Reovirus tenosynovitis in a flock of layer breeders

Abstract

The present paper describes a reovirus infection with clinical course in a flock of layer breeders. Lameness and tenosynovitis of flexor tendons were observed in approximately 15% of the cockerels and 3% of the hens from 17 weeks of age onwards. Affected birds did not die; on the contrary, most of them recovered clinically within a period of 8 weeks. Two other breeds of layer parents that were housed in close contact with the affected flock did not develop clinical signs, although serology indicated that infection with reovirus had taken place. These field observations constitute the first report of clinical reovirus tenosynovitis in layer parents and indicate different susceptibilities of layer parent breeds in developing clinical signs following reovirus infection.

Introduction

Recently a high prevalence of reovirus infections was reported in Belgium between 2001 and 2007 through the relatively frequent isolation of the agent from clinically diseased broilers (De Herdt et al., 2008). In the same period, no clinical signs related to reovirus infections were observed in layer-type chickens, notwithstanding the absence of vaccination in these birds. Attempts to isolate a causal agent were negative. These findings are consistent with the literature. Indeed, experimental studies have indicated a lower susceptibility of layer breeds for reovirus infection compared with meat-type chickens (Jones & Kibenge, 1984). Furthermore only a few reports (Schwartz et al., 1976; Jones & Onunkwo, 1978) describe reovirus arthritis in layers under field conditions and there are none on this condition in layer breeders, thereby confirming their lower sensitivity. In October 2008 however, reovirus infection was diagnosed in a flock of layer breeders suffering from tenosynovitis.

Materials and Methods

Case history

Three different breeds of layer parents were simultaneously reared on the same farm in two adjacent houses. Breed A was reared in the first house and Breed B in the second. Breed C was evenly divided over the two houses, separated from the other birds by only a wire fence. No specific problems occurred during the rearing period. At the age of 17 weeks the birds were all transferred to the same production farm, where each breed was placed in separate houses, located immediately next to each other with one central hallway. Each flock consisted of approximately 8500 hens and 850 cockerels. At 2 days after arrival some of the Breed C birds showed difficulties in walking. Affected chickens were unsteady or were limping. Within a period of 2 weeks these signs extended to approximately 15% of the cockerels and 3% of the hens in the flock. No spontaneous mortality occurred, nor were any other clinical signs seen. Affected birds always had a discrete swelling of the digital flexor tendons and eventually of the gastrocnemius tendon. Unexpectedly, almost all of the hens and most of the cockerels recovered clinically within the following weeks. At 25 weeks of age, all male birds that were still presenting clinical signs were killed; their number amounted to approximately 2% of those in the flock. Later on in this flock no negative effects were observed on egg production, fertility or performance of the progeny. In the other two breeder flocks, no clinical signs were observed at any time during their life.

Post-mortem examinations

At regular intervals between 18 and 24 weeks of age, 20 clinically affected birds were killed and examined post mortem. Gross lesions were noted after careful and systematic examination of the legs and organs. Lesions to flexor tendons were evaluated upon comparison with unaffected congeners through visual inspection and palpation. Additionally, samples were collected for cytological, histological, bacteriological, polymerase chain reaction (PCR) and virological examinations (see below).

Standard diagnostic procedures in six birds included cytological examination of the proventriculus, pancreas, liver and flexor tendons. For this purpose, organs were cut aseptically and impression smears were stained with the Hemacolor (Merck) staining reagent. They were observed microscopically at a magnification×1000.

Samples from flexor tendons, brains and liver were taken from four, three and one chickens, respectively, and fixed for histological examination in a 4% phosphate-buffered formaldehyde solution, paraffin-embedded, and sectioned at 5 µm. All sections were stained with haematoxylin and eosin. This allowed examination for the presence of lesions often associated with reovirus infections in broiler breeds (Van De Zande & Kühn, 2007; Jones, 2008).

Routine bacteriological examinations were done on three samples from swollen flexor tendons and on one sample from the liver and spleen. These included inoculations on Columbia blood agar, McConkey agar and agar containing polymyxin and gentamicin appropriate for the selective isolation of Orthinobacterium rhinotracheale (van Empel et al., 1997). PCR (Adiagene) to detect the presence of M. synoviae was performed on samples collected from flexor tendons and hock joints of three birds.

Pooled samples of flexor tendons and of the liver, pancreas and proventriculus obtained from four chickens at 21 weeks of age were inoculated as described elsewhere (De Herdt et al., 2008) in order to examine them for the presence of reovirus in the locomotory and/or intestinal systems. Briefly, the organ samples were homogenized in tryptose solution with penicillin and streptomycin added. The supernatant obtained after centrifugation of the homogenized tissues was filtered and inoculated onto freshly prepared primary chicken embryo liver cells. After 4 to 5 days incubation, the monolayers were inspected for the presence of a typical reovirus syncytial cytopathic effect. Reovirus was identified by the immunofluorescence technique using monoclonal anti-reovirus antibodies.

Serology

Sera collected from 20 birds in the affected flock at 20 weeks of age were examined for the presence of antibodies against reovirus by enzyme-linked immunosorbent assay (ELISA) (IDEXX). The same test was repeated at 33 weeks of age, this time not only in the affected flock but also in the two other breeds that had remained clinically healthy. Fifteen samples were taken per flock. Titres of the tested sera were calculated from optical densities read at 650 nm in relation to positive and negative controls, by the Manufacturer's software.

Results

Post-mortem examinations

At post-mortem examination of affected birds, lesions always consisted of discrete swelling and oedema of the tendons of the M. flexor perforatus and the M. gastrocnemius. Swelling and accumulation of inflammatory fluid was seen in the sternal bursa of three chickens. Unilateral femoral head necrosis occurred in one cockerel, concurrently suffering from tenosynovitis.

No abnormalities at all were found during cytological and/or histological examination of the proventriculus, pancreas, liver and brains. There was, however, a diffuse infiltration of lymphocytes and heterophilic granulocytes between the connective tissue fibres of the flexor tendons as well as mild lymphocytic infiltrates and oedema of the synoviae. Histologically these findings were considered compatible with lesions resulting from reovirus infections (Jones, 2008).

Routine bacteriology did not reveal the presence of any bacteria nor did PCR detect Mycoplasma synoviae.

Virological examination demonstrated the presence of reovirus in samples from the flexor tendons. Pooled samples of liver, pancreas and proventriculus were negative for the presence of this virus.

Serology

ELISA demonstrated the presence of antibodies against reovirus in all 20 birds examined from the affected flock at 20 weeks of age. Titres ranged from 1882 to 27,343, the average being 15,129. Comparative reovirus serology at 33 weeks of age demonstrated mean ELISA titres of 6068 in the flock that had suffered from clinical signs and of 2560 and 2869 in the two clinically healthy flocks.

Discussion

Spontaneous cases of reovirus infections with a clinical course in layer type of chickens seem to be rare. Indeed, most literature data refer to outbreaks in broilers and broiler breeders (De Herdt et al., 2008; Jones, 2008) and only a few reports exist on reovirus infection in layers (Schwartz et al., 1976; Jones & Onunkwo, 1978). The present article seems to be the first report on clinical reovirus tenosynovitis in layer breeders.

It is generally accepted that meat-type chickens are more susceptible to clinical reovirus tenosynovitis compared with layer breeds (Jones & Kibenge, 1984; Jones, 2008). Observations from the present case appear to confirm this. Indeed within a period of 6 weeks the majority of affected birds had recovered clinically. This is in contrast to the situation in broilers where usually no clinical recovery is seen (Jones & Kibenge, 1984; De Herdt et al., 2008). The estimated morbidity in the affected flock amounted to 15% in the cockerels and 3% in the hens. This could indicate a higher sensitivity of male birds. Furthermore, the present data strongly indicate differences in susceptibility to the clinical course of a reovirus infection between breeds of layer parents. Indeed, since reovirus-associated clinical signs were observed already at 2 days following transportation of the layer breeders from the rearing farm to the production farm, it seems evident that the infection occurred during the rearing period. Although by then the affected breed was housed together with the two other breeds, only the former developed clinical signs and lesions. Also, in the production farm, the three breeds were kept under almost identical circumstances in adjacent houses. Comparative serology demonstrated that the two breeds that did not develop clinical signs had nevertheless been in contact with reovirus.

Summarizing, this paper describes a case of reovirus tenosynovitis in a flock of layer breeders. Despite the tendon lesions, most affected birds recovered spontaneously. This outbreak was unusual since clinical reovirus infections occur almost exclusively in broiler breeds that always suffer serious consequences.