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Comparative suitability of the feathers, peripheral blood and spleen tissue samples of chickens for the detection of Marek’s disease virus by polymerase chain reaction

A total of 24 clinical specimens (10 feathers, 8 peripheral bloods and 6 spleens) were collected from 10 suspected outbreaks of Marek’s disease (MD) from Mymensingh, Tangail, Gazipur and Pabna districts of Bangladesh. A polymerase chain reaction (PCR) protocol originally described by Silva (1992) was adopted to detect Marek’s disease virus (MDV) genome in these specimens. All the tested peripheral blood buffy coat samples (100%) were positive for MDV in PCR, while 70% of feather samples and 66.6% of spleen samples were positive. A band of 317 bp size was found in all positive samples. A few samples also yielded additional bands of 185 bp size and/or multiple bands of larger than 317 bp size, indicating the presence of both virulent MDV and the vaccine virus. The study suggests that peripheral blood and feathers from live birds, and feathers from dead birds are the samples of choice for the detection of MDV by PCR. ( Bangl. vet . 2018. Vol. 35, No. 1&2, 1 – 6)


Introduction
Marek's disease (MD) is a complex, immunosuppressive disease characterized by paralysis, chronic wasting, lymphoma development in the viscera and musculature, and blindness in chickens (Davison and Nair, 2004;Jarosinski et al., 2006). The disease is caused by Marek's disease virus (MDV) belonging to the family Herpesviridae under the genus Mardivirus. MD has been successfully controlled by vaccination during the last decades (Churchill et al., 1969;Rispens et al., 1972). However, it is still a matter of great concern for the poultry industry because often the efficacy of MD vaccines is compromised by the constant evolution of MDV towards greater virulence (Witter, 1997). In addition to the evolution of MDV, other factors may reduce the efficacy of MD vaccines. Problems associated with the handling and storing of MD vaccines, early challenge of MDV in the farm, and co-infection with other immunosuppressive agents can greatly decrease the efficacy of MD vaccines. As a result, there are still sporadic outbreaks of MD that cause significant economic losses.
Marek's disease is prevalent in Bangladesh (Mosleuddin and Dewan, 1974;Suma et al., 2017). While histopathology is the gold standard for the diagnosis of MD, advanced techniques of plaque assay and real-time PCR are also used for the diagnosis of MD and monitoring of MDV vaccines (Abdul-Careem et al., 2006;Islam *Corresponding author:-E-mail: mrislam_bau@yahoo.com The Bangladesh Veterinarian (2018) 35(1&2): 1 -6 et al., 2004;Baigent et al., 2005;Cortes et al., 2011). However, in Bangladesh, the diagnosis of MD is largely dependent on clinical and necropsy findings, which greatly vary among different outbreaks and is often confused with other lymphoproliferative diseases. The present study was designed to adopt a PCR technique for the molecular detection of MDV. Besides, we investigated the suitability of feathers, peripheral blood and spleen tissue for the molecular detection of MDV from field outbreaks.

Samples
We received sick or dead birds for investigation from 10 suspected outbreaks of Marek's disease in commercial layer farms of Mymensingh, Tangail, Gazipur and Pabna districts from March to December 2011. All the birds were reportedly vaccinated against MD at the hatchery at day-old with HVT vaccine. The age of the chickens was 8 to 35 weeks. Most of the affected birds showed anorexia, progressive emaciation, lameness, paralysis and twisted neck. Primary diagnosis of Marek's disease was made on the basis of typical clinical signs, necropsy findings and histopathology (Suma et al., 2017). A total of 24 samples (10 feather follicles, 8 blood samples and 6 spleens) of MD suspected sick or dead chickens were available. A live vaccine containing Serotype 1 MDV strain CVI 988 (Rispens) (Merial Ltd, France, marketed by Advance Animal Science Company Bangladesh Ltd., Dhaka) was used as the positive control.

DNA extraction
DNA from clinical samples and the vaccine virus was extracted by conventional phenol-chloroform-isoamyl alcohol method. For that, approximately 200 mg of tissue (the proximal end of the feather shaft or a part of the spleen) or 200 µl of buffy coat cells was taken in an Eppendorf tube, mixed with 200 µl of digestion buffer and 2 µl of proteinase K to prepare a digestion mixture. The samples were digested overnight at 56C with shaking on a thermo block. On the next day, 400 µl of phenol-chloroformisoamyl alcohol was added to the digestion mixture, mixed with vigorous vortexing and incubated on ice for 2 minutes. The mixture was then centrifuged at 15000 rpm for 5 minutes at 4C and the supernatant was collected in a fresh Eppendorf tube. Again 400 μl of phenol-chloroform-isoamyl alcohol was added to the tube and centrifuged as above to collect the supernatant. The supernatant was transferred to 95% ethanol and 3M sodium acetate solution @ 2.5 times and 1/10 th of the supernatant volume, respectively and centrifuged at 13000g for 5 minutes. The supernatant was discarded, and the DNA pellet was washed twice with 500 µl of 70% ethanol by centrifugation as above. The DNA pellet was dried at 37C on a thermo block. Finally, the DNA pellet was reconstituted in 50 μl of nuclease-free water and stored at -20C.

Molecular detection of the virus
The PCR protocol for the detection of MDV was adopted from published literature (Silva, 1992). PCR was carried out using the PCR Master Mix kit (Promega Corporation, WI, USA) as per the manufacturer's instruction. The extracted DNA was used as a template to amplify 132 base pair tandem repeats in the 'internal repeat long' of the MDV genome using the primer pair Oligo 1 (5′-TGCGATGAAAGTGCTA TGGAGG-3′) and Oligo 2 (5′-GAGAATCCCTATGAGAAAGCGC-3′) as described by Silva (1992). The thermal profile was as follows: initial denaturation at 95ºC for 2 min, followed by 35 cycles of PCR consisting of denaturation at 94ºC for 1 min, annealing at 55ºC for 1 min, and extension for 3 min at 72ºC, followed by final elongation for 5 min at 72ºC. The amplified products were visualized by electrophoresis using 1.4% agarose gel and the image was documented and stored in a computer.

Adoption of PCR for the molecular detection of MDV
The PCR thermal profile originally suggested by Silva (1992) successfully amplified MDV DNA. On electrophoresis, two strong bands of expected 185 and 317 bp, flanking one or two 132-bp tandem repeats, respectively, were observed. In addition, several weak bands of larger sizes, containing multiple repeats, were also observed (Fig. 1).

Suitability of clinical specimens for molecular detection of MDV
Next, we studied which clinical specimens produced the best results during molecular detection of the MDV. For that, we examined 24 clinical samples including 10 feather follicles, 8 buffy coats and 6 spleen samples from 10 suspected MD outbreaks. The results are presented in Fig. 2 and summarized in Table 1. The eight buffy coat samples were all (100%) positive in PCR (Fig. 2a). Of the 10 feather follicles, 7 (70%) produced positive amplification of the MDV genome (Fig. 2b). Four of the six spleen tissue samples (67% produced positive amplification of MDV in PCR (Fig. 2c). Taken together, buffy coat samples appeared to be the most suitable for molecular detection of the MDV from clinical specimens, followed by feather samples. However, Cortes et al (2011) observed that for the detection of serotype 1 MDV, solid tumor tissues and feathers were more suitable than blood. The number of bands observed on electrophoresis in the PCR products varied between samples. One band of 317 bp size was found in all positive samples. A few samples also yielded additional bands of 185 bp size and/or multiple bands larger than 317 bp size along with a smear at a higher molecular weight position. The primers used in this study amplify 132 bp repeat sequences in the 'internal repeat long region' of the MDV genome. Virulent MDV field strains usually contain one or two repeats, while the attenuated strains of MDV have multiple repeats (Silva 1992).
Thus the PCR protocol based on the abundance of 132 bp repeats has been used to differentiate attenuated strains from the virulent strains (Becker et al., 1992;Zhu et al., 1992;Davidson et al., 1995). In the present study, all the samples were collected from clinically affected birds with a history of MDV vaccination; hence it is very likely that both vaccine virus and the field virus were detected in the PCR. This situation demands the development of a PCR protocol that would specifically detect the virulent MDV only. In summary, the PCR protocol developed by Silva (1992) was successfully used for the detection of MDV in clinically affected birds. The possible presence of both virulent MDV and the vaccine virus in the samples was reflected by variable electrophoretic patterns of the PCR products, which limits the use of this PCR protocol in the situation where MDV infection occurs in chickens despite vaccination. Therefore, a PCR protocol is required that would exclusively detect virulent MDV. In our study, the buffy coat extracted from the blood samples appeared to be the most suitable followed by the feathers and spleen. Therefore, if live birds are submitted for investigation, peripheral blood samples as well as feathers should be collected for PCR. In case of dead birds, feathers may be the sample of choice.