ES2239891B1 - IMMUNOLOGICAL METHOD FOR THE DIAGNOSIS OF AVIAR ASCARIDIASIS. - Google Patents

Abstract

Method for in vitro diagnosis of avian ascaridiasis through the use of Ascaris summ antigen complexes, as antigens in ligand enzyme immunosorbent assay (ELISA) techniques and diagnostic kit.

Description

Immune method for the diagnosis of avian ascaridiasis.

The present invention relates to an in vitro diagnostic method, in particular a method for the in vitro diagnosis of avian ascaridiasis.

Prior art

Avian ascaridiasis is a disease caused by nematodes of the genus Ascaridia galli , one of the most common intestinal parasites in poultry farming. This nematode affects the intestine of both poultry (chicken, turkey, goose) and wild birds (partridge, capercaillie, duck, pheasant and common vulture), developing in the mucosa and intestinal lumen of the bird.

The preparation period, time that passes from that the bird ingests the embryonic eggs of the parasite until this reaches the adult state and produce eggs that appear in the Bird feces, it is approximately 50 days.

The females of this parasite produce great number of eggs (about 5,000 per day), which are expelled,  without embryo, along with feces. The larva, inside the egg, reaches its infective state in a period of 2 to 3 weeks, in about environmental conditions of good humidity and high temperature.

The symptoms and signs of the disease are enteritis, diarrhea and anemia, symptoms that are associated with presence of worms in the digestive tract, but other symptoms such as the decrease of glycogen and proteins contained in muscles, they indicate the ability of parasites to modify the metabolism of its host, and the consequences are the reduction of  growth and weight loss of affected birds. The big ones concentrations of laying hens and existing chickens on farms they facilitate the spread of the parasite among birds, since these are infected by the ingestion of parasite eggs  present on land or garbage, or eating worms, which They act as parasite deposits.

In this way ascariadisis is considered, at present, as an important economic problem. Several studies, from different parts of the world, on the epidemiology of Ascaris galli have been published in recent years indicating this problem. (Eshetu et al., Rev. Sci. Tech . 2001; 20: 791-796; Gauly et al., Vet. Parasitol . 2000; 96: 301-307; Magwisha et al., Trop. Anim. Health Prod . 2002 ; 34: 205-214; Mpoame & Agbede, Rev. Elev. Med. Vet. Pays Trop . 1995; 48: 147-151; Permin et al., J. Helminth . 1997; 71: 233-240. Permin et al. ., Br. Poult. Sci . 1999; 40: 439-443; Poulsen et al., Prev. Vet. Med . 2000; 45: 237-245; Wilson et al., Avian Dis . 1994; 38: 158-160 ).

The detection methods, at present, of Ascaridiasis are performed by stool analysis to Identify the parasite's eggs. This implies that when it detects the infection, the damage it causes is already occurring The parasite In addition, eggs ejected by the host infected constitute a risk to the rest of the birds that They live with her.

Therefore, there is a need for a method of analysis that allows to detect the presence of the parasite before That adult worms begin to produce eggs. Taking like this preventive measures on the affected farms and avoid, by less in part, the damage caused by the level parasite intestinal.

After thorough research, the authors of the present invention have discovered that antigenic complexes of adult Ascaris suum vermes can be used as antigens in immunosorbent ligand enzyme (ELISA) assay techniques for the diagnosis of avian ascaridiasis.

Explanation of the invention.

In accordance with an object of the present invention, a new method is provided for the detection of avian ascaridiasis comprising the use of Ascaris suum antigen complexes, as antigens in ligand enzyme immunosorbent assay (ELISA) techniques. These antigenic complexes from complete extracts of adult Ascaris suum vermes share numerous antigens with Ascaridia galli , allowing the diagnosis of avian ascaridiasis before the parasite's eggs begin to be eliminated through the feces of infected birds. This will allow preventive measures to be taken on affected farms and avoid damage caused by the parasite at the intestinal level.

The ELISA method allows the simultaneous analysis of numerous serum or blood samples, therefore, both the diagnostic method and the kit of the present invention imply an improvement in terms of diagnostic time and performance, where the antigens used are obtained. industrially in a standardized manner, increasing the sensitivity and specificity of the diagnosis of the infection caused by Ascaridia galli .

The diagnostic method according to the invention is performed " in vitro " by means of the ELISA technique on serum or whole blood from the animal suspected of suffering from avian ascaridiasis, and comprises, in a preferred embodiment, the steps of:

to.

upholster the wells of a polystyrene plate with the antigenic extract of adult vermes of Ascaris suum , by incubating the plate with a solution of the antigens diluted in PBS (phosphate buffered saline);

b.

poststap with BSA (serum albumin bovine) in PBS;

C.

add the serum problem sample or whole blood diluted in diluent buffer and incubate;

d.

add secondary antibody anti-IgG anti-chicken marked with an enzyme generally used in an immunoenzymatic analysis of diagnosis selected from the group formed by peroxidase, alkaline phosphatase and glucoxidase, diluted in diluent buffer of section c) and incubate;

and.

reveal the reaction by addiction of a colorless substrate solution specific to the enzyme conjugate used in the previous stage and incubate;

F.

read the absorbance at 492 nm.

The diagnostic method of the invention is preferably carried out so that step b) is post-taped with 1% BSA; in step c) the test sample is diluted 1/200 in diluent buffer and said diluent buffer is composed of NaCl 1.7 gr, Na 2 HPO 4 0.426 gr, NaH 2 PO 4 0.078 gr, BSA 2 gr, Tween 20 0.05 ml and distilled H2O 200 ml; in stage d) the secondary antibody is diluted 1: 8000 in the buffer diluent

In another preferred embodiment, the method of Diagnosis of the invention comprises the steps of:

to.

cover the wells of a polystyrene plate with the antigenic extract of adult vermes of Ascaris suum , by incubating the plate at a temperature of 4 ° C for 16 hours, with 200 µl / well of a solution of 0.8 µg / well of the antigen diluted in PBS (pH 7.2);

wash 3 times the plate with PBS (pH 7.2), to separate antigens that have not been bound;

b.

posttap with 1% BSA in PBS (pH 7.2), for 1 hour at 37 ° C;

wash again as in the previous stage;

C.

add 100 µl / sample well serum or whole blood problem diluted problem 1/200 in buffer diluent (consisting of NaCl 1.7 gr, Na 2 HPO 4 0.426 gr, NaH 2 PO 4 0.078 gr, BSA 2 gr, Tween 20 0.05 ml and H 2 O distilled 200 ml) and incubate for 1 hour;

wash the plates with 0.05% PBS / Tween 20 mixture 3 times.

d.

add 100 µl / well of secondary anti-IgG antibody anti-chicken labeled with an enzyme usually used in a diagnostic immunoenzymatic analysis selected from the group consisting of peroxidase, phosphatase alkaline and glucoxidase, diluted 1: 8000 in the diluent buffer of section c) and incubate 2 hours at 37 ° C;

wash the plates with 0.05% PBS / Tween 20 mixture 3 times.

and.

reveal the reaction by addiction of a colorless substrate solution specific to the enzyme of the conjugate used in step (g), and incubate for 10 to 15 minutes at room temperature from 22 to 25 ° C;

F.

stop the enzymatic reaction with 1 N sulfuric acid and read the absorbance at 492 nm.

According to another preferred embodiment, the Diagnostic method of the invention comprises:

Stages a) - b)

In step a) of the method of the present invention, the polystyrene microtiter plate is treated previously by generally known methods. In practice, 0.8 mug / well of the antigen extract is dissolved in PBS buffer (0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.2). 200 µl / well of said solution being introduced into all wells of a microtiter plate. The plates with incubated for 16 hours at a temperature of 4 ° C, time needed to that the antigens are absorbed in the walls of the wells. The plates are washed with PBS buffer to separate antigens that are not have linked.

A post-upholstery is done with 1% BSA in PBS buffer pH 7.2, for 1 hour at 37 ° C.

Stage C)

The samples used can be serum or blood. The samples are diluted in 1/200 proportion in diluent buffer consisting of NaCl 1.7 gr, Na2 HPO4 0.426 gr, NaH2PO4 0.078 gr, BSA 2 gr, Tween 20 0, 05 ml and distilled H2O 200 ml and incubate for 1 hour. The plates are washed with the 0.05% PBS / Tween 20 mixture. In general, 3 washes are performed. By adding the serum or blood test sample onto the upholstered plates with the antigens, the Ascaridia galli antibodies, if present in the sera, will bind to the antigen fixed on the plate.

Stage d)

In step d) the secondary anti-chicken IgG antibody labeled with an enzyme suitable for the purposes of the present invention is added to each well, in general any enzyme generally used in a diagnostic immunoenzymatic analysis such as peroxidase is valid , alkaline phosphatase and glucoxidase. In accordance with a preferred embodiment of the present invention, the peroxidase labeled anti-chicken IgG antibody diluted 1/8000 is used in the diluent buffer of section (c). Plates treated in this way are incubated for 2 hours at a temperature of 37 ° C. The plates are then washed with 0.05% PBS / Tween 20. By adding the enzyme-anti-IgG conjugate, it binds to any anti- Ascaridia galli antibody that could have bound to the plate-bound antigen. If the serum sample does not contain any anti- A. Galli antibody, the conjugate will remain free or in suspension and will be removed during washing.

Stages e) - F)

In these stages, a well is added to each well colorless substrate solution specific for the enzyme conjugate used in step (d). The substrate solution used When the enzyme is peroxidase it has the following composition: OPD buffer (orthophenylenediamine) 10 ml (citric acid 2.14 gr, Na 2 HPO 4 x 12 H 2 O 6.54 gr, H 2 O distilled 400 ml, adjust to pH 7.5) + OPD 0.0028 gr + H 2 O 2 4 µl.

The presence of the immobilized enzyme within the bound conjugate is revealed by adding the specific substrate, producing a reaction that occurs with a Color change.

For a quantitative determination, the plaque is read in an ELISA reader to measure absorbance (A) for each well at a wavelength that is specific to each type of enzymatic substrate, in the case of peroxidase and the substrate described above, the measurement is made at 492 nm. They are considered positive sera with an optical density (D.O.) equal to or greater than 0.85. This limit of positivity has been set analyzing a wide range of sera from healthy animals from a non-endemic exploitation of ascaridiasis (sera normal) and adding to your D.O. 3 times its deviation standard.

Another object of the invention is a kit of ELISA diagnosis to detect avian ascaridiasis in a animal serum or blood sample, characterized in that understands:

to.

a polystyrene plate previously treated with Ascaris suum adult vermes extract for binding to the anti- Ascaridia galli antibodies present in serum samples or whole blood to be analyzed.

b.

a secondary anti-IgG antibody anti-chicken labeled with an enzyme usually used in a diagnostic immunoenzymatic analysis selected from the group consisting of peroxidase, phosphatase alkaline and glycoxidase.

C.

a colorless substrate solution specific for the enzyme conjugate used in the previous stage (b).

Brief description of the chart

In Fig. 1 the graphs are shown Optical density (D.O.) results, obtained in the analysis carried out by the ELISA method on blood samples from of seven farms of laying hens (Exp.).

Where the meaning of the symbols is the next:

The dark rectangle corresponds to the range of optical density (D.O.) of blood samples analyzed in each holding Where the line that divides it into two represent the arithmetic mean of said optical densities (D.O.).

The upper and lower dimensions of the rectangles are the standard deviations of optical densities  (D.O.) of the blood samples analyzed.

Black dots correspond to values of optical density (D.O.) of analyzed blood samples that were They leave the standard deviation.

Examples

The following experimental trials illustrate the invention made by the inventors and highlights the specificity of the method. For these examples 7 were studied Laying chicken farms.

Example 1

A sample was made in each of the seven farms or farms studied, in which they were taken in Total 140 blood samples. These samples were analyzed by the ELYSA method for IgG antibodies. Table 1 indicates the number of samples analyzed in each farm; the life time of the animals analyzed; and the number of samples they have given positive in the assay, giving an optical density (D.O.) greater than 0.85, calculating the prevalence (% of positive samples in the samples tested) in each of the farms.

In Fig. 1, as described previously, the results of optical density (D.O.) obtained in blood samples analyzed in each holding (Exp.).

TABLE 1

one

Example 2

A sampling of the faeces of the hens in each of the farms was also carried out, analyzing the abundance of ascaridia galli eggs by the flotation method in zinc sulfate.

Due to the nature of the samples to analyze, in four of the seven farms analyzed (Nº 1, 4,  5 and 7), the samples were joint and in each of these feces 10 samples of 1 gr each are analyzed, in different areas of the fecal mass.

It was found that farms No. 1 and 2 were not endemic to ascaridiasis, no parasite eggs found in the samples analyzed.

Claims (9)

1. In vitro diagnostic method of avian ascaridiasis characterized by the use of Ascaris suum antigen complexes, as antigens in ligand enzyme immunosorbent assay (ELISA) techniques. 2. Method according to claim 1, characterized in that it comprises the steps of:

to.

cover the wells of a polystyrene plate with the complete extract of Ascaris suum , by incubating the plate with a solution of the antigens diluted in PBS;

b.

post-taper with BSA in PBS;

C.

add the serum problem sample or whole blood diluted in diluent buffer and incubate;

d.

add secondary antibody anti-IgG anti-chicken marked with an enzyme generally used in an immunoenzymatic analysis of diagnosis selected from the group formed by peroxidase, alkaline phosphatase and glucoxidase, diluted in diluent buffer of section c) and incubate;

and.

reveal the reaction by addiction of a colorless substrate solution specific to the enzyme of the conjugate used in the previous stage, and incubate;

F.

read the absorbance at 492 nm.

3. Method according to any of the preceding claims, characterized in that in step b) it is post-taped with 1% BSA; in step c) the test sample is diluted 1/200 in diluent buffer and said diluent buffer is composed of 1.7 g NaCl, Na 2 HPO 4 0.426 gr, NaH 2 PO 4 0.078 gr , BSA 2 gr, Tween 20 0.05 ml and distilled H2O 200 ml; in step d) the secondary antibody is diluted 1: 8000 in the diluent buffer. 4. Method according to any of the preceding claims, characterized in that the enzyme used is peroxidase. 5. Method according to any of the preceding claims, characterized in that the substrate solution used has the following composition: OPD buffer 10 ml (citric acid 2.14 gr, Na 2 HPO 4 x 12 H 2 O 6 , 54 gr, H 2 O distilled 400 ml, adjust to pH 7.5) + OPD 0.0028 gr + H 2 O 2 4 µl. Method according to any of the preceding claims, characterized in that the reaction is considered positive when the recorded optical density exceeds the value of 0.85. 7. A diagnostic kit for in vitro detection of avian ascaridiasis in a serum or whole blood sample by the ELISA method, characterized in that it comprises:

to.

a polystyrene plate previously treated with complete Ascaris suum extract for binding to the anti- Ascaridia galli antibodies present in serum samples or whole blood to be analyzed.

b.

A secondary anti-IgG antibody anti-chicken labeled with an enzyme usually used in a selected diagnostic immunoenzymatic analysis between the group formed by peroxidase, alkaline phosphatase and glycoxidase

C.

A colorless substrate solution specific for the enzyme conjugate used in the previous stage (b).

8. An ELISA diagnostic kit according to claim 7 characterized in that the enzyme used is peroxidase. 9. An ELISA diagnostic kit according to any of claims 7 to 8, characterized in that the substrate solution used has the following composition: OPD buffer 10 ml (citric acid 2.14 gr, Na 2 HPO 4 x 12 H2O 6.54 gr, H2O distilled 400 ml, adjust to pH 7.5) + OPD 0.0028 gr + H2O2 4 µl.