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DIAGNOSIS OF CANINE DEMODICOSIS BY ELISA

KrishiKosh

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Title DIAGNOSIS OF CANINE DEMODICOSIS BY ELISA
 
Creator LUBNA FATHIMA
 
Contributor Dr.G. S. SREENIVASA MURTHY
 
Subject demodex, diseases, biological phenomena, elisa, hides and skins, biological interaction, fruits, concentrates, proteins, sampling
 
Description Canine demodicosis is a common inflammatory skin condition of dogs characterized by the presence of Demodex mite overpopulation and development of cutaneous lesions. An alteration in the immune system of skin or immunosuppression allows the mites to proliferate in hair follicles and sebaceous glands. The present study was aimed to develop ELISA for diagnosis of canine demodicosis.
ABSTRACT
The preliminary study was conducted in 146 dogs by skin scrapings examination with dermatological disorder to investigate the prevalence of canine demodicosis at Teaching Veterinary Clinical Complex (TVCC), College of Veterinary Science located at Bhoiguda, Rajendranagar and Mailardevpally and also in stray dogs of Animal Birth Control (ABC) centre at Hyderabad, Telangana during the period of March 2016 to August 2016, out of which 17 were diagnosed as suffering from canine demodicosis.
The overall prevalence of canine demodicosis recorded was 11.64 % (17 out of 146) by skin scraping examination. Highest prevalence of canine demodicosis was observed in males as 64.7 % (11 out of 17). 8-12 months of age group dogs recorded highest prevalence 35.3 % (6 out of 17) and higher prevalence 35.3 % (6 out of 17) was observed in breed of Labrador retriever.
The dermatological lesion area was sterilized using 70 % alcohol, then skin fold over the lesion was squeezed between the fingers and then deep skin scrapings were collected by using rounded 10 number scalpel blade scrapping the blade back and forth over the skin until capillary bleeding is evident. Acetate tape impressions were collected from the areas of dermatological lesions.
Morphological characterization of mites was done by microscopic examination and micrometry. Cigar shaped mites whose body divided into gnathosoma bearing paired palps and chelicerae, and unpaired hypostome; podosoma consisting of four pairs of stumpy legs and elongated opisthosoma were observed. Stubby Demodex cornei with rounded terminal end was observed. Spindle shaped egg and larva with three pairs of legs were also noticed during the study.
Upon measurement of Demodex canis, mean body length, mean body width, the mean length of gnathosoma, podosoma and opisthosoma were 216.95 ± 2.07 μm, 37.07 ± 0.18μm, 19.79 ± 0.18 μm, 62.62 ± 0.2 μm and 134.67 ± 1.69 μm respectively. Measurement of Demodex cornei revealed mean body length, mean body width, mean length of gnathosoma, podosoma and opisthosoma as 148.02 ± 1.12 μm, 37.37 ± 0.54 μm, 17.57 ± 0.16 μm, 62.28 ± 0.46 μm and 67.83± 0.89 μm respectively. The mean length and mean width of Demodex spp. eggs; larva were 81.15 ± 0.32μm and 31.09 ± 0.64 μm; 96.08 ± 0.15 μm and 31.45 ± 0.04 μm. Lengths of total body, gnathosoma and opisthosoma of
both types of mites differed statistically significant (P0.05).
Antigenic material was placed in 15 ml centrifuge tube and was washed several times in sterile PBS (pH 7.4) until the supernatant fluid became clear. The mites suspension in PBS (pH 7.4) was mixed well by using a pipette. Before settle down of the mites, the suspension was taken and placed on a clean glass slide covered with cover slip and the mites were counted and then entire volume was counted. The protein concentration of crude somatic antigen was 0.448 mg/ml. The crude somatic antigen extracted was used for standardization of ELISA.
For standardization of ELISA checker board titration was followed for optimization of Rabbit anti-dog IgG HRPO conjugate, Demodex canis antigen concentration and serum dilution. Then after standardizing the Demodex canis antigen concentration at 0.5 μg against 1:50 anti- Demodex positive sera and 1:5000 Rabbit anti-dog IgG HRPO conjugate dilution a cut off value was determined using 10 negative sera samples at antigen concentration of 0.5 μg against 1:50 sera dilution and 1:5000 conjugate dilution. The cut-off value was determined as 0.58 (Mean + 2SD) and the samples which gave OD above this value were considered as positive for Demodex canis antibody. The overall sensitivity of ELISA was determined as 70.58% (12 out of 17 true positive) and specificity was 76.92% (10 out of 13 negative sera).
In the present study, per cent positives of dog sera detected were 24.37 % (39 out of 160) using Crude Somatic Antigen of Demodex canis with ELISA. Out of 88 stray dogs, 17 were positive and out of 72 pet dogs, 22 were detected as positive with ELISA.
The result between pet dog sera and stray dogs did not show a significant difference in incidence of canine demodicosis.
Demodectic mange in dogs can be confused with many dermatological conditions. Traditionally skin scrapping is the only reliable and gold standard diagnostic method available. An accurate diagnosis will be more helpful for successful treatment of dogs with canine demodicosis. The results of present study have shown that with the ELISA standardized it is possible to demonstrate antibodies to Demodex canis mites in dogs with an acceptable level of accuracy and if further characterization and purification of immunodominant Demodex canis antigen is done then the sensitivity and specificity of the assay can further be improved.
 
Date 2016-12-31T13:36:17Z
2016-12-31T13:36:17Z
2016-12-30
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/93801
 
Relation D;508
 
Format application/pdf
 
Publisher PVNR TVU