Case Report

Marek’s disease virus associated ocular lymphoma in Roulroul partridges (Rollulus

rouloul)

Running Title: Marek’s Disease in Roulroul partridges

Roel HaesendonckA,*, An GarmynA, Gerry M. DorresteinB, Tom HellebuyckA, Gunther

AntonissenA, Frank PasmansA, Richard DucatelleA, An MartelA

A Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary

Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.

B Dutch Research Institute for Birds and Exotic Animals, Wintelresedijk 51, 5507 PP

Veldhoven, Netherlands

*Roel Haesendonck: Roel.Haesendonck@Ugent.be, Tel.: +32 9 264 73 76, Fax: +32 9 264

74 94

An Garmyn: An.Garmyn@Ugent.be

Gerry M. Dorrestein: info@noivbd.nl

Tom Hellebuyck: Tom.Hellebuyck@Ugent.be

Gunther Antonissen: Gunther.Antonissen@Ugent.be

Frank Pasmans: Frank.Pasmans@Ugent.be

Richard Ducatelle : Richard.Ducatelle@Ugent.be

An Martel : An.Martel@Ugent.be

* Corresponding author.

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Abstract

Two 1-year old Roulroul partridges (Rollulus rouloul), one male and one female, were

presented because of eye problems and anorexia. Already 20 of the 30 Roulroul partridges in

the owner’s collection had died. The affected animals stopped eating, became thinner, and

eventually died. Antibiotic treatment, started because of the suspicion of a septicemic

process, was unsuccessful. At clinical examination of the two partridges it was found that in

both birds, 1 eye ball was filled with a whitish yellow amorphous material and the other eye

ball of the female showed a distinct corneal opacity. Both presented animals were euthanized.

Necropsy revealed no significant abnormalities besides the eye lesions. Histology and

immunohistochemistry of the female’s eye revealed an infiltrate of T-lymphocytes

corresponding with ocular lymphoma. Herpesvirus genus-specific PCR, followed by Sanger

sequencing confirmed the presumptive diagnosis of Marek’s disease in both animals. To our

knowledge, this is the first confirmed Gallid Herpesvirus 2 (Marek’s disease) case in

partridges and the first case in this specific species.

Key words: Gallid Herpesvirus 2, Marek’s disease, Marek’s disease virus, ocular

lymphoma, Roulroul partridge, T-lymphocytes

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Introduction

Marek’s disease is a well-known disease in poultry (Marek, 1907; Biggs, 1967) caused by a

cell-associated lymphotropic alpha-herpesvirus (Parcells et al., 2003; Biggs & Nair, 2012)

which can induce tumours in different organs (e.g. liver, lungs, ovary) including the eyes

(Smith et al., 1974; Pandiri et al., 2008). Chronic Marek’s disease is known to affect nerves,

particularly of the lumbo-sacral plexus (Biggs & Nair, 2012). The ocular form of the disease

consists of different types of lesions depending on the anatomical structures involved. These

lesions may result in blindness leading to death caused by starvation (Pandiri et al., 2008).

The disease was first described in chickens (laying hens as well as broilers) and thoroughly

studied in this species (Marek, 1907). Cases in turkeys (Davidson et al., 2002; Pennycott &

Venugopal, 2002; Blake-Dyke & Baigent, 2013), quail (Coturnix coturnix japonica)

(Pradhan et al., 1985; Imai et al., 1990; Pennycott et al., 2003) and pheasants (experimental)

(Phasianus colchicus) (Lesnik, 1981), and one case in a flock of geese (Anser albifrons)

(Murata et al., 2007) have been reported. According to Murata et al. (2012) Marek’s disease

virus is widespread among waterfowl without causing symptoms. These species could be

considered a reservoir for other avian species. Pettit et al. (1976) described macroscopic and

histopathological lesions similar to those caused by Marek’s disease in a black francolin

(Francolinus francolinus) without the confirmation of the etiologic agent. Jennings (1954)

reported a case of neural lymphomatosis in a partridge (Perdix perdix) in the UK. This bird

showed enlargement of the lumbo-sacral plexus in combination with corresponding

histological lesions, similar to those described in chickens (Biggs, 1967), but, again, an

etiologic agent could not be assigned.

Roulroul partridges (Rollulus rouloul) are medium size partridges originating from Thailand

and Malaysia, which are frequently kept in private and zoo collections. Marek’s disease virus

has not been reported previously in this species.

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Materials and Methods

History

In a breeding group of 30 adult Roulroul partridges (Rollulus rouloul), over a period of 2.5

months, 20 animals died after developing a whitish yellow, amorphous material in their eyes

or an opaque cornea. These Roulroul partridges were bought at the age of 2-3 months from a

breeding facility in which previously chickens have been kept for several years. Other species

such as black francolins (Francolinus francolinus), blue scaled quails (Callipepla squamata),

European partridges (Perdix perdix) and Chinese bamboo partridges (Bambusicola

thoracicus) were kept in separate cages in the same room and showed no symptoms nor

mortality. These species were bought from another breeding facility. The Roulrouls became

anorectic and died approximately 10 days after the first symptoms. They were unsuccessfully

treated orally with enrofloxacine (Baytril®, Bayer Animal Health Care) via drinking water

and locally with chloramphenicol ointment (unknown origin), because of the suspicion of

septicaemia after a bacterial infection. Two chicks from the affected animals (eggs laid at the

onset of the eye symptoms), which were artificially incubated and reared (no vaccination was

performed), were completely normal and in good health at 10 weeks of age (the moment of

presenting the adults).

Clinical examination

Two of the birds, one male and one female, both 1-year old, were presented. The animals

displayed a poor body condition (210g, normal bodyweight 230-250g), were depressed and

showed eye lesions, resulting in reduced eyesight. At the left side the female had

exophthalmia and a whitish yellow, amorphous granular material in the anterior eye chamber

that seemed to be attached to the cornea (Figure 1) and at the right side an opacity of the

cornea (Figure 2). The male had exophthalmia at the right side and similar material as

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described in the female. The left eye appeared normal. Because of the high mortality, poor

prognosis and the importance of a correct diagnosis, the birds were euthanized by an

intravenous injection in the vena ulnaris of sodiumpentobarbital 0.5 ml/kg body weight

(Natrium Pentobarbital®, Kela Laboratoria, Belgium) for necropsy and further examination.

Necropsy and further diagnostic procedures

The two birds were submitted for necropsy. On both animals, a macroscopic evaluation of the

organs and cytology (Hemacolor®, VWR International, Leuven, Belgium) was done on

smears from lung, spleen, kidney, liver, crop and eye. Small and large intestines, as well as

caecal content were evaluated for endoparasites.

A swab of the eyes (cornea and anterior eye chamber) and faecal material from the female

were collected and routinely processed for bacteriological and mycological examination.

Faecal material of the same bird was examined for the presence of Salmonella sp. Eyes

including optical nerve, spleen, liver, lung, kidney, heart, proventriculus, ventriculus,

intestines and adrenal glands, were sampled and fixed in 10% buffered formalin. After

fixation, the samples were processed for histological examination. Paraffin sections were

stained with haematoxylin-eosin. Paraffin sections of the eye of the female were also stained

for CD-3 (T-lymphocytes) (Polyclonal Rabbit Anti-Human CD3, Dako, Glostrup, Denmark)

and CD-20 (B-lymphocytes) (Polyclonal Rabbit anti-Human CD20, Thermo Scientific,

Fremont, USA) immunohistochemistry. The former polyclonal antibody has been tested in

our laboratory and shows cross-reactivity with chicken B-lymphocytes. The latter was tested

by Jones et al. (1993) and appropriate to use on chicken tissue.

A swab from the eye of the female and samples from the liver of both animals were preserved

at -20°C for further molecular diagnostic procedures. DNA from these samples was extracted

using the DNeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). A nested Herpesvirus

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genus-specific polymerase chain reaction (PCR) was done as described by VanDevanter et al.

(1996) with adjustment of the annealing temperature to 43°C and 48°C for the first and

second assay, respectively. This assay targeted a region of the herpes viral DNA directed

DNA polymerase gene. DNA from an avian herpesvirus (Columbid Herpesvirus 1) served as

a positive control in these assays. All PCR assays were done using a Mastercycler thermal

cycler (Eppendorf, Hamburg, Germany). Secondary PCR products were run on a 1.5%

agarose gel stained with gelred for 75 min at 170 volt and visualized under UV-light to

evaluate the PCR results. Positive PCR products were submitted for Sanger sequencing

(GATC-Biotech, Constance, Germany) using the primers from the second PCR assay.

Reticuloendotheliosis virus (REV) PCR, which targeted the gp90 gene, was done as

described previously by Li et al. (2012). REV antigen concentrate (Charles Rivers

Laboratories, Wilmington, USA) served as a positive control and water as a negative.

Equipment and gel electrophoresis were similar as mentioned above.

Results

Gross pathologic examination of both Roulrouls revealed no abnormalities except for the eye

lesions. Cytology of the internal organs and the eyes of the female showed no abnormalities.

Cytology of the right eye of the male showed heterophils, lymphocytes and coccoid bacteria,

however bacteriological and mycological examination of the eyes of both animals were

negative. The faecal material tested negative for Salmonella sp.

Histopathological examination of the eyes revealed a diffuse infiltration of the iris with round

cells with a large central nucleus and a narrow rim of cytoplasm (Figure 3). There was

moderate anisokaryosis and anisocytosis. There were an average of 2 mitoses per high power

field (HPF). These cells were also infiltrating in the corneal stroma and the corpus ciliare.

Additionally, paraffin sections of the eye ball were stained with a CD-3 and CD-20 specific

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staining. The CD-3 specific staining was positive (Figure 4A) and the CD-20 staining

negative (Figure 4B), meaning that the eye was infiltrated by a monomorphic population of

T-lymphocytes in the absence of B-lymphocytes. Histology of the other organs revealed an

infiltration of lymphoblasts in the optic nerve, ventriculus, heart, kidney, lung and adrenal

glands.

REV PCR was negative and herpesvirus genus-specific PCR positive for the female eye

swab, female and male liver. These 3 latter PCR products revealed a single band on agarose

gel. To confirm the diagnosis of Marek’s disease, the PCR products were sequenced. The eye

revealed a sequence of 240 basepairs (bp) and the liver one of 245 bp. These sequences were

compared with known sequences using the on-line Basic Local Alignment Search Tool

(BLAST). Both sequences matched for 99% with the Gallid Herpesvirus 2 (Marek’s Disease

virus type 1).

Discussion

Ocular neoplasia in birds is a rare disease, with ocular lymphomatosis in chickens being the

most prevalent (Cho, 1974; Dukes & Pettit, 1983). Previous cases describing clinical signs

and histologic characteristics suggestive for Marek’s disease in partridges or closely related

birds such as quail and francolins are rare and the aetiology has never been confirmed

(Jennings, 1954; Biggs, 1967; Pettit et al., 1976). With recent techniques, and especially

PCR, confirming the diagnosis of Marek’s disease should be easier. To our knowledge, this is

the first confirmed diagnosis of Marek’s disease in partridges. It is remarkable that this virus

has a tropism for ocular tissue in this species and that there were no macroscopic

abnormalities noticed at the internal organs, although an infiltration of lymphoblasts was

present in may organs and the birds’ livers tested positive in the PCR. Ocular lesions as the

only gross anomaly in Marek’s disease has been reported previously in chickens (Ficken et

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al., 1991). It appears to be caused by specific isolates. But in quail, a bird species closely

related to partridges, nerve lesions and ocular lesions due to Marek’s disease are rare (Kenzy

& Cho, 1969; Imai et al., 1991).

Pandiri et al. (2008) reported that the distribution of the lymphoid infiltrates in the eye differs

according to the time after infection. Lymphocytic infiltration of the iris is classified as an

early lesion while late lesions consist of aggregates of lymphocytes and macrophages in the

anterior chamber resulting in granular material often attached to the cornea. In this case

however, both lesions were present at the same time in one bird. Additionally, corneal

oedema was present. Most likely early eye lesions were present but obviously it was only the

granular material which drew the owner’s attention. These ocular changes most likely result

in impaired vision, followed by the inability to find food, resulting in wasting and eventually

death. Blindness due to Marek’s disease associated miosis and grey iris discoloration has

been described in chickens (Ficken et al., 1991), but was not present in this case.

Differential diagnosis in these cases includes Salmonella sp., Pasteurella multocida and

Mycoplasma gallisepticum septicaemia (Bayón et al., 2007) and intraocular aspergillosis

(Beckman et al., 1994). P. multocida associated ophthalmia has been reported in Turkeys

(Olson, 1981) resulting in similar granular material in the anterior chamber. Beckman et al.

(1994) reported intraocular aspergillosis in chicks which resulted in similar lesions as in the

present case. Nunya et al. (1995) reported a corneal opacity in layer chickens infected with

M. gallisepticum. Salmonella Typhimurium has been reported as the causative agent of eye

changes in young broilers (Hinz & Kaleta, 1970). The authors described similar material in

the anterior chamber as reported in this study. Bacteriological and mycological examination

of the eye swab and faecal material obtained from the female was negative. Furthermore, the

high morbidity and mortality, combined with the fast onset of symptoms and progression of

the infection, are more likely associated with a viral pathogen. Reticuloendotheliosis virus

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(REV), an oncogenic retrovirus has been described in a number of species including chickens

(Robinson & Twiehaus, 1974), quail (Coturnix coturnix japonica) (Carlson et al., 1974) and

partridges (Perdix perdix) (Trampel et al., 2002) and can cause similar gross lesions as

Marek’s disease virus, but often limited to the intestinal tract, liver and spleen (Carlson et al.,

1974; Trampel et al., 2002; Cheng et al., 2007). Eye lesions caused by REV are not

mentioned. In the present case there were no gross lesions noticed at the internal organs as

described in REV cases. Besides, both liver samples and the eye sample tested negative in the

REV PCR assay.

Coccoid bacteria were observed in cytology smears of the male’s eye but cultures were not

obtained. These bacteria could be indigenous to the conjunctival flora (Zenoble et al., 1983)

or could be secondary to the viral primary pathogen.

In the present case, it was not possible to identify the source of infection with certainty. The

other species and specifically the other partridges showed no clinical symptoms. Most likely,

the Roulroul partridges were infected at a young age in the breeding facility from which the

animals were bought. In this breeding facility chickens were kept during the previous years.

Pradhan et al. (1985) already described the occurrence of Marek’s disease in quail located at

the same farm where there was a problem of recurrent Marek’s disease among chickens.

In the present outbreak, chicks from these infected parents showed no problems (at the

moment of diagnosis 10 weeks old). Artificial incubation and rearing is a good preventive

measure as vertical transmission of this virus in not seen (Solomon et al., 1970). The other

partridges showed no symptoms, probably because they came into contact with the virus from

the Roulrouls when they already gained age-resistance. Besides, these partridges were bought

from another breeding facility than the Roulrouls.

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In conclusion, we can state that partridges are indeed susceptible to Marek’s disease virus. In

the present case, noteworthy is the presence of different ocular lesions in different animals in

absence of any other symptoms or macroscopic lesions.

Acknowledgements

We would like to thank Dr. C. Adriaensen and Dr. P. Van Rooij for their skilful technical

assistance. This research was supported by the Research Fund of Ghent University, Belgium

(BOF Grant 01D20312).

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Figure 1: Left eye of the female showing the whitish yellow, amorphous granular material in

the anterior eye chamber.

Figure 2: Right eye of the female with distinct corneal opacity.

Figure 3: Histopathological section (HE) of the female’s iris (I) showing a diffuse infiltration

with round cells with a large central nucleus and a large amount of apoptotic cell bodies (C:

Cornea).

Figure 4: Immunohistochemistry of the female’s eye shows a T-lymphocyte infiltration in the

iris (I) (CD-3 immunohistochemistry) (A) and an absence of B-lymphocyte infiltration in the

(CD-20 immunohistochemistry) (B) (L: lens).

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