ISOLATION, ANATOMICAL DISTRIBUTION AND SUSCEPTIBILITY OF AFFECTING HORSES IN KWARA STATE, NIGERIA

By

RASHIDAT BOLANLE BALOGUN

DEPARTMENT OF VETERINARY MICROBIOLOGY FACULTY OF VETERINARY MEDICINE AHMADU BELLO UNIVERSITY, ZARIA NIGERIA

JUNE, 2015

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ISOLATION, ANATOMICAL DISTRIBUTION AND ANTIFUNGAL SUSCEPTIBILITY OF DERMATOPHYTES AFFECTING HORSES IN KWARA STATE, NIGERIA

By

RashidatBolanle BALOGUN, DVM 2010 (ABU) MSc/Vet Med/5162/2011-2012

A THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE IN VETERINARY MICROBIOLOGY

DEPARTMENT OF VETERINARY MICROBIOLOGY AHMADU BELLO UNIVERSITY, ZARIA NIGERIA

JUNE, 2015

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DECLARATION

I declare that the work in this Thesis entitled “Isolation, anatomical distribution and antifungal susceptibility of dermatophytes affecting horses in Kwara State, Nigeria” has been carried out by me in the Department of Veterinary Microbiology under the supervision of Dr. (Mrs.) C. N. Kwanashie and Prof. H. M. Kazeem. The information derived from the literature has been duly acknowledged in the text and a list of references provided. No part of this thesis was previously presented for another degree or diploma at this or any other institution.

RashidatBolanle BALOGUN ______Student Signature Date

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CERTIFICATION

This Thesis entitled “ISOLATION, ANATOMICAL DISTRIBUTION AND ANTIFUNGAL SUSCEPTIBILITY OF DERMATOPHYTES AFFECTING HORSES IN KWARA STATE, NIGERIA” by RashidatBolanle BALOGUN meets the regulations governing the award of the degree of Master of Science of the Ahmadu Bello University, Zaria and is approved for its contribution to knowledge and literary presentation.

Dr. (Mrs.) C. N. Kwanashie Chairman, Supervisory Committee Signature Date

Prof. H. M. Kazeem Member, Supervisory Committee Signature Date

Dr. (Mrs.) C. N. Kwanashie Head of Department Signature Date

Prof. A. Z. Hassan Dean, School of Postgraduate Studies Signature Date

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DEDICATION

This work is dedicated to, my husband and children.

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ACKNOWLEDGMENTS

I wish to express my sincere gratitude to the Almighty Allah (Subhanahuwata‟alah) for giving me the strength and vigor and to members of my supervisory committee, Dr. (Mrs.) C. N. Kwanashie and Prof. H. M. Kazeem for their guidance and encouragement throughout the course of this study.

I am grateful to the staff members of the Department of Veterinary Microbiology, for their support and encouragement throughout the period of this study. My special thanks to Dr Paul Habila, DrAnkeli, Mallam Dodo, mallamTanko, mallamSalisu and HajiaSalamat for the help and concern they demonstrated to see the completion of this work.

My profound gratitude is to my professional colleagues in the University of Ilorin, especially Dr G. Atoyebi, Dr A. Obalowu, Dr Adam Mohd., Dr L. Jegede, Dr Paul and my brother, Kabir who all assisted me during my numerous trips to seek permission to collect skin samples from horses in Kwara state. I am grateful for the understanding and support of all members of my family, my dad, Shaid, Fati, Aisha, Meena and espeacially my mom and sister, Shaidat who have been patient enough to take care of my children in my absence.

My special thanks also go to Prof. Sackey, A.K.B., Dr. AdamuJibril, Dr. Alam L. and Dr. M.T. Salawudeen for their help and contributions toward the success of this work.

I would also like to acknowledge the support and encouragement given to me by the Dean of Faculty of Veterinary Medicine, Unilorin, Prof. S. F, Ambali and Director of V. T. H. University of Ilorin, Prof. E. O. Oyedipe, I am very grateful. Finally, I give my most special word of deep appreciation to my loving husband in the person of Suleiman ZakariyauJimoh, who stood by me in all ramifications, I am deeply indebted, and to my children (Faiza, Suleiman and Mohd- Khalid) for their patience and understanding.

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ABSTRACT

Dermatophytes are fungi that colonize keratinized tissues of humans and animals. This study was conducted to isolate dermatophytes from both clinical and asymptomatic cases of in horses in seven Local Government Areas (LGA) of Kwara State and to determine the susceptibility pattern of the dermatophytes isolated from different anatomical sites on horses to five antifungal agents. Ninety-one samples of plucked hair or scales and scrapings from the skin of horses were initially examined directly using microscopy prior to culture for isolation and identification usingSabouraud‟s dextrose agar (SDA), with 5% NaCl and potato dextrose agar as media for culture and isolation, respectively. Identification of each isolate was through observation of colonial morphology and microscopic appearance of lactophenol cotton blue stained fungal specimen obtained from culture. The assessment of antifungal susceptibility patterns of the dermatophytes isolated was by broth microdilution assay using ketoconazole, fluconazole, amphotericin B, griseofulvin and as antifungal agents. From all the samples obtained (91 samples), 14(15.4%) were dermatophytes out of which one was from an asymptomatic horse. These dermatophytes were identified as members of

Trichophyton(T.)andMicrosporum(M.)genera. The dermatophyte species isolated were T. tonsurans(7.14%) and T. soudanense (7.14%) which are anthropophylicdermatophytes, T. verrucosum(35.7%), M. gypseum (7.14%), M. persicolor (14.2%), M. equinum (7.14%) and M. fulvum (21.4%). Twelve of the 14 dermatophytes were isolated from 85 male horses while the remaining two were from six female horses. Based on anatomical sites, the highest isolation rate was from the limbs (18.7%) and the lowest from the abdomen (10%). However, the differences between the dermatophytes isolated from male and female horses or the different anatomical sites were not statistically significant (p ˃ 0.05). With regards to samples from the seven LGAs,

vii samples collected from Ilorin-East LGAyielded the highest isolation rate (25%) whilst those fromBarutenLGA had the lowest isolation rate (9.1%). The antifungal susceptibility test showed that terbinafine was the most potent drug with the lowest range of MIC values (0.2-6.5µg/ml) followed by amphotericin B which had MIC range of 0.6-4.0µg/ml and then ketoconazole (0.3-

9µg/ml), whereas griseofulvin and fluconazole showed the highest MIC ranges of 1.5-8.0µg/ml and 0.6-19.2µg/ml, respectively,indicating that terbinafine was the most efficacious of the five antifungal agents used in this study. Culture and sensitivity tests should be carried out for effective diagnosis and treatment of fungal infections especially in horses.

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TABLE OF CONTENT Page Cover page ------i Title Page ------ii Declaration ------iii Certification ------iv Dedication ------v Acknowledgements ------vi Abstract ------vii Table of Contents ------ix List of Tables ------xii List of Figures ------xiii List of Plates ------xiv List of Abbreviations and Symbols ------xv

CHAPTER 1: INTRODUCTION------1 1.1 Background of the Research ------1 1.2 Statement of the Research Problem ------4 1.3 Justification of the Study------5 1.4 Aim of the Study------6 1.5 Objectives of the Study ------6 1.6 Research Questions ------7

CHAPTER 2: LITERATURE REVIEW------8 2.1 Fungal Infections------8 2.2 Historical Background ------10 2.3 Nomenclature------12 2.4 Virulence of Dermatophytes------13 2.5 IsolationTechniques------14 2.6 Cultural Characteristics------15 2.6.1 Identifying dermatophytes ------15

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2.6.2 Microscopic identification process ------15 2.6.3 Macroscopic/colonial morphology ------16 2.6.4 Microscopic morphology ------17 2.7 Etiological Agents of Dermatophytes ------18 2.8 Growth and Nutritional Requirement ------21 2.9 Effective Chemicals and Physical Agents ------21 2.10 Serological Properties/Characteristics------22 2.11 Susceptible Host Range and Resistance------27 2.11.1 Transmission ------28 2.11.2 Clinical signs and anatomical location of lesions ------29 2.12 Dermatophytosis in Other Domestic Animals------30 2.12.1 Morbidity in other domestic animals ------33 2.13 Distribution of Lesions of Dermatophytes------35 2.13.1 Histopathological findings ------36 2.13.2 Clinical diagnosis ------37 2.14 Dermatophytosis in Humans------39 2.15 Classes of Antifungal Agents and their Resistance------43 2.15.1 Antifungal agents ------43 2.15.2 Flouropyrimidines ------43 2.15.3 Polyenes ------45 2.15.4 Azoles ------47 2.15.5 Echinocandins ------51 2.15.6 Other antifungal agents ------53 2.16 Current Synopsis of Dermatophyte Species and Congeners: Ecological Classification and Endemicity------54 2.17 Case Reports of Animal Dermatophytosis in Nigeria------54 2.18 Case Reports of Animal Dermatophytosis in Other Parts of the World------57 2.19 Reports of Dermatophytosis in Humans in Nigeria------60 2.20 Reports of Dermatophytosis in Africa and Other Parts of the World------64 2.21 Antifungal Susceptibility Testing of Dermatophytes------65

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CHAPTER 3: MATERIALS AND METHODS------69 3.1 Study Area ------69 3.2 Sampling and Sample Size------69 3.3 Sample Collection ------69 3.4 Direct Microscopic Examination of Samples ------72 3.5 Laboratory Culture of Dermatophytes------72 3.6 Identification of Isolates ------72 3.7 Slide Culture Preparation------73 3.8 Antifungal Drug Dilution------74 3.9 In-vitro SusceptibityTesting------74 3.10 Preparation of Innoculum------74 3.11 Antifungal Susceptibility Test Procedure------75 3.12 Data Presentation and Analysis------75

CHAPTER 4: RESULTS------76 4.1 Occurrence of Dermatophytes in Horses with Skin Lesions------76

4.2 Colonial Morphology and Microscopic Appearance of tonsurans, Trichophytonverrucosum, Trichophytonsoudanense, Microsporumgypseum, Microsporumpersicolor, Microsporumequinum andMicrosporumfulvum------82 4.3 Minimum Inhibitory Concentrations of ketoconazole, Amphotericin B, Terbinafine, Griseofulvin and Fluconazole against DermatophytesIsolated fromHorses in Seven Local Government Areas of Kwara State------89

CHAPTER 5: DISCUSSION------95

CHAPTER 6: SUMMARY, CONCLUSIONS ANDRECOMMENDATIONS------98 6.1 Summary ------98 6.2 Conclusions------99 6.3 Recommendations------100

REFERENCES------101

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LIST OF TABLES

Page Table 2.1: Current synopsis of dermatophyte species and congeners: ecological classification and endemicity------55

Table 4.1: Percentage distribution of dermatophytes isolated from samples obtained from horses in seven Local Government Areas (LGA) of Kwara State------77

Table 4.2: Distribution of dermatophytes based on anatomical site of lesions------79

Table 4.3: Anatomical locations of different dermatophytes isolated from horses in seven Local Government Areas of Kwara State------80

Table 4.4: Frequency of dermatophytespecies isolated from horses fromseven Local Government Areas in Kwara State------81

Table 4.5: Sex distribution of dermatophytes isolated from horses in seven Local Government Areas of Kwara State ------83

Table 4.6: Dermatophytes isolation rates from horses with dermatophytic lesions or asymptomatic in Kwara State ------84

Table 4.7: Minimum Inhibitory Concentrations of fiveantifungal drugs tested on 14 dermatophytesisolated from horses in sevenLocal Government Areasof Kwara State------93

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LIST OF FIGURES

Page Figure 3.1: Map of Kwara State. Arrows are indicating LGAs where samples were collected.------71

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LIST OF PLATES Page

PlateI: Circumscribed,dermatophytic lesion (at point of arrow) on the forelimb Ofa horse.------78

Plate II: Colonies of Trichophytontonsurans (a) withsuede like to powdery yellow surface,raised center and radial grooves (C).Lactophenol cotton blue stained smear ofTrichophytontonsurans (b and c)×400. Note thethin walledhyphae withnumerous septa and irregular branching(H), clavatemacroconidia and chlamydospores at terminal ends of hyphae (S).------85

Plate III: Small white button shape colony ofTrichophytonverrucosumwith a raised center(C),velvet surfaceand flat submerged periphery (a). Lactophenol cotton blue stained smear ofTrichophytonverrucosum×400 (b and c) with broad irregular hyphae (H) and chains ofbroad, club shaped intercalary chlamydospores (S). ------86

Plate IV: Colony of Trichophytonsoudanensewith yellow folded suede like surface, raised center (C) and broad white fringed edges (a).Lactophenol cotton blue stained smear ofTrichophytonsoudanense×400 withreflexivebranching hyphae (H)and pyriform(M) microconidi a (b). ------87

Plate V : Colony of Microsporumgypseum with flat spreading suede like, deep cream surface, central dome (C) and narrow white peripheral border (a).Lactophenol cotton blue stained smear of Microsporumgypseum×400 showing 4 celled macroconidia (M)with truncated distal ends (b).------88 : Plate VI: Pink tinged colonies of Microsporumpersicolor (C) with granular texture and irregular advancing edges (VIa). Lactophenol cotton blue stained smearof Microsporumpersicolor×400 (VIb) with thin walled, cigar shaped, 7 celled macroconidia (M) and chlamydospores (S). ------90

Plate VII: Colonies of Microsporumequinum(C) withflat, spreading to suede like surface and pale buff to salmon colour (VIIa).Lactophenol cotton blue stained smear ofMicrosporumequinum×400 showing small spindle shaped macroconidia (M) with few septae (VIIb). ------91

Plate VIII: Profuse white colony of Microsporunfulvum(C) withraised center (VIIIa). Lactophenol cotton blue stained smear of Microsporumfulvum ×400 (VIIIb) showingnumerous spiral hyphae (H). ------92

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LIST OF ABBREVIATIONS AND SYMBOLS

ABC -ATP binding Cassette AmB-Amphotericin B AIDS-Acquired Immunodefeciency Syndrome C - Candida CMI - Cell mediated Immunity CLSI - Clinical Laboratory Standards Institute DTH - Delayed Type Hypersensitivity DTM -Dermatophyte Test Medium DNA - Deoxyribonucliec Acid EMEA - European Agency For the Evaluation of Medicinal Products ERG II gene - Ergosterol II gene E - FDA - Food and Drug Administration G - gramme H&E - Hematoxylin& Eosin Stain HIV - Human Immune Deficiency Virus IgA - Immunoglobulin A IgE - Immunoglobulin E IgG - Immunoglobulin G IgM - Immunoglobulin M ITS - Internal Transcribed Spacer K+ - Potassium ion KOH - Potassium hydroxide LGA - Local Government Area M - Micropsorum MIC - Minimum Inhibitory Concentration Ml - Millilitre n/NO - Number P - Prevalence PAS - Periodic acid Schiff PDA - Potato Dextrose Agar Ph - Acidity/ Alkalinity RNA - Ribonucleic acid RPMI - Roswell Park Memorial Institute SDA - Sabouraud‟s Dextrose Agar Spp - species T - Trichophyton TRM - TrichophytonrubrumMannan µg - Microgramme

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UPRT - UridinePhosphoribosylTransferase 5-FC - 5-Flourocytosine 5-FU - 5-Flourouracil 5-FUMP - 5-Flourouracil Monophosphate

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CHAPTER ONE

INTRODUCTION

1.1 Background of the Research

Dermatophytes are a group of keratinophilic fungi that cause dermatophytosis which is a highly contagious fungal infection of the skin that affects horses and other animals of all ages and breeds. Dermatophytes produce proteolytic , keratinases, which are able to hydrolyze the main protein constituent of hair, nails and skin, the infection can be mild to severe, depending on the host immune response (Akcaglar et al., 2011). The keratin, collagen and elastin constitute 25% of the mass of mammals. The required to hydrolyze these macromolecules is found in infected tissues and is therefore considered essential to the virulence of dermatophytes (Simpanya, 2000). Colonization by a dermatophyte, and its ability to cause an infection in the host, depends on several factors, among which are the escape mechanisms of the host resistance, including dry skin, a slightly acidic pH, the continuous regeneration of the skin, the fungicidal effect of fatty acids, the state of the keratinized layer and other factors, such as competition with the normal skin microbiota (Cabanes, 2000).

Establishment of an infection is initiated by the inoculation of arthrospores deposited on the skin, favored by a pre-existing skin lesion or abrasion and the microorganism‟s remarkable enzymatic ability to degrade keratin (Simpanya, 2000; Abdel-Rahman, 2001;

Macêdo et al., 2005). Dermatophytosis can also infect several animal species, creating generally dry, rounded and usually pruritic lesions, distributed focally on the skin

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(Sobestiansky, 2001). Animals serve as reservoirs of zoophilic dermatophytes and their zoonotic infections have considerable importance ((Sidrim et al., 2004; Baranova et al.,

2003).

Dermatophytosis is a mycotic disease known as ringworm or tinea caused by dermatophytes which comprises a group of closely related fungi in the Genera

Microsporum, Trichophyton and Epidermophyton (Emmons, 1955; Weitzmann and

Summerbell, 1995). They are ecologically classified into zoophilic (animal loving), anthropophilic (human loving) and geophilic (soil occurring) with Trichophyton,

Microsporum and Epidermophyton constituting the prominent dermatophytes globally

(Weitzmann and Summerbell, 1995; Rudy, 1999). By segmentation and fragmentation of the hyphae, dermatophytes produce arthrospores which adhere strongly to keratin and are highly resistant, surviving in a dry environment for 12 months or longer. In a humid environment, however, arthrospores are short-lived and high temperatures (100°C) destroy them quickly (Sparkes et al., 1994).

Dermatophytes belong to the class Ascomycetes and the order Hyphomycetes, these fungi are cosmopolitan and occur widely in the soil and other keratin containing substrate such as birds nest and thus the soil serves as a source of infection (Ainsworth and Austwick,

1973; Beneke and Rogers, 1980). The dermatophyte structure commonly associated with contagion is the oblong to rounded and persistent spore, arthroconidium or chlamydospore found within or attached to the exterior of infected hairs, fur, and within skin scales. These structures may persist for years in the environment (Rippon, 1998) and are highly heat resistant, particularly when embedded in hair, fur or skin scales (Sinski et al., 1980).

Dermatophytes have been reported worldwide, though with variation in distribution,

2 incidence and epidemiology. Etiology targets host from one location to another with the passage of time. A number of factors including geographic location, prevailing climate

(temperature, humidity, wind etc.), overcrowding, health care, immigration, environmental hygiene, culture and socioeconomic disposition have great implication for the proliferation of dermatophytes (Hay, 2003; Havlickova et al., 2008). Dermatophytes have the ability to invade the stratum corneum of the epidermis and keratinized tissues derived from it, such as skin, hair and nails of humans and animals (Weitmann and Summerbell, 1995). It is one of the most common cause of cutaneous infections all over the world (Nweze and Okafor,

2005; Ameen, 2010.). Dermatophytosis causes superficial fungal infection that poses public health problems to man and animals (Havlickova et al., 2008) and can be disfiguring and recurrent and generally need long term treatment with antifungal agents

(Nweze et al., 2007).

Clinical signs of dematophytosis in horses include variable skin lesions and do not necessarily form a ring. There will be hair loss, usually in small patches at first. There might be scratching due to itchiness (Muller et al., 1989). Other signs include papular reaction, pustules, epidermal collarets, erythema, crusting and scaling. Hypersensitivity reactions may be more severe, including pruritus, edema, suppuration and necrosis. Also in horses, most dermatophyte lesions are found in areas of contact with saddles or other tack.

T. equinum lesions are usually pruritic, with exudative lesions and areas of alopecia, thickened skin. M. equinum lesions are usually less severe and consist of small scaly areas with brittle hairs. Early dermatophyte lesions may resemble papular urticaria (OIE, 2005).

Young animals are affected most often and asymptomatic infections are common, particularly in adult animals (Nweze, 2010).

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Dermatophytes grow best in warm and humid environment and are therefore more common in tropical and subtropical regions and this probably explains why they are very common in Africa, for instance, some species of dermatophytes such as Trichopyton mentagrophytes var interdigitale, Microsporum canis, Epidermophyton fluccosum and

Trichophyton rubrumare distributed all over the world, however some species probably have partial geographic restriction.. Example, Trichophyton schoenleinii and Trichophyton soudanense are found in Africa (Weizmann and Summerbell, 1995).

In tropical and sub-tropical areas, the disease can be epizootic and can result in considerable economic losses as a result of lost production, public health concern, premature culling, treatment costs, and down grading of hides and skins (Pandey and

Cabaret, 1980; Ogbonna et al., 1986; Wabacha et al., 1998; Nweze, 2010). Nigeria located in the tropic with wet humid climate falls into the category of regions with high prevalence of dermatophytosis (Gugnani et al., 1995; Rudy, 1999).

1.2 Statement of the Research Problem

Dermatophytosis is a disease that affects many species of livestock and occurs as an acute or chronic skin disease (Svejgaard, 1986; Chermette et al., 2008). Close confinement, host factors (age, immuno- competence, type of breed, host grooming behavior), dietary factors deficiencies, and condition of exposed skin surfaces (Moretti et al., 1998; Papini et al.,

2009). Affected animals initially develop characteristically discrete, scaling patches of hair loss with grey-white crust that later become thickly suppurated crust whose location is highly variable (Radostits etal., 2000).

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In tropical and sub-tropical areas, the disease can be epizootic and can result in considerable economic losses, as a result of lost production such as premature culling, treatment costs and down grading of hides and skins (Pandey and Cabaret, 1980; Ogbonna etal., 1986; Wabacha etal., 1998; Nweze, 2010). Although dermatophytosis is worldwide in distribution, it is more prevalent in hot humid climates than in cold dry regions

(Emmons, 1955; Scott, 1988; Macura, 1993b). Dermatophytosis is however not reportable or notifiable disease in Nigeria and in the tropical areas because the disease is usually self- limiting i.e. usually, produces benign skin lesions (Mackenzie, 1963; Adekeye et. al.,

1989; Macura, 1993b) and as a result, actual prevalence figures for dermatophytosis is unknown in many endemic areas.

Fungal infections have been described in connection with poor quality hooves and several diseases such as white line disease or laminitis (Chapman, 1993; Kuwano et al. 1998;

Jahns and Dietz, 2000; Keller, et al., 2000) Also, several reports indicate that domestic animals constitute important reservoirs of human ringworm epidemics (La Touch, 1955;

Blank and Craig, 1977; Gugnani, 1982).

1.3 Justification of the Study

Animals serve as reservoirs for zoophilic dermatophytes, and their infections have considerable zoonotic importance. Zoophilic dermatophytes such as M. canis, T. mentagrophytes and T. verrucosum are significant causal agents in human ringworms in many areas of the world (Nweze, 2011). The incidence of dermatophytosis varies according to climate and natural reservoirs. However, the pattern of the species of dermatophytes involved in dermatophytosis may be different in similar geographic

5 conditions both in humans and animals. This has been related among many factors to the decline in the incidence of animal ringworm in some areas or the degree of closeness of animals to human contact (Pier et al., 1994).

This research is important because dermatophytes are the most common cause of cutaneous infections in domesticated animals. They are known to serve as reservoirs of the zoophilic dermatophytes and these infections have important zoonotic implication. In

Nigeria and especially Kwara State, there are not many studies on the incidence of dermatophytosis in domesticated animals especially in horses. This study is also relevant because of the role played by horses in the socio-economic aspects of human life in Kwara

State, the zoonotic nature of the disease and also because, fungal infections are reported to produce the most common lesions in HIV infections (Amen et al., 2004; Scott, 1988;

Macura, 1993a). This investigation was carried out to determine the incidence of dermatophytosis and the susceptibility of fungal isolates to 5 selected antifungal agents

(Fluconazole, Ketoconazole, Griseofulvin, Amphotericin B, and Terbinafine).

1.4 Aim of the Study

To determine the occurrence and antifungal susceptibility of the isolates obtained from

horses in Kwara state.

1.5 Objectives of the Study

1. To isolate and identify dermatophytes from horses in Kwara state using Sabouraud

Dextrose Agar.

2. To determine antifungal susceptibility of the isolates to selected antifungal agents.

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3. To determine the anatomical distribution of dermatophytes isolated from horses in

Kwara state.

1.6 Research Questions

1. Are there dermatophytes in horses from Kwara state, Nigeria?

2. What are the species of dermatophytes isolated from horses in Kwara State?

3. Are the dermatophytes isolated from horses in Kwara state susceptible to the commonly

available antifungal agents?

4. Does sex of horses have any significant influence on dermatophyte infection?

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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Fungal Infections

The fungal kingdom encompasses an enormous diversity of taxa with varied ecological niches, life-cycle strategies, and morphologies. Dermatophytic and keratinophilic fungi can attack eyes, nails, hair, and especially skin and cause local infections such as ringworm and athlete‟s foot. Fungal spores are also a cause of allergies, and fungi from different taxonomic groups can provoke allergic reactions.

At the beginning of the 20th century, bacterial epidemics were a global and important cause of mortality. In contrast, fungal infections were almost not taken into account. Since the late 1960s when antibiotic therapies were developed, a drastic rise in fungal infections was observed, and they currently represent a global health threat. This increasing incidence of infection is influenced by the growing number of immunodeficient cases related to

AIDS, cancer, old age, diabetes, cystic fibrosis, and organ transplants and other invasive surgical procedures.

These infections are caused by two types of microorganisms: primary and opportunistic pathogens. Primary pathogens are naturally able to establish an infection in the healthy population. In contrast, opportunistic pathogens, among them commensal microorganisms of the healthy population, are able to develop infectious colonization of the human body when particular criteria, such as immunosuppression, are met. Fungal pathogens can be divided into two major groups: filamentous fungi and yeasts. Most of the primary

8 pathogens are filamentous fungi, while most of the opportunistic pathogens are yeasts and some species of filamentous fungi are increasingly identified as opportunistic pathogens. It is also important to note that fungal infections can be classified in function of the tissue infected

Cutaneous and subcutaneous mycoses caused by dermatophytes fungi affect keratinized structures of the body. The most frequently involved dermatophyte genera are

Trichophyton, Epidermophyton, and Microsporum. In most cases, cutaneous fungal infections require a challenge of immune system, and their incidence varies depending on the site of infection. For example, onychomycoses are very frequent in the global population, with an incidence varying from 5 to 25% (Diamond et al., 1991).

Mucosal infections are mostly caused by opportunistic yeasts, and those belonging to the

Candida genus are by far the most frequent. Vaginal, esophageal, oropharyngal, and urinary tract candidiasis are very frequent in immunocompromised patients. For example, esophageal candidiasis is associated with the entry into the clinical phase of AIDS and during the 1980s more than 80% of seropositive patients developed such an infection.

Fungal infections, of the eye are also classified as mucosal fungal infections, but are caused more frequently by Fusarium or Aspergillus species rather than Candida species.

Theoretically systemic mycoses may involve any part of the body, and a lot of species formerly considered as nonpathogenic are now recognized opportunistic pathogens responsible for deep-seated mycoses. These infections, with symptoms ranging from a simple fever to a severe and rapid septic shock, are very common in immunocompromised patients and are frequently associated with an elevated mortality rate. The most frequent

9 pathogens involved in systemic fungal infections are Candida, Pneumocystis, Histoplasma,

Aspergillus, Cryptococcus, Mucor, Rhizopus, and Coccidioidomyces (Arendrup et al.,

2010).

2.2 Historical Background

Historically, medical mycology specifically relating to human disease began with the discovery of the fungal etiology of favus and centered around three European physicians in the mid-19th century: Robert Remak, Johann L. Scho¨nlein, and David Gruby. According to Seeliger (Seeliger, 1985). Remak in 1835 first observed peculiar microscopic structures appearing as rods and buds in crusts from favic lesions. He never published his observations, but he permitted those observations to be cited in a doctoral dissertation by

Xavier Hube in 1837. Remak claimed that he did not recognize the structures as fungal

(Remak, 1942) and credited this recognition to Scho¨nlein, who described their mycotic nature in 1839 (Scho¨nlein, 1839).

However, Remak established that the etiologic agent of favus was infectious, cultured it on apple slices, and validly described it as Achorion schoenleinii, in honor of his mentor and his initial discovery (Remak, 1945). The real founder of dermatomycology was David

Gruby on the basis of his discoveries during 1841 to 1844, his communications to the

French Academy of Science, and his publications during this period (Gruby, 1843).

Independently, and unaware of the work of Remak and Scho¨nlein, he described the causative agent of favus, both clinically and in microscopic details of the crusts, and established the contagious nature of the disease (Gruby, 1841). He also described ectothrix invasion of the beard and scalp, naming the etiologic agent of the latter

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Microsporum(referring to the small spores around the hair shaft) audouinii, and described endothrix hair invasion by Herpes (Trichophyton) tonsurans. In addition to his observations on dermatophytes, he also described the clinical and microscopic appearance of thrush in children (Remak, 1845). Raimond Sabouraud, one of the best known and most influential of the early medical mycologists, began his scientific studies of the dermatophytes around 1890, culminating in the publication of his classic volume, Les

Teignes (Sabouraud, 1910). In 1910, Sabouraud‟s contributions included his studies on the , morphology, and methods of culturing the dermatophytes and the therapy of the dermatophytoses. He classified the dermatophytes into four genera, Achorion,

Epidermophyton, Microsporum, and Trichophyton, primarily on the basis of the clinical aspects of the disease, combined with cultural and microscopic observations. The medium that he developed is in use today for culturing fungi (although the ingredients are modified) and is named in his honor, Sabouraud glucose (dextrose) agar (Odds, 1991).

Sabouraud‟s treatment of by a one-dose, single-point roentgenologic epilation achieved cures in 3 months as opposed to the then current therapy of manual epilation and topical application of medications (Kwon-chung, 1992). In 1934, Chester Emmons modernized the taxonomic scheme of Sabouraud and others and established the current classification of the dermatophytes on the bases of spore morphology and accessory organs. He eliminated the genus Achorion and recognized only the three genera

Microsporum, Trichophyton, and Epidermophyton on the basis of mycologicalprinciples.(Emmons, 1955).

Nutritional and physiological studies of the dermatophytes pioneered at Columbia

University by Rhoda Benham and Margarita Silva and at the Center for Disease Control, in

11

Georgia, by Libero Ajello, Lucille K. Georg, and coworkers, simplified the identification of dermatophytes and led to reduction of the number of species and varieties. The discovery of the teleomorphs (perfect or sexual state) of Trichophyton (Keratinomyces) ajelloi in 1959 by Dawson and Gentles (Dawson, 1961) using the hair bait technique of

Vanbreuseghemii led to the rapid discoveries of the teleomorphs of many dermatophytes and related keratinophilic fungi. Griffin in 1960 and Stockdale in 1961 and 1963 independently obtained the teleomorphs of the complex, thereby vindicating Nannizzi‟s original observation. The discovery of sexual reproduction in the dermatophytes opened the door to classical genetic studies with these fungi, e.g., determining the cause of pleomorphism (Weitzman, 1965) and clarifying the taxonomy and understanding of the incompatibility systems operating in these fungi (Weitzman,

1964).

The successful oral therapy with griseofulvin of experimental dermatophytosis in guinea pigs reported by Gentles in 1958 revolutionized the therapy of dermatophytosis and initiated the first major change in the therapy of tinea capitis since the work of Sabouraud .

2.3 Nomenclature

Organisms may be assigned different names depending on whether the source is using traditional identification methods or genetic typing. Diagnostic laboratories have traditionally identified dermatophytes based on their colony and microscopic morphology, nutritional and biochemical characteristics, and other factors. Such methods, together with the ecology of an organism (Hanaa et al., 2003) (e.g., its adaptation to a particular host) have given rise to a number of species names. However, some organisms that appear to be

12 different species, based on conventional typing and/or ecology, may be very closely related genetically. Furthermore, the traditional typing methods have given rise to a situation where a single anamorph can have two different teleomorphs, suggesting that such

“species” actually contain more than one species.

A taxonomic method first proposed in 1999 defines dermatophyte species by genetic techniques, specifically the sequencing of highly variable internal transcribed spacer (ITS) regions of the ribosomal DNA. Some authors have adopted the ITS scheme. (Rebell et al.,

1974) Others feel that its adoption is premature and based on limited data. Internal

Transcribed Spacer (ITS) taxonomy has been criticized because it may place organisms into the same species even when they seem to be ecologically distinct based on their adaptation to different hosts; zoophilic, anthropophilic or geophilic nature; or distinctive characteristics such as opposite mating types or ability to penetrate hair in vitro. In addition. The results of ITS typing may not agree with the results of genetic analyses based on other genes (Hebert et al., 1985). Some sources also use traditional typing schemes for practical reasons: genetic typing is not widely used in diagnostic laboratories, and some species defined by ITS sequencing can be difficult or impossible to identify by conventional methods.

2.4 Virulence of Dermatophytes

Rippon (Rippon, 1967) demonstrated close linkage between the gene for elastase activity

(a suggested virulence factor) and the gene for mating type in A. fulvum (N. fulva).

Furthermore, Rippon and Garber (Rippon, 1969) suggested an association of dermatophyte pathogenicity as a function of mating type and associated enzymes in A. benhamiae.

13

However, Cheung and Maniotis (Cheung, 1973) demonstrated in A. benhamiae that elastolytic activity segregated independently of mating type but was closely linked to the locus governing colonial morphology. Hejtma´nek and Lenhart (Hejtma´nek et al., 1970) studied the genetic basis for virulence in A. incurvatum (N. incurvata) and demonstrated that multiple chromosomal genes were involved. The locus for virulence was independent of colonial morphology but was related to growth rate. All cultures with a normal growth rate were virulent, whereas those with a lower growth rate were avirulent. In a later study they obtained genetic complementation of virulencein avirulent mutants by heterokaryon formation in a nutrient agar medium (Hejtma´nek et al., 1972 ) and on soil (Hejtma´nek et al., 1973) they did not address mating type or enzymes in their studies.

2.5 Isolation Techniques

Dermatophytes can be cultured on various fungal media, including Sabouraud agar (with cycloheximide and antibiotics) and dermatophyte test medium (DTM). Cultures are usually incubated at room temperature (20–28ºC), but higher temperatures can be used when certain organisms like T. verrucosum are suspected. Colonies often become visible within

1-2 weeks but, some species grow more slowly and may require longer time to appear.Colony morphology can differ with the medium; descriptions are usually based on

Sabouraud agar.Dermatophyte test medium (DTM) contains a pH indicator (phenol red) that will turn the medium red when a dermatophyte is growing. However, the mycelial growth must also be examined microscopically, as this color change alone is not diagnostic and could be produced by other fungal or bacterial organisms. In addition, the color change may be delayed with certain dermatophytes such as M. persicolor in asymptomatic

14 animals; caution must be used to distinguish infection from contamination of the coat with organisms from the environment (Chermette et al., 2008).

2.6 Cultural Characteristics

Dermatophyte cultures can be challenging to perform and interpret correctly. However, knowing how to best collect samples for culture, select and incubate culture media, and identify media culture changes and fungal colony morphology will help to avoid a misdiagnosis.

2.6.1 Identifying dermatophytes

Understanding macroscopic fungal colony morphology is an important first step in determining whether a dermatophyte is present. Microscopic evaluation of suspect fungal growth is also important since some environmental fungi can mimic dermatophytes in gross colony morphology and in their ability to turn the media red and because some strains of Microsporum canis may not produce media color change.Microscopic examination can be done in the clinic, or the entire culture plate can be sent to a reference laboratory for fungal identification (Abdullah et al., 1995).

2.6.2 Microscopic identification process

Dermatophytosis is a zoonotic infection therefore gloves should be worn to avoid transmitting dermatophyte spores to the hands. Gently touch a small piece of clear acetate tape to the surface of the fungal colony, and then apply the tape to a glass slide over a drop of blue stain (methylene blue, lactophenol cotton blue, or the blue Diff-Quik solution

15

(basophilic thiazine dye]) . Examine the slide under 10X and 40X objective lens to identify the characteristic dermatophyte features (Rebell et al., 1974)

In the early stages of growth, only fungal hyphae with no macroconidia may be seen, especially in cases of Trichophyton species infections. Incubating these cultures longer allow spore development for more reliable identification.

2.6.3 Macroscopic/colonial morphology

Growth occurs on Sabouraud dextrose agar containing chloramphenicol, cyclohexamide

(Actidione) and nicotinic acid and incubated at 370C for 3-4 weeks. Trichophyton verrucosum grows better at 37oC. Colonies may be circular, asteroid, polygonal or lobulate. Colour of the colony may be white to gray, rosy, vinaceous, reddish, apricot, orange, yellow, violet and the back of the colony varies in colour from yellow to dark red to purplish black. Some dermatophytes need vitamins for growth such as inositol and thiamine (T. verrucosum), nicotinic acid (T. equinum) and histidine (T. megnini).

Dermatophytes produce colonies that vary in texture and rate of growth. There are three forms of colonies (Ajello et al., 1968). a. The membranous form (glabrous, waxy, faviform): the aerial mycelium is entirely absent and the vegetative mycelium is in compact masses e.g. M. ferrugineum, T. concentricum, T. schoenleinii, T. violaceum and T. verrucosum. b. The filamentous form (cottony, fluffy, hairy, velvety, woolly): the aerial mycelium is more or less high and dense e.g. E. floccosum, M. audouinii, M. canis, M. distortum, M. nanum and T. rubrum.

16 c. The granular-powdery form: characterized by excessive conidia and absence of aerial filamentous elements e.g. M. equinum, T. mentagrophytes and T. megnini (Ajello et al.,

1968).

2.6.4 Microscopic morphology

The main microscopic structures of dermatophytes are micro and macroconidia, chlamydospores, septated hyphae, racquet hyphae spirals. The microconidia are usually single-celled, sessile or on a stack, single or in groups. The macroconidia are spindle, pencil or club-shaped. (Hasegawa, 2000) Some dermatophytes consist of only sterile hyphae and rarely produce spores. The sexually reproducing fungi show cleistothecia and . The main diagnostic structures in dermatophytes are the macroconida,

In case of the genus Epidermophyton the macroconidia are broadly clavate with typically smooth, thin to moderately thick walls and one to nine septa. They are usually abundant and borne singly or in clusters. Microconidia are absent. The genus is represented by two species, only E. floccosum is pathogenic.

In Microsporum, the macroconidia are characterized by the presence of rough walls which may be asperulate, echinulate or verrucose. They are spindle-shaped or fusiform, obovate or cylindrofusiform. (Feuerman et al., 1975) Microconidia are sessile or stalked and clavate and usually arranged singly along the hyphae. The genus comprises at least 12 species, M. audouinii, M. canis, M. equinum, M. ferrugineum, M. fulvum, M. gallinae , M. gypseum, M. nanum, M. persicolor, M. preecox, M. racemosum and M. vanbreuseghemii.

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In Trichophyton, the macroconidia, when present, have smooth, usually thin walls and one to 12 septa, are born singly or in clusters and may be elongated and pencil shaped, clavate, fusiform or cylindrical. Example Trichophyton mentagrophyte macroconidia are Cigar- shaped with thin smoothed walls, Trichophyton verrucosum macroconidia are rare, long, thin and smooth wall with many chlamydospore,Trichophyton equinum macroconidia are rare, clavate, thin and smooth wall,3 to 5 cell. Trichophyton microconidia, usually are more abundant, may be globose, pyriform, or clavate, sessile or stalked, and are borne singly along the sides of the hyphae or in grape-like clusters. The genus comprises about

15 species, e.g. T. concentricum, T. equinum, T. gourvilii, T. kane, T. megninii, T. mentagrophytes, T. raubitschekii, T. rubrum, T. schoenleinii, T. simii, T. soudanense, T. tonsurans, T. verrucosum, T. violaceum and T. yaoundei. (Feuerman et al. 1975)

2.7 Etiological Agents of Dermatophytosis

Etiological agents of dermatophytosis are divided into 2 according to their mode of reproduction.

Anamorphs

The etiologic agents of dermatophytosis are classified into three anamorphic (asexual or imperfect) genera, Epidermophyton, Microsporum, and Trichophyton, of anamorphic class

Hyphomycetes of the Deuteromycota (Fungi Imperfecti). (Abdullah et al., 1995) The descriptions of the genera essentially follow the classification scheme of Emmons

(Emmons,1955) on the bases of conidial morphology and formation of conidia and are updated following the discovery of new species (Ajello,1968; Ajello,1977; Matsumoto and

Ajello,1987). The genera and their descriptions are as follows:

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1. Epidermophyton spp. The type species is Epidermophyton floccosum. The

macroconidia are broadly clavate with typicallysmooth, thin to moderately thick

walls and one to nine septa,20 to 60 by 4 to 13 mm in size. They are usually

abundant andborne singly or in clusters. Microconidia are absent. This genushas

only two known species to date, only E. floccosum ispathogenic and E. stockdaleae

is apathogenic.

2. Microsporum spp. The type species is Microsporum audouinii. Macroconidia are

characterized by the presence of rough walls which may be asperulate, echinulate,

or verrucose. Originally, the macroconidia were described by Emmons as spindle

shaped or fusiform, but the discovery of new species extended the range from

obovate (egg shaped) as in Microsporum nanum (Fuentes, 1956) to

cylindrofusiform as in Microsporum vanbreuseghemii (Georg et al., 1962). The

macroconidia may have thin, moderately thick to thick walls and 1 to 15 septa and

range in size from 6 to 160 by 6 to 25 mm. Microconidia are sessile or stalked and

clavate and usually arranged singly along the hyphae or in racemes as in

Microsporum racemosum, a rare pathogen (Borelli, 1965).

3. Trichophyton spp. The type species is Trichophyton tonsurans. Macroconidia,

when present, have smooth, usually thin walls and one to 12 septa, are borne

singly or in clusters, and may be elongate and pencil shaped, clavate, fusiform, or

cylindrical. They range in size from 8 to 86 by 4 to 14 mm. Microconidia, usually

more abundant than macroconidia, may be globose, pyriform or clavate, or sessile

or stalked, and are borne singly along the sides of the hyphae or in grape-like

clusters.

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Descriptions of the species and related keratinophilic fungi may be found in several publications (Kwon-Chung and Bennett, 1992; Rebell and Taplin, 1970; Rippon, 1998;

Weitzman, 1964. 1991). Since the classification of the dermatophytes by Emmons, as a result of the discovery of new species and variants, the rigid morphological distinction among the three genera has become a morphological continuum based on overlapping characteristics; e.g., Trichophyton kanei (Summerbell, 1987), Trichophytonlongifusum and a variant of T. tonsurans lack microconidia, and therefore are more suggestive of the genus

Epidermophyton, whereas isolates of Microsporum spp. Producing smooth-walled macronidia are more suggestive of Trichophyton spp.

Teleomorphs

Some dermatophytes, mostly the zoophilic and geophilic species of Microsporum and

Trichophyton, are also capable of reproducing sexually and producing ascomata with asci and ascospores. These species are classified in the teleomorphic genus Arthroderma

(Weitzman et al., 1986), family of the , phylum

Ascomycota. Previously, the teleomorphs of the sexually reproducing Microsporum and

Trichophyton species and related keratinophilic fungi had been classified in the genera

Nannizzia and Arthroderma, respectively (Ajello, 1977). However, on the basis of a careful evaluation of the morphological characteristics used to define these two genera,

Weitzman et al. (1986) concluded that the species making up these genera represented a continuum and that their minor differences did not merit maintaining them in two separate genera. Nannizzia and Arthroderma are considered to be congeneric, with Arthroderma having taxonomic priority.

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2.8 Growth and Nutritional Requirement

The physiology of dermatophytes has always been studied for a better understanding of their mode of infection, host-parasite relationship and specialized nature of parasitism to evolve better therapeutic measures and improved diagnostic approaches (Sieseno and

Bohm, 1995) With respect to utilization of carbon, nitrogen and vitamins, dermatophytes present a versatile nutritional range.

Dermatophytes have a common preference for carbohydrates among carbon sources, growing best in the presence of glucose (George, 1952). Although they can well utilize carbon from other sources, they are not able to utilize the trisaccharide melezitose probably due to lack of specific enzymes. Specificity to particular amino acids as a source of nitrogen has been observed in some dermatophytes. T. tonsurans is specific for ornithine, citrulline or arginine and T.mentagrophytes is specific for methionine. The majority of the dermatophytes are autotrophic for vitamins. Pereiro-Miguens (1955) demonstrated the growth inhibition of T. rubrum by vitamin C and of T. quinckeanum and T. mentagrophytes by folic acid. Specificity to nutritional characteristics in certain species of dermatophytes has led to suggestions by some authors on the use of nutritional properties for the identification of certain species of dermatophytes (Philpot, 1967; Shome, 1967).

There is no specific differentiation among dermatophytes with respect to metal ion utilization (Stockdale, 1967).

2.9 Effective Chemicals and Physical Agents

Dermatophyte spores are susceptible to benzalkonium chloride, dilute chlorine bleach (1% sodium hypochlorite), enilconazole (0.2%), formaldehyde and some strong detergents. In

21 one study, a 10% solution of alkyldimetylbenzylammonium chloride prevented the growth of M. canis from 97% of contaminated hairbrushes, and Virkon-S® was effective on 87%.

Another study found that a preparation containing benzylammonium bromide and ethoxyllauric alcohol was effective against the anthropophilic fungi usually found on swimming room floors. Dermatophytes are also reported to be susceptible to iodophors, glutaraldehyde and phenolic compounds; however, some agents may have limited efficacy in “real life” environmental disinfection.

2.10 Serological Properties/Characteristics

Dermatophyte colonization is characteristically limited to the dead keratinized tissue of the stratum corneum and results in either a mild or intense inflammatory reaction. Although the cornified layers of the skin lack a specific immune system to recognize this infection and rid itself of it, nevertheless, both humoral and cell-mediated reactions and specific and nonspecific host defense mechanisms respond and eventually eliminate the , preventing invasion into the deeper viable tissue. This array of defense mechanisms thought to be active against dermatophytes consists of a 2-macroglobulin keratinase inhibitor (Yu et al., 1972), unsaturated transferrin (King et al., 1975), epidermal desquamation (Berk, 1979), and lymphocytes, macrophages, neutrophils, and mast cells

(Calderon, 1989).

There are two major classes of dermatophyte antigens: glycopeptides and keratinases. The protein portion of the glycopeptides preferentially stimulates cell-mediated immunity

(CMI), whereas the polysaccharide portion preferentially stimulates humoral immunity

(Dahl, 1993). Keratinases, produced by the dermatophytes to enable skin invasion, elicit

22 delayed-type hypersensitivity (DTH) responses when injected intradermally into the skin of animals (Grappel and Blank, 1972).

Although the host develops a variety of antibodies to dermatophyte infection, i.e., immunoglobulin M (IgM), IgG, IgA, and IgE, they apparently do not help eliminate the infection since the highest level of antibodies is found in those patients with chronic infection (Dahl, 1987). IgE, which mediates immediate hypersensitivity, appears to play no role in the defense process (Dahl, 1993; Jones, 1993). Rather, the development of CMI which is correlated with DTH is usually associated with clinical cure and ridding the stratum corneum of the offending dermatophyte (Dahl, 1993; Jones, 1993). In contrast, the lack of CMI or defective CMI prevents an effective response and predisposes the host to chronic or recurrent dermatophyte infections (Jones, 1986; 1993; Jones et al., 1975).

Several in vitro systems have been studied to assess CMI in dermatophyte-infected hosts, e.g., lymphocyte transformation (Helander, 1978;Svejgaard et al., 1976), leukocyte migration inhibition (Hay and Brostoff, 1977; Hay et al., 1983), and leukocyte adherence inhibition (Hay and Brostoff, 1977; Hay et al., 1983; Walters et al., 1974;Walters et al.,

1976). Lymphocyte transformation is a widely used in vitro assay of cellular immune function (Calderon, 1989).

Experimental animal models have been used to study the role of CMI during dermatophytosis, and the results are summarized by Calderon (1989). Clearance of infection was found to correlate with DTH to dermatophyte antigens on skin testing. Green et al. (1983) showed that athymic (nude) rats that lack T-cell-mediated immunity could not clear T. mentagrophytes infections compared with genetically matched euthymic control

23 rats. Calderon (Calderon, 1989) demonstrated in experiments with mice that the T-helper lymphocytes bearing the phenotype Thy-11 Ly2 mediate immunity to dermatophytosis.

Immunity to dermatophyte infection in experimentally infected mice could be achieved by adoptive transfer of lymphoid cells but not serum from infected donors (Calderon, 1989;

Calderon and Hay, 1984).

The classical studies of Jones and coworkers (Jones, 1986; 1993; Jones et al., 1973; 1974) in human volunteers suggested that CMI is the major immunologic defense in clearing dermatophyte infections. Experimentally infected volunteers deliberately infected with T. mentagrophytes who developed CMI associated with intense inflammation accompanied by T-cell-mediated DTH to the trichophytin skin test (glycoprotein skin test antigen) achieved a mycologic cure. A protective immunologic memory was indicated by the rapid inflammatory response and elimination of the fungus on reinoculation and a continued positive trichophytin test. A single volunteer, who was atopic, characterized another group of individuals having a second type of reaction: i.e., development of a chronic or recurrent infection, high immediate- type (anti-Trichophyton IgE mediated) hypersensitivity, and low or waning DTH to trichophytin (Jones, 1993). These individuals, however, had a normal response to other skin test antigens, indicating a selective or induced immune deficit that was found in 10 to 20% of the population in temperate climates (Jones et al.,

1973). An association between chronic dermatophytosis and atopy (asthma or allergic rhinitis) is well recognized (Hanifin et al., 1974; Hay, 1982; Hay and Brostoff, 1977; Hay and Clayton, 1982; Jones et al., 1973), and several mechanisms explaining this association have been suggested by Jones (Jones, 1993). Approximately 80 to 93% of chronic or recurrent dermatophyte infections are estimated to be caused by T. rubrum; these patients

24 often fail to express a DTH reaction to trichophytin when injected intradermally (Blake et al., 1991; Hay, 1982).

Infections by anthropophilic fungi, like T. rubrum, often elicit less of an inflammatory response and are less likely to elicit an intense DTH response than infections caused by geophilic or zoophilic dermatophytes which characteristically evoke an intense inflammatory reaction. Much of this inflammation is produced by activated lymphocytes and macrophages which are involved in the DTH reaction to the trichophytin glycopeptides. Enhanced proliferation of the skin in response to the inflammation may be the final mechanism that removes the fungus from the skin by epidermal desquamation

(Dahl, 1987; Berk et al., 1976) had earlier reported that dermatophytes can be removed from the skin by accelerated epidermal turnover. There are some evidence that certain dermatophytes, like T. rubrum, produce substances that diminish the immune response.Mannan, a glycoprotein component of the fungal cellwall, may suppress the inflammatory response especially inatopic or other persons susceptible to the mannan- inducedsuppression of CMI (Dahl, 1987; Blake et al., 1991) demonstrated thatincubation of purified T. rubrum mannan (TRM) with peripheralblood mononuclear cells suppressed lymphoblast formationand inhibited the lymphocyte proliferation response tomitogens and a variety of antigenic stimuli. Also, it has been shown that TRM inhibits keratinocyte proliferation, thusslowing epidermal turnover and allowing for a more persistentchronic infection.

Grando et al. (1992) identified the monocyte as the likely target cell for the immunosuppressive influence of TRM on the basis of observations made by using a fluorescein conjugate of TRM (fluorescein isothiocyanate-TRM) in conjunction with

25 fluorescence microscopy and flow cytometry. They found that monocytes, not lymphocytes, bound fluorescein isothiocyanate- TRM and that the surface-bound ligand appeared to be internalized and digested over time. They suggested that this binding, which appeared to be receptor cell mediated, interferes with accessory cell functions of the monocyte in CMI (Grando et al., 1992).

Blake et al. (1991) compared the abilities of the cell wall mannan glycoproteins from two dermatophyte species to inhibit CMI in vitro. He used a zoophilic dermatophyte (M.canis), which causes an intense inflammatory reaction, and T.rubrum, which is associated with a chronic, non inflammatory reaction. Although mannan from both species significantly inhibited OKT3 antibody-stimulated lymphoproliferation, which was dose dependent,

TRM was isolated in a greater amount than was M. canis mannan and was more inhibitory.

The investigators speculated that the increased amount and potency of TRM compared with that of M. canis may explain why T.rubrum elicits less inflammation and causes a more chronic infection than M. canis (Carfachia et al., 2013).

Chronic dermatophytosis may also be caused by the anthropophilic form of T. mentagrophytes, T. mentagrophytes var. interdigitale (T. interdigitale) (Gregurek-Novak et al., 1993). Gregurek-Novak et al. (1993) studied primary chronic trichophytosis in Croatia and found it to be mostly caused by this fungus (T. mentagrophytes). They found that this clinical entity was associated with defective phagocytosis by peripheral blood leukocytes, i.e., impaired random mobility, and ingestion and digestion of foreign material. The patients were not abnormal in their skin test reactions with Mycobacterium tuberculosis purified protein derivative, the numbers of T and B lymphocytes in their peripheral blood, or their concentrations of immunoglobulins in serum. They concluded that primary chronic

26 trichophytosis appears to be associated with defective phagocytosis of peripheral blood leukocytes and that this defect is probably caused by the fungus itself.

Although there are no serological kits commercially available to specifically detect and identify antibodies to dermatophytes, studies of dermatophyte antigens by monoclonal antibodies indicate a potential use of such reagents in the immunoidentification of dermatophytes (De Haan et al., 1989; Polonelli and Morace, 1989). Polonelli and Morace

(1989) suggested that the effectiveness of monoclonal antibodies may be enhanced by using the Western blotting (immunoblotting) technique and those difficulties in finding specific monoclonal antibodies devoid of cross-reacting antibodies may be overcome by newer methods such as affinity chromatography. A compilation of serological procedures to detect dermatophyic antigens may be found in the review by Polonelli and Morace

(1989).

The early literature on the immunology and immunochemistry of dermatophytosis is reviewed by Grappel et al. (1974); another method of characterization of dermatophyte antigens was presented by De Haan et al. (1989).

Descriptions of the immunoregulation and immunology of dermatophytosis may be found in review articles by Dahl (1993), Calderon (1989), and Jones (1993). An update on the suppression of immunity by dermatophytes is given by Dahl (1993).

2.11 Susceptible Host Range and Resistance

Animals with immune suppression such as those treated with glucocorticoids for other conditions.are more prone to establishment of fungal infection. Horses contaminated by

27 other skin conditions, such as prevalence of lice or those in an environment with a high incidence of biting flies are also prone to fungal infections. Those housed in crowded, moist or filthy conditions are highly susceptible, too. The presence of skin abrasions, however slight, or horses with areas subject to skin friction such as girth or bridle irritation, are also at risk for ringworm.

2.11.1 Transmission

People and animals become infected by dermatophytes after contact with spores (conidia).

Dermatophytes growing in a vertebrate host normally form only arthrospores

(arthroconidia), asexual spores that develop within the hyphae. In the environment (e.g., in laboratory culture), they can also produce microconidia and macroconidia, asexual spores that develop outside the hyphae. Initially, dermatophyte infects a growing hair or the stratum corneum of the skin. These organisms do not usually invade resting hairs, since the essential nutrients they need for growth are absent or limited. Hyphae spread in the hairs and keratinized skin, eventually developing infectious arthrospores. (MacKenzie et al.,

1986)

Anthropophilic and zoophilic dermatophytes are mainly transmitted between hosts by arthrospores in hairs or skin scales. Other asexual or sexual spores formed by the environmental stages may also be infectious. Fomites such as brushes and clippers are important in transmission. Spores may remain viable in suitable environments for up to 12-

20 months, and some spores were also reported to persist for at least a year in salt water.

Certain types of spores (e.g., microconidia) might be dispersed by airborne means.

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2.11.2 Clinical signs and anatomical location of lesions

Dermatophyte lesions in animals are characterized by areas with varying degrees of alopecia, scaling, crusts and erythema, and may or may not be pruritic. Hairs in the affected area are usually brittle and break near the skin surface, often giving the lesion a

“shaved” appearance; truncated hair shafts may be seen through the scales and crusts.

(Hofbauer et al., 2002) Occasionally, dermatophytes may die at the center of a lesion and that area resolves, leaving a circular lesion with central crusts or hair re-growth. Some degree of folliculitis occurs in most cases; papules or pustules involving the hair follicle or conical dilation of the hair follicle ostium are suggestive of dermatophytosis in small animals. Asymptomatic infections are also common, particularly in adult animals (OIE,

2005).

In horses, most dermatophyte lesions are found in areas of contact with saddle and girth.

They usually begin as small patches of raised hairs, and progress to hair loss, with variable amounts of scaling, erythema, crusting and exudation. M. canis lesions are reported to be milder, in most cases, than T equinum. Kerions may occur in some animals, especially on the face. Miliary dermatitis may also be seen, with small crusted lesions especially on the flanks. Early dermatophyte lesions can sometimes resemble papular urticaria, but more characteristic signs develop within a few days. Lesions may coalesce, especially where the skin is abraded from tack.

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2.12 Dermatophytosis in Other Domestic Animals

Cats

Many cats infected with dermatophytes have few or no lesions. Long-haired adults, in particular, can be subclinical carriers or have only minimal signs, such as patchy areas of alopecia, erythematous plaques, visible only on close inspection. More apparent cases tend to be seen in kittens, with the early lesions often found on the face, ears and paws. (Nuttall et. al., 2008) In addition to focal alopecia and scales, affected areas may develop a thin, grayish white crust or a thick, moist scab. They may or may not be pruritic. The cat‟s grooming behavior may eventually spread the infection to the entire body. Other presentations that have been reported in cats include miliary dermatitis and recurrent chin acne. Severe cases of dermatophytosis, with large, erythematous, alopecic, exudative lesions, may be seen in debilitated cats, or in animals that have been treated with corticosteroids (Brilhante, 2006). Onchomycosis can occur concurrently in cats with dermatophytosis; the nails may be opaque, with whitish mottling, and shredding of the nail surface (Cafarchia et al., 2010).

Uncomplicated dermatophyte lesions are usually self- limiting within a few weeks to a few months in short-haired cats; however, the organisms may persist, either symptomatically or asymptomatically, in long-haired cats. (Holzworth , 1987).

Dogs

Dermatophytosis is seen most often in puppies. The lesions frequently develop on the face and limbs, although they may occur on any part of the body. M. canis tends to appear as

30 small circular areas of alopecia. The hairs are typically broken at the base, giving the appearance of having been shaved. (Cervantes, 2003)The center of the lesion usually contains pale skin scales in the early stage, giving it a powdery appearance, and the edges are generally erythematous. Vesicles and pustules may also be seen. In later stages, the area is often covered by a crust and the edges swollen. Individual lesions may coalesce to form large, irregular patches. (Chah et al., 2012). Lesions caused by T. mentagrophytes and T erinacei tend to be more thickened and inflammatory than those caused by M. canis while M persicolor typically causes localized or generalized scaling with little erythema and minimal alopecia. Other forms of dermatophytosis can include kerions (localized severe inflammation with swollen, boggy skin oozing pus) and pseudomycetomas.

Onchomycosis may occur concurrently with dermatophytosis (Cafarchia et al., 2010).

Although dermatophytosis is often self-limited in dogs, some animals can develop severe, chronic cases with widespread lesions, and severe inflammation and alopecia. Generalized cases in adult dogs usually occur in immunosuppressed animals, especially those that have hyperadrenocortictropism or have been treated with corticosteroids (Seebacher et al.,

2008).

Cattle

In cattle, dermatophytosis varies from small focal lesions to extensive generalized skin involvement. The initial lesions may be discrete, scaly and alopecic with grayish-white crusts, and tend to appear on the face and neck in calves. (Kawasaki , 2011) Cows and heifers may have lesions more often on the chest and limbs, and bulls on the dewlap and intermaxillary skin. Some areas may become suppurative and thickly crusted. Lesions

31 resembling light brown scabs may also be seen; when these scabs fall off, they leave an area of alopecia. The clinical signs usually resolve spontaneously in 2 to 4 months

(Seebacher et al., 2010).

Sheep and goats

The most noticeable signs are usually circular, alopecic areas with thick scabs on the head, face and non-wooled areas of the legs; however, widespread lesions may be found under the wool. In healthy lambs, the disease is usually self-limiting. (Brilhante, 2006)

Swine

Pigs may develop a wrinkled lesion covered by a thin, brown, easily removed scab, or a spreading ring of inflammation Dermatophyte infections are often asymptomatic in adult swine (Chermette et al., 2008).

Rodents

Most rodents infected with Trichophyton mentagrophytes are asymptomatic or have few clinical signs. There may be areas of partial or complete alopecia, erythema, scales, and crusts in symptomatic animals (Harkness et al., 1983). In guinea pigs, the lesions tend to appear first on the face, then spread to the back and limbs. In mice, the lesions are often found on the tail (Cafarchia et al., 2010).

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Rabbits

Focal alopecia, with erythema, crusts, scales and scabs, is initially seen mainly around the eyes, nose, ears and dorsal neck. The lesions may later spread to other areas of the body.

The disease is usually self-limiting (Cafarchia et al., 2010).

Birds

In cage birds, there may be alopecia and scales, particularly on the face, head, neck and chest, as well as auto-mutilation and feather plucking. The head and neck, especially the comb, are often affected in fowl .The lesions may include white crusts or plaques and hyperkeratosis, although feathers may be lost in birds (Koski, 2002).

Reptiles

Reptiles are not usually affected by dermatophytes of mammals or birds; however, there are rare case reports of dermal lesions in lizards and green anacondas. Clinical signs reported during a Trichophyton outbreak in iguanas included scaling, crusting, thickening of the skin and ulcerative dermatitis (Miller, 2004). Trichophyton spp. infection was associated with papular and pustular cutaneous lesions in a Tenerife lizard. This animal died of an undetermined illness, soon afterward. Trichophyton spp. was also detected by immunohistochemistry from systemic lesions in a moribund sea turtle (Miller, 2004).

2.12.1 Morbidity in other domestic animals

Whether an animal becomes infected after contact with a dermatophyte may depend on the animal‟s age, the condition of its exposed skin, general health and grooming behavior

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(Borman et al.,, 2007). Young animals, including puppies, kittens, calves, lambs and young camelids, are more likely to have symptomatic infections than adults. (Pascoe,

1984) Clinical dermatophytosis is also thought to be more common in immunosuppressed animals. Most infections in healthy animals heal spontaneously within one to a few months. Hair loss is not permanent unless the follicle has been destroyed by inflammation.

Infections can be more persistent or widespread in young or sick animals (Pascoe, 1984).

Dermatophytes can be isolated from animals with or without clinical signs (asyptomatic infections). Highly variable infection rates, between 6% and 100%, have been reported in surveys of cats, which are thought to carry these organisms more often than dogs (Espinel-

Ingroff et al., 2002) The estimated prevalence among pet cats and dogs in individual households is still unclear. While some surveys suggest that many cats are infected with these organisms, one University of Wisconsin study did not detect dermatophytes in any of

182 asymptomatic pet cats that lived alone with their owners (Pascoe, 1984).

Among livestock, dermatophytes are particularly common in cold climates where animals are stabled for long periods of time. This disease usually becomes endemic in cattle herds, where it most often affects animals under a year of age. (Borman et al.,, 2007). The lesions tend to develop in cattle when they are stabled indoors in winter, and to resolve when they are turned out in the spring. Clinical cases do not seem to be common in sheep and goats, with the exception of show lambs; however, M. canis caused some outbreaks that affected

20-90% of sheep herds in Australia. It is possible that cases are under diagnosed in small ruminants. Infected animals are reported to be common on rabbit farms in some countries.

Clinical cases seem to be infrequent in birds (Borman et al.,, 2007).

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2.13 Distribution of Lesions of Dermatophytes

Distribution of the gross lesions on cattle, sheep and horses usually correlates with the predisposing factors that reduce or permeate the natural barriers of the integument. In cattle, the lesions can be observed in three stages:

1) hairs matted together as paint-brush lesions

2) crust or scab formation as the initial lesions coalesce, and

3) accumulations of coetaneous keratinized material forming wart- like material

lesions that are 0.5-2cm in diameter. (Menges and Georg,1955)

Typical lesions consist of raised, matted tufts of hair. Most lesions associated with prolonged wetting of the skin are distributed over the head, dorsal surfaces of the neck and body, and upper lateral surfaces of the neck and chest. (Katoh et al., 1990) Cattle that stand for long periods in deep water and mud develop lesions in areas such as skin folds of the flexor surfaces of the joints. Dairy cows may present with papular crusted lesions on the udder. Lesions initiated by biting flies (mechanical vectors) are found primarily on the back, whereas lesions induced by ticks are primarily on the head, ears, axillae, groin, and scrotum.

Chronic lumpy wool infections are characterized by pyramid-shaped masses of scab material bound to wool fibers. The crusts are primarily on the dorsal areas of the body and prevent the shearing of sheep; spiny plants often predispose to lesions on the lips, legs, and feet. Strawberry footrot is a proliferative dermatitis affecting the skin from the coronet to the carpus or hock. (Sidrim and Rocha, 2004)

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Lesions on horses with long winter hair coats are similar to those of cattle, developing with matted hair and paint-brush lesions leading to crust or scab formation with yellow- green pus present under larger scabs. With short summer hair, matting and scab formation is uncommon; loss of hair with a fine paint-brush effect can be extensive. (Ajello, 1968;

1977; Matsumoto and Ajello,1987) Persistent wetting of pasterns in wet yards, stables, or at pasture leads to lower limb infection; white legs and the white-skinned areas of the lips and nose are more severely affected. Generalized disease is also associated with prolonged wet weather. Outbreaks occur on farms with previously affected horses.

2.13.1 Histopathological findings

Histopathologic examination reveals the characteristic branching hyphae with multidimensional septations, coccoidal cells, and zoospores in the epidermis. The organisms are usually abundant in active lesions but can be sparse or absent in chronic lesions. The most common histopathological findings include:

1) Perifolliculitis, folliculitis, and furunculosis more specifically, infiltrative lymphocytic mural folliculitis, suppurative luminal folliculitis, and pyogranulomatous furunculosis;

2) Hyperplastic or spongiotic superficial perivascularor interstitial dermatitis with prominent parakeratotic or orthokeratotic hyperkeratosis of the epidermis and hair follicles;

3) Intraepidermal pustular dermatitis (suppurative, neutrophilic epidermitis).

Arthroconidia and hyphae can be detected in hair shafts with H&E staining, but special staining such as Periodic acid-Schiff (PAS) (Menges and Georg, 1955).

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2.13.2 Clinical diagnosis

Physical examination of the skin of animals and a complete clinical examination of all affected animals should always be performed. Evaluation of the general state of the animals, temperature, pulse rate, respiratory rate, appetite and morbidity rates should be recorded. The shape, size, position, distribution and time of the appearance of skin lesions as well as the age of the animals should also be recorded (Al-Ani et al., 2002.).

Diagnosis is based on the history, physical examination, and microscopic examination of scrapings and hairs from the lesions, sometimes in conjunction with fungal culture and other techniques such as Wood‟s lamp examination and histology of the tissues.

(Svejgaard, 1986; Chermette et al., 2008).

Microscopic examination of skin scrapings or plucked hairs may reveal hyphae or arthroconidia. Hyphae rounding up into arthroconidia are diagnostic; however, hyphae alone could be caused by other fungi, including contaminants. Samples should be selected from the margins of active lesions, or from the entire lesion if there is no inflammatory margin. The best hairs to select are those that fluoresce under a Wood's lamp, or are broken or scaly. Samples are usually cleared with potassium hydroxide (KOH) to help visualize the organism. A longer clearing time can be helpful when the hair is thicker and more heavily pigmented, or if the sample is taken from a thick, crusted lesion. (Mackenzie,

1963; Adekeye et. al., 1989; Macura, 1993) Various stains such as chlorazol black E,

Parker blue-black ink, Swartz-Lamkin stain, Congo red stain or Giemsa can aid the visualization of fungal structures. In practice where fluorescence microscopy is available, calcofluor white staining can be used. Skin scrapings or plucked hair samples for culture

37 should be taken from active lesions, as for microscopic examination. Nail beds and claws are cultured in cases of onchomycosis. Swabbing dermatophyte lesions first with alcohol may decrease contaminants, especially in livestock (Abo El-Yazeed et al., 2013).

Additional collection methods, which are especially helpful in asymptomatic animals suspected of being carriers, include brushing the fur with a disinfected toothbrush or other small brush, or rubbing it with a sterile piece of carpet. Dermatophytes can be cultured on various fungal media, including Sabouraud agar (with cycloheximide and antibiotics) and dermatophyte test medium (DTM). Cultures are usually incubated at room temperature

(20–28ºC), but higher temperatures can be used when T. verrucosum is suspected.

Colonies often become visible within 1-2 weeks but, some species grow more slowly and may require longer to appear. Colony morphology can differ with the medium; descriptions are usually based on Sabouraud agar. DTM contains a pH indicator (phenol red) that will turn the medium red when a dermatophyte is growing. However, the mycelial growth must also be examined microscopically, as this color change alone is not diagnostic and could be produced by other fungal or bacterial organisms. In addition, the color change may be delayed with certain dermatophytes such as M. persicolor. In asymptomatic animals, caution must be used to distinguish infection from contamination of the coat with organisms from the environment (Batte et al., 1981)

Dermatophyte species can be identified by the colony morphology; the appearance of microconidia, macroconidia and other microscopic structures. Microconidia and macroconidia can be used to distinguish the genera Microsporum, Trichophyton and

Epidermophyton. Members of Microsporum spp. produce microconidia and rough-walled, multiseptate macroconidia. The thickness of the wall, shape and number of macroconidia

38 vary with the species (Lund et al., 2008) Trichophyton spp. produce microconidia and smooth, thin-walled, cigar shaped macroconidia. Macroconidia are rarely seen with some species. E. floccosum, which is anthropophilic and has very rarely been reported in animals, (Gehrt et al., 1995.) produces large, thin-walled, multicellular, club-shaped, clustered macroconidia. This organism does not produce micro-conidia. Specialized tests, such as the ability to penetrate hairs in vitro or mating tests performed at reference laboratories, may occasionally be used in the differentiation process. Differential media

(e.g. bromocresol purple - milk solids glucose) can also be helpful (Al-Ani et al., 2002.).

2.14 Dermatophytosis in Humans

Traditionally, infections caused by dermatophytes have been named according to the anatomic locations involved by appending the Latin term designating the body site after the word tinea, e.g., tinea capitis for ringworm of the scalp. The clinical manifestations are as follows:

(i) tinea barbae (ringworm of the beard and mustache)

(ii) tinea capitis (scalp, eyebrows, and eyelashes)

(iii) (glabrous skin)

(iv) tinea cruris (groin); (v) tinea favosa (favus)

(vi) tinea imbricata (ringworm caused by T. concentricum)

(vii) tinea manuum (hand)

(viii) tinea pedis (feet); and

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(ix) tinea unguium (nails)

Tinea Barbae

Tinea barbae, an infection of the bearded area, may be mild and superficial or a severe inflammatory pustular folliculitis,the latter form more commonly caused by the zoophilic dermatophytes Trichophyton verrucosum, T. mentagrophytes var.mentagrophytes, and T. mentagrophytes var. erinacei (Kwon-Chung et al.,1992).

Tinea Capitis

Tinea capitis, an infection commonly involving the scalp, is usually caused by members of the genera Microsporum and Trichophyton. The infection may range from mild, almost subclinical, with slight erythema and a few patchy areas of scaling with dull gray hair stumps to a highly inflammatory reaction with folliculitis, kerion formation, and extensive areas of scarring and alopecia, sometimes accompanied by fever, malaise, and regional lymphadenopathy (Rippon, 1985). Both the skin surface and hairs are involved. Infection of the hair may be described as ectothrix (sheath of arthroconidia formed on the outer side of the hair shaft) or endothrix (arthroconidia formed within the hairshaft). The current predominant cause of tinea capitis in most of North, Central, and South America is T. tonsurans (endothrix) replacing M. audouinii (ectothrix) (Rippon, 1985).

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Tinea Corporis

Ringworm of the body, usually involving the trunk, shoulders, or limbs, and occasionally the face (excluding the bearded area), may be caused by any dermatophyte. The infection may range from mild to severe, commonly appearing as annular, scaly patches with sharply marginated, raised erythematous vesicular borders ( Kwon-Chung et al.,1992).

Tinea Cruris (‘‘Jock Itch’’)

Infection of the groin, perianal, and perineal areas, and occasionally the upper thighs, is usually seen in adult men. T.rubrum and E. floccosum are the most frequent etiologic agents. Lesions are erythematous to tawny brown and coveredwith thin, dry scales. They are usually bilateral and often asymmetric,extending down the sides of the inner thigh and exhibiting a raised, sharply marginated border that is frequently studded with small vesicles ( Kwon-Chung et al.,1992).

Tinea Favosa

Tinea favosa, usually caused by Trichophyton schoenleinii, is severe and chronic, characterized by the presence on the scalp and glabrous skin of yellowish, cup-shaped crusts called scutula, which is composed of epithelial debris and dense masses of mycelium. The disease is most common in Eurasia and Africa (Rippon, 1988).

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Tinea Imbricata

Tinea imbricata, the chronic infection which is a specialized manifestation of tinea corporis, is characterized by concentric rings of overlapping scales scattered throughout the body. It is geographically restricted to certain of the Pacific islands of Oceana, Southeast

Asia, Mexico, and Central and South America (Rippon, 1988). T. concentricum, a strictly anthropophilic dermatophyte, is the only etiologic agent (Rippon, 1988).

Tinea Manuum

The palmar and interdigital areas of the hand are usually involved in tinea manuum, most frequently presenting as unilateral diffuse hyperkeratosis with accentuation of the flexural creases. Most infections are caused by T. rubrum (Rippon, 1988).

Tinea Pedis (‘‘Athlete’s Foot’’)

The feet, especially the soles and toe webs, are most frequently involved in tinea pedis.

The most common clinical manifestation is the intertriginous form, which presents with maceration, peeling, and fissuring, mainly in the spaces between the fourth and fifth toes.

Another common presentation is the chronic, squamous, hyperkeratotic type in which fine silvery scales cover pinkish skin of the soles, heels, and sides of the foot (moccasin foot).

An acute inflammatory condition, characterized by the formation of vesicles, pustules, and sometimes bullae, is most frequently caused by T. mentagrophytes. The more chronic agents of tinea pedis are T. rubrum, T.mentagrophytes var. interdigitale, and E. floccosum(Rippon, 1985).

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Tinea Unguium

Invasion of the nail plate by a dermatophyte is referred to as unguium while infection of the nail by nondermatophytic fungi is called onychomycosis. The latter word is often used as a general term for a nail infection. There are two main types of nail involvement: invasive subungual (distal and proximal) and superficial white mycotic infection

(leukonychia trichophytica). T. rubrum and T. mentagrophytes, respectively, are the most common dermatophytes isolated in this infection (Rippon, 1988).

2.15 Classes of Antifungal Agents and their Resistance

2.15.1 Antifungal agents

Despite extensive research dedicated to the development of new therapeutic strategies, there are only a limited number of available drugs to fight against invasive fungal infections. Indeed, only four molecular classes that target three distinct fungal metabolic pathways are currently used in clinical practice to treat essentially systemic fungal infections: fluoropyrimidine analogs, polyenes, azoles, and echinocandins. Several other classes, such as morpholines and allylamines are only used as topical agents due to either poor efficacy, or severe adverse effects when administered systemically (Walsh et al..

1996).

2.15.2 Fluoropyrimidines

Fluoropyrimidines, of which only 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU) are used in human medicine, are synthetic structural analogs of the DNA nucleotide cytosine.

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5-FC was synthesized in 1957 by Duschinsky initially as an antitumor therapy. In 1963,

Grunberg and his co-workers discovered its antifungal potential by means of murine models of cryptococcosis and candidiasis. Several years later 5-FC was successfully used for the treatment of systemic candidiasis and of cryptococcal meningitis (Grunberg, 1963).

5-FC possesses a broad range of activity. This drug is active against Candida and

Cryptococcus genera. 5FC activity on Phialophora, Cladosporium, and Aspergillus genera is much less limited. 5-FC is also active against protozoa belonging to Leishmania and

Acanthamoeba genus (Walsh et al.. 1996).

Due to its high hydrosolubility and small size, 5-FC possesses interesting pharmacokinetic properties, since it diffuses rapidly throughout body even when orally administered.

Generally, it produces negligible side effects, despite some severe adverse effects, such as hepatotoxicity or bone marrow lesions, occurring in rare cases. (Eucker et al., 2001).

Surprisingly, these side effects are identical to those observed with 5-FU treatment, despite the fact that humans do not possess a cytosine deaminase enzyme that is responsible for the conversion from 5-FC into 5-FU in fungal cells. Some data suggest that the intestinal microbiome could be responsible for the 5-FU production and the observed side effects

(Eucker et al., 2001).

Despite its numerous pharmacological advantages, the use of 5-FC in clinical practice is decreasing because of the frequent occurrence of innate or acquired resistance to this drug in fungal pathogens. Thus, with few exceptions, 5-FC is never used as monotherapy but always in combination with another antifungal, such as amphotericin B. However, the elevated renal and liver toxicities of amphotericin B, that further increase 5-FC

44 hepatotoxicity, has led to combination therapy of 5-FC more frequently with azole drugs

(Walsh et al., 1996).

5-FC itself has no antifungal activity, and its fungistatic properties are dependent upon the conversion into 5-FU. The drug rapidly enters the fungal cell through specific transporters, such as cytosine permeases or pyrimidine transporters, before it is converted into 5-FU by the cytosine deaminase. 5-FU itself is converted into 5-fluorouracil monophosphate (5-

FUMP) by another enzyme, uridine phosphoribosyltransferase (UPRT). 5-FUMP can then be either converted into 5-fluorouracil triphosphate, which incorporates into RNA in place of UTP and inhibits protein synthesis, or converted into 5-fluorodeoxyuridine monophosphate, which inhibits a key enzyme of DNA synthesis, the thymidylate synthase, thus inhibiting cell replication (Duschinsky et al., 1960).

2.15.3 Polyenes

More than 200 molecules belonging to the chemical class of polyenes have an antifungal activity, most of them being produced by Streptomyces bacteria. However, only three possess a toxicity allowing their use in clinical practice: amphotericin B (AmB), nystatin, and natamycine.

Streptomyces bacteria synthesize polyenes through a gene cluster phylogenetically conserved within these species. This cluster contains genes coding for several polyketide synthases, ABC (ATP-binding cassette) transporters, cytochrome P450-dependent enzymes, and enzymes responsible for the synthesis and the binding of the mycosamine group. Although it is possible to synthesize polyenes chemically, they are still produced from Streptomyces cultures for economic reasons (Duschinsky et al., 1960).

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Polyenes are cyclic amphiphilic organic molecules known as macrolides. Most of them consist of a 20 to 40 carbons macrolactone ring conjugated with a d-mycosamine group.

Their amphiphilic properties are due to the presence of several conjugated doublebounds on the hydrophobic side of the macrolactone ring, and to the presence of several hydroxyl residues on the opposite, hydrophilic side. Polyene drugs target ergosterol, the main sterol component of fungal membranes. Their amphiphilic structure allows them to bind the lipid bilayer and form pores. Magnetic nuclear resonance data suggest that eight AmB molecules bind eight ergosterol molecules through their hydrophobic moieties, with their hydrophilic sides forming a central channel of 70–100 nm in diameter (Duschinsky et al.,

1960).

Pore formation promotes plasma membrane destabilization, and channels allow leakage of intracellular components such as K+ ions, responsible for cell lysis .While structural data suggest that polyenes target ergosterol, and despite the fact that their binding to ergosterol was experimentally demonstrated, controversy remains over a possible intracellular mode of action. Some research has suggested that polyene drugs are able to induce an oxidative stress (particularly in C. albicans as well as their activity seems to be reduced in hypoxic conditions.

Polyenes possess a lower but non-negligible affinity for cholesterol, the human counterpart of ergosterol. This slight affinity for cholesterol explains the high toxicity associated with and is responsible for several side effects (Eucker et al., 2001). For this reason, only AmB is given systemically, while nystatin and natamycin are only used locally or orally. These two last molecules fortunately possess a very limited systemic activity, since their absorption trough gastrointestinal mucosa is almost nonexistent (Eucker et al., 2001).

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For these reasons, AmB is the most used polyene antifungal for systemic infections. Due to its high hydrophobicity and poor absorption through the gastrointestinal tract, it is necessary to administer AmB intravenously (Walsh et al., 1996). However, AmB administration is accompanied with adverse effects, mostly at the level of kidneys and liver. New AmB formulations, such as liposomal AmB or lipid AmB complexes, minimize such side effects (Arendrup et al., 2010).

For more than 40 years, AmB was one of the goldstandards for the treatment of systemic fungal infections due to the low occurrence of acquired or innate resistance to this drug and also because of its broad range of activity. Indeed, AmB is active against most yeasts and filamentous fungi. It is recommended for the treatment of infections caused by Candida,

Aspergillus, Fusarium, Mucor, Rhizopus, Scedosporium, Trichosporon, (Duschinsky et al.,

1960).

Cryptococcus, and so on. AmB is also widely used to treat parasitic infections such as leishmaniasis and amibiasis. Natamycin and nystatin are active against fungi belonging to the genera Cryptococcus, Candida, Aspergillus, and Fusarium. Nystatin is frequently used for the treatment of cutaneous, vaginal, and esophageal candidiasis, and natamycin can be used for the treatment of fungal keratosis or corneal infections (Walsh et al., 1996)].

2.15.4 Azoles

Azoles are by far the most commonly used antifungals in clinical practice, and consequently, they are also the mostly studied by the scientific community regarding their mode of action, their pharmacological properties, and the resistance mechanisms developed by microorganisms. Azole antifungals are also largely studied by

47 pharmaceutical companies, who seek to enhance their efficacy and to develop the perfect antifungal (Arendrup et al., 2010).

Azoles are cyclic organic molecules which can be divided into two groups on the basis of the number of nitrogen atoms in the azole ring: the imidazoles contain two nitrogen atoms, and the triazoles contain three nitrogen atoms Azole drugs target the ergosterol biosynthetic pathway by inhibition of a key enzyme, the lanosterol 14 alpha demethylase, encoded be the erg11 gene. This inhibition occurs through the binding of the free nitrogen atom of the azole ring to the iron atom of the heme group of the enzyme. The resulting accumulation and metabolism of 14alpha methylated sterol species leads to the synthesis of toxic compounds, which are unable to successfully replace ergosterol (Grunberg et al.,

1963).

The first azole was synthesized in 1944 by Woolley, but it was not until 1958 that scientific community began to consider azoles as potential antifungal agents. In late 1960s, clotrimazole, econazole, and miconazole became available for treatment. However, their use was restricted to external application due to their high toxicity when administered orally. In 1968, miconazole became the first antifungal available for parenteral injection, but due to its toxicity and relatively limited range among fungal species, its use decreased until it was no longer commercialized (Grunberg et al., 1963).

In 1981, the Food and Drug Administration (FDA) approved a new antifungal, ketoconazole, developed by Heeres and his coworkers. This drug was the only antifungal available for treatment of systemic fungal infections caused by yeasts for the following ten years. However, there are several drawbacks to this drug. It is poorly absorbed when

48 administered orally, and no ketoconazole form has ever been developed for intravenous injection. Moreover, it cannot cross the cerebrospinal barrier and is less active in immunosuppressed patients. It causes some severe side effects such as a decrease in testosterone or glucocorticoids production and liver and gastrointestinal complications.

Lastly, numerous interactions with other drugs were described. For these reasons, the triazoles were developed (Grunberg et al., 1963).

Fluconazole became available for use by clinicians in 1990 and provided many advantages over the use of imidazoles. Fluconazole is highly hydrosoluble and therefore can be easily injected intravenously. It is almost completely absorbed through the gastrointestinal tract, and it diffuses throughout the whole body, including cerebrospinal fluid. Fluconazole is suitable for the treatment of superficial candidiasis (oropharyngal, esophageal, or vaginal), disseminated candidiasis, cryptococcal meningitis, coccidioidomycosis, and cutaneous candidiasis. Due to its good pharmacokinetic properties as well as its broad spectrum of activity, fluconazole was the gold-standard treatment of fungal infections during the 1990s.

Unfortunately, the overprescription of this drug by physicians for prophylaxis or treatment led to an increase in resistance to azole drugs. Moreover, fluconazole is almost ineffective against most molds (Tassel et al., 1968)

Itraconazole was approved and made available by the FDA in 1992. This triazole possesses a broad spectrum of activity across fungal species comparable to this of ketoconazole and wider than fluconazole. Moreover, it is less toxic than ketoconazole and replaced it for treatment of histoplasmosis, blastomycosis, and paracoccidioidomycosis. Contrary to fluconazole, it is also used for the treatment of infections due to species belonging to the genera Aspergillus and Sporothrix. However, itraconazole is hydrophobic and is thus more

49 toxic than fluconazole. Itraconazole is only indicated for the treatment of onychomycosis, of superficial infections, and in some cases for systemic aspergillosis. A new itraconazole formulation with an enhanced absorption and a decrease toxicity was approved by FDA in

1997. An injectable formulation of itraconazole was made available in 2001 (Stiller et al.,

1983).

Fluconazole and itraconazole are still not the perfect antifungals, since they have some non

-negligible drug interactions with such drugs that are used in chemotherapy or with AIDS treatment. These interactions can result in a decrease in azole concentration or even to an increase in toxicity. Furthermore itraconazole and fluconazole are ineffective against some emerging pathogens like Scedosporium, Fusarium, and Mucorales, and resistance to azoles is increasingly reported (Stiller et al., 1983).

New generation triazoles have also been developed. Voriconazole and posaconazole were approved by FDA in 2002 and 2006, respectively. Ravuconazole is currently under clinical trial phase of drug development. They possess a wide range of activity, since they are active against Candida, Aspergillus, Fusarium, Penicillium, Scedosporium, Acremonium, and Trichosporon, and dimorphic fungi, dermatophytes, and Cryptococcus neoformans

(Sabo et al., 2000). While new generation triazoles were shown to be more effective against Candida and Aspergillus, compared to classical triazoles, their side effects and drug interactions are similar to those observed with fluconazole and itraconazole. Likewise, fungal isolates resistant to classical triazoles are also cross-resistant to new generation triazoles (Stiller et al., 1983).

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2.15.5 Echinocandins

Echinocandins constitute the only new class of antifungals made available for clinicians to fight invasive fungal infections within the past 15 years. Three echinocandins were approved for clinical use by the FDA in United States and later by the European Agency for the Evaluation of Medicinal Products (EMEA): caspofungin in 2001 by the FDA and in

2002 by the EMEA, micafungin in 2005, and lastly anidulafungin in 2006.

Echinocandins are synthetic derivatives of lipopeptides. These lipopeptides are naturally produced by several fungal species: Aspergillus rugulovalvus synthesizes caspofungin B,

Zalerion arboricola synthesizes pneumocandin B, and Papularia sphaerosperma synthesizes papulacandin. Echinocandins are noncompetitive inhibitors of β(1-3)-glucan synthase, an enzyme that catalyzes the polymerization of uridine diphosphate-glucose into β(1-3) glucan, one of the structural components responsible for the maintenance of fungal cell- wall integrity and rigidity. β(1-3)-glucan synthase consists of an activating and a catalytic subunit encoded by FKS genes. In most fungi, two FKS genes are found within the genome. It has been shown in the model organism Saccharomyces cerevisiae that FKS1 is expressed during the vegetative growth phase and FKS2 during sporulation. Echinocandins are able to inhibit both isoforms of the enzyme. Inhibition of β(1-3)-glucan synthase leads to cell wall destabilization and to the leakage of intracellular components, resulting in fungal cell lysis (Arendrup et al., 2010).

These drugs are poorly absorbed in the gastrointestinal tract because of their high molecular weights and are therefore only used intravenously. Their pharmacologic properties are one of the reasons responsible for the approval of echinocandins by the FDA

51 and the EMEA. These molecules possess a low toxicity (very rare side effects were reported) and are slowly degraded, and a daily injection is sufficient, and contrary to other antifungals, interactions between echinocandins and other drugs are rare. Combined therapy between echinocandins and AmB or another azole often leads to a synergistic effect or at least to an additive effect (Arendrup et al., 2010).

Another reason for which the echinocandins were approved is their activity spectrum.

Indeed, echinocandins are active against most fungal species, including Candida and

Aspergillus. For still unclear reasons, these molecules are fungicidal in Candida but only fungistatic in Aspergillus. Moreover, fungicidal activity of echinocandins is species and isolate dependent within the Candida genus (Sabo et al., 2000). There exist several species within the fungal kingdom for which the echinocandins are ineffective. Such species include Cryptococcus neoformans or species belonging to Trichophyton and Fusarium genera. Other species have an intermediate susceptibility to echinocandins, such as

Scedosporium apiospermum, S. prolificans, and Cladophialophora bantiana. However, echinocandins constitute a good alternative to fight against fungal infections and most of treatment of infections for which classical therapy with azoles or polyenes failed are successfully managed with echinocandins (Grunberg et al., 1963). Therefore, caspofungin is indicated for the treatment of candidemia and invasive candidiasis, for fungal infection prophylaxis, and for the treatment of invasive aspergillosis for which itraconazole, voriconazole, or AmB is ineffective. Micafungin is used for treatment of candidemia and is particularly indicated for fungal infection prophylaxis in bone-marrow transplant patients.

Anidulafungin has no particular indications, but its main advantage is its slow degradation

52 in the body without liver or kidney involvement, thus it can be used in patients with liver and/or kidney insufficiencies (Arendrup et al., 2010).

What makes echinocandins unique is their fungal target. For many years, the fungal cell wall was considered to be a promising target for the development of new antifungal molecules. The fungal cell wall contains elements that have no equivalents in human. Its integrity is necessary for the fungal survival, since it provides a physical barrier against the host immune cells or against other microorganisms. Cell wall integrity is also responsible for osmolarity homeostasis and the maintenance of cell shape and size. Cell wall is also indispensable to essential enzymatic reactions and as an important role in cell-cell communication. The internal layer of the cell wall is composed of a β (1-3)-glucans and chitin web, in which are included some mannoproteins, while external layer is composed of mannoproteins (Arendrup et al., 2010).

2.15.6 Other antifungal agents

Three minor ergosterol biosynthesis inhibitors are used as topical antifungals. The allylamines and thiocarbamates, such as terbinafine and tolnaftate, both inhibit the ERG1- encoded enzyme, squalene epoxidase. The morpholines such as amorolfine act by inhibiting two different enzymes of the pathway, the 7, 8-isomerase (encoded by ERG24) and the C14-reductase (encoded by ERG2). Despite their wide spectrum of activity, these antifungal agents are essentially used to treat dermatophyte infections such as tinea capitis, tinea pedis, and onychomycosis, because they do present numerous side effects (Tassel et al., 1968).

53

Ciclopirox is also used as a topical antifungal agent, but its mode of action remains poorly understood in fungi. Another drug, griseofulvin, inhibits mitosis by interfering with microtubules function (Vermes et al., 2000).

2.16 Current Synopsis of Dermatophyte Species and Congeners: Ecological

Classification and Endemicity

Several anatomic sites may be infected by a single dermatophyte species, and different species may produce clinically identical lesions. The major etiologic agents may be global, such as T. rubrum, while the distribution of others may vary geographically (Table 2.1).

2.17 Case Reports of Animal Dermatophytosis in Nigeria

Anyanwu et al. in 2013 reported a case of a five-year-old Nigerian part Arab horse kept in the University of Nigeria, Nsukka demonstration farm and was observed to have non- exudative, scaly circumscribed lesions on its body. Two dermatophytic fungi,

Trichophyton mentagrophytes and Trichophyton verrucosum, were isolated from its hair plucking and skin scrapings. This report describes the trend in the diagnosis and differentiation of the two species of dermatophytes in a con-current infection of horse.

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Table 2.1: Current synopsis of dermatophyte species and congeners: ecological classification and endemicity

Anthropophylic dermatophytes Zoophylic Geophylic (Endemic regions) dermatophytes dermatophytes (Endemic regions) E. floccosum M. canis E. stockdaleae

M. audouinii (Africa) M. equinum M. amazonicum

M. ferrugineum (East Asia, East Europe) M. gallinae M.cookiellum (Central America, T. concentricum (Southeast Asia, Melanesia M. persicolor Mexico)

T. gourvilii (Central Africa) M. boullardii T. equinum T. kanei M. cookei T.mentagrophytes T. megninii (Portugal, Sardinia) M. gypseum T. sarkisorii T. mentagrophytes (complex of two species) M. nanum T. simii T. raubitschekii (Asia, Africa, Mediterranean) M. persicolor T. verrucosum T. rubrum M. racemosum

T. schoenleinii M. ripariae

T. tonsurans M.vanbreuseghemii

T. violaceum (North Africa, Middle East, T. soudanense (Subsaharan Africa) Mediterranean) T. ajelloi T. vanbreuseghemii T. flavescens T. gloriae,

T. longifusum

T. phaseoliforme,

T. terrestre

T.vanbreuseghemii

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Chah et al. (2012) conducted a study to determine the occurrence and species of dermatophytes in skin lesions in domestic animals in Nsukka Agricultural Zone of Enugu

State, Nigeria using forty-six domestic animals (dogs, goats, sheep and pigs) presented for sale in the local markets with suspected skin lesions of dermatophytosis. Of the 46 animals with suspected lesions of dermatophytosis, six (13.0%) were positive for a dermatophyte, and the following dermatophytes were identified: Microsporum gypseum, two of 12 sheep;

Microsporum audouinii, one of 16 dogs; Trichophyton mentagrophytes, one of 16 dogs and one of 12 sheep; and Trichophyton schoenleinii, one of 13 goats.

In 2011, Nweze carried out an investigation on 538 domesticated animals with clinically suggestive lesions for dermatophytes. Identification of dermatophyte species was performed by macro- and micro morphological examination of colonies and by biochemical methods. In the cases of isolates that had atypical morphology and/or biochemical test results, the rDNA internal transcribed spacer region 2 (ITS 2) sequencing was performed. Out of this number, 214 (39.8%) were found to be colonized by a variety of ten species of dermatophytes. M. canis was the most frequently isolated species

(37.4%), followed by T. mentagrophytes (22.9%) and T. verrucosum (15.9%). M. persicolor and T. gallinae were jointly the least species isolated with a frequency of 0.55% respectively.

An outbreak of ringworm in young calves was reported from Vom in Nigeria by Dalis et al. 2014. Twelve out of fourteen calves were observed to have skin lesions consistent with dermatophytosis. Lesions were seen mostly around the eyes and neck. Skin scrapings were collected from the affected areas and processed for mycology. Trichophyton verrucosum was isolated from all the affected calves.

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2.18 Case Reports of Animal Dermatophytosis in Other Parts of the World

Rakhi et al. carried out a study on one hundred and twenty canine samples obtained during a period of three years (2008-2011) from dogs suffering from different dermatological disorders and were in-vitro processed for dermatophytes detection at the Department of

Microbiology, Apollo College of veterinary medicine Agra Road, Jaipur. Out of these, eighty nine samples were positive respectively for Microsporum gypseum 55.83%,

Trichophyton mentagrophytes 18.3% and other fungal isolate Alternaria spp. sporadic in

15 samples (0.12%).

Gugnani et al. (2011) investigated the occurrence of keratinophilic fungi including dermatophytes on feathers of domestic and wild birds in the islands of St Kitts and Nevis from 94 birds. The authuors recovered four isolates of the dermatophyte, Microsporum gypseumcomplex (two each of M gyspeumand M fulvum) from feathers of birds.

A retrospective study of 16 cases of dermatophytosis due to Microsporum persicolor in hunting dogs was reported by Arnaud et al. (2013). Skin lesions were observed on the face in all cases, but also on other locations (limbs, neck). The lesions included alopecia

(15/16), erythema (13/16), scales (14/16), and crusts (13/16).

Shams et al. 2009 sampled a total number of 3,540 cattle in different age groups at three major farms of Mashhad including Kenebist (KB) in the east, Mazraeh Nemooneh (MN) in the south, and Moghoofat Malek (MM) in the north-east. Skin scrapings were prepared from all animals clinically suspected to have dermatophytosis. Among 684 suspected cases

(19.3%) selected from a total number of 3,540 cattle based on clinical signs, 604 case

(88.3%) were KOH positive in direct microscopy, while 490 cases (71.6%) were culture

57 positive on selective agar for pathogenic fungi (SAPF) medium. The most frequent dermatophyte isolated was Trichophyton verrucosum (495 isolates accounting for 99% of total isolates) which was obtained from all culture positive cases except five cases (1.0%) infected with Trichophyton mentagrophytes.

In a survey on dermatophytes among farm and pet animals in Suez Canal Area by Abou-

Eisha et al., 2008. T. verrucosum was the main aetiological agent isolated from clinically diagnosed cattle, buffaloes, sheep, goats and horses with ringworm lesions at rates of 75%,

50%, 71.4%, 65% and 25%, respectively. While, M. canis was the only dermatophyte species isolated from clinically affected dogs and cats at a rate of 41.7% and 56.7%, respectively. Dermatophyte infections among the examined animals reached its peak obviously during autumn and winter months and the lowest rates were in summer and spring months. Young aged animals are more susceptible for dermatophytic infection than older ones. On the other hand, out of 142 apparently healthy animals examined, dermatophytes were isolated from only 10 (7.04%) animals. The isolated dermatophytes of the apparently healthy animals were T. mentagrophytes var. mentagrophytes that was isolated from cattle, sheep and goat (4% of each animal species), T. verrucosum from cattle

(8%) and M. gypseum (15%) and M. canis (10%) of the examined cats. While, the apparently healthy buffaloes, horses, donkeys and dogs were culturally negative for dermatophytes.

Swai et al 2012 reported an outbreak of acute bovine dermatophytosis in a dairy herd in

Arusha region of Tanzania is described. Clinical history of the condition complimented with detailed examination of the affected animals revealed that animals were pastured

58 during the day and padlocked at night. 14 cattle out of 42 (33.3%) yielded positive on cultural isolation and Trychophyton verrucosum was isolated from all the 14 cattle.

Copetti et al. 2006 reported a survey evaluating fungal and parasitic aetiology of skin diseases through the analysis of 1,240 fur, nails and skin scraping specimens from dogs and cats with clinical suspicion of dermatophytosis. Samples collected in several veterinary clinics of the Santa Catarina, Paraná and Rio Grande do Sul states, mainly of the Santa Maria city in Rio Grande do Sul, were processed at theMycology

Research Laboratory of the Federal University of Santa Maria, Southern Brazil, between

1998 and 2003. Among canine and feline samples, the percentages of positive dermatophyte specimens were 10.2% and 27.8%, respectively. The most prevalent fungal specie in both cats and dogs was Microsporum canis, which was isolated in 68.5% of the positive cultures for dermatophytes in dogs‟ samples, being the only species recovered from cats‟ cultures. Malassezia pachydermatis was the most commonly isolated yeast from the skin of dogs. Acari, mainly Demodex canis, were found in 5.0% of all samples with suspected

Rosa et al. 2011, isolated and identify dermatophytes from skin scales collected from household cats and dogs sent to veterinary clinics in Alfenas city, Minas Gerais State,

Brazil. The clinical material was collected from the areas of the head, back and abdomen of 40 cats and 40 dogs. The isolation of dermatophytes occurred in 13 dogs (32.5%) and 14 cats (35%), and only two (7.4%) animals presented lesions of dermatophytosis. The fungi were identified as Microsporum canis (52.2%), Microsporum gypseum (14.9%) and

59 species of the genus Trichophyton (31.9%). M. canis was the predominant species among cats (67.8%) and Trichophyton spp among dogs (57.9%)

During five years (2000-2004), 1298 samples were analyzed by Bernardo et al. 2005, being 978 from clinical material (skin scrapings and hair), obtained from dogs and 320 samples from cats. These samples for dermatophytes detection. One thousand and sixty four samples were negatives for dermatophytes (82.0%) and 588, presented contamination with saprophytes fungi (45.3%) . Mycological culture revealed ringworm in 146 samples of dogs (11.2%) and 89 samples in cats (6.9 %). Microsporum canis was isolated in 63.7% of infected dogs and 82.0 % of cats. The other dermatophytes isolated belong to different species: Microsporum gypseum (5.5% in dogs and 5.6% in cats), Microsporum vanbreuseghemii (1.4% in dogs), and Microsporum nanum (2.2% in cats). Trichophyton mentagrophytes was isolated in 24.0% of the positive samples from dogs and 7.9% in cats.

In dogs Trichophyton ajelloi and T. terrestre were found 2.0% and 3.4% respectively. In

588 samples other contaminants fungi were found. The most prevalent were Alternaria spp. (22.6%), Candida sp. (14.5%), Cladosporium spp.(17.2%) and Penicillium spp.

(11.4%).

2.19 Reports of Dermatophytosis in Humans in Nigeria

In Nigeria, there are varying reports of dermatophytosis in different cities and communities

(Nweze et al., 2005).Considering its human and socioeconomic diversity and the staggering population, this is understandable. Nweze carried out an extensive survey of dermatophytosis in Nigeria‟s Northeastern state of Borno. The study involved 2193 children aged between 4-16 years in different local and urban communities of the state.

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Seven percent were proved to be positive for dermatophytosis. Incidence was significantly higher in young children aged 7-11 years and 4-6 years than in older children aged 12-16 years. Moreover, there was a significant difference in the incidence of dermatophytoses amongst children in urban and rural areas, thereby emphasizing the role of locality in dermatophytoses.

Tinea capitis was the predominant clinical type followed by tinea corporis. Trichophyton schoenleinii was the most prevalent etiological agent (28.1%), followed by T. verrucosum

(20.2%) and M. gallinae (18.4%). Other species recovered included T. mentagrophytes

(16.7%), T. tonsurans (10.5%), T. yaoundei (4.4%) and M. gypseum (1.8%) (Nweze,

2001).. In a similar study carried out in Kano State Nigeria, 2150 quranic scholars were screened (Adeleke et al., 2008).. Only 9.5% were found to be infected and the age group

10-14 years was most affected. T. rubum (50.2%) was the most prevalent followed by M. audouinii (26.5%). T. rubrum was the only dermatophyte that was recovered from all sites apart from the buttocks (Adeleke et al., 2008)..

A total of 6987 primary school children were sampled across 4 schools in Jos, Plateau state

Nigeria, only 3.4% were found to be infected by dermatophytosis (Ogbonna et al., 1985)..

There was a high incidence of both scalp and foot ringworms among the infected children.

A large spectrum of fourteen species of dermatophytes was isolated from the ringworm cases. The scalp ringworm had the highest number of fungal isolates. Trichophyton mentagrophyte and T. rubrum had the highest frequencies of occurrence (Ogbonna et al.,

1985).. .

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Ten years later, Ayanbimpe et al., 1994 found that Trichophyton soudanense was the major aetiological agent in the same area, indicating a shift in the pathological spectrum of the species. He latter carried out a more more expanded involving several states in Central

Nigeria, a total of 28505 primary school children aged between 3 and 16 years were sampled from 12 primary schools in 2008 (Ayanbimpe et al., 2008). Tinea capitis was found to be the most prevalent superficial mycoses. The most common aetiological agent was T. soudanense, (30.6%), followed by M. ferrugineum, (7.7%) and M. audouinii,

(7.7%) (Ayanbimpe et al., 2008).

In Ogun state, South Western part of Nigeria, a total of 2772 randomly selected junior secondary school pupils between the ages of 8-14 years from 60 schools were examined

(Popoola et al., 2006). The prevalence of dermatophytosis was 23.21%. Etiological agents identified with infection were M. canis (30.19%), M. audouinii (32.92%), T. interdigitale

(14.37%), T. soudanense (9.73%) and T. tonsurans (12.05%). Most of the dermatophytes encountered were anthropophilic species with M. canis being the only zoophilic dermatophyte (Popoola et al., 2006).

In a much older study in Lagos Southwest Nigeria (Adetosoye, 1977). 3860 school children were screened. The prevalence at the time was just 2.1%. Seven species of dermatophytes were recovered from specimens collected from the hair, skin and scalp scrapings of 81 school children. T. soudanense was the most prevalent etiological agent followed by M. canis.

Years later, another study in Lagos involving patients attending the Lagos State University

Teaching Hospital, Lagos found that 162 (41% ) of the patients were infected by

62 dermatophytoses. Microsporum species were the most common species (74.1%), followed by E. floccosum (4.3%) (Nwobu and Odugbemi, 1990). Superficial of the skin was the most prevalent (59.3%), followed by that of the hairs (27.2%) while infection of the nails (13.6%) was the least. Those aged five years and below had the lowest isolation rate (3.7%) (Nwobu and Odugbemi, 1990).

In Mid-western state of Edo state Nigeria, another group found a prevalence of 13.4% among primary school children infected with dermatophytosis (Enweani et al., 1996)..

In one of the earlier studies carried out between 1974-1977 in the old Anambra state of

Nigeria, (now subdivided into three States: Enugu, Ebonyi and Anambra states), 3478 primary school children aged 4-13 years made up of 1868 males and 1610 females were screened for dermatophytoses (Gugnani and Njoku-Obi, 1986). A total of 303 (8.7%) mycologically proven cases of tinea capitis were detected. Microsporum audouinii was the commonest etiological agent (48.3%) followed next by T. soudanense (26.6%) and T. tonsurans (15.2%). Other dermatophytes occasionally represented were M. ferrugineum

(3.4%), T. violaceum (3.7%), T. yaoundei (1.2%), T. mentagrophytes (0.9%) and T. schoenleinii (0.6%). Investigation of scalp carriage of dermatophytes by the authors showed that approximately 9% of children without any clinical signs of tinea capitis harbor dermatophytes in their scalp hair (Gugnani and Njoku-Obi, 1986)..

In 2005, Nweze et al. studies conducted a study in Anambra State by screening 1624 children with clinically suggestive lesions. These children aged between 4 and 16 years were sampled in selected urban and rural areas of the State. The data showed that tinea capitis was the predominant clinical type. T. tonsurans was the most prevalent etiological

63 agent while M. audouinii was the least in occurrence (Nweze et al., 2005)). Emele and

Oyeka, 2008 in another larger study which involved a total of 47723 primary school children residing in different regions of Anambra State, found that 4498 (9.4%) had tinea capitis. The highest prevalence of the disease occurred in the Southern region of the state

(12.6%). Schools in urban areas recorded lower prevalence of the disease. Moreso, tinea capitis occurred significantly more in children below 10 years of age than in those above this age. This agrees in part with the findings in Anambra state by Nweze and Okafor

(2005) M. audouinii was the most prevalent (42%), followed by M. ferrugineum (17%) and

T. mentagrophytes (16%).

In Aba, Abia State in Southeast Nigeria, T. mentagrophytes was however observed to be predominant in primary school children (Okafor et al., 1998)In a neighbouring local community of Arochukwu, also in Abia state Nigeria, it was found that tinea capitis was the predominant clinical type of dermatophytoses affecting 13.7% of the total population studied (Ngwogu and Otokunefor, 2007) T. soudanense and T. tonsurans were the predominant etiological agents of dermatophytoses with a prevalence of 26.2 and 21.6%, respectively (Ngwogu and Otokunefor, 2007).

2.20 Reports of Dermatophytosis in Africa and Other Parts of the World

In Africa and several other countries in Latin America and the Middle East, there is a kind of variability and geographical/regional associations in the pattern of dermatophytic infections. For instance, tinea capitis is known to be very common in Western Africa especially among children and several species of dermatophytes are known to be responsible. Tinea cruris, tinea pedis, tinea corporis and tinea unguium are caused by T.

64 rubrum in many urban areas of developing countries (Hernandez-Salazar, 2007) and even in developed (Borman et al., 2007: Foster et al., 2004). Microsporum audouinii is the predominant dermatophyte species in many parts of Africa. T. violaceum is reportedly endemic in several parts of South and Northern Africa and T. soudanense in central

Northwestern parts of Africa (Ellabib et al., 2002; Morat et al., 2004; Wondemanuel et al.,

2005). Conversely, M. canis predominates other dermatophytes in Southern and Central

European countries as the most common cause of tinea capitis while T. mentagrophytes and T. rubrum are the cause of increasing cases of tinea unguium and pedis, respectively

(Tao-Xiang et al., 2005; Tan, 2005).

2.21 Antifungal Susceptibility Testing of Dermatophytes

Antifungal susceptibility testing is a dynamic field of medical mycology (Sevtap, 2007).

Development and standardization of antifungal susceptibility tests have shown remarkable progress in the field of medical mycology. Despite the many guide lines that NCCLS have published for susceptibility tests of moulds (M-27A, M- 28A), there is no clear method and routine test for the evaluation of dermatophyte antifungal activity (Sevtap, 2007; NCCLS,

2002).

There are several antifungal drugs used to treat dermatophytosis. Some infections respond well to topical antifungal therapy, whereas others like tinea capitis, tinea unguium (nail infection), and more extensive or severe types may require systemic therapy (Esteban, et al., 2005). Occasionally, in some cases, antifungal therapy is a failure because of resistance to the antifungal drugs by the fungi. Therefore, it is believed that it is essential to evaluate the resistant dermatophytes using a standardized, simple and reproducible in vitro assay to

65 determine the antifungal activity of drugs against isolates (Fernández-Torres, et al, 2001).

In-vitro antifungal susceptibility tests are now mainly used for epidemiological surveys, determination of the degree of antifungal activity, and the prediction of clinical outcome based upon an optimization of antifungal therapy (Sevtap, 2007). Several methods have been developed for testing antifungal agents against this group of pathogens (Sevtap, 2007;

Santos, 2006). Multicenter studies to develop a standardized antifungal susceptibility assay were initiated by the Clinical and Laboratory Standards Institute (CLSI, formerly „National

Committee for Clinical Laboratory Standards‟, NCCLS) in 1983. Dilution tests are widely used in macro- and micro-assays, but these methods are difficult to be used in most laboratories. Studies have been done to establish a simple method to solve this problem

(Sevtap, 2007; NCCLS, 2002).

The agar-based disk diffusion (DD) susceptibility method for dermatophytes is simple, inexpensive, and does not require specialized equipment (Singh, 2007). The disk diffusion method has a good correlation with the reference dilution assay (Esteban, et al., 2005;

Rubio, 2003; Macura, 1993). The agar diffusion method is a practical, agar-based method which enables the determination of the activity of various antifungal drugs against various fungal genera and species. Broth macro- and micro-dilution assays can be used to determine antifungal susceptibility of dermatophytes, but these methods are expensive and require specific media and equipment such as RPMI, MOPS buffer, and micro plate trays.

The standard disk diffusion assay constitutes a good model to be used for investigational purposes to test other fungal genera and drugs as well.

Agar-based disk diffusion (DD) can be adapted for routine diagnosis in the laboratory and for assessment of dermatophyte resistance against antifungal drugs. Some studies have

66 focused on the comparison of the disk diffusion method with the reference micro-dilution method. These studies suggest that disk diffusion is a reproducible method which in general shows good correlation with the reference method for micro-dilution antifungal susceptibility test (Barry, et al., 2002; Karace et al., 2004) Other studies such as the one done by Singh et al. (2007) could not find a significant correlation between micro-dilution and disk diffusion methods, probably due to their use of Dermasel agar medium. This medium is unacceptable for antifungal susceptibility testing. In a study carried out by

Singh et al. 2007, clotrimazole, terbinafine and ketoconazole had large inhibition zones around the disks; clotrimazole had the best activity against the isolates. Clotrimazole is one of the oldest antifungal drugs formulated for topical use against dermatophytosis.

Although clotrimazole is effective against most cases of dermatophytosis, it is not suitable for severe infections involving hair and nail, which need additional systemic therapy.

Terbinafine was the second most effective antifungal drug obtained This drug has two forms (topical and oral) and has many advantages (Singh et al., 2007; Rubio, 2003). It was believed that this drug should be considered as the first choice for treatment of dermatophytosis, especially in nail and hair invasion when its side effects are tolerated.

Fluconazole, in spite of using drug potency up to 50 microgram per disk, had poor activity on isolates tested. In most isolates, no inhibition zones were observed around the disks

(Singh et al., 2007).

There are many studies indicating that fluconazole had less activity against dermatophytes

(Singh et al., 2007; Rubio, 2003; Favre, 2003). This is perhaps because fluconazole is a triazole, and Sabouraud dextrose agar has components that can interfere with the test.

Moreover, in vitro determination of antifungal activity of fluconazole against

67 dermatophytes has variable results because of the use of different methods and media

(Singh et al., 2007; Rubio, 2003; Karaca et al, 2004; Fernandes-Torres, 2005). Such variations were seen even between broth macro- and micro-dilution methods in studies.

Their results lacked a good and significant correlation with each other. It was then recommended that the other susceptibility test methods with RPMI medium should be used instead of disk diffusion for fluconazole (Siqueira et al., 2008).

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CHAPTER THREE

MATERIALS AND METHODS

3.1 Study Area

The study area is Kwara State which covers an area of 34,407.5 square kilometer and lies at latitude 80 North and longitude 50 East (Fadeyi et al., 2009). It has a population of

2,365,353 (2006 census figures) and accounts for 1.6% of the country‟s population. Kwara

State has 16 LGAs, which are grouped into three Senatorial Districts, namely Northern,

Southern and Central Senatorial Districts (Figure 3.1). Agriculture is the major occupation of the people in the State (Fadeyi et al., 2009).

3.2 Sampling and Sample Size

Based on availability of horses and period of sampling, purposive sampling method was used. Ninety-one skin scrapings and hair samples were taken from both clinical and asymptomatic cases of dermatophytosis in horses between March - June from different farms, homes and horse stables from seven LGA in Kwara State namely: Pategi, Oyun,

Baruten, Offa, Ilorin East, Ilorin West and Irepodun.

3.3 Sample Collection

Skin scrapings and swabs as well as plucked hair were collected from the margins of the lesions after cleaning and disinfecting with 70% alcohol as described by (Elewski, 1995).

Hairs were plucked by pulling them (Quinn et al., 1994).On the other hand, hair and scale specimens were collected form apparently healthy animals using Mackenzie's hairbrush

69 technique (Mackenzie, 1963). All collected animal samples were accompanied by data involving location, sex, and anatomical sites of collection of samples from the animals, in addition to date of sample collection.

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LEGEND

Senatorial zones:

Pink= Kwara-North

Yellow= Kwara-Central

Blue- Kwara-South

Kaiama

Baruten

Edu Moro

Ilorin-East Pategi

Ifelodun Ilorin-West

Asa Ilorin-South

Isin Oyun Oke-Ero

Offa Irepodun Ekiti

Figure 3.1: Map of Kwara State (www.the-nigeria.com).

Arrows are indicating LGAs where samples were collected.

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Collected samples were placed in envelops in separate polythene bags, and transported as dry packet (Gempieri, 2004) to the microbiology laboratory of the Faculty of Veterinary

Medicine, Ahmadu Bello University, Zaria.

3.4 Direct Microscopic Examination of Samples

Small samples of each scrapping were placed on a microscope slide and 1 to 2 drops of

10% potassium hydroxide added. A cover slip was applied and the slide gently heated over a flame as described by Hainer (2003). Each treated slide was carefully examined under low objective (×10) and high (×40) power objectives to observe for presence of diagnostic fungal forms.

3.5 Laboratory Culture of Dermatophytes

Sabouraud dextrose agar (SDA) containing chloramphenicol (40mg/L), cycloheximide

(500mg/L) and nicotinic acid (100µg/ml) which is a selective media was used for primary isolation. Cycloheximide prevents growth of majority of molds and yeasts, chloramphenicol is an antibacterial agent and nicotinic acid promotes the growth of

Trichophyton equinum (Raymond and Piphet, 2008). The SDA slants were inoculated with the sample and incubated at room temperature for one to four weeks.

3.6 Identification of Isolates

Suspected growths were sub-cultured on PDA (Oxoid, UK) to facilitate distinctive spore formation for identification and pigment production. The subcultures were incubated at room temperature for one to four weeks (Raymond and Piphet, 2008). Identification was based on colony and microscopic characteristics after staining with lactophenol cotton blue

72 and using the Fungal color Atlas (Wolf et. al., 1975, Evans and Richard, 1989, Baron et. al., 2003).

3.7 Slide Culture Preparation

Dermatophytes that could not be identified conclusively due to lack of sporulation were further subjected to slide culture. This was essential to observe the precise arrangement of the conidiophores and the way in which spores were produced (conidial ontogeny). A simple modification of Riddel‟s method of slide culturing (1950) as described below was used.

Potato dextrose agar was used. In glass petri-dish, cotton wool, two small sticks were arranged and a clean glass slide and cover slip were placed on them and sterilized in a hot air oven. The cotton wool was moistened with sterile water. Using a sterile blade an agar block (1x 1cm) was cut and flipped up onto the surface of a sterile glass slide. The four slides of the agar were inoculated with spores or mycelia fragments of the fungus and cover slip placed centrally upon the agar block, leaving space between the agar and the edge of the cover slip. The plates were covered and incubated at room temperature until growth and sporulation occurred, the cover slip were from the agar block and gently onto a small drop of Lactophenol cotton blue on a clean glass slide and observed for conidia.

This technique is based on the fact that when a dermatophyte grows in the 1cm by 1cm agar provided even unto the glass slide cover, it meets an empty space. Fearing starvation, it starts to produce spores for survival. These spores are usually diagnostic for each species and can then be recorded.

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3.8 AntifungalDrug Dilution

Five antifungal agents were tested; Amphotericin B, 50mg (Neon Laboratories Limited,

India), Ketoconazole,100mg (Oxoid, UK) Fluconazole,50mg (Pfizer International

Newyork), Terbinafine,100mg (Novartes Research Institute, Vienna, Austria) and

Griseofulvin, 5mg (oxoid, UK). A stock solution for each antifungal agent was prepared.

Amphotericin B, Terbinafine, and Griseofulvin were dissolved in 100% dimethylsulphuroxide while Fluconazole and Ketoconazole were dissolved in sterile distilled water into a working solution of 256μg/ml for Fluconazole, 64μg/ml for each of

Ketoconazole, Terbinafine, Amphotericin B and 32μg/ml for Griseofulvin.

3.9 In -vitro Susceptibity Testing

The broth microdillusion assay for antifungal susceptibility testing of dermatophytes was performed according to Nweze (2007).

3.10 Preparation of Inoculum

The inoculums were prepared according to Norris et al., (1995). Freshly growing cultures on the SDA were sub-cultured on Potato Dextrose Agar (PDA) slants for 4 days to enhance conidial production. Conidial suspension of dermatophytes were made by flooding the slants with sterile distilled water supplemented with 0.1% tween 20 and the conidia were dislodged with a sterile loop and the suspension collected with a syringe into a sterile tube.

The suspension was then shaken to homogenize and allowed to settle for twenty minutes then the upper layer was removed and adjusted with sterile distilled water to match the opacity of a standard control (McFarland standard).

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3.11 Antifungal Susceptibility Test Procedure

The tests were performed in a plastic microtiter plates with flat bottom wells. By using a multichannel micropipette with disposable pipette tips, 100 μl of RPMI 1640 medium was dispensed into wells 1-12, 100μg of drug dilution was added to well 1 of each plate and a two-fold serial dilution was carried out to well 11. 100μl of the leftover from the last well was discarded, after which 100μl of the inoculum was then dispensed into well 1-10 with well 11 as the media control and well 12 the solvent control. The plates were incubated at room temperature and Minimum Inhibitory Concentrations (MICs) read after 48 hours.

Endpoint determination was done according to the class of drugs (Araujo et al., 2009).

Growths were compared to that of drug free controls and scored by visual observation as follows. Growth same as control = 4. Slight decrease in growth (90% growth) = 3.

Significant reduction in growth (50% growth) = 2. Slight growth (few visible hyphae fragments) = 1. The MIC is represented as concentrations in which 50% of the growth is inhibited for fluconazole, ketoconazole, terbinafine, griseofulvin and complete growth inhibition observed for amphotericin B (Araujo et al., 2009).

3.12 Data Presentation and Statistical Analysis

The results obtained were described using percentages and presented in charts, tables and plates. Fisher‟s exact test was used to measure the association between positively tested samples and anatomical sites and also positively tested samples and sex of the horses.

Levels of P<0.05 were considered significant.

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CHAPTER FOUR

RESULTS

4.1 Occurrence of Dermatophytes from Horses with Skin Lesions The highest dermatophyte isolation rate per total samples collected from each of the seven different LGA was 25%, for Ilorin-East followed by Offa with 20%, 16.7% for each of

Irepodun and Oyun. Ilorin-West had the largest number of samples collected (30) but with

13.3% dermatophyte isolation rate. Pategi and Baruten had dermatophyte isolation rates of

12.5% and 9.1%, respectively. Statistically significant differences (p < 0.05) were observed in the number of dermatophytes isolates that were obtained from the seven different LGA (Table 4.1). Dermatophytic lesions were observed on four anatomical sites of the body of horses that were sampled. These sites were the limbs (Plate I), tail, head and abdominal region with dermatophyte isolation rate per total samples collected being

18.7%, 16%, 15% and 10%, respectively (Table 4.2). However, there was no statistically significant association (p ˃ 0.05) between the number of dermatophytes obtained and the anatomical sites from where samples were collected. The distribution of the dermatophyte species in relation to the four anatomical sites of isolation from horses was as follows. The limbs: M. fulvum (2) and T. verrucosum (1). The tail: M. gypseum (1), M. equinum (1) and

T. verrucosum (2). The head: M. fulvum (1), M. persicolor (2), T. verrucosum (2) and T. soudanense (1). The abdominal region: T. tonsurans (1). Two Anthropophylic dermatophytes were isolated, namely T. soudanense from the head and T. tonsurans from the abdominal region (Table 4.3). Seven diferent dermatophyte species were identified from the 14 isolates that were obtained in the study (Table 4.4). These are one T. tonsurans, five T. verrucosum, one T. soudanense, one M. gypseum, two M. persicolor, one M. equinum and three M. fulvum.

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Table 4.1: Percentage distribution of dermatophytes isolated from samples obtained from horses in seven Local Government Areas (LGA) of Kwara State

LGA Number of samples Positive samples % positive

Ilorin East 12 3 25

Offa 10 2 20

Irepodun 6 1 16.7

Oyun 6 1 16.7

Ilorin West 30 4 13.3

Pategi 16 2 12.5

Baruten 11 1 9.1

Total 91 14 15.4

P = 0.0028

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Plate I: Circumscribed, dermatophytic lesion (arrow point) on the forelimb of a horse

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Table 4.2: Distribution of dermatophytes based on anatomical site of lesions

Anatomical site Number of samples Dermatophyte % positive for of lesion collected positive samples dermatophytes Limbs 16 3 18.7

Tail 25 4 16

Head 40 6 15

Abdomen 10 1 10

Total 91 14 15.4

P = 0.9456

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Table 4.3: Anatomical locations of different dermatophytes isolated from horses in seven Local Government Areas of Kwara State

Sample Anatomical site Dermatophyte number of isolation species 5 Head M. persicolor

6 ,, T. verrucosum

7 ,, M. persicolor

22 ,, T. verrucosum

43 ,, M. fulvum

12 ,, T. soudanense

49 Abdomen T. tonsurans

10 Limb T. verrucosum

48 ,, M. fulvum

80 ,, M. fulvum

8 Tail T. verrucosum

11 ,, T. verrucosum

75 ,, M. equinum

91 ,, M. gypseum

Total 14

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Table 4.4: Frequency of dermatophyte species isolated from horses from seven Local Government Areas in Kwara State

Dermatophyte Frequency %

species T. verrucosum 5 35.7

M. fulvum 3 21.4

M. persicolor 2 14.3

M. equinum 1 7.14

T. soudanense 1 7.14

M. gypseum 1 7.14

T. tonsurans 1 7.14

Total 14 100

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Out of 85 male horses sampled 12 were positive, and out of the six female horses sampled, two were positive. However, there was no statistically significant difference (p ˃ 0.05) between the total dermatophytes isolated from male (14.1%) or female (33.3%) horses from the seven LGA in Kwara state (Table 4.5). Out of 62 horses with dermatophytic lesions and 19 asymptomatic horses, 14 dermatophytes (15.4%) were isolated with only one dermatophyte from asymptomatic horses (Table 4.6).

4.2 Colonial Morphology and Microscopic Appearance of Trichophyton tonsurans, Trichophyton verrucosum, Trichophyton soudanense, Microsporum gypseum, Microsporum persicolor, Microsporum equinum and Microsporum fulvum Colonies of T. tonsurans showedsuede like to powdery yellow surfacewith raised center and radial grooves (Plate IIa). Microscopically, T. tonsuransshowed irregular, branched hyphae with thin walled numerous septae and irregular clavate macroconidia (Plate IIb).

Arrows indicating irregular, branched hyphae, numerous septa of hyphae, thin walled irregular clavate macroconidia and chlamydospores at terminal ends of hyphae (Plate IIc).

Colonies of T. verrucosum revealed small button-like shape, white to creamy velvety surface with raised center and flat periphery with submerged growths (Plate IIIa).

Microscopic features of T. verrucosum were broad and irregular hyphae with club shaped tips and chains of intercalary terminal chlamydospores (Plates IIIb and c). Colonies of T. soudanense showedyellow, folded suede like surface with raised center and broad white fringed edges (Plate IVa). Microscopically, T. soudanense hyphae showed reflexive hyphae with branching and pyriform microconidia (Plate IVb). Colonies of M. gypseum were flat, spreading, suede like with deep cream surface, central white downy umbo

(dome) and narrow white peripheral border (Plate Va). Microscopically, M. gypseum revealed 4 celled macroconidia with truncated (arrow) distal end (Plate Vb).

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Table 4.5: Sex distribution of dermatophytes isolated from horses in seven Local Government Areas of Kwara State

Sex of horse Number of samples Positive samples % positive

Female 6 2 33.3

Male 85 12 14.1

Total 91 14 15.4

P=0.2293

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Table 4.6: Dermatophytes isolation rates from horses with dermatophytic lesions or asymptomatic in Kwara State

Dermatophytic Number of samples Positive samples % positive lesion on horses Present 62 13 21

Absent 19 1 5.2

Total 91 14 15.4

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C

a H

S H

b c

Plate II: Colonies of Trichophyton tonsurans (IIa) withsuede like to powdery yellow surface,raised center and radial grooves (C). Lactophenol cotton blue stained smear ofTrichophyton tonsurans (IIb and c)×400. Note thethin walledhyphae withnumerous septa and irregular branching(H), clavate macroconidia and chlamydospores at terminal ends of hyphae (S).

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C

a

S H

b c

Plate III: Small white button shape colony of Trichophyton verrucosumwith a raised center(C),velvet surfaceand flat submerged periphery (IIIa). Lactophenol cotton blue stained smear of Trichophyton verrucosum ×400 (IIIb and c) with broadirregular hyphae (H) and chains ofbroad, club shaped intercalary chlamydospores (S).

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C

a

M

H b

Plate IV: Colony of Trichophyton soudanensewith yellow folded suede like surface, raised center (C) and broad white fringed edges (IVa). Lactophenol cotton blue stained smear ofTrichophyton soudanense×400 withreflexivebranching hyphae (H)and pyriform (M) microconidia (IVb).

87

C

a

M

b

Plate V:Colony of Microsporum gypseumwith flat spreading suede like, deep cream surface, central dome (C) and narrow white peripheral border (Va).Lactophenol cotton blue stained smear of Microsporum gypseum×400 showing 4 celled macroconidia (M)with truncated distal ends (Vb).

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Microsporum persicolor colonies hadpink tinged colour, granular texture and irregular advancing edges (Plate VIa).Distinct thin walled, cigar shaped macroconidia (4-7 cells) and chains of chlamydospores of M. persicolor were observed under the microscope (Plate

VIb). Colonies of M. equinum were flat, spreading to suede like and pale buff to salmon colour (Plate VIIa). Small spindle shaped macroconidia with few septae of M. equinum were observedunder the microscope (Plate VIIb). Microsporun fulvum showed white profuse colony with raised center (Plate VIIIa) and numerous spiral hyphae microscopically (Plate VIIIb).

4.3 Minimum Inhibitory Concentrations of Ketoconazole, Amphotericin B,

Terbinafine, Griseofulvin and Fluconazole against Dermatophytes Isolated

from Horses in Seven Local Government Areas of Kwara State

There was limited data correlating susceptibility in vitro with outcome in vivo to define interpretive breakpoints for ketoconazole, amphotericin B, terbinafine, griseofulvin and fluconazole against the seven dermatophyte species isolated from horses in this study.

However, the MICs were determined and presented in comparison with ranges of susceptibility data for 50 T. rubrum isolates (Santos and Hamdan, 2005) and T. equinum

CBS127.97 strain (Coelho et al., 2008), with ketoconazole, terbinafine, griseofulvin, fluconazole and amphotericin B (Table 4.7). Ketoconazole gave a MIC range of 0.3-9.0

µg/ml when tested against the 14 dermatophytes with 50% of the isolates test results within the range that was reported for T. rubrum. Apart from one Microsporum persicolor

(9.0µg/ml), the remaining dermatophytes had only 1-1.5 values above ketoconazole MIC range reported against 50 T. rubrum isolates.

89

C

a

S M

b

Plate VI: Pink tinged colonies of Microsporum persicolor (C) with granular texture and irregular advancing edges (VIa). Lactophenol cotton blue stained smearof Microsporum persicolor×400 (VIb) with thin walled, cigar shaped, 7 celled macroconidia (M) and chlamydospores (S).

90

C

a

M

b

Plate VII: Colonies of Microsporum equinum(C) withflat, spreading to suede like surface and pale buff to salmon colour (VIIa).Lactophenol cotton blue stained smear of Microsporum equinum×400 showing small spindle shaped macroconidia (M) with few septae (VIIb).

91

C

a

H

b

Plate VIII:Profuse white colony of Microsporun fulvum(C) withraised center (VIIIa). Lactophenol cotton blue stained smear of Microsporum fulvum ×400 (VIIIb) showingnumerous spiral hyphae (H).

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Table 4.7: Minimum Inhibitory Concentrations of five antifungal drugs tested on 14 dermatophytes isolated from horses in seven Local Government Areas of Kwara State Sample Minimum Inhibitory Concentration (µg/ml) of antifungal drugs number Dermatophyte species Ketoconazole Amphotericin B Terbinafine Griseofulvin Fluconazole * Trichophyton rubrum (n=50) 0.031-2.0 < 0.031 0.25-2.0 2.0-32.0 ** T. equinum CBS127.97 0.1 0.2 4.0 100 6 Trichophyton verrucosum 0.8 0.8 0.8 7.0 6.0 8 Trichophyton verrucosum 4.3 0.8 0.2 2.3 2.0 10 Trichophyton verrucosum 3.5 3.5 0.6 4.5 5.0 11 Trichophyton verrucosum 3.5 3.0 1.0 3.8 6.5 22 Trichophyton verrucosum 1.5 2.5 1.5 5.0 4.6 12 Trichophyton soudanense 0.3 3.0 4.0 4.0 19.2 49 Trichophyton tonsurans 3.3 4.0 0.3 2.3 1.6 43 Microsporum fulvum 4.5 0.8 1.4 2.5 4.0 48 Microsporum fulvum 0.4 0.8 0.8 3.5 3.0 80 Microsporum fulvum 0.3 0.6 3.6 2.0 0.6 5 Microsporum persicolor 9.0 4.0 0.4 2.0 5.0 7 Microsporum persicolor 3.0 0.6 0.6 1.5 4.0 75 Microsporum equinum 3.3 1.6 6.5 2.0 12.8 91 Microsporum gypseum 1.6 4.0 3.3 8.0 19.2 *Santos and Hamdan (2005) **Coelho et al. (2008)

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Amphotericin B susceptibility of the 14 dermatophyte isolates with MIC range of 0.6-4.0 µg/ml was higher than the value for M. equinum strain of the CBS Fungal Biodiversity Centre,

Netherlands (0.1 µg/ml). The highest MIC (4.0 µg/ml) was against T. tonsurance, an isolate of

M. persicolor and M. gypseum. Terbinafine produced a range of MIC values of 0.2-6.5 µg/ml with 50% of the isolates test results < 1µg/ml. However, all the terbinafine MICs were greater than the upper limit (0.031µg/ml) demonstrated against 50 T. rubrum isolates. Also, the highest terbinafine MIC obtained (6.5 µg/ml), against M. equinum, was greater than that reported for M. equinum strain of the CBS Fungal Biodiversity Centre, Netherlands (0.2 µg/ml). Griseofulvin

MIC range was 1.5-8.0 µg/ml when tested against the 14 isolated dermatophytes. The highest

MIC (8.0 µg/ml) was against M. gypseum followed by an isolate of T. verrucosum (7.0 µg/ml) while the lowest (1.5 µg/ml) was against an isolate of Microsporum persicolor. Only two T. verrucosum isolates and M. gypseum had MIC values higher than the MIC for M. equinum strain of the CBS Fungal Biodiversity Centre, Netherlands (4.0 µg/ml). Fluconazole MIC range of 0.6-

19.2 µg/ml for was obtained from the 14 dermatophyte isolates that were tested. The highest

MIC (19.2 µg/ml) was against M. gypseum and T. soudanense followed by M. equinum (12.8

µg/ml) while the lowest (0.6 µg/ml) was against one M. fulvum isolate. Only this isolate of M. fulvum and M. tonsurans had fluconazole MIC lower than the range that was reported for 50 T. rubrum isolates.

1

CHAPTER FIVE

DISCUSSION

Cultivation of the collected specimens revealed seven isolates made up of four Microsporum species and three Trichophyton species, with isolation rate of 15.4% (14 out of 91 positive samples) which is closely similar to the result obtained by Chah et al (2012) who examined 46 domestic animals (sheep,dogs and goats) and found 6 (13%) positive for dermatophytes but lower than the report of Nweze ( 2011), who examined 25 horses out of which 11 samples (44

%) were positive as well as Hassan (2011), who isolated dermatophytes from 36.5% of total horse samples in Cairo, Egypt and also Abo El-Yazeed (1990) who obtained an isolation rate of

(49%) for dermatophyte species from horses. The lower isolation rate obtained in this study can be attributed to the fact that samples were collected from both clinical and asymptomatic cases coupled with the management practice by most horse groomers in Kwara State with most horses being kept in separate stalls and with different grooming equipments.

The 14 isolates made up of five T. verrucosum (35.7%), one T. tonsurans (7.1%), one T. soudanense (7.1%), one M. gypseum (7.1%), two M. persicolor (14.3%), one M. equinum

(7.15%) and three M. fulvum (21.4%)confirm the etiological agents of equine dermatophytosis as reported by Abo El-Yazeed (1990); Pilsworth and Knottenbelt (2007) and Nweze (2011).

The observation in this study of Trichophyton verrucosum being the most prevalent etiological agent of dermatophytosis in equine (5 isolates out of 14) is in contrast with the reports of Abo El-

Yazeed (1990); Kane et al., (1997) and Nweze (2011) who observed that T. equinum and T.

2 equinum var autotrophicum were the most commonly isolated dermatophyte species from horses and Trichophyton verrucosum in cattle. This can be due to the management practice by most horse owners in Kwara State as they keep many species of animals together including horses and cattle and this play an effective role in cross infection through several routes from cattle to horses as suggested by Mantovani et al. (1978). Microsporum fulvum is a cosmopolitan geophilic dermatophyte species and with similar clinical disease is similar to that of M. gypseum but less common. However it was the second most causative agent of dermatophytosis in this study and this is a rare occurrence and could have been contacted by the affected horses rolling in the sand as they do sometimes and this sand could have been contaminated by anthrospores and infection aided by skin abrasions, followed by M. persicolor which is a zoophilic and geophilic fungus.

Based on anatomical location, the limbs showed the highest distribution rate (18.7%) than the other body locations where samples were collected from (tail, head and abdomen). This is contrary to OIE (2005) report which stated that most dermatophyte lesions are found in areas on the back of horses in contact with saddle. The reason for this higher distribution on the limbs may be due to contamination of the hay given to the horses and also the floor of the stalls as confirmed by the isolation of two M. fulvum a geophilic dermatophyte, from the limbs.

The observation of Ilorin-East having the highest incidence rate of 25% is possibly due to the high concentration of stables in that LGA as this can facilitate the spread of infections.

The observation of fluconazole having the highest MIC against M. gypseum in this study agrees with the report of Carrillo et al., (2003). Also, MIC ranges obtained in this study for all the used drugs were similar to ranges obtained by Araujo et al. (2009). The finding of terbinafine showing

3 an excellent in vitro potency and broad-spectrum activity against all the isolated species and therefore being considered as the most active agent. agrees with the observations made by

Fernandez-Torres et al. (2001) and Santos et al. (2005) suggestting that terbinafine can be used to treat most dermatophytic infections in horses.

It will be of interest to state that all isolates used in this study were obtained from animals not previously on any antifungal treatment. Although some of the antifungal agents recorded relatively higher MIC values against the tested dermatophyte isolates this was not enough to confirm resistance. This study also showed that amphotericin B comes next after terbinafine as the next most effective antifungal agent followed by ketoconazole. Thus clinicians can now have the confidence of choosing effective medications for treating animal dermatophytosis especially in horses in Nigeria where this condition is endemic and also of public health significance.

CHAPTER SIX

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

6.1 Summary

In this study, 91 samples were collected from clinical and asymptomatic cases of dermatophytosis in horses from March-June 2014 from Offa, Pategi, Irepodun, Ilorin-West,

Ilorin-East, Baruten and Oyun LGAs in Kwara State. Fourteen samples were positive for dermatophytes with isolation rate of 15.4%. Of the 10 samples from Offa, two were positive for dermatophytes, 16 samples from Pategi, gave two positive isolates, six samples from Irepodun produced one positive isolate, 30 samples were from Ilorin-West which yeilded four positive isolates, 12 samples from Ilorin-East produced three positive isolates, 11 samples from Baruten,

4 produced one positive isolate and six samples from Oyun, with one positive for dermatophyte.

There was significant association between the dermatophytes isolated and the local government areas from where samples were taken from (P=0.0028) (P< 0.05). Based on anatomical sites, highest incidence rate was seen on the limbs (18.7%) and lowest was seen on the abdomen

(10%). There was no significant association between dermatophytes and anatomical sites from which samples were collected (P=0.9456) (P> 0.05). The dermatophytes isolated were three

Trichophyton species namely T. tonsurans(one) (7.14%), T. verrucosum(five) (35.7%), T. soudanense (one) (7.14%), and four Microsporum species namely M. gypseum (one) (7.14%),

M. persicolor (two) (14.2%), M. equinum (one) (7.14%), and M. fulvum (one) (7.14%). Eighty- five male horses were sampled, with 12 (14.1%) being positive for dermatophytes; six females were sampled and two were positive (33.3%) for dermatophytes though there was no significant differences between the sexes and positive isolation (P< 0.05) (P=0.2293). Antifungal susceptibility test was carried out on the 14 dermatophyte isolates using five antifungal agents:

Ketoconazole (0.03-64µg/ml), fluconazole (0.125-64µg/ml), amphotericin B (0.03-64µg/ml), griseofulvin (0.03-32µg/ml) and terbinafine (0.03-64µg/ml), using, the broth microdilution method according to Nweze (2007). The susceptibility test showed that terbinafine was the most potent drug with the lowest range of MIC values of 0.2-6.5µg/ml followed by amphotericin B which had MIC range of 0.6-4.0µg/ml and then ketoconazole (0.3-9µg/ml), whereas griseofulvin and fluconazole showed the highest MICs of 1.5-8.0µg/ml and 0.6-19.2µg/ml, respectively. Indicating that terbinafine was the most efficacious of the five antifungal agents used in this study.

5

6.2 Conclusions

This study has indicated that:

:the dermatophytes affecting horses in the seven LGAs of Kwara State were

T.verrucosum, T. tonsurans, T. soudanense, /M. equinum, M.gypseum, M.

persicolor and M. fulvum.

Ilorin-East LGA had the highest distribution (25%) of suspected cases of

dermatophytosis while Baruten LGA had the lowest (9.1%).

female horses had higher rate of infection (33.3%) than male horses (14.1%).

anatomically, the limbs had the highest frequency of dermatophyte infection

(18.7%) while the abdomen had the lowest frequency (10%).

most of the dermatophytes isolated were susceptible to the 5 antifungal drugs

tested against them, with terbinafine being the most potent drug with the lowest

MIC range of 0.2-6.5 µg/ml whilefluconazole had the highest MIC range value of

0.6-19.2µg/ml.

6.3 Recommendations

The outcome of this study indicates that equine dermatophytosis is prevalent in the seven sampled LGAs of Kwara State. In view of the zoonotic (public health) and economic implications of dermatophytosis in both humans and animals, the following recommendations are therefore being made:

1. Newly acquired horses must be screened before being introduced into stables, followed

by regular check-up.

6

2. Horse owners should be educated on the zoonotic significance of dermatophytosis.

3. Horse owners/groomers should also be educated on the importance of good sanitation

(cleaning and disinfection) in horse stables as well as grooming equipments.

4. Culture and sensitivity tests should be carried out for effective diagnosis and treatment of

fungal infections especially in horses.

7

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