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Serological evidence of avian encephalomyelitis virus infection associated with vertical transmission in chicks
Biologicals 43 (2015) 512e514
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Short paper
Serological evidence of avian encephalomyelitis virus infection associated with vertical transmission in chicks Xiao-hui Yu, Jing Zhao, Xiu-hui Qin, Guo-zhong Zhang* Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2015 Received in revised form 11 August 2015 Accepted 23 September 2015 Available online 20 October 2015
Avian encephalomyelitis virus (AEV) can be transmitted both horizontally and vertically. In the present study, we report a typical case of AEV infection in broiler breeder chickens and their progeny identified by clinical survey of the disease, antibody detection, and reverse transcription (RT)-polymerase chain reaction (PCR) assay. © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Avian encephalomyelitis virus Chicken Serology Vertical transmission
Avian encephalomyelitis virus (AEV) was first identified as the cause of avian encephalomyelitis (AE) in the 1930s [1,2]. Based on the characteristics of the AEV genome, the virus has been provisionally assigned to the genus Hepatovirus within the family Picornaviridae [3]. AE is an important disease of chickens, pheasants, quails, and turkeys, and is observed worldwide [4e6]. In young chickens, typical symptoms include characteristic neurological disorders such as tremors, paralysis, ataxia and muscular dystrophy, and subsequently blindness or cataract formation may be seen in some of the affected chicks. In older chickens, infection is usually subclinical, resulting in declines in egg production and hatchability [7e9]. AEV can be transmitted horizontally, and infection of chickens by the fecal-oral route is common [10]. Vertical transmission is an important route of AEV infection, and the virus can be transmitted from infected hens to their progeny via the egg, either on the surface of the egg (transovum transmission), or within the egg (transovarian transmission) [10,11]. This can result in severe diseases in progeny, characterized by tremors and/or ataxia between 1 and 14 days of age [9,12].
* Corresponding author. Tel.: þ86 10 62733660; fax: þ86 10 62732984. E-mail address: [email protected] (G.-z. Zhang).
In this study, we report a typical case of AEV infection in broiler breeder chickens and their progeny. Infection was confirmed by clinical survey of the disease, measurement of antibodies to AEV using an enzyme-linked immunosorbent assay (ELISA), and antigen detection using a reverse transcription (RT)-polymerase chain reaction (PCR) assay. A broiler breeder flock showed a decline in egg production from 84.8% to 71.8% beginning at 32 weeks of age (Fig. 1a). A decline in the hatch rate of the eggs was also observed during this period (Fig. 1b). As no other clinical signs or changes were observed, the changes were predicted to be caused by AEV infection. Blood samples were collected aseptically from the wing vein of birds and transferred to the laboratory for antibody detection of AEV using a commercial ELISA kit (IDEXX, Westbrook, Maine, USA) in accordance with the manufacturer's instructions. In brief, plates were coated with AEV antigen, and upon incubation of the diluted samples in the coated well, antibody specific to AEV formed a complex with the coated antigens. Following removal of unbound material by washing, a horseradish peroxidase conjugate (Goat Anti-Chicken) was added to the wells, which bound to any attached chicken antibody. Unbound conjugate was washed away, and enzyme substrate (1,2,4,5-tetramethoxybenzene) was added. The absorbance values were measured using a microplate photometer (Labsystems, Vantaa, Finland) at a wavelength of 650 nm. A titer above 396 was considered positive. ELISA results confirmed that the flock experiencing egg productivity problems had serological
http://dx.doi.org/10.1016/j.biologicals.2015.09.003 1045-1056/© 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
X.-h. Yu et al. / Biologicals 43 (2015) 512e514
513
Fig. 1. (A) Changes in the laying rate of breeder hens. (B) Changes in the hatch rate of eggs.
Fig. 2. (A) Measurement of antibodies to AEV by ELISA in sequential serum samples in the breeder flock. (B) Measurement of antibodies to AEV by ELISA in sequential serum samples in commercial broilers.
evidence of infection with AEV, and the antibody titer increased in sequential serum samples (Fig. 2a). At 36 weeks of age, the sera had an average titer of 1500 with a 63.2% coefficient of variation (CV), at 37 weeks the average titer was 3200 with a 59.8% CV, and at 39 weeks the average titer was 4000 with a 23.3% CV. Although the mean titer gradually increased, the variable levels of AEV antibody declined over the same period. This appears to be evidence of a natural outbreak of the infection as the chickens had no history of vaccination against AEV after 12 weeks of age. An outbreak of neurological disease also occurred in chicks on a commercial farm associated with the diseased breeder flock. The disease was first seen in the 13th hatching of chicks on the farm. Affected chicks showed trembling and incoordination from the time of hatching, and blindness was observed in some of the affected chicks at 3 weeks of age. Detection of antibody against AEV confirmed AEV infection, as shown in Fig. 2b. As maternal antibodies have disappeared by 20e30 days of age, a high titer of AEVspecific antibodies in serum must be the result of infection. Brain tissue samples from deceased birds were also collected and tested using RT-PCR for AEV. Total RNA was obtained using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), as recommended for tissue samples. RT-PCR was performed to amplify a 619-bp cDNA product using a pair of primers designed based on the VP2 gene of the AEV genome, as described previously [13]. The RT-PCR analysis of the brain samples of sick chickens further confirmed AEV infection (data not shown). Combined with the clinical presentation of the breeder chickens, these results indicated that the disease in the chickens of this age group most likely resulted from vertical transmission. AEV infections in laying breeder flocks usually influence the chicks hatching from any eggs from 10 days prior to egg drop to ten days after egg drop, although the period of egg drop is particularly susceptible [9,10]. Therefore, farmers must be careful about whether to hatch or sell the chicks during this time. Studies have shown that breeder flock vaccination programs designed to provide progeny with maternal antibody can result in better performance of progeny and prevent transovarian transmission during the periods of greatest susceptibility in the
first 2e3 weeks postehatch [10,14e18]. Therefore, it is very important to control the disease by vaccination of breeding flocks during the growing period to prevent vertical transmission. In China, the most widely used vaccination program for chicken breeder flocks is the administration of live AE vaccines to 12e15week-old chickens, either orally or by wing-web inoculation. However, breeder flocks frequently have low antibody titers against AEV, or are serologically negative, and can become infected with AEV during or just prior to the onset of production, thereby shedding virus to any progeny. Breeder flock vaccination programs should be optimized to improve the resistance of the hens to AEV, and to provide maternal antibody-mediated protection to their progeny. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgments This study was supported by the China Agriculture Research System Poultry-related Science and Technology Innovation Team of Peking (CARS-PSTP). References [1] Jones EE. An encephalomyelitis in the chicken. Science 1932;76:331e2. [2] Jones EE. Epidemic tremor, an encephalomyelitis affecting young chickens. J Exp Med 1934;59:781e98. [3] Bakhshesh M, Groppelli E, Willcocks MM, Royall E, Belsham GJ, Roberts LO. The picornavirus avian encephalomyelitis virus possesses a hepatitis C viruslike internal ribosome entry site element. J Virol 2008;82:1993e2003. [4] Tannock GA, Shafren DR. Avian encephalomyelitis: a review. Avian Pathol 1994;23:603e20. [5] Toplu N, Alcigir G. Avian encephalomyelitis in naturally infected pigeons in Turkey. Avian Pathol 2004;33:381e6. [6] Welchman DB, Cox WJ, Gough RE, Wood AM, Smyth VJ, Todd D, et al. Avian encephalomyelitis virus in reared pheasants: a case study. Avian Pathol 2009;38:251e6. [7] Berger RG. An in vitro assay for quantifying the virus of avian encephalomyelitis. Avian Dis 1982;26:534e41.
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X.-h. Yu et al. / Biologicals 43 (2015) 512e514
[8] Meroz M, Elkin N, Hadash D, Abrams M. Egg drop associated with avian encephalomyelitis virus. Vet Rec 1990;127:532. [9] Taylor LW, Lowry DC, Raggi LG. Effects of an outbreak of avian encephalomyelitis (epidemic tremor) in a breeding flock. Poult Sci 1955;34: 1036e45. [10] Calnek BW, Taylor PJ, Sevoian M. Studies on avian encephalomyelitis. IV. Epizootiology. Avian Dis 1960;4:325e47. [11] Schaaf K, Lamoreux WF. Control of avian encephalomyelitis by vaccination. Am J Vet Res 1955;16:627e33. [12] Van Roekel H, Bullis KL, Clarke MK. Transmission of avian encephalomyelitis. J Am Vet Med Assoc 1991;99:220. [13] Xie Z, Khan MI, Girshick T, Xie Z. Reverse transcriptase-polymerase chain reaction to detect avian encephalomyelitis virus. Avian Dis 2005;49: 227e30.
[14] Davison S, Gingerich EN, Casavant S, Eckroade RJ. Evaluation of the efficacy of a live fowlpox-vectored infectious laryngotracheitis/avian encephalomyelitis vaccine against ILT viral challenge. Avian Dis 2006;50:50e4. [15] Garrett JK, Davis RB, Ragland WL. Correlation of serum antibody titer for avian encephalomyelitis virus (AEV) in hens with the resistance of progeny embryos to AEV. Avian Dis 1985;29:878e80. [16] Gharaibeh S, Mahmoud K. Decay of maternal antibodies in broiler chickens. Poult Sci 2013;92:2333e6. [17] Sumner FW, Jungherr EL, Luginbuhl RE. Studies on avian encephalomyelitis. I. Egg adaptation of the virus. Am J Vet Res 1957;18:717e9. [18] Westbury HA, Sinkovic B. The pathogenesis of infectious avian encephalomyelitis. 4. The effect of maternal antibody on the development of the disease. Aust Vet J 1978;54:81e5.
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Short paper
Serological evidence of avian encephalomyelitis virus infection associated with vertical transmission in chicks Xiao-hui Yu, Jing Zhao, Xiu-hui Qin, Guo-zhong Zhang* Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2015 Received in revised form 11 August 2015 Accepted 23 September 2015 Available online 20 October 2015
Avian encephalomyelitis virus (AEV) can be transmitted both horizontally and vertically. In the present study, we report a typical case of AEV infection in broiler breeder chickens and their progeny identified by clinical survey of the disease, antibody detection, and reverse transcription (RT)-polymerase chain reaction (PCR) assay. © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Avian encephalomyelitis virus Chicken Serology Vertical transmission
Avian encephalomyelitis virus (AEV) was first identified as the cause of avian encephalomyelitis (AE) in the 1930s [1,2]. Based on the characteristics of the AEV genome, the virus has been provisionally assigned to the genus Hepatovirus within the family Picornaviridae [3]. AE is an important disease of chickens, pheasants, quails, and turkeys, and is observed worldwide [4e6]. In young chickens, typical symptoms include characteristic neurological disorders such as tremors, paralysis, ataxia and muscular dystrophy, and subsequently blindness or cataract formation may be seen in some of the affected chicks. In older chickens, infection is usually subclinical, resulting in declines in egg production and hatchability [7e9]. AEV can be transmitted horizontally, and infection of chickens by the fecal-oral route is common [10]. Vertical transmission is an important route of AEV infection, and the virus can be transmitted from infected hens to their progeny via the egg, either on the surface of the egg (transovum transmission), or within the egg (transovarian transmission) [10,11]. This can result in severe diseases in progeny, characterized by tremors and/or ataxia between 1 and 14 days of age [9,12].
* Corresponding author. Tel.: þ86 10 62733660; fax: þ86 10 62732984. E-mail address: [email protected] (G.-z. Zhang).
In this study, we report a typical case of AEV infection in broiler breeder chickens and their progeny. Infection was confirmed by clinical survey of the disease, measurement of antibodies to AEV using an enzyme-linked immunosorbent assay (ELISA), and antigen detection using a reverse transcription (RT)-polymerase chain reaction (PCR) assay. A broiler breeder flock showed a decline in egg production from 84.8% to 71.8% beginning at 32 weeks of age (Fig. 1a). A decline in the hatch rate of the eggs was also observed during this period (Fig. 1b). As no other clinical signs or changes were observed, the changes were predicted to be caused by AEV infection. Blood samples were collected aseptically from the wing vein of birds and transferred to the laboratory for antibody detection of AEV using a commercial ELISA kit (IDEXX, Westbrook, Maine, USA) in accordance with the manufacturer's instructions. In brief, plates were coated with AEV antigen, and upon incubation of the diluted samples in the coated well, antibody specific to AEV formed a complex with the coated antigens. Following removal of unbound material by washing, a horseradish peroxidase conjugate (Goat Anti-Chicken) was added to the wells, which bound to any attached chicken antibody. Unbound conjugate was washed away, and enzyme substrate (1,2,4,5-tetramethoxybenzene) was added. The absorbance values were measured using a microplate photometer (Labsystems, Vantaa, Finland) at a wavelength of 650 nm. A titer above 396 was considered positive. ELISA results confirmed that the flock experiencing egg productivity problems had serological
http://dx.doi.org/10.1016/j.biologicals.2015.09.003 1045-1056/© 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
X.-h. Yu et al. / Biologicals 43 (2015) 512e514
513
Fig. 1. (A) Changes in the laying rate of breeder hens. (B) Changes in the hatch rate of eggs.
Fig. 2. (A) Measurement of antibodies to AEV by ELISA in sequential serum samples in the breeder flock. (B) Measurement of antibodies to AEV by ELISA in sequential serum samples in commercial broilers.
evidence of infection with AEV, and the antibody titer increased in sequential serum samples (Fig. 2a). At 36 weeks of age, the sera had an average titer of 1500 with a 63.2% coefficient of variation (CV), at 37 weeks the average titer was 3200 with a 59.8% CV, and at 39 weeks the average titer was 4000 with a 23.3% CV. Although the mean titer gradually increased, the variable levels of AEV antibody declined over the same period. This appears to be evidence of a natural outbreak of the infection as the chickens had no history of vaccination against AEV after 12 weeks of age. An outbreak of neurological disease also occurred in chicks on a commercial farm associated with the diseased breeder flock. The disease was first seen in the 13th hatching of chicks on the farm. Affected chicks showed trembling and incoordination from the time of hatching, and blindness was observed in some of the affected chicks at 3 weeks of age. Detection of antibody against AEV confirmed AEV infection, as shown in Fig. 2b. As maternal antibodies have disappeared by 20e30 days of age, a high titer of AEVspecific antibodies in serum must be the result of infection. Brain tissue samples from deceased birds were also collected and tested using RT-PCR for AEV. Total RNA was obtained using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), as recommended for tissue samples. RT-PCR was performed to amplify a 619-bp cDNA product using a pair of primers designed based on the VP2 gene of the AEV genome, as described previously [13]. The RT-PCR analysis of the brain samples of sick chickens further confirmed AEV infection (data not shown). Combined with the clinical presentation of the breeder chickens, these results indicated that the disease in the chickens of this age group most likely resulted from vertical transmission. AEV infections in laying breeder flocks usually influence the chicks hatching from any eggs from 10 days prior to egg drop to ten days after egg drop, although the period of egg drop is particularly susceptible [9,10]. Therefore, farmers must be careful about whether to hatch or sell the chicks during this time. Studies have shown that breeder flock vaccination programs designed to provide progeny with maternal antibody can result in better performance of progeny and prevent transovarian transmission during the periods of greatest susceptibility in the
first 2e3 weeks postehatch [10,14e18]. Therefore, it is very important to control the disease by vaccination of breeding flocks during the growing period to prevent vertical transmission. In China, the most widely used vaccination program for chicken breeder flocks is the administration of live AE vaccines to 12e15week-old chickens, either orally or by wing-web inoculation. However, breeder flocks frequently have low antibody titers against AEV, or are serologically negative, and can become infected with AEV during or just prior to the onset of production, thereby shedding virus to any progeny. Breeder flock vaccination programs should be optimized to improve the resistance of the hens to AEV, and to provide maternal antibody-mediated protection to their progeny. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgments This study was supported by the China Agriculture Research System Poultry-related Science and Technology Innovation Team of Peking (CARS-PSTP). References [1] Jones EE. An encephalomyelitis in the chicken. Science 1932;76:331e2. [2] Jones EE. Epidemic tremor, an encephalomyelitis affecting young chickens. J Exp Med 1934;59:781e98. [3] Bakhshesh M, Groppelli E, Willcocks MM, Royall E, Belsham GJ, Roberts LO. The picornavirus avian encephalomyelitis virus possesses a hepatitis C viruslike internal ribosome entry site element. J Virol 2008;82:1993e2003. [4] Tannock GA, Shafren DR. Avian encephalomyelitis: a review. Avian Pathol 1994;23:603e20. [5] Toplu N, Alcigir G. Avian encephalomyelitis in naturally infected pigeons in Turkey. Avian Pathol 2004;33:381e6. [6] Welchman DB, Cox WJ, Gough RE, Wood AM, Smyth VJ, Todd D, et al. Avian encephalomyelitis virus in reared pheasants: a case study. Avian Pathol 2009;38:251e6. [7] Berger RG. An in vitro assay for quantifying the virus of avian encephalomyelitis. Avian Dis 1982;26:534e41.
514
X.-h. Yu et al. / Biologicals 43 (2015) 512e514
[8] Meroz M, Elkin N, Hadash D, Abrams M. Egg drop associated with avian encephalomyelitis virus. Vet Rec 1990;127:532. [9] Taylor LW, Lowry DC, Raggi LG. Effects of an outbreak of avian encephalomyelitis (epidemic tremor) in a breeding flock. Poult Sci 1955;34: 1036e45. [10] Calnek BW, Taylor PJ, Sevoian M. Studies on avian encephalomyelitis. IV. Epizootiology. Avian Dis 1960;4:325e47. [11] Schaaf K, Lamoreux WF. Control of avian encephalomyelitis by vaccination. Am J Vet Res 1955;16:627e33. [12] Van Roekel H, Bullis KL, Clarke MK. Transmission of avian encephalomyelitis. J Am Vet Med Assoc 1991;99:220. [13] Xie Z, Khan MI, Girshick T, Xie Z. Reverse transcriptase-polymerase chain reaction to detect avian encephalomyelitis virus. Avian Dis 2005;49: 227e30.
[14] Davison S, Gingerich EN, Casavant S, Eckroade RJ. Evaluation of the efficacy of a live fowlpox-vectored infectious laryngotracheitis/avian encephalomyelitis vaccine against ILT viral challenge. Avian Dis 2006;50:50e4. [15] Garrett JK, Davis RB, Ragland WL. Correlation of serum antibody titer for avian encephalomyelitis virus (AEV) in hens with the resistance of progeny embryos to AEV. Avian Dis 1985;29:878e80. [16] Gharaibeh S, Mahmoud K. Decay of maternal antibodies in broiler chickens. Poult Sci 2013;92:2333e6. [17] Sumner FW, Jungherr EL, Luginbuhl RE. Studies on avian encephalomyelitis. I. Egg adaptation of the virus. Am J Vet Res 1957;18:717e9. [18] Westbury HA, Sinkovic B. The pathogenesis of infectious avian encephalomyelitis. 4. The effect of maternal antibody on the development of the disease. Aust Vet J 1978;54:81e5.