Detection and molecular analysis of Pseudorabies virus from free-ranging Italian wolves (Canis lupus italicus) in Italy - a case report.

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.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF AMBLYOMMA SCUTATUM (ACARI: IXODIDAE) ACCIDENTALLY INTRODUCED IN ITALY.

Eight ticks were found in Comacchio (FE), Italy parasitizing a young black iguana (Ctenosaura similis) that had been accidentally transported in a commercial plant container from Costa Rica. Specimens were identified morphologically as Amblyomma scutatum and then confirmed by the barcoding of the mitochondrial cytochrome c oxidase subunit 1 gene. Amblyomma scutatum is a common tick known to infest reptiles in Central America, Mexico, and Venezuela, but not in Europe. In Italy, the possibility for this tick to become endemic is unlikely because of the absence of its principal hosts. Nevertheless, this finding confirms the high risk of introducing exotic species that is linked with global commerce and therefore the need for veterinary control of shipments.

Identification and phylogenetic analysis of Taenia spp. parasites found in wildlife in the Emilia-Romagna region, northern Italy (2017-2022).

The recent expansion of the habitat of several wildlife species, comprising anthropized areas, is a relevant risk factor for many zoonotic diseases and should be considered in national and regional sanitary monitoring systems. We evaluated adult intestinal Taenia spp. parasites isolated from wild carnivores and cystic larval forms isolated from wild mammals analysed at the Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna (IZSLER) as part of the regional wildlife sanitary surveillance plan. Then, we assessed parasite species through molecular analysis (multiplex PCR followed by ribosomal 12S subunit gene sequencing) in order to update the epidemiological situation on Taeniids infection in the Emilia-Romagna wildlife, reporting the prevalence of each isolated species. The most commonly isolated species was Taenia serialis, which we detected in both wolves and foxes as definitive hosts and in roe deer as intermediate host. More attention on the distribution of Taeniids in wildlife should be paid, considering their potential zoonotic role: several Taenia spp. (Taenia solium, Taenia multiceps, Taenia serialis, Taenia brauni, Taenia glomerulatus) are known for causing coenurosis in humans, with possible severe or fatal outcomes.

Detection of mosquito-only flaviviruses in Europe.

The genus Flavivirus, family Flaviviridae, includes a number of important arthropod-transmitted human pathogens such as dengue viruses, West Nile virus, Japanese encephalitis virus and yellow fever virus. In addition, the genus includes flaviviruses without a known vertebrate reservoir, which have been detected only in insects, particularly in mosquitoes, such as cell fusing agent virus, Kamiti River virus, Culex flavivirus, Aedes flavivirus, Quang Binh virus, Nakiwogo virus and Calbertado virus. Reports of the detection of these viruses with no recognized pathogenic role in humans are increasing in mosquitoes collected around the world, particularly in those sampled in entomological surveys targeting pathogenic flaviviruses. The presence of six potential flaviviruses, detected from independent European arbovirus surveys undertaken in the Czech Republic, Italy, Portugal, Spain and the UK between 2007 and 2010, is reported in this work. Whilst the Aedes flaviviruses, detected in Italy from Aedes albopictus mosquitoes, had already been isolated in Japan, the remaining five viruses have not been reported previously: one was detected in Italy, Portugal and Spain from Aedes mosquitoes (particularly from Aedes caspius), one in Portugal and Spain from Culex theileri mosquitoes, one in the Czech Republic and Italy from Aedes vexans, one in the Czech Republic from Aedes vexans and the last in the UK from Aedes cinereus. Phylogenetic analysis confirmed the close relationship of these putative viruses to other insect-only flaviviruses.

Etiological [corrected] agents of rickettsiosis and anaplasmosis in ticks collected in Emilia-Romagna region (Italy) during 2008 and 2009.

Ticks are the main vectors of rickettsiae of the spotted fever group, as well as of a variety of other Rickettsiales, including bacteria of the genus Anaplasma, that might cause diseases in humans and animals. Here we present the result of a survey for ticks and for tick-associated Rickettsiales in the Emilia Romagna region (Northern Italy). The study was focused on ticks collected from wild-hunted animals. Out of 392 ticks collected from these animals, 282 (72%) were identified as Ixodes ricinus, 110 (28%) as Dermacentor marginatus. The former was found on four vertebrate species, whereas the latter appeared more specific for wild boar. The presence of rickettsiae was demonstrated in 22.5% of I. ricinus (57/253) and in 29% of D. marginatus (32/110). Five ticks of the species I. ricinus were also positive for Anaplasma phagocytophilum (2%). In addition, we collected ticks by dragging in a natural park of the same region. All of the ticks captured by dragging were identified as I. ricinus. Thirty-six out of 200 analyzed ticks proved positive for Rickettsia monacensis and R. helvetica (16.5 and 1.5%, respectively). Our results highlight that that ticks present in wild areas, widely exploited for recreation and hunting in Emilia-Romagna, represent a risk for the transmission of spotted fevers and anaplasmosis to humans.

Outbreak of type C botulism in birds and mammals in the Emilia Romagna region, northern Italy.

Over a 7-day period beginning 8 August 2011, a large number of wild birds of several species were found dead or with neurologic clinical signs along the shore of Crostolo stream, in the Emilia Romagna region, Italy. Twenty-eight Mallards (Anas platyrhynchos), two Hooded Crows (Corvus corone cornix), and three coypus (Myocastor coypus) were found moribund on the Crostolo stream bank, collected, and sent to Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Reggio Emilia Section. The cause of mortality was determined to be Clostridium botulinum type C toxin. The toxin was identified by a mouse bioassay for botulinum toxins and confirmed in bird sera and blowfly larvae (Lucilia caesar) collected from the stomachs of birds.

Mosquito, bird and human surveillance of West Nile and Usutu viruses in Emilia-Romagna Region (Italy) in 2010.

BACKGROUND: In 2008, after the first West Nile virus (WNV) detection in the Emilia-Romagna region, a surveillance system, including mosquito- and bird-based surveillance, was established to evaluate the virus presence. Surveillance was improved in following years by extending the monitoring to larger areas and increasing the numbers of mosquitoes and birds tested. METHODOLOGY/PRINCIPAL FINDINGS: A network of mosquito traps, evenly distributed and regularly activated, was set up within the surveyed area. A total of 438,558 mosquitoes, grouped in 3,111 pools and 1,276 birds (1,130 actively sampled and 146 from passive surveillance), were tested by biomolecular analysis. The survey detected WNV in 3 Culex pipiens pools while Usutu virus (USUV) was found in 89 Cx. pipiens pools and in 2 Aedes albopictus pools. Two birds were WNV-positive and 12 were USUV-positive. Furthermore, 30 human cases of acute meningoencephalitis, possibly caused by WNV or USUV, were evaluated for both viruses and 1,053 blood bags were tested for WNV, without any positive result. CONCLUSIONS/SIGNIFICANCE: Despite not finding symptomatic human WNV infections during 2010, the persistence of the virus, probably due to overwintering, was confirmed through viral circulation in mosquitoes and birds, as well as for USUV. In 2010, circulation of the two viruses was lower and more delayed than in 2009, but this decrease was not explained by the relative abundance of Cx. pipiens mosquito, which was greater in 2010. The USUV detection in mosquito species confirms the role of Cx. pipiens as the main vector and the possible involvement of Ae. albopictus in the virus cycle. The effects of meteorological conditions on the presence of USUV-positive mosquito pools were considered finding an association with drought conditions and a wide temperature range. The output produced by the surveillance system demonstrated its usefulness and reliability in terms of planning public health policies.

First reports of Trichinella pseudospiralis in wild boars (Sus scrofa) of Italy.

Trichinella pseudospiralis is a non-encapsulated species infecting both mammals and birds. In Italy, this parasite was reported only in two night-birds of prey of Central Italy. In January 2010, Trichinella larvae were detected in three wild boars (Sus scrofa) of two regions of Northern Italy by enzymatic digestion. The parasites were identified as T. pseudospiralis by multiplex-PCR. The first infected wild boar was hunted in the Emilia Romagna region and the other two infected wild boars were bred outdoors in a small family farm of the Friuli Venezia Giulia region. These new epidemiological data reinforce the role of the wild boar as the main reservoir of T. pseudospiralis in Europe.

Evidence of simultaneous circulation of West Nile and Usutu viruses in mosquitoes sampled in Emilia-Romagna region (Italy) in 2009.

BACKGROUND: In recent years human diseases due to mosquito-borne viruses were increasingly reported in Emilia-Romagna region (Italy), from the chikungunya virus in 2007 to the West Nile virus (WNV) in 2008. An extensive entomological survey was performed in 2009 to establish the presence and distribution of mosquito arboviruses in this region, with particular reference to flaviviruses. METHODOLOGY/PRINCIPAL FINDINGS: From May 6 to October 31, a total of 190,516 mosquitoes were sampled in georeferenced stations, grouped in 1,789 pools according date of collection, location, and species, and analyzed by reverse transcription polymerase chain reaction (RT-PCR) to detect the presence of RNA belong to Flavivirus genus. WNV was detected in 27 mosquito pools, producing sequences similar to those of birds and human strains obtained in 2008 outbreak, pointed out the probable virus overwintering. Isolation of WNV was achieved from one of these pools. Moreover 56 pools of mosquitoes tested positive for Usutu virus (USUV). Most PCR positive pools consisted of Culex pipiens, which also was the most analyzed mosquito species (81.4% of specimens); interestingly, USUV RNA was also found in two Aedes albopictus mosquito pools. Simultaneous circulation of WNV and USUV in the survey area was highlighted by occurrence of 8 mosquito WNV- and USUV-positive pools and by the overlaying of the viruses "hot spots", obtained by kernel density estimation (KDE) analysis. Land use of sampled stations pointed out a higher proportion of WNV-positive Cx. pipiens pool in rural environments respect the provenience of total sampled pool, while the USUV-positive pools were uniformly captured in the different environments. CONCLUSIONS/SIGNIFICANCE: Obtained data highlighting the possible role of Cx. pipiens mosquito as the main vector for WNV and USUV in Northern Italy, and the possible involvement of Ae. albopictus mosquito in USUV cycle. The described mosquito-based surveillance could constitute the foundation for a public health alert system targeting mosquito borne arboviruses.

Arboviral survey of mosquitoes in two northern Italian regions in 2007 and 2008.

Recently, Italy-particularly the Emilia-Romagna region-was the location of consecutive outbreaks of human diseases caused by the arboviruses chikungunya virus and West Nile virus. The two outbreaks, spread by different species of mosquitoes, were not related, but pointed out the lack of an arboviral surveillance program in this region. Beginning in 2007 entomological surveillance was initiated in the Emilia-Romagna region, and in 2008 the program was improved and extended at Lombardia region. Using CO(2)-baited traps, 65,292 mosquitoes were collected; pooled by date of collection, location, and species; macerated manually; and tested by reverse transcription (RT)-polymerase chain reaction for the presence of alphaviruses, orthobunyaviruses, and flaviviruses. Amplicons were sequenced and employed for identification of viral RNA by basic local alignment search tool search in GenBank. Results of these assays showed (1) the presence of West Nile virus in two pools of Culex pipiens mosquitoes, (2) the presence of RNA of two orthobunyaviruses, Tahyna virus in a pool of Ochlerotatus caspius mosquitoes and Batai virus in a pool of Anopheles maculipennis mosquitoes, and (3) the presence of flavivirus RNAs in pools of Oc. caspius, Aedes albopictus, and Aedes vexans mosquitoes; the sequences of these amplicons were most closely related to flaviviruses that have been detected only in mosquitoes and had no recognized vertebrate host (Aedes flavivirus, Culex flavivirus, and Kamiti River virus).

Occurrence of Rickettsia felis in dog and cat fleas (Ctenocephalides felis) from Italy.

Rickettsia felis is an obligate intracellular bacterium belonging to the spotted fever group, suspected to cause a murine typhus-like illness in humans, with a cosmopolitan distribution. This study was designed to estimate presence and occurrence of this pathogen in fleas collected from dogs and cats in different areas of Italy. Two species of fleas were identified, Ctenocephalides felis (80.3%) and Ctenocephalides canis (19.7%).Overall, 320 fleas (257 C. felis and 63 C. canis) collected from 117 animals (73 dogs and 44 cats) were tested. Thirty-eight (11.9%) C. felis fleas, 13 from cats (17.6%) and 25 from dogs (10.2%) were positive for R. felis. No C. canis was positive. Fleas from cats showed a tendency to be more positive than fleas from dogs. Prevalence of R. felis among areas and within provinces of the same area was extremely variable, ranging from 0 to 35.3%. Overall, prevalence in north-eastern Italy (23.2%) was significantly higher than in south-western Italy (7.1%). This study confirmed the occurrence of R. felis in cat and dog fleas (C. felis) from Italy, similar to other European countries. The results also suggest that R. felis should be considered in the human differential diagnosis of any spotted-like fever in Italy, especially if the patient is known to have been exposed to flea bites.