Occurrence and Anti-Fungal Sensitivity Testing of Candida Species Isolated from Canine Conjunctivitis


Olabode, H.O.K, Ameh, J.A, Egwu, G.O, Bello, R.H, Nwniyi, F.C

Olabode, H.O.K 1 

Ameh, J.A 1 Egwu, G.O 1 Bello, R.H 4
Nwniyi, F.C 5

  1. Department of Veterinary Microbiology, Faculty of Veterinary Medicine, University of Abuja 1

  2. Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, University of Ilorin 4

  3. Department of Veterinary Pharmacology, Faculty of Veterinary Medicine, University of Abuja 5

Pages: 8-14

DOI: 10.18488/journal.110/2016.2.2/110.2.8.14

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Article History:

Received: 03 June, 2016
Revised: 10 November, 2016
Accepted: 16 November, 2016
Published: 21 November, 2016


Abstract

Fungal infection in dogs andother domestic pets caused by Candida species have been previously reported. Several available antifungal drugs for treatment of yeast associated infections have been used with diverse effects. This study establishes antifungal susceptibility profile on identified Candida species isolates associated with canine conjunctivitis using disks diffusion method as described by NCCLS (M44-A). The antifungal disks used include Amphotericin-B (20mcg), Nystatin (100units), Itraconazole (10mcg), Ketaconazole (10mcg), Fluconazole (25mcg). The identified Candida species were C. albicans, C. krusei, C. tropicalis andC. parapsilosis. Percentage susceptibility was high in both Amphotericin B (77.27%) andNystatin (58.18%), while Itraconazole showed (54.54%), Ketoconazole (55.45%) andFluconazole was least (11.82%). The sensitivity ranking showed Amphothericin B > Nystatin > Itraconazole > Ketoconazole > Fluconazole andincreased resistance amongst the Azoles especially Fluconazole (70.90%) in comparison with Polyenes. C. albicans showed the highest resistance (84.51%) to both Fluconazole andketoconazole (67.61%). C. parapsilosis showed 63.64% and61.54% percentage resistance to Nystatin andItraconazole respectively. In conclusion, this finding showed that Amphotericin B andNystatin were most effective against isolated Candida species. Proper diagnosis of the underlying ailment is necessary in conjunction with routine anti-fungal susceptibility testing to avoid drug abuse andresistance.

Keywords: Anti-fungal sensitivity testing, Candida species, Canine conjunctivitis.

Received: 3 June 2016/ Revised: 10 November 2016/ Accepted: 16 November 2016/ Published: 21 November 2016

Contribution/ Originality

This study contributes to the existing literature of canine Candida species epidemiology andcontrol using conventional cultural, characterization andantifungal sensitivity test methodology. This first preliminary study conducted in Abuja-FCT, Nigeria revealed effectiveness of Amphotericin B andNystatin on Candida species isolates associated with canine conjunctivitis.


1. INTRODUCTION

Candidiasis is an infection caused by yeast of the fungal genus called Candida [1] characterized by whitish, chessy discharges accompanied with flakes from infected sites such as vagina, eye, ear, andmouth of man andanimals. Systemic candidiasis is however rare in animals with scanty literatures describing multi-sys­temic infection [2]. Candida species are ovoid budding yeast cells, 2 - 4 µm in diameter with thin walls occurring in chains. They produce pseudohyphae with attached blastospores andtrue regular filamentous hyphae post budding [3]. The cultural morphology of C. albicans is circular, round, smooth, glabrous to waxy surface with creamy yeast like appearance andcharacteristic microscopic gram positive chained cocci [4]. Other virulent species in dogs include C. tropicalis, and C. stellatoides, while C. parapsilosis, C. guillermondi, and C. krusei are less virulent [5]. Candida albicans is an opportunistic dimorphic, sugar digesting yeast [6] anda natural inhabitant of the genital, alimentary andupper respiratory tract mucous membranes in animals [3]. Superficial infections have been limited to mucous membranes of intestinal tracts in pigs, foals andChickens [7]. Identification andcharacterization of yeast species are based on morphological traits andphysiological capabilities [8]. The major biochemical characteristic is the ability to ferment sugars for the production of ethanol andhydrolysis of citrates andurea [4]. The increasing incidence of human candidisis have been associated with greater use of cytoxic, immunosuppressive andantibiotic therapies [3]. Amphotericin B, Nystatin, 5-Fluoro-cytosine, andFluconazoles have been used for candidiasis treatment [9]. However, Amphotericin B showed superior effect over 5-Fluoro-cytosine in combating candida infections [10]. Ketoconazole indicated promising antifungal effect, with little available clinical trial data on its use for systemic candidiasis [11]. In domestic pets Candida albicans has been reported as the etiological agent of multi-systemic infections in dogs [12]. The zoonotic nature of C. albicans especially in canine species [13] andtreatment failures due to drug resistance [14] necessitated the assessment of some commercially available anti-fungal drugs on Candida isolates associated with canine conjunctivitis in Abuja andits environs.

2. MATERIAL andMETHODS

2.1. Study Design

This study was conducted between November andDecember 2012 to report only isolates of Candida species predominately associated with suspected canine conjunctivitis cases presented to some veterinary clinics in Abuja andits environs. The isolates was subjected to in-vitro antifungal susceptibility testing to establish the best drug of choice for treatment in order to minimize cost andavoid indiscriminate drug use. All dogs during the period of study with complains of inflamed eyes were also considered andsampled.

2.2. Sample Collection

Ocular swabs were collected using sterile swab sticks (pre-moistened with sterile saline) from tentatively diagnosed dogs with canine conjunctivitis presented to the clinics. A total of 150 swab samples were collected, packed andimmediately taken to the laboratory for mycological investigation.

2.3. Isolation of Candida

Collected samples were inoculated on Sabouraud Dextrose Agar (SDA) andincubated at 37oC for 48 hours as described [15]; [16]. Plates were inoculated under aseptic conditions andincubated aerobically. They plates were later observed macroscopically at intervals for yeasty growth. Plates that yielded creamy, mucoid andmusty colonial appearance post 48 hr aerobic incubation were maintained on SDA slant at 4oC andused for further characterization.

2.4. Identification andCharacterization of Isolates

Pure Isolates on Sabouraud Dextrose Agar (SDA) were identify andcharacterized by sub-culturing on CHROMagar Candida (CHROMagar Candida®, France) plates andincubated at 37°C for 48hours. All isolates were identified based on their colonial morphology (Pigment formation) on CHROMagar Candida, germ tube pro­duction at 45°C, sugar fermentation reactions andgram staining to observe for the thick walled, round andbudding clamydospores [15].

2.5. Preparation of Yeast Inocula

Yeast inocula were prepared using the Mcfarlandturbidity standard as described [15]; [17]; [18]. Pure isolate of yeast were inoculated into Sabouraud Dextrose Broth (SDB) andincubated at 37oC for 5 hours until the turbidity was 0.5 McFarlandturbidity scale. This turbidity scale was prepared by adding 0.6ml of 1%w/v aqueous solution of barium chloride in 9.4ml of 1% v/v sulphuric acid giving an approximate equivalent bacterial density of 1-5 x 106 CFU/ml.

2.6. Yeast Susceptibility Test

Susceptibility testing was conducted using disk diffusion technique on Mueller Hilton Agar (MHA) plates as described [19]; [20]. On MHA, Methylene blue-glucose solution (assisted yeast growth andenhanced visualization of inhibition zones) was added to the surface of the agar andallowed to air dry prior to the addition of yeast inocula. Inoculation was carried out by dipping a sterile swab into the inoculum suspension adjusted to turbidity of 0.5 McFarland standards (106cells/ml) andstreaking was done across the agar surface in four directions. The plates were dried at ambient temperature for 15 minutes in a Laminar flow cabinet before applying the commercially impregnated anti-fungal disk drugs [Itraconazole (10mcg), Fluconazole (25mcg), Amphotericin B (20mcg), Ketoconazole (10mcg), Nystatin (100 U)]. The diameter zone of inhibitions was measured in millimeter 24hrs post-incubation.

The interpretation criteria for susceptibility andresistance of the anti-fungal used (Fluconazole, Nystatin, Amphotericine B, Ketoconazole, andItraconazole disks) were as indicated in the Table below [2, 7].

2.7. Interpretation Criteria for Susceptibility andResistance of An­tifungal Disks Used

Anti-fungal Agent
Zone of Inhibition (mm)
Sensitive
Dose- Dependent
Resistant
Amphotericin B
>15
10 -14
< 9
Ketoconazole
> 30
23 – 29
< 22
Fluconazole
> 19
15 -18
< 16
Nystatin
> 25
17 – 24
< 16
Itraconazole
>16
10 – 15
< 9

Source: Pakshir, et al. [21] and Pfaller, et al. [22]

2.8. Statistical Analysis

All the data obtained in this study were expressed in simple descriptive statistics (frequency andpercentages). The number of isolates was expressed as frequency, while the susceptibility andresistance patterns were presented in percentages.

3. RESULTS

Out of one hundred andfifty (150) swabs analyzed, 110 showed typical yeasty appearance while 40 samples showed no evidence of microbial culture andor yeast growth. Further identification andcharacterization of Candida isolates showed highest number (71) of Candida albicans isolates 64.55%, C. krusei 14.55% (16), C. tropicalis 9.09% (10) and C. parapsilosis 11.82% (13) as shown in Table I.

Antifungal susceptibility pattern indicated all isolates of Candida species 77.27% (85) were highly susceptible to Amphotericin B while Nystatin, Fluconazole, Ketoconazole, andItraconazole showed varied percentage susceptibility with different Candida species. This sensitivity ranking was represented as Amphotericin B (AMP) > Nystatin, (NSY) > Itraconazole (ICZ) > Ketoconazole (KCZ) > Fluconazole, (FCZ) as shown in Table II

The percentage antifungal drugs resistance indicated the occurrence of varied levels of resistance amongst all the Candida species especially against the Trizoles in a sensitivity ranking represented as Fluconazole, (FCZ) > Ketoconazole (KCZ) >Itraconazole (ICZ) as shown in Table III.

Table-1. Distributions of Candida species isolates from Canine ocular swabs in Abuja –FCT, Nigeria

Species
Number of Isolates
% of Isolates
Candida albicans
71
64.55
C. krusei
16
14.55
C. tropicalis
10
9.09
C. parapsilosis
13
11.82

N = 110

Prevalence Rate: 73.33%

Table-2. Percentage susceptibility profile of Candida species isolated from ocular swabs in Abuja (n=110) %

Antifungal Agents
C.albicans(n=71)
C. krusei (n=16)
C.tropicalis (n=10)
C.paraplosis (n=13)
Total
Amphotericin B
54
13
7
11
85
(76.06)
(81.25)
(70)
(84.62)
(77.27)
Nystatin
47
11
2
4
64
(66.20)
(68.75)
(20)
(36.36)
(58.18)
Fluconazole
11
7
5
9
32
(15.49)
(43.75)
(50)
(69.23)
(29.09)
Ketoconazole
23
9
8
10
50
(32.39)
(56.25)
(80)
(76.92)
(45.45)
Itraconazole
38
7
10
5
60
(53.52)
(43.75)
(100)
(38.46)
(54.54)

Table-3. Percentage resistance profile of Candida species isolated from ocular swabs in Abuja (n=110) %

Antifungal Agents
C.albicans(n=71)
C. krusei (n=16)
C.tropicalis (n=10)
C.paraplosis (n=13)
Total
Amphotericin B
17
3
3
2
25
(23.94)
(18.75)
(30)
(15.38)
(22.73)
Nystatin
24
5
8
9
46
(33.80)
(31.25)
(80)
(63.64)
(41.82)
Fluconazole
60
9
5
4
78
(84.51)
(56.27)
(50)
(30.77)
(70.90)
Ketoconazole
48
7
2
3
60
(67.61)
(43.75)
(20)
(23.08)
(54.55)
Itraconazole
33
9
0
8
50
(46.48)
(56.25)
(0.00)
(61.54)
(45.45)

4. DISCUSSION

In this study, Candida species associated with canine conjunctivitis were C. albicans, C. krusei, C. tropicalis and C. parapsilosis with overall prevalence of 73.3%. Candida albicans was most predominant while C. krusei and C. tropicalis were least isolated. The occurrence of Candida associated with canine conjunctivitis in this finding is similar to the mycotic endopthalmitis reported in dogs [23] caused by same species. All the Candida species isolated in this study, especially Candida albicans and Candida parapsilosis, conforms with previous Candida isolated in the ears, nose, oral cavity, andanus of clinically healthy dogs [3]. Candida tropicalis, andCandida krusei that were associated with immunocompromised dogs as well as Candida parapsilosis, reported in cats [24] were also identified in this study. This suggests that samples analyzed were either obtained from uncontrolled healthy dogs with immune disorders [25] or dogs exposed to chronic antibacterial therapy [26] andor dogs that co-exist with cats andhumans under poor management andenvironmental conditions.

The Antifungal sensitivity testing of selected Triazoles (Fluconazole, Ketoconazole andItraconazole) andPolyenes (Amphothericin B andNystatin) test against Candida species indicates Amphothericin B as the most effective antifungal agent with percentage susceptibility 77.27%, Nystatin (58.18%) andfluconazole (29.09%) resistance. This finding is similar to the reports of Nweze [27]; Apurva, et al. [28]. This high efficacy of Amphotericin B andNystatin on the isolates (C. albicans, Candida krusei, C. tropicalis andC. parapsilosis) following anti-fungal sensitivity testing is also in-line with other earlier reports [9] as previous candida treatment with fluconazole (FCZ) [10] terbinafine (TER) Favre, et al. [29] andflucytosine (FCY) [3] were ineffective andunsuccessful.

The highest resistance was observed in C. albican against fluconazole with a percentage resistance of 84.50% and67.61% against Ketoconazole. C. parapsilosis also showed percentage resistance of 63.64% and61.54% against Nystatin andItraconazole respectively. This finding is in line with Manfredi, et al. [14] that reported increasing resistance of C. albicans to antifungal medicines especially azoles. This study indicates Amphotericin B andNystatin high effectiveness in management of Candida albicans andother Candida species associated canine conjunctivitis. In addition, Itraconazole andKetoconazole could also be helpful as shown in this study. Although, treatment of fungal infection in veterinary medicine is being limited to conventional polyenes andtriazoles, these drugs have adverse effects andare relatively of high cost, especially when used in animals [30].

In conclusion, this study provides a preliminary report on the in-vitro antifungal susceptibility testing of Candida isolates associated with suspected cases of canine ocular conjunctivitis. Hence further specific Ocular Candida case-control study anddrug clinical trial is thus recommended. Definitive etiology of canine conjunctivitis alongside routine anti-bacterial susceptibility testing post cultural investigation is also suggested, to reduce drug misuse, cost anddevelopment of resistance.

5. ETHICAL APPROVAL

The research procedure was approved by the staff andseminar committee of the Faculty of Veterinary Medicine, University of Abuja, FCT, Nigeria. The samples were collected, stored andanalyzed using standard laboratory protocols as approved by the committee andrecommended by Clinical Laboratory standards US.

Funding: This study received no specific financial support.
Competing Interests: All authors indicated their consent prior to the commencement of this study. We hereby declare no conflict andcompeting interests.
Contributors/Acknowledgement: The authors appreciate the efforts of Mr. Hakeem Onigbanjo, Dr. Ifeanyi Casmir andMadam Grace Popoola for their assistance during the preliminary stages of this research as well as the staff of Microbiology Department, University of Ilorin for characterization andinterpretation of this work.

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