OJVRTM Online Journal of Veterinary Research Volume 17 (4): , 2013

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1 MAIN All Rights Reserved. Online Journal of Veterinary Research. You may not store these pages in any form except for your own personal use. All other usage or distribution is illegal under international copyright treaties. Permission to use any of these pages in any other way besides the before mentioned must be gained in writing from the publisher. This article is exclusively copyrighted in its entirety to OJVR. This article may be copied once but may not be, reproduced or re-transmitted without the express permission of the editors. This journal satisfies the refereeing requirements (DEST) for the Higher Education Research Data Collection (Australia). Linking: To link to this page or any pages linking to this page you must link directly to this page only here rather than put up your own page. OJVRTM Online Journal of Veterinary Research Volume 17 (4): , 2013 Detection of avian Metapneumovirus infection in broilers by nested RT-PCR Hesami G 1, Seyfi Abad Shapouri MR, DVM PhD, 2 Mayahi M.DVM PhD 3 2 Departments of Pathobiology and Clinical Sciences, 1 School of Veterinary Medicine, Shahid Chamran University, Ahwaz, Iran. ABSTRACT Hesami G, Seyfi Abad Shapouri MR, Mayahi M., Detection of avian Metapneumovirus infection in broilers by nested RT-PCR, Onl J Vet Res., 17 (4): , Avian Metapneumovirus (ampv) is the casual agent of Turkey Rhinotracheitis (TRT) which also causes a respiratory infection in chickens, resulting in Swollen Head Syndrome. ampv infection in chicken flocks gives rise to considerable economic losses. ampv infections were detected by RT-PCR in broiler flocks in Ahwaz, Iran. Fifty broiler flocks were sampled at slaughter via tracheal swabbing. RNA was extracted and RT-PCR based on viral N gene sequence specific primers (Nd and Nx) was performed. Positive results were confirmed by a semi-nested PCR. ampv was detected in 28% of samples. We report prevalence ampv infections and describe a novel semi nested PCR to confirm Nd/Nx RT-PCR. Keywords: Avian Metapneumovirus, N gene, RT-PCR, Semi-nested PCR, Broiler flocks, Ahwaz INTRODUCTION Avian Metapneumovirus (ampv) is classified in the genus Metapneumovirus, within the subfamily Pneumovirinae of the Paramyxoviridae family (Pringle et al, 1998). The human metapneumovirus that causes mild to severe respiratory tract disease in humans also has been classified in this genus (Van den Hoogen et al., 2001). Like other paramyxoviruses, ampv contains a negative-sense, single stranded RNA which code for the viral nucleocapsid, phosphoprotein, matrix, fusion, second matrix, small hydrophobic, glycoprotein and large polymerase proteins. (Gough and Jones, 2008).

2 This virus causes a widespread upper respiratory tract infection in turkeys (turkey rhinotracheitis), chickens and some other avian species. The severity of the clinical signs is largely influenced by the intervention of secondary pathogens (Jones, 1996; Cook et al, 2000). In chickens, ampv can be one of the predisposing factors to swelling head syndrome (SHS), characterized by swelling of the periorbital tissues and infraorbital sinuses, torticollis, cerebral disorientation, and opisthotonos. Mortality caused by ampv in chickens is less than 2%, and less than 4% of the flock will show swelling of the head (Gough and Jones, 2008). Infection of broiler breeders and commercial layers cause a drop in egg production with associated loss of egg quality, causing important economic loss (Cook et al, 2000). Isolates of ampv have been classified into four subtypes A, B, C and D based on the nucleotide sequence of the virus attachment glycoprotein (G) (Toquin et al, 2000; Alvarez et al, 2003). Subtype A was initially isolated in South Africa and England, whereas subtype B virus was first identified in continental European countries. Subtype C was isolated in the United States (Seal, 1998) and presence of the forth subtype (D) was reported in France (Bayon-Auboyer et al, 2000). Infections caused by ampv can be diagnosed by virus isolation in chicken or turkey tracheal organ cultures (TOCs), or alternatively in Vero cell cultures (Cook and Cavanagh, 2002). Identification and characterization of ampv isolates can be achieved by immunofluorescence staining or virus neutralisation of the isolate with specific antibodies (Baxter-Jones et al,1989), or demonstration of seroconversion in chickens inoculated by the virus (Grant et al, 1987 and Heckert et al, 1993) Molecular methods, such as reverse transcriptase-polymerase chain reaction (RT-PCR), allow a rapid, sensitive and specific detection of MPV (Bäyon-Auboyer et al, 1999; D Arce et al, 2005; Dani et al, 1999; Cook and Cavanagh, 2002) Serological evidence indicated that ampv is widespread in broilers and broiler breeder flocks in different regions of Iran. Earlier positive samples belonged to 1995 from broiler breeder flocks in north east of Iran (Sheikhi, 2010). In this study, the use of RT-PCR to determine the prevalence of ampv infections in non-vaccinated broiler flocks in Ahwaz slaughterhouses was investigated. MATERIALS AND METHODS Sampling: Fifty broiler flocks were sampled during slaughtering in two slaughterhouses in Ahvaz from January to November The flocks were in 6 to 8 weeks of age and were located in different regions of Khouzestan province. Ten birds in every population of each flock were sampled via tracheal swabs. The swabs were allowed to dry at ambient temperature and then were stored at 4 C until RNA was extracted. All of the broiler flocks weree non-vaccinated for ampv. RNA extraction: The swabs were first soaked in normal saline, then dipped and rotated for 10 sec into 1 ml RNX-Plus TM (CinnaGen Co.), a commercial RNA extraction solution. Swabs belonging to each flock were processed in 1 pool of 10. Then, RNA was precipitated according to RNX-Plus manufacturer s instructions and dissolved in 15 µl double distilled water, treated by diethyl pyrocarbonate (sigma, USA). RT-PCR method: Complementary DNA (cdna) was constructed in a final volume of 20 µl containing 12.5 µl of extracted RNA, 4 µl 5 Reverse transcription buffer (Fermentas, Lithuania),

3 200 units of RevertAid TM M-MuLV reverse transcriptase enzyme (Fermentas, Lithuania), 50 pmols of Random Hexamer oligonucleotide (5 ' -NNNNNN-3'), 2 µl 10 mm dntp mix (Cinnagen, Iran) and 20 units RNase inhibitor (Fermentas, Lithuania). RNA and primer were mixed and incubated at 65 C for 5 min and then chilled on ice, before adding other reagents. Reverse transcription was performed at 42 C for one hour and terminated by 10 min incubation at 70 C. PCR was carried out by the nucleocapsid gene specific primers (Nd: 5 AGC AGG ATG GAG AGC CTC TTT G 3 and Nx: 5 CAT GGC CCA ACA TTA TGT T 3 ) described by Bayon-Auboyer et al. (1999). In brief, 5 µl of cdna was added to 45 µl of PCR mix containing 2.5 units Taq DNA polymerase (Gefanavaran, Iran), 5 µl 10 Taq PCR buffer, 1.5 µl 50 mm MgCl 2, 1 µl 10 mm dntps mix and 50 pmol of each primer. Thermal cycling program consisted on 94 C for 5 min; then 30 PCR cycles at 94 C for 30 s, 51 C for 45 s, and 72 C for 45 s, with a final extension cycle at 72 C for 7 min. PCR products were visualized under UV light in ethidium bromide stained 2% agarose gels. Nemovac APV subtype B live vaccine (Merial, France) and RNase free water were used as the positive and negative controls, respectively. Semi-nested PCR: A semi-nested PCR was designed to confirm the PCR positive results. An internal forward primer (Nd : 5' ACA TCT TCA TGC AAG CTT ATG 3') was designed based on the nucleotide sequence of the Nd/Nx PCR product and used with Nx as reverse primer in the reaction of semi-nested PCR. One µl of positive PCR products was used as the template in seminested PCR. Reagents and thermal cycling program of semi-nested PCR were similar to those described in the first round PCR. RESULTS Nd/Nx PCR was expected to produce a DNA band of 115 bp. Agarose gel electrophoresis of the RT-PCR products revealed that tracheal swab samples of 14 out of 50 broiler flocks had resulted in the amplification of a DNA band, similar to that expected (Figure 1). To confirm the identity of the amplified products as being related to ampv, a semi-nested PCR was carried out by Nd /Nx primers. Nd /Nx PCR would have to produce a DNA band of about 90 bp. Based on the results of Nd /Nx semi-nested PCR, all the samples positive in the first round PCR, showed the amplification of a DNA band of about 90 bp (Figure 2). Therefore, it was concluded that 28% of broiler flocks sampled in Ahwaz slaughterhouses had been infected by ampv.

4 Figure 1. Electrophoresis analysis (2% agarose gel) of three PCR positive samples; A) 100 bp DNA Ladder, B) Positive control, C) Positive field samples, D) Negative control. Figure 2. Electrophoresis analysis (2% agarose gel) of semi-nested PCR products; A) 100 bp DNA Ladder, B) Positive control, C) Negative control. Other lanes correspond to 6 field positive samples. DISCUSSION Previous studies have exploited enzyme linked immunosorbent assay (ELISA) to reveal the seroprevalence of ampv in different regions of Iran. Aali Mehr et al. (2006) showed that the broiler breeder flocks located in Urmia city were serologically positive for ampv specific antibodies. Sheikhi et al. (2010) reported the occurrence of ampv infections in grandparent, breeder, commercial broiler and layer flocks in some central and western regions of Iran by ELISA. The study performed in Kermanshah province showed that 48.1% of broilers and 93.2% of broiler breeder flocks were serologically positive for ampv (Rahimi, 2011). In a recent work in Ahwaz at south west of Iran, Mayahi et al. (2012) reported 55.5% seropositivity against ampv in broiler flocks. For the first time in Iran, the present study was planned to investigate the prevalence of ampv infections in broiler flocks via a molecular method (RT-PCR). Hess et al. (2004) concluded that the use of single RT-PCR for detection of ampv was as sensitive as virus isolation. Nested RT-PCR has been found to be more sensitive that single RT- PCR (Cavanagh et al. 1999; Cook & Cavanagh, 2002). However, the nested RT-PCR has the

5 disadvantage of requiring an additional step prior to obtaining results and also has an increased risk of false-positive results due to carryover DNA contamination (Dani et al., 1999). Bäyon-Auboyer et al. (1999) reported that RT-PCR using primer set Nd/Nx, specific for nucleocapsid gene (N), represents both a specific and a widely applicable tool for ampv detection and could be useful for rapid detection of ampv in field samples. On the other hand, the N gene is the promoter closest gene in the viral genome and the transcription process produces more N mrna than other genes (Barik, 1992). In accordance to its characteristics, RT- PCR based on the N gene had the highest detection limit when compared with fusion protein (F) and glycoprotein (G) genes based RT-PCR assays (Ferreira et al. 2007). Therefore, in present study, the primer set Nd/Nx was applied for molecular detection of ampv. The difference between the results of this study (28% infections) and the seroprevalence results reported by Mayahi et al. (2012) (55.5% infection in broiler flocks in Ahwaz) can be explained by the fact that APV replicates poorly in the infected host and can be detected for only a few days after initial infection (Cook et al. 1993; Naylor et al. 1997), whereas the antibody response after infection lasts longer (Jones et al., 1988) and previously infected flocks will remain positive by ELISA but not by RT-PCR testing. Gharaibeh and Algharaibeh (2007) have also found that RT-PCR results indicated a lower prevalence of infection in all types of flocks compared with ELISA results. Despite this apparent disadvantage of RT-PCR in determining the overall prevalence of ampv, RT-PCR can be applied for subtyping of ampv isolates circulating in the field. In fact, one of the objectives of present study was to prepare cdna of ampv positive samples by random primer, in order to subtype the virus field isolates in the near future. Conclusively, based on our results and serological studies in other parts of the country, ampv infections are widespread in poultry flocks and more attention should be paid to this virus, due to its effect on chicks performance. Moreover, continuing epidemiological and molecular typing surveys are necessary for better prevention of ampv infections in chicken flocks. The paper also represents a novel semi nested PCR to confirm the results of Nd/Nx RT-PCR. REFERENCES Aali mehr, M., M. Tabatabae, and A. Mamaghani, Seroprevalence study of avian pneumovirus infection in broiler chickens. Journal of veterinary research. 61:

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