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The aim of this study was to molecularly investigate the presence of antimicrobial resistance genes (ARGs) in organ samples from 11 Apennine wolves (Canis lupus italicus) collected in Central Italy. Samples from lung, liver, spleen, kidney, tongue and intestine were investigated by PCRs targeting the following genes: tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tetA(P), tet(Q), tet(S), tet(X), sul1, sul2, sul3, blaCTX−M, blaSHV, blaTEM and mcr-1. A PCR positivity was highlighted for 13 out of the 21 tested genes; no positive results were obtained for tet(C), tet(D), tet(E), tet(G), sul3, blaCTX, blaSHV and mcr-1 genes. All 11 animals sampled showed positivity for one or more resistance genes. The results confirm the potential role of the wolf as an indicator and/or vector of antimicrobial-resistant bacteria or ARGs. [ABSTRACT FROM AUTHOR]

Abstract Background The only natural hosts of Pseudorabies virus (PRV) are members of the family Suidae (Sus scrofa scrofa). In mammals, the infection is usually fatal and typically causes serious neurologic disease. This study describes four Aujeszky’s disease cases in free-ranging Italian wolves (Canis lupus italicus). In Italy, the wolf is a strictly protected species and is in demographic expansion. Case presentation Three wolves (Wolf A, B, and C) were found in a regional park in Northern Italy, and one (Wolf D) was found in Central Italy. Wolf A and D were alive at the time of the finding and exhibited a fatal infection with epileptic seizures and dyspnoea, dying after a few hours. Wolf B presented scratching lesions under the chin and a detachment of the right earlobe, whilst Wolf C was partially eaten. The wolves showed hepatic congestion, diffuse enteritis, moderate pericardial effusion, severe bilateral pneumonia, and diffuse hyperaemia in the brain. The diagnostic examinations included virological analyses and detection of toxic molecules able to cause serious neurological signs. All four wolves tested positive for pseudorabies virus (PrV). The analysed sequences were placed in Italian clade 1, which is divided into two subclades, “a” and “b”. The sequences of Wolf A, B, and C were closely related to other Italian sequences in the subclade b, originally obtained from wild boars and hunting dogs. The sequence from Wolf D was located within the same clade and was closely related to the French hunting dog sequences belonging to group 4. Conclusion Results showed the presence of PrV strains currently circulating in wild boars and free-ranging Italian wolves. The genetic characterisation of the PrV UL44 sequences from the four wolves confirmed the close relationship with the sequences from wild boars and hunting dogs. This fact supports a possible epidemiological link with the high PrV presence in wild boars and the possibility of infection in wolves through consumption of infected wild boar carcasses or indirect transmission. To the best of our knowledge, this study is the first detection of Pseudorabies virus in free-ranging Italian wolves in northern and central Italy.

Background: The only natural hosts of Pseudorabies virus (PRV) are members of the family Suidae (Sus scrofa scrofa). In mammals, the infection is usually fatal and typically causes serious neurologic disease. This study describes four Aujeszky's disease cases in free-ranging Italian wolves (Canis lupus italicus). In Italy, the wolf is a strictly protected species and is in demographic expansion. Case presentation: Three wolves (Wolf A, B, and C) were found in a regional park in Northern Italy, and one (Wolf D) was found in Central Italy. Wolf A and D were alive at the time of the finding and exhibited a fatal infection with epileptic seizures and dyspnoea, dying after a few hours. Wolf B presented scratching lesions under the chin and a detachment of the right earlobe, whilst Wolf C was partially eaten. The wolves showed hepatic congestion, diffuse enteritis, moderate pericardial effusion, severe bilateral pneumonia, and diffuse hyperaemia in the brain. The diagnostic examinations included virological analyses and detection of toxic molecules able to cause serious neurological signs. All four wolves tested positive for pseudorabies virus (PrV). The analysed sequences were placed in Italian clade 1, which is divided into two subclades, "a" and "b". The sequences of Wolf A, B, and C were closely related to other Italian sequences in the subclade b, originally obtained from wild boars and hunting dogs. The sequence from Wolf D was located within the same clade and was closely related to the French hunting dog sequences belonging to group 4. Conclusion: Results showed the presence of PrV strains currently circulating in wild boars and free-ranging Italian wolves. The genetic characterisation of the PrV UL44 sequences from the four wolves confirmed the close relationship with the sequences from wild boars and hunting dogs. This fact supports a possible epidemiological link with the high PrV presence in wild boars and the possibility of infection in wolves through consumption of infected wild boar carcasses or indirect transmission. To the best of our knowledge, this study is the first detection of Pseudorabies virus in free-ranging Italian wolves in northern and central Italy. [ABSTRACT FROM AUTHOR]

Tapeworms are trophically-transmitted and multi-host parasites with a complex indirect life cycle, strictly depending on predator-prey interactions. Their presence in a free-living population, mainly definitive hosts, is arduous to study due to the complexity of collecting fecal samples. However, epidemiological studies on their frequency are crucial from a public health perspective, providing information on food habits and prey selection of predators. The present study aims to update the frequency of tapeworms detected in stool samples by molecular analysis in Italian wolf populations of Umbria and Marche regions collected from 2014 to 2022. Tapeworm's total frequency was 43.2%. In detail, Taenia serialis was detected in 27 samples (21.6%), T. hydatigena in 22 (17.6%), and Mesocestoides corti (syn. M. vogae) in 2 (1.6%). Three samples were identified as M. litteratus and E. granulosus s.s. (G3) and T. pisiformis, with a proportion of 0.8%, respectively. The low frequency of E. granulosus in a hyperendemic area is discussed. The results show for the first time a high frequency of Taenia serialis not comparable to other Italian studies conducted on wild Carnivora; thus, a new ecological niche is conceivable. These findings suggest a plausible wolf-roe deer cycle for T. serialisin the investigated area.

Tapeworms are trophically-transmitted and multi-host parasites with a complex indirect life cycle, strictly depending on predator-prey interactions. Their presence in a free-living population, mainly definitive hosts, is arduous to study due to the complexity of collecting fecal samples. However, epidemiological studies on their frequency are crucial from a public health perspective, providing information on food habits and prey selection of predators. The present study aims to update the frequency of tapeworms detected in stool samples by molecular analysis in Italian wolf populations of Umbria and Marche regions collected from 2014 to 2022. Tapeworm's total frequency was 43.2%. In detail, Taenia serialis was detected in 27 samples (21.6%), T. hydatigena in 22 (17.6%), and Mesocestoides corti (syn. M. vogae) in 2 (1.6%). Three samples were identified as M. litteratus and E. granulosus s.s. (G3) and T. pisiformis , with a proportion of 0.8%, respectively. The low frequency of E. granulosus in a hyperendemic area is discussed. The results show for the first time a high frequency of Taenia serialis not comparable to other Italian studies conducted on wild Carnivora; thus, a new ecological niche is conceivable. These findings suggest a plausible wolf-roe deer cycle for T. serialis in the investigated area. [Display omitted] • Italian wolves in Central Italy act as definitive host for Taenia serialis. • The role of Italian wolves on echinococcosis is negligible. • The tapeworms detected provided information on the wolves' diet and zoonotic risk. • Italian wolves in Central Italy mainly prefer wild preys. [ABSTRACT FROM AUTHOR]

Resource selection analyses based on detection data are widely used to parametrize resistance surfaces used to identify ecological corridors. To successfully parametrize resistance, it is crucial to decouple resident and disperser behaviours yet to date connectivity studies using detection data have not addressed this issue. Here, we decoupled data of resident and dispersing wolves by analysing detection data collected within a natural corridor crossing a human dominated plain in Italy. To decouple residents and dispersers, we ran a Kernel Density analysis to investigate whether clusters of wolf detection points characterized by sharply higher points' density exist and checked whether the areas outlined by these clusters (core areas) hold specific characteristics. Habitat selection analysis was then performed to compare the intensity of habitat selection carried out by putative residents and dispersers. We identified a high-density cluster of 30 detection points outlining a small core area stably located in the central part of the park. The dramatic differences of the R2 and the AUC of the habitat selection models performed inside (R2 = 0.506; AUC = 0.952) and outside (R2 = 0.037; AUC = 0.643) the core area corroborated the hypothesis that the core area effectively encloses detection points belonging to residents. Our results show that through simple space use analyses it is possible to roughly discriminate between detection points belonging to resident-behaving and disperser-behaving individuals and that habitat selection models separately performed on these data have extremely different results with strong possible effects on resistance surfaces parametrized from these models. Highlights We decoupled data of resident and dispersing wolves by analyzing detection data collected within a natural ecological corridor. Through space use analyses on detection data, it is possible to roughly discriminate between resident-behaving and disperser-behaving individuals. Habitat selection carried out by resident-behaving and disperser-behaving individuals is dramatically different. [ABSTRACT FROM AUTHOR]

Wolves (Canis lupus), like most wild carnivores, are elusive and usually live at low density; this makes it challenging to monitor specific individuals through time. Non-invasive genetic sampling, when protracted over a long period and a sufficiently large geographic scale, has revealed an effective tool to follow individual patterns in a population. During a long-term project focused on the monitoring of presence and distribution of wolf packs in the Tuscan Apennines in Italy, we have followed the destiny of a single female wolf (F82) exclusively by the analysis of microsatellite multilocus genotypes obtained from non-invasive samples (n = 118 including scats, hair and blood residuals) collected from 2003 to 2014. From the natal pack, she dispersed to a neighboring area where she established a new territory together with an unrelated male. The pair persisted for 8 years, mating at least twice and occupying a territory of a minimum size of 60–80 km2. In December 2014, the carcass of F82 was found victim of a car accident. Her estimated age at death was at least 11 years (the time elapsed between the first and last samplings). This record represents one of the very few instances of free-ranging wild wolves monitored for such a long period in Europe and her lifespan represents the longest ever recorded in the old continent. [ABSTRACT FROM AUTHOR]

Over the last 40 years the gray wolf (Canis lupus) re-colonized its historical range in Italy increasing human-predator interactions. However, temporal and spatial trends in wolf mortality, including direct and indirect persecution, were never summarized. This study aims to fill this gap by focusing on the situation of Tuscany and Emilia-Romagna regions, hosting a significant proportion of the Italian wolf population, by: (i) identifying the prevalent causes of wolf mortality, (ii) summarizing their temporal and spatial patterns and (iii) applying spatially-explicit Generalized Linear Models to predict wolf persecution. Between October 2005 and February 2021, 212 wolf carcasses were collected and subjected to necropsy, being involved in collisions with vehicles (n = 104), poisoned (n = 45), wounded with gunshot (n = 24) or blunt objects (n = 4) and being hanged (n = 2). The proportion of illegally killed wolves did not increase through time. Most persecution events occurred between October and February. None of our candidate models outperformed a null model and covariates such as the density of sheep farms, number of predations on livestock, or human density were never associated to the probability of having illegally killed wolves, at the municipal scale. Our findings show that conventional correlates of wolf persecution, combined with a supposedly high proportion of non-retrieved carcasses, fail to predict illegal wolf killings in areas where the species have become ubiquitous. The widespread spatial distribution of illegal killings indicates that persecution probably arises from multiple kinds of conflicts with humans, beyond those with husbandry. Wolf conservation in Italy should thus address cryptic wolf killings with multi-disciplinary approaches, such as shared national protocols, socio-ecological studies, the support of experts’ experience and effective sampling schemes for the detection of carcasses.

After centuries of massive decline, the recovery of the wolf (Canis lupus italicus) in Italy is a typical conservation success story. To learn more about the possible role of parasites in the wolves' individual and population health and conservation we used non-invasive molecular approaches on fecal samples to identify individual wolves, pack membership, and the taeniids present, some of which are zoonotic. A total of 130 specimens belonging to 54 wolves from eight packs were collected and examined. Taeniid eggs were isolated using a sieving/flotation technique, and the species level was identified by PCR (gene target: 12S rRNA and nad1). Taeniid prevalence was 40.7% for Taenia hydatigena, 22.2% for T. krabbei, 1.8% for T. polyachanta and 5.5% for Echinococcus granulosus. The prevalence of E. granulosus is discussed. Our results show that the taeniid fauna found in wolves from the Foreste Casentinesi National Park is comparable to that described for other domestic and wild Italian canids and provides insights into the wolves’ diet and their relationship with the environment.

The canine adenovirus type 2 (CAdV-2) is associated with the infectious tracheobronchitis commonly called "kennel cough", cosmopolitan in dogs but little explored in gray wolves. Our goals were (i) to evaluate the presence and circulation of CAdV-2 in free-ranging Italian wolves (Canis lupus italicus), through the analysis of spleens and tongues collected from 56 carcasses sampled in three Italian regions between August 2017 and July 2020, and (ii) to support the validity of a matrix such as the tongue, which was never used before. Samples were screened for the presence of CAdV-2 DNA using both PCR and real-time PCR assay. Positive results were related to sampling year, location, sex, age, genetic determination of species, and matrices tested. Three male wolves (5.4%) tested positive in tongue samples, demonstrating that the tongue is an excellent matrix for the detection of CAdV-2. To the best of our knowledge, no studies were performed to evaluate the usability of tongue samples to detect CAdV-2 DNA in grey wolves or other wild animals. The number of wolves tested positive suggests that, during the studied years, the circulation of CAdV-2 in Italian wolves showed a low frequency, consistent with irregular introductions of the virus by dogs or other wild carnivores in these populations. This preliminary study provides new data on the ecology of CAdV-2 in Italian wolves, although future studies are needed to fully understand its real circulation at a national scale, its pathogenetic role in gray wolves, and its risk of transmission to other wild carnivores. [ABSTRACT FROM AUTHOR]

Canids are definitive hosts of Taenia and Echinococcus species, which infect a variety of mammals as intermediate or accidental hosts including humans. Parasite transmission is based on domestic, semi-domestic and wildlife cycles; however, little is known of the epidemiological significance of wild large definitive hosts such as the wolf. In this study, 179 scats of wolves (Canis lupus italicus) collected throughout the Italian region of Liguria were analyzed for the detection of taeniid infection. Taeniid egg isolation was performed using a sieving/flotation technique, and the species level was identified by PCR (gene target: 12S rRNA and nad 1) followed by sequence analyses. Based on sequence homologies of ≥99%, Taenia hydatigena was identified in 19.6%, Taenia krabbei in 4.5%, Taenia ovis in 2.2%, Taenia crassiceps in 0.6%, Hydatigera taeniaeformis in 0.6% and Echinococcus granulosus in 5.6% of the samples. According to these results, Canis lupus italicus can be considered as involved in the wild (including cervids and rodents) and semi-domestic cycles (including sheep and goats) of taeniids in this area.

In the past century the Italian wolf has been repeatedly indicated as a distinct subspecies, Canis lupus italicus , due to its unique morphology and its distinctive mtDNA control region (CR) monomorphism. However, recent studies on wolf x dog hybridization in Italy documented the presence of a second mtDNA CR haplotype (W16), previously found only in wolves from Eastern Europe, casting doubts on the genetic uniqueness of the Italian wolves. To test whether this second haplotype belongs to the Italian wolf population, we genotyped 92 wolf DNA samples from Italy, Slovenia, Greece and Bulgaria at four mtDNA regions (control-region, ATP6, COIII and ND4 genes) and at 39 autosomal microsatellites. Results confirm the presence of two mtDNA multi-fragment haplotypes (WH14 and WH19) in the Italian wolves, distinct from all the other European wolves. Network analyses of the multi-fragment mtDNA haplotypes identified two strongly differentiated clades, with the Italian wolf WH14 and WH19 multi-fragment haplotypes rooted together. Finally, Bayesian clustering clearly assigned all the wolves sampled in Italy to the Italian population, regardless of the two different multi-fragment haplotypes. These results demonstrate that the W16 CR haplotype is part of the genetic pool of the Italian wolf population, reconfirming its distinctiveness from other European wolves. Overall, considering the presence of unique mtDNA and Y-linked haplotypes, the sharply different frequencies of genome-wide autosomal alleles and the distinct morphological features of Italian wolves, we believe that this population should be considered a distinct subspecies. [ABSTRACT FROM AUTHOR]

After centuries of massive decline, the recovery of the wolf ( Canis lupus italicus ) in Italy is a typical conservation success story. To learn more about the possible role of parasites in the wolves' individual and population health and conservation we used non-invasive molecular approaches on fecal samples to identify individual wolves, pack membership, and the taeniids present, some of which are zoonotic. A total of 130 specimens belonging to 54 wolves from eight packs were collected and examined. Taeniid eggs were isolated using a sieving/flotation technique, and the species level was identified by PCR (gene target: 12S rRNA and nad1 ). Taeniid prevalence was 40.7% for Taenia hydatigena , 22.2% for T . krabbei , 1.8% for T. polyachanta and 5.5% for Echinococcus granulosus . The prevalence of E. granulosus is discussed. Our results show that the taeniid fauna found in wolves from the Foreste Casentinesi National Park is comparable to that described for other domestic and wild Italian canids and provides insights into the wolves’ diet and their relationship with the environment. [ABSTRACT FROM AUTHOR]

Canids are definitive hosts of Taenia and Echinococcus species, which infect a variety of mammals as intermediate or accidental hosts including humans. Parasite transmission is based on domestic, semi-domestic and wildlife cycles; however, little is known of the epidemiological significance of wild large definitive hosts such as the wolf. In this study, 179 scats of wolves ( Canis lupus italicus ) collected throughout the Italian region of Liguria were analyzed for the detection of taeniid infection. Taeniid egg isolation was performed using a sieving/flotation technique, and the species level was identified by PCR (gene target: 12S rRNA and nad 1 ) followed by sequence analyses. Based on sequence homologies of ≥99%, Taenia hydatigena was identified in 19.6%, Taenia krabbei in 4.5%, Taenia ovis in 2.2%, Taenia crassiceps in 0.6%, Hydatigera taeniaeformis in 0.6% and Echinococcus granulosus in 5.6% of the samples. According to these results, Canis lupus italicus can be considered as involved in the wild (including cervids and rodents) and semi-domestic cycles (including sheep and goats) of taeniids in this area. [ABSTRACT FROM AUTHOR]

The article presents a case study of a wild 29-kilogram male Italian wolf suffering from gait abnormality in the pelvic limbs. It underwent neurological examinations that revealed thoracic kyphosis, decreased proprioception in pelvic limbs and normal spinal reflexes. Other findings discussed include thoracolumbar spinal cord lesions, leukocytosis by pathological tests and ventral spondylosis by spinal radiographs.