a nested PCR protocol optimized for animal tissues. Our results demonstrated that most (over 90%) of L. infantum infections in roe deer were due to the strain circulating in humans in the Emilia-Romagna region.