In 1981 a case of MCF was reported on the basis of clinical signs and symptoms (see Additional File 1) (18). The disease was reported in a buffalo heifer in a village near Faisalabad. The animal showed all the signs and symptoms of MCF and died after an illness of 19 days. No postmortem examination of the carcass was performed (18). In neighbouring countries like India (15, 24), China (23) and Iran (16, 17) documented reports of MCF and the prevalence of OvHV-2, have been published from time to time. However, since the report from Ashfaque and Mohammad in 1981, no reports on cases of MCF in Pakistan have been published. This present study is therefore the first confirmed report of the prevalence of OvHV-2 amongst the national herd of Pakistan.
It is a well-established fact that sheep act as asympatomatic carriers of OvHV-2 and in Pakistan it is a common practice to keep sheep, goats, cattle and buffaloes in close proximities to each other, often with shared common feeding areas2 (8, 18),. In the present study, heminested PCR assays was used to find the presence of OvHV-2 DNA in blood of apparently healthy animals. The hemi-nested PCR used to detect OvHV-2 DNA is highly sensitive, specific and is the diagnostic tool for SA-MCF recommended by the OIE (9, 25). DNA Sequence analysis confirmed the similarity of our Sequences with other OvHV-2 tegument protein gene and POL gene. Our results revealed an overall prevalence of OvHV-2 of 40% in the Rawalpindi District and Islamabad study area. Of the samples taken from apparently healthy animals, 48% of the sheep, 52% of the goats, 39% of the cattle, and 26% of the buffalo tested were found to be positive for OvHV-2 using PCR (Table 1).
Previous studies into the prevalence of OvHV-2 from around the world have indicated that goats, believed to be the natural hosts of Caprine Herpesvirus 2 (CpHV-2), are also susceptible to infection by OvHV-2 (16, 24–29). Studies on wild ruminants in Iran, indicated an OvHV-2 prevalence in goats of 10% (16). The prevalence of OvHV-2 amongst the goats in our study area is higher, however it is consistent with that reported by sampling goats tested in the Kashmir valley (61%) where mixed grazing of goats and sheep is practiced (25, 28). Whilst, goats have been shown to transmit CpHV-2, to susceptible species (5, 29) it has not yet been demonstrated that goats can transmit OvHV-2 to naïve animals (30). Though the majority of cases of OvHV-2 infection of goats appear to be subclinical, reports have tied OvHV-2 infection with clinical symptoms in goats including corneal opacity and pyrexia with neurological signs (31, 32). Further research is required to understand both the impact of OvHV-2 on the health and productivity of goats and whether goats can act as a reservoir animal, and source of infection for susceptible animals for OvHV-2.
Results of our study also indicated high levels of samples taken from apparent healthy cattle (40%) and buffalos (26%) to be positive for OvHV-2. This may suggest that under natural exposure conditions sub-clinical OvHV-2 infections regularly occur in cattle and buffaloes. This observation is consistent with experimental studies, using OvHV-2, that have demonstrated infection can occur in cattle without concurrent development of clinical MCF (33, 34). Furthermore, experimental evidence has indicated that whether an animal develops clinical signs of MCF may depend on the infectious dose of OvHV-2 that the animal receives (35) .
Several studies in Pakistan focused on diseases in cattle and buffaloes such as Rinderpest, peste des petits ruminants (PPR), foot and mouth disease (FMD), theileriosis and babesiosis (18, 36), which share clinical symptoms with MCF, have reported large percentages of symptomatic animals that tested negative for the presence of the pathogens under investigation. Veterinarians in Pakistan do not test for MCF routinely and so its impact on the national herd and economy may be underestimated. Movements of animals between cities for trade purposes during religious and social festivals caused mixing of different animal species which provides opportunity for transmission of infections like MCF. In Pakistan and neighbouring countries free movement of goats and sheep across the borders make MCF a transboundry disease (15). Phylogenetic analysis has shown that the OvHV-2 detected in Pakistan, showed no geographic separation from isolates from around the world. Diagnostic tests, using nested PCR, should therefore be able to identify all cases of MCF, caused by OvHV-2 whether they have resulted from within the national herd or from transboundary cases. Furthermore, the development of improved laboratory diagnostic capability through availability of sensitive and specific molecular tools like PCR, which enables detection of viral DNA, provides a reliable test to distinguish MCF cases from other diseases that induce similar clinical symptoms in affected species.
MCF causes significant economic losses worldwide in major ruminant species. MCF also poses threat to other susceptible species which are housed in close proximity to infected species (15). Whilst transmission of OvHV-2 over distances of five kilometres between lambs and bison without any physical contact has been reported (8, 37), close proximity of susceptible species with infected sheep, and potentially goats, on mixed pasture provides a greater risk of transmission of virus and development of MCF. Since there are no vaccines available for the disease so far and on control measures is the best strategy (15), such as segregating animals by 5 km or housing lambs separate from susceptible species, to reduce the incidence (38).
Continued investigation of the epidemiology and patho-physiology of OvHV-2, particularly in regards to cellular tropism, virus replication, and viral protein expression, is necessary for a more complete understanding of the virus and its association with MCF in cattle and buffaloes. Development of penside testing for MCF and other related with similar clinical symptoms diseases (like FMD and PPR) could help in the early diagnosis of the diseases and lead to better husbandry/ planning during potential outbreaks for veterinarians and government officials.