L. O. Wosu and M. C. O. EzeibeDepartment of Veterinary Medicine, University of Nigeria, Nsukka, Nigeria
Abstract
Introduction
Materials and methods
Results and discussion
Acknowledgements
References
A simple and rapid serological method for definitive identification for Kata virus or peste des petite ruminants (PPR) virus specific antibody is described. The technique is based on adsorbing out the cross-reacting antibodies to rinderpest antigen from a PPR serum and leaving the specific antibody to PPR which is determined by haemagglutination-inhibition test.
Utilisation de la réaction d'inhibition de l'hémagglutination pour la détection de l'anticorps contre le virus de la peste des petits ruminants
Résumé
Cette étude décrit une méthode sérologique simple et rapide utilisée pour l'identification définitive de l'anticorps contre le virus Kata ou contre le virus de la peste des petits ruminants. La technique est basée sur l'adsorption des anticorps réagissant à l'antigène de la peste bovine dans un sérum de peste des petits ruminants. L'anticorps spécifique de la peste des petits ruminants qui reste dans ce sérum est ensuite déterminé au moyen de la réaction d'inhibition de l'hémagglutination.
The causative viral agents of both peste des petite ruminants (PPR) disease and rinderpest disease belong to the same Morbillivirus subgroup of the genus Paramyxovirus and show a great deal of cross-reactivity. They cross-react in the immunodiffusion and complement fixation tests (Johnson and Ritchie, 1968; Ihemelandu et al, 1976; Nawathe, 1983), immunoosmo-precipitation test (Majiyagbe et al, 1984) and indirect haemagglutination-inhibition test using measles virus and monkey red blood cells (Nawathe, 1983).
Even the serum neutralisation test (SNT) currently being used as the confirmatory diagnosis for PPR and rinderpest also shows cross-reactivity (Mornet et al, 1956; Taylor, 1979; Obi, 1984). Wosu (1985) first demonstrated the haemagglutinin or PPR homogenate antigen, to porcine erythrocytes.
Wosu (1977) employed an adsorption technique to make a definitive differentiation between very closely related and cross-reacting strains of the protozoan Trichomonas foetus var. brisbane, var. belfast and var. manley. It was therefore thought desirable to try the adsorption technique in this virological system to see if it could eliminate the cross-reactivity between PPR virus (Kate virus) and rinderpest virus (RV) and make a definitive diagnosis of the PPR specific antibody in a single test. This is of special interest in West Africa where both rinderpest and PPR are endemic.
This report details the technique used in eliminating the cross-reactivity between PPR virus and rinderpest virus serologically.
A known lymph node homogenae PPR antigen (Tambo), a known rinderpest cultured antigen and a known PPR immune serum were obtained from the National Veterinary Research Institute, Vom, Nigeria. Porcine red blood cells (RBC) collected in Alsevers anticoagulant were used. The porcine red blood cell suspension (0.6%) was prepared as described by Wosu (1984).
The diluent was phosphate buffered saline (PBS) at pH 6.8. The adsorption technique described by Johnson (1967) and Wosu (1977) was used as the basis for the removal of antibodies cross-reacting with rinderpest virus from known PPR serum.
A known PPR immune serum was divided into two portions A and B. Complement was removed from A and B by heating at 56°C for 30 min. In addition, natural agglutinins and non-specific serum inhibitors were removed from portion B by adsorption with porcine erythrocytes and kaolin respectively, as described by Joo (1976). The treatment of the PPR immune sera A and B involved adding 0.5 ml of known rinderpest antigen (Tambo) to 0.5 ml of each of the sera and 0.5 ml of each of the known PPR antigens to another 0.5 ml of each of the sera. Another 0.5 ml of each sera was untreated and used as a control.
All the portions were incubated at 37°C for two hours and centrifuged for 10 minutes at 3000 rpm. The supernatants were used for the haemagglutination-inhibition (HI) test as described by Wosu (1985), involving V-bottomed microtitre plates and microtitre pipettes. Four haemagglutinating units (HAU) of the PPR homogenate antigen were used.
The procedure for the HI test involved adding 0.05 ml of PBS (pH 6.8) to each well of the V-bottomed microtitre plate. Serial double dilutions of 0.05 ml of each of the test sera were made on the same plate. Next 0.05 ml of the PPR homogenate antigen containing four HAU was added to each well. This was incubated at room temperature (25°C) for one hour. Then 0.05 ml of 0.6 % porcine RBC suspension (the same porcine RBC sample used in determining the four HAU) was added to each well. Controls for the four HAU of the PPR virus and the porcine erythrocytes were included in the protocol. The plates were incubated at 4°C for six hours. Five repeats of the HI test were carried out immediately and the treated sera A and B stored at-20°C for preservation. After a month, HI tests were carried out on the preserved serum portions.
The HI titre of the PPR immune serum adsorbed with rinderpest antigen was less than the non-adsorbed portion and the titre of the serum adsorbed with PPR antigen was zero. Thus adsorption with rinderpest antigen neutralised the common antibodies leaving only antibodies specific to PPR antigen. This was confirmed by the fact that the HI titre of a portion of the same serum adsorbed with PPR was zero. The PPR antigen therefore neutralised both the antibodies it shared with rinderpest and those which were specific to the PPR virus.
Differences between treatments were maintained even after the removal of serum natural haemagglutinins and non-specific serum inhibitors as shown in Table 1. This eliminated the possibility that the difference in titre could have arisen from serum non-specific haemagglutinins or non-specific inhibitors.
Table 1. HI titres of PPR immune serum.
|
Serum treatment |
Titre |
Remarks |
|
|
Serum A |
Serum B |
||
|
Adsorption with rinderpest antigen |
1:1024 |
1:512 |
Control for four HAU and RBC were good |
|
Adsorption with PPR antigen |
Zero |
Zero |
- |
|
No adsorption |
1:4096 |
1:2048 |
- |
Repeats of the HI test, carried out on the same treated serum samples immediately after the adsorption, gave the same titres as above.
The treatment of serum A alone is quite adequate for a rapid qualitative survey or epidemiological work, but for quantitative work treatment of serum B would be necessary. Since the result was obtained, the technique has been used on many PPR and rinderpest serum samples with the same outcome.
Definitive tests for antibodies due to either antigen have generally been based on carrying out at least two tests with the two antigens by SNT. It was evident that the antibody was due to the virus which gave the higher titre due to cross-reactivity between the two antigens by SNT (Mornet et al, 1956; Taylor, 1979; Obi, 1984). However this procedure was rather laborious. More importantly, when the results of the two tests were close, it was very difficult to ascertain the correct antigen. Moreover, it was frequently necessary to know whether a known serum would neutralise and consequently identify an unknown antigen or vice-versa.
The technique described in this paper appears to have elucidated the problem of cross-reactivity which exists in the diagnosis of PPR and rinderpest. Indeed, this HI technique was used in a virological system. An immune serum was made more specific by adsorption with a heterologous virus of the same group. Adsorbed serum tested for haemagglutination-inhibition (HI) activity reacted only with homologous and not with heterologous strains of arboviruses, thereby facilitating the identification of strains (Jawetze et al, 1978). It is suggested that this treatment of serum samples be applied to the other tests in order to eliminate cross-reactivity between PPR and RV. A comparative study could be made to determine the optimal test for identifying the specific antibody to PPR antigen.
The disappearance of differences in HI titres after the tested sera were stored at-20°C for one month was a chance finding (Table 2). While the cause of the differences was not investigated, it may be attributed to viral elusion from antibodies (Nathalie et al, 1973). An investigation is needed to determine how long the treated sera can be stored at-20°C without elusion.
Table 2. HI titres of PPR serum portions as in Table 1 after storage at-20°C for one month.
|
Serum treatment |
Titre |
Remarks |
|
|
Serum A |
Serum B |
||
|
Adsorption with rinderpest antigen |
1:4096 |
1:2048 |
Control for four HAU and RBC were good |
|
Adsorption with PPR antigen |
1:4096 |
1:2048 |
|
|
No adsorption |
1:4096 |
1:2048 |
|
The HI test, especially in less sophisticated laboratories, is a very useful, quick and accurate method for determining the antibody levels in a given immune serum (Johnson, 1971). Where there are closely related antigens which cross-react, the test cannot readily be a definitive diagnosis of a specific antibody unless it is possible to eliminate the cross-reactivity due to the other related antigens.
It is possible to make a definitive serological diagnosis of PPR specific antibodies by haemagglutination-inhibition test using the adsorption technique.
We appreciate the co-operation we received from the National Veterinary Research Laboratory (NVRI), Vom, Nigeria. We are grateful to the University of Nigeria Senate Research Grant Committee for funding the work. We also thank the secretarial and other staff of the Department of Veterinary Medicine for their help.
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