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Case Report
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Marek’s disease virus associated ocular lymphoma in Roulroul partridges (Rollulus
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rouloul)
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Running Title: Marek’s Disease in Roulroul partridges
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Roel HaesendonckA,*, An GarmynA, Gerry M. DorresteinB, Tom HellebuyckA, Gunther
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AntonissenA, Frank PasmansA, Richard DucatelleA, An MartelA
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A
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Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary
Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
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Dutch Research Institute for Birds and Exotic Animals, Wintelresedijk 51, 5507 PP
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Veldhoven, Netherlands
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*Roel Haesendonck: [email protected], Tel.: +32 9 264 73 76, Fax: +32 9 264
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An Garmyn: [email protected]
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Gerry M. Dorrestein: [email protected]
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Tom Hellebuyck: [email protected]
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Gunther Antonissen: [email protected]
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Frank Pasmans: [email protected]
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Richard Ducatelle : [email protected]
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An Martel : [email protected]
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* Corresponding author.
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Abstract
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Two 1-year old Roulroul partridges (Rollulus rouloul), one male and one female, were
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presented because of eye problems and anorexia. Already 20 of the 30 Roulroul partridges in
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the owner’s collection had died. The affected animals stopped eating, became thinner, and
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eventually died. Antibiotic treatment, started because of the suspicion of a septicemic
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process, was unsuccessful. At clinical examination of the two partridges it was found that in
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both birds, 1 eye ball was filled with a whitish yellow amorphous material and the other eye
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ball of the female showed a distinct corneal opacity. Both presented animals were euthanized.
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Necropsy revealed no significant abnormalities besides the eye lesions. Histology and
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immunohistochemistry of the female’s eye revealed an infiltrate of T-lymphocytes
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corresponding with ocular lymphoma. Herpesvirus genus-specific PCR, followed by Sanger
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sequencing confirmed the presumptive diagnosis of Marek’s disease in both animals. To our
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knowledge, this is the first confirmed Gallid Herpesvirus 2 (Marek’s disease) case in
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partridges and the first case in this specific species.
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Key words: Gallid Herpesvirus 2, Marek’s disease, Marek’s disease virus, ocular
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lymphoma, Roulroul partridge, T-lymphocytes
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Introduction
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Marek’s disease is a well-known disease in poultry (Marek, 1907; Biggs, 1967) caused by a
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cell-associated lymphotropic alpha-herpesvirus (Parcells et al., 2003; Biggs & Nair, 2012)
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which can induce tumours in different organs (e.g. liver, lungs, ovary) including the eyes
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(Smith et al., 1974; Pandiri et al., 2008). Chronic Marek’s disease is known to affect nerves,
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particularly of the lumbo-sacral plexus (Biggs & Nair, 2012). The ocular form of the disease
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consists of different types of lesions depending on the anatomical structures involved. These
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lesions may result in blindness leading to death caused by starvation (Pandiri et al., 2008).
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The disease was first described in chickens (laying hens as well as broilers) and thoroughly
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studied in this species (Marek, 1907). Cases in turkeys (Davidson et al., 2002; Pennycott &
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Venugopal, 2002; Blake-Dyke & Baigent, 2013), quail (Coturnix coturnix japonica)
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(Pradhan et al., 1985; Imai et al., 1990; Pennycott et al., 2003) and pheasants (experimental)
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(Phasianus colchicus) (Lesnik, 1981), and one case in a flock of geese (Anser albifrons)
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(Murata et al., 2007) have been reported. According to Murata et al. (2012) Marek’s disease
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virus is widespread among waterfowl without causing symptoms. These species could be
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considered a reservoir for other avian species. Pettit et al. (1976) described macroscopic and
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histopathological lesions similar to those caused by Marek’s disease in a black francolin
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(Francolinus francolinus) without the confirmation of the etiologic agent. Jennings (1954)
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reported a case of neural lymphomatosis in a partridge (Perdix perdix) in the UK. This bird
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showed enlargement of the lumbo-sacral plexus in combination with corresponding
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histological lesions, similar to those described in chickens (Biggs, 1967), but, again, an
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etiologic agent could not be assigned.
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Roulroul partridges (Rollulus rouloul) are medium size partridges originating from Thailand
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and Malaysia, which are frequently kept in private and zoo collections. Marek’s disease virus
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has not been reported previously in this species.
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Materials and Methods
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History
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In a breeding group of 30 adult Roulroul partridges (Rollulus rouloul), over a period of 2.5
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months, 20 animals died after developing a whitish yellow, amorphous material in their eyes
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or an opaque cornea. These Roulroul partridges were bought at the age of 2-3 months from a
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breeding facility in which previously chickens have been kept for several years. Other species
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such as black francolins (Francolinus francolinus), blue scaled quails (Callipepla squamata),
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European partridges (Perdix perdix) and Chinese bamboo partridges (Bambusicola
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thoracicus) were kept in separate cages in the same room and showed no symptoms nor
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mortality. These species were bought from another breeding facility. The Roulrouls became
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anorectic and died approximately 10 days after the first symptoms. They were unsuccessfully
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treated orally with enrofloxacine (Baytril®, Bayer Animal Health Care) via drinking water
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and locally with chloramphenicol ointment (unknown origin), because of the suspicion of
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septicaemia after a bacterial infection. Two chicks from the affected animals (eggs laid at the
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onset of the eye symptoms), which were artificially incubated and reared (no vaccination was
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performed), were completely normal and in good health at 10 weeks of age (the moment of
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presenting the adults).
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Clinical examination
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Two of the birds, one male and one female, both 1-year old, were presented. The animals
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displayed a poor body condition (210g, normal bodyweight 230-250g), were depressed and
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showed eye lesions, resulting in reduced eyesight. At the left side the female had
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exophthalmia and a whitish yellow, amorphous granular material in the anterior eye chamber
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that seemed to be attached to the cornea (Figure 1) and at the right side an opacity of the
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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
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prognosis and the importance of a correct diagnosis, the birds were euthanized by an
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intravenous injection in the vena ulnaris of sodiumpentobarbital 0.5 ml/kg body weight
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(Natrium Pentobarbital®, Kela Laboratoria, Belgium) for necropsy and further examination.
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Necropsy and further diagnostic procedures
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The two birds were submitted for necropsy. On both animals, a macroscopic evaluation of the
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organs and cytology (Hemacolor®, VWR International, Leuven, Belgium) was done on
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smears from lung, spleen, kidney, liver, crop and eye. Small and large intestines, as well as
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caecal content were evaluated for endoparasites.
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A swab of the eyes (cornea and anterior eye chamber) and faecal material from the female
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were collected and routinely processed for bacteriological and mycological examination.
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Faecal material of the same bird was examined for the presence of Salmonella sp. Eyes
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including optical nerve, spleen, liver, lung, kidney, heart, proventriculus, ventriculus,
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intestines and adrenal glands, were sampled and fixed in 10% buffered formalin. After
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fixation, the samples were processed for histological examination. Paraffin sections were
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stained with haematoxylin-eosin. Paraffin sections of the eye of the female were also stained
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for CD-3 (T-lymphocytes) (Polyclonal Rabbit Anti-Human CD3, Dako, Glostrup, Denmark)
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and CD-20 (B-lymphocytes) (Polyclonal Rabbit anti-Human CD20, Thermo Scientific,
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Fremont, USA) immunohistochemistry. The former polyclonal antibody has been tested in
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our laboratory and shows cross-reactivity with chicken B-lymphocytes. The latter was tested
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by Jones et al. (1993) and appropriate to use on chicken tissue.
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A swab from the eye of the female and samples from the liver of both animals were preserved
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at -20°C for further molecular diagnostic procedures. DNA from these samples was extracted
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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.
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(1996) with adjustment of the annealing temperature to 43°C and 48°C for the first and
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second assay, respectively. This assay targeted a region of the herpes viral DNA directed
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DNA polymerase gene. DNA from an avian herpesvirus (Columbid Herpesvirus 1) served as
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a positive control in these assays. All PCR assays were done using a Mastercycler thermal
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cycler (Eppendorf, Hamburg, Germany). Secondary PCR products were run on a 1.5%
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agarose gel stained with gelred for 75 min at 170 volt and visualized under UV-light to
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evaluate the PCR results. Positive PCR products were submitted for Sanger sequencing
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(GATC-Biotech, Constance, Germany) using the primers from the second PCR assay.
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Reticuloendotheliosis virus (REV) PCR, which targeted the gp90 gene, was done as
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described previously by Li et al. (2012). REV antigen concentrate (Charles Rivers
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Laboratories, Wilmington, USA) served as a positive control and water as a negative.
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Equipment and gel electrophoresis were similar as mentioned above.
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Results
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Gross pathologic examination of both Roulrouls revealed no abnormalities except for the eye
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lesions. Cytology of the internal organs and the eyes of the female showed no abnormalities.
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Cytology of the right eye of the male showed heterophils, lymphocytes and coccoid bacteria,
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however bacteriological and mycological examination of the eyes of both animals were
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negative. The faecal material tested negative for Salmonella sp.
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Histopathological examination of the eyes revealed a diffuse infiltration of the iris with round
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cells with a large central nucleus and a narrow rim of cytoplasm (Figure 3). There was
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moderate anisokaryosis and anisocytosis. There were an average of 2 mitoses per high power
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field (HPF). These cells were also infiltrating in the corneal stroma and the corpus ciliare.
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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
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negative (Figure 4B), meaning that the eye was infiltrated by a monomorphic population of
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T-lymphocytes in the absence of B-lymphocytes. Histology of the other organs revealed an
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infiltration of lymphoblasts in the optic nerve, ventriculus, heart, kidney, lung and adrenal
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glands.
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REV PCR was negative and herpesvirus genus-specific PCR positive for the female eye
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swab, female and male liver. These 3 latter PCR products revealed a single band on agarose
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gel. To confirm the diagnosis of Marek’s disease, the PCR products were sequenced. The eye
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revealed a sequence of 240 basepairs (bp) and the liver one of 245 bp. These sequences were
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compared with known sequences using the on-line Basic Local Alignment Search Tool
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(BLAST). Both sequences matched for 99% with the Gallid Herpesvirus 2 (Marek’s Disease
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virus type 1).
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Discussion
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Ocular neoplasia in birds is a rare disease, with ocular lymphomatosis in chickens being the
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most prevalent (Cho, 1974; Dukes & Pettit, 1983). Previous cases describing clinical signs
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and histologic characteristics suggestive for Marek’s disease in partridges or closely related
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birds such as quail and francolins are rare and the aetiology has never been confirmed
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(Jennings, 1954; Biggs, 1967; Pettit et al., 1976). With recent techniques, and especially
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PCR, confirming the diagnosis of Marek’s disease should be easier. To our knowledge, this is
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the first confirmed diagnosis of Marek’s disease in partridges. It is remarkable that this virus
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has a tropism for ocular tissue in this species and that there were no macroscopic
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abnormalities noticed at the internal organs, although an infiltration of lymphoblasts was
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present in may organs and the birds’ livers tested positive in the PCR. Ocular lesions as the
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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
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related to partridges, nerve lesions and ocular lesions due to Marek’s disease are rare (Kenzy
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& Cho, 1969; Imai et al., 1991).
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Pandiri et al. (2008) reported that the distribution of the lymphoid infiltrates in the eye differs
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according to the time after infection. Lymphocytic infiltration of the iris is classified as an
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early lesion while late lesions consist of aggregates of lymphocytes and macrophages in the
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anterior chamber resulting in granular material often attached to the cornea. In this case
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however, both lesions were present at the same time in one bird. Additionally, corneal
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oedema was present. Most likely early eye lesions were present but obviously it was only the
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granular material which drew the owner’s attention. These ocular changes most likely result
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in impaired vision, followed by the inability to find food, resulting in wasting and eventually
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death. Blindness due to Marek’s disease associated miosis and grey iris discoloration has
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been described in chickens (Ficken et al., 1991), but was not present in this case.
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Differential diagnosis in these cases includes Salmonella sp., Pasteurella multocida and
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Mycoplasma gallisepticum septicaemia (Bayón et al., 2007) and intraocular aspergillosis
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(Beckman et al., 1994). P. multocida associated ophthalmia has been reported in Turkeys
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(Olson, 1981) resulting in similar granular material in the anterior chamber. Beckman et al.
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(1994) reported intraocular aspergillosis in chicks which resulted in similar lesions as in the
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present case. Nunya et al. (1995) reported a corneal opacity in layer chickens infected with
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M. gallisepticum. Salmonella Typhimurium has been reported as the causative agent of eye
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changes in young broilers (Hinz & Kaleta, 1970). The authors described similar material in
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the anterior chamber as reported in this study. Bacteriological and mycological examination
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of the eye swab and faecal material obtained from the female was negative. Furthermore, the
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high morbidity and mortality, combined with the fast onset of symptoms and progression of
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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
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(Robinson & Twiehaus, 1974), quail (Coturnix coturnix japonica) (Carlson et al., 1974) and
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partridges (Perdix perdix) (Trampel et al., 2002) and can cause similar gross lesions as
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Marek’s disease virus, but often limited to the intestinal tract, liver and spleen (Carlson et al.,
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1974; Trampel et al., 2002; Cheng et al., 2007). Eye lesions caused by REV are not
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mentioned. In the present case there were no gross lesions noticed at the internal organs as
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described in REV cases. Besides, both liver samples and the eye sample tested negative in the
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REV PCR assay.
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Coccoid bacteria were observed in cytology smears of the male’s eye but cultures were not
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obtained. These bacteria could be indigenous to the conjunctival flora (Zenoble et al., 1983)
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or could be secondary to the viral primary pathogen.
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In the present case, it was not possible to identify the source of infection with certainty. The
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other species and specifically the other partridges showed no clinical symptoms. Most likely,
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the Roulroul partridges were infected at a young age in the breeding facility from which the
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animals were bought. In this breeding facility chickens were kept during the previous years.
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Pradhan et al. (1985) already described the occurrence of Marek’s disease in quail located at
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the same farm where there was a problem of recurrent Marek’s disease among chickens.
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In the present outbreak, chicks from these infected parents showed no problems (at the
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moment of diagnosis 10 weeks old). Artificial incubation and rearing is a good preventive
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measure as vertical transmission of this virus in not seen (Solomon et al., 1970). The other
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partridges showed no symptoms, probably because they came into contact with the virus from
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the Roulrouls when they already gained age-resistance. Besides, these partridges were bought
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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
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the present case, noteworthy is the presence of different ocular lesions in different animals in
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absence of any other symptoms or macroscopic lesions.
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Acknowledgements
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We would like to thank Dr. C. Adriaensen and Dr. P. Van Rooij for their skilful technical
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assistance. This research was supported by the Research Fund of Ghent University, Belgium
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(BOF Grant 01D20312).
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Figure 1: Left eye of the female showing the whitish yellow, amorphous granular material in
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the anterior eye chamber.
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Figure 2: Right eye of the female with distinct corneal opacity.
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Figure 3: Histopathological section (HE) of the female’s iris (I) showing a diffuse infiltration
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with round cells with a large central nucleus and a large amount of apoptotic cell bodies (C:
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Cornea).
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Figure 4: Immunohistochemistry of the female’s eye shows a T-lymphocyte infiltration in the
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iris (I) (CD-3 immunohistochemistry) (A) and an absence of B-lymphocyte infiltration in the
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(CD-20 immunohistochemistry) (B) (L: lens).