Occurrence of Bovine Dermatophilosis in Huambo Province, Angola
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Rev. Salud Anim., Vol. 39, No. 2 (mayo-agosto 2017), ISSN: 2224-4697 Artículo Original Occurrence of bovine dermatophilosis in Huambo province, Angola Ocurrencia de dermatofilosis bovina en la provincia Huambo, Angola Aires Walter1, Armindo Paixão1, Siomara Martínez2, Ivette Espinosa2 1 Faculty of Veterinary Medicine, Jose Eduardo Dos Santos University, Angola. 2 Departament of Microbiology-Epidemiology, National Center for Animal and Plant Health (CENSA), Apartado 10, San José de las Lajas, Mayabeque, Cuba. ABSTRACT: Dermatophilosis is an exudative pustular dermatitis that affects cattle, sheep, horses, goats, dogs, cats, reptiles, and occasionally humans. This disease is caused by Dermatophilus congolensis, a pleomorphic Gram-positive actinomycete. According to the Veterinary Services, the highest incidence of the disease in Angola was identified in the provinces Huambo, Kwanza Sul and Malang during May 2013 and April 2015, but only supported on clinical sings. In the present work ninety crust samples were taken from animals with lesions on the skin in Huambo province, in the period May 2013-April 2015. The bacteria were isolated following Haalstra´s method. Presumptive colonies of the Dermathophilus genus were subjected to Gram stain, biochemical identification, and molecular detection by PCR. Eight Dermathophilus congolensis isolates were tested for their sensitivity to eleven different antibiotics. A total of 30 samples showed typical colonies of the Dermatophilus genus. The amplicon of 817 bp corresponding to the 16S rRNA region was obtained in the 30 isolates suspecting of being D. congolensis. Drug sensitivity test revealed that Amoxicillin/clavulanate, Clindamycin, Erythromycin, Lincomycin, Azithromycin, Rifampicin, and Enrofloxacin were highly effective against the eight D. congolensis isolates. Observation of lesions and microbiologic diagnosis using biochemical and molecular techniques allowed confirming bovine dermatophilosis in the region of Huambo, Angola. Key words: Dermatophilus congolensis, bovine, diagnosis, antimicrobial resistance. RESUMEN: Dermatofilosis es una dermatitis pustular exudativa que afecta al ganado bovino, ovejas, caballos, cabras, perros, gatos, reptiles y, ocasionalmente, a humanos. Esta enfermedad es causada por Dermatophilus congolensis, un actinomiceto pleomórfico Gram-positivo. Según los Servicios Veterinarios, la mayor incidencia de la enfermedad en Angola se identificó en las provincias Huambo, Huila, Kwanza Sul y Malang durante mayo de 2013 y abril de 2015, pero solo se basó en lesiones clínicas. En el presente trabajo se obtuvieron 90 muestras de costra de animales con lesiones en la piel en Huambo, Angola, en mayo de 2013 y abril de 2015. Las bacterias se aislaron siguiendo el método de Haalstra. A las colonias presuntivas del género Dermathophilus se les realizó tinción de Gram, identificación bioquímica y detección molecular por PCR. Se seleccionaron ocho aislamientos de Dermathophilus congolensis para su sensibilidad a 11 antibióticos diferentes. Un total de 30 muestras mostraron colonias típicas del género Dermatophilus. El amplicón de 817 pb correspondiente a la región ARNr 16S se obtuvo en treinta cepas sospechosas de D. congolensis. La prueba de sensibilidad a las drogas reveló que Amoxicilina/clavulanato, Clindamicina, Eritromicina, Lincomicina, Azitromicina, Rifampicina y Autor para correspondencia: Ivette Espinosa. E-mail: [email protected] Recibido: 6/2/2017 Aceptado: 9/6/2017 1 Rev. Salud Anim., Vol. 39, No. 2 (mayo-agosto 2017), ISSN: 2224-4697 Enrofloxacina fueron altamente eficaces contra los ocho aislados de D. congolensis. La observación de lesiones y la identificación por técnicas bioquímicas y moleculares confirmaron la dermatophilosis bovina en la región de Huambo, Angola. Palabras clave: Dermathophilus congolensis, bovino, diagnóstico, resistencia antimicrobiana. INTRODUCTION General Directory of the Veterinary Services of Dermatophilosis is a contagious skin disease Angola, (10). However, this information has caused by Dermatophilus congolensis. Such been supported only on clinical examination, disease is characterized by exudative, but there are also other diseases affecting cattle proliferative or hyper keratotic dermatitis, and producing similar injuries to the bovine skin. For these reasons, the application of a accompanied by the production of crusts and foliculitis (1,2,3). The causative organism is an polyphasic diagnosis combining phenotypic aerobic Gram-positive bacterium that produces and molecular assays has been necessary. motile zoospores. This bacterium invades the This work was aimed at confirming the skin and causes an acute, subacute or chronic presence of D. congolensis in animals with skin disease. This disease affects cattle, sheep, clinical lesions, similar to the dermatophilosis horses, goats, wild animals, and man, being in the bovine farms in Huambo province, considered a zoonosis (4,5,6). Angola, by using microbiological, phenotypic This disease, which has a worldwide and molecular methods. distribution, prevails in tropical areas and it is MATERIALS AND METHODS associated to humid environments and other A randomized, cross-sectional and factors. The first case was reported in Belgian prospective study was conducted on farms and Congo, the current Democratic Congo, by Van communities with traditional systems and Saceghem in 1915, and since then, the extensive breeding located in five bacterium has been isolated from animal municipalities in Huambo province, Angola. infections chiefly in Africa (Kenya, Ethiopia, The total cattle population consisted of 752 Tanzania, Nigeria, and South Africa), Asia animals, which included the Nelore race and (Turkey, India and China), and Central and indigenous cattle. The samples were taken in South America (Argentina, Uruguay and two stages in the period May 2013–April 2015. Brazil). The disease has also been isolated in Animals with skin lesions were selected Australia, the United States (New York, specifically for the production of crust, purulent Kentucky, Florida, and Texas), Canada, and discharge and alopecia, similar to those Europe (France, Spain, and Germany), but as a described by Topa and Iseensee (11) and chronic endemic disease or, more rarely, as an Tavanaeimanesh et al. (12). acute and epidemic infection (7,8,9). A prevalence of 30 % based on previous In Angola, the dermatophilosis is considered studies was considered (13), and the minimum as a re-emerging disease. The costs are sample size was calculated with 95 % calculated in terms of productivity losses, confidence and 5 % estimated error. The reproduction problems in cows with severe following equation was used: n=Nz2pq/dz(N- vulva infection, and also chemotherapy and 1)+z2pq (14). Based on this equation, the chemoprophylaxis costs, as well as losses due minimum sample size was 73 bovines for to culling and death of the infected animals. The obtaining the ratio of animals positive for D. disease has occurred in Angola with the highest congolensis. incidence in the provinces of Huambo, Kwanza Sul and Malang, as it was reported by the 2 Rev. Salud Anim., Vol. 39, No. 2 (mayo-agosto 2017), ISSN: 2224-4697 Isolation of Dermatophilus congolensis from observed for five days for any activity by the the infected scabs organisms. The samples were collected in sterile plastic Antibiotic susceptibility test tubes by scraping the lesions located on The cultures of the eight strains were different parts of the animal body and performed on Muller Hinton Agar medium conserved in refrigeration, maintaining the (Oxoid) supplemented with 5 % blood. The biosecurity in the whole process of specimen antibiotics used were Amoxicillin + clavulanate collection. Then they were transported to the (10 ug, Oxoid), Clindamycin (2 ug, Oxoid), Veterinary Medical Microbiology Laboratory, Streptomycin (10 ug, Oxoid), Sulfazotrim (30 Faculty of Agriculture and Veterinary Medicine ug, Oxoid) Erythromycin (15 ug, Oxoid), of the University of Brasilia, Brazil. The Lincomycin (2 ug, Oxoid), Azithromycin (15 containers with specimen were properly closed ug, Oxoid), Rifampicin (5 ug, Oxoid), and decontaminated in each farm. Enrofloxacin (10 ug, Oxoid), and Sulfonamides Dermatophilus species isolation was carried (250 ug). The inhibition zone diameter was out by the Haalstra’s method with minor measured after 48 h incubation at 37°C. The modifications (15). The samples were results were analyzed according to the Clinical fragmented with a scalpel, moistened with and Laboratory Standards Institute (16), sterile saline solution, and stained by the Gram specifically for Gram positive microorganisms. stain. Small pieces of scab were placed in a vial with 1 ml of sterile distilled water and stored at D. congolensis detection by PCR room temperature for 3-4 hours. The opened Bacterial DNA was extracted from colonies vial was placed for 15 minutes in a candle jar. which had been resuspended in 200 µl of The samples of the liquid surface were nuclease-free water. Proteinase K (20 mg/ml) extracted with a bacteriological loop and was added and incubated at 37oC for 90 min. cultured on Blood Agar Base (Oxoid) Then, phenol-chloroform extractions and supplemented with 5 % sheep blood and ethanol precipitation were made. The incubated at 37°C under 5 % CO2 tension in an concentration and purity of the DNA from all incubator for 72 hours. samples were determined by Colibri The colonies with the presumptive Microvolume Spectrophotometer (Titertek- characteristics of the Dermatophilus