Investigation of bacterial and fungal agents from cutaneous lesions in canine Leishmaniasis

B-696 [1-6] Indian J. Anim. Res., Print ISSN:0367-6722 / Online ISSN:0976-0555 AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.arccjournals.com/www.ijaronline.in Investigation of bacterial and fungal agents from cutaneous lesions in canine Leishmaniasis Ugur Parin*, Goksel Erbas 1, Kerem Ural 2, Serap Savasan 1, Hafize Tugba Yuksel 1, Gamze Balat 1 and Şukru Kirkan 1 Department of Microbiology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydın, Turkey Received: 18-10-2017 Accepted: 21-11-2017 DOI: 10.18805/ijar.B-696 ABSTRACT The objective of the present study was to determine the bacterial and fungal agents accompanying with cutaneous lesions of dogs suffering from Canine Visceral Leishmaniasis (CVL). Sterile swap samples were taken from cutaneous lesions inspected of ears, dorsal, collar, pectoral, inguinal and interdigital spaces of 20 dogs, in which diagnosis were based on a combination of clinical symptoms, anti-leishmania antibody titers by use of Immunofluorescence antibody test (IFAT), and dogs were further classified according to the Leishvet Guidelines involving serological, hematological, serum biochemical, and urinalysis. Swap samples were inoculated to culture media for isolation and identification of bacterial and mycotic agents. 48 Staphylococcus aureus, 4 Bacillus cereus, 8 Staphylococcus epidermidis, 24 Candida albicans, 24 Microsporium canis and 4 Aspergillus niger were isolated and identified in both group of dogs. Regarding antibiotic susceptibility test, the isolates were found 100% susceptible to cefoperazone and amoxicillin-clavulanic acid, 53% to danofloxacin, 46% of the isolates were susceptible to enrofloxacin, and all of the isolates were resistant (100%) to penicillin G and gentamicin. In conclusion, the antibacterial and antifungal therapy should be provided in accordance with antibiotic susceptibility tests in skin lesions of dogs suffering with CVL. Key words: Bacteria,Canine visceral leishmaniasis, Cutaneous lesion, Dog, Fungi INTRODUCTION Leismaniasis has been recognized as a protozoal disease caused by the intracellular agents of the genus Leishmania (Blavier et al., 2011). The latter parasite is transmitted to man and animals by blood-sucking sandflies of the genus Lutzomyia in America and Phlebotomus in Asia, Africa and Europe. Infected domestic dogs serve as reservoirs of the disease, namely Canine Visceral Leishmaniasis (CVL), for people in several areas where the infection is endemic (Gramiccia, 2011). A number of different species of Leishmania cause cutaneous (CL) or mucocutaneous (MCL) leishmaniasis, although not fatal, and responsible for a reasonable morbidity in a number of people residing within endemic focus (Tanner et al., 2000). The significance of leishmaniasis on human health has been classified by the World Health Organization (WHO) as one of the most neglected tropical diseases with a probable yearly incidence of 1 1.5 million cases of CL and 500,000 cases of VL (Desjeux, 1996). Dogs are very susceptible to this parasite and may suffer from a complex syndrome, CVL, one of the emerging zoonoses globally causing severe fatal disease. In most countries, dogs are common companion animals and their health is of great concern, therefore management of CVL possesses similarity to those of human leishmaniasis. The CVL disease causes cutaneous skin lesions in dogs, and as a consequence, bacterial and mycotic pyoderma cases may occur as secondary infections. The secondary deficiency of specific immunity may lead to deep infectious pyoderma in dogs, and CVL is a good example for occurrence of these types of bacterial and mycotic skin lesions (Mason et al., 1996; Ciaramella et al.,1997). The aim of this study was to identify the infectious agents that arise in cutaneous skin lesions in dogs with CVL. MATERIALS AND METHODS Dogs: The microbiological and serological samples were taken from dogs that were brought to clinics of Adnan Menderes University, Faculty of Veterinary Medicine, Department of Internal Diseases and pre-diagnosed with CVL according to clinical symptoms from March 2014 to December 2015. The exact diagnosis of CVL was based on a combination of clinical symptoms, anti-leishmania antibody titers by use of IFAT, and dogs were further classified according to the Leishvet Guidelines involving serological, hematological, serum biochemical and urinalysis (Solano- *Corresponding author s e-mail: uparin@adu.edu.tr 1 Department of Microbiology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydýn, Turkey 2 Department of Internal Medicine, Adnan Menderes University Faculty of Veterinary Medicine, 09016, Aydýn, Turkey

2 INDIAN JOURNAL OF ANIMAL RESEARCH Gallego et al., 2009). A total of 20 CVL positive dogs met the criteria for involvement in the present study. The dogs were divided into two groups with regard to the disease stages. Sample collection: Sterile swap samples were withdrawn from a total of 120 cutaneous lesions of ears, dorsum, collar, pectoral, inguinal and interdigital spaces of 20 dogs, which were serologically diagnosed as CVL, and were brought to laboratory under cold chain. Blood samples were collected from vena cephalica antebrachium in the volume of 10 ml for haematological and serum biochemical tests. Serum biochemical values were not subjected to statistical analysis, as this was not purpose of the study. The authors of this research hereby declare that collection of specimen was carried out in accordance with the guidelines laid down by the National Institute of Health (NIH) in the US regarding the care and use of animals for experimental procedures or with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Bacterial and mycotic identification: Each swab samples were streaked onto blood agar, MacConkey agar and Eosin Methylene Blue agar for bacterial examination and the agar plates were incubated at 37 C for 24 h under aerobic conditions. Mycotic examinations were performed by streaking to SDA (Sabouraud Dextrose agar) and incubating at 25 C for 7 days under aerobic conditions. After 24-48 h growth period, the preliminary evaluation of the bacterial colonies was made on the grounds of colony appearance and Gram staining method. The bacterial colonies were identified with conventional biochemical and carbohydrate fermentation tests by standard procedures (Holt and Krieg, 1993). All isolates were confirmed with API (biomérieux, France) Identification System. Mycotic diagnoses of the colonies were performed by the preliminary macroscopic evaluation of the fungal colonies grown on SDA after 7 days of incubation period. Suspicious mycotic colonies were identified according to microscopic colony morphologies by Lactophenol Cotton Blue staining and susceptible mycotic colonies by Gram staining method (Koneman et al., 1997), and were later identified by API (biomérieux, France) Yeast Identification System. Antimicrobial sensitivity of the bacterial strains: All strains were tested for their sensitivity against amoxicillinclavulanic acid, penicillin G, cefoperazone, gentamicin, enrofloxacin and danofloxacin as per standard procedure (CLSI, 2011). RESULTS AND DISCUSSION Classification to the groups: According to Leishvet Guidelines dogs were staged as stage I mild disease (n=10) in group I and stage II moderate disease in group II (n=10), as described previously (Solano-Gallego et al., 2009). Haematological and serum biochemical results: There were significant differences among mean (±SD), RBC, Hb and PCV values between group I and group II of dogs infected with CVL (Table 1). Serum biochemical analysis showed individual alterations with min-max values for ALT (29-90 U/L), AST (39-126 U/L), creatinine (0,72-4,53 mg/ dl) and BUN (17,5-84 mg/dl). Microbiological analysis: 56 strains were identified as Gram positive round shaped, 4 strains were identified as Gram positive rod shaped bacteria. A total of 60 gram positive bacterial strains were isolated through the collected 120 specimen from both groups. A total of 48 out of 60 bacterial isolates (80%) were identified as Staphylococcus aureus, 8 (13,3%) isolates were identified as S. epidermidis and 4 (6,7%) isolates were identified as Bacillus cereus. A total of 52 mycotic agents were isolated through the collected 120 Table 1: Haematological results of dogs with CVL were presented as mean ±SD, and min-max values Group N Mean±SD Min-Max P Value WBC(x10 9 /L) Group I 10 16,983±15,879 5,89-59,05 0,529 Group II 10 15,887±7,268 6,40-31,66 RBC(x10 12 /L) Group I 10 4,724±0,774 3,76-5,74 0,007 Group II 10 6,033±1,350 4,67-9,27 HGB(g/dL) Group I 10 10,910±2,331 7,10-13,70 0,005 Group II 10 14,550±3,158 10,20-21,80 PCV(%) Group I 10 30,415±4,947 23,02-38,14 0,009 Group II 10 38,667±8,117 28,57-57,54 MCV(fL) Group I 10 64,500±3,689 60,00-72,00 0,912 Group II 10 64,100±2,234 61,00-68,00 MCHC(g/dL) Group I 10 35,610±2,580 30,90-38,30 0,123 Group II 10 37,580±1,124 35,60-39,20 RDWc(%) Group I 10 16,730±1,880 15,10-21,30 0,971 Group II 10 16,690±1,611 15,30-20,50 PLT(x10 9 /L) Group I 10 284,300±161,452 32,00-681,00 0,190 Group II 10 451,800±200,305 177,00-717,00 WBC: white blood cell; RBC: red blood cell; HGB: hemoglobin; PCV: packet cell volume; MCV: mean cell volume; MCHC: mean cell hemoglobin concentration; RDWc: red cell width distribution; PLT: platelet

Case no Vol. Issue, () Table 2: Distribution of microbiological agents in relation to groups of dogs with Leishmaniasis Pectoral Dorsal Collar Right ear Leftear Inguinal Interdigita 1, 2 S. epidermidis S. epidermidis S. epidermidis S. epidermid 3,, 4 Group I 5 B. cereus 6 S. epidermidis S. epidermid 7 B. cereus 8 9 S. epidermidis 10 11,, 12 S. epidermidis 13 Group II 14 15 continue Table 2...

4 INDIAN JOURNAL OF ANIMAL RESEARCH continue Table 2... 16 17 18 19 B. cereus A. niger A. niger 20 B. cereus A. niger A. niger specimen. 24 (46%) as Candida albicans, 24 (46,1%) as Microsporium canis, 4 (7,7%) isolates were identified as Aspergillus niger. The findings present that bacterial and mycotic agents are intensively colonized especially in ear, dorsal and pectoral region. The distribution of bacterial and mycotic agents in dogs with CVL according to groups and isolation location was mentioned on Table 2 and it is also seen that in case numbers 1, 3, 4, 8, 11, 15, 17; bacterial and mycotic infections occur together in same anatomical region. In this study, the isolation numbers of, B. cereus, and are close to each other. Since, in group I, S. epidermidis is isolated in the ratio of 11.4% while it is isolated as 2% in group II. It is noteworthy that A. niger was isolated in both group I and group II. The number of isolated species of the bacterial and mycotic agents in relation to groups of dogs with Leishmaniasis is shown on Table 3. Antibiotic susceptibility tests (Table 4) revealed that all of the bacterial isolates (n=60) were found 100% susceptible to cefoperazone and amoxycilin-clavulanic acid, and 100% resistant to penicillin G and gentamicin. The results indicated that bacterial and mycotic agents may be isolated from the skin lesions of dog patients with CVL in both group of dogs with stage I (mild) and stage II (moderate) disease. According to the authors knowledge this is the first study involving microbiological and dermatological analysis in dogs with CVL as per Leishvet guidelines. There was no significant difference between groups for distribution of selective microorganisms. Various bacterial species, that are well adapted to the microenvironment of the superficial Stratum corneum and the hair follicles, may colonize within the skin surface in animals. By this extent, the normal flora contributes to skin immunity (Lloyd, 1992). In this study, Staphylococcus sp. was identified as one of the most frequent colonizing infectious agents, among canine skin lesions. The vast majority of the healthy dogs harbor these potential microbial pathogens in low number on the skin surface, but it is likely that they have transient infections rather than chronic diseases status (Saijonmaa-Koulumies and Lloyd, 1996). Bacterial skin lesions in dogs occur subordinately to any type of skin inflammation and hypersensitivity reactions. The result of this research indicates the higher incidence level for agents colonizing on skin in lesions of CVL positive cases. Furthermore, it is determined Table 3: The number of isolated species of the bacterial and mycotic agents in relation to groups of dogs with Leishmaniasis Groups Identified bacterial Sampling region in dogs and mycotic Pectoral Dorsal Collar Right ear Leftear Inguinal Interdigital Total agents Group I 5 4 4 4 4 2 2 25 S. epidermidis - 3-1 1-2 7 B. cereus - 2 - - - - - 2 3 4-1 1 3 1 13 2 4 3 - - 2 3 14 A. niger - - - - - - - - Group II 5 4 4 3 3 2 2 23 S. epidermidis - 1 - - - - - 1 B. cereus - 2 - - - - - 2 3 4 - - - 3 1 11 2 4 1 - - 2 1 10 A. niger - - - 2 2 - - 4

Vol. Issue, () Table 4: The antibiotic sensitivities of the bacterial isolates Bacterial Isolates Gentamicin Cefoperazone Amoxycilin-clavulanic acid Danofloxacin Enrofloxacin Penicillin G n S I R S I R S I R S I R S I R S I R 48 - - 48 48 0 0 48 0 0 32 8 8 28 8 12 0 0 48 S. epidermidis 8 - - 8 8 0 0 8 0 0 0 8 0 8 0 0 0 0 8 that, and A. niger was accompany these bacterial skin lesions in dogs. Many skin diseases have been reported to accompany with CVL and to complicate its clinical picture and diagnosis (Saridomichelakis, 2009). Bacterial skin infections, mycotic dermatitis and dermatophytosis have all been diagnosed in CVL cases (Blavier et al., 2011; Solano- Gallego et al., 2011). However, cutaneous lesions and their relation with bacterial and mycotic cultures were not analyzed in details according to the stages of the disease, as per Leishvet guidelines. The current study reveals that secondary bacterial and mycotic diseases may develop in animals suffering from CVL both in stage I and II of the disease. The suppression of immune system may lead the alteration of the integumentary flora, as soon the opportunistic pathogen bacteria and mycotic agents may cause skin lesions. Our research indicated that bacterial and mycotic agents both emerge in skin lesions of dogs with CVL and dogs are more susceptible to S. epidermidis and A. niger infections in Stage I of the disease also. It seems to be a challenging condition since antibiotics have no effect on yeasts and mold. Animal owners and veterinary practitioners should take care about the hygienic conditions of dogs with CVL, thus the damage of cutaneous tissue may be reduced. In the present study the antimicrobial susceptibility tests indicated that cefoperazone and amoxycilin-clavulanic acid are significant choices for antibacterial therapy. These types of chemotherapeutics have a broad range of spectrum and they are resistant to penicillinase. Fluoroquinolones like enrofloxacin have an excellent tissue penetration and this type of antimicrobial may be also effective for treatment of bacterial skin lesions (Lloyd, 1992). The present study showed that the bacterial strains are quite susceptible to enrofloxacin. On the other hand, responsible agent was found resistant against penicillin G and gentamicin, which have narrow spectrums for treatment of staphylococcal infections. Given that mycotic agents are resistant to antibiotics, the therapy procedure should be followed by determining the stage and identification of causative agents. CONCLUSION The altered cutaneous homeostasis and the dysregulation of the immune system, including the depression of cell-mediated immunity, imposed by CVL may predispose to secondary infections, such as bacterial pyoderma (Koutinas et al., 2001). The similar reason may exist in the increased frequency of mycotic isolation from the skin of dogs with CVL. The latter disease may cause a group of various bacterial and mycotic skin diseases and dogs are prone to these types of cutaneous skin lesions. Therefore an accurate diagnosis is obligatory for treatment. It was determined that bacterial and mycotic agents may colonize in the cutaneous lesions of the dogs with Leishmaniasis. An appropriate wide range antibacterial and antifungal therapy should be provided in accordance with antibiotic susceptibility tests in skin lesions of dogs suffering with CVL. ACKNOWLEDGEMENT The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed and the researches were conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. REFERENCES Blavier, A., Keroack, S., Denerolle, P.A. (2011). Typical forms of canine leishmaniasis. Veterinary Journal, 162: 108-120. Ciaramella, P., Oliva. G., De Luna, R. (1997). A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum. Veterinary Record, 22: 539-543. CLSI, (2011). Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals. Approved Standard. 3 rd ed. Document M31-A3, vol 28 No. 8. Villanova. Pa. 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