Malignant catarrhal fever-like disease in Barbary red deer (Cervus elaphus barbarus) naturally infected with a virus resembling alcelaphine herpesvirus 2

Abstract

Eight Barbary red deer (Cervus elaphus barbarus) developed clinical signs suggestive of malignant catarrhal fever (MCF) over a 28-day period. These animals were housed outdoors with four other species of ruminants. Affected red deer had lethargy, ocular signs, and nasal discharge and were euthanatized within 48 h. Lesions included ulcers of the muzzle, lips, and oral cavity associated with infiltrates of neutrophils and lymphocytes. Serologically, six of seven red deer tested during the outbreak were positive by competitive enzyme-linked immunosorbent assay for antibodies to a shared MCF virus antigen. PCR using oligonucleotide primers designed for a conserved protein of alcelaphine herpesviruses 1 (AlHV-1) and 2 (AlHV-2) and for conserved regions of a herpesvirus DNA polymerase gene was positive for tissues from all eight clinically affected animals and negative for eight out of eight red deer without clinical signs of MCF. DNA sequencing of PCR amplicons from the diseased red deer indicated that they were infected with a novel herpesvirus closely related to AlHV-2; immunohistochemistry using polyclonal anti-AlHV-2 serum and in situ hybridization demonstrated the presence of virus within salivary glands adjacent to oral lesions of affected animals. A survey of other ruminants near the outbreak subsequently showed that normal Jackson's hartebeest (Alcelaphus buselaphus jacksoni) that were cohoused with the diseased red deer were infected with the same virus and were shedding the virus in nasal excretions. These findings suggest that a herpesvirus closely related to AlHV-2 caused the MCF-like disease epizootic in Barbary red deer and that the virus may have originated from Jackson's hartebeest.

FIG. 1.

(A and B) Photomicrographs of immunohistochemistry for AlHV-2 demonstrating positive staining in buccal salivary gland cells (A) and no staining in negative control (B). Diaminobenzidine tetrahydrochloride with Gill's hematoxylin counterstain, bar = 50 μm. (C and D) In situ hybridization with an AlHV-2-like viral PCR probe from Barbary red deer with a similar pattern of virus detection in submucosal salivary glands (C) and lack of staining in negative control (D). Nitroblue tetrazolium chloride-5-bromo-4-chloro-3-indolyl phosphate was used with Gill's hematoxylin counterstain. Bar = 50 μm.

FIG. 2.

Alignment of homologous DNA sequences from the AlHV-1/AlHV-2 PCR for Barbary red deer, Jackson's hartebeest, topi AlHV-2, and previously characterized MCF viruses. Identical nucleotides in the majority of aligned sequences for a given position are enclosed in a box. Sequence identity between the Barbary red deer virus amplimers and other MCF viruses is shown in bold adjacent to the ends of the sequences. The GenBank accession number (and reference) for AlHV-1 is AF005370 (6). The source of the AlHV-2 isolate was topi (Damaliscus lunatus jimela), isolate 840412 (44).

FIG. 3.

Ethidium bromide-stained agarose gel (A) and film exposure of Southern blot hybridization (B) of Barbary red deer AlHV-1/AlHV-2 PCR for selected unaffected animals, including Jackson's hartebeest. Lanes 1 and 20, AlHV-1 isolate WC11 (31) from blue wildebeest; lanes 2, 9, and 12, 100-bp ladder; lane 3, Jackson's hartebeest 697068 PBL sampled in October 2001; lane 4, Jackson's hartebeest 697068 nasal swabs sampled in October 2001; lane 5, Jackson's hartebeest 697068 PBL sampled in January 2002; lane 6, Jackson's hartebeest 697068 nasal swabs sampled in January 2002; lane 7, Jackson's hartebeest 601119 PBL; lane 8, Jackson's hartebeest 601119 nasal swabs; lane 10, affected Barbary red deer 696250 nasal swabs; lane 11, Jackson's hartebeest 697064 lymph node sampled in October 1998; lane 13, Ankole PBL; lane 14, scimitar-horned oryx PBL; lanes 15 to 18, mule deer intestine, liver, lymph node, and spleen, respectively; lane 19, no-DNA negative control.

FIG. 4.

Alignment of homologous DNA sequences for the Herpesviridae DNA polymerase PCR from Barbary red deer, Jackson's hartebeest, and known MCF viruses. Identical nucleotides in the majority of aligned sequences for a given position are enclosed in a box. Sequence identity between the Barbary red deer virus amplimers and other MCF viruses is shown in bold adjacent to the ends of the sequences. GenBank database numbers (and references) for AlHV-1, AlHV-2, OvHV-2, and the deer MCF virus are AF005370 (6), AF275942 (19), AF031812 (42), and AF181468 (18), respectively.

FIG. 5.

Alignment of predicted amino acid sequences from the Herpesviridae DNA polymerase PCR from Barbary red deer, Jackson's hartebeest, and known MCF viruses. Similar residues between aligned sequences for a given position are enclosed in a box. Identity between the Barbary red deer virus predicted amino acid sequence and other MCF viruses is shown in bold adjacent to the ends of the sequences. GenBank numbers (and references) for AlHV-1, AlHV-2, OvHV-2, and the deer MCF virus are AF005370 (6), AF275942 (19), AF031812 (42), and AF181468 (18), respectively.