E. Kukhar, V. Kiyan, A. Khalikova
S. Seifullin Kazakh Agro Technical University, Astana, Kazakhstan

IMMUNOCHEMICAL CHARACTERISTICS ANTIGENS
DERMATOMYCETES MICROSPORUM CANIS

Microsporia - (from the Greek. mikrуs – small and sporo – seed sowing), microsporoz, ringworm, a contagious disease (mycosis) of animals, caused by fungi Microsporum, characterized by lesions of the skin and its derivatives. Microsporia is one of the most common dermatological pathologies in domestic animals. Microsporia get sick more often cats, dogs, fur-bearing animals, rabbits, at least - horses, sheep, goats, pigs, deer, monkeys, tigers. The problem of Microsporia, caused by zooantropogenic mushrooms, is still one of the urgent problems of modern dermatomycology in connection with the observed epidemic outbreaks of epizootic in humans and domestic animals in several countries [1].

In Kazakhstan, the incidence of zooantroponosis microsporia of the Republic of the public was in the 90s. up to 42,6-44,3%. According to the Ministry of Health the incidence of dermatomycoses in Kazakhstan is a stable level, while microsporia registers everywhere. Intensive microsporia incidence in 2007 was 79.8 per 100 thousand population (Figure 1).

Figure 1 - Incidence microsporia population of Kazakhstan for 2000-2007

(the figures indicate the number of patients)

At present, diagnosis of dermatomycoses, includes methods such as microscopy of pathological material, cultural and fluorescent diagnostics. These methods do not allow a fairly high degree of accuracy the correct diagnosis, not to mention the specific identification of the pathogen. Traditional diagnostic methods do not meet modern requirements, and highly effective methods of diagnosis of pathogenic dermatomycoses in Kazakhstan has not yet been developed. Therefore, before the medical and veterinary mycology is an issue on the development of more sophisticated methods that allow to quickly diagnose fungal infections.

One of the important areas of modern biotechnology, both in this country and abroad, is the development and improvement of technological processes of production of diagnostic biologics against zooantroponosis disease [2].

These requirements are fully in line immunoassay diagnostic test kits. In order to develop diagnostic test systems are required antigens that differ high specificity and activity. Thus, the aim of our study was to obtain antigenically-active components of the cell wall dermatomycetes Microsporum canis and the study of their immunological properties.

Methods and materials

In the experimental work was used dermatomycetes strain of M. canis, isolated from pathological material. In order to obtain antigenically-active components of the cell walls of submerged cultivation was carried out dermatomycetes M. canis in 500 ml flasks on Sabouraud nutrient mediums not within 15 days.

As the antigens were used: protein components of the cell wall dermatomycetes M. canis, isolated in L. Tabatabai (1979) (antigen №1); protein components of the cell wall dermatomycetes M. canis, isolated by the method of freezing and thawing (antigen №2); culture medium antigen (CM). The antigens were purified by low-speed centrifugation and gel-filtration chromatography [3].

Gel-filtration chromatography columnar separation of protein antigens was performed on the equipment of the company "Rharmacia" in the sorbent in the quality used vehicle brand Sephadex and Sephacryl (“Sigma”) [4].

The degree of purification of antigens was monitored by polyacrylamide gel electrophoresis by the method of V.K. Laemmli (1970) on the unit to a vertical electrophoresis («Compact dual mini code», England) using a Tris-glycine buffer, and 12.5% polyacrylamide gel in the presence of sodium dodecyl sulfate.

To evaluate the antigenic activity of protein antigens using immunoblotting method, the reaction RMA, IDR and indirect ELISA.

Results of the study

Biomass dermatomycetes got deep cultivation within two weeks. At the end of the growth of mycelial mass was separated from the liquid medium by filtration through a paper filter and used to isolate antigenically-active components.

The average yield of biomass dermatomycetes M. canis was 5.2 g per 100 ml of liquid medium (Figure 2).

Figure 2 - The yield of biomass dermatomycetes M. canis

Output antigen №1 was 0.375 mg, and antigen №2 – 0.325 mg with 1 g of fungal biomass. In the obtained preparations was determined the concentration of proteins and carbohydrates.

The result is a visually into account (Table 1).

Table 1 - Characteristics of antigens dermatomycetes M. canis

The antigen	Concentration (in mg/ml)		Ratio carbohydrates to proteins
	Carbohydrates	Proteins
Antigen №1	0,005	0,250	1:50
Antigen №2	0,025	0,250	1:10
Culture medium antigen (СM)	1,0	0,030	1:0,03

Further analysis of the antigens was performed by column chromatography, gel-equipment of «Pharmacia». As the sorbent used Sephadex G-25 and Sephacryl S-200, as eluate – 0.9% NaCl solution and 0,02 M Tris-HCl.

The results of the separation of antigens in samples of gel-filtration column chromatography, the separation into fractions were recorded on an automatic recorder (Figure 3).

1 2 3

Note: 1 – antigen №1, 2 – antigen №2, 3 – antigen-CM

Figure 3 – Chromatogram of purification of antigens dermatomycetes M. canis

As seen in Figure 3, the cell wall protein antigens of M. canis contain six, The culture of the antigen contains four components are well separated into fractions.
Analysis of protein antigens was performed using polyacrylamide gel electrophoresis by the method of V.K. Laemmli (1970) on the vertical electrophoresis device (Hoefer Scientific, USA) using a Tris-glycine buffer, 12% polyacrylamide gel in the presence of sodium dodecyl sulfate at capacity – 120 V, a current - 30 mA, voltage – 50W.

The resulting protein fraction had the following molecular weights: a protein antigen is №1 – 116 kDa, 110 kDa, 98 kDa and 36 kDa; protein antigen №2 – 116 kDa, 110 kDa, 98 kDa and 66 kDa, 36 kDa; the antigen-CM – 110 kDa (Figure 3).

Electrophoregram dermatomycetes protein antigens were transferred to nitrocellulose membrane (Schleicher & Schuller, Germany) for 3 h at a power of - 120 V, a current – 45 mA, voltage – 50W by means of the device company Scientific (USA) by the method of H. Towbin et al.

Free media sites blocked with 1% solution of bovine serum albumin. Nitrocellulose membrane was washed and then incubated for 1 hour with the patient's blood serum microsporia man, and then kept in a working dilution of antibodies against human immunoglobulins labeled with horseradish peroxidase. Then performed immunochemical manifestation of a nitrocellulose membrane, which plunged it into the substrate solution, prepared immediately before use.
A positive reaction was characterized by the appearance of lines, painted in gray-purple color (Figure 4).

30 кДа

14 кДа

20 кДа

24 кДа

32 кДа

45 кДа

66 кДа

1 2 3 4

Note: 1 – markers, 2 – antigen №1; 3 – antigen №2, 4 – antigen

Figure 4 – Results of immunoblotting of protein antigens dermatomycetes M. canis

As seen in Figure 4, the molecular weight antigenic protein antigen fraction number 1 that interacts with the antibody is 30 kDa.

To study the specific activity of antigen immunized albino mice. In further studies, we tested antigens derived on the activity and specificity with the sera of mice in indirect ELISA.

We found that both the antigen and immunogenes quite active. They identify specific antibodies by indirect ELISA in sera diluted 1:800 (antigen №1) and 1:1600 (antigen №2).

The presence of antigen precipitating properties dermatomycetes M. canis was studied in the reaction of immunodiffusion (IDR). As a result, we found that the precipitating antigen №1 has properties of that was characterized by the appearance of a clear precipitin line with the corresponding immune serum (Figure 5).

AG – the antigen №1 M. canis; 1 – mouse serum immunized with the antigen №1; 2 – mouse serum immunized with the antigen №2, 3 – mouse serum immunized with the antigen-CM; 4 - mouse serum immunized with the protein antigen T. verrucosum; 5 – serum mice, immunized with LTP-130; 6 – negative control.

Figure 5 – The reaction of immunodiffusion in agarose gel protein antigen №1 dermatomycetes M. canis

To identify the properties of the obtained agglutinated antigen reacted droplet agglutination and micro agglutination reaction.

In the reaction of agglutination (RA) on glass droplet formation characteristic of flakes was observed after 3-5 seconds after the introduction of serum. This indicates the presence of marked agglutinating properties of the derived antigens.

In setting up the reaction with micro agglutination (RMA) derived antigens, the titer of specific antibodies detected in relatively high dilutions of sera (Table 2).

Table 2 - Results of testing sera of people in the micro agglutination reaction with protein antigens of M. canis

Antigens	Antibody titers of sera of blood
	К ^+	К^-	К^+ to T. verrucosum
Protein antigen №1	1:1024	РО	1:2
Protein antigen №2	1:512	РО	1:16
Notes: K^+ – human serum, with a clinically confirmed diagnosis of Microsporia, K^- – a negative control

As can be seen from Table 2, agglutinating antibodies were found in the RMA at a dilution of 1:1024 sera before – with the protein antigen №1, 1:512 – to a protein antigen №2.

Antigenic activity of antigenic preparations obtained by us in a indirect ELISA, IDR, RMA and RA is presented in Table 3.

Table 3 – Results of the study of antigenic properties of antigens dermatomycetes M.canis in various serological tests n = 3, P<0.05

Serological Reactions	Of antibody titers in sera, identified antigens of the fungus M. canis
	Protein antigen №1	Protein antigen № 2	Antigen-СM
IDR	+	-	-
РА	+	+	-
RMA	1:1024	1:512	1:2
Indirect ELISA	1:1200 ± 0,25	1:1200 ± 0,25	1:100 ± 0,2

From Table 3 it can be concluded that the isolated protein antigeny fungus M.canis, have distinct antigenic properties. The maximum titer of antibodies detectable protein antigens in the ELISA is 1:1600, 1:1024 and PMA.

Conclusions

Thus, the investigation resulted in two protein antigen of M. canis. A study of immunochemical characteristics of antigens dermatomycetes revealed the presence of sufficient activity in the ELISA and PMA, as well as the presence of precipitating properties.

References:

1. Pozdnyakov, O.N., Makhnovets, E.N., Reshetnikov T.B., Nemchaninova O. Epidemiology of zooantropophyles dermatomycoses in Novosibirsk // Problems of medical Mycology. – St-P., 2003. – V.5, №2. – S. 64.

2. Yelinov, N.P., Vasilyeva N.V., Raznatovsky K.I. Ringworm, or tinea surface of the skin and its appendages - the hair and nails. Laboratory diagnosis of // Problems of medical Mycology. – St-P., 2008. – T.10, №1. – S. 27-34.

3. Toleutaeva. S.T. Manufacturing technology of antigens from cultures of dermatophytes for the production of serological reactions // Actual problems in the diagnosis of animal diseases: Sat. Mater. II Int. Scientific-Practical. Conference. – Almaty, 2005. – S. 325-326.

4. Osterman, L.A., Chromatography of proteins and nucleic acids. – Moscow: Nauka, 1985. – S. 65-91, 145-166.