Identificar espécies de Candida isoladas do trato respiratório superior de equinos.
Avaliar a sensibilidade antifúngica in vitro destas leveduras.
Investigar a presença de IgG anti-C. posadasii e IgG anti-H. capsulatum em amostras de soro obtidas de equinos do Estado do Ceará.
48 6. CAPÍTULO 1
Fungos do trato respiratório de equinos: destaque para o isolamento de espécies de Candida não-albicans
Equine respiratory fungi: an outline for the isolation of Candida non-albicans species
49 RESUMO
As infecções respiratórias são um problema comum em equinos e demonstram morbidade e mortalidade variáveis. Embora algumas espécies de fungos sejam consideradas os principais agentes de infecção respiratória em diversas mamíferos, a sua relevância nas doenças respiratórias de equinos é frequentemente negligenciada. Neste estudo, foi realizada uma busca ativa de Candida spp. na cavidade nasal de equinos, bem como uma pesquisa imunológica para coccidioidomicose e histoplasmose nesses animais. A presença de Candida spp. foi investigada por meio de swabs nasais e posterior semeadura em meios de cultura. Estas leveduras foram identificadas por testes fisiológicos e caracterizados quanto ao perfil de sensibilidade a antifúngicos in vitro. A pesquisa sorológica foi realizada por meio do teste de imunodifusão dupla para detecção de anticorpos IgG específicos. A análise do material da cavidade nasal de 97 equinos, escolhidos aleatoriamente, permitiu o isolamento de 56
Candida spp. em 35 animais (36,08%), sendo 18 (32,14%) C. famata, 14 (25%) C. parapsilosis, 12 (21,42%) C. guilliermondii, 11 (19,64%) C. tropicalis e 1 (1,78%) C. pelliculosa. Os valores de Concentração Inibitória Mínima (CIM) variaram de 0,03125 a
1µg/mL para anfotericina B; de 0,03125 a >16µg/mL e 0,125 a >64µg/mL para itraconazol e fluconazol. Resistência ao fluconazol e itraconazol foi observada em C. tropicalis (n=3) e C.
guilliermondii (n=1). Não foi detectada reatividade aos antígenos de Coccidioides spp. e Histoplasma capsulatum em 177 animais investigados. Os dados demonstram um predomínio
de espécies de Candida não-albicans na microbiota da cavidade nasal de equinos, inclusive com isolados resistentes a antifúngicos, e reforçam a importância do monitoramento de patógenos fúngicos nesses animais.
Palavras-chave: Cavalos, Candida spp., sensibilidade antifúngica, histoplasmose, coccidioidomicose
50 Medical Mycology – Original Paper
Equine respiratory fungi: an outline for the isolation of antifungal resistant Candida non-albicans strains
Rossana de Aguiar Cordeiro1, Paula Vago Bittencourt2, Raimunda Sâmia Nogueira Brilhante1, Carlos Eduardo Cordeiro Teixeira1, Débora de Souza Collares Maia Castelo- Branco1, Sabrina Tainah da Cruz Silva², André Jalles Monteiro3, José Júlio Costa Sidrim1, Marcos Fábio Gadelha Rocha1,2
1
Department of Pathology and Legal Medicine, School of Medicine, Specialized Medical Mycology Center, Postgraduate Program in Medical Microbiology, Federal University of Ceará, Fortaleza-CE, Brazil.
2
School of Veterinary, Postgraduate Program in Veterinary Science, State University of Ceará, Fortaleza-CE, Brazil.
3
Department of Statistics and Applied Mathematics, Federal University of Ceará, Fortaleza- CE, Brazil.
Corresponding Author. R. S. N. Brilhante. Rua Barão de Canindé, 210; Montese. CEP: 60.425-540. Fortaleza, CE, Brazil. Fax: 55 85 3295-1736 E-mail: brilhante@ufc.br
51 Abstract
Respiratory infections are a common problem among equines and occur with variable rates of morbidity and mortality. Although some fungal species are considered primary agents of respiratory tract infections in several mammals, their relevance in respiratory diseases of equines is frequently neglected. In the present study, we performed an active search for
Candida spp. in the nasal cavity of horses and also conducted an immunological survey for
coccidioidomycosis and histoplasmosis on these animals. The presence of Candida spp. was investigated through the use of nasal swabs that were streaked on culture media. These yeasts were identified through physiological testing and the in vitro antifungal susceptibility was also characterized. The serological survey was carried out through double immunodiffusion testing for the detection of specific IgG antibodies. The analysis of the material from the nasal cavity of 97 randomly chosen horses resulted in the isolation of Candida spp. in 35 animals (36.08%), out of which 18 (32.14%) were C. famata, 14 (25%) C. parapsilosis, 12 (21.42%)
C. guilliermondii, 11 (19.64%) C. tropicalis and 1 (1.78%) C. pelliculosa. The minimum
inhibitory concentration (MIC) values ranged from 0.03125 to 1µg/mL for amphotericin B; and from 0.03125 to >16µg/mL and 0.125 to >64 µg/mL for itraconazole and fluconazole, respectively. Resistance to fluconazole and itraconazole was observed in C. tropicalis (n = 3) and C. guilliermondii (n = 1). Reactivity to Histoplasma capsulatum and Coccidioides spp. antigens was not detected in the 177 investigated animals. The data show a predominance of non-albicans Candida species in the microbiota of the nasal cavity of equines including antifungal resistant isolates, reiterating the importance of monitoring fungal pathogens in these animals.
Keywords: Horse, Candida spp., antifungal susceptibility, histoplasmosis, coccidioidomycosis
52 Introduction
Yeasts of the Candida genus are considered commensal microorganisms associated with skin and mucosae of the urogenital, respiratory and gastrointestinal tracts of humans and various animal species [1,2,3,4,5]. Among the 25 species of yeasts that are considered emerging pathogens, 20 are Candida spp. [6]. Generally, these microorganisms do not cause any harm to their hosts, but they can become pathogenic when changes occur in physical, chemical and immunological barriers, culminating in an illness called candidiasis [2,5].
In horses, more specifically in foals, the Candida species are opportunistic pathogens that can cause gastroesophageal ulcerations, diarrhea, meningitis, omphalophlebitis and less often systemic infections [7,8]. In adult horses, C. famata has been associated with septic arthritis, while C. albicans has been isolated from mares suffering from keratitis and endometritis [9,10].
The dimorphic fungi Histoplasma capsulatum var. capsulatum and Coccidioides
posadasii are the causative agents of histoplasmosis and coccidioidomycosis, respectively,
both of which can affect several species of animals including horses. In Brazil, histoplasmosis is an endemic disease that occurs mainly in patients with AIDS and it is highly prevalent in the state of Ceará [11]. More recently, we described clinical and epidemiological aspects of three cases of feline histoplasmosis isolated in this state [12]. Despite the lack of reports of this disease in horses in Brazil, histoplasmosis has been diagnosed in these animals in the United States [13,14,15]. Concerning coccidioidomycosis, this disease is considered endemic to Northeastern Brazil, as evidenced by the occurrence of autochthonous cases in humans [16,17] and dogs [16]. Unlike Brazil, in the United States, coccidioidomycosis has been diagnosed in horses [18,19,20,21].
Because of the small number of studies investigating fungi from the equine respiratory tract, we performed an active search for Candida spp. in the nasal cavity of horses and also
53 conducted an immunological survey in these animals for coccidioidomycosis and histoplasmosis, which are endemic mycoses that primarily infect the lungs.
Materials and Methods Animals
One hundred and seventy-seven clinically healthy horses, from farms in the municipalities of Fortaleza (3° S-40° 41 ' 20 ' W), Eusébio (3 º 38° 53 ' S – 28 ' W), Caucaia (3° 43 ' S 38° 39 ' W), Jaguaribe (5 º 38° 53 ' S – 37 ' W), Sobral (3° 41 ' S – 40° 29 ' W) and Santa Quitéria (04° 19 ' S-40° 09 ' W), Ceará, Northeastern Brazil, were included in this survey, during the period from March to November 2011. These animals were used for the serological investigation and 97 of them were randomly chosen for the detection of Candida spp. Samples were collected from females and males (castrated and stallions), both juveniles (≤4 years) and adults (>5 years), of seven different breeds (Quarter horses, Arabian horses, Brazilian Jumper horses, Lusitano, Breton, British Thoroughbred and mixed breed horses). For each animal, a clinical-epidemiological form was completed, including information on the origin, age, sex, breed and clinical features. This study was approved by the animal research ethics committee of State University of Ceará (protocol number 10610172-2/55).
Sample collection, isolation and identification procedures
Microbiological samples were collected from the nasal cavity through the use of sterile cotton swabs. The first swab was inserted in the rostral region, approximately 10 cm from the nostril orifice, and the second in its caudal portion, approximately 30 cm from the entrance. After collection, the swabs were stored in glass slants with sterile saline supplemented with chloramphenicol (50 mg/L), stored and transported in isothermal trays to the Specialized Medical Mycology Center of Federal University of Ceará. Blood samples (5 mL) were
54 obtained through venipuncture of the jugular vein, with sterile Vacutainer® tubes, without anticoagulant. These were stored in isothermal trays and transported to the laboratory.
The swabs were inoculated in Petri dishes containing 2% Sabouraud dextrose agar (SGA, Difco Laboratories) with chloramphenicol and incubated for up to 5 days at room temperature (28 oC), with daily reading for evaluation of the presence of fungal colonies. Then colonies suggestive of Candida spp. were streaked on chromogenic medium (CHROMagar
CandidaTM), on which the colonies were incubated for 48 hours. The isolated colonies were inoculated in Petri dishes containing corn meal agar supplemented with Tween 80 and incubated for up to 5 days at 28 ºC, and later were observed with an optical microscope. Biochemical analyses with each isolate were also performed, such as carbohydrate assimilation tests, and the results were interpreted according to standards [22]. In inconclusive situations, the strains were identified through VITEK 2 automated system (BioMérieux Vitek, Hazelwood, France), according to the manufacturer’s instructions. Data concerning the collection site and the isolated species were analyzed through Pearson’s Chi-Square test, considering P< 0.05 as the smallest significance level.
Antifungal susceptibility testing
The minimum inhibitory concentration (MIC) was determined by the broth microdilution method, standardized by the Clinical and Laboratory Standards Institute [23], for all the isolates of Candida spp. An isolate of Candida parapsilosis ATCC 22019 was included as quality control. The fungal inoculum was prepared from 24-hour old colonies grown on potato dextrose agar at 28 °C for 24 hours.
For this purpose, 5 mL of saline solution (NaCl 0.9%) was added in sterile glass slants, after which a sample of the colony was added and the concentration was adjusted to 0.5 on the MacFarland scale. For the test, the suspensions were diluted at a ratio of 1:100 and then 1:20
55 with RPMI 1640 medium, buffered to pH 7 with MOPS (acid 2-[N-morfolin]- propanesulfonic) at 0.165 M, to obtain inocula varying from 1 to 2.5 103 CFU/mL [23].
The final concentration of antifungal drugs (amphotericin B, fluconazole and itraconazole) was obtained as recommended by CLSI [23]. All drugs were diluted and resuspended in RPMI 1640 medium. The range of concentration tested was from 0.125 to 64 µg/mL for fluconazole and from 0.03125 µg/mL to 16 µg/mL for amphotericin B and itraconazole [2,4].
The samples were analyzed in duplicate. The trays were incubated at 35 ºC for 48 hours. The MIC for the azole derivatives (fluconazole and itraconazole) was considered to be the lowest concentration able to inhibit 50% growth when compared to the growth control well. For amphotericin B, the MIC was considered to be the lowest concentration capable of inhibiting growth completely. MIC values of >1, > 64 and ≥ 1 µg/mL indicated in vitro resistance to amphotericin, fluconazole and itraconazole, respectively [23]. For the species C.
parapsilosis and C. tropicalis, MIC values of ≥ 8 µg/mL indicated resistance to fluconazole
[24].
Immunodiffusion test
The serum samples were obtained by centrifuging the blood samples at a speed of 1,500 rpm for 15 minutes at a temperature of 28 °C, after which they were stored at -20 °C. The double immunodiffusion technique in agarose gel was used to detect anti-C. posadasii IgG and anti-H. capsulatum IgG. The antigens Histoplasma ID (H&M) and Coccidioides IDCF were used according to the manufacturer's recommendations (Immy Immunodiagnostics, Inc., USA). Positive human sera for coccidioidomycosis and histoplasmosis (Immy Imunodiagnostics, USA) were used as controls for the immunodiffusion reactions. The results were initially read after 24 hours of incubation in a
56 moist chamber, and the final reading was made after 48 hours. The quality control of the test was assured by the presence of the precipitation line obtained between the antigen and the positive control serum.
Results
From the total of 97 horses assessed, 35 (36.08%) were positive for Candida spp. Out of these positive animals, 19 were females (18 adults and 1 juvenile), while 16 were males (14 adults and 2 juveniles). A total of 56 isolates of Candida spp. were recovered. The rostral nasal cavity was the anatomical site with the highest isolation rate, with 36 isolates (64.29%), belonging to five species (10 C. tropicalis, 10 C. famata, 9 C. guilliermondii and 7 C.
parapsilosis). From the caudal nasal cavity, 20 (35.71%) isolates were obtained, belonging to
the species C. famata (n = 8), C. parapsilosis (n = 7), C. guilliermondii (n = 3), C. tropicalis (n = 1) and C. pelliculosa (n=1) (Table 1).
As for the antifungal susceptibility assays, the MIC values for Candida isolates are detailed in Table 2. The MIC for amphotericin B ranged from 0.03125 to 1 µg/mL for all isolates. The MIC for itraconazole ranged from 0.03125 to >16 µg/mL and for fluconazole from 0.125 to >64 µg/mL. Three isolates of C. tropicalis were resistant to itraconazole and fluconazole (MIC ≥1 µg/mL and ≥8 µg/mL, respectively). Also, two isolates of C.
guilliermondii were resistant to fluconazole (MIC >64 µg/mL). There was no statistically significant difference between the analyzed anatomical sites, sex, age, breeds and cities.
Finally, all 177 serum samples were negative for anti-Histoplasma and anti-Coccidioides antibodies.
57 Discussion
Despite the clinical reports identifying Candida species as a cause of infection in animals, the number is still considered small when compared to human infections [25]. Because of this lower number of cases, there are still some questions about which species are part of the microbiota, how they become pathogenic and their prevalence.
This study suggests that the species found (C. parapsilosis, C. guilliermondii, C.
tropicalis, C. famata, C. pelliculosa) are commensal of the equine nasal cavity. Although the
literature reports C. albicans as an important component of the microbiota of animals [3,25], it was not isolated from any sampled horse. The most frequently isolated species in our study was C. famata (n = 18), similar to what was found by another author [8], who studied the microbiota of the oropharynx and rectum of healthy and debilitated foals. The second most isolated species was C. parapsilosis, which has gained importance in recent years as a pathogen of humans and animals [1,2,26].
The species C. pelliculosa is commonly isolated from fruit, vegetables, tree exsudate and soil, being considered an environmental yeast. However, in recent years it has been occasionally reported as a causative agent of fungemia in pediatric patients, mainly newborns in intensive care [27,28]. Despite the lack of case reports concerning the occurrence of clinical cases in animals, this species has been isolated from birds [29] and feces of bats [30]. In our study, a single sample of this species was recovered, but its phenotypical identification through manual methods was not conclusive. Therefore, the confirmation of its identity was performed via the VITEK 2 automated system.
The rostral portion of the nasal cavity showed a higher rate of isolation (64.29%), with the species C. tropicalis, C. famata and C. guilliermondii as the most prevalent ones. On the other hand, the recovery rate from the caudal portion of the nasal cavity was lower and C.
58 isolation rates of the two anatomical sites may be related to environmental factors. Furthermore, even though it is not the focus of this survey, it is important to highlight that other yeasts, e.g., Rhodotorula spp. and Trichosporon spp., were found. However, the isolation of these microorganisms was hampered by the excessive growth of filamentous fungi, which are commonly found in the nasal cavity, considering the anatomical, physiological and biological characteristics of equines.
In our study, three strains of C. tropicalis were resistant to fluconazole and itraconazole and two strains of C. guilliermondii were resistant to fluconazole. These findings are relevant since the animals were healthy and had no history of previous antifungal treatment. The phenomenon of in vitro antifungal resistance in Candida spp. isolated from animals has been frequently reported by our research group [2,4,12,31]. This phenomenon is possibly associated with the use of azole derivatives in agriculture [32].
In our survey, all animals were negative for the presence of anti-H. capsulatum and anti-
C. posadasii antibodies. Several serological techniques can be used to diagnose these
mycoses, such as complement fixation, latex agglutination, ELISA and agarose gel immunodiffusion (ID). ID is the most commonly used technique due to its quick and easy implementation, low cost, fast results and high specificity [33].
Histoplasmosis in the state of Ceará has high incidence in humans [11]. However, the epidemiological data regarding animal infections are scarce and the prevalence of the disease is unclear in this region [12,34]. There are no reports of histoplasmosis in horses in Brazil, in spite of having been described in other countries [13,14,15,35]. As for coccidioidomycosis, it is considered endemic in some Northeastern states, including Ceará [16,36], with reports in humans and animals [16,37]. In Brazil, there are no reports of this mycosis in horses, but cases of this disease in these animals have been reported in the USA [21]. Despite this evidence, found that only 4% of healthy horses, in endemic areas, had positive serology
59 according to the agarose gel immunodiffusion test and the authors observed that serological titers tended to decrease within 2 to 6 months [38]. Even though all animals presented negative serological results for histoplasmosis and coccidioidomycosis, this study is relevant because it was the first to investigate the role of horses in the epidemiology of these systemic mycoses in Brazil.
Conclusion
The data show a predominance of non-albicans Candida species in the nasal cavity of equines, including antifungal resistant strains, and that horses are not good sentinel animals for histoplasmosis and coccidioidomycosis in this geographical region, when tested through double immunodiffusion.
Acknowledgements
This work was supported by grants from the National Council for Scientific and Technological Development (CNPq; Brazil; Processes 302574/2009-3 and 562296/2010-7) and the Coordination Office for the Improvement of Higher Education Personnel (CAPES/PNPD 2103/2009).
Transparency Declaration
Nothing to declare.
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