Antigenic diversity and similarity of Newcastle disease viruses isolated from unvaccinated free-range rural chickens characterised by polyvalent and monoclonal antibodies

M G S Yongolo, A P Muhairwa*, D J Alexander**, K J Handberg***, R. J Manvell** and U M Minga****

Animal Disease Research Institute, Ministry of Agriculture and Co-operatives, PO Box 9254, Dar es Salaam, Tanzania
myongolo@yahoo.co.uk
*Department of Veterinary Medicine and Public Health, PO Box 3021, Morogoro, Tanzania
**Avian virology, VLA, Weybridge, Addlestone, Surrey KT15 3NB, UK 4 Department of Pathobiology
***The Royal Veterinary and Agricultural University, Frederiksberg C
****Open University of Tanzania, PO Box 23409 Dar-es Salaam, Tanzania

Abstract

Antigenic heterogeneity of 33 Newcastle disease virus isolates from healthy and sick free-range rural chickens and ducks was determined by analysis of their binding patterns to a panel of 27 monoclonal antibodies (mAb). The studied viruses had been isolated from apparently healthy and sick unvaccinated village chickens and ducks from four regions and different times of the year.

All isolates were positive to polyvalent APMV-1 antisera and to mAb U85 and negative to the pigeon mAb 161/617. Four isolates had high HI titres to mAb 7D4, which is specific for La Sota vaccine. Five antigenic binding profiles A, B1, EL, EB, and G segregated into 14 antigenic groups. Five antigenic groups had 100% antigenically similar isolates from different regions, indicating a spread from the same source. Isolates with EL and EB binding patterns probably had vaccine strains as progenitor virus. Nine were composed of isolates exclusively from the same region, indicating that isolates had not spread to other regions. Isolation of 100% antigenically similar strains from the same region suggested the persistence of NDV there. Detection of virulent isolates was clear with groups A, B and EB, but failed in group G and one isolate in group EL, possibly due to mixed infection. The use of mAb e, l and H33A for detection of virulent NDVs is recommended.

It is concluded that mAb binding test is a potentially useful tool in poor resource country laboratories, where routine use of in vivo tests and molecular pathotyping are difficult to sustain.

Keywords: Monoclonal antibodies, NDV pathoptyping, Newcastle disease

Introduction

Newcastle disease (ND) caused by an ND virus (NDV) serotype 1 is one of the most serious disease of poultry worldwide (Spradbrow 1999; Alexander 2001). Therefore, efficient and prompt diagnosis, which requires assessment of virulence, is important for timely containment of outbreaks and spread of NDVs. The disease and the virus were first reported in late 1920s (Alexander 2001). From then until the early 1980s using polyvalent antiserum, NDVs were considered homogeneous, evidenced by the use of a single vaccine strain for protection against different field strains (Alexander 1997). However, in the advent of monoclonal antibodies antigenic differentiation of NDVs was made possible and useful for diagnosis, strain differentiation and epidemiology of ND (Russell and Alexander 1983; Alexander et al 1987). Newcastle disease virus [avian paramyxovirus type 1] belongs to the genus Avulavirus, has a non-segmented single stranded RNA genome, contained in an envelope (Mayo 2002). Viruses with such genomes have inexact replication of the RNA, which frequently leads to production of variants with differences, often subtle differences, in phenotype from the parent particle (Spradbrow 1999). Thus, they are considered as quasi-species and the populations that spread in the field, or the populations that make up a vaccine stock, are not clonal. Furthermore, selection pressure can alter the average behaviour of the population (phenotype). Thus, NDVs with similar antigenicity gives indication of an epidemiological link.

In many countries of Africa and Asia, ND outbreaks are common in FRC and the disease can be considered endemic (Spradbrow 1999). These flocks are often composed of birds at different ages, not vaccinated, no biosecurity and mixed with other species (Yongolo et al 2002). It is however not known whether ND viruses circulating in FRC are subjected to selection pressure or not. Antigenic characteristics of NDVs circulating in the field could give an indication on the existence of selection or not. Recently, vaccination against ND has been recommended to protect FRC from ND outbreaks (Spradbrow 1999). However, for any success in the control of ND the background knowledge of its epidemiology is a prerequisite. Ironically, little is documented on the spread and introduction of ND in FRC populations in most African and Asian countries and the little information available is based on observational data. Monoclonal antibodies [mAbs] have been used for virus identification of antigenically variant NDVs (Aldous et al 2003) and have proved useful in epidemiological considerations of the disease (Alexander et al 1984). Furthermore, differentiation of strains with virulent characteristics has been reported (Alexander et al 1992). Thus, data from mAb analysis of NDV can be used in determining the source and explain its spread (Alexander et al 1997, Panshin et al 2000; Panshin et al 2001; Panshin et al 2002).

In FRC populations in Tanzania velogenic, mesogenic and lentogenic NDVs have been isolated from different sources (Yongolo et al 2002). However, there have been no studies on characteristics of NDVs from FRC in Tanzania, which could be of epidemiological importance. The presence and characteristics of NDV can be demonstrated by a number of methods. Currently, genetic characterisation is the method of choice to achieve three needs at one go; pathotyping, strain differentiation and epidemiological links (Aldous and Alexander 2001). Furthermore, in vivo virulence determination needs eggs and chicks from SPF flocks and a high level of laboratory sterility to curb contaminations. However, these techniques are expensive, difficult to maintain and require specialised facilities which are difficult to attain in poor resource countries. Haemagglutination and haemagglutination inhibition tests have traditionally been used to determine presence of NDV (Alexander 2000). They are uncomplicated, require minimum inputs in terms of equipment and reagents, and as a result can be afforded by poor resource laboratories. Despite the fact that monoclonal antibodies have a limited capacity to differentiate viruses that are antigenically similar but genetically distinguishable (Aldous et al 2003), they can still be used in HI tests to rapidly confirm and characterise NDVs and give some information relating to their epidemiology and origins (Aldous et al 2003; King and Seal 1998).

This paper focuses on antigenic variations manifested by NDVs from FRCs in Tanzania, which can be used in virulence assessment and epidemiological inference. Furthermore, to avoid the use of in vivo tests, this paper advocates the use of monoclonal antibodies as a tool for early detection of NDV similarities and pathotyping. Taking in consideration that ND is a trans-boundary problem, achievement of this would enhance early warning and early reaction, which would lead to enabling effective control of Newcastle disease.

Materials and methods

Virus

Allantoic fluid harvests of thirty-three Newcastle diseases virus isolates were used in this study. The isolates were obtained from unvaccinated free-range rural chickens and ducks. Of the 33, two were from apparently healthy ducks reared together with chickens and the remaining 31 were from chickens. Of the 31 from chickens 10 were from sick chickens from an ND suspected outbreak and 21 from apparently healthy chickens from ND free flocks. Characterisation with mAbs was done to all isolates after five passages in 9 - 11 days embryonated chicken eggs. Before characterisation confirmation of NDV was done at the Veterinary Laboratories Agency, Weybridge, Surrey, United Kingdom. Identity of each isolate indicated information on the source of the virus (geographical location, month of isolation and host). The first letters showed the regions in Tanzania where it was isolated, the middle number shows first the month when it was isolate, followed by a dot and the laboratory serial number. The last letters show the host from which it was isolated. Thus, all isolates with MG are from Morogoro, TB are from Tabora, MB from Mbeya and MS from Kilimanjaro. 1 to 12 of the first numbers before the dot indicates months from January to December respectively.

Antibodies

Newcastle disease polyvalent antisera and a panel of 27 monoclonal antibodies were used to characterise the isolates. The ability of each mAb to bind to the NDVs was carried out according to the procedure described by Alexander et al (1999) without modification. Abbreviations used for the identity of mAbs are also according to Alexander et al (1999). Before using the panel of 27 mAbs preliminary NDV identification was done using monoclonal antibody U85 a mAb specific to APMV-1 and not APMV-2 to 9, 7D4 a mAb specific for La Sota vaccine like NDVs, 617/161 a mAb specific for the pigeon variant NDV and polyvalent APMV-1 antiserum were used as a 1:32 dilution for the monoclonal antibodies and 1:16 dilution for the polyclonal antiserum. The haemagglutination inhibition tests procedure was as described by CEC (1992).

Analysis of the data

Serological test results of negative or positive were transformed to two level numerical scale. Where negatives were scored as 1 and positives as 2. The taxonomy programme NYTSYS (Rohlf 1993) commonly used for analysing data from biochemical test reactions of bacteria (Angen et al 1997) was adopted and used to perform the analysis of mAbs reactions of different isolates. Cluster analyses were performed on the distance matrix using the unweighted pair group with arithmetic averages (UPGMA).

Results

Haemagglutination inhibition test results to polyclonal APMV-1 antisera, mAb U85, 7D4 and the mAb 161/617 are shown in Table 1. All 33 isolates reacted positive to APMV-1 antiserum and mAb U85. However, eight isolates had partial inhibition to mAb U85 (Table 1). Results with mAb 7D4 showed that only four isolates were positive and all isolates were HI negative to mAb 617/161 (Table 1). Six isolates MG10.03C, MG6.16C, MG3.35C, TB5.20D, MS8.39C and MG6.7C had the highest titres to mAb U85, and two of these MG10.3C and TB5.20D had the highest HI titres to mAb 7D4. Whereas isolate TB5.22C had the lowest HI titres to all tested antiserum and mAbs (Table 1).

The binding pattern of the study isolates as a results of the ability of the 27 mAbs to bind (+) or not to bind (-) to Vero cells are shown in Table 2. Ten different mAbs (e, f, h, I, j, l, w, x, £, and H339A) had no ability to bind to some isolates as shown by the (-) sign in Table 2. Comparing the binding profiles of the study isolates and that of La Sota and those from previous findings and designation by Alexander et al (1997), our isolate were then judged to fall under five antigenic patterns, eight isolates in A, two in B, five in E/L, three in E/B and 16 in G (Table 2).