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The detection of H5N1 highly pathogenic avian influenza virus (HPAIV) in lactating dairy cattle in the United States, with high viral titers in raw milk, has raised concerns about zoonotic transmission through unpasteurized milk and dairy products. While viral inactivation during pasteurization is documented, data on persistence in raw-milk cheeses remain limited. This study evaluated the survival of avian influenza viruses (AIVs), both low pathogenic (LPAIV, H1N1) and highly pathogenic (HPAIV, H5N1), during the production and ripening of Grana-type hard cheeses from raw cow’s milk. Experimental cheesemaking was carried out with milk artificially contaminated with A/duck/Italy/281904-2/06 (H1N1; 107.75 EID50/mL) or A/duck/Italy/326224-2/22 (H5N1 clade 2.3.4.4b; 106.75 EID50/mL). Cheeses were manufactured under Parmigiano-Reggiano standards and ripened 30 days at 5–6 °C. Viral detection in finished cheeses was performed using inoculation in specific-pathogen-free embryonated chicken eggs (SPF-ECEs), hemagglutination (HA) assay, and monoclonal antibody-based ELISA. No infectious virus was detected in cheese samples after two blind passages in SPF-ECEs. Both HA and ELISA tests were negative, indicating complete viral inactivation. These results demonstrate that Grana-type cheese processing, including cooking, acidification, and ripening, effectively inactivates LPAIV and HPAIV. Findings support the microbiological safety of raw-milk hard cheeses regarding AIV, contributing to risk assessment and food safety policies.

Genetic testing of animal biological material has become a valuable tool in forensic investigations, and it is successfully used to identify unknown crime perpetrators, to unmask food frauds, or to clarify cases of animal attacks on humans or other animals. When DNA profiling is not possible due to inadequate amounts of nuclear DNA, mitochondrial DNA (mtDNA) testing is the only viable alternative, as in the case of shed hair samples. In this case, a dog was allegedly killed by wild animals while being hosted in a boarding house. Extraneous hair fragments recovered from the dog’s mouth and paws were subjected to genetic analysis: the cytochrome b gene located on mtDNA was amplified and sequenced in order to determine the species responsible for the killing. The mtDNA analysis provided evidence that the dog was killed by other dogs, thus unmasking a false wild animal attack and putting the case in an entirely different perspective.

We recently described the genetic antimicrobial resistance and virulence profile of a collection of 279 commensal E. coli of food-producing animal (FPA), pet, wildlife and human origin. Phenotypic antimicrobial resistance (AMR) and the role of commensal E. coli as reservoir of extra-intestinal pathogenic Escherichia coli (ExPEC) virulence-associated genes (VAGs) or as potential ExPEC pathogens were evaluated. The most common phenotypic resistance was to tetracycline (76/279, 27.24%), sulfamethoxazole/trimethoprim (73/279, 26.16%), streptomycin and sulfisoxazole (71/279, 25.45% both) among the overall collection. Poultry and rabbit were the sources mostly associated to AMR, with a significant resistance rate (p > 0.01) to quinolones, streptomycin, sulphonamides, tetracycline and, only for poultry, to ampicillin and chloramphenicol. Finally, rabbit was the source mostly associated to colistin resistance. Different pandemic (ST69/69*, ST95, ST131) and emerging (ST10/ST10*, ST23, ST58, ST117, ST405, ST648) ExPEC sequence types (STs) were identified among the collection, especially in poultry source. Both ST groups carried high number of ExPEC VAGs (pandemic ExPEC STs, mean = 8.92; emerging ExPEC STs, mean = 6.43) and showed phenotypic resistance to different antimicrobials (pandemic ExPEC STs, mean = 2.23; emerging ExPEC STs, mean = 2.43), suggesting their role as potential ExPEC pathogens. Variable phenotypic resistance and ExPEC VAG distribution was also observed in uncommon ExPEC lineages, suggesting commensal flora as a potential reservoir of virulence (mean = 3.80) and antimicrobial resistance (mean = 1.69) determinants.

The gray wolf ( Canis lupus ) expanded its distribution in Europe over the last few decades. To better understand the extent to which wolves could re-occupy their historical range, it is important to test if anthropization can affect their fitness-related traits. After having accounted for ecologically relevant confounders, we assessed how anthropization influenced i ) the growth of wolves during their first year of age (n = 53), ii ) sexual dimorphism between male and female adult wolves (n = 121), in a sample of individuals that had been found dead in Italy between 1999 and 2021. Wolves in anthropized areas have a smaller overall variation in their body mass, during their first year of age. Because they already have slightly higher body weight at 3–5 months, possibly due to the availability of human-derived food sources. The difference in the body weight of adult females and males slightly increases with anthropization. However, this happens because of an increase in the body mass of males only, possibly due to sex-specific differences in dispersal and/or to “dispersal phenotypes”. Anthropization in Italy does not seem to have any clear, nor large, effect on the body mass of wolves. As body mass is in turn linked to important processes, like survival and reproduction, our findings indicates that wolves could potentially re-occupy most of their historical range in Europe, as anthropized landscapes do not seem to constrain such of an important life-history trait. Wolf management could therefore be needed across vast spatial scales and in anthropized areas prone to social conflicts.

Background Transport to slaughterhouses is a critical phase in pig production, affecting welfare and economics. Mortality during transport, or dead-on-arrival (DOA), is a direct indicator of welfare compromise. This study evaluated the association between estimated transport distance, ambient temperature, and consignment size with mortality in commercial pig journeys lasting ≤ 8 h. Results Data from 34,239 consignments from 434 farms transported to a high-throughput slaughterhouse were analyzed. Consignment size ranged from 5 to 164 pigs, estimated transport distances from 5 to 605 km, and ambient loading temperatures ranged from 0.9 °C to 31.2 °C. Overall average mortality rate was 0.062%, with 93.1% of consignments showing no DOA pigs. Seasonal patterns were pronounced, with summer exhibiting the highest mortality and winter/spring the lowest. Mixed-effects logistic regression showed that ambient temperature and estimated transport distance were positively associated with mortality (OR = 1.354 and 1.086, respectively), while larger consignments exhibited a negative association (OR = 0.910). Each 1 °C increase in temperature corresponded to a 3.86% rise in the odds of pigs being DOA. Random farm effects showed substantial between-farm variability. Average transport mortality per farm categorized 36.9% of farms as being at very low risk, 13.1% as low, 24.9% as medium, and 25.1% as high risk. Best Linear Unbiased Predictors (BLUP) estimates identified 38 farms with significantly higher-than-average mortality, while funnel plot analysis highlighted 26 outliers, providing complementary approaches to benchmark farm performance and prioritize high-risk holdings for welfare monitoring. Among 664 (1.95%) consignments inspected by official veterinarians, mortality was 0.036%, and inspections were evenly distributed across seasons, ambient temperatures, and consignment characteristics. Only 86 (12.95%) of inspections targeted the 26 high-risk farms identified by the funnel plot. These results suggest that inspection efforts were not preferentially directed toward farms with elevated predicted mortality. Conclusions Pig mortality during journeys lasting ≤ 8 h transports is mainly associated with ambient temperature and, to a lesser extent, estimated transport distance, with summer as a high-risk period. Larger consignments modestly reduce risk, and mortality per farm highlight the potential for targeted interventions. Current veterinary inspections are not systematically aligned with risk, suggesting scope to optimize monitoring and welfare outcomes.

Background Domesticated and wild swine play an important role as reservoir hosts of Trichinella spp. and a source of infection for humans. Little is known about the survival of Trichinella larvae in muscles and the duration of anti- Trichinella antibodies in pigs with long-lasting infections. Methods Sixty pigs were divided into three groups of 20 animals and infected with 10,000 larvae of Trichinella spiralis , Trichinella britovi or Trichinella pseudospiralis . Four pigs from each group were sacrificed at 2, 6, 12, 18 and 24 months post-infection (p.i.) and the number of larvae per gram (LPG) of muscles was calculated. Serum samples were tested by ELISA and western blot using excretory/secretory (ES) and crude antigens. Results Trichinella spiralis showed the highest infectivity and immunogenicity in pigs and larvae survived in pig muscles for up to 2 years p.i. In these pigs, the IgG level significantly increased at 30 days p.i. and reached a peak at about 60 days p.i., remaining stable until the end of the experiment. In T. britovi- infected pigs, LPG was about 70 times lower than for T. spiralis at 2 months p.i. and only very few infecting larvae were detected at 6 months p.i., whereas no larvae were detected at 12, 18 and 24 months p.i. At 6 months p.i., degenerated/calcified larvae and cysts were detected in the muscles by trichinoscopy and histology. The IgG pattern showed by T. britovi -infected pigs was similar to that of T. spiralis -infected pigs, although seroconversion occurred some days later. The larval burden of T. pseudospiralis was slightly greater than for T. britovi at 2 months p.i., but no larvae were detected at 6 and 12 months p.i. In T. pseudospiralis -infected pigs, seroconversion occurred slowly, as in T. britovi -infected pigs. The IgG level showed a significant drop at 6 months p.i. and declining to the cut-off value at 12 months p.i. Conclusions The longer survival of T. spiralis in pigs in comparison with the other two species highlights its exceptional dissemination potential. These results provide an explanation of the controversial data collected by parasitological and serological tools in the course of epidemiological investigations.

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.

Visceral leishmaniasis (VL) caused by Leishmania infantum is an ongoing health problem in southern Europe, where dogs are considered the main reservoirs of the disease. Current data point to a northward spread of VL and canine leishmaniasis (CanL) in Italy, with new foci in northern regions previously regarded as non-endemic. Multilocus microsatellite typing (MLMT) was performed to investigate genetic diversity and population structure of L. infantum on 55 samples from infected humans, dogs and sand flies of the E-R region between 2013 and 2017. E-R samples were compared with 10 L. infantum samples from VL cases in other Italian regions (extra E-R) and with 52 strains within the L. donovani complex. Data displayed significant microsatellite polymorphisms with low allelic heterozygosity. Forty-one unique and eight repeated MLMT profiles were recognized among the L. infantum samples from E-R, and ten unique MLMT profiles were assigned to the extra E-R samples. Bayesian analysis assigned E-R samples to two distinct populations, with further sub-structuring within each of them; all CanL samples belonged to one population, genetically related to Mediterranean MON-1 strains, while all but one VL cases as well as the isolate from the sand fly Phlebotomus perfiliewi fell under the second population. Conversely, VL samples from other Italian regions proved to be genetically similar to strains circulating in dogs. A peculiar epidemiological situation was observed in northeastern Italy, with the co-circulation of two distinct populations of L. infantum; one population mainly detected in dogs and the other population detected in humans and in a sand fly. While the classical cycle of CanL in Italy fits well into the data obtained for the first population, the population found in infected humans exhibits a different cycle, probably not involving a canine reservoir. This study can contribute to a better understanding of the population structure of L. infantum circulating in northeastern Italy, thus providing useful epidemiologic information for public health authorities.

In the last two years, there have been three major epidemic seasons in the territory of the European Union and the HPAI epizootic in 2021–2022 is the most severe in recent history. In Italy, the disease was introduced to dense poultry areas with serious economic consequences for the entire sector. In Lombardy, the analysis of the risk factors was carried out, also taking into account the density of domestic birds. In the most affected areas, 66.7% of the outbreaks occurred in the areas with the highest poultry density and the likelihood of an outbreak occurring increased with an increase in the density of birds per km2. In cells 10 × 10 km with a density greater than 10,000 birds/km2, the probability of outbreak occurrence was over 66.7%. The provinces involved in the last epidemic were the same involved in previous epidemics and, given the risk factors present in the area, it is plausible that the risk remains high also for future epidemic seasons. Therefore, to avoid the repetition of similar events, certain control measures shall be strengthened and vaccination considered as a complementary tool for the control of HPAI virus in risk areas.

In this study, internal organs (tongue, intestine, and spleen) of 23 free-ranging Italian wolves (Canis lupus italicus) found dead between 2017 and 2019 were tested for Carnivore protoparvovirus 1, Canine adenovirus (CAdV), and Canine circovirus (CanineCV) using real-time PCR assays. Genetic characterisation of the identified viruses was carried out by amplification, sequencing, and analysis of the complete viral genome or informative viral genes. All the wolves tested positive for at least one of the DNA viruses screened, and 11/23 were coinfected. Carnivore protoparvoviruses were the most frequently detected viruses (21/23), followed by CanineCV (11/23) and CAdV (4/23). From the analysis of the partial VP2 gene of 13 carnivore protoparvoviruses, 12 were canine parvovirus type 2b, closely related to the strains detected in dogs and wild carnivores from Italy, and one was a feline panleukopenia-like virus. Of the four CAdV identified, two were CAdV-1 and two were CAdV-2. The complete genome of seven CanineCVs was sequenced and related to the CanineCV identified in dogs, wolves, and foxes worldwide. Close correlations emerged between the viruses identified in wolves and those circulating in domestic dogs. Further studies are needed to investigate if these pathogens may be potentially cross-transmitted between the two species.