APV in Poultry

Avian pneumovirus (APV) is the causative agent of turkey rhinotracheitis (TRT) and swollen head syndrome (SHS) in turkeys and chickens, respectively (Buys et al., 1989). The APV belongs to the family paramyxoviridae which has two subfamilies, the paramyxovirinae and pneumovirinae (Regenmortel et al., 2000). The first isolate of APV was obtained in South Africa in 1979 (Buys et al., 1989) and later the virus was detected in other countries around the world (Alexander, 1997). One APV serotype has been identified and within this serotype there are three subgroups designated A, B, and C based on molecular and antigenic differences (Cook et al., 1999; Harlow & Lane, 1988; Seal, 2000).
Turkeys and chickens, of any age, are known to be natural hosts of APV. The APV causes respiratory diseases in young birds and a in egg production in breeder flocks (Alexander, 1997; Cook et al., 1999; Jones et al., 1988). Although, all ages of turkeys are susceptible to APV, the severity of the disease varies. Alexander (1997) reported that the disease was more severe in day old poults than 6-week-old turkeys. The clinical disease is characterized by nasal and ocular discharge, swollen infraorbital sinuses and sneezing (Jones et al., 1986; McDougall & Cook, 1986, Wyeth et al., 1986). These clinical signs have been observed in different countries and found to be similar but different names were given to the disease, which reflects the complexity of the etiology when associated with secondary infections. Serological tests such as enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test are the most commonly used methods to diagnose the APV infection. The VN test is performed in a variety of systems, including TOC, CEF, or Vero cells. Although, ELISA and VN test show similar sensitivity (Cook et al., 1999), ELISA is the most commonly used assay (Chettle & Wyeth, 1988; Eterradossi et al., 1995, O´Loan et al., 1989). Therefore, serological survey was conducted to detect avian pneumovirus (APV) antibodies in commercial poultry farms using ELISA and VN test in Saudi Arabia.

Materials & Methods

Samples
Eighty seven serum samples were collected from different commercial chicken farms flocks in Qassium area, Saudi Arabia in a period from 2007- 2008. Serum samples were collected from birds showing respiratory symptoms, with different ages ranging from one day to 62 weeks old.

Antigen preparation for ELISA
The preparation of APV antigen for ELISA was similar to that described (Chettle & Wyeth, 1988) with some modifications. Briefly, the supernatant of infected Vero cells was collected and treated by centrifugation at 100,000x g for three hours at 4 C (Beckman L7-55 ultracentrifuge, rotor SW 41. Palo Alto, CA). The pellets were resuspended in PBS (pH 7.2) and placed on a sucrose gradient (35% and 55%) and centrifuged at 100,000x g for three hours at 4 ºC. The virus was harvested from the sucrose interphase and diluted with PBS (pH 7.2), then centrifuged at 100,000x g for three hours at 4 ºC. The concentration of the virus protein was determined by electrophotometry at wavelengths of 260 and 280 nm (Harlow & Lane, 1988). A negative control of Vero cells was treated the same as the APV-infected Vero cells. The ELISA procedures were performed as described by O´Loan et al. (1989). Positive control sera were prepared in specific pathogen free (SPF) turkeys that were inoculated with inactivated virus.

Virus neutralization (VN) test
The procedure for conducting the VN test was described by O´Loan et al. (1989) with some modification as follows: the serum samples were serially diluted two fold in serum free tissue culture medium (MEM) starting from 1:10 up to 1: 1280. A volume of 50 ml containing 100 tissue culture infective dose50 (TCID50) of APV (Minnesota/turkey/2a/97) was added to an equal volume of each serum dilution contained in sterile 96-well flat-bottom plates (Corning Incorporated, Corning, NY). A volume of 50 ml of the virus/serum mixture of each dilution was transferred to a duplicate of monolayer of Vero cells contained in 96-well flat-bottom plates. The cells were incubated at 37 ºC for 5-6 days and checked every day for cytopathic effect (CPE) consisting of large syncytial formation and rounded cells.

Turkeys and chickens, of any age, are known to be natural hosts of APV. The APV causes respiratory diseases in young birds and a in egg production in breeder flocks (Alexander, 1997; Cook et al., 1999; Jones et al., 1988). Although, all ages of turkeys are susceptible to APV, the severity of the disease varies. Serological tests such as ELISA and VN test are the most commonly used methods to diagnose the APV infection. Baxter-Jones et al. (1989) reported the detection of antibodies to APV five days after the clinical signs appeared using VN test in chicken embryo fibroblast (CEF) then the antibody titer declined by days 13 after the appearance of clinical sings.
In this study, results of serological survey on APV in commercial poultry farms with different ages using ELISA and VN tests were summarized in table 1. It was observed that the antibody against APV were detected in 50% (8/16) in both ELISA and VN of serum samples collected from 11 - 18 weeks old chickens. No antibody was detected in younger or older poultry. This result disagrees with the finding of Alexander (1997), who reported that the disease was more severe in day old chicks than 6-week-old chickens.

This study indicates the presence of antibody against APV in commercial poultry farms with age range from 11 - 18 weeks of age in Saudi Arabia.

Table 1: Serological survey against avian pneumovirus (APV) in serum samples collected from commercial poultry farms in Saudi Arabia using VN and ELISA test

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