First National Prevalence in Italian Horse Population and Phylogenesis Highlight a Fourth Sub-Type Candidate of Equine Hepacivirus.

Equine hepacivirus (EqHV, Flaviviridae, hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95% CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, also suggesting a possible fourth sub-type that, however, requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis, and in quality control protocols for blood derived products due to its parental transmission.

Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus.

Starting from October 2021, several outbreaks of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 were reported in wild and domestic birds in Italy. Following the detection of an HPAIV in a free-ranging poultry farm in Ostia, province of Rome, despite the lack of clinical signs, additional virological and serological analyses were conducted on samples collected from free-ranging pigs, reared in the same holding, due to their direct contact with the infected poultry. While the swine nasal swabs were all RT-PCR negative for the influenza type A matrix (M) gene, the majority (%) of the tested pigs resulted serologically positive for the hemagglutination inhibition test and microneutralization assay, using an H5N1 strain considered to be homologous to the virus detected in the farm. These results provide further evidence of the worrisome replicative fitness that HPAI H5Nx viruses of the 2.3.4.4b clade have in mammalian species. Moreover, our report calls for additional active surveillance, to promptly intercept occasional spillover transmissions to domestic mammals in close contact with HPAI affected birds. Strengthened biosecurity measures and efficient separation should be prioritized in mixed-species farms in areas at risk of HPAI introduction.

Complete Genome of African Swine Fever Virus Genotype II in Central Italy.

We report here the whole-genome sequence of the African swine fever virus (ASFV) genotype II, strain 20355/RM/2022_Italy, identified in a wild boar in the city of Rome (Lazio region, Italy) in April 2022.

A retrospective study on dioxins and dioxin-like polychlorinated biphenyls in milk and dairy products from the Latium region (Italy) over a 7-year study period (2011-2017).

This study reports the data of polychlorinated dibenzo-p-dioxins (PCDDs), -furans (PCDFs), and polychlorinated biphenyls dioxin-like (dl PCBs) measured in a total of 260 samples of the dairy supply chain collected over a period of 7 years (2011-2017) in the Latium region (Italy). Levels and average profiles of congeners were reported for each group of the analyzed dairy matrices, and any differences between different sampling strategies were considered (around likely pollutant sources or casual sampling). Of the samples, 95.4% resulted compliant with the regulated levels; only samples belonging to the "sheep bulk milk" matrix were found to be above either the action levels or the maximum levels (tot. 12 samples). Raw milk of the sheep species showed the highest averages (PCDD/F 0.248 and dl PCB 0.966 WHO TEQ pg/g of fat) compared to the milk of other species. The buffalo milk showed a content of dl PCB significantly lower (dl PCB: 0.371 WHO TEQ pg/g of fat) than the sheep milk (p<0.05). Dioxins were found to be superior to furans in almost all dairy products, except in the noncompliant samples where furans were higher. The OCDD was found to be the most abundant congener in almost all dairy products. This study provides a first list of reference values for background contamination of the dairy supply chain in the Latium region. These pre-existing values will be useful in all cases of environmental pollution to identify critical situations.

Sero-Epidemiological Survey of Orthopoxvirus in Stray Cats and in Different Domestic, Wild and Exotic Animal Species of Central Italy.

Orthpoxvirus infection can spread more easily in a population with a waning immunity with the subsequent emergence/re-emergence of the viruses pertaining to this genus. In the last two decades, several cases of Orthopoxvirus, and in particular Cowpoxvirus infections in humans were reported in different parts of the world, possibly due to the suspension of smallpox vaccinations. To date, in Italy, few investigations were conducted on the presence of these infections, and because of this a serosurvey was carried out to evaluate Cowpoxvirus infection in feline colonies situated in the province of Rome, since these are also susceptible to other zoonotic viruses belonging to Orthopoxvirus, and from which humans may contract the infection. The sample design was set at an expected minimum seroprevalence of 7.5%, a 5% standard error and 95% confidence level. In parallel, a serological investigation was conducted using convenience sampling in domestic, exotic and wild susceptible animals of the Latium and Tuscany Regions, which are areas in the jurisdiction of the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, coordinating this study. The serological methods employed were indirect immunofluorescence for 36 sera of nonhuman primate and virus neutralization for 1198 sera of different species. All the 1234 sera examined were negative for the presence of antibodies against Cowpoxvirus, indicating its limited circulation in the areas of investigation. The methodology applied for the serosurveillance could be adopted in the case of outbreaks of this infection and for the evaluation of the spread of this infection in the area of interest, to obtain essential information crucial for animal and public health policies according to the One Health concept.

West Nile and Usutu viruses co-circulation in central Italy: outcomes of the 2018 integrated surveillance.

BACKGROUND: West Nile (WNV) and Usutu (USUV) are emerging vector-borne zoonotic flaviviruses. They are antigenically very similar, sharing the same life cycle with birds as amplification host, Culicidae as vector, and man/horse as dead-end host. They can co-circulate in an overlapping geographic range. In Europe, surveillance plans annually detect several outbreaks. METHODS: In Italy, a WNV/USUV surveillance plan is in place through passive and active surveillance. After a 2018 WNV outbreak, a reinforced integrated risk-based surveillance was performed in four municipalities through clinical and serological surveillance in horses, Culicidae catches, and testing on human blood-based products for transfusion. RESULTS: Eight WNV cases in eight equine holdings were detected. Twenty-three mosquitoe catches were performed and 2367 specimens of Culex pipiens caught; 17 pools were USUV positive. A total of 8889 human blood donations were tested, and two asymptomatic donors were USUV positive. CONCLUSIONS: Different surveillance components simultaneously detected WNV only in horses and USUV only in humans and mosquitoes. While in endemic areas (i.e. northern Italy) entomological surveillance is successfully used as an early detection warning, this method in central Italy seems ineffective. To achieve a high level of sensitivity, the entomological trapping effort should probably exceed a reasonable balance between cost and performance. Besides, WNV/USUV early detection can be addressed by horses and birds. Further research is needed to adapt the surveillance components in different epidemiological contexts.

Farms at risk from environmental pollution: a proposal for a risk ranking procedure.

OBJECTIVES: to produce a methodology for the risk ranking of farms located around proven or potential sources of contamination to prioritize the monitoring procedures in food production. DESIGN: environmental factors that can favour the risk of contamination of products of animal origin (POAO) around a pollutant source were identified. A scale of standardized scores was produced for each risk factor (RF) in order to obtain a risk-based classification. Subsequently, an algorithm was developed to obtain a relative risk assessment (RRA). SETTING AND PARTICIPANTS: in order to validate the proposed methodology, data of 80 livestock farms in a polluted area, enrolled in a previous study, were subjected to the RRA. Results of RRA were compared with contamination data. MAIN OUTCOME MEASURES: seven RFs were included in a mathematical formula used to classify each farm. RESULTS: a scoring system was produced for each RFs and an algorithm was developed to obtain RRA which is an estimate of the farm probability of having POAO contamination in comparison to other farms located in the same area potentially affected by chemical pollution based exclusively on site-specific environmental characteristics. In the case study used for validation, a significant relationship between RRA and contamination data was found. CONCLUSIONS: the proposed methodology is a useful tool to support the authorities responsible for the food safety in carrying out the monitoring of POAO in areas subject to environmental risks. It helps to rationalize resources and make controls more effective.

Management of Mycobacterium avium subsp. paratuberculosis in dairy farms: Selection and evaluation of different DNA extraction methods from bovine and buffaloes milk and colostrum for the establishment of a safe colostrum farm bank.

The aim of this study was to develop and validate different innovative DNA extraction methods to detect Mycobacterium avium subsp. paratuberculosis (MAP) DNA from bovine and buffalo colostrum. Paratuberculosis is a chronic inflammatory infection of domestic and wild animals, especially ruminants, caused by MAP. The primary route of disease transmission is feces, but MAP can also be excreted in milk and colostrum. In 2015, the Italian Ministry of Health has issued a voluntary control plan of MAP in order to allow risk-based certification of bovine and buffaloes farms. In addition to the annual diagnostic screening and to the clinical surveillance of animals the plan includes the adoption of biosecurity and management measures to progressively mitigate the incidence of MAP. To achieve this goal it is crucial to ensure the accuracy of the methods used to detect the presence of MAP in bovine and buffaloes milk and colostrum, in order to: (1) support a "safe colostrum farm-bank" set-up and thus prevent the main within-farm MAP transmission route and (2) to allow the MAP-free certification of milk products for export purposes. To achieve these goals, seven different DNA extraction protocols were identified from bibliography, out of which three methods were finally selected after the adoption of an evaluation procedure aimed at assessing the efficiency of extraction of DNA, the purity of DNA and the adaptability of the DNA amplification: NucleoSpin® Food Kit (Macherey-Nagel), NucleoSpin® Food Kit (Macherey-Nagel) combined with the magnetic beads, and QIAamp Cador Pathogen Mini kit (QIAGEN). In particular, the NucleoSpin® Food Kit (Macherey-Nagel) and the QIAamp Cador Pathogen Mini kit (QIAGEN) were tested on bovine and buffalo colostrum, showing a LOD between 4 × 104 (2.6 × 106  cfu/ml) and 4.08 (26.7 cfu/ml) IS900 target copies and a LOD between 5.3 × 105 (4.1 × 106  cfu/ml) and 53 (4.1 × 103  cfu/ml) IS900 target copies, respectively.

A cross-sectional serosurvey in a sheep population in central Italy following a bluetongue epidemic.

Bluetongue (BT) is a viral disease that affects ruminants and is transmitted by midges of the genus Culicoides spp. The seroprevalence, the clinical form and the occurrence rates significantly differ in relation to several factors such as bluetongue virus (BTV) serotype, host species, breed susceptibility, specific previous exposure, vector ecology, husbandry and health status. Following the 2001-2006 BTV2 and BTV16 epidemics in central Italy, a new epidemic caused by BTV1 occurred in 2013-2015 causing 398 outbreaks in a susceptible population of about 1 million ruminants. The present study assessed the BTV1 seroprevalence in the sheep population of central Italy by conducting two cross-sectional surveys, in the proximity of and within BT outbreak farms. A total of 2,984 sheep from 437 farms were sampled. The animal-level prevalence was 19% (95% CI: 17-21%), the between-herd prevalence was 46% (95% CI: 41-51%) and the within-herd prevalence was 21% (95% CI: 16-26%). Risk factors were investigated by logistic regression models. Living on a farm where an outbreak occurred and the number of outbreaks in proximity of the farm were identified as risk factors, while herd size was identified as a protective factor. This study represents the first BT survey in southern Europe and reports valuable findings on BTV epidemiology. Despite intensive virus circulation, the estimated seroprevalences were low. The assessment of the population immunity level is crucial for defining an efficient vaccination strategy and for predicting the impact of future virus circulation. In view of the low seroprevalence detected albeit an extensive BTV1 circulation, the population immunity was likely to be inadequate in preventing new BTV1 epidemics. Moreover, considering the recurrent introduction of new serotypes from North Africa and the Balkans, the control of multi-serotype BTV infections will continue to present a challenge in the near future.

Monitoring for the possible introduction of Crimean-Congo haemorrhagic fever virus in Italy based on tick sampling on migratory birds and serological survey of sheep flocks.

Crimean-Congo haemorrhagic fever (CCHF), endemic in Africa, Asia, Eastern Europe and the Middle East, is caused by a tibovirus (CCHFV) transmitted in particular by the Hyalomma genus of the Ixodidae family that can remain attached to the host for up to 26days, which in case of migratory birds allows long distance carriage. Although CCHF in domestic ruminants is usually subclinical, they may become reservoirs and act as sentinels for the introduction and/or circulation of CCHFV. In this study, possible CCHFV introduction and circulation in Italy were monitored by tick sampling on migratory birds and by a serosurvey conducted on sheep. While bird tick sampling was conducted in thirteen ringing sites of Central and Southern Italy, the serosurvey was performed on flocks grazing in coastal provinces of Central Italy that are stop over areas for birds flying from Africa, where Hyalomma ticks and CCHFV are endemic, to Central and Northern Europe. A total of 282 ticks (80.8% were Hyalomma spp.) were collected from 139 (0.28%) migratory birds of the 50,325 birds checked with 0.22% infested by Hyalomma spp., involving 22 avian species with a mean number of 1.6 Hyalomma spp. per infested bird. For the serosurvey, 540 sheep sera were randomly collected that resulted all negative when examined by an indirect IgG ELISA, employing a recombinant antigen coded by the CCHFV S gene. While the present study confirmed the introduction of CCHFV potential vectors in Central Italy, transported by migratory birds arriving from endemic areas, the serosurvey results did not put in evidence the concomitant arrival of the virus in the study area during the survey period. In general, in areas potentially at risk of CCHFV introduction and circulation, structured serological monitoring of susceptible domestic animals represents a rational system for an early detection of virus circulation.

Completeness of the dog registry and estimation of the dog population size in a densely populated area of Rome.

In most European countries, registration and identification of dogs is compulsory. In Italy, the national dog registry is composed of regional dog registries. Although dog registries have been established for many years, the issue related to completeness of data has not been addressed so far. The objective of this study was twofold: first to assess the completeness of data of the dog registry through telephone interview of a sample of dog owners drawn from the dog registry, then to estimate the total owned dog population in 4 boroughs of Rome. For the second objective, a capture-recapture method was applied using data from the dog registry and data from a face-to-face questionnaire submitted to people waiting in the sitting room of 5 points of access for booking and payment of primary and specialist care. Different scenarios are proposed to verify the assumptions of the estimation procedure and potential biases are discussed. The completeness of data of the dog registry was 88.9% (95% CI: 85.8-91.9%) and the owned-dog population was estimated at 26,244 dogs (95% CI: 24,110-28,383). The dog registry is an important source of information especially when it is properly updated and completeness of data is known.

A spatial, statistical approach to map the risk of milk contamination by β-hexachlorocyclohexane in dairy farms.

In May 2005, beta-hexachlorocyclohexane (ß-HCH) was found in a sample of bovine bulk milk from a farm in the Sacco River valley (Latium region, central Italy). The primary source of contamination was suspected to be industrial discharge into the environment with the Sacco River as the main mean of dispersion. Since then, a surveillance programme on bulk milk of the local farms was carried out by the veterinary services. In order to estimate the spatial probability of ß- HCH contamination of milk produced in the Sacco River valley and draw probability maps of contamination, probability maps of ß-HCH values in milk were estimated by indicator kriging (IK), a geo-statistical estimator, and traditional logistic regression (LR) combined with a geographical information systems approach. The former technique produces a spatial view of probabilities above a specific threshold at non-sampled locations on the basis of observed values in the area, while LR gives the probabilities in specific locations on the basis of certain environmental predictors, namely the distance from the river, the distance from the pollution site, the elevation above the river level and the intrinsic vulnerability of hydro-geological formations. Based on the ß-HCH data from 2005 in the Sacco River valley, the two techniques resulted in similar maps of high risk of milk contamination. However, unlike the IK method, the LR model was capable of estimating coefficients that could be used in case of future pollution episodes. The approach presented produces probability maps and define high-risk areas already in the early stages of an emergency before sampling operations have been carried out.

[Beta-hexachlorocyclohexane contamination in dairy farms of the Sacco River Valley, Latium, Italy, 2005. A retrospective cohort study]. / Contaminazione da beta-esaclorocicloesano delle aziende bovine da latte della Valle del Sacco (Lazio). Gestione dell'emergenza 2005 e conseguente studio di coorte retrospettivo.

OBJECTIVE: in March 2005, the Italian National Monitoring System on Chemical Residuals in Food of Animal Origin detected levels of the pesticide beta-hexachlorocyclohexane (ß-HCH) that were 20 times higher than the legal limit of 0.003 mg/kg in bulk milk from a dairy farm in the Sacco River valley. ß-HCH, a lindane isomer and possible human carcinogen, was subsequently found in milk from several neighboring farms. A study was therefore undertaken to evaluate the extent and risk factors for contamination. DESIGN: all dairy cattle farms in the valley were enrolled in a retrospective cohort study and their bulk milk analyzed for ß-HCH. A questionnaire was administered to farmers to evaluate possible exposure factors. SETTING AND PARTICIPANTS: cases: dairy farms with at least one result indicating ß-HCH ≥ 0.002 mg/kg in bulk milk during the period april-june 2005; exposure: feeding animals on fodder cultivated in soils watered with and/or flooded by river water; participants: IZSLT, RMG Local Health Unit, FR Local Health Unit. MAIN OUTCOME MEASURES: attack rate, relative risk, attributable proportion among exposed. RESULTS: of 244 farms tested, 34 met the case definition (attack rate 14%). The exposure to fodder cultivated in soils watered with and/or flooded by river water was observed in 33/34 (97%) case-farms and in 23/210 (10.9%) of those with contamination <0.002 mg/kg in bulk milk (RR 110.8; 95%CI 15.5- 792). Attributable proportion among exposed was more than 99%. CONCLUSION: fodder cultivated near a contaminated river was the main risk factor for ß-HCH contaminated milk. On the basis of the epidemiologic evidence and laboratory testing, watering local fields with river water and production of fodder in farms with contaminated soil was banned, and all the animals from positive farms were culled.

Feline injection-site sarcoma: recurrence, tumour grading and surgical margin status evaluated using the three-dimensional histological technique.

The three-dimensional histology technique is used in human medicine for the evaluation of complete lateral and deep surgical margins. In this study, the technique was applied to 48 excised feline injection-site sarcoma specimens. The predictive value of the histological margin status and tumour grading on local recurrence was investigated. In 32/48 cases, the margins were non-infiltrated, whilst in the remaining 16 cases, they were infiltrated. Overall, 6/32 (19%) tumours with non-infiltrated margins and 11/16 (69%) with infiltrated margins recurred. Tumours with infiltrated margins recurred about 10 times more frequently compared to tumours with non-infiltrated margins (P=0.0011). No statistically significant correlation was observed between grading and recurrence. The assessment of margin status using the 3D histology technique showed a good predictivity for post-surgical tumour recurrence. Extensive application of the 3D histology technique is recommended to standardise the evaluation of histological margins and to allow comparison between results from different laboratories.

Comparison of indirect fluorescent antibody test and modified agglutination test for detecting Toxoplasma gondii immunoglobulin G antibodies in dog and cat.

The present study describes the comparison between a modified agglutination test (MAT) and the indirect fluorescent antibody test (IFAT) for the detection of Toxoplasma specific IgG antibodies in dog and cat sera. MAT showed an "almost perfect" agreement with IFAT in detecting positive and negative results in cat sera, where as only a "substantial" agreement was observed in dog sera due to false negative results. Differences relative to sample dilution were recorded and serological titres obtained by MAT and IFAT are not directly comparable in cat and dog sera.

Protective effect of the AT137RQ and ARQK176 PrP allele against classical scrapie in Sarda breed sheep.

The susceptibility of sheep to scrapie is under the control of the host's prion protein (PrP gene and is also influenced by the strain of the agent. PrP polymorphisms at codons 136 (A/V), 15 (R/H) and 171 (Q/R/H) are the main determinants of susceptibility/resistance of sheep to classical scrapie. They are combined in four main variants of the wild-type ARQ allele: VRQ, AHQ, ARH and ARR. Breeding programmes have been undertaken on this basis in the European Union and th USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report th results of a multi-flock study showing the protective effect of polymorphisms other than those a codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 37 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the Pr gene. The distribution of variations other than those at the standard three codons, between scrapie cases and negative controls, was statistically different in all flocks. In particular, the AT(137)RQ an ARQK(176) alleles showed a clear protective effect. This is the first study demonstrating a protective influence of alleles other than ARR under field conditions. If further investigations in other sheep breeds and with other scrapie sources confirm these findings, the availability of various protective alleles in breeding programmes of sheep for scrapie resistance could be useful in breeds with a low frequency of the ARR allele and would allow maintaining a wider variability of the PrP gene.

Abomasal nematode community in an alpine chamois (Rupicapra r. rupicapra) population before and after a die-off.

Abomasa of 185 chamois shot during 5 consecutive hunting seasons were collected as part of a health monitoring program in an alpine area of Italy and examined for nematodes. The data were obtained during both the preceding period and that following a severe die-off caused by a pneumonia outbreak. Prevalence, mean abundance, mean intensity, and Thul Importance index were consistently high, in particular for Haemonchus contortus, having a low host specificity and high pathogenic potential. Species typical of cervids were also consistently detected. The abomasal nematode community showed an isolationist structure, suggesting its composition was primarily determined by external factors such as interspecific interaction among host species and environmental conditions. The effect of different factors (host sex, sampling site, and time) on nematode counts and aggregation were analyzed and discussed considering the peculiarities of the study site and the chamois population crash. In the light of parallel results for health monitoring, abomasal parasitism could represent a predisposing factor for the observed die-off.

Dog filariosis in the Lazio region (Central Italy): first report on the presence of Dirofilaria repens.

BACKGROUND: Epidemiological investigations were carried out in the Lazio Region to assess the status of canine filariosis and to evaluate the actual risk for veterinary and medical public health. METHODS: Since August 2001 to June 2003, a total of 972 canine blood samples, collected in public kennels and from private owners animals of the 5 Provinces of the Region, were tested. The presence of filarial parasites was evaluated by microscopy and bio-molecular techniques; the species identification was performed by means of the same diagnostic tools. RESULTS: A total of 17/972 (1.75%; 95%CI 1.06%-2.85%) blood samples were parasitized by D. repens,13 out them drawn by dogs resident in the Province of Roma, and 4 in the other provinces. Multivariate analysis was performed in order to evaluate the association between filariosis and risk factors. The origin from coastal territories seems to be a significant risk factor to acquire the infection. CONCLUSION: This is the first report of canine filariosis in the Lazio Region, where D. repens was before reported only in foxes. The risk of human zoonotic infection is stressed, and the absence of other filarial species is discussed.