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Malignant catarrhal fever (MCF) is a severe and usually fatal disease of ungulates, including domestic cattle, deer, bison and, occasionally, pigs (Reid and Buxton 1984, Loken and others 1998, Schultheiss and others 2000). The majority of documented cases of clinical MCF in domestic cattle are caused by specific and distinct gamma-herpesviruses, including wildebeest-associated MCF (WA-MCF) due to alcelaphine herpesvirus 1 (AlHV-1), and sheep-associated MCF (SA-MCF) due to ovine herpesvirus 2 (OvHV-2) (Plowright and others 1960, Baxter and others 1993). A long-held belief about these viruses is that they do not produce clinical disease in their natural host. However, this report describes a case of severe clinical disease in a domestic lamb that, on histological postmortem examination and sample testing, strongly resembles MCF.
A six-month-old Lleyn cross commercial lamb was referred to the Scottish Centre for Production Animal Health and Food Safety in Glasgow in early October 2011 for progressive respiratory distress that had previously received treatment with long-acting penicillin with no response. On presentation, the animal was in very poor body condition and was tachypnoeic (respiratory rate of 80 breaths per minute) and dyspnoeic. The heart rate was also markedly increased (170 beats per minute) and harsh lung sounds were heard bilaterally on auscultation of the thorax. The animal's body temperature was 40.2°C. No change in lymph node size was detected on palpation. Haematological parameters were all within the reference ranges, and no significant abnormalities were observed on a biochemistry profile. The differential diagnoses considered were bacterial pneumonia, viral pneumonia, lungworm or Ovine Pulmonary Adenocarcinoma (Jaagsiekte). Jaagsiekte was excluded based on the signalment and a negative wheelbarrow test. The animal was treated with intramuscular long-acting oxytetracycline on the suspicion of a bacterial pneumonia. A McMaster faecal egg count revealed 4200 strongyle and 700 nematodirus eggs per gram of faeces, so this animal was also treated with oral fenbendazole. Unfortunately, despite treatment, the animal's condition deteriorated over the next five days and she became recumbent, at which point she was euthanazed.
No striking lesions were observed on gross postmortem examination, though several organs, including the lungs, were noted to be diffusely pale. Histological examination of the internal organs revealed a widespread lymphocytoclastic arteritis within the heart, kidney and gastrointestinal tract, as well as a marked histiocytic interstitial pneumonia with BALT hyperplasia and multinucleated cells, which are characteristic of herpesvirus replication (World Organisation for Animal Health 2008). Such widespread arteritis is quite rare in sheep, but can be caused by several viruses, including Bluetongue virus, Border Disease virus, and Maedi-Visna virus (Zakarain and others 1975, Cutlip and others 1988, Maclachlan and others 2009). Serum ELISA results, as part of a routine screening protocol, were negative for Maedi-Visna virus. We did not test for Bluetongue virus as it has never circulated within Scotland.
As the lesions were markedly similar to those seen in MCF in cattle, samples were sent to the Moredun Research Institute, Midlothian, to be analysed for OvHV-2. Tissues (lung, heart and kidney) were homogenised using a gentleMACS dissociator with M tubes (both Miltenyi Biotec Ltd, Surrey, UK) in virus transport medium. DNA was extracted from the homogenate using a commercial DNA extraction system (DNAeasy, Qiagen, UK) as per the manufacturer's directions. Samples were tested for the presence of OvHV-2 DNA by real-time PCR (Hussy and others 2001, Traul and others 2007). All OvHV-2 real-time PCR assays were duplexed with an endogenous internal control to confirm successful nucleic acid extraction and absence of PCR inhibitory factors. This control comprised a pan-species β-actin assay, using oligonucleotide primers (Actin_F CAC CTT CCA GCA GAT GTG GA and Actin_R CTA GAA GCA TTT GCG GTG GAC) and a VIC dye labelled minor grove binding (MGB) oligonucleotide probe (Actin_Probe VIC-AGC AAG CAG GAG TAC G-MGB). The kinetics of the β-actin and OvHV-2 real-time PCRs are similar, allowing an estimation of virus genome load per cell to be made. All assay runs included three dilutions of OvHV-2 plasmid standards as positive controls, and no template controls for the extraction and reaction stages. This assay is in routine use as a diagnostic test for clinical disease in cattle.
Ovine herpesvirus 2 DNA was detected in all three tissues (Table 1). Comparisons of the Ct values with those of β-actin suggest a high virus load in all three tissues (between 2.46 and 4.56 virus genomes per cell genome), the highest of which being present within the lung, the primary site of OvHV-2 replication in infected sheep (Li and others 2008). While no age-matched controls were available, our previous experience with the use of this assay in ovine tissue and heparinised blood samples is that either OvHV-2 is not detected (70 per cent) or, where virus genome is detected (30 per cent), the Ct values for OvHV-2 are much higher (range 5–14 Cts above the β-actin value; 3.0×10−2–4.4×10−5 virus genomes per cell genome), suggesting low virus loads considered to be consistent with detection of latent infection (virus carriage).
The current tissue samples were also tested for pestivirus RNA using real-time RT-PCR (Willoughby and others, 2006), and no Border Disease virus, Bovine Viral Diarrhoea Virus type 1 or type 2 RNA were detected.
The pathological and virus genome detection findings could support a diagnosis of MCF in this animal, suggesting that it is possible for OvHV-2 to cause clinical disease in domestic sheep. This MCF-like syndrome had only been previously documented in naïve wild ovine species, and within the laboratory setting after direct exposure of naïve animals to high-titre virus (Buxton and others 1985, Yeruham and others 2004, Li and others 2005, Himsworth and others 2008). Exposure to lower virus titres via aerosolization of infected ovine nasal secretions in naïve lambs of 9–10 months of age has been shown to cause infection, but did not produce clinical signs of disease (Taus and others 2005). This suggests there may be various factors contributing the development of clinical disease in the animal presented in this paper, including the virus titre at exposure and, potentially, the age of exposure. Currently, there are insufficient samples of organ tissue from age-matched lambs to establish for certain what the ‘normal’ range of values for virus levels would be in clinically normal latently infected sheep. Further study of tissue samples from all sheep are needed to compare the levels of virus found in infected ‘normal’ animals with those showing signs of disease; particularly at 6–9 months of age, which is considered the peak time for shedding the virus (Li and others 2004).
The skilled technical services of Mr Richard Irvine, University of Glasgow and Madeleine Maley, Moredun Research Institute are gratefully acknowledged. The Virus Surveillance Unit at the Moredun Research Institute is funded by the Scottish Government. Dr Rob Moeller for providing an insightful second opinion on the histology slides.
Provenance: Not commissioned; externally peer reviewed
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