Characterization of ovine gammaherpesvirus 2 in a goat by nanoplate digital PCR and other diagnostic methods

Abstract

The Macavirus, ovine gammaherpesvirus 2 (OvGHV2), is the cause of sheep-associated malignant catarrhal fever (SA-MCF). Although SA-MCF occurs in a wide range of mammalian hosts, there are few descriptions of this disease and/or infection in goats. This report describes the findings observed in a goat that was infected by OvGHV2 and adds to the rare description of this infection in this animal species. A 6.5-year-old, female, Anglo Nubian goat, with a neurological syndrome, that was euthanized after severe esophageal obstruction was investigated to determine the cause of the brain disease. Histopathology revealed cerebral cortical edema, hemorrhagic rhombencephalitis, severe hepatic necrosis, and atrophic enteritis. An immunohistochemical (IHC) assay identified intracytoplasmic antigens of a malignant catarrhal fever virus (MCFV) within epithelial cells of the intestine, liver, lungs, and kidneys. A semi-nested PCR assay amplified the partial fragment of the OvGHV2 tegument protein gene from the intestine, confirming that the MCFV identified by IHC was OvGHV2. A qPCR assay that targeted the OvGHV2 polymerase gene revealed an elevated quantification cycle (Cq), while nanoplate-based digital PCR (dPCR) detected low viral copy load within the OvGHV2 DNA. Furthermore, the nucleic acids of several disease pathogens associated with diseases in ruminants were not amplified. However, the exact cause of the neurological syndrome remained obscure since nucleic acids of neurological disease pathogens such as bovine viral diarrhea virus, bovine alphaherpesvirus 1 and 5, Histophilus somni, and OvGHV2 were not detected from the brain. Collectively, the results of the Cq and dPCR confirmed that this goat was infected with a low viral load of OvGHV2, which probably was insufficient to induce the typical histopathological alterations and subsequent clinical manifestations associated with SA-MCF and/or infections by OvGHV2. Therefore, elevated viral loads of OvGHV2 would have been required for the development of histological lesions and/or clinical manifestations of SA-MCF in this goat. Furthermore, the dPCR methodology can be used for the efficient detection and quantification of OvGHV2 DNA in animals with or without clinical and/or histopathological evidence of SA-MCF. Additionally, since previous cases of OvGHV2 infections in goats did not have the typical clinical manifestations of SA-MCF, one wonders if this Macavirus can induce SA-MCF in goats.

Keywords: Macavirus; Pathogenesis; Sheep-associated malignant catarrhal fever; Viral load; dPCR; qPCR.

Fig. 1

Immunohistochemical detection of the MAb-15A antigen in a goat infected with OvGHV2. There is positive, intracytoplasmic immunoreactivity of MCFV antigen within epithelial cells of the small intestine (A), kidneys (B), bile duct (C), and lungs (D). Immunoperoxidase counterstained with hematoxylin. Bar (A–D), 20 µm

Fig. 2

Phylogenetic analysis of the OvGHV2 strain identified in goat based on the tegument protein gene. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The strain identified in this study is highlighted ( ) and compared with similar strains derived from diverse animal species identified in several countries. Bovine alphaherpesvirus 1 was used as the outgroup

Fig. 3

Efficiency and standard curves for qPCR of OvGHV2, based on tenfold serial dilutions (1 to 1 × 10⁻⁴) of a field sample (A) and tenfold serial dilutions (1 to 1 × 10⁻⁵) of gBlocks® (B)

Fig. 4

One-dimensional scatterplots of digital PCR for absolute quantification of field sample, pool of positive controls, and OvGHV2 synthetic DNA. Legend: Fluorescence thresholds for the FAM channel for samples #1 (1:100); #1 (1:1000); # 2 (1:100); # 2 (1:1000); # 3 (1:100); # 3 (1:1000); A, Pool of pure positive controls, (1:100); B, OvGHV2 pure synthetic sample; C, OvGHV2 synthetic DNA, 1:10; and No Template Control (NTC) were 24.71. Fluorescence thresholds for the FAM channel for samples # 4 (pure) and #5 (pure) were 26.59