Investigation of the Vaginal and Clitoral Fungi in One-Humped Camels (Camelus Dromedarius)

Introduction
In the Middle East and other regions, the one-humped camel (Camelus dromedarius) is a local source of milk, meat, and wool. Camel racing is a multi-dollar industry in some regions that emphasizes the overall high value of camel breeding (Noakes et al., 2019). As for other production and sport animals, such as cattle and horses, successful reproduction of camels plays a crucial role in maintaining continuous and good production and subsequent income for breeders.
A normal structure and a functional genital tract is a prerequisite for normal fertility in female camels. Genital tract infection during breeding reduces fertility due to lack of fertilization or embryonic death (Noakes et al., 2019; Gideon et al., 2014; Enany et al., 1990). Investigating fungal contamination in the reproductive systems of animals has received less attention. However, most studies have been conducted in genital systems of animals, such as dairy cows (Dabiri et al., 2020; Talebkhan Garoussi et al., 2008; Talebkhan Garoussi, 2007), buffaloes (Singh et al., 1992), horses (Azarvandi et al., 2017a; Azarvandi 2017b; Różański ski et al., 2013a; Różański et al., 2013b), cats (Garoussi et al., 2016), and dogs (Cleff et al., 2005. Khosravi et al., 2008). Fungal agents can affect the placenta, fetus, or both and cause abortion in domestic animals (Antoniassi et al., 2013; Fourichon et al., 2000). However, some investigations have been conducted in bulk milk tanks on dairy farms (Hosseinabadi et al., 2022). Fungal agents can invade the genital system and cause reproductive disorders under abnormal conditions (Noakes et al., 2019; Rebhun et al., 1995). However, identification of abnormal microflora requires knowledge of the normal flora. Several bacteriological studies have been conducted on the reproductive system of camels (Ali et al., 2010; Pal, 2015) but the studies conducted on fungal infections of one humped camel reproduction are few (Shokri et al., 2010) although fungi constitute a crucial part of the vaginal microenvironment in animals (Azarvandi et al., 2017a; Azarvandi et al., 2017b; Garoussi et al., 2016; Hopper, 2015; Talebkhan Garoussi et al., 2008; Talebkhan Garoussi et al., 2007; Youngquist & Threlfall, 2006; Heuwieser et al., 2000).
To obtain a basic understanding of the mucosal fungi of the caudal genitals of female one-humped camels, we cultured paired swabs of the vaginal and clitoral mucosa of 110 animals to determine the fungi and study whether the fungi depended on the age of the animal.

Materials and Methods

Study population
From March 2018 to August 2019, dairy camel farms were sampled in Khorasan Razavi, Semnan, Tehran, Qom, and Isfahan Provinces located in central and northeast Iran (Figure 1). These provinces represent major camel-rearing regions in Iran. The study population included 550 clinically healthy female one-humped camels of which 20% were included randomly using a lottery approach, according to Thrusfield and Christley (2018). The age of the sampled camels was determined based on dental characteristics (Hillson, 2009).
The camels were kept in semi-intensive dairy systems with access to pastures. A typical diet consisted of alfalfa hay, corn silage, and concentrates in various proportions using a mixed ration. Average milk production was 5 kg/day with dual milking. Approximately 45% of camels were non-lactating at the time of sampling. However, pregnant camels within the herds were diagnosed using ultrasonography, transrectal palpation, and persistently raised tail (called tashweel) (Noaks et al., 2009; Enany et al., 1990; Merkt et al., 1990; Chen & Yuen, 1984). Vaccination against foot and mouth diseases, Clostridium chauvoei, and Clostridium Septicum, were performed regularly. All herds used natural breeding.

Sampling and fungal culture
The external genitalia was thoroughly washed with a 5% povidone-iodine solution, and the vulva opening was cleaned with a sterilized tampon before sample collection. The vaginal walls were manually separated to allow the deep insertion of a simple sterile cotton swab without contamination. The cranial part of the vaginal mucosa was gently swabbed before removal of the cotton swab. Then a second swab was taken from the clitoral mucosa using the same technique. The swabs were transferred to a sterilized Stuart transport medium (Difco Laboratories, Detroit, MI, USA) and transported on ice to the Mycology Research Centre, Tehran.
Swabs were spread onto Sabouraud dextrose agar with chloramphenicol (Merck Co., Darmstadt, Germany) and incubated at 32 °C for 48 h under aerobic conditions and on CHROMagarTM Candida (CHROMagar, Paris, France) and incubated at 35 °C for 48 h in the dark. Sugar fermentation and assimilation tests were performed using the RapIDTM Yeast Plus System (Thermo Fisher Scientific, Lenexa, KS, USA) according to the manufacturer’s instructions for the presumptive identification of isolated yeasts. Final identification was performed using the germ tube formation test, urease test, and culturing on CHROM agar and corn meal agar medium with Tween 80.

Statistical analysis
The data were analyzed using the Genmod procedure, including the function link logit in the model. Differences at P<0.05 were considered statistically significant.

Results
Culturing revealed fungal growth in 31% of the examined camels. In 11% of camels, fungi were found on both the vaginal and clitoral mucosa, while fungi were only isolated from the clitoral or vaginal mucosa in 11% and 9% of camels, respectively (Table 1). 
Several fungal species were found, and in many cases, they were present as monoculture (Table 2). The fungi of the vaginal and clitoral mucosa seemed to be similar but varied between animals. No single species was consistently isolated from all or most camels. No correlation was found when analyzing the culture results (culture positive vs. negative) against the animals’ age (Table 3). 

Discussion 
A range of different fungal species was isolated from the vaginal and clitoral mucosa of the examined one-humped camels. All species were isolated from both locations, indicating that these fungi can normally inhabit the mucosa of the posterior genital tract. As all camels included in the study were healthy in the genital tract, the isolated fungi probably represent a part of the normal vaginal fungi in camels. However, some fungi are opportunistic pathogens, such as Aspergillus spp. and Candida spp., and may be involved in the development or progression of an infection. In animals, Aspergillus can cause abortion (Constable et al., 2017). Aspergillus spp. and Mucor spp. have been isolated from the uterus of one-humped camels with endometritis (Refaat et al., 2020; Tibary et al., 2006). As endometritis can originate from ascending transcervical migration of pathogens, therefore, normal vaginal mucosal fungi can cause post-partum uterine fungal infection. Usually, fungi are not vital in the endometritis of camels or other animals, but they sometimes become crucial (Karstrup et al., 2017). Some of the Aspergillus spp. that were isolated in this study, such as Aspergillus niger and Aspergillus flavus are well-known causes of aspergillosis (Sugui et al., 2015), including mycotic placentitis in cattle and horses (Hopper, 2015; Knudtson & Kirkbride, 1992; Murase et al., 2015; Orellana-Guerrero et al., 2019; Youngquist & Threlfall, 2006). Therefore, vaginal and clitoral fungi may serve as sources of genital tract infections in certain circumstances.
Yeast commonly inhabits the mucosa of the caudal genital tract of camels with the highest load in the vestibulum, and the numbers decrease towards the uterine horns (Shokri et al., 2010). Candida spp. are particular prevalent, with Candida zeylanoides being the most common in the vagina (Shokri et al., 2010; Sobel, 1988). In the present study, Candida spp. was cultured from the vaginal mucosa in 3% of the camels and from the clitoris in 15% of camels and, therefore, not as prevalent as expected from the study by Shokri et al. (2010). The presence of filamentous fungi has not been previously reported, but they occur in both the vaginal and clitoral mucosa at an equal prevalence (Table 2).
In the present study, the fungal isolates showed progressive involvement of different structures of external organs in the female camel reproduction system, thus confirming the locally invasive nature of different isolates consistent with previous reports that infection did not disseminate (Moradi et al., 2024. Garoussi et al., 2016). Therefore, this fungal group may demonstrate tropism towards this anatomical site in camels of different ages (Table 3). A. niger, A. flavus. (No. 3, 9%) and Cladosporium spp. A. flavus (No. 3, 9%), the most frequent fungal species, located in cervical and vaginal tissues, respectively (Table 2). Therefore, they may spread through the male genital system in camels. Candida can localize to mucous membranes and skin. It is distributed worldwide in different animals and is most commonly caused by species of yeast-like fungus, Candida albicans, Candida Krusei, and others (Garoussi et al., 2016).

This survey showed that the amount of fungal contamination of external organs of the reproductive system in female camels is much lower than in mares (Azarvandi et al., 2017a, Azarvandi et al., 2017b). This may be due to the camel’s innate and general immune systems’ resistance to fungal infection.
Despite the importance of camels, scientific knowledge of their reproduction and reproductive pathology is limited. Establishing basic knowledge is therefore crucial, but efforts should also be made to improve reproductive efficiency and to increase our knowledge of infections causing reproductive failure, such as embryonic losses, abortion, and subfertility, to prevent such conditions.

Ethical Considerations

Compliance with ethical guidelines
The Animal Experimentation Ethics Committee of the Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, approved all protocols and procedures under the recommendations for proper care and use of laboratory animals (Code: 28903. 1.2).

Funding
This study was financially supported by the Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran (Grant No.: 28903.1.2).

Authors' contributions
Conceptualization, visualization, and funding acquisition: Massoud Talebkhan Garoussi: Methodology, and software: Hosein Kochakzadeh Omran; Investigation: Mehran Dabiri, and Massoud Talebkhan Garoussi; Validation, and formal analysis: Jørgen Steen Agerholm; Writing the original draft: Hosein Kochakzadeh Omran and Massoud Talebkhan Garoussi; Supervision, review and editing: Massoud Talebkhan Garoussi, and Ali Reza Khosravi.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors would like to appreciate the vice-chancellor for Research & Technology, and Education & Graduate Studies of the Faculty of Veterinary Medicine, University of Tehran (FVM-UT, Tehran, Iran). The authors thank Deldar Talebkhan Garoussi for English language editing of the manuscript.

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