Diagnosis

2.1.3.1. Virus isolation systems

The most widely used system for the isolation of aMPV-A and aMPV-B is the embryo tracheal organ culture (TOC), prepared from turkey or chicken embryos shortly before hatch (Cook et al., 1976). Similar to other viral respiratory pathogens, such as infec-tious bronchitis virus (IBV), aMPV subtypes A and B induce ciliostasis in TOC at four to five days after inoculation. If aMPV-titres in the sample are sufficient, ciliostasis can already be visible during the first TOC passage (Jones et al., 1986; McDougall & Cook, 1986; Wilding et al., 1986; Cook et al., 1999; Lee et al., 2007). In contrast aMPV-C replicates in TOC without induction of ciliostasis, making the system less suitable for the isolation of this subtype. The exclusive use of TOC for the isolation of aMPV can therefore not be recommended (Cook et al., 1999; Cook, 2000). aMPV has been

shown to retain full virulence after continuous propagation in TOC for at least 25 to 100 passages (Jones et al., 1986; Buys et al., 1989b; Williams et al., 1991a).

The first isolation of aMPV has been performed in embryonated chicken and turkey eggs via the yolk sac route, resulting in embryo mortality and stunted embryos after few passages (Buys et al., 1980).

Cell culture systems have been widely used for aMPV-C isolation and less often for other subtypes. Successful virus isolation has been achieved in the African green monkey VERO cell line, primary chicken embryo fibroblasts (CEF), a chicken embryo rough (CER) cell line and the continuous quail tumour cell line QT-35 (Giraud et al., 1986; Hafez & Weiland, 1990; Chiang et al., 1998; Bennett et al., 2002). Multiple blind passages are necessary, before the virus produces a typical cytopathic effect (CPE), which is characterized by rounding and destruction of cells and development of large syncytia (Hafez & Weiland, 1990; Gough et al., 1994).

CEF and VERO cells are also commonly used for the attenuation and propagation of aMPV-strains for diagnostic purposes and for the development of attenuated live vac-cines (Buys et al., 1989b; Cook et al., 1989a; Cook et al., 1989b; Williams et al., 1991a; Williams et al., 1991b; Gulati et al., 2001b; Patnayak et al., 2002; Patnayak &

Goyal, 2004a; Patnayak & Goyal, 2004b). Patnayak et al. (2005) and Tiwari et al.

(2006a) also found several additional cell lines of avian and mammalian origin to be permissive for replication of aMPV-C.

Identification and characterization of aMPV isolates in TOC and cell cultures can be achieved by immunofluorescence test (IFT), immuno-peroxidase (IPO) staining or RT-PCR (see chapters 2.1.3.2 and 2.1.3.3 ).

The time of sampling is crucial for attempted isolation of aMPV. Experimental infec-tions revealed that infectious virus is recovered for no more than five to seven days post inoculation, which is even before cessation of clinical signs (see chapters 2.1.4.1 and 2.1.4.2). Virus isolation should be attempted as early as possible after the onset of the disease and sampled birds should display acute clinical signs. Swabs or tissue samples collected from upper respiratory tract organs, such as the trachea, sinus or

nasal turbinates, are considered to be the most promising materials for virus isolation (Van de Zande et al., 1999; Pedersen et al., 2001).

2.1.3.2. Detection of aMPV antigen

aMPV antigen in tissue samples and cell and organ cultures can be detected by IFT (Baxter-Jones et al., 1986; Jones et al., 1987; Majo et al., 1995; Majo et al., 1996; Jirjis et al., 2002b) and IPO staining (O´Loan & Allan, 1990; Majo et al., 1995; Jirjis et al., 2001; Alvarez et al., 2004b). Both techniques have been shown to be equally sensitive (Majo et al., 1995). IPO staining provides the advantage of microscopic identification of antigen-positive cell-types, making this technique a valuable tool for pathogenesis studies (see chapter 2.1.4.1). Antigen detection by IFT is predominantly used for identi-fication of aMPV isolates in cell or organ cultures.

2.1.3.3. Molecular-biological detection

Several RT-PCR assays have been developed for detection of aMPV-RNA directly from samples as well as for identification and characterization of isolates. PCR is more sensitive than virus isolation (Shin et al., 2000c; Pedersen et al., 2001; Cecchinato et al., 2004). Furthermore viral RNA is detectable for up to 21 days after infection (Jing et al., 1993; Pedersen et al., 2001; Velayudhan et al., 2005; Liman & Rautenschlein, 2007; Aung et al., 2008), compared to detection of aMPV-antigen and live virus for no longer than five to eight days (see chapter 2.1.4.1). PCR techniques are also less time consuming than virus isolation, allowing the testing of high sample numbers within few ours after sampling (Cavanagh et al., 1997; Cavanagh et al., 1999). The choice of the optimal PCR test is crucial for diagnosis. It should be considered, that not all assays are suitable to detect more than one or two subtypes (Bäyon-Auboyer et al., 1999).

Amplification with N-gene-specific primers has been shown to allow detection of all currently identified subtypes (Bäyon-Auboyer et al., 1999; Toquin et al., 1999). Given the conserved nature of the N-gene this technique may also detect potential new sub-types. Subtype-specific nested or hemi-nested PCR assays based on the G-gene are

widely used for identification of aMPV-A and B (Cavanagh et al., 1997; Bäyon-Auboyer et al., 1999; Cavanagh et al., 1999). However, due to the high variability of the G-gene, strains of subtypes C and D can not be detected by this test. It can not be excluded that potential new subtypes of aMPV as well as aMPV-strains with altered G-gene se-quences will remain undetected, if this test is used as the only diagnostic tool for aMPV detection. Specific detection of aMPV-C can be achieved by PCR assays amplifying the F- or M-gene of this subtype (Ali & Reynolds, 1999; Shin et al., 2000c; Dar et al., 2001b). More recently a quantitative real time PCR (qPCR) has been described for detection of aMPV-C (Velayudhan et al., 2005) and for simultaneous detection of sub-types A to D (Guionie et al., 2007).

An in situ hybridization assay has been established for the localization of aMPV-C RNA in tissue samples (Velayudhan et al., 2005).

2.1.3.4. Serology

VNT, ELISA and indirect immunofluorescence test (iIFT) have been established for detection of aMPV-specific antibodies from sera and respiratory secretions. Despite the antigenic cross-reactivity between the aMPV subtypes (see chapter 2.1.1.3), the choice of test antigen used in serological assays is crucial. Antibodies directed against aMPV-C, which is antigenically most distinct from the other identified subtypes, are not detectable by tests based on aMPV-A or B (Cook et al., 1999). Due to this fact, sero-logical diagnosis was not possible during the first month of the aMPV-C outbreak in Colorado in 1996 (Seal, 2000).

Numerous aMPV-specific ELISA systems have been developed for in-house use as well as for commercial distribution, coated with whole-antigen preparations of either one subtype or mixtures of different subtypes (Grant et al., 1987; Chettle & Wyeth, 1988; Eterradossi et al., 1992; Heckert et al., 1994; Tanaka et al., 1996a; Mekkes & de Wit, 1998; Chiang et al., 2000). In addition ELISA systems using recombinant M- or N-protein of aMPV-C have been established (Gulati et al., 2000; Gulati et al., 2001a). The aMPV-C M-protein ELISA also detected antibodies directed against aMPV-A and B (Lwamba et al., 2002b). Most ELISA systems have been designed to detect antibodies

in chicken and turkey sera, using either chicken-immunoglobulin G (IgG) or anti-turkey-IgG as detection antibodies. Anti-chicken conjugates have been demonstrated to be cross-reactive with turkey antibodies, but sensitivity may be decreased compared to the use of anti-turkey antibodies (Heckert et al., 1994; Chiang et al., 2000; Jirjis et al., 2000). ELISA systems for detection of aMPV-specific IgA or IgM have been used for experimental purposes (Ganapathy et al., 2005; Cha et al., 2007; Kapczynski et al., 2008). Cadman et al. (1994) adapted a commercial aMPV ELISA to detection of os-trich antbodies by replacing the conjugate with anti-osos-trich-IgG. Competitive ELISA systems have been developed for detection of aMPV-specific antibodies in various bird species (Mekkes & de Wit, 1998; Welchman et al., 2002; Turpin et al., 2003;

Gharaibeh & Algharaibeh, 2007; Turpin et al., 2008).

Indirect IFT has been described for detection of aMPV-specific IgG in research (Baxter-Jones et al., 1986; Baxter-Jones et al., 1989; O´Loan et al., 1989). VNT can be performed in TOC, CEF, chicken embryo liver cells (CEL), VERO cells and MA-104 cells (Baxter-Jones et al., 1989; O´Loan et al., 1989; Toquin et al., 2000; Alkhalaf et al., 2002a). VNT provides the advantage to be accessible for detection of antibodies from all bird species. Both techniques are laborious and time consuming and therefore less suitable for testing large numbers of samples for diagnostic purposes.

Results of all three serological techniques show good correlation with each other (Baxter-Jones et al., 1989; Alkhalaf et al., 2002a). However, virus neutralizing (VN) antibodies can be detected about two days earlier than aMPV-specific IgG detected by ELISA or iIFT (Baxter-Jones et al., 1989; O´Loan et al., 1989; Liman & Rautenschlein, 2007).