Serological and Molecular Detection of Avian Pneumovirus in Chickens with Respiratory Disease in Jordan

Serological and Molecular Detection of Avian Pneumovirus in Chickens with Respiratory Disease in Jordan S. M. Gharaibeh1 and G. R. Algharaibeh Department of Pathology and Animal Health, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid 22110, Jordan tected in 5 out of 23 broiler flocks (21.7%), 6 out of 8 layer flocks (75%), and 7 out of 7 broiler breeder flocks (100%). Avian pneumovirus nucleic acid was detected in 17 broiler flocks (12.8%) and 3 layer flocks (42.9%). None of the broiler breeder flocks tested by reverse-transcription PCR was positive. All of the 20 detected APV isolates were subtype B. This is the first report of APV infection in Jordan. In conclusion, the Jordanian poultry industry, vaccination programs should be adjusted to include the APV vaccine to aid in the control of this respiratory disease.

Key words: avian pneumovirus, chicken, enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, Jordan 2007 Poultry Science 86:1677–1681

INTRODUCTION Avian pneumovirus (APV), also called turkey rhinotracheitis, is an upper respiratory tract infection of turkeys and some other avian species (Cook and Cavanagh, 2002), including chickens (Shin et al., 2000a). Avian pneumovirus belongs to the family Paramyxoviridae, subfamily Pneumovirinae, genera Metapneumovirus, and has an unsegmented single-stranded negative sense RNA (Pringle, 1998). It was first reported in South Africa in the late 1970s (Buys and Du Preez, 1980), initially in turkeys and later in chickens (Buys et al., 1989). Several years later, a disease with similar clinical signs was reported in France (Giraud et al., 1986) and then in the United Kingdom, where the causal agent was isolated (McDougall and Cook, 1986) and characterized as a pneumovirus (Cavanagh and Barrett, 1988). Two different subgroups, designated A and B, exist within a single serotype. Recently, subgroup C was reported in the United States (Seal, 1998) and subgroup D was reported in France (Toquin et al., 2000). Clinical signs of APV infection in chickens may include swelling of the periorbital tissue and infraorbital sinuses, torticollis,

cerebral disorientation, and opisthotonos. Although widespread respiratory signs are usually observed, mortality caused by APV is less than 2%, and less than 4% of the flock will show swelling of the head (Gough, 2003). In broiler breeders and commercial layers, egg production and egg quality are frequently affected (Cook, 2000). In Jordan, the chicken industry is the most developed industry in the animal sector. Annual investment in this industry is around US $1 billion (Anonymous, 2005). This industry is composed of broilers, layers, and broiler breeders, with the total number of chickens ranging from 25 to 30 million. There are no turkey farms in Jordan, and rearing of turkeys is restricted to very small individual backyard flocks. There is a serious respiratory disease in chickens in Jordan causing catastrophic economic losses to the chicken industry. In many of the flocks with this respiratory disease, some chickens exhibit swelling of the heads. Although APV has not been reported in Jordan, we believe that APV is part of this multifactorial respiratory disease. This study was designed to document the involvement of APV as a cause of this respiratory disease in chickens in northern Jordan, and to subtype APV in Jordan if detected.

MATERIALS AND METHODS ©2007 Poultry Science Association Inc. Received January 28, 2007. Accepted April 8, 2007. 1 Corresponding author: [email protected]

Sample Collection During the period August 2004 to January 2005, we examined 150 commercial chicken flocks (133 broiler 1677

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ABSTRACT Avian pneumovirus (APV) causes upper respiratory tract infection in chickens and turkeys. There is a serious respiratory disease in chickens, resulting in catastrophic economic losses to chicken farmers in Jordan. The objective of this study was to investigate the role of APV as a factor in the respiratory disease of chickens in Jordan by serological and molecular methods. Thirtyeight chicken flocks were examined by competitive ELISA (23 broilers, 8 layers, and 7 broiler breeders), and 150 chicken flocks were examined by reverse-transcription PCR (133 broiler flocks, 7 layer flocks, and 10 broiler breeder flocks). Avian pneumovirus antibodies were de-

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GHARAIBEH AND ALGHARAIBEH Table 1. Polymerase chain reaction primers used for avian pneumovirus molecular detection and typing Primer

Gene

Sequence

Reference

Nd Nx Ga G2G12C1 C2 G150 G-1005

N N G G G M M G G

5′ 5′ 5′ 5′ 5′ 5′ 5′ 5′ 5′

Bayon-Auboyer et Bayon-Auboyer et Bayon-Auboyer et Bayon-Auboyer et Bayon-Auboyer et Shin et al., 2000b Shin et al., 2000b Bayon-Auboyer et Bayon-Auboyer et

AGC AGG ATG GAG AGC CTC TTT G 3′ CAT GGC CCA ACA TTA TGT T 3′ CCG GGA CAA GTA TCT CTA TGG 3′ CCA CAC TTG AAA GAT CTA CCC 3′ CAG TCG CCT GTA ATC TTC TAG GG 3′ GAT GAC TAC AGC AAA CTA GAG 3′ CTT CAG GAC ATA TCT CGT AC 3′ GCG ATG CCC AGT TAA TGA A 3′ CCC CTT ACA AAC ACT GTT C 3′

Serology The 15 serum samples from each flock were pooled into 5 tubes. The presence of antibodies against APV in serum samples was evaluated by competitive ELISA with the turkey rhinotracheitis/swollen head syndrome Avian Pneumovirus ELISA kit (Svanova Biotech, Lyon, France) according to the manufacturer’s instructions. Positive and negative control antisera were provided in the kit and used in each run. Absorbance was read at a wave length of 405 nm on an ELx800 ELISA reader (BioTek Instruments Inc., Winoski VT). The mean optical density (OD) and the percent inhibition (PI) values for each pooled sample were calculated {PI = [(ODNegCtrl − ODSample/PosCtrl) × 100]/OD NegCtrl}. According to the manufacturer’s procedure, cases with PI values of less than 30 were considered negative. Cases with PI values of between 30 and 40 were considered doubtful. Cases with PI values of greater than 40 were considered positive.

RNA Extraction Nasal turbinates from the frozen group of heads from each flock were swabbed with sterile swabs (Heinz Herenz Medizin GmbH, Hamburg, Germany). Swabs were placed in PBS and were scraped on the side of the tube to facilitate removal of contents from the swab head. Extraction of RNA was performed on the pooled material for swabs from each flock with a Purescript RNA purification kit (Gentra Systems, Minneapolis, MN) according to the manufacturer’s procedure. The RNA yield was usu-

1999 1999 1999 1999 1999

al., 2000 al., 2000

ally approximately 120 ␮g/mL with the ratio of RNA:DNA (260:280 nm) ≥1.

Identification of APV by ReverseTranscription PCR The APV detection primers used in this study were previously evaluated by Bayon-Auboyer et al. (1999) and are listed in Table 1. The screening of flocks was performed with the primer pair Nd/Nx. Reverse-transcription PCR (RT-PCR) produces a 115-bp fragment in positive samples. One-step RT-PCR was performed using an Access RT-PCR System kit (Promega Corp., Madison, WI) according to the manufacturer’s procedure. Briefly, a 50-␮L reaction volume per sample was prepared by adding 10 ␮L of avian myeloblastosis virus/reverse transcriptase/Thermus filiformis DNA polymerase 5× reaction buffer, 1 ␮L of deoxy nucleotide 5′-triphosphate mixture (10 mM each deoxy nucleotide 5′-triphosphate), 1 ␮L (50 pmol/␮L) of each downstream and upstream primer (Alpha DNA, Montreal, Quebec, Canada; Table 1), 2 ␮L of 25 mM MgSO4, 1 ␮L of avian myeloblastosis virus reverse transcriptase (5 u/␮L), 1 ␮L of T. filiformis DNA polymerase (5 u/␮L), 28 ␮L of nuclease-free water, and 5 ␮L of RNA template. Reverse-transcription PCR was carried out for 1 RT cycle of 45 min at 45°C, followed by 94°C for 2 min, then 35 PCR cycles at 94°C for 30 s, 51°C for 45 s, and 68°C for 60 s, with a final extension cycle at 68°C for 10 min. The PCR products were separated on a 2% agarose gel (Promega Corp.) and stained by ethidium bromide (Promega Corp.). Agarose gel electrophoresis was run for 40 min at 100 V, 400 mA, and the gel was visualized under UV light (AlphaImager, Alpha Innotech, San Leandro, CA). Nemovac APV subtype B live vaccine (Merial, Lyon, France) was used as a positive control for Table 2. Polymerase chain reaction primer pairs used for avian pneumovirus (APV) molecular detection and typing, annealing temperature, and expected band size

Gene N G G G M

Primer

APV type

Annealing temperature, °C

Nd/Nx Ga/G2Ga/G12G150/G-1005 C1/C2

All A B D C

51 54 54 57 51

Band size, bp 115 504 312 956 468

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flocks, 7 layer flocks, and 10 broiler breeder flocks) located in northern and central Jordan, in which the chickens were suffering from respiratory disease. Broiler flocks had mild respiratory signs, with a few birds exhibiting swelling of the heads. Layer and breeder flocks had respiratory signs, a decrease in feed intake, and a decrease in egg production (10 to 11%). Three to 5 chicken heads per flock were collected at an acute phase of the respiratory disease. Heads were transported on crushed ice to the laboratory and stored at −80°C until used for RNA extraction. Fifteen serum samples per flock were collected from 38 different chicken flocks (23 broiler flocks, 8 layer flocks, and 7 broiler breeder flocks) that showed disease signs similar to those described above. All the flocks were more than 4 wk of age and were not vaccinated against APV.

al., al., al., al., al.,

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AVIAN PNEUMOVIRUS SUBTYPES IN JORDAN Table 3. Avian pneumovirus ELISA results in different chicken flocks tested Type of bird Broilers Layers Broiler breeders Total

Number

Average PI values1

Negative, n

Doubtful, n

23 8 7 38

49.8 63.3 49.8 —

15 1 0 16

3 1 0 4

Positive, n (%) 5 6 7 18

(21.7) (75) (100) (47.4)

Percent inhibition (PI) values for each sample = [(ODNegCtrl − ODSample/PosCtrl) × 100]/ODNegCtrl.

1

RNA extraction and RT-PCR. Negative control (nucleasefree water) was also used in each trial.

Molecular Subtyping of APV by RT-PCR

RESULTS AND DISCUSSION Eighteen out of the 38 chicken flocks (47.4%) tested by competitive ELISA were found to have positive antibody titer for APV. The positive flocks comprised 21.7, 75, and 100% of broilers, layers, and broiler breeders, respectively (Table 3). Because all studied flocks were more than 4 wk of age and none was vaccinated, our data suggest field exposure of these flocks to APV and exclude the possibility that the detected antibodies were due to maternal antibodies or vaccination. This confirms the endemic nature of the disease in Jordan. Seroprevalence of APV has been found in other countries in the region (Weisman et al., 1988; El Houadfi et al., 1991). Detection of anti-APV antibodies among broilers (21.3%) was significantly lower than among layer (75%) and broiler breeder (100%) flocks. This may be due to the short life span of broiler flocks and the time of blood sample collection, which was at the acute stage of the respiratory disease. This detection level might have been higher if we had tested convalescent serum samples from the same flocks. The higher average PI values in layers (63.3) and broiler breeders (70.9) compared with broilers (49.8; Table 3) also supports the previous point that the long life span of layers and breeders allows them to develop a stronger immune response detectable by ELISA. Pooling of serum samples was done to reduce the cost of testing with the ELISA kit. According to Maherchandani et al. (2004), pooling of serum is more advantageous and cost-effective than testing individual samples and does not interfere with the sensitivity of the ELISA kit. Thus, pooling of

Table 4. Reverse-transcription PCR results for avian pneumovirus in different chicken flocks tested Type of bird Broilers Layers Broiler breeders Total

Tested, n

Positive, n (%)

133 7 10 150

17 (12.8) 3 (42.9) 0 20 (13.3)

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The APV molecular typing primers used in this study were evaluated previously (Mase et al., 2003) and are listed in Table 1. The Ga-G2-, Ga-G12-, and GG-1005 primers are specific for APV subtypes A, B, and D, respectively (Bayon-Auboyer et al., 1999, 2000). In addition, the ClC2 primers are specific for APV subtype C (Shin et al., 2000b). Extraction of RNA and the RT-PCR program were performed as described above except for the annealing temperatures. Primers specific for each subtype, their annealing temperature, and the expected band size are listed in Table 2.

the serum samples may also explain the difference in PI values among the flocks because pooled broiler serum samples may not all be positive at the time of testing. In this study, our strategy was to use the Nd/Nx primers for initial detection of APV. Positive cases were then RT-PCR subtyped using subtype-specific primers for the G gene (subtypes A, B, and D) and the M gene (subtype C). We used Nemovac APV subtype B live vaccine as a positive control for detection and subtyping by RT-PCR. Unfortunately, positive controls for other subtypes were unavailable for use in this study. Twenty out of 150 cases (13.3%) tested positive for APV using the Nd/Nx primer set (Table 4). Seventeen broiler flocks (12.8%) and 3 layer flocks (42.9%) were positive by RT-PCR. None of the broiler breeder flocks tested by RT-PCR was positive. All cases testing positive by RT-PCR were of subtype B (Figure 1). Reverse transcription-PCR was found to be a very useful diagnostic test for APV screening as well as for subtyping. Although the flocks examined by RT-PCR were different from the flocks examined by ELISA, RT-PCR results indicated a lower prevalence of infection in all types of flocks compared with ELISA results. The lower detection rate by RT-PCR may be because APV is present for only a few days after initial infection and because it replicates poorly in the infected host (Cook et al., 1993; Naylor et al., 1997; Catelli et al., 1998), whereas the antibody response after infection lasts longer than that (Jones et al., 1988) and previously infected flocks will remain positive by ELISA but not by RT-PCR testing. In addition, it is important to mention that some samples for RT-PCR testing were received with massive secondary bacterial infection, when the virus could no longer be detected. As reported in Europe, and because of our geographic location, we expected to diagnose APV subtypes A, B, or both in Jordan. Characterization of the Jordanian strain showed that all detected viruses were APV subtype B. Similarly, APV subtype B was characterized earlier in Israeli poultry (Banet-Noach et al., 2005).

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Most of the cases tested in this study had a history of respiratory illness for broilers or a drop in egg production for layers and broiler breeders, or both. A few chickens with swollen heads were also seen in those flocks. These signs are usually associated with APV infection in chickens. Most of the positive broiler cases were more than 4 wk of age. This is the period when most of the respiratory problems begin in broiler flocks in Jordan. This may indicate, in one way or another, the involvement of APV in the respiratory disease we are seeing in chickens in Jordan. The drop in egg production in layer and breeder flocks may be due to APV infection alone or to other concurrent infections, although investigating other concurrent infections was not the aim of this study. In addition, because of the long life span of these flocks, it is possible that they were exposed to APV earlier in life and that the current drop in egg production may be due to other causes, such as Newcastle disease virus or Mycoplasma gallisepticum infections. This is the first study conducted on APV infection in chickens in Jordan, and it will pave the way for further studies on this virus. Future work may and should include the use of RT-PCR-positive nasal turbinate homogenates for isolation of the virus. Isolation of APV will allow the possibility of making autogenous vaccines. In conclusion, vaccination programs in the Jordanian poultry industry should be adjusted to include the APV vaccine to aid in the control of this respiratory disease.

ACKNOWLEDGMENTS I would like to thank the Deanship of Research at Jordan University of Science and Technology for supporting this work by grant number 85/2005.

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Figure 1. Electrophoresis analysis (2% agarose gel) for avian pneumovirus (APV) detection and subtyping. Lanes 1 and 7: 100-bp DNA ladder marker (Promega Corp., Madison, WI). Lane 2: Nd/Nx primers set for general detection (positive; band at 115 bp). Lane 3: Ga/G2- primers set for subtype A detection (negative; no band). Lane 4: Ga/G12- primers set for subtype B detection (positive; band at 312 bp). Lane 5: C1/C2 primers set for subtype C detection (negative; no band). Lane 6: G150/G-1005 primers set for subtype D detection (negative; no band).

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