Short Communication

Isolation and Identification of Fungi from Reproductive Tract of Healthy Bali Cows

Putu Henrywaesa Sudipa1*, Hapsari Mahatmi1, Ketut Tono Pasek Gelgel1, I Gusti Ketut Suarjana1, I Nengah Kerta Besung1, Romy Muhammad Dary Mufa2 , I Wayan Sukernayasa3

1Laboratory of Bacteriology and Mycology, Faculty of Veterinary Medicine, Udayana University, Bukit Jimbaran 80361- Indonesia; 2Laboratory of Veterinary Public Health, Faculty of Veterinary Medicine, Udayana University, Bukit Jimbaran 80361- Indonesia; 3Laboratory of Veterinary Reproduction, Faculty of Veterinary Medicine, Udayana University, Bukit Jimbaran 80361- Indonesia.

Received | May 23, 2023; Accepted | June 20, 2023; Published | November 01, 2023

*Correspondence | Putu Henrywaesa Sudipa, Laboratory of Bacteriology and Mycology, Faculty of Veterinary Medicine, Udayana University, Bukit Jimbaran 80361- Indonesia; Email: [email protected]

Citation | Sudipa PH, Mahatmi H, Gelgel KTP, Suarjana IGK, Besung INK, Mufa RMD, Sukernayasa IW (2023). Isolation and identification of fungi from reproductive tract of healthy bali cows . J. Anim. Health Prod. 11(4): 337-343.

DOI | http://dx.doi.org/10.17582/journal.jahp/2023/11.4.337.343

ISSN | 2308-2801

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Bali cattle are iconic animals from Bali Island and are the best choice for livestock farmers among several breeds. Bali cattle have unique characteristics such as red brick color on females and adult males with a blackish color of fur, white on the lower limbs, the back of the pelvis (buttocks), and good reproduction performance (Merdana et al., 2020). The reproductive health of Bali cattle is also critical for their preservation (Puja et al., 2018). Abortion, especially in cattle, is fundamental to study because it is one of the factors inhibiting the government’s plan to increase the cattle population to meet the demand for meat consumption (Gholib and Ahmad, 2013). Reproductive disorders can be caused by various microorganisms, including bacteria (Brucella spp., Vibrio spp., and Staphylococcus spp.), viruses, mycoplasma, and fungi (Hariadi et al., 2011). Bacterial and fungal microflora that inhibit the reproductive tract are among the causes of abortion. In Indonesia, of the 20.4% of cases of reproductive disorders in dairy cows, 2-5% are thought to be caused by bacterial infections, while those caused by fungal infections are not yet quantified (Hariadi et al., 2011).

According to Wienanto (2008), cases of mycotic abortion are mainly caused by Aspergillus fumigatus and Mucor species. The infection resulting in abortion begins with the entry of fungal spores into the animal’s body through the respiratory and digestive organs, which are then carried to the placenta through the bloodstream, causing inflammation in pregnant cattle and deteriorating fetal growth. From an ecological point of view, host microflora are not separate environments but are a network of interconnected communities that are continually exchanging (Neckovic et al., 2020). Therefore, microorganisms can enter the reproductive tract from other anatomical sites; for example, at birth, the physical cervical barrier is decreased, allowing microorganisms to be transported from the vagina, feces, animal skin, or from the environment via the vagina to the genital tract (Piersanti and Bromfield, 2019). Studies of the postpartum bovine uterine microflora have mainly focused on bacteria, but the presence of fungi has also been mentioned, although not discussed in detail (Bonnet et al., 1991). According to Ahmed and Bhattacharyya (2015), the prevalence of pathogenic fungi in dairy cows and buffaloes was reported at 17.98%, and the most common isolated fungi were Aspergillus fumigatus and Penicillium spp. in cattle and buffalo. Derakhshandeh et al. (2015) reported 8.7% (25–35 days postpartum) and 5.5% (39–49 days postpartum) prevalence of mycotic endometritis in Holstein cattle, and the most frequently isolated fungus was Aspergillus spp. (60.0%), Penicillium spp. (26.0%), and Yeast (13.0%).

There is a growing interest in the microflora of the reproductive tract, and its relationship with disease and health is a new and rapidly evolving field of study. Although the microbiome can have a significant influence on host biology, relatively little is known about microbial communities in the reproductive tracts of dairy and beef cattle (Appiah et al., 2020). Because of the large number of fungal infections that can cause reproductive disorders in cattle and the absence of initial data on the prevalence and identification of fungi in the reproductive tract, especially in female Bali cattle, it is necessary to identify the fungus and the prevalence of fungi inhabiting the reproductive tract of Bali cattle.

MATERIALS AND METHODS

Sample collection

Vaginal swabs were aseptically collected from female Bali cattle (n = 22) under normal health conditions. Sterile swabs (without transport media) were used for sample collection. Samples were collected in triplicates. The cows selected for sample collection were from various locations in Bali province, Indonesia. General data such as age, weight and sample location were recorded during sample collection.

Isolation and identification procedures

Sabouraud’s dextrose agar (Oxoid CM41) medium was prepared according to manufacturer instructions (Oxoid, UK). In brief, sixty-five grams of Sabouraud’s dextrose agar was mixed with 1000 ml of distilled water and put into an Erlenmeyer. The solution was homogenized using a digital magnetic stirrer. The Gentamicin antibiotic (40 mg/ml) was added to the medium to prevent the bacterial growth. The media was sterilized using an autoclave for 15 minutes at 121C° and then poured into the Petri dishes. Then the vaginal swabs were seeded to the media and plates were incubated at 25 C° for 3 days. After 3d, the yeast were sub-cultured to get pure culture for identification.

Fungi identification was conducted both macroscopically and microscopically. The macroscopic examination was carried out by observing the presence of fungal growth in a petri dish and confirmed directly with the naked eye. The microscopic examination was made by slide preparation to see under the microscope for the hypha, sporangia, oogonia and reproduction structures (Yudiarti et al., 2012). The tape smear method using Methylene Blue staining was used for microscopic identification.

Data Analysis

The data were tabulated and explained descriptively. The prevalence percentages of various fungal organisms was expressed in percentages.

RESULTS AND DISCUSSION

Thirty-two fungi belonging to seven genera were isolated from vaginal swabs of Bali cattle (Table 1). The isolated fungal organisms included Aspergillus, Fusarium, Cladosporium, and Curvularia spp., with prevalence rates of 41, 25, 19, and 6%, respectively. Candida, Mucor, and Penicillium spp. were also found, with prevalence rates of 3% each.

Table 1: The Prevalence of Fungi in the reproductive tract of healthy Bali cows.

Aspergillus spp. are considered environmental fungi that are transmitted by airborne conidia. Their colony could be observed as white, yellow, green, red, or black molds (Kaur, 2017). Identifying characteristics of Aspergillus spp. include the size and arrangement of conidial heads, size, and shape of vesicles, and phialide arrangement, as well as the color, size, and length of spores produced (Kaur, 2017). The isolated Aspergillus spp. formed a black-colored colony Fig. 1a) and macroconidia that were microscopically observed in long dry chains that may diverge or coalesce in compact columns (Fig. 1b) (Saleemi et al., 2017). Aspergillus species were found in abnormal cervical mucus discharge of Holstein-Friesian cows and heifers with a 4% incidence rate (Ata et al., 2010). In another study, Aspergillus fumigatus was the most commonly isolated fungi in repeat breeding cows and buffaloes, with 17.98% prevalence rate (Ahmed & Bhattacharyya, 2015). Nevertheless, in this current study used healthy cows, most likely, the fungi that were isolated are contaminants from the soil, animal excreta, and even in the vegetative parts of the plants or feed waste. The fungi may also enter the reproductive tract of animals most commonly from skin or feces (Stout, 2008).

The genus Fusarium consists of more than 80 species of fungi, which can infect plants, vertebrates, insects, humans, or even other fungi (Ivic et al., 2009). It is the most diverse group that can infect even food and feed grains. Fusarium species produce long, multicellular, canoe-shaped, or banana-shaped macroconidia. These large asexual conidia are the morphological characteristic of the genus. Many species also produce small, generally single-celled microconidia ranging from fusiform to oval to spherical shapes (Glenn, 2007). Colonies proliferate and may be pale (whitish to cream) or brightly coloured in yellow, brownish, pink, reddish, violet, or lilac shades (Abdel-Azeem et al., 2019). The isolated fungi exhibited pale white colonies (Fig.2a) with banana-shaped macroconidia (Fig.2b) as the definitive characteristic (Glenn, 2007; Abdel-Azeem et al., 2019). Furthermore, Fusarium is a typical soil-borne genus, widely distributed and generally abundant in all types of soils around the world (Backhouse et al., 2001). These vaginal swab samples showed Fusarium spp., most likely because the vagina has contact with the contaminated soil in the surrounding cowshed. High concentration of these airborne spores is usually associated with working environments and tasks, like uploading of grain or hay (Jestoi, 2008). This fungi is called as “storage fungi” because it grown in stored grains when adequately dried prior to storing, or stored in high humid environments. The taxa that are most importantly included in the storage pathogen category are those belonging to the genera of Aspergillus and Penicillium (Magan et al, 2010).

The genus Cladosporium is hygiene standard in many parts of the world; their spores can be found in air, soil, and water (Domsch et al., 1980). Some species are described as a cause of opportunistic phaeohyphomycosis, including subcutaneous and deep infections in humans and animals (Sandoval-Denis et al., 2015). Cladosporium morphology showed solitary to fasciculate conidiophores, proliferating, sympodial, and forming unbranched or branched acropetal conidial chains (Bensch et al., 2012). The colony characteristic is grey-green to deep green, flat or folded, velvety to dusty or granular, aerial mycelium scarce, sometimes showing cottony to floccose white to grey cushions, with a regular margin; reverse dark green to black (Sandoval-Denis et al., 2016). The current research showed that the fungi have a dark green colony (Fig.3a), and the microscopic findings represent branched and chain-type conidia (Fig.3b). These characteristics match Cladosporium fungi morphology in the previous research (Bensch et al., 2012). 

This fungus is a ubiquitous pigmented fungus that is usually recovered from vegetative matter and soil (Domsch et al., 1980). The color of the fully grown Curvularia colonies was dark brown to black on the obvious side. On the reverse side, the colonies appeared black. The texture of the colonies was hairy and, to some extent, woolly, and the colonies appeared to be slightly raised (Subapriya et al., 2015). Curvularia microscopically shows the characteristics of brown hyphae and brown conidiophores and produces branched septa. As the Curvularia fungus matures, one of the central cells in the conidia grows larger and darker than the lateral cells, which produces a characteristic curvature and gives it a croissant-like appearance (Paterson & Lima, 2015). Current research result is related to previous findings that showed that macroscopy colony of Curvularia have a characteristics of hairy and dark brown to black color (Fig.4a). The microscopic examination also matches the characteristic of general Curvularia spp. with branched septa and curvature croissant-like conidia (Fig. 4b). 

As a saprophyte, Candida can be found on the skin, genital tract, upper respiratory tract, and digestive tract, including the oral cavity (Zaremba et al., 2006). One of the species of this fungi, Candida albicans, are important opportunistic yeast identified as the cause of endometritis in water buffalo (Pal, 2002). Candida species colonies are white to cream in color, smooth, and shaped like yeast (Hamid et al., 2014), matching the result in this research, which shows the growth of white-cream colonies with yeast-like shapes. According to Yilmaz et al. (2012), the uterine culture showed 12.96% Candida spp. in healthy Anatolian Water Buffaloes. However, in this research, only 3% of Candida spp. are isolated. Candida are opportunistic fungi, occurring as normal inhabitants of the digestive tract, oral cavity, and vagina of humans and many of our domestic animal (Chengappa et al, 1984). The difference in prevalence rate in our and previous researches might be due to the difference in degree of colonization of these fungi in different animal species/breeds.

Mucor species

These fungi can be easily isolated from soil, dung, water, stored grains, and plants (Nguyen et al., 2016). These fungi also play the role of endophytic fungi; it has a round colony shape and flat colony margins, the surface of the colony is fine fibrous like thick cotton, the mycelium is white, the color of the colony on the front surface, and the color on the back surface is white. At the beginning of growth, the mycelium is white, and it turns black after the spores appear (Yuliana, 2016). Mucor spp. has a branched sporangiophore (sympodial or monopodial), and the columella is pear-shaped, round, or elliptical. Sporangiospores are elliptical to semi-spherical in shape and have a 5 – 10 µm diameter. This species can grow to sporulate at temperatures of 5 – 20°C but not at 37°C (Gandjar, 1999). In this research, the fungi that were isolated has a similar characteristic to that of Mucor spp. with white to the black colony and ellipse or round microscopic sporangiospores appearance (Figure not showed).

Penicilium species

The occurrence of this fungi in cervicovaginal fluids of Holstein dairy cows has been observed from the cervix and vagina at 27.14% and 28.57% respectively in infertile/repeat breeder cows (Garoussi et al., 2007). However, in this research, the samples were taken from healthy Bali cattle with no reproductive problems, but this finding should become attention to farmers. Specific characteristics of Penicillium are insulated or septate hyphae, branched mycelium, usually colorless, conidiophores insulated or septate and appear above the surface originating from hyphae below the surface of branched or unbranched hyphae, the head of the hyphae carrying spores is shaped like a broom, with sterigmata appear in clusters, conidia are chain-shaped because they arise one by one from the sterigmata and for their conidium, when young color is green, then turns bluish or brown (Fardiaz, 1992). Penicillium spp. on the SDA medium had septate hyphae. Conidia are spherical. Initially white, then changed to blue-green, greenish grey, and the opposite color is usually pale yellow. At the same time, the microscopic form of the fungus Penicillium has hyphae, round conidia, and a collection of phialides (Ristiari et al., 2018). In this research, the characteristic of the isolated Penicillium fungi was similar and has a green-colored colony with conidia forming in chains when observed in a microscope (Figure not shown).

Fungi contamination can be pathogenic that excrete some pathogen protein to infect the host and toxigenic fungi that produce toxin for their virulence, but not all molds are classified as that type. Pathogenic molds include, among others, Aspergillus (A. fumigatus, A. terreus, A, nidulans, A. niger., Mucor spp., Absidia spp., Trichoderma spp. and others). In contrast, toxigenic molds include A. flavus, Penicillium spp., Fusarium spp., and others (Ahmad & Gholib, 2017). However, in this research, the Bali cattle were healthy and have no reproductive system issues. Many of the isolated fungi are environmental fungi that can be easily found in soil, air, or feed close to the cow shed. Various predisposing factors help the fungus to cause infection in the reproductive tract, the exact conditions that make the fungus capable of colonizing the uterus or reproductive tract are still unknown (Stout, 2008). Although the primary reservoir of the fungal pathogens is the caudal reproductive tract, sometimes contamination from feces may also occur due to neovagina (Dascanio, 2000). It is presumed to cause ascending reproductive tract infection (Zafracas, 1975), and due to the opportunistic nature of the fungal pathogen, disease results when it accidentally penetrates host barriers and during immunologic deficiency or debilitating conditions which helps the fungus to enter and grow inside the host (Hogan et al., 1996). 

Feed and fodder are also significant sources of contamination. Mycotoxins are secreted from Aspergillus and Penicillium that grow on foods in humid weather (85%) and temperatures between 12°C and 25°C. Fungal spores are found in good-quality fodder material at 106/g but more in low-quality fodder (Laing et al., 1988). Such contaminated food material can act as a direct or indirect source of fungal pathogens to the bovines. With the findings of high Aspergillus spp. prevalence, the farmers and related parties should be concerned about this research result. However, the function of these opportunistic fungal pathogens, which play a role in the various processes, requires further investigation. The microflora in the cattle vaginal tract has also recently been revealed to play a significant role as biomarkers of reproductive success and failure (Deng et al., 2019). 

CONCLUSION

Morphological identification of fungi isolated from the Bali cattle reproductive tract showed that the genus Aspergillus was dominant, followed by Fusarium, Cladosporium, and Curvularia, while Candida, Mucor, and Penicillium species had the lowest prevalence. These findings add important information to the literature on the fungal microbiota in healthy Bali cows. In the future, more advanced techniques such as molecular methods for precise identification should be adopted.

ACKNOWLEDGEMENT

The authors thank the Udayana University Research and Community Services, Laboratory of Bacteriology and Mycology, Faculty of Veterinary Medicine, and fellow veterinarian colleagues for providing research facilities and support.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

NOVELTY STATEMENT

The morphological identification and types of Bali cattle reproductive tract fungi have yet to be researched. With many possibilities of fungal disease that can occur to the Bali cattle, this data offers basic information as stepping stone for the following research.

AUTHORS CONTRIBUTION

Putu Henrywaesa Sudipa and Romy Muhammad Dary Mufa conducted the research process in the laboratory. Hapsari Mahatmi, Ketut Tono Pasek Gelgel, I Gusti Ketut Suarjana, and I Nengah Kerta Besung designed the research and provided media for the research. I Wayan Sukernayasa, helped in the data collection process.

REFERENCES

Abdel-Azeem A. M., Abdel-Azeem M. A., Darwish A. G., Nafady N. A., Ibrahim N. A. (2019). Fusarium: Biodiversity, Ecological Significances, and Industrial Applications. In A. N. Yadav, S. Mishra, S. Singh, & A. Gupta (Eds.), Recent Advancement in White Biotechnology Through Fungi (pp. 201–261). Springer International Publishing. https://doi.org/10.1007/978-3-030-10480-1_6

Ahmad R. Z., Gholib D. (2017). Cemaran Kapang pada Pakan Sapi dan Uji In Vitro Sirih terhadap Pertumbuhan Kapang Aspergillus flavus (mold contamination in cattle feed and in vitro assay of piper betel againts growth of mold contaminant aspergillus flavus). J. Vet., 18(3): 453. https://doi.org/10.19087/jveteriner.2017.18.3.453

Ahmed K., Bhattacharyya D.K. (2015). Isolation of pathogenic fungi associated with repeat breeder bovine. Intas. Polivet. 16(2), 459.

Ata A., Turutoglu H., Kale M., Gulay M.S., Pehlivanoglu F. (2010). Microbial flora of normal and abnormal cervical mucous discharge associated with reproductive performance of cows and heifers in Estrus. Asian Aust. J. Anim. Sci. 23(8), 1007– 1012.

Appiah M. O., Wang J., Lu W. (2020). Microflora in the reproductive tract of cattle: A review (running title: The microflora and bovine reproductive tract). Agriculture (Switzerland), 10(6): 1–27. https://doi.org/10.3390/agriculture10060232

Backhouse D., Burgess LW, Summerell B. (2001). Biogeography of Fusarium.

Bensch K, Braun U, Groenewald JZ, et al. (2012). The genus Cladosporium. Studies in Mycology 72: 1– 401.

Bonnett BN, Martin SW, Gannon VP, Miller RB, Etherington WG. (1991). Endometrial biopsy in Holstein-Friesian dairy cows. III. Bacteriological analysis and correlations with histological findings. Can. J. Vet. Res. 55:168–73.

Chengappa MM, Maddux RL, Greer SC, Pincus DH, Geist LL. (1984). Isolation and identification of yeasts and yeastlike organisms from clinical veterinary sources. J. Clin. Microbiol. 19:427-428.

Dascanio J. (2000). How to diagnose and treat fungal endometritis. In the Proceedings of the Annual Convention of AAEP (American Association of Equine Practitioners 2000), San Antonio, TX, vol. 46, pp: 316–318.

Deng F., McClure M., Rorie R., Wang X., Chai J., Wei X., Lai S., Zhao J. (2019). The vaginal and fecal microbiomes are related to pregnancy status in beef heifers. J. Anim. Sci. Biotechnol. 2019, 10: 1–13.

Derakhshandeh, A., Aghamiri, S.M., Ahmadi, M.R. and Mirzaei, A. (2015). Prevalence of opportunistic fungi and their possible role in postpartum endometritis in dairy cows. Vet. Sci. Dev. 5, 5977.

Domsch KH, Games W, Anderson TH. (1980). Compendium of soil fungi. Academic Press. London, 201-208.

Fardiaz DS (1992). Mikrobiologi Pangan 1. Gramedia Pustaka Utama, Kompas Gramedia, Indonesia.

Gandjar I.,R.A. Samson., K. Van den Tweel-Vermeulen., A. Oetari dan I. Santoso. (1999). Pengenalan Kapang Tropik Umum. Jakarta: Yayasan Obor Indonesia.

Garoussi M.T, Khosrave A.R, Havareshti P. (2007). Mycoflora of cervicovaginal fluids in dairy cows with or without reproductive disorders. Mycopathologia. 164:97–100.

Gholib, Djaenuddin., Ahmad, Riza Zainuddin. (2013). Cendawan Penyebab Abortus Dalam Alat Reproduksi Sapi Betina. Berita Biologi 12(2).

Glenn AE. (2007). Mycotoxigenic Fusarium species in animal feed, Animal Feed Science and Technology, Volume 137, Issues 3–4, 2007, Pages 213-240, ISSN 0377-8401, https://doi.org/10.1016/j.anifeedsci.2007.06.003.

Hariadi M, Hardjopranjoto S, Wurlina W, Hermadi HA, Utomo B, Rimayanti R, Triana IN, Ratnani H. (2011). Ilmu Kemajiran Pada Ternak. Cetakan 1. Airlangga University Press. Surabaya.

Hamid ME, Assiry MM, Joseph MR, Haimour WO, Abdelrahim IM, Al-Abed F, Fadul AN, Al-Hakami AM (2014). Candida and other yeasts of clinical importance in Aseer region, southern Saudi Arabia. Presentation of isolates from the routine laboratory setting. Saudi Med. J. 35: 1210–1214.

Hogan L.H., Klein B.S., Levitz S.M. (1996). Virulence factors of medically important fungi. Clin. Microbiol. Rev. 9, 469–488.

Ivic D, Domijan A, Peraica, M. (2009). Fusarium spp. contamination of wheat, maize, soybean, and pea in Croatia. ArhHigRadaToksikol 60: 435-442.

Jestoi M. (2008). Emerging Fusarium-mycotoxins fusaproliferin, beauvericin, enniatins, and moniliformin, a review. Crit. Rev. Food Sci. Nutrit., 48 (1).

Kaur O. (2017). Aspergillus and Cervicovaginal Papanicolaou Smear: A Review. Int. Clin. Pathol. J., 4(1). https://doi.org/10.15406/icpjl.2017.04.00086

Laing J.A, Morgan W.J.B, Wagner W.C. (1988). Fertility, and infertility in veterinary practice. 4th. UK: Baillier Tindal; pp. 228–232.

Magan N., Aldred D., Mylona K., Lambert R. J. (2010). Limiting mycotoxins in stored wheat. Food Addit. Contamin. 27(5): 644.

Merdana IM, Sulabda IN, Tiasnitha NMWA, Gunawan IWNF, Sudira IW. (2020). Erythrocyte, hemoglobin and hematocrit profile of Bali cattle during the various periods of parturition. J. Anim. Health Prod. 8(2): 75-79. http://dx.doi.org/10.17582/journal.jahp/2020/8.2.75.79

Neckovic A., van Oorschot R. A., Szkuta B., Durdle A. (2020). Investigation of direct and indirect transfer of microbiomes between individuals. Foren. Sci. Int. Genet., 45: 102212.

Nguyen TT, Lee SH, Bae S, Jeon SJ, Mun HY, Lee HB. (2016). Characterization of two new records of zygomycete species belonging to undiscovered taxa in Korea. Mycobiology.; 44:29-37

Pal M. (2002). Endometritis in a water buffalo due to Candida albicans. Buffalo Bull ;21(1):10–11.

Paterson RRM, Lima N, (2015). Molecular biology of food and water borne mycotoxigenic and mycotic fungi. CRC Press. New York.

Piersanti R.L., Bromfield J.J. (2019). The Consequence of Postpartum Uterine Disease on Dairy Cow Fertility. EDIS 2019, 1–4. 

Puja IK, Sulabda IN, Wandia IN. (2018). Microsatellite polymorphisms and its relationship with calving interval and gestation period in bali cattle. Adv. Anim. Vet. Sci. 6(5): 197-200. DOI | http://dx.doi.org/10.17582/journal.aavs/2018/6.5.197.200

Ristiari NPN, Ketut SMJ, Ida APS. (2018). Isolaai dan Identifikasi Jamur Mikroskopis pada Rizosfer Tanaman Jeruk Siam (Citrus nobilis Lour.) Di Kecamatan Kintamani, Bali. J. Pendidikan Biol. Undiksha. 6: 12-17. http://dx.doi.org/10.23887/jjpb.v6i1.21921.g13564

Saleemi MK, Khan MZ, Khan A, Hameed MR, Khatoon A, Abidin Z, Hassan ZU (2017). Study of fungi and their toxigenic potential isolated from wheat and wheat-bran. Toxin Rev.36: 80-88. http://dx.doi.org/10.1080/15569543.2016.1233890

Sandoval-Denis M, Sutton DA, Martin-Vicente A, et al. (2015). Cladosporium species recovered from clinical samples in the United States. J. Clin. Microbiol. 53: 2990– 3000.

Sandoval-Denis M., Gené J., Sutton D. A., Wiederhold, N. P., Cano-Lira J. F., Guarro J. (2016). New species of Cladosporium associated with human and animal infections. Persoonia - Molecul. Phyl. Evol. Fungi., 36(1): 281–298. https://doi.org/10.3767/003158516X691951

Subapriya S, Nagarajan B, Kavitha S, Senthil NR, Padmanath K, Vairamuthu S (2015). Emerging Incidence of Fungal Dermatitis in Canines Caused by Curvularia spp: An Opportunistic Fungal Pathogen. Int. J. Adv. Res. Biolog. Sci. 2: 264-267.

Stout T. (2008). Fungal endometritis in the mare. Pferdeheilkunde. 24. 83-87. https://doi.org/10.21836/PEM20080117.

Wienanto R. (2008). Hubungan antara Infeksi Brucella abortus dengan somatic cell count pada Sapi Perah di Daerah Pusat Penghasil Susu Jawa Timur (Skripsi). Universitas Airlangga. http://id.wikipedia.org/wiki/Abortus_pada _sapi (diakses 22-04-2010).

Yilmaz O, Kuyucuoglu Y, Sevimli A, Yazici E, Ucar M. (2012). Uterine microbiology and histopathology in repeat breeder anatolian water buffaloes. Kafkas Univ. Vet. Fak. Derg., 18(5):791–798.

Yudiarti T., Yunianto V. D., Murwani R., Kusdiyantini E. (2012). Isolation of Fungi from the Gastrointestinal Tract of Indigenous Chicken. J.Indonesian Trop.Anim.Agric. 37(2)

Yuliana, Anik Karimatu. (2016). Potensi Fungi Endofit pada Kulit Buah Naga Super Merah (Hylocereus costaricensis) sebagai Penghasil Senyawa Antioksidan. Skripsi. Jurusan Bilologi Fakultas Sains dan Teknologi Universitas Islam Negeri Maulana Malik Ibrahim Malang.

Zafracas A.M. (1975). Candida infection of the genital tract in Thoroughbred Mares. J. Reprod. Fertil. 23, 349–351.

Zaremba ML, Daniluk T, Rozkiewicz D, Cylwik-Rokicka D, Kierklo A, Tokajuk G. (2006). Incidence rate of Candida spesies in oral cavity of middle-aged and elderly subjects.Adv. Med. Sci.; 1 (51): 233-6.