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The global decline in amphibian populations is a major environmental issue. Chytridiomycosis, Ranaviruses and the red-leg syndrome have been identified in unusual mortality events. However, these infections do not account for all causes of declining amphibian populations. Moreover, several cases of amphibian mortality are difficult to solve without resorting to an interdisciplinary approach. Two cases of unusual mortality in Rana temporaria occurred at two high-mountain ponds (northwest Italy) in April and May 2021. Water and frog samples were analysed to understand the possible causes responsible for the unusual mortalities. Results of the main physicochemical (pH, conductivity, dissolved oxygen, chemical and biochemical oxygen demand) and nutrient (ammonia/ammonium, nitrite, nitrate, total phosphorus) parameters revealed a good condition of the water quality, with the absence of the main cyanotoxins (microcystins/nodularins). However, unseasonably high spring water temperatures were recorded in both ponds (12.73 °C and 14.21 °C for Frog Pond and Selleries Pond, respectively). Frogs (n = 50; snout-vent length: 7.0-9.8 cm; body mass: 85-123 g) collected from Frog Pond mainly presented bumps on the ventral cavity and dermal ulceration associated with the isolation of Carnobacterium maltaromaticum. On the other hand, frogs (n = 5; snout-vent length: 8.0-9.1 cm; body mass: 87-92 g) from Selleries Pond presented petechiae and dermal ulcerations on the rear limbs associated with the isolation of Aeromonas salmonicida and A. sobria. In both mortality events, the interdisciplinary approach revealed an association between frog mortalities and the isolation of bacteria. Isolated bacteria are considered opportunistic pathogens, and the high values of the water temperature has certainly led a stress on the frogs, favouring the spread of bacteria and the death of the frogs. Further studies are needed to assess the pathophysiological effects of the opportunistic bacteria here isolated, clarifying the interactions between emerging pathogens and climate change.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have emerged worldwide as zoonotic pathogens. Data on LA-MRSA in veal calf production in Italy are lacking; the aim of thi...

Swine farms are considered a hotspot of antimicrobial resistance and may contribute to the spread of antibiotic-resistant and/or pathogenic bacteria into the environment as well as to farm workers. In this study, swine fecal samples have been collected over the primary production, selecting three categories, i.e., "Suckling piglets", "Weaning pigs" and "Fatteners", in six intensive swine farms, for two years. Feces were analysed for the detection and abundance of class 1 integrons (used as proxy of antibiotic resistance and of anthropogenic pollution), and of enterococci [fecal indicator bacteria (FIB) and potentially pathogenic for humans] by quantitative Real Time PCR. Furthermore, Enterococcus faecalis and Enterococcus faecium were isolated, analysed for the presence of the intI1 gene by Real Time PCR and genetically typed by Pulsed-Field Gel Electrophoresis. Both enterococci and class 1 integrons were significantly more abundant in the Suckling piglets (p = 0.0316 and 0.0242, respectively). About 8% of the isolated enterococci were positive for the intI1 gene by Real Time PCR. E. faecalis and E. faecium were found genetically heterogeneous and no specific pattern could be identified as the driver for their presence along the pig primary production. These findings suggest that the "Suckling piglets" category of production represents the key point where to mitigate the risk of transmission of enterococci and class 1 integrons with associated antibiotic resistance genes to humans and spread into the environment.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic proliferative enteritis found in ruminants, known as paratuberculosis (PTB). The spread of PTB is increasing in countries with advanced animal husbandry practices, leading to significant economic losses. Moreover, a supposed zoonotic role of MAP in Crohn’s disease (CD) in humans has been discussed by the scientific community, however, although the association between MAP and CD has generally been accepted, it is still up for debate if MAP is the main cause of CD, a contributing factor, or merely a commensal organism for the development of CD. The aim of this study was to assess the survival of MAP during the entire production process of a traditional Italian goat’s raw milk fresh cheese, the “Robiola di Roccaverano”, assessing the survival rate and persistence of MAP in the final product. A mix of MAP field isolates from goats of the Roccaverano area and a reference ATCC strain were used to carry out milk in experimental inoculation. Samples of milk, curd and cheese were taken in two consecutive batches of production. Microbiological challenge tests, evaluated by f57-qPCR, showed a significant decrease in MAP charge during the cheesemaking process for both batches, suggesting the productive process has an impact on MAP survival.

The sub-Alpine lakes of Switzerland, Italy and France have long been reported as an endemic area of diphyllobothriosis, a parasitic zoonosis aused by Dibothriocephalus latus. With this study, we explored the hypothesis for a relationship between the prevalence of D. latus in Perca fluviatilis and the Escherichia coli load in lake water. To do this, we identified eleven sampling sites in three areas (north, centre and south) of Lake Iseo (north Italy) to determine E. coli load and the prevalence of D. latus in P. fluviatilis. Prevalence and 95% confidence interval (CI) of D. latus infestation ranged from 0% (95% CI: 0.71–0.0) in Sarnico (southern area) to 20% (95% CI: 33.0–11.2) in Pisogne (northern area). There were significant differences in prevalence be-tween the sites (χ2= 31.12; p- value= .0006) and in E. coli load (Kruskal–Wallis test; p- value= .0005). There was decreasing gradient of E. coli load and prevalence of D. latus infestation from north to south. A significant positive correlation (r= .881; p- value= .003) was found between E. coli load and prevalence of D. latus. Also, linear regression showed a significant relationship between E. coli load and prevalence of infestation (R2= .775). Our findings offer an explanation for the link between E. coliload in water and D. latus prevalence. The potential factors in this link are the ef-ficiency of the local wastewater treatment plant, the bathymetric profile of the lake and the life cycle of D. latus, which is mainly affected by light and water temperature.KEYWORDSItaly, Lake Iseo, Perca fluviatilis, prevalence of infestation, wastewater treatment plant, the bathymetric profile of the lake and the life cycle of D. latus, which is mainly affected by light and water temperature.

Livestock-associated methicillin-resistant

Diseases are natural regulating factors of wildlife populations, but some pathogens may become an important threat in wildlife conservation, especially for endangered species. The presence of domestic animals may foster the spread of diseases in natural population, although their role in the dynamic of infections in wildlife is not clear. In this study, we investigated the presence and prevalence of a range of multi-host pathogens in wild species (red fox, Eurasian badger, beech marten, pine marten, stoat for a total of 89 carcasses analysed) and domestic animals (n = 52 shepherd and n = 25 companion dogs) living in a protected area of the Alps (the Gran Paradiso National Park) and discussed the role of domestic dogs as possible source of infection for wild species. Our results showed that domestic dogs are potential shedder of three important pathogens: Canine distemper virus, Toxoplasma sp. and Neospora caninum. In particular, shepherd dogs seem to represent a threat for wildlife as they are exposed to multiple pathogens because of free-roaming, scavenging lifestyles and close proximity to livestock. However, also companion dogs more subject to veterinary care may foster the spread of pathogens. Our results highlight the importance of regulating the access of domestic dogs to protected areas that aim at preserving biodiversity and enhancing the conservation of endangered species.

PCR ribotypes (RTs027 and 078) are known causes of Clostridioides difficile infection (CDI) in humans. Molecular typing and characterization of 39 C. difficile strains isolated from samples from humas and animals in 2016–2018 indicated an overlap of RTs between community-acquired patients (CA-CDI) and domestic animals from the same geographical area; 14 RTs were identified: 12 RTs were positive for toxins A/B; RT078, RT080 and RT126 were also positive for binary toxin (CDT). Most of the RTs from the animals (RTs020, 078, 106, 126) were also detected in the samples from humans. Strains grouped into three clusters: cluster I included prevalently human strains, mainly RT 018; clusters II and III included strains from humans and animals, mainly RT078 and RT020. The CA-CDI strains suggested animals as a reservoir of C. difficile isolated together with other microorganisms from animals, highlighting the association of enteric pathogens as a cause of infection and death.

We report the first case of Mycobacterium bovis infection in a free-living fallow deer (Dama dama) in northwest Italy, the epidemiological analysis (i.e., tracing source and dissemination during outbreaks), and the potential source of infection in a historically animal tuberculosis (aTB)-free area. Gross lung and lymph node lesions were histologically consistent with a severe parasitic bronchopneumonia due to lungworms associated with severe mycobacterial infection. The lesions contained numerous densely packaged, acid-fast bacilli, raising suspicion of an active, open form of aTB. Acid-fast organisms were characterized as M. bovis SB0120-ETR 45533, one of the most common profiles in Italy. Epidemiological investigation into the most recent outbreaks caused by M. bovis SB0120-ETR 45533 within a 50-km radius of where the fallow deer was retrieved disclosed two different situations: one case involving a water buffalo (Bubalus bubalis) in 2008 and one involving a donkey in 2016. Analysis of spoligotype and VNTR-type pattern circulating and recorded in northwest Italy from 1999 to 2014 suggested that in the cases of the donkey and the fallow deer, the source of infection was most likely attributable to spillover from outbreaks in domestic species: cattle and water buffalo, respectively. According to the European Commission, aTB status of livestock does not depend on aTB cases in wildlife (Council Directive 64/432/33C of 26 June 1964); nevertheless, the primary aim of aTB eradication should include global monitoring of all susceptible species if the re-emergence of M. bovis from established wildlife reservoir is to be prevented.

Animal and bait poisoning data for northwest Italy collected between 2012 and 2017 were described and analyzed to estimate the risk of exposure to hazardous substances by animals. In about 4% of animals necropsied (n = 356/9512), the cause of death was poisoning and domestic pets (9.5%) and synanthropic animals (12.2%) appear to be the most involved. Furthermore, 294 out of 728 baits (40.4%) were positive for toxic substances and/or inert hazardous material. Application of a mixed-effects Poisson regression model and local cluster analysis evidenced increased risk of exposure to poisoning with altimetry (>600 m.a.s.l.) and municipality (PR = 1.6, 95%CI 1.2–2.1 for poisoning, PR = 2.2, 95%CI 1.2–4.2 for poisoning by insecticides and PR = 2.9, 95%CI 1.4–6.2 for poisoning by metaldehyde). Since the mountainous areas in the region are mostly devoted to pasture and extensive farming, the high frequency of animal and bait poisoning events may be related to farmers' need to protect their livestock and crops against foxes, wolves, rodents or wild boars. Summarizing, the type of land use and context may influence the frequency and type of toxin chosen to kill animals considered a nuisance for hunting, farming, agriculture and apiculture. Despite bans and limitations, the use of harmful substances is not perceived as an environmental threat but rather as routine pest control. Animal and bait poisoning constitute a public health concern because it is potentially harmful to humans and the environment. Our findings may inform risk communication strategies, as well as prevention and control measures for the reduction of illegal and non-targeted species poisoning.

The Apennine chamois (Rupicapra pyrenaica ornata) is a geographically isolated subspecies of chamois currently living only in limited areas of Central Italy. It is included in the Red List of the International Union for Conservation of Nature (IUCN). To assure long-term conservation of the species, the number of individuals needs to be increased creating a consistent and viable population. Accordingly, relocation for reintroduction programs is being planned. However, this could lead to the introduction of infectious diseases. Thus, knowledge on the health status of the Apennine chamois is important for the management of this species. In the absence of existing data, retrospective investigation is useful. A serological retrospective survey on 119 Apennine chamois, captured for routine marking or reintroduction operations from 1990 to 2008 at the National Park of Abruzzo, Lazio, and Molise (PNALM), was performed to detect antibodies against pestiviruses, bovine parainfluenza 3 virus (PI-3), bluetongue virus (BTV), bovine herpesvirus type 1 (BHV-1), Brucella spp., Chlamydophila spp., Coxiella burnetti, and Leptospira spp. Serums were negative for PI-3, BTV, BHV-1, Brucella spp., Chlamydophila spp., C. burnetti, and Leptospira spp., while three animals in 1992 and five in 2008 were positive for pestivirus antibodies. Although the number of samples per year was often limited, inference to evaluate the infectious status of the overall population in the PNALM was attempted. Better planning of sampling activities and a specific surveillance program for infectious diseases are needed, especially in a small and limited population like the Apennine chamois, in which reintroduction programs are considered strategic for conservation and risk factors, like sharing of grazing with wild and domestic animals, exist.

Canine distemper (CD) may pose a serious threat to Alpine wild carnivores and affect their population dynamics. Since 2006, the strain Europe Wildlife 2006-09, a distinct CD virus subgroup within viral lineage Europe 1 (EU1) characterized by increased virulence and host range expansion, has been linked to multiple CD outbreaks in Alpine wild carnivores. The aim of this study was to fill knowledge gaps about ongoing Alpine outbreaks of CD. To do this, we report on the circulation of canine distemper virus (CDV) and outbreaks of CD in Alpine wild carnivores in northwest Italy. A specific diagnostic protocol applied to a sample of 548 wild carnivores collected between January 2013 and December 2015 revealed the circulation of CDV belonging to the EU1 lineage. All isolates were carriers of amino-acid mutations defining the cluster Europe Wildlife 2006-09. A self-maintained multihost pathogen system may have developed in northwest Italy in which interspecies transmission from red foxes (Vulpes vulpes) to other noncanid species enhanced pathogen maintenance in the system.

Thymoquinone, a naturally derived agent, has been shown to possess antioxidant, antiproliferative and proapoptotic activities. In the present study, we explored thymoquinone effects on the proteasomal complex, the major system involved in the removal of damaged, oxidized and misfolded proteins. In purified 20S complexes, subunit-dependent and composition-dependent inhibition was observed, and the chymotrypsin-like and trypsin-like activities were the most susceptible to thymoquinone treatment. U87 MG and T98G malignant glioma cells were treated with thymoquinone, and 20S and 26S proteasome activity was measured. Inhibition of the complex was evident in both cell lines, but predominantly in U87 MG cells, and was accompanied by accumulation of ubiquitin conjugates. Accumulation of p53 and Bax, two proteasome substrates with proapoptotic activity, was observed in both cell lines. Our results demonstrate that thymoquinone induces selective and time-dependent proteasome inhibition, both in isolated enzymes and in glioblastoma cells, and suggest that this mechanism could be implicated in the induction of apoptosis in cancer cells.

BackgroundHealth of mammary glands is fundamental for milk and dairy products hygiene and quality, with huge impacts on consumers welfare.MethodsThis study aims to investigate the microbial agents (bacteria, fungi and lentiviruses) isolated from 89 macroscopically healthy udders of regularly slaughtered small ruminants (41 sheep, 48 goats), also correlating their presence with the histological findings. Multinomial logistic regression was applied to evaluate the association between lesions and positivity for different microbial isolates, animal age and bacteria.ResultsTwenty-five samples were microbiologically negative; 138 different bacteria were isolated in 64 positive udders. Coagulase-negative staphylococci were the most prevalent bacteria isolated (46.42%), followed by environmental opportunists (34.76%), others (10.14%) and pathogens (8.68%). Most mammary glands showed coinfections (75%). Lentiviruses were detected in 39.3% of samples. Histologically, chronic non-suppurative mastitis was observed in 45/89 glands, followed by chronic mixed mastitis (12/89) and acute suppurative mastitis (4/89). Only 28 udders were normal. Histological lesions were significantly associated with the animal species and lentiviruses and coagulase-negative staphylococci infections. Goats had significantly higher risk to show chronic mixed mastitis compared to sheep. Goats showed a significantly lower risk (OR = 0.26; 95% CI [0.06–0.71]) of being infected by environmental opportunists compared to sheep, but higher risk (OR = 10.87; 95% CI [3.69–37.77]) of being infected with lentiviruses.DiscussionThe results of the present study suggest that macroscopically healthy glands of small ruminants could act as a reservoir of microbial agents for susceptible animals, representing a potential risk factor for the widespread of acute or chronic infection in the flock.

Thymoquinone, a naturally derived agent, has been shown to possess antioxidant, antiproliferative and proapoptotic activities. In the present study, we explored thymoquinone effects on the proteasomal complex, the major system involved in the removal of damaged, oxidized and misfolded proteins. In purified 20S complexes, subunit-dependent and composition-dependent inhibition was observed, and the chymotrypsin-like and trypsin-like activities were the most susceptible to thymoquinone treatment. U87 MG and T98G malignant glioma cells were treated with thymoquinone, and 20S and 26S proteasome activity was measured. Inhibition of the complex was evident in both cell lines, but predominantly in U87 MG cells, and was accompanied by accumulation of ubiquitin conjugates. Accumulation of p53 and Bax, two proteasome substrates with proapoptotic activity, was observed in both cell lines. Our results demonstrate that thymoquinone induces selective and time-dependent proteasome inhibition, both in isolated enzymes and in glioblastoma cells, and suggest that this mechanism could be implicated in the induction of apoptosis in cancer cells.