Detection and Quantification of Antibodies to Newcastle Disease Virus in Ostrich and Rhea Sera Using a Liquid Phase Blocking Enzyme-Linked Immunosorbent Assay

Abstract

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutinationinhibition (HI) test was regarded as the reference method. The cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r ‫؍‬ 0.875). The two tests showed good agreement ( ‫؍‬ 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.Newcastle disease (ND) is caused by an avian paramyxovirus (APMV-1 serotype) that belongs to the genus Rubulavirus of the family Paramyxoviridae (19). Newcastle disease virus (NDV) occurs worldwide and has a considerable economic impact on the world poultry industry, ranging from losses due to disease and the expense of vaccination to the significant cost of diagnostic laboratory investigations (14). The breeding of ratites (ostriches, emus, and rheas) has expanded considerably all over the world in recent years. They are susceptible to several diseases of domestic fowl, including ND (15,20). Efforts to control and prevent ND through efficient vaccination programs and corresponding serological monitoring are constant.The hemagglutination-inhibition (HI) test is still the most widely used conventional serological method for measuring anti-NDV antibody levels in poultry sera, and it is considered the standard laboratory test for this disease (30). However, sera from other species tend to give a high incidence of falsepositive results. And although the number of nonspecific agglutination reactions can be reduced by pretreatment with heat and kaolin, these procedures decrease the sensitivity of this test (28).Indirect enzyme-linked immunosorbent assays (I-ELISA) have been developed, evaluated, and well correlated to the HI test for serodiagnosis of NDV in poultry (4,8,18). In spite of their high sensitivity, easy standardization, lack of requirement for serum pretreatment, and possible computerization of the system, these assays have the disadvantage of not being applicable to the testing of ratite sera in a single system unless anti-ratite species conjugates are used in place of an antichicken conjugate (5, 28).An APMV-1-specific monoclonal antibody blocking ELISA with the ability to test sera from exotic or wild avian species for NDV-specific antibodies in serial twofold dilutions or a single dilution has been described (9, 13). However, production and maintenance of hybridoma cells are time-consuming and sometimes expensive for laboratories with limited facilities. Moreover, assays with a single serum dilution are faster and more ...

Serological and Virological Studies of Newcastle Disease and Avian Influenza in Slaughter-Age Ostriches (Struthio camelus) in Japan

“…The sensitivity, specificity, and predictive accuracy of the B-ELISA and the HI test relative to the VN test were similar. Moro et al [18] reported that a liquid phase B-ELISA and HI test with heat and kaolin treatment showed good agreement. False positive reaction in the HI test might arise from ostrich species.…”

“…Each test serum sample was treated at 56°C for 30 min and then incubated with kaolin at 37°C for 30 min to extract nonspecific inhibitors [18,23]. Subsequently, the treated sera were incubated with 10% chicken red blood cells at room temperature for 10 min [5].…”

ADAPTATION OF HEMAGGLUTINATION INHIBITION TECHNIQUE (HI) FOR THE DIAGNOSIS OF NEWCASTLE DISEASE IN OSTRICHES (Struthio camellus)

“…When tested by HI assay or ELISA, the antibodies titers after treatment with kaolin did not vary either (Sousa et al 2000).…”

“…However, HI test tends to give false positive results in serum samples of some species including ostriches, due to the presence of nonspecific hemagglutination inhibitors (Willians et al 1997, Alexander 1997, Koch et al 1998, Sousa et al 2000. According to the OIE (2004), serum samples can be incubated with 10% chicken red blood cells (RBCs) suspension at room temperature, for 10 min, to eliminate these agents.…”

“…The breeding of ratites (ostriches, emus and rheas) has expanded considerably all over the world in recent years (Sousa et al 2000). In the 1990s, a worldwide renaissance has been observed in ostrich production arising from an increased demand for leather and…”