Experimental Infections of Broilers with Avian Metapneumovirus Subtype a and B
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Experimental infections of broilers with avian Metapneumovirus subtype A and B Y. H. AUNG1, M. LIMAN1 and S. RAUTENSCHLEIN1* 1Clinic for Poultry, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany. *Corresponding author: [email protected] Avian Metapneumovirus (aMPV) affects both turkeys and chickens. The virus is associated with swollen head syndrome (SHS) in broilers. Most of the studies regarding aMPV have been done in turkeys. Not much is known about the pathogenesis and immune response to aMPV in broilers. Therefore, our objectives were to study the pathogenesis and immune responses of broilers experimentally infected with aMPV subtype A and B. Three groups of 16-day-old commercial broilers were inoculated oculonasally with 104 ciliostatic dose50 (CD50) of turkey isolates aMPV subtype A or aMPV subtype B. Control birds were inoculated with virus free trachea organ culture (TOC) supernatant. Clinical signs started to appear at 4 days post infection (dpi), and reached peak levels at 6-dpi. At 15 and 17 dpi subtype A and B-infected broilers were free of respiratory signs, respectively. Subtype B-infected broilers showed significantly more severe clinical signs than subtype A- infected ones comparing the clinical score index (P < 0.05). The distribution of aMPV in different tissues was investigated by nested RT-PCR. The viral genome was detected in aMPV subtype A infected chickens at 3 and 6-dpi in the upper respiratory tract tissues such as nasal turbinate, Harderian gland and trachea. In subtype B infected chickens the viral genome was detected not only in the upper respiratory tract tissues but also in the lung, spleen and bursa cloacalis. Virus neutralizing antibodies in tracheal washes of infected broilers started to appear at 3 dpi, reached peak levels at 14 dpi and then declined gradually. Serum virus neutralizing antibody titres reached peak levels at 11 dpi, and persisted throughout the experimental period up to 24 dpi. The effect of aMPV-infection on T cell activity was determined. Splenic T cells were stimulated with concanavalin A (ConA), a T cell mitogen, and the ability of T cells to release IFN gamma was detected by ELISA. At 3 and 6 dpi, aMPV-infected birds showed a significant enhanced T cell response following ConA-stimulation in comparison to non-infected broilers (P < 0.05). Overall, our study demonstrates that broilers are susceptible to infection with turkey isolates aMPV subtype A and B, and the upper respiratory tract was the main target tissue for aMPV in broilers. aMPV B was detected in other systemic tissues such as lung, spleen and bursa cloacalis. Both subtypes stimulated not only systemic immunity but also local antibody production in the respiratory tract of broilers. Keywords: Avian Metapneumovirus; pathogenesis; immune response; commercial broilers Introduction Avian Metapneumovirus (aMPV) causes Turkey Rhinotracheitis (TRT) in turkeys. The virus is also associated with swollen head syndrome (SHS) in broilers and broiler breeders. Initially, SHS in broiler was hypothesized to be due to combined infection of coronavirus and Escherichia coli (Morley and Thomson, 1984). Later TRT-like virus was isolated from hens and guinea fowl in France and from broilers in South Africa (Picault et al., 1987, Buys et al., 1989). 2 SHS due to aMPV has been reported in broilers and broiler breeders in United Kingdom (O’ Brien, 1985, Pattison et al., 1989, Jones et al., 1991, Gough et al., 1994), in Israel (Perelman, 1988), in Canada (Zellen, 1988), in Germany (Hafez and Löhren, 1990) in South Africa (Maharaj et al., 1994), in Taiwan (Lu et al., 1994), in Japan (Tanaka et al., 1995, Mase et al., 2003), and in Saudi Arabia (Al- Ankari, 2004). Although SHS in broilers due to aMPV has been reported from many countries, immune mechanism and pathogenesis of aMPV infections in broilers have not been well understood. Therefore, our objectives were to investigate pathogenesis and immune responses to aMPV subtype A and B infections in commercial broilers. Materials and methods Chickens One hundred and twenty one-day-old commercial broilers (Ross type) were kept in the isolation house, Clinic for Poultry, University of Veterinary Medicine Hannover. Viruses A virulent aMPV subtype A strain (BUT 8544) and a virulent Italian subtype B strain (kindly provided by Dr. R. C. Jones, Liverpool, UK), both isolated from turkeys, were propagated and titrated in chicken tracheal organ culture (TOC) (Cook et al., 1976). For virus neutralization (VN) test aMPV subtype A strain BUT 8544 (Wilding et al., 1986), which was adapted to chicken embryo fibroblasts (CEF), was used (Hafez, 1990). Detection of aMPV in tissue samples Detection of aMPV was done by subtyping nested RT-PCR (Cavanagh et al., 1999). Virus neutralization test and ELISA Sera and tracheal washes (TW) were tested for aMPV-specific neutralizing antibodies following previously published procedures (Baxter-Jones et al., 1989; Obi et al., 1997). In addition, sera were tested for aMPV-specific antibodies using a commercially available aMPV-ELISA system (ART Ab Test Kit©, BioCheck B.V.). Isolation and in vitro stimulation of splenic leukocytes Leukocytes were isolated by density centrifugation (Archambault et al., 1976) from single cell suspensions of spleens. For in vitro culture, triplicates of 106 spleen cells/well were incubated in 96- well tissue culture plates with 5 µg Concanavalin A (ConA)/ml medium (RPMI 1640 supplemented with 10 % foetal bovine serum, antibiotics and L-glutamine) or medium without ConA. After 48 h of incubation at 41°C and at 5 % CO2, supernatants were harvested and tested for IFN-γ using a commercially available IFN-γ capturing ELISA-system (Cytosets©, BioSource). Samples were diluted 8-fold to obtain OD-values in the linear range of IFN-γ standard dilution series’ regression curve. The IFN-γ-concentrations are expressed in pg/ml. Experimental Trial At 16 days of age, when maternally derived aMPV antibodies of birds had waned, the chickens 4 were divided into three groups (n=40). Group A chickens were inoculated oculonasally with 10 CD50 4 of aMPV subtype A, group B chickens were inoculated with 10 CD50 of aMPV subtype B, group C chickens received virus-free TOC supernatant. Three groups of chickens were maintained in separate isolation units, where feed and drinking water were available ad libitum. Clinical signs in broilers were observed daily up to 24 dpi. The clinical scores were determined according to the scoring system previously described (Jones et al., 1992, Naylor and Jones, 1994): score 0: no clinical signs; score 1: clear nasal exudates; score 2: turbid nasal exudates; and score 3: swollen infraorbital sinus and/or frothy eyes. Five chickens from each group were exsanguinated at 3, 6, 11, 14, 17, 20, and 24 dpi. Spleen, bursa cloacalis, lung, trachea, Harderian gland, and nasal turbinate were collected, and RNA was isolated from each sample. Pools from isolated total RNA were made per organ, per group, and per day and aMPV was detected by subtyping nested RT-PCR. Tracheas were removed carefully, and tracheal washings with 500µl of PBS supplemented with antibiotics were conducted thoroughly. Eight serum samples and 5 tracheal washes per group were 3 investigated for VN and ELISA antibodies against aMPV. Spleens were harvested for ex vivo culture of leukocytes. Statistical analysis Group responses of infected and virus-free birds were analyzed by student’s T-test or analysis of variance (ANOVA). P-values < 0.05 were considered as significant. Results and discussion In this study we compared the pathogenesis of aMPV-A and aMPV-B turkey isolates in broilers. Both infected groups began to develop mild clinical signs such as depression, nasal exudates, swollen infraorbital sinus and frothy eyes at 4 dpi. The peak of clinical signs was seen at 6 dpi, when 15 to 27 % of aMPV A and aMPV Binfected birds showed clinical signs, respectively. Clinical signs ceased at 14 dpi and 16 dpi in group A and B, respectively. Chickens from virus free group did not show any clinical signs throughout the experiment (Figure 1). 50 45 40 35 30 Group A 25 Group B Group C 20 15 10 Percent of birds showing clinical signs 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Days Post Infection Figure 1 Percentage of chickens showing clinical symptom after experimental infection with aMPV 4 Group A: Chickens were inoculated oculonasally with 10 CD50 of aMPV subtype A, Group B: Chickens were inoculated 4 oculonasally with 10 CD50 of aMPV subtype B, Group C: Chickens received TOC media only. aMPV-A infected birds showed less severe clinical signs than aMPV-B infected broilers with a maximum clinical score index of 0.36 and 0.56 at 6 dpi, respectively (data not shown). In comparing the clinical score index throughout the observation period of 24 days aMPV subtype B infected chickens showed significantly more clinical signs than aMPV subtype A inoculated birds (P<0.05). Both aMPV-subtypes induced mild clinical lesions in broilers under experimental conditions, but these were milder than in turkeys as it has been shown with the same virus strains before (Liman et al., 2004). 4 The viral genome of aMPV was detected using a subtyping nested RT-PCR (Table 1). Table 1. Detection of the aMPV viral genome by nested RT-PCR Groups at days post infection Tissue 3 6 11 14 samples A B C A B C A B C A B C Nasal turbinate + + - + + - - - - - - - Harderian gland + + - + + - - - - - - - Trachea - + - + + - - + - - - - Lung - - - - + - - - - ND ND ND Spleen - - - - + - - - - ND ND ND Bursa clocalis - - - - + - - - - ND ND ND 4 + = aMPV positive, - = aMPV negative, ND = Not done, A: Chickens were inoculated oculonasally with 10 CD50 of aMPV 4 subtype A, B: Chickens were inoculated oculonasally with 10 CD50 of aMPV subtype B, C: Chickens received TOC media only.