Appendix 27

Appendix 27

Diagnosis of A22 Mahmatlı and O1 Manisa FMD viruses from field samples: Development of a latex agglutination test kit

Abstract

Nilgün Özdural*, Aysel Candaş, Melahat Cengiz *Sap Institute, PK 714,Ulus, 06044 Ankara, TURKEY

Foot-and-mouth disease (FMD) is a severe highly contagious and acute disease of swine and cloven-hoofed animals. Among other diagnosis test methods, complement fixation test (CFT) and ELISA are widely use for FMD diagnosis. Recently, Latex Agglutination Test (LAT) has found an ever increasing application in the diagnosis of some types of bacterial, viral and parasitical infections. In this research a new LAT kit was developed as an alternative method to the other diagnosis tests. First two different size mono-disperse polystyrene latex particles were prepared with a modified emulsion polymerisation of styrene monomer. Subsequently these latex particles were coated with two different rabbit anti FMD IgG, which were against A22 Mahmatlı and O1 Manisa FMD viruses. In this research, the virus detection was successfully achieved. Due to high sensitivity of LAT, prepared in this work, special attention should be given to the time of the observation of agglutination. Prolong time, results some cross-reactions.

Keywords: Rapid diagnosis, Foot-mouth disease, Latex agglutination test

1. Introduction

Rapid confirmatory diagnosis of FMD is necessary to initiate procedures to control the spread of disease and to reach the source of an outbreak. For this purpose laboratory based test are necessary to detect and differentiate the FMD from vesicular viruses and also to differentiate FMD virus types.

State owned SAP Institute (Ankara,Turkey), with a prime mission of preparation of FMD vaccine and FMD diagnosis, receives field samples for FMD diagnosis. All field samples are routinely tested and serotyped by CFT, ELISA combined with virus isolation in cell culture. The shipment of the epithelium or vesicular fluid from the scene of the suspected outbreak plays a vital role on the precision of diagnosis. Delays and unproper shipment conditions in the transportation of samples to the laboratory might cause degradation of virus antigen.

In this laboratory, the positive specimens are diagnosed by CFT and ELISA, the remaining of field samples being typed after virus amplification in cell culture. The virus amplification procedure may require up to 4 days additionally. Virus isolation also requires laboratory cell culture facilities which can be difficult and expensive to maintain. RT-PCR procedure while extremely useful as a diagnostic technique along with ELISA and virus isolation (Reid et al., 1998, 1999), require skilled personnel, contamination-free laboratory space and specialized equipment.

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* Corresponding author: Tel.: 00-90-312-287 36 00/157; fax: 00-90-312 287 36 06 e-mail : nilgunozdural@hotmail.com

FMD is one the most contagious animal virus disease of cloven-hoofed livestock and its most dramatic effects can be seen in FMD-free countries which have a well developed export-trade. In these conditions, the population of non-immun animals provides the ideal conditions for rapid spread of disease after introduction. The early reporting disease, followed rapid diagnosis in the laboratory, are very important to prevent spread of disease.

A confirmatory diagnosis at the point of suspected outbreak of FMD would be very useful for faster diagnosis. The faster diagnosis would increase disease awareness and would be improve epidemiological information.

The methods of diagnosis might generally be given as following: 1. Clinical diagnosis 2. Serological diagnosis 3. Virological diagnosis a. Virus isolation b. İmmunological method (CFT, antigen capture ELISA) c. Nücleic acid recognition method (Nücleic acid hybridization assay, RT-PCR, Nested PCR) 4. Field tests

These tests mainly rely on agglutination assays. Consequently, serotyping is easy to perform with these assays.

An example such a test is Stapylococus aureus naturally bound to FMD virus guinea-pig antibody via the protein A (Montassier et al., 1994) and this test was successfully applied on field samples in Turkey and display high sensitivity for oesophago-pharyngeal samples (Ünver et al, 2000)

Another one is coloured latex particles coupled to a monoclonal antibody in a chromatographic strip test. Favorable results were obtained in a laboratory-based study and this test also showed higher sensitivity than the ELISA for epithelial suspensions and nasal swabs (Reid et al., 2001)

The principle of LAT kit developed in this work is based on latex agglutination. Uniform latex particles (ULPs) have an intriguing history. The Dow Chemical Company had been manufacturer of polystyrene, including latex used to formulate paints. In 1947, lot no. 3584 was manufactured among others, and a Dow chemist sent a sample of it to a colleague at the University of Michigan. He wanted to get a picture of it using the electron microscope. The electron micrograph showed spherical particles of astonishing and unexpected uniformity. This finding became even more significant because no other lot of latex had particles of similar uniform diameter. By 1956 the technology was complete, including the method of building larger diameter particles from smaller ones.

Latex Agglutination Tests or Latex Immunoassays both start with latex particles. These are tiny plastic spheres suspended in water, which have same very special properties for test development.

First there are ‘billions and billions’ of particles in a single millilitre of latex. The light scattering from all these particles causes latex to look milky.

Second, they are all perfect spheres of the same size (uniform diameter). They all have the same surface properties (uniform surface chemistry). Therefore, they all behave the same and do things together.

They can be uniformly coated with antibodies (or other proteins) due to hydrophobic interaction of portions of the protein with the polystyrene surface of the particles. After coating the particles are said to be ‘sensitized’ (they still look milky).

If a drop of antibody coated particles is mixed with a drop of sample (urine, serum, etc) containing antigen, then the latex will become ‘agglutinated’; the milky latex looks ‘curdled’. The sub-microscopic particles aggregate to form large visible clumps, resembling chunks of chalk rolling around on a plate. This is principle of all latex agglutination tests. Simple latex agglutination slide tests are performed on a glass or paper slide to give a yes/no answer. A simple slide test does not require a fancy instrument it does not even require electricity. Latex test are inexpensive as compared with the other techniques (Bangs, 1988).

The research of our group on rapid diagnosis test of FMD dates back to the year 1991. The development of the method was carried out in three stages during the year 1991-1996 at the laboratories of SAP Institute.

The first stage comprises the preparation of uniform latex particles a modification of well known emulsion polymerisation process were elaborated, where styrene monomer was polymerised. After the preparation of latex particles, they are coated with rabbit anti FMD IgG, which were against A22 Mahmatlı and O1 Manisa FMD viruses. The latex particles are coated with antibody via van der Waals forces and they are store in 2 mL sized capped tubes, to be used test reactive. During the first stage of this research, some preliminary agglutination tests were also carried out. The detailed description of latex preparation and coating procedures are given elsewhere. (Ozdural et al., 1994).

The second stage of this research was mainly focused on differentiation of types of FMD virus. A22 Mahmatlı and O1 Manisa FMD viruses were taken as test viruses so as to demonstrate the type detection (Ozdural et al., 1994).

The logical extension and the third stage of this work was the comparison of the results of this newly developed latex agglutination technique with well established CFT technique (Ozdural et al., 1996).

2. Material and methods

2.1. Preparation of latex particles

A modified emulsion polymerisation technique is used for polymerisation of styrene monomer (Petkim A.S., Turkey). Long chain water-soluble polymers were added to the emulsion polymerisation medium. So as to obtain mono-sized latex particles. Detailed description of the procedure is given elsewhere (Ozdural et al., 1994).

2.2. Coating (sensitising) of latex particles

The rabbit anti FMD IgG, which were against A22 Mahmatlı and O1 Manisa FMD viruses were obtained from Pirbrigth Laboratory (UK). Lowry protein determination technique is

used for determining the IgG concentration (Peterson, 1983). The optimum amount of coated IgG were found by trying different initial IgG concentrations.

2.3. Latex agglutination Test

The agglutination procedure is carried out on a black coloured analysis plate with a three detection spot.

2.3.1. Detection of agglutination

a) The concentration of IgG coated latex (test reactive) were as follows: Latex concentration (D= about 1µm) 5.68 mg latex/1 mL latex suspension

IgG concentration at coated latex suspension 0.95 mg IgG/1mL latex suspension 10 µL of test reactive placed on one of the detection spot of the analysis plate b) 10 µL of field sample (prepared for CFT) dropped on to the test reactive and mix for 15-20 second c) Tilt the analysis plate carefully a couple of times and look for agglutination d) If the result is ambiguous control the test results by using control reagent (BSA solution 0.1% by weight).

2.3.2. Differentiating the FDA virus types

For this purpose all three detection spots of the analysis plate are used.

a) 10 µL of field sample (prepared for CFT) placed on all three detection spots of the analysis plate b) - Drop 10 µL of BSA solution (0.1% by weigth) to the first detection spot of analysis plate - Drop 10 µL of test reagent prepared against O1 Manisa FMD virus to the second detection spot of analysis plate - Drop 10 µL of test reagent prepared against A22 Mahmatlı FMD virus to the third detection spot of analysis plate c) Tilt the analysis plate carefully a couple of times and look for agglutinations

3. Results and Discussion

106 field samples were simultaneously cross-tested with using both CFT and LAT (developed in this work) techniques. Table 1. summarises the results.

Table 1. Cross Test Results of Field Samples

CFT LAT

Number of A22 O1 Number of A22 O1 samples positive positive samples positive positive

95 - + 94 - +

4 + - 10 + -

7

_____ 106 - - 2 - -

_____ 106

Table 1 illustrated the close agreement between CFT and LAT (developed in this work) results. Thus one might conclude that the LAT is good candidate for being routinely used in the field for FMD virus diagnosis. Furthermore, the LAT technique does not require a fancy and complicated instrument and it is cost effective as compared with the other techniques. Due to the high sensitivity of LAT, prepared in this work, special attention should be given to the time of observation of agglutination. Prolonged time, results cross-reactions. Alternatively cross-reactions can be minimized by diluting the sample, so as to generate specific protocols for different types of FMD virus diagnosis.

References

Bangs, L.B., 1988. Latex agglutination tests. Amer. Clin. Lab. News, 7, 20-26

Montassier, H.J., Araujo, J.P., Pinto, A.A., 1994. Rapid co-agglutination test for the detection and typing of foot-and-mouth disease. J. Virol. Methods 50, 29-42

Ozdural, N., Yigit, A., Çelik, N., 1994. Sap virusu tayinine yönelik lateks aglutinasyon test kiti geliştirilmesi. 1. Ulusal Veteriner Mikrobiyoloji Kongre özet kitapcığı, Ankara, Turkiye, 61

Ozdural, N., Candas, A., Cengiz, M.,1996. Diagnosis of samples came from fields with polystyrene latex particles coated with antibodies against O1 Manisa and A22 Mahmatlı FMD virus. 1. International Veterinary Microbiology Congress Abstract, İstanbul, Turkey, 47

Reid, S.M., Ferris, N.P., Brüning, A., Hutchings, G.H., Kowalska, Z., Akerblam, L., 2001. Development of a rapid chromatographic strip test for the pen-side detection of foot-and-mouth disease virus antigen. J. Virol. Methods 96,189-202

Peterson, G.L. 1983. Determination of total protein. Methods Enzymol. 91, 95-121

Reid, S.M., Fortsyth, M.A., Hutchings, G.H., Ferris, N.P.,1998. Comparison of reverse transcription polymerase chain reaction, enzyme linked immunusorbent assay and virus isolation for the routine diagnosis of foot-and-mouth disease. J. Virol. Methods 70, 213-217

Reid, S.M., Hutchings, G.H., Ferris, N.P., De Clercq, K., 1999. Diagnosis of foot-and-mouth disease by RT-PCR: evaluation of primers for serotypic characterisation of viral RNA in clinical samples. J. Virol. Methods 83, 113-123

Ünver G., Alkan F., 2000. Serotyping of FMDV with coagglutination test as an alternative to CFT and ELISA. Report of the session of the FAO research group of the standing technical committee of the European Commission for the control of foot-and-mouth disease. Borovets, Bulgaria, 152-158.