Dogs and cats have coexisted with humans for thousands of years, and as domesticated animals, they provide significant psychological benefits for our contemporary, city-centered society. In addition, there are recognized links between companion animals and human infections caused by various zoonotic agents, including bacteria, fungi, viruses and parasites (1). Whilst certain pathogens may be widely recognized, others are newly emerging, under-researched or have unexplored potential to spread between animals and humans, yet are not currently subject to extensive reporting or scientific analysis. A number of zoonoses affecting pets are well known in Italy [e.g., leptospirosis (2–4), leishmaniosis (5–9), dermatophytosis (10), echinococcosis (11, 12), bartonellosis (13–15), gastrointestinal parasites and vector-borne diseases (16, 17)], however the lack of official surveillance programs results in a scarcity of information about the prevalence of these diseases and pathogens. There are also emerging or rare infections for which the prevalence is completely unknown. For example, Capnocytophaga canimorsus, commonly part of the oral bacterial microbiota of dogs and cats, is reported to cause fatal infections in humans (18–21). Furthermore, the zoonotic role of antibiotic-resistant bacteria (22–25) or other pathogens such as Brucella canis are still poorly investigated (26–28). Hepatitis E virus (HEV) detected in both domestic and wild species (e.g., swine, wild boar, dog, cat, deer, rabbit, mongoose) (29–39), and Cowpox virus recently identified (40–42), are pathogens agents with unknown prevalence among pets in Italy. In the context of the recent SARS-CoV-2 pandemic, both experimental evidence and observations of natural infections have demonstrated the susceptibility of companion animals (43–49). In addition, emerging and potential zoonotic pathogens responsible for respiratory or gastrointestinal syndromes in humans, including Influenza A viruses (50, 51), Rotaviruses (1, 52, 53), Mammalian Orthoreovirus (54) and Noroviruses (55, 56), need to be further investigated. For these reasons, the lack of official reporting of emerging and neglected zoonoses calls attention to the need to improve surveillance and promptly identify potential outbreaks and spillovers. These animals can serve as environmental sentinels or reservoirs for numerous potential zoonotic diseases, either through direct or indirect transmission pathways, which are often overlooked (57–59). As previously reported, fatal epizootic and/or potentially epidemic infectious diseases such as respiratory, neurological and systemic streptococcal infections (in dogs and cats), canine influenza, haemorrhagic respiratory Escherichia coli infection (in dogs) and virulent systemic feline Calicivirus infection, have all emerged within animal shelter populations over the past decade (60–62).

Recent official data about the population of stray dogs and cats in North-East Italy (Veneto region) reported a number of 2,706 accesses in dogs’ sanitary shelters, 790 accesses in kennel shelters. Among these dogs, 1,279 dogs were returned to the owner, and 867 were adopted. The data reported that in Veneto the Veterinary Services neutered 8,842 cats as part of the stray prevention campaign (catch and release).¹ Despite this scenario, for companion animals, there are no organized infectious and zoonotic diseases surveillance plans according to updated public veterinary services. Few studies have reported specific surveys of shelter demographics, infection control practices and policies, as well as awareness and concern about infectious and zoonotic diseases (63–65). The day-to-day management of the facilities primarily falls upon non-specialized personnel. The facilities pose a significant health and hygiene risk for both animals and humans due to the high turnover of animals, varying in ages, breeds, and origins, confined in a small space. Additionally, the frequent presence of volunteer staff, often inadequately trained, exacerbates these risks. Staff may expose themselves directly to health risks, and/or unintentionally spreading infections.

The aim of the present study was to investigate the prevalence and the circulation of different known, emergent, neglected or potential zoonotic pathogens among cats and dogs sheltered, in order to update the information on these zoonotic agents and suggest a prioritization for surveillance programs should be implement. In addition, this study reports the implementation of protocols for screening and prevention of zoonotic diseases, in order to improve the management for health protection in a One Health perspective.

Zoonoses, despite being recognized for centuries, have recently garnered heightened attention, particularly in the post-COVID-19 era. Consequently, the prevention and control of zoonotic diseases must be revised by focusing on the complex interaction between humans, animals, and the ecosystem through the One Health approach. In a context of routinely health monitoring of kennel dogs and free-roaming cats in North-East Italy, this study updates about the epidemiology and the circulation of known, neglected, potential, emerging and re-emerging zoonoses among the sheltered dogs and cats. The updated acknowledgement of the presence and prevalence of zoonotic agents is pivotal for a correct optimization of any surveillance action (91). Companion animal shelters present a challenge in balancing animal health and welfare, staff organization and training, and economic resources. It is critical to implement effective surveillance for infectious or zoonotic agents in companion animal kennels, dog and cat colonies or stray communities where animals of different ages, breeds and origins, often with poorly known medical histories, live together in close contact (92–94).

The present results confirmed the data about leptospirosis as previously reported in dogs and wild animals in the North-East Italy (2, 3, 95, 96), and that except for L. interrogans serogroups Pomona and Sejroe, the serovars most commonly found were those already targeted by vaccines commercially available, specifically serogroups Grippotyphosa, Icterohaemorrhagiae, Bratislava, and Canicola. Animals with the highest MAT titers (≥1:800) were typically those that had not received vaccination against the specific serogroup. As published data is limited regarding the incidence of naturally occurring leptospirosis in fully vaccinated dogs (97, 98), it is interesting to report that two apparently healthy dogs, one of which received regular vaccination against leptospirosis, had asymptomatic shedding of Leptospira (urine). The outstanding information about the carrier dogs is that they were clinically healthy, outdoors and young (1 y/o and 2 y/o). This finding thus indicates the necessity of implementing monitoring protocols for kennel resident animals or those newly admitted to the shelters and of emphasizing the importance of operators’ observance of biosecurity measures.

The seroprevalence data for leishmaniosis were consistent with recent studies conducted in the same areas (6, 99) indicating a significant prevalence of exposure to the pathogen (6, 8, 16, 100). The majority of the sampled animals were asymptomatic, and for a small percentage data on antiparasitic treatment was available, mainly oral medication (11.15%; CI 7.33–14.98). Although most positive dogs reported low IFAT titers (from 1:40 to 1:80), indicating contact with the pathogen rather than clinical active infection, it is pivotal to acknowledge that the North-East Italy includes areas of gradually increasing endemicity (99, 101). This situation highlights the necessity of continuous monitoring, not only to safeguard animal health but also to prevent zoonotic risks (102). In addition, our results confirm that animal shelters play a critical role in identifying positive subjects traveling from highly endemic regions (i.e., southern Italy) (103, 104).

Furthermore, this study reported a significant prevalence of Bartonella spp., exceeding that which was previously reported (5, 13, 15, 105), both for active infection (Bartonella spp. DNA) and serological positivity. Only seven cats were PCR-positive for B. henselae, but tested negative in serology, suggesting recent infection/reinfection. In addition, by comparing the positivity between the molecular and serological tests, our results indicate that the IFAT analysis for B. henselae gave positive results in animals where there was evidence of only B. clarridgeiae DNA. This finding evokes serological cross-reactivity among various Bartonella species, which have been reported also for other agents and bacterial species (106–108). This aspect can suggest that the serology for Bartonella sp. could be a useful screening tool in the large population; however, as with human medicine (109), molecular analysis is the confirmatory test. Most of the cats positive for Bartonella species on direct DNA testing were also positive on serology, although statistical significance was not reported between the Bartonella species identified by molecular analysis and IFAT titers. The presence of the arthropod vector Ctenocephalides felis is closely associated with Bartonella sp. infection in cats and free-living or colony animals are frequently re-exposed, as they do not receive regular anti-parasite prophylaxis. In felines, the infection can have a host-adapted-reservoir course showing either an asymptomatic or a paucisymptomatic form (15, 74, 110). Conversely, in humans, the infection can exhibit severe forms of the disease, especially in young or immune-compromised people (111–116). Hence, it is high-priority to ascertain the latest prevalence and spread of the infection, and effectively communicate the associated zoonotic risk to companion animals’ shelters operators and the general public (110, 117). Treatment of this infection in feral cats with drugs is not feasible and may prove ineffective. Instead, preventive measures must center on flea prophylaxis to diminish the likelihood of infection in cats (117–121). In addition, training for health and ethology professionals reduces the probability of exposure to bite and scratch wounds.

The antibiotic-resistant bacteria are significant sources of hospital-acquired infections (122–124), reported low prevalence within the studied population: no methicillin-resistant coagulase-positive Staphylococci (MRCPS) or vancomycin-resistant Enterococci (VRE) were isolated. According to previous studies, the prevalence of ESBL-producing Enterobacteria and Pseudomonas aeruginosa was higher in dogs than in cats. The significant number of positive subjects, mostly asymptomatic, highlights the need to implement the surveillance of this pathogen to prevent the emergence and spread of multidrug-resistant strains, particularly in shelter and kennel situations (24, 59, 125–127). The low prevalence of antibiotic resistance among animals in shelters and/or free-living cats may be attributed to their limited exposure to antibiotics (128, 129), as well as some of them not living constantly close to humans. Additionally, veterinary professionals responsible for the facilities follow antibiotic stewardship guidelines, contributing to the low prevalence observed (130, 131). Nevertheless, the aforementioned is a dynamic circumstance, and the findings imply that surveillance of these microorganisms should be considered, especially in scenarios that might promote their proliferation such as overcrowding, inadequate hygiene, and recurring bacterial illnesses.

Brucella canis is a zoonotic pathogen considered to be emerging because of the movement of animals, including trade and legal/illegal transfer, from countries where this disease is more prevalent or endemic (132, 133). The diagnosis of Brucella canis remains challenging owing to the inadequacy of current diagnostic methods; indeed, the diagnosis demands multiple tests to be run in parallel, including different serological methods, molecular analysis, and bacterial isolation (75). Furthermore, the bacterium’s biological and pathophysiological characteristics complicate diagnostic accuracy (134), and uncertain clinical manifestations may lead to underdiagnosed of this disease. In this study, none of the five serological positivities were followed by confirmation by direct pathogen detection analysis. Specifically, we reported a case of clinically asymptomatic dog (7-year-old mixed-breed male) with anti-Brucella canis mSAT antibody titer of 1:40 and concurrently positive for Trichuris spp. at the coprological examination. One month later, the dog was retested using serological methods and was found to be seronegative. The second case involved a Rottweiler, a male dog 5 years old and reported both testes reduced in size. Serology reported mSAT 1:20, CFT 1:10 and IF 1:80. However, bacterial isolation proved negative. After a month, this dog was retested, reporting mSAT 1:20, CFT negative, IF 1:40, negative bacterial culture (in both urine and blood), and borderline Brucella spp. PCR positivity. Indeed, our results reported a low overall seroprevalence for anti-Brucella canis antibodies and no positivity was confirmed on direct research of the pathogen. Nevertheless, there is a potential zoonotic risk to consider and communicate. An intensified collaboration with the veterinary medical professionals would be desirable to include this pathogen more frequently in the list of differential diagnoses, particularly in clinical pictures consistent with an acute or chronic infection caused by this microorganism (26, 28, 135).

Gastrointestinal parasites reported a high prevalence in both investigated species, especially in cats, according with recent literature (16, 101, 120, 136). Possible reasons for the lower occurrence of parasitosis found in dogs include increased control of confined dogs and more frequent use of anti-parasite treatment in refuge areas and kennels. Cats that have access to large outdoor areas are at a higher risk of being exposed frequently to environments contaminated with intestinal helminth eggs and consequently at a greater risk of re-infestation (120). The majority of parasite species identified in cats belong to the nematode family, are specific to certain species and pose limited zoonotic potential, mainly in ‘fragile’ categories or in children or immunocompromised people (1, 137–140). In addition, our study highlights that parasites in dogs and cats may require a specific prevention and control protocol, as these infestations can lead to acute, chronic and long-term health problems that affect shelter management from a medical, economic and managerial perspective (64, 120, 141). Notably, this study reported reassuring data about the prevalence of Echinococcus among the sheltered or free-roaming companion animals, which yielded no positive results in the investigated area. However, given the dangerous nature of the parasite (94, 142) and its prevalence among wildlife (11, 78), it may be useful to include this agent within a screening panel for zoonotic pathogens surveillance, or a specific and systematic prophylaxis. Additionally, it is recommended that training programme are provided for kennel and cat colony operators, as well as auxiliary staff in biosecurity, to reduce the risk of animal infestation (143). Differently to common perception, cases of dermatophytosis were rarely reported among our study population. The infected animals were either immunocompromised or living in overcrowded conditions. This result is particularly noteworthy, as previous studies have traditionally associated highly populated areas like shelters and catteries with a higher prevalence of dermatophytosis (144), whether it be through asymptomatic carriers or clinical signs, especially in cats (10, 17, 145). Low positivity to dermatophytes may be consistent with good and efficient management of the facilities in which the animals are housed. Moreover, we want to highlight that Italian legislation protects free-living feral cats and does not provide for close catteries except for health reasons and for a short time (Italian Law 281/1991; Agreement of 6 February 2003 between the Ministry of Health, the regions and the autonomous provinces of Trento and Bolzano ‘on the welfare of pets and pet-therapy’; D. Lgs n°134–05/08/2022). Therefore, the cat population lives in free colonies and crowding is limited.

The potential or emerging zoonotic viruses, such as Hepatitis E virus, Influenza A, Norovirus, and Cowpoxvirus, were also investigated. Although no positive results were recorded, it is important to note that the majority of the animals sampled in this study did not show any clinical symptoms. Therefore, it is recommended to include among the differential diagnoses these agents, when managing animals with medical history, clinical presentation and epidemiological data, suggestive of zoonotic diseases related to the aforementioned microorganisms.

A different scenario has opened up for two familiae of viruses, for which positivities have been observed, in particular in cats: Mammalian Orthoreovirus (MRV) and Rotavirus A.

MRV is an emerging zoonotic agent (146) that is responsible for causing gastrointestinal diseases in both humans and animals. Although some positive cases have been found in wildlife in areas adjacent to those covered in this study (147, 148), no data on the prevalence of this microorganism among dogs and cats in Italy have been recorded to date. This study reported positive samples in cats, where the viral RNA was detected primarily in fecal samples, although both OP swabs and feces yielded two MRV-positive cats. It is noteworthy that the positive cats were part of the same feline colony and were sampled in a single session, possibly indicating a cluster outbreak as previously reported in other animal species (147, 148). The Rotavirus genus is classified into eight serogroups denoted as A to H, with four of them (A, B, C and H) having the potential to cause diseases in humans (52, 86, 149). Significantly, serogroup A accounts for over 90% of cases associated with gastrointestinal illnesses in people (53, 150–152). According to previous studies (93, 152), our results reported that cats from the same feline colony exhibited positive results during the same sampling period. Unfortunately, the clinical features for both MRV and Rotavirus A are difficult to be documented in free-living cats.

During the SARS-CoV-2 pandemic, research has aimed to investigate the role of dogs and cats in sheltered housing (82). Among the positive animals, dogs exhibit the highest serological positivity, possibly due to their higher level of interaction with humans in sheltered housing environments. On the other hand, PCR positivity was recorded in two cats, confirming the higher receptivity to the infection of this species. Details of this investigation have been already published (82). Due to its emergence, zoonotic nature, and virus characteristics, it is crucial to adhere to the guidelines and preventive measures proposed by both international and national institutions,^2,3 eventually prompt screening for zoonotic diseases is imperative.

Of the emerging zoonotic agents, Capnocytophaga spp. (C. spp) is a microorganism characterized as commensal bacterium. Some serotypes, mainly belonging to the species C. canimorsus, can lead to infections in humans, particularly in immunocompromised patients (18, 20, 153, 154). Several species of Capnocytophaga spp. have been reported in humans (C. ochracea, C. sputigena, C. gingivalis), while others have been described in animals (C. cynodegmi, C. canimorsus, C. canis, C. felis) (21, 80). Taxonomic classification may rapidly evolve as this is a recently discovered microorganism and there could be unknown species. Infection is transmitted through contact with infected dogs and cat’s saliva or by being bitten (55). This study examines the presence of these emerging pathogens with zoonotic potential in the oral flora of companion animals through microbiological and molecular techniques. The two methods yield different outcomes, but neither of them can easily differentiate species and serotype: while the bacterial culture identifies the living organism, the rPCR detects the genetic material. Our findings confirm the presence of the microorganism in both dogs and cats; however, due to limitations in diagnostic methods and a lack of knowledge regarding the bacteria’s pathogenicity and biological behavior, an accurate diagnostic screening protocol cannot yet be defined. In front of outstanding C. spp. prevalence (82.5% in dogs and 64.8% in cats), the real risk to enter in contact with a dog or cat carrier of dangerous strains is probably extremely low. The results have enabled the authors to implement identification methods, but further in-depth studies are still underway. Considering the potential for the bacteria to spread to humans, particularly immunocompromised persons, it would be useful to implement collaboration with physicians, so it will be possible to gather extensively data and information on the bacterial strains that cause infections in humans.

Previous studies highlight the need of implementing communication and awareness programmes targeted toward the general public on the subject of zoonotic risk in the context of human-pet interaction (57, 58, 91, 141, 155). Recently, a study conducted in the Northeast of Italy, highlighted the ongoing need to enhance owners’ understanding of zoonoses affecting their pets and also the protective role of vaccines (156). This emphasizes the importance of adopting behaviors that promote the preservation of both the health and welfare of animals and humans alike. Furthermore, as previously reported (58, 63, 65, 157), there is a clear requirement to implement mechanisms that support the ongoing training of all companion animals shelter operators, thereby facilitating the dissemination and implementation of guidelines and best practices across the remit of each operator’s activities.

The collected data may help to prioritize zoonotic diseases, by implementing the periodical update, considering a shared algorithm that includes various parameters, such as the current and potential spread of the zoonotic agent, the severity of the infection in humans, the availability of therapeutic treatment, the probability of contracting it for operators and the public (59). The absence of specific and continuous training programmes for kennel operators or volunteers that cover topics such as infectious agents, biosecurity protocols, and basic ethological knowledge can lead to low or incorrect perception of zoonotic risk. In addition, the awareness about emergent or neglected zoonosis among the general population is usually scarce. A future perspective should be the implementation of sheltered companion animals’ health monitoring and ad hoc prophylactic programmes, as well as encouraging correct behavior from medical and veterinary doctors, hospital staff and citizens, as important outputs for recognizing possible emergent or neglected zoonoses. In conclusion, we want to stress the need of communication programmes in a One Health view about zoonoses and pets, and harmonized guidelines and operational protocols for better management and health approaches in dogs and cats in shelters and feline colonies. In addition, encouraging correct behavior both of operators within hospital facilities and of citizens in their daily lives, are also desirable outputs in the medium and long term to control the spread of these pathogens.

This study investigated the epidemiology of known, neglected, potential, emerging and re-emerging zoonotic agents. The findings yielded data on the prevalence of zoonotic diseases in the population of animals admitted to shelters, as well as integrated medical and organizational management aspects. Prevalence data confirms the incidence of several known zoonoses and sheds light on agents that are rare or only potentially zoonotic, such as MRV and Rotavirus A. This research emphasizes the necessity for more understanding on the dissemination of the emergent and neglected microorganisms and their potential for zoonotic transmission, which is presently unconfirmed, especially in contexts of human-animal interaction.