OCCURRENCE AND CHARACTERISTICS OF MYCOTIC AGENTS ISOLATED FROM SKIN LESIONS OF TRADE HORSES AND DONKEYS IN OBOLLO-AFOR, ENUGU STATE, NIGERIA

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OCCURRENCE AND CHARACTERISTICS OF MYCOTIC AGENTS ISOLATED FROM SKIN LESIONS OF TRADE HORSES AND DONKEYS IN OBOLLO-AFOR, ENUGU STATE, NIGERIA

ABSTRACT 

This study was conducted to document the occurrence and characteristics of mycotic agents isolated from trade horses and donkeys in Obollo-Afor, Enugu State. A total of 497 horses and 65 donkeys presented for sale at Obollo-Afor horse/donkey market during the wet season (June to August 2014) and dry season (January to March 2015) were examined for skin lesions. For each horse/donkey examined, the location of skin lesion as well as the sex of the animal were recorded. Each skin lesion was disinfected with 70% ethanol and skin scrapings collected.  Each sample was inoculated onto the surface of SDA plates containing 0.05mg/ml of chloramphenicol and 0.5mg/ml of cycloheximide. Inoculated plates were incubated at room temperature (27oC) for up to 4 weeks and examined daily for fungal growth. Laboratory identification of the fungal isolates was based on macroscopic and microscopic features. Wet mount preparations of fungal growth were examined for microscopic features of the isolates (presence, shape, arrangement and relative abundance of micro and macroconidia). The cultural and microscopic features of the fungi isolated were compared with those in the reference colour atlas. One hundred and fifty (30.18%) horses and 11 (16.92%) of the donkeys had skin lesions. Forty seven (9.5%) of the 497 horses and 2 (3.1%) of the 65 donkeys examined yielded fungal growth. Of the 150 horses with skin lesions, 87 horses had lesions on the dorsal trunk with 27 (31%) being positive for fungal isolation; 11(52.4%) of the 21 lesions on the head yielded fungal growth. Six (6.9%) of the 18 lesions on the hind limb were positive for fungal isolation while 2 (18.2%) of the 11 lesions on the forelimb yielded fungal growth. Fungal agents were isolated from one of the 3 lesions on the tail of the horses examined. No mycotic agent was isolated from the lesions located on the neck and ear of the horses. Fungi were isolated from 2 (18.2%) of the 11 donkeys with lesions on the dorsal trunk.  Out of 97 male horses, 43 (44.3%) were positive for fungal isolation while 4 (7.5%) of the 53 female horses were positive. Fungi were isolated from none of the 8 males and 2 (66.7%) of the 3 female donkeys examined. There was a significant association (P>0.05) between sex and the prevalence of fungal isolation in both animal species. Fungi were isolated from 45 (32.1%) of the 140 horses with lesions observed during the wet season. Of the 10 horses with skin lesions recorded during the dry season, 2 (20%) were positive for fungal isolation. Fungi were isolated from 2 (20%) of the 10 donkeys with lesions observed during the wet season but from none that were observed during the dry season. There was also a significant association (P>0.05) between season and the prevalence of fungal isolation in horses but no significant association (P>0.05) in the donkeys. The fungi genera identified in order of descending rate of isolation were Aspergillus (24.5%), Trichophyton (14.3%), Microsporum (10.2%), Penicillium (8.2%), Curvularia (6.1%), Fusarium (4.1%) and Coccidoides (2%); 22.4% of the fungal isolates (moulds) were not identified. The fungal species identified include: Aspergillus niger (10.2%), Aspergillus fumigatus (8.2%), Aspergillus flavus (6.1%) and Fusarium subglutinans (4.1%). All the seven Trichophyton spp tested were resistant to fluconazole while none of them was resistant to voriconazole and nystatin. All the five isolates of Microsporum spp, 3 of Curvularia, 2 of Fusarium and 12 of Aspergillus were resistant to fluconazole while none of them was resistant to voriconazole and nystatin. The four isolates of Penicillium were all resistant to fluconazole, voriconazole and nystatin. This study revealed that a wide range of fluconazole resistant mycotic agents, covering all ecological groups infect horses and donkeys in the study area, all of them with the potential of being zoonotic. Preventive measures should be put in place by veterinarians and groups to mitigate the spread of the infectious agent.

 

TABLE OF CONTENT

Page

Cover page—————————————————————————————-i

Title page —————————————————————————————–ii

Declaration —————————————————————————————iii

Certification—————————————————————————————iv

Dedication —————————————————————————————–v

Acknowledgements——————————————————————————-vi

Table of contents——————————————————————————– vii-ix

List of Tables————————————————————————————–x

List of Plates—————————————————————————————xi-xii

List of Appendix——————————————————————————— xiii

Abstract ——————————————————————————————–xiv-xv

CHAPTER ONE: INTRODUCTION

1.1 Background of the study——————————————————————- 1

1.2 Statement of the research problem—————————————————— 4

1.3 Research questions————————————————————————-5

1.4 Aim and objectives of the study———————————————————6

CHAPTER TWO: LITERATURE REVIEW

2.1 Brief history of mycology——————————————————————7

2.2 Classification of fungi ———————————————————————-9

2.3 Characteristics of fungi——————————————————————–16

2.4 Dermatomycoses—————————————————————————-17

2.4.1 Dermatophytoses————————————————————————-17

2.4.2 Aetiologic agents————————————————————————–18

2.4.2.1 Anamorphs ——————————————————————————18

2.4.2.2 Teleomorphs—————————————————————————–23

2.5 Ecology of dermatophytes—————————————————————–23

2.5.1 Geophilic dermatophytes—————————————————————-24

2.5.2 Zoophilic dermatophytes—————————————————————-26

2.5.3 Anthropophilic dermatophytes———————————————————28

2.6 Epidemiology of dermatophytosis ——————————————————30

2.6.1 Epidemiology in human —————————————————————30

2.6.2Epidemiology of  in animals———————————————————–33

2.7 Transmission of dermatophytoses——————————————————-34

2.9 Other dermatomycoses in horses and donkeys—————————————-35

2.10 Laboratory diagnosis of dermatomycosis——————————————–42

2.10 Antifungal therapy———————————————————————–47

2.10.1 Azoles———————————————————————————–47

2.10.2 Polyenes——————————————————————————–48

2.10.3 Allylamines—————————————————————————–48

2.10.4 Echinocandins————————————————————————–48

2.11 Antifungal susceptibility testing methodologies ———————————-49

2.12 Resistance to antifungal drugs———————————————————52

2.12.1 Mechanisms of resistance to azoles————————————————-53

2.12.2 Mechanism of resistance to polyenes ———————————————-53

CHAPTER THREE: MATERIAL AND METHODS

3.1 Study animals——————————————————————————-55

3.2 The study population and sampling —————————————————–55

3.3 Sample collection —————————————————————————55

3.4 Materials and reagents ———————————————————————56

3.5 Media preparation ————————————————————————–56

3.5.1 Sabouraud dextrose agar (SDA) (Oxoid®) ——————————————–56

3.5.2 Potato dextrose agar (PDA) (Oxoid®) ————————————————-57

3.6 Fungal isolation——————————————————————————-57

3.7 Fungal identification————————————————————————–57

3.7.1 Slide culture technique ——————————————————————–58

3.8 Antifungal susceptibility test of fungal isolates——————————————59

3.8.1 Preparation of inocula ———————————————————————59

3.8.2 Antifungal susceptibility testing procedure ——————————————–60

3.9 Data presentation and analysis ————————————————————60

CHAPTER FOUR: RESULTS

4.1 Frequency of occurrence of mycotic agents on skin lesions of trade horses

and donkeys at Obollo-Afor  ——————————————————————-61

4.2 Fungal agents isolated from skin lesions of trade horses and donkeys at

Obollo-Afor—————————————————————————————-62

4.3 Antifungal resistance profile of dermatomycotic agents from horses/donkeys—–64

CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 Discussion————————————————————————————-94

5.2 Conclusion————————————————————————————-98

5.3 Recommendations—————————————————————————-99

REFERENCES———————————————————————————–101 Appendix——————————————————————————————–124

 

            CHAPTER ONE

           INTRODUCTION

1.1 Background of the study

Horses and donkeys are domesticated mammals used worldwide for draft,  transport, sports, ceremonial exhibitions, research purposes, warfare, crowd control, food (meat) and medicinal products as well as source of variety of leather products (Edwards, 1994; Bush and Marczak, 2005; Wikipedia, 2011). There are over 300 breeds of horses and 185 breeds of donkeys in the world (Hedge and Wagoner, 2004). In Nigeria, horses are mainly of the Arewa breed and their crosses with Arabian, Dongola, and Sudanese breeds found mainly in the Northern part of the country (Blench et al., 2004). Similarly, there are about four breeds of donkeys that are owned by both pastoralists and settled farmers in Nigeria (Blench et al., 2004).

Horses are known to live beyond 30 years with good care while donkeys live between 30-50 years (Blocksdorf, 2005). However, feed shortage and diseases are major constraints to their life span, productivity and work performance (Starkey and Starkey, 2004). The skin is the largest and most visible part of the body and the anatomic and physiologic barrier between animal and environment. A horse’s skin is its largest body organ, ranging from 12-24% of the animal’s weight; depending on age (Blocksdorf, 2005).The skin consists of various cellular and tissue components. There is an epidermis, an appendageal system, dermis, errector pili muscle, twitch muscle (panniculus carnosus), and a fatty subcutaneous layer known as the panniculus adiposus. Another cell type in the epidermis is the Langerhans’ cell, a non-neural cell that is active in the immunologic process and possibly in the regulation of keratin formation (Blocksdorf, 2005).

The main activity of the epidermis is to produce two types of protein–keratin and melanin. Keratin is a scleroprotein, which is the principal constituent of the epidermis. Scleroprotein is a simple protein characterized by its insolubility and fibrous structure, and which usually serves a supportive or protective function in the body. Melanin is the dark, shapeless pigment of the skin and hair (Blocksdorf, 2005). The most important part of the epidermis is the superficial layer, since much of the functional activity of the skin resides here. The proper functioning of this superficial layer is dependent on the structural arrangement of the keratin it contains and perhaps on its lipids (fats and fatlike substances characterized by being water-insoluble). In addition, the skin is synergistic with internal organ systems and thus reflects pathologic processes that are either primary elsewhere or shared with other tissues (Blocksdorf, 2005).

Fungal skin infections in horses range from some very common superficial disorders such as dermatophytosis (ringworm) which are almost ubiquitous and caused by two genera: Trichophyton and Microsporum (Pilsworth and Knottenbelt. 2007) to the exotic and difficult disorders related to secondary wound infections such as pythiosis due to Pythium spp. and conidiobolomycosis (black-grained mycetoma) which is a subcutaneous infection caused by Conidiobolus coronatus (Al-Ani, 1999) are some very common superficial disorders.

Dermatophytosis in equines is commonly caused by Trichophytonequinum (Trichophytosis) and Microsporum equinum (Microsporosis) (Pilsworth and Knottenbelt. 2007). Trichophyton is characterized by typical numerous small and rounded patches covered by small bran like asbestos colored scales while Microsporumequinum (microsporosis) is characterized by irregular limited patches, often overlapping by pronounced desquamation and accumulation of large, lime white scales firmly adheres to the base (Pilsworth and Knottenbelt. 2007). The reaction to dermatophytes can be variably pruritic and ranges from mild to severe according to the host reaction to metabolic products of the fungus, virulence of the species, anatomic location of infection and local environmental factors (Weitzman and Summerbell, 1995). Fungal lesions are most commonly present on the face, neck, thorax and girth with the legs are less commonly affected (Moriello, 2004). The mane and tail are rarely, if ever affected. The clinical lesions according to Radostits etal. (1997) and Pal (1987) are alopecia and/or circular circumscribed grayish-white, crusty, raised lesions which were most commonly found.

Less frequently, yeasts have been under the spotlight in equine dermatology since they cause dermatitis in immunocompromised individuals (White et al., 2006; Kim et al., 2011). Mycotic infection in horses has been associated with lipophilic yeast of the species Malassezia furfur (Cabanes, 2007). This yeast can be isolated from axilla, interbulbar region, groin, external ear canals of healthy horses (Nell et al., 2002). Out of the 14 species ranked within the genus, only M. furfur, M. slooffiae, M. obtusa, M. globosa, M. restricta, M. equina and M. pachydermatis have been isolated from horses and donkeys (Cabanes, 2007). Furthermore many species of Candida, namely C. albicans,C. krusei, and C. parapsilosis (Reilly and Palmer, 1994; Stout, 2008), have been described as causative agents of cutaneous disease in foals. Other fungi such as the genus Geotrichum has been implicated in both human and animal dermatomycosis, (Reppas and Snoeck, 1999; Chahota etal., 2001, Conti Diaz et al., 2003, Henrich et al., 2009).

Keratinopathogenic fungi can cause infections in horses resulting in conditions such as abnormalities of the hoof. Notable examples of such fungi are Scopulariopsisbreviacaulis, Alternataalternate, Geotrichumcandidum and Accremoniumblochii (Keller etal., 2000; Apprich etal., 2006). Risk factors for horse infections include an underlying status of immunodeficiency, prolonged administration of antibiotics and/or corticosteroids, alteration of normal microbial flora of the skin (Reilly and Palmer, 1994).

Fungal diseases are major public and veterinary health problem reported from different parts of the world and cause great economic losses to the nation (Calderone, 1989). The disease appears to be more common in tropical than temperate climates, particularly in countries having hot and humid climatic conditions (Pascoe, 1976). Studies have shown that the distribution pattern of mycotic infection vary considerably among different countries (Ayadi et al., 1993; Staats and Korstanje, 1995). This distribution pattern of fungal agents in different parts of the world has been attributed to climate and life style among other factors such as the prevalence of immunodeficiency disease (Hashem Al Sheikh, 2009).

Although, the role of drug resistance in treatment failure is not clearly known, invitro susceptibility testing could help the clinicians to select the proper antifungal agent (Norris etal., 1999). Shah et al. (1988), reported clinical cases of dermatophytosis in which the agents were resistant to the antifungal drugs griseofulvin and the azoles. With an increasing variety of drugs available for the treatment of dermatophytosis, the need for a reference method for the testing of the antifungal susceptibilities of dermatophytes has become apparent.

1.2 Statement of the research problem

Fungal diseases represent a serious threat to horses and donkeys (Fisher etal., 2012) due to their extreme contagious potential and challenging diagnosis. Fungal infections are usually neglected by public health authorities and often by the scientific community; this has serious impacts on the availability of epidemiological data and on knowledge of their clinical features.

Ringworms are highly contagious and can spread quickly through a yard by direct and indirect contact between horses and between horses and humans. Hot and humid climate in tropical and subtropical countries like Nigeria makes infections by dermatophytes or other fungal agents very common. Several studies on the prevalence and etiologic aspects of superficial mycosis in humans have been conducted in various parts of Nigeria (Nweze, 2011, Enemuor and Amedu, 2009; Mbata and Nwajagu, 2007; Anosike etal; 2005; Ameh and Okolo, 2004; Ive, 1996; Obasi and Clayton, 1989; Ogbonna etal., 1986). Documented information on dermatophytes from skin lesions of domestic animals in Southeastern region of Nigeria is scanty (Chah etal., 2012). Successful treatment of dermatophytoses depends on the ability of a given antimycotic agent to eliminate the fungi isolate (Santos et al., 2006). In order to predict this ability, invitro susceptibility testing becomes helpful because it can help clinicians to choose the correct treatment for their patients. Increased use of antifungals, often for prolonged periods has led to the recognition of acquired antifungal resistance (Alexander and Perfect, 1997). Furthermore, the rapid increase in fungal infections and the growing number of new antifungal agents (Andriole, 2000) indicate an increase need for rapid and accurate methods for antifungal susceptibility testing (Cormican and Pfaller, 1996)

Currently, information in available literature on dermatophytic infections and other fungal agents on the skin of horses and donkeys in Nigeria is lacking. Information on the antifungal susceptibility of drugs to cutaneous mycotic agents in horses and donkeys in Nigeria is currently lacking. There is therefore, need to provide information on the frequency of occurrence and characteristics of these mycotic agents on skin lesions of horses and donkeys.

1.3 Research questions

Based on the problems highlighted above, the following questions guided the study:

  1. Are fungal agents associated with skin lesions in trade horses and donkeys?
  2. If yes, what are the genera and species of these mycotic agents?
  3. What is the prevalence rate of these mycotic agents in trade horses and donkeys at Obollo Afor horse/donkey market?
  4. Are these mycotic agents susceptible to commonly available antifungal agents?
  5. Is there any association between the occurrence rate of the mycotic agents and body site, sex and season.

1.4 Aim and objectives of the study

This study was designed to determine the occurrence and characteristics of mycotic agents in skin lesions in trade horses and donkeys in Obollo-Afor, Enugu State, Nigeria. Specifically the study sought to:

  1. document the sex of horses and donkeys, sites and seasons of occurrence of these mycotic agents.
  2. determine the frequency of occurrence of dermatophytes in skin lesions of trade horses and donkeys.
  3. identify the genera and species of other fungal agents associated with skin lesions in horses and donkeys in Nigeria.
  4. determine the antifungal susceptibility profile of the cutaneous mycotic agents.

 

OCCURRENCE AND CHARACTERISTICS OF MYCOTIC AGENTS ISOLATED FROM SKIN LESIONS OF TRADE HORSES AND DONKEYS IN OBOLLO-AFOR, ENUGU STATE, NIGERIA

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