Introduction

Marek’s disease (MD) is a highly contagious oncogenic and neuropathic disease of chickens responsible for great economic losses to the poultry industry all around the world and characterized by development of CD4+ T cell lymphomas as well as infiltration of nerves and visceral organs by lymphocytes. It is a lymphoproliferative disease of chickens caused by cell associated MD herpes virus (MDV), a member of subfamily Alphaherpesvirinae of the family Herpesviridae (Calnek and Witter, 1997). MD poses a big challenge to the welfare and wellbeing of the poultry with increased condemnation of carcass, loss of productivity and quality products leading to huge economic losses. Though the development of effective vaccines and breeding lines of chickens resistant to the disease have been an enormous success, but new and more virulent strains continue to emerge till today (Venugopal, 2000). Marek’s disease virus as an evolving pathogen as the virus itself has changed in the 30 years since its first isolation, so also have many other aspects of the disease and its control (Witter, 1998).  MD is also an immunosuppressive disease and causes increased susceptibility to other concurrent infections. In addition, the highly oncogenic nature of MDV particularly the mechanisms of protection and of pathogenesis can be used as an excellent model for studying the transformation of lymphocytes and the metastasis of lymphoid tumors in other animals including humans, in addition to its unique cell tropism and transmission traits. Swathi et al. (2012) reported that out of 72 cases grossly, 25% were suspected for Marek’s disease and histopathologically, out of 189 tissue samples of 72 cases, 120 (63.49%) were confirmed as Marek’s disease. The histological incidence of Marek’s disease was highest followed by Lymphoid leucosis and other tumours in and around Hyderabad.

Effective preventive measures with prompt diagnostic procedure are of prime importance for this devastating disease. Although advanced diagnostic techniques like PCR, ELISA and immunhistochemistry helps in very rapid and sensitive diagnosis, there is always a need for a simple, inexpensive and reliable diagnostic method under field conditions. Histopathology is still considered as a good and reliable aid in determining the extent of damage at the tissue level and diagnosis of tumours. Therefore, the present study was carried out to diagnose Marek’s disease by adopting and correlating various clinicopathological changes such as clinical signs, gross pathology, cytological and histopathological examinations.

Materials and Methods

The birds of various age groups presented to the Department of Veterinary Pathology for necropsy examination, samples collected from two organized Government poultry farms (one was college poultry farm, CVSc & AH, OUAT and the second was poultry farm of Animal husbandry Dept. at Laxmisagar, Bhubaneswar of Govt. of Odisha), various organized private poultry farms in and around Bhubaneswar within 15 km radius suspected for Marek’s disease during the period of 7 months from December 2014 to June 2015 were included in the study. As the tissue samples were collected from dead birds presented for necropsy examination, there were no ethical issues. The information pertaining to the study such as detailed history of clinical signs, mortality, age and meat type or egg type chickens were recorded. For haematological analysis, 15 blood samples were collected from suspected live birds along with six blood samples from apparent healthy birds (control). Blood samples (2-3ml) were collected through venipuncture of wing vein into an EDTA (ethylenediamine-tertraacetic acid) vial according to the guidelines for ethical treatment to animals and birds without any deviation. Haematological parameters were studied using standard methods such as haemoglobin (Hb) by Sahli’s acid haematin method, total erythrocyte count (TEC) and total leukocyte count (TLC) by haemocytometer, differential leukocyte count (DLC) by Wright-Giemsa stain which is type of modified Giemsa stain and Packed Cell Volume (PCV) by Wintrobe’shaematocrit tube method (Coles, 1986). A total of 62 suspected tumour cases were necropsied systematically as per the standard procedure. All the organs were examined for any tumourous growths and were recorded with reference to location, size, shape, color, consistency etc. A total of 174 representative tissue samples from tumourous lesions of various organs of 62 suspected cases of 20-30 weeks age were collected to carry out cytological and histopathological examinations. The impression smears from affected tissues were collected and prepared as per the standard procedure described by Meinkoth and Cowell (2002). Then the impression smears were stained with Leishman’s or Giemsa stain for cytological examination and interpretation. The representative tissue samples were collected and preserved in 10% formal saline solution for histopathological studies. The formalin fixed tissues were processed by routine histological techniques. Paraffinised tissue sections were prepared with 4-5 µm thickness stained by routine Haematoxylin and Eosin method as per the standard procedure described by Brar et al. (2000). Stained slides were then mounted with cover slip by DPX mountant. Then slides were examined under Olympus microscope for histopathological examination and interpretation. The results of the study were recorded and statistically analyzed by one way ANOVA test as per the methods suggested by Cochran and Snedecor (1994).

Results and Discussion

Clinical Signs

Detailed history regarding the clinical signs of dead birds submitted for necropsy and some suspected live birds was collected and recorded. Affected birds had reduced growth rates, showed inappetence, depression, inactivity, anorexia, decreased egg production, with reduced size of eggs and mortality causing severe economic losses. Some birds had neurologic disorders, paralysis (lameness), blindness, stunting and skin nodular lesions. The birds were thin, dehydrated with pale and anaemic combs and wattle. These findings are in agreement with the findings of earlier studies by Sung (2002), Santin et al. (2006), Kalyani et al. (2010), Arulmozhi et al. (2011), Xu (2011), Singh et al. (2012), Musa (2013) and Sawale et al. (2014). The clinical manifestations might be due to viral infection with multiple lymphoid tumours in visceral organs and neurological degeneration leading to paralysis and nervous symptoms.

Heamatology

Analysis of 15 cases suspected for Marek’s disease on the basis of age (20-30 weeks), belonging to the same flock (in which pathological examination of dead birds had lesions suggestive of MD) and with above mentioned clinical signs such as stunted growth, anemic comb & wattle of decreased size, neurologic disorders etc. showed that there was highly significant increase (p < 0.01) in the TLC values in affected birds as compared to normal birds. There was no significant difference observed between the mean values of other haematological parameters between the two groups. None of the birds died during the study period for which other pathological examinations couldn’t be carried out. No comparative literatures could be traced out regarding haematological study of Marek’s disease affected birds. The details are given in Table 1.

Gross Lesions

A total of 62 suspected cases were examined thoroughly at necropsy for the presence of gross lesions in various organs. Sixteen (25.80%) cases had variable sized grayish white to yellow obvious tumour-like nodular lesions in various visceral organs such as Liver, Spleen, Kidney, Heart, Proventriculus, Ovary, Lungs, Trachea and also nodular lesions in skin which were suggestive of Marek’s disease infections. Liver was enlarged, friable with diffusely distributing variable sized discrete grayish white nodules, few nodules coalesced with each other forming big nodules (Fig. 1).

Cut surface revealed the involvement of parenchyma. Spleen was enlarged to 3-4 times than its normal size with a diffuse white or grayish discolouration. Ovaries were grayish white in colour with multiple nodular growths and marked enlargement with cauliflower like appearance (Fig. 2). Hearts were pale, enlarged, flabby and had single or multiple nodular tumours in the myocardium with loss of coronary fats (Fig. 3). Kidneys were enlarged, pale and had pin point whitish nodules. Proventriculus was thickened with tumourous growth. In some cases lungs and trachea were also affected with similar type of enlargement. The tumours were soft, smooth, grayish and creamy white in colour with or without areas of necrosis. Cutaneous form was characterized by nodular lesions at the base of feather follicles (Fig. 5). The observations are supported by various workers viz. Benton and Cover (1957), Biggs (1973), Ahmed (1982), Panda et al. (1983), Kobayashi et al. (1986); Ghosh et al. (1989); Panneerselvam et al. (1990); Kalyani (2006), Kamaldeep (2007), Balachandran et al. (2009), Gopal et al. (2012) and Swathi et al. (2012) who reported similar type of lesions in Marek’s disease affected birds. There was also occurrence of unilateral or bilateral severe enlargement and edema of nerves particularly sciatic nerve and brachial nerve with loss of cross striations and glistening appearance and grey or yellow discolouration in some cases (Fig. 4). This may be due to infiltration of lymphoblasts. This is in accordance with earlier reports of Clanek et al. (1970), Frank (2001) and Sawale et al. (2014).

Cytology

The cytological examination of impression smears taken from tumourous growths of affected tissues revealed lymphoid cells with thin rim of cytoplasm and vesicular nucleus having a fine network of chromatin and marked pleomorphism with increased cellularity (Fig. 6). ). In the present study, based on cytological evaluation of smears 24 (38.70%) cases were diagnosed as Marek’s disease. These findings are in the agreement with the reports of Biggs (1973) and Swathi et al. (2012).

Histopathological Lesions

A total of 174 representative tissue samples of various affected organs from 62 suspected cases were subjected to histopathological examination and interpretation. Twenty seven (43.54%) cases had lesions suggestive of Marek’s disease based upon histopathological lesions in any of the representative tissue samples. The details are given Table 2. The histological diagnosis of MD in the present study was made based on the pleomorphism of lymphoid series and the same were described by Burmster and Witter (1996) and Biggs (1967). The detailed histopathological observations are organ wise described below:

Liver

Sections revealed infiltration of pleomorphic lymphoid cells including small, medium and large lymphocytes, lymphoblasts and reticulum cells, compressing and obliterating the normal structure of organ (Fig. 7-8). In most of liver sections marked degree of enlargement of vessels with presence of tumour emboli indicated metastasis to various organs and malignancy. In few sections, in addition to neoplasic changes, infiltration of heterophils, variable degrees of degenerative changes, necrosis, oedema were also observed. These findings are almost similar to the lesions described by Benton and Cover (1957), Arulmozhi et al. (2011) Kamaldeep (2007), Balachandran et al. (2009), Gopal et al. (2012) and Swathi et al. (2012).

Spleen

In spleen sections, a clear demarcation between the normal lymphocytes and neoplastic cells was lost and were found mostly perivascular and much thickening of the blood vessels was noted (Fig. 9). Biggs (1973) stated that the proliferative lymphoid involvement is characteristically perivascular in Marek’s disease.

Nerve

The lesions found in the peripheral nerves were infiltration of pleomorphic lymphoid cells, mononuclear cells and plasma cells (Fig. 10) which is in accordance with earlier reports of Biggs (1975), Balachandran et al. (2009), Kalyani et al. (2010), Singh et al. (2012) and Sawale et al. (2014).

Heart & Kidney

Proliferative lymphoid infiltrations with pleomorphism were observed in kidney (Fig. 11) and heart. Proventriculus

Hyperplasia of lymphoid nodules and infiltration of lymphoid cells in submucosa were observed in proventriculi and these findings were in agreement with the reports of Prakash and Rajya (1970) and Swathi et al. (2012).

Ovary

The lesions observed in ovaries were infiltration of pleomorphic lymphoid cells (Fig. 12) and are in accordance with the reports of Payne and Biggs (1967); Pradhan and Nayak (1972), Reddy (1990), Balachandran et al. (2009) and Swathi et al. (2012).

Skin

Sections prepared from skin showed cornification, oedema, keratinization and discrete lymphoid aggregation in feather follicles. Similar observations were also reported by Sharma et al. (1972), Balachandran et al. (2009) and Singh et al. (2012). Similar lesions were also observed in duodenum and lungs in some cases which are in agreement with earlier reports of Balachandran et al. (2009), Carvallo et al. (2011) and Swathi et al. (2012).

Conclusions

From the present study, it can be concluded that despite advancement of molecular biology and virology with development of vaccines, Marek’s disease is still prevalent in many organized poultry farms. The incidence is highest in female egg type birds of age group 20- 30 weeks. Affected birds had decreased breeding potential, reduced growth rates, inappetence, depression and inactivity. Based on necropsy, cytology and histopathological examinations, 25.80%, 38.70% and 43.54% suspected cases of poultry were diagnosed as marek’s disease respectively. There is 88.88% correlation between cytological and histopathological method of diagnosis of Marek’s disease in poultry. Cytological and histopathological examination along with thorough postmortem examination can be profitably utilized for early diagnosis and differential diagnosis of Marek’s disease from other neoplastic diseases in poultry.

Acknowledgements

The authors duly acknowledge the Dean, CVSc & AH, Orissa University of Agriculture and Technology and for providing the support and facility for carrying out the research work.

References

1. Ahmed SI. 1982. Pathology of spontaneous cases of Marek’s disease with special reference to nervous system and experimental studies on some aspects of pathogenesis, PhD Thesis.           University of Agricultural Sciences, Hebbal, Bangalore.
2. Ajinkya SM and Sardeshpande PD. 1969. Observations on Occurrence of Neoplasms in the Domestic Birds. The Indian Veterinary Journal. 46 (5): 380-385.
3. Arulmozhi A, Saravanan, Mohan B and Balasubramaniam GA. 2011.Marek’s disease in vaccinated poultry flocks in and around Namakkal region of TamilNadu. Indian Journal of Veterinary Pathology. 35(1): 45-47.
4. Balachandran C, Pazhanivel N, Vairamuthu S and MuraliManohar B. 2009. Marek’s Disease and Lymphoid Leucosis in Chicken- A Histopathological Survey. Tamilnadu Journal of Veterinary Animal Science. 5(4):167-170.
5. Benton WJ and Cover MS. 1957. The increased incidence of visceral lymphomatosis in broiler and replacement birds. Avian Diseases. 1: 320-327.
6. Bergmann V, Scheer J, Valentine A and Schwarz P.1984.Occurrence of Neoplatic diseases in slaughter hens. Poultry Abstracts. 10(5):131.
7. Biggs PM. 1975. Marek’s disease–The disease and its prevention by vaccination.Br. J. Cancer. 2:152–155.
8. Brar RS, Sandhu HS and Singh A. 2000. Histopathological techniques. In: Veterinary Clinical Diagnosis by Laboratory Methods. Kalyani Publishers, New Delhi. pp.193-195.
9. Calnek BW, Adldinger HK, Kahn DE. 1970. Feather follicle epithelium: a source of enveloped and infectious cell-free herpesvirus from Marek’s disease. Avian Diseases. 14: 219–233.
10. Carvallo FR, French RA, Gilbert-Marcheterre K, Risatti G, Dunn JR, Forster F, Kiupel M and Smyth JA.2011. Mortality of One-Week-Old Chickens during Naturally Occurring Marek’s Disease Virus Infection. Veterinary Pathology. 48(5): 993-998.
11. Deka BC and Grewal GS. 1982. Non Leukotic Tumours in Domestic Fowl in the Punjab. Tropical Animal Health & Production. 14: 59-60.
12. Frank F. 2001. Marek’s disease: History, actual and future Perspectives. Lohmann\Inform. 25:1-5.
13. Gopal S, Manoharan P, Kathaperumal K, Balachandran C and Divyaa KC. 2012. Differential Detection of Avian Oncogenic Viruses in Poultry Layer Farms and Turkeys by Use of Multiplex PCR. Journal of Clinical Microbiology. 50: 2668–2673.
14. Kalyani IH, Tajpar MM, Jhala MK, Bhanderi BB, Nayak J B and  Purohit JH. 2010. Characterization of the ICP4 gene in pathogenic Marek’s disease virus of poultry in Gujarat, India, using PCR and sequencing. Vet. Arhiv. 80: 683-692.
15. Kamaldeep, Sharma P C, Jindal N, and Narang G. 2007.Occurrence of Marek’s Disease in Vaccinated Poultry Flocks of Haryana (India). International Journal of Poultry Science. 6 (5): 372-377.
16. Kobayashi S, Kobayashi K and Mikami T.1986. A study of Marek’s disease in Japanese quails vaccinated with herpes virus of turkeys. Avian Diseases. 30: 816-819.
17. Meinkoth JH and Cowell RL. 2002. Sample collection and preparations in cytology: Increasing diagnostic yield and recognition of basic cell types and criteria of malignancy. Vet. Clin. North Am. Small Anim. Pract. 32 : 1187-1235.
18. Musa IW, Bisalla M, Mohammed B, Saidu L and Abdu PA. 2013. Retrospective and clinical studies of Marek’s disease in Zaria. Nigeria.Journal of Bacteriology Research. 5(2):13-21.
19. Panda BK, Arya SC, Pradhan HK and Johri DC. 1983. Marek’s disease in chicken in various strains of White Leghorn. Indian Journal of Poultry Science. 18: 100.
20. Panneerselvam S, Dorairajan N, Balachandran C, ManoharMurali B. 1990. Incidence of Marek’s disease in Namakkal, Tamil Nadu. Cheiron. 19: 143-144.
21. Pradhan HK and Nayak BC. 1972. Studies on pathology of the female reproductive tract of domestic fowl, I. Naturally occurring oophoritis and salpingitis. Indian Journal of Poultry science. 7: 45-49.
22. Reddy MR. 1990. Studies on etiopathology of reproductive disorders in poultry, M. V. Sc. Thesis. Indian Veterinary Research Institute, Izatnagar.
23. Reece RL.1996. Some Observation on naturally occurring neoplasms in birds in the state of Victoria, Australia. Avian Pathology. 21: 407-447.
24. Santin ER, Shamblin CE, Pigge JT, Arumugaswami V, Dienglewicz RL and Parcells MS. 2006. Examination ofnaturally occurring mutation in glycoprotein L on Marek’s disease virus pathogenesis. Avian Diseases. 80: 96-103.
25. Sawale GK, Shelke VM and Kinge GS. 2014. Observation on occurrence of neural form of Marek’s disease in desi chickens. Indian J. Vet. Pathol. 38(2): 94-97.
26. Sharma DN and Singh CM. 1968.Studies on pathology of female Genital tract of poultry with special Reference to Egg peritonitis- Neoplasms of ovary and oviduct. The Indian Veterinary Journal. 45:388- 391.
27. Singh SD, Barathidasan R, Kumar A, Deb R, Verma AK and Dhama K. 2012. Recent trends inIntroduction

    Marek’s disease (MD) is a highly contagious oncogenic and neuropathic disease of chickens responsible for great economic losses to the poultry industry all around the world and characterized by development of CD4+ T cell lymphomas as well as infiltration of nerves and visceral organs by lymphocytes. It is a lymphoproliferative disease of chickens caused by cell associated MD herpes virus (MDV), a member of subfamily Alphaherpesvirinae of the family Herpesviridae (Calnek and Witter, 1997). MD poses a big challenge to the welfare and wellbeing of the poultry with increased condemnation of carcass, loss of productivity and quality products leading to huge economic losses. Though the development of effective vaccines and breeding lines of chickens resistant to the disease have been an enormous success, but new and more virulent strains continue to emerge till today (Venugopal, 2000). Marek’s disease virus as an evolving pathogen as the virus itself has changed in the 30 years since its first isolation, so also have many other aspects of the disease and its control (Witter, 1998).  MD is also an immunosuppressive disease and causes increased susceptibility to other concurrent infections. In addition, the highly oncogenic nature of MDV particularly the mechanisms of protection and of pathogenesis can be used as an excellent model for studying the transformation of lymphocytes and the metastasis of lymphoid tumors in other animals including humans, in addition to its unique cell tropism and transmission traits. Swathi et al. (2012) reported that out of 72 cases grossly, 25% were suspected for Marek’s disease and histopathologically, out of 189 tissue samples of 72 cases, 120 (63.49%) were confirmed as Marek’s disease. The histological incidence of Marek’s disease was highest followed by Lymphoid leucosis and other tumours in and around Hyderabad.

    Effective preventive measures with prompt diagnostic procedure are of prime importance for this devastating disease. Although advanced diagnostic techniques like PCR, ELISA and immunhistochemistry helps in very rapid and sensitive diagnosis, there is always a need for a simple, inexpensive and reliable diagnostic method under field conditions. Histopathology is still considered as a good and reliable aid in determining the extent of damage at the tissue level and diagnosis of tumours. Therefore, the present study was carried out to diagnose Marek’s disease by adopting and correlating various clinicopathological changes such as clinical signs, gross pathology, cytological and histopathological examinations.

    Materials and Methods

    The birds of various age groups presented to the Department of Veterinary Pathology for necropsy examination, samples collected from two organized Government poultry farms (one was college poultry farm, CVSc & AH, OUAT and the second was poultry farm of Animal husbandry Dept. at Laxmisagar, Bhubaneswar of Govt. of Odisha), various organized private poultry farms in and around Bhubaneswar within 15 km radius suspected for Marek’s disease during the period of 7 months from December 2014 to June 2015 were included in the study. As the tissue samples were collected from dead birds presented for necropsy examination, there were no ethical issues. The information pertaining to the study such as detailed history of clinical signs, mortality, age and meat type or egg type chickens were recorded. For haematological analysis, 15 blood samples were collected from suspected live birds along with six blood samples from apparent healthy birds (control). Blood samples (2-3ml) were collected through venipuncture of wing vein into an EDTA (ethylenediamine-tertraacetic acid) vial according to the guidelines for ethical treatment to animals and birds without any deviation. Haematological parameters were studied using standard methods such as haemoglobin (Hb) by Sahli’s acid haematin method, total erythrocyte count (TEC) and total leukocyte count (TLC) by haemocytometer, differential leukocyte count (DLC) by Wright-Giemsa stain which is type of modified Giemsa stain and Packed Cell Volume (PCV) by Wintrobe’shaematocrit tube method (Coles, 1986). A total of 62 suspected tumour cases were necropsied systematically as per the standard procedure. All the organs were examined for any tumourous growths and were recorded with reference to location, size, shape, color, consistency etc. A total of 174 representative tissue samples from tumourous lesions of various organs of 62 suspected cases of 20-30 weeks age were collected to carry out cytological and histopathological examinations. The impression smears from affected tissues were collected and prepared as per the standard procedure described by Meinkoth and Cowell (2002). Then the impression smears were stained with Leishman’s or Giemsa stain for cytological examination and interpretation. The representative tissue samples were collected and preserved in 10% formal saline solution for histopathological studies. The formalin fixed tissues were processed by routine histological techniques. Paraffinised tissue sections were prepared with 4-5 µm thickness stained by routine Haematoxylin and Eosin method as per the standard procedure described by Brar et al. (2000). Stained slides were then mounted with cover slip by DPX mountant. Then slides were examined under Olympus microscope for histopathological examination and interpretation. The results of the study were recorded and statistically analyzed by one way ANOVA test as per the methods suggested by Cochran and Snedecor (1994).

    Results and Discussion

    Clinical Signs

    Detailed history regarding the clinical signs of dead birds submitted for necropsy and some suspected live birds was collected and recorded. Affected birds had reduced growth rates, showed inappetence, depression, inactivity, anorexia, decreased egg production, with reduced size of eggs and mortality causing severe economic losses. Some birds had neurologic disorders, paralysis (lameness), blindness, stunting and skin nodular lesions. The birds were thin, dehydrated with pale and anaemic combs and wattle. These findings are in agreement with the findings of earlier studies by Sung (2002), Santin et al. (2006), Kalyani et al. (2010), Arulmozhi et al. (2011), Xu (2011), Singh et al. (2012), Musa (2013) and Sawale et al. (2014). The clinical manifestations might be due to viral infection with multiple lymphoid tumours in visceral organs and neurological degeneration leading to paralysis and nervous symptoms.

    Heamatology

    Analysis of 15 cases suspected for Marek’s disease on the basis of age (20-30 weeks), belonging to the same flock (in which pathological examination of dead birds had lesions suggestive of MD) and with above mentioned clinical signs such as stunted growth, anemic comb & wattle of decreased size, neurologic disorders etc. showed that there was highly significant increase (p < 0.01) in the TLC values in affected birds as compared to normal birds. There was no significant difference observed between the mean values of other haematological parameters between the two groups. None of the birds died during the study period for which other pathological examinations couldn’t be carried out. No comparative literatures could be traced out regarding haematological study of Marek’s disease affected birds. The details are given in Table 1.

     

    Gross Lesions

    A total of 62 suspected cases were examined thoroughly at necropsy for the presence of gross lesions in various organs. Sixteen (25.80%) cases had variable sized grayish white to yellow obvious tumour-like nodular lesions in various visceral organs such as Liver, Spleen, Kidney, Heart, Proventriculus, Ovary, Lungs, Trachea and also nodular lesions in skin which were suggestive of Marek’s disease infections. Liver was enlarged, friable with diffusely distributing variable sized discrete grayish white nodules, few nodules coalesced with each other forming big nodules (Fig. 1).

    Cut surface revealed the involvement of parenchyma. Spleen was enlarged to 3-4 times than its normal size with a diffuse white or grayish discolouration. Ovaries were grayish white in colour with multiple nodular growths and marked enlargement with cauliflower like appearance (Fig. 2). Hearts were pale, enlarged, flabby and had single or multiple nodular tumours in the myocardium with loss of coronary fats (Fig. 3). Kidneys were enlarged, pale and had pin point whitish nodules. Proventriculus was thickened with tumourous growth. In some cases lungs and trachea were also affected with similar type of enlargement. The tumours were soft, smooth, grayish and creamy white in colour with or without areas of necrosis. Cutaneous form was characterized by nodular lesions at the base of feather follicles (Fig. 5). The observations are supported by various workers viz. Benton and Cover (1957), Biggs (1973), Ahmed (1982), Panda et al. (1983), Kobayashi et al. (1986); Ghosh et al. (1989); Panneerselvam et al. (1990); Kalyani (2006), Kamaldeep (2007), Balachandran et al. (2009), Gopal et al. (2012) and Swathi et al. (2012) who reported similar type of lesions in Marek’s disease affected birds. There was also occurrence of unilateral or bilateral severe enlargement and edema of nerves particularly sciatic nerve and brachial nerve with loss of cross striations and glistening appearance and grey or yellow discolouration in some cases (Fig. 4). This may be due to infiltration of lymphoblasts. This is in accordance with earlier reports of Clanek et al. (1970), Frank (2001) and Sawale et al. (2014).

    Cytology

    The cytological examination of impression smears taken from tumourous growths of affected tissues revealed lymphoid cells with thin rim of cytoplasm and vesicular nucleus having a fine network of chromatin and marked pleomorphism with increased cellularity (Fig. 6). ). In the present study, based on cytological evaluation of smears 24 (38.70%) cases were diagnosed as Marek’s disease. These findings are in the agreement with the reports of Biggs (1973) and Swathi et al. (2012).

    Histopathological Lesions

    A total of 174 representative tissue samples of various affected organs from 62 suspected cases were subjected to histopathological examination and interpretation. Twenty seven (43.54%) cases had lesions suggestive of Marek’s disease based upon histopathological lesions in any of the representative tissue samples. The details are given Table 2. The histological diagnosis of MD in the present study was made based on the pleomorphism of lymphoid series and the same were described by Burmster and Witter (1996) and Biggs (1967). The detailed histopathological observations are organ wise described below:

    Liver

    Sections revealed infiltration of pleomorphic lymphoid cells including small, medium and large lymphocytes, lymphoblasts and reticulum cells, compressing and obliterating the normal structure of organ (Fig. 7-8). In most of liver sections marked degree of enlargement of vessels with presence of tumour emboli indicated metastasis to various organs and malignancy. In few sections, in addition to neoplasic changes, infiltration of heterophils, variable degrees of degenerative changes, necrosis, oedema were also observed. These findings are almost similar to the lesions described by Benton and Cover (1957), Arulmozhi et al. (2011) Kamaldeep (2007), Balachandran et al. (2009), Gopal et al. (2012) and Swathi et al. (2012).

    Spleen

    In spleen sections, a clear demarcation between the normal lymphocytes and neoplastic cells was lost and were found mostly perivascular and much thickening of the blood vessels was noted (Fig. 9). Biggs (1973) stated that the proliferative lymphoid involvement is characteristically perivascular in Marek’s disease.

    Nerve

    The lesions found in the peripheral nerves were infiltration of pleomorphic lymphoid cells, mononuclear cells and plasma cells (Fig. 10) which is in accordance with earlier reports of Biggs (1975), Balachandran et al. (2009), Kalyani et al. (2010), Singh et al. (2012) and Sawale et al. (2014).

     

     

    Heart & Kidney

    Proliferative lymphoid infiltrations with pleomorphism were observed in kidney (Fig. 11) and heart. Proventriculus

    Hyperplasia of lymphoid nodules and infiltration of lymphoid cells in submucosa were observed in proventriculi and these findings were in agreement with the reports of Prakash and Rajya (1970) and Swathi et al. (2012).

     

     

    Ovary

    The lesions observed in ovaries were infiltration of pleomorphic lymphoid cells (Fig. 12) and are in accordance with the reports of Payne and Biggs (1967); Pradhan and Nayak (1972), Reddy (1990), Balachandran et al. (2009) and Swathi et al. (2012).

    Skin

    Sections prepared from skin showed cornification, oedema, keratinization and discrete lymphoid aggregation in feather follicles. Similar observations were also reported by Sharma et al. (1972), Balachandran et al. (2009) and Singh et al. (2012). Similar lesions were also observed in duodenum and lungs in some cases which are in agreement with earlier reports of Balachandran et al. (2009), Carvallo et al. (2011) and Swathi et al. (2012).

    Conclusions

    From the present study, it can be concluded that despite advancement of molecular biology and virology with development of vaccines, Marek’s disease is still prevalent in many organized poultry farms. The incidence is highest in female egg type birds of age group 20- 30 weeks. Affected birds had decreased breeding potential, reduced growth rates, inappetence, depression and inactivity. Based on necropsy, cytology and histopathological examinations, 25.80%, 38.70% and 43.54% suspected cases of poultry were diagnosed as marek’s disease respectively. There is 88.88% correlation between cytological and histopathological method of diagnosis of Marek’s disease in poultry. Cytological and histopathological examination along with thorough postmortem examination can be profitably utilized for early diagnosis and differential diagnosis of Marek’s disease from other neoplastic diseases in poultry.

    Acknowledgements

    The authors duly acknowledge the Dean, CVSc & AH, Orissa University of Agriculture and Technology and for providing the support and facility for carrying out the research work.

    References

    1. Ahmed SI. 1982. Pathology of spontaneous cases of Marek’s disease with special reference to nervous system and experimental studies on some aspects of pathogenesis, PhD Thesis.           University of Agricultural Sciences, Hebbal, Bangalore.
    2. Ajinkya SM and Sardeshpande PD. 1969. Observations on Occurrence of Neoplasms in the Domestic Birds. The Indian Veterinary Journal. 46 (5): 380-385.
    3. Arulmozhi A, Saravanan, Mohan B and Balasubramaniam GA. 2011.Marek’s disease in vaccinated poultry flocks in and around Namakkal region of TamilNadu. Indian Journal of Veterinary Pathology. 35(1): 45-47.
    4. Balachandran C, Pazhanivel N, Vairamuthu S and MuraliManohar B. 2009. Marek’s Disease and Lymphoid Leucosis in Chicken- A Histopathological Survey. Tamilnadu Journal of Veterinary Animal Science. 5(4):167-170.
    5. Benton WJ and Cover MS. 1957. The increased incidence of visceral lymphomatosis in broiler and replacement birds. Avian Diseases. 1: 320-327.
    6. Bergmann V, Scheer J, Valentine A and Schwarz P.1984.Occurrence of Neoplatic diseases in slaughter hens. Poultry Abstracts. 10(5):131.
    7. Biggs PM. 1975. Marek’s disease–The disease and its prevention by vaccination.Br. J. Cancer. 2:152–155.
    8. Brar RS, Sandhu HS and Singh A. 2000. Histopathological techniques. In: Veterinary Clinical Diagnosis by Laboratory Methods. Kalyani Publishers, New Delhi. pp.193-195.
    9. Calnek BW, Adldinger HK, Kahn DE. 1970. Feather follicle epithelium: a source of enveloped and infectious cell-free herpesvirus from Marek’s disease. Avian Diseases. 14: 219–233.
    10. Carvallo FR, French RA, Gilbert-Marcheterre K, Risatti G, Dunn JR, Forster F, Kiupel M and Smyth JA.2011. Mortality of One-Week-Old Chickens during Naturally Occurring Marek’s Disease Virus Infection. Veterinary Pathology. 48(5): 993-998.
    11. Deka BC and Grewal GS. 1982. Non Leukotic Tumours in Domestic Fowl in the Punjab. Tropical Animal Health & Production. 14: 59-60.
    12. Frank F. 2001. Marek’s disease: History, actual and future Perspectives. Lohmann\Inform. 25:1-5.
    13. Gopal S, Manoharan P, Kathaperumal K, Balachandran C and Divyaa KC. 2012. Differential Detection of Avian Oncogenic Viruses in Poultry Layer Farms and Turkeys by Use of Multiplex PCR. Journal of Clinical Microbiology. 50: 2668–2673.
    14. Kalyani IH, Tajpar MM, Jhala MK, Bhanderi BB, Nayak J B and  Purohit JH. 2010. Characterization of the ICP4 gene in pathogenic Marek’s disease virus of poultry in Gujarat, India, using PCR and sequencing. Vet. Arhiv. 80: 683-692.
    15. Kamaldeep, Sharma P C, Jindal N, and Narang G. 2007.Occurrence of Marek’s Disease in Vaccinated Poultry Flocks of Haryana (India). International Journal of Poultry Science. 6 (5): 372-377.
    16. Kobayashi S, Kobayashi K and Mikami T.1986. A study of Marek’s disease in Japanese quails vaccinated with herpes virus of turkeys. Avian Diseases. 30: 816-819.
    17. Meinkoth JH and Cowell RL. 2002. Sample collection and preparations in cytology: Increasing diagnostic yield and recognition of basic cell types and criteria of malignancy. Vet. Clin. North Am. Small Anim. Pract. 32 : 1187-1235.
    18. Musa IW, Bisalla M, Mohammed B, Saidu L and Abdu PA. 2013. Retrospective and clinical studies of Marek’s disease in Zaria. Nigeria.Journal of Bacteriology Research. 5(2):13-21.
    19. Panda BK, Arya SC, Pradhan HK and Johri DC. 1983. Marek’s disease in chicken in various strains of White Leghorn. Indian Journal of Poultry Science. 18: 100.
    20. Panneerselvam S, Dorairajan N, Balachandran C, ManoharMurali B. 1990. Incidence of Marek’s disease in Namakkal, Tamil Nadu. Cheiron. 19: 143-144.
    21. Pradhan HK and Nayak BC. 1972. Studies on pathology of the female reproductive tract of domestic fowl, I. Naturally occurring oophoritis and salpingitis. Indian Journal of Poultry science. 7: 45-49.
    22. Reddy MR. 1990. Studies on etiopathology of reproductive disorders in poultry, M. V. Sc. Thesis. Indian Veterinary Research Institute, Izatnagar.
    23. Reece RL.1996. Some Observation on naturally occurring neoplasms in birds in the state of Victoria, Australia. Avian Pathology. 21: 407-447.
    24. Santin ER, Shamblin CE, Pigge JT, Arumugaswami V, Dienglewicz RL and Parcells MS. 2006. Examination ofnaturally occurring mutation in glycoprotein L on Marek’s disease virus pathogenesis. Avian Diseases. 80: 96-103.
    25. Sawale GK, Shelke VM and Kinge GS. 2014. Observation on occurrence of neural form of Marek’s disease in desi chickens. Indian J. Vet. Pathol. 38(2): 94-97.
    26. Sharma DN and Singh CM. 1968.Studies on pathology of female Genital tract of poultry with special Reference to Egg peritonitis- Neoplasms of ovary and oviduct. The Indian Veterinary Journal. 45:388- 391.
    27. Singh SD, Barathidasan R, Kumar A, Deb R, Verma AK and Dhama K. 2012. Recent trends in diagnosis and control of Marek’s disease (MD) in poultry. Pakistan Journal of Biological Science. 15(20): 964-970.
    28. Sung HW. 2002. Recent increase of Marek’s disease in Korea related to virulence increase of virus. Avian Diseases. 46:517-524.
    29. Swathi B, Kumar, Anand and Reddy MR. 2012. Histological and molecular diagnosis of poultry tumours. Indian Jurnal Veterinary Pathology. 36(1): 41-48.
    30. Xu Ming, Zhang Huanmin, Lee Lucy, GaoHongwei, Sharif Shayan, Silva Robert F and Heidari Mohammad. . 2011. Gene Expression Profiling in rMd5- and rMd5meq-Infected Chickens. Avian Diseases. 55(3): 358-367.

    and control of Marek’s disease (MD) in poultry. Pakistan Journal of Biological Science. 15(20): 964-970.

28. Sung HW. 2002. Recent increase of Marek’s disease in Korea related to virulence increase of virus. Avian Diseases. 46:517-524.
29. Swathi B, Kumar, Anand and Reddy MR. 2012. Histological and molecular diagnosis of poultry tumours. Indian Jurnal Veterinary Pathology. 36(1): 41-48.
30. Xu Ming, Zhang Huanmin, Lee Lucy, GaoHongwei, Sharif Shayan, Silva Robert F and Heidari Mohammad. . 2011. Gene Expression Profiling in rMd5- and rMd5meq-Infected Chickens. Avian Diseases. 55(3): 358-367.