Epidemiologic and molecular analysis of sarcoid cases in a herd of donkeys in Northern Italy

Status
Status: 
Completed
Applicants
Collaborators
Details
When conducted: 
1 October 2011 - 1 February 2013
Location: 
Il Rifugio degli Asinelli
Country: 
Italy
Core methodology: 
Samples collected in 2011 and 2012 (skin scurf, swabs, tumour material, hair roots, insect tissue) from affected (n=13), possibly affected (n=5) and unaffected (n=26) donkeys, PCR sequencing to isolate BPV DNA. 20 tabanid flies collected from locality for dissection and further processing including DNA purification, homogenization or mounting on slides.
Aims: 
To investigate; 1.1) the link between current sarcoid cases and BPV -1/2 1.2) if clinically healthy animals are infected with the virus 1.3) using viral sequence analysis, to investigate if the disease is transmitted between animals 1.4) if the caseload constitutes an outbreak and constitutes a true epidemic and can a viral source be identified 2.1) can viral DNA and/or virus like structures be detected in affected individuals 2.2) do lesion-derived BPV DNA isolates harbour the same genetic variants 2.3) whether scurf obtained from BPV –positive animals as well as grooming kits harbour virion-like structures/virions 2.4) whether flying insects; particularly tabanid flies, feeding on lesions harbour virion-like structures/virions 2.5) analyse sarcoid epidermis for the presence of viral protein and virion-like structures.
Results: 
1) Viral DNA detected in vast majority of samples from affected donkeys 2) Viral DNA also found in old dandruff from affected grooming brush. 3) No viral DNA found in material from unaffected samples 4) 17 of 20 tabanid samples tested negative for BPV1/BPV2 DNA 5) Gene sequencing is still in progress, initial results show E5 sequences are not identical but vary by one or more silent or non-synonymous point mutations. 6) Variants of BPV1 and BPV2 type E5 detected. 7) So far there is evidence of a productive BPV 1/BPV2 infection having occurred in a subset of investigated material.
Conclusions: 

Viral infection demonstrated for all tumour bearing individuals on a DNA level, adding evidence to the widely accepted concept that BPV1/2 is chiefly involved in onset and progression of sarcoids in equids.
Failure to trace viral genome in some affected samples is likely to be due to low viral copy numbers escaping detection.
Analysis of 5 tumours excised for clinical reasons showed intralesional expression of major oncogene E5 providing further evidence for its active role in cell transformation.
Intralesional expression of major capsid protein L1 is suggestive of production infection ie assembly of infectious virion, in these lesions.
Two tabanids scoring positive for BPV DNA failed to yield positive results from PCR, providing no evidence for tabanids transferring infection. However, low sample numbers require further analyses to clarify this.
Sequencing to date yielded variations indicative that the outbreak is more accidental rather than from one single common source. This concept is strengthened by the fact that the caseload has dropped since 2011.
The mode(s) of transmission still remain unknown, and pending further research. In lieu of this spatial separation of sarcoid bearing donkeys and mules is recommended as a prophylactic.