The Comparison of Scraping , Trichogram , and Taping Techniques for Diagnosis of Demodicosis in Dog ( PERBANDINGAN TEKNIK SCRAPING

Demodicosis is a parasitic disease which is predilected in skin. Demodicosis in dogs may present local or general clinical symptoms. The success of demodicosis treatment is highly dependent on dog immune conditions, nutritional status, disease status, and routine treatment. The success of the treatment is also affected by the accuracy in the diagnostic technique used. The purpose of this study was to compare the three diagnostic skin examination techniques namely scraping, trichogram, and taping to diagnose cases of demodicosis. A total of 20 dog samples was taken from dog patients that came to Bali Veterinary Clinic, at Prerenan, Badung, Bali, with symptoms of itching, hair loss, skin redness, scale, and hyperpigmentation. Sampling was done by technique of scraping, trichogram, and taping. Scraping technique was done by scraping the skin, trichogram technique was done by pulling hair, and taping technique was done by attaching the tape. The result of Demodex mite isolation from the three diagnostic techniques performed showed scraping technique 5.45 ± 1.05; trichogram technique 1.10 ± 0.91; and taping technique 3.50 ± 0.83 dogs. Its Concluded that scraping technique provides the best diagnostic value for the isolation of Demodex mites compared to the other two.


INTRODUCTION
Demodicosis is a parasitic disease caused by Demodex mite which is predilected in skin.The diseases firstly reported in year 1842 (Sastre et al., 2012).Normally Demodex mites live in dog skin (Fondati et al., 2010;Ravera et al., 2013).This mite can transmit from mother to puppies during lactation period in 2-3 days old (Miller et al., 2013).There are three strain of Demodex who infected dog i.e.Demodex canis (Hillier et al., 2002), D. conei (Carlotti et al., 2006), and D. injai (Sastre et al., 2012).Clinical sign of demodicosis in dog are in local and general form.Local demodicosis is a lesions which find in less than six locations, with diameter less than 2.5cm, while, general demodicosis is a lesion which find in 12 or more locations and even in all body parts (head, face and body) (Miller et al., 2013).However, no absolute definitions are agreed to define the shape of local and general lesions of demodicosis (Waisglass, 2015).Isolation of blood biomarkers i.e. serum Creactive protein level and haptoglobin, can be used as an indicator that demodecosis is general (Martinez-Subiela et al., 2014).
Juvenile demodicosis is a disease found in small races dogs under 12 months, under 18 months of age in large breeds, and less than two years of age in giant dog races.While, demodicosis is categorized in adult dogs if the dog has never suffered from skin problems since the dog was born until the dog was four years old (Waisglass, 2015).These groupings often have difficulty when the medical record of the dog is not recorded completely.
The success of demodicosis treatment depends on the isolation of the causative agent, and the introduction of clinical demodicosis at the location of the body first attacked (Saridomichelakis et al., 2007).Other factors determining the success of demodicosis treatment are immune dog conditions, nutritional status, disease status, and routine therapy (Ferrer et al., 2014).Cooperation and understanding of the owner is necessary because treatment requires a longer time and intensive (Waisglass, 2015).Early onset of infection also affects the speed of cure of demodicosis treatment.In dogs that have suffered from demodicosis since juvenile require longer treatment period, compared to dogs suffering from demodicosis as adults.
Rapid, easy, and simple diagnostic techniques of demodicosis are necessary in veterinary practice.The speed of diagnosis provides a good added value for practitioners in veterinary medical services.The technique of scraping and trichogram is a common way of diagnosis of demodicosis (Saridomichelakis et al., 2007).One other demodicosis diagnostic technique that can be used is taping (Pereira et al., 2012).This paper would like to compare the implementation of three diagnostic techniques in diagnosing cases of demodicosis at the veterinary practice.The purpose of this study was to compare the three diagnostic skin examination techniques namely scraping, trichogram, and taping to diagnose cases of demodicosis.

RESEARCH METHODS
The samples were taken from dog patients who came to Bali Veterinary Clinic, Prerenan, Badung-Bali.Patients used as samples were those who had skin problems and showed symptoms of itching, hair loss, skin redness, scale and hyperpigmentation.Sampling was performed at three different sites of the lesion (head, body, and leg).At each location sampling was done with all three diagnostic techniques (scraping, trichogram, and taping).The number of dogs taken were 20 samples, without regard to gender, age, and race.

Scraping Technique
Scraping was done by using scalpel blade.Prepare; mineral oil dripped in the middle of the glass object.Area of the skin sampled, massage with finger and thumb as much as 10 times, and continue when doing scraping.Avoid cutting skin when doing scraping.Scraping was done on the skin until it bleeds.Samples are placed on mineral oil droplets on glass objects.The skin scrap sample was flattened with a syringe needle, covered with a glass cover and observed under a light microscope with 400x magnification.Note the number of mites, eggs, and Demodex larvae observed (Saridomichelakis et al., 2007).

Trichogram Technique
In areas of the skin that indicate a lesion, the hair on it was removed with the forefinger and thumb.Prepare an object glass and spilled mineral oil on it.Removed hair was placed on mineral oil droplets and then spread and flattened with a syringe needle.Cover the object glass with the cover glass then examined under a light microscope with 400x magnification.Note  c).This can be due to the help of massage and scraping the skin until bloody.Massage helps release Demodex mites from the keratin tissue of the hair follicle.Scrape until it bleeds as a scrape marker until the dermis layer of the skin, the predilection site of the Demodex mite (Beco et al., 2007).
In the trichogram technique found the least amount of Demodex mites.This can be caused by Demodex mites still left in the skin during hair removal process.In the trichogram technique was not recommended to do hair root massage, so keratin on the hair root is not released, which is where the predilection of Demodex mite (Beco et al., 2007).Trichogram was less sensitive than scraping when mite infestation is low (Saridomichelakis et al., 2007).However, if a Demodex mite is found, it can't be indicated as a normal condition (Fondati et al., 2010).Further examination is needed.It was reported that for the isolation of Demodex mite in dogs is more difficult than isolating Demodex mite in humans, so isolation of a single human Demodex mite may be indicated as a normal condition.
In taping technique found fewer Demodex mites than scraping methods and more from the trichogram technique.In taping technique was also done a massage on the skin before sampling process, so that the mites in the dermis layer rise to surface and taped to the tape used.In the taping technique was not followed by scraping the skin, so that mites that are still more deep in the skin can't be attached to tape.Taping techniques can be used as an alternative to replace scraping techniques (Pereira et al., 2012;Miller et al., 2013).Taping techniques can isolate Demodex canis in dogs more sensitive than scraping techniques (Pereira et al., 2015).the number of mites, eggs, and Demodex larvae observed (Beco et al., 2007).

Taping Technique
Tapping was done by using clear tape with length 6 cm and width 2 cm.In the area of the skin that have lesion, massage with finger and thumb as much as 10 times.Press the sticky side of the tape firmly to the skin area to be sampled.Pinch up the taped skin and place fingers on top of the tape; roll and squeeze the skin firmly, as if trying to squeeze the follicle contents onto the sticky portion of the tape.Remove the tape and place sticky side down onto the object glass.Examined the sample under a light microscope with 400x magnification.Note the number of mites, eggs, and Demodex larvae observed (Pereira et al., 2012).Data of Demodex mites obtained were analyzed with SPSS 13 for windows program.

RESULT AND DISCUSSION
The mean number of Demodex sp mites observed in each field of view of microscope, respectively; technique of scraping, trichogram, and taping was 5.45 ± 1.05; 1.10 ± 0.91; 3.50 ± 0.83.The results of Demodex sp mite isolation, each data of the three diagnostic techniques are presented in Table 1.
Demodicosis is a skin disorder that takes a long time and perseverance in the treatment.The inaccuracy of the diagnostic technique used has an effect on the accuracy of the diagnosis of demodicosis.Difficulties in the diagnosis also due to clinical symptoms caused by demodicosis often overlap with other diseases that attack the skin.Symptoms become less specific if there is a secondary infection of bacteria.Inexpensive, quick, and easy diagnostic techniques for isolation of Demodex mites are scraping, trichogram and taping (Saridomichelakis et al., 2007;Beco et al., 2007;Fondati et al., 2010).Another technique that can be used is biopsy (Fondati et al., 2010) and isolation techniques from feces (Waisglass, 2015).Biopsy techniques are more difficult and take longer time.When making histologic preparations in biopsy techniques, Demodex mites are often frequently drift.
In this study, dogs used as samples showed symptoms of itching, hair loss, skin redness, scale, and hyperpigmentation (Fig 1 a-b).Of the three techniques used, all were able to isolate

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Nyoman Suartha, et al Jurnal Veteriner the demodex mite (Fig 2 a-c).Scraping technique obtained the most Demodex mite isolation, which was significantly different from two other techniques (P<0.05).In the scraping technique can also be isolated eggs and pupa of Demodex mites (Fig 3 a-

Figure 1a .
Figure 1a.Dog samples with hair loss symptoms in the face and leg areas Figure 1b.Dog samples with hair loss symptoms around the eyes

Figure 3b .
Figure 3b.Result of Demodex mites isolation on taping technique, under light microscope with 400x magnification.

Figure 3c .
Figure 3c.Result of Demodex mites isolation on scraping technique, under light microscope with 400x magnification

Table 1 .
Average number of Demodex mites isolated from all three diagnostic techniques