The Potential of Antigenic Protein of Sarcoptes scabiei as a

Serological Diagnostic Candidate for Scabies in Goats

Nunuk Dyah Retno Lastuti

1,2

, Dony Chrismanto

, Poedji Hastutiek

and Agus Sunarso

Postgraduate School, Universitas Airlangga, Surabaya

Dept. of Parasitology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya

Animal Health Program, Faculty of Vocational Education, Universitas Airlangga, Surabaya

Keywords: Antigenic Protein, Goat, Sarcoptes scabiei, Serological diagnostic.

Abstract: In Indonesia, the prevalence of scabies on goat cattle is still high, which shows that scabies is still not

handled properly. It is now considered as an emerging/re-emerging parasitic disease that threatens human

and animal health globally. The scabies disease has been known for a thousand years, and it is a persistent

problem on public health and also livestock. Yet, until today a serological diagnostic tool is sensitive and

specific is not available, especially for goat cattle in Indonesia. In recent years, an indirect antibody enzyme-

linked immunosorbent assay (ELISA) has been available, which has higher sensitivity and specificity than

traditional diagnostic methods. In order to overcome those problems preventive actions need to be

undertaken through research for serological diagnostic development as an alternative for scabies prevention

in goats in Indonesia. The purpose of this research is doing sensitivity and specificity tests towards the

antigenic protein of Sarcoptes scabiei isolated from goat as diagnostic material for scabies in goats. The

method was comprised of deciding the gold standard for positive and negative scabies, checkerboard test

toward antigenic protein, and ELISA test for measuring sensitivity and specificity. The research results

showed that S. scabiei antigenic protein with molecular weight of 57.3 kDa can be recognized by serum

antibody of goat that infested with scabies with sensitivity level of 96% and specificity of 86,6%. From that

result, it can be recommended that S.scabiei with a molecular weight of 57.3 kDa is the specific antigenic

protein that can be used as a candidate of serological diagnostic material for scabies isolated from

Indonesian local goat.

1 INTRODUCTION

Scabies disease is already known for thousand years

ago and is persistently harming the health of people

as well as cattle. However, until today a sensitive

and specific tool for serological diagnostic which is

specially provided for goat livestock in Indonesia

has not been found yet. Diagnosis of scabies

nowadays is still conventional, which is based on

clinical symptoms and microscopic examination

from the skin scraping results done by scraping until

the deep layer skin is peeled off. The diagnosis is not

very practical if the number of livestock is high and

the livestock is less sensitive because the clinical

symptoms are similar to other skin disease like

caused by other mites (psoroptes, notoedres and

chorioptes), fungus, and ticks, which cause atopic

dermatitis, itching, and alopecia (Soulsby, 1986;

Walton and Currie, 2007; Yu Zheng, 2016). A

definite diagnosis (skin scraping) by finding

Sarcoptes scabiei mites will meet difficulties,

especially if the number of mites is low in the

infected animals and the success level is only 30-

50% (Arlian, 2000; Lower et al., 2001; Tarigan,

2004; Walton and Currie, 2007). As an effort to

resolve the problem, it is necessary to develop

diagnostic material serologically to enable early

therapy to prevent broader transmission. Some

countries, such as Australia, Germany and the

Uniteds States, have developed serological diagnosis

(ELISA) for dogs and pigs, it is very possible

because S.scabiei could induce a humoral antibody

response on the infected host (Lower et al., 2001;

Arlian et al., 2004; Tarigan, 2004; Vercruysse, 2004;

Walton and Currie, 2007; Lastuti, 2017; Lastuti,

2018).

Lastuti, N., Chrismanto, D., Hastutiek, P. and Sunarso, A.

The Potential of Antigenic Protein of Sarcoptes scabiei as a Serological Diagnostic Candidate for Scabies in Goats.

DOI: 10.5220/0007546505370540

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 537-540

ISBN: 978-989-758-348-3

537

2 MATERIAL AND METHODS

2.1 Determination of Gold Standard

The determination of gold standard for positive

control and negative controls was based on

microscopic examination of goat skin scraping

infected with scabies. Positive results were declared

if the researchers found S.scabiei mites under the

goat skin by scraping examination, which would

then be used as positive control. Meanwhile, the

negative control comes from healthy goats, which

were previously examined by skin scraping and did

not contain any mites. The number of samples for

gold standard was 40 samples consisting of 25

positive samples of scabies and 15 negative samples

of scabies (Lastuti, 2017).

2.2 Indirect ELISA assay

The checkerboard result of antigenic protein of

S.scabiei (57.3 kDa) was examined through indirect

ELISA test to test the ability to detect antibody

reaction towards positive control and negative

control performed as follows: a microtiter plate

consisting of 96 wells, with each well being coated

with 100 μl of antigen solution with a concentration

of 10 μg/ml in buffer coating, was incubated at 4 ºC

overnight. The next day, the well was washed with

buffer washing (NaCl-Tween) 200μl three times.

Furthermore, the well was blocked using a 4%

creamer (in PBS-Tween) of 200 μl/well and

incubated at 37 ºC for one hour, then microtiter plate

will be washed the same way three times. The next

step is that the well was added with goat serum from

positive control and negative controls and also added

with PBS tested on 1/100 dilution for as much as

100 μl per well. Work was done in duplo. As per the

standard for counting antibody titers, the dilution of

antibody for which the positive control antibody titer

was known was done at dilutions of 1/25 to 1/51200

and 100 μl was added per well. Then, the plate was

incubated at 37 ºC for an hour and washed again.

The next step is the addition of anti-goat conjugate

(IgG anti-goat) at1/5000 dilution for as much as 100

μl each well and incubated at 37 ºC for one hour.

The plate was washed again and added pNPP

substrate in substrate buffer (diethanolamine 1

mg/ml) of 100 μl per well. Then, the well was

incubated at room temperature in a dark room within

15 to 45 minutes. The reaction was stopped by the

addition of 50 μl NaOH 3N solution per well, then

the plate was read using the ELISA reader with a

wavelength of 405 nm (Lastuti, 2018). The value of

OD obtained in positive and negative controls would

determine the sensitivity and specificity of the tested

antigens.

3 RESULTS AND DISCUSSION

The results of the indirect ELISA test showed that

the antigenic protein identified by the gold standard

antibody sample was a molecular weight protein of

57.3 kDa. The number of samples was 40, which

consists of positive controls with 25 samples and

negative controls with 15 samples. The average

value of Optical Density (OD) and the antibody titer

were listed in table 1 below.

Table 1. Average OD values and gold standard sample

antibody titer which recognized the S.scabiei protein 57.3

kDa

Sample

Value of OD

(mean  SD)

Antibody Titer

(mean  SD)

Positive

0.281

 0.096

712,000



451.220

Negative

0.166

 0.020

26,666



70,373

Annotation: A different superscript on the same column

showed a very significant difference (p <0.01).

Based on the ELISA indirect test, the value of

OD and antibody titer was used as the basis for

testing the sensitivity and specificity of proteins with

a molecular weight of 57.3 kDa. The test results

showed that 57.3 kDa protein serum antibodies

could be recognized by a goat, with the following

results: out of 25 positive samples of gold standard,

24 samples were positive (true positive) and one

sample was negative (false positive). Meanwhile,

from 15 negative samples of gold standard, 13

negative samples and 2 positive samples (false

negative) were found. The results are summarized in

Table 2 below.

Table 2: Sensitivity and Specificity Tests of S.scabiei

Protein 57.3 kDa.

Skin Scraping Examination

Elisa

test

Total

Note: Skin scraping: conventional method for scabies

diagnostic ELISA test: serological test used to develop

scabies diagnostic.

The calculation results of sensitivity test, which

is the positive number of ELISA test divided by the

positive number of scraping examination, is: 24/25 =

ICPS 2018 - 2nd International Conference Postgraduate School

538

96%, while the specificity test result is the negative

number of ELISA test divided by the number of total

negative examination of gold standard scraping is:

13/15 = 86.6%. These results indicated that S.scabiei

antigen protein with a molecular weight of 57.3 kDa

could be identified by scabies infected goat serum

antibody with 96% sensitivity level and specificity

level of 86.6%. From the results, it can be

recommended that the S.scabiei protein goat isolates

with a molecular weight of 57.3 kDa is a specific

antigenic protein that could be used as a serological

diagnostic candidate for scabies in goats.

Based on the results of Tarigan's research

(2004a) that goat infected with S.scabiei showed a

high IgG response ten days after infection and the

high level of IgG could be maintained for up to 20

days after receiving ivermectin. The antibody was

able to recognize antigen with molecular weight of

43 to 220 kDa with four highly prominent antigens

being 180, 60, 38 and 37 kDa. Sensitivity test results

of 96% and a specificity of 86.66% showed an

accurate diagnosis result exceeding 80% (Bornstein,

2006; Lower et al., 2001). The development of

serological diagnostic tests that have been performed

to diagnose scabies in various animals have been

undertaken by researchers, including a diagnosis

developed by ELISA techniques to detect S.scabiei

antibody. Serological test results showed not much

different levels of sensitivity and specificity as they

did Lower et al (2001) for serologic diagnosis in

dogs with ELISA assay, which found a sensitivity

level of 84.2% and specificity of 89.5%, in which

antigen was used to detect antibodies in dogs who

had received scabies treatment for 1 to 4.5 months

and the ELISA test was recommended for the

diagnosis of scabies in dogs. The same test had been

evaluated by serological test with ELISA indirect

test against scabies in red fox (Vulpes vulpes), which

showed a sensitivity level of 95.4% and specificity

level of 100% and based on these results it was

concluded that ELISA test was used for diagnosing

and studying the epidemiology on scabies on red fox

(Bornstein, 2006). Similarly, Rambozzi et al (2004)

performed a serological test for detecting antibodies

induced by S. scabiei in chamois (Rupicapra spp)

with asymptomatic symptoms in the outbreak region

of the scabies showing 97% sensitivity level with

ELISA assays. Based on the exploratory results of

S.scabiei protein, it has been proved that S.scabiei

contains a protein, which is capable of inducing

humoral and cellular immune responses and has high

sensitivity and specificity level (> 80%), which can

be used as serological diagnostic candidate kits for

scabies in goats.

4 CONCLUSIONS

Antigenic protein of S. scabiei with a molecular

weight of 57.3 kDa could be recognized by serum

antibody of goat infested with scabies and had high

sensitivity and specificity level (> 80%). From the

results, it can be recommended that S.scabiei with a

molecular weight of 57.3 kDa is the specific

antigenic protein that can be used as a candidate of

serological diagnostic material for scabies isolated

from Indonesian local goat.

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