Pet Rabbits (Oryctolagus Cuniculus) and Guinea Pigs (Cavia Porcellus) are Vehicles of Pathogenic and Allergenic Fungi

Background: Nowadays, rabbits and guinea pigs are frequently adopted as companion animals, representing a vehicle for the dissemination of potentially pathogenic and allergenic fungi to their tutors. This study aimed to characterize the cutaneous mycobiota of these species and evaluate the association between mycological cultures results and several variables related to these animals’ husbandry. Hair and scales samples (n=102) were collected from 32 rabbits and 19 guinea pigs: 51 by pulling hairs surrounding lesions and collecting scales (if lesions present) or along the body of the animal (if absent); the other 51 samples were collected using Mackenzie’s technique. Samples were inoculated in Sabouraud Chloramphenicol Agar and Dermatophyte Test Media and observed daily during the incubation period. Isolated fungal species were identied based on their macro and microscopic morphology. A questionnaire was provided to the animal’s tutors to collect information on animal husbandry. Results: Most frequently isolated species corresponded to saprophytic fungi, such as Aspergillus spp., Penicillium spp., Scopulariopsis spp.; Candida sp. and Rhodotorula sp. were also found. Statistical analysis showed that a positive mycological culture was related with animal’s age and the administration of ongoing medication, while the number of isolated fungal species was related with animal’s species and outdoor access. Conclusions: These fungi have already been reported as responsible for mycotic infections in humans and animals, including dogs and cats, although they usually affect immunocompromised individuals. Therefore, these animals can represent a zoonotic risk, which may be related with animals age, species, ongoing medication and outdoor access.

Some studies have been carried regarding the characterization of the cutaneous mycobiota of rabbits (6,8,20,21) and guinea pigs (6,8,(20)(21)(22)(23)(24), but such studies are not available in Portugal, where there is no information on the mycobiota of these pets, its role on these animals health, and on the in uence of husbandry on fungal colonization. Such data would contribute to evaluate the role of these pets as potential carriers of fungal species with zoonotic potential. Therefore, the present study aimed to characterize the skin and hair mycobiota of pet rabbits and guinea pigs presented for consultation in the University Teaching Hospital of Faculty of Veterinary Medicine, University of Lisbon, Portugal, and to determine the association between animals related factors and their colonization by fungi.
Animals age was between one month and a half (0.2 years) and 10 years and 2 months (10.3 years), with an average of 3.70 years and a median of 3.10 years.
Animals were presented for consultation due to several reasons. Approximately half of the animals (n = 24; 47.1%) were presented for a preventive medicine consultation, with the remaining being presented for consultation due to problems in other organic systems. The most frequent consultations were of dermatology, neurology and dentistry, with the same number of cases (9.8% each). Despite that, dermatological signs were found in 14 animals presented for non-related consultations. In total, 19 animals (37.3%) showed diverse dermatological lesions at the moment of sampling. The most frequent were scaling (n = 7, 28.0%), alopecic lesions (n = 6, 24.0%), and pododermatitis (n = 5, 20.0%), followed by moist dermatitis (n = 2, 8.0%) and mites, pruritus, lipoma, otitis externa and thinned skin (n = 1 each, 4.0% each).

Isolated fungal species
Eleven fungal species were isolated from the collected samples. It was not possible to isolate any dermatophytes. Nine genera of saprophytic moulds were identi ed, representing 93.5% of the total number of fungal isolates obtained, and also 2 species of yeasts, representing 6.5% of the isolates obtained (Table 1). It was also possible to obtain 11 yeast isolates, identi ed as Candida sp. (n = 9, 5.4%) and Rhodotorula sp. (n = 2, 1.2%). The table represents the results obtained after regression analysis between the dependent variable positivity in mycological culture and the independent variables. Symbols presented in front of the p-value indicates that the variable is signi cant for the most usual levels of statistical signi cance: "." -0,1; "*" -0,05; "**" -0,01; "***" -0,001. Legend: The table represents the results obtained after regression analysis between the dependent variable positivity in mycological culture and the independent variables. Symbols presented in front of the p-value indicates that the variable is signi cant for the most usual levels of statistical signi cance: "." -0,1; "*" -0,05; "**" -0,01; "***" -0,001. Legend: The table represents the results of the regression analysis between the dependent variable number of isolated fungal species and the independent variables. Symbols presented in front of the pvalue indicates that the variable is signi cant for the most usual levels of statistical signi cance: "." -0,1; "*" -0,05; "**" -0,01; "***" -0,001. Legend: The table represents the results of the regression analysis between the dependent variable number of isolated fungal species and the independent variables. Symbols presented in front of the pvalue indicates that the variable is signi cant for the most usual levels of statistical signi cance: "." -0,1; "*" -0,05; "**" -0,01; "***" -0,001.

Statistical analysis
The number of isolated fungal species was signi cantly related with the animal species; in fact, since the regression coe cient for the rabbit category was found to be negative, rabbit samples are more likely to originate cultures with a lower number of fungal species than guinea pig samples (reference category).
Animal age was signi cantly related with a positive mycological culture; the coe cient for the age category was found to be positive indicating that older animals are more likely to produce a positive fungal culture.
Results from animals' which origin was a petshop were signi cantly related with a positive mycological culture. However, all these animals were adults at the time of sample collection, a long time period after their last contact with the petshop, which reduces the relevance of this relation for further discussion.
The number of isolated fungal species is related with the outdoor access. The coe cients of the categories backyard and public garden were found to be positive, suggesting a higher probability that samples obtained from animals with outdoor access, especially those who have access to a backyard, will originate cultures with a higher number of fungal species than those obtained from animals without outdoor access (reference category). A positive mycological culture was also related to outdoor access, speci cally the public garden. However, only one observation contributed to this result, and therefore this result was not considered for further discussion. The same was observed for the number of isolated fungal species and the contact with other animals (dogs).
Finally, a positive mycological culture was found to be related with animals ongoing medication. The coe cients of the categories anti-in ammatory, antinematodal drugs and antibiotic were found to be positive, indicating that animals taking these medications have a higher probability of originating a positive mycological culture than samples obtained from animals that were not taking any medication (reference category).

Discussion
Pet rabbits and guinea pigs can be potential vehicles for zoonotic or allergenic fungi to their tutors. Results revealed that the cutaneous mycobiota of the rabbits and guinea pigs under study was mainly composed by environmental lamentous saprophytic fungi and yeasts, as described in other studies performed in these animal species (6,7,21,22), as well as in others, such as dogs and cats (21,22,(25)(26)(27)(28)(29)(30)(31)(32). Saprophytic fungi were expected to be found in these animals because of their permanent contact with organic matter present in the hay, food and substrate and with the environment. The yeast species identi ed were also expected, as they are commensals of animals and humans microbiota (Candida) (33) or humid environments (Rhodotorula) (34). As far as we know, this study allowed the isolation of three fungal genera in rabbits and guinea pigs for the rst time, namely Chaetomium sp., Phoma sp. and Rhodotorula sp., which were already isolated from dogs and cats (25,26,29,30,35).
It was not possible to isolate dermatophytes from the animals under study; however, the other fungi identi ed were already reported as responsible for cutaneous lesions in humans, dogs and cats. In humans, Aspergillus sp., Alternaria sp., Candida sp., Chaetomium sp., Cladosporium sp., Mucor sp., Penicillium sp., Phoma sp., Rhizopus sp. and Scopulariopsis sp. have been reported to be responsible for cutaneous infections (36)(37)(38)(39)(40)(41)(42). In dogs and cats, Alternaria sp., Aspergillus sp., Candida sp., Cladosporium sp., Mucor sp. and Penicillium sp. have also been reported as responsible for cutaneous infections (36,(43)(44)(45)(46). Most of them were not previously associated with infections in pet rabbits and guinea pigs, except for Aspergillus sp., that was already related with cutaneous infections in rabbits (12). However, it is important to refer that Scopulariopsis sp. was isolated from one animal under study, which had alopecia and crusting lesions on the extremities. Several factors need to be taken into consideration before relating this species to infection, namely the concentration of isolated fungi, the clinical status of the animal and the possibility of contamination of the sample or culture (47). Although the hypothesis of colonization of a pre-existing wound cannot be ruled out, results seem to indicate that this fungus may have been responsible for this lesion, especially since its association with lesions very similar to dermatophytosis in humans has been described (41). In fact, identi ed species may have the ability to cause disease in rabbits and guinea pigs, not only in animals with immunosuppression or de cient nutritional status, but also in healthy individuals after abrasive or perforating injury (36). Therefore, veterinarians should consider non-dermatophytic fungal infections as a differential diagnosis for cutaneous lesions.
Many of the variables under study are frequently referred as predisposing factors for dermatophytosis in guinea pigs and rabbits (4). Dermatophytes were not isolated, but the association between positive mycological cultures and number of fungal species isolated with several independent variables was evaluated.
It was possible to observe that samples collected from guinea pig are likely to originate cultures with a higher number of fungal species than those from rabbits, which seems to indicate that the self-cleaning habits of guinea pigs are less e cient than the ones from rabbits; also, unlike rabbits, mutual grooming is less frequent between different guinea pigs (48). Self-cleaning allows the maintenance of animal hygiene by removing dirt and parasites (49). If not performed, the skin surface presents a higher probability of fungi colonization and dermatological disease development, which may also explain the higher tendency of guinea pigs for dermatophytosis (4,6,8,20,21,50). In fact, a study in cats with dermatophytosis reported that collared cats show more generalized lesions comparing with animals without Elizabethan collar that could perform their self-cleaning, suggesting that the cleaning behaviour may limit the development of lesions (51).
Age was found to be related with positive mycological cultures. Older animals may suffer from diseases that could impair cleaning behaviours, such as dental disease associated with oral pain, or musculoskeletal disorders that reduce mobility and cause pain (52,53).
Regarding outdoor access, it was already reported that fungal species present in cats' hair may vary depending on the environment (54). Considering that the organic matter present in gardens and backyards constitutes a relevant substrate for saprophytic fungi, it is expected that samples collected from animals with outdoor access to originate cultures with a higher number of fungal species.
Prolonged antimicrobial therapy can in uence the composition of the commensal microbiota of mucosa and facilitate yeast proliferation (33), being reported that the skin and hair microbiota may vary according to the immune state (54). In this study, samples from animals which were under drug therapy (antiin ammatory, antinematodal drugs and antibiotic) presented a higher probability of originating positive cultures. These animals were mainly diagnosed with advanced dental disease or presented neurological signs compatible with Encephalitozoon cuniculi. These two situations generally promote a decrease in body condition, poor nutritional status, and an altered degree of activity (51)(52)(53), which may explain this result.
Concluding, this is the rst study regarding cutaneous mycobiota of pet rabbits and guinea pigs performed in Portugal. Despite of not being possible to isolate dermatophytes, results showed that these animals can carry several lamentous fungi and yeasts in their hair and skin. Three of these genera were isolated for the rst time in these animals, namely Chaetomium, Phoma and Rhodotorula. All the isolated fungi are frequently present in the environment and usually do not cause disease. However, infections related with most of the identi ed fungi were already described in animals and humans, especially in immunocompromised individuals. Therefore, these animals can represent a zoonotic risk, which may be related with animals age, species, ongoing medication and outdoor access.

Conclusion
Currently, pet rabbits and guinea pigs are frequently adopted as companion animals, being a potential vehicle for the dissemination of pathogenic and allergenic fungi to their owners. A characterization of animal's skin mycobiota and its relationship with their medical records was performed. Results showed that these animals can carry several lamentous fungi and yeasts in their hair and skin. The most frequently isolated species corresponded to saprophytic fungi, such as Aspergillus spp., Penicillium spp., Scopulariopsis spp.; Candida sp. and Rhodotorula sp. In addition, the three fungal genera Chaetomium sp., Phoma sp. and Rhodotorula sp. were identi ed for the rst time in pet rabbits and guinea pigs.
Animal's age or the presence of ongoing medication were related to a positive mycological culture, while the number of isolated fungal species was related with the animal's species and outdoor access.
In conclusion, infections related with most of the identi ed fungi were already described in animals and humans, reinforcing the importance of the mycological assessment in these animals, as they may represent a potential zoonotic risk.

Materials And Methods
Inclusion criteria

Sample collection
In each animal, samples were collected by two methods. The rst method consisted of pulling hairs and collecting scales from the periphery of lesions, if present, with a sterile Halsted Mosquito haemostatic forceps; in animals with no lesions, samples were collected from the area's most frequently associated with dermatophytosis lesions in the rabbit and guinea pig, such as the head and extremities in both species and the thorax in guinea pigs (4,5). Samples were also collected through the Mackenzie's technique, which consists in passing a sterilized toothbrush through the entire coat of the animal thirty times or until it has hairs and visible skin residues (55). A total of 102 samples were collected, 51 by the pulling method and 51 by Mackenzie's technique.
Samples were collected by trained veterinarians following standard routine procedures with consent of the owner, and no ethics committee approval was needed.
During the incubation period, inoculated media were observed daily. Colonies obtained were identi ed at the genus level based on their macro and microscopic morphology (57).
A questionnaire was provided to the tutors with the goal of collecting detailed information about the history, husbandry and characterization of the animals.
The statistical analysis was performed in the R software (58). Fit models were used to evaluate the relation between dependent variables (positivity for dermatophytes, positivity in mycological culture and number of isolated fungal species) and independent variables (species, gender, age, origin, dental disease, contact with other animals, hygiene, outdoor access, food and ongoing medication), accessed through anamnesis and the questionnaire.
Two different regression models were used, since dependent variables with different structures were observed. First, for the variable's positivity for dermatophytes and positivity in the mycological culture, a logistic regression model was used, which considered the value 1 to represent the presence of dermatophytes or fungal species and the value 0 to represent their absence. Second, a linear regression model was used for the variable number of isolated fungal species.
For each independent variable, the regression coe cient, standard deviation and p-value were determined, after de ning a reference category, used for comparison. The variable species was divided into two categories, rabbit and guinea pig, with the reference category corresponding to guinea pig; the variable outdoor access was divided into ve categories, street, backyard, public garden, terrace and none, with the reference category corresponding to none; the variable ongoing medication refers to the medication the animal was taking at the time of sampling and was divided into eight categories, antiin ammatory, antinematodal drugs, antibiotic, antifungal, analgesic, vitamins, probiotics and none, with the reference category corresponding to none. involving the manipulation of animals were consented by the owners, and the necessary information about the study was provided to all the participants before obtaining their consent. ARRIVE guidelines were considered in this study.

Consent to publish:
Not applicable.
Availability of data and material: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests:
The authors declare that they have no competing interests. The funding sources were not involved in the conduct of the research and in the preparation of the article.

Authors' contributions:
RA performed the experiments, analysed the data and wrote the manuscript. AR helped to perform the experiments. SP and TM contributed to the analysis and interpretation of data. EC and ATR helped to analyse the data and to draft and revise the manuscript. MO conceived the study and participated in its coordination, helped to draft the manuscript and supervision throughout. All authors read and approved the nal manuscript.