ABSTRACT
Examination of carcasses of Himalayan goral (Naemorhedus goral) revealed nodular, pox-like eruptions in the skin. Similar disease was also seen in domestic goats (Capra aegagrus hircus) in the same area. Goatpox virus was identified as the etiology of the disease in both cases, with probable transmission between the species.
The genus Capripoxvirus (CaPV) of the family Poxviridae includes three members, namely sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus causing sheeppox, goatpox, and lumpy skin disease of cattle, respectively (Babiuk et al. 2008), which are World Organization for Animal Health notifiable diseases (Bhanuprakash et al. 2011). Goatpox and sheeppox infections are endemic in India and regular reports of outbreak episodes are available (Bhanuprakash et al. 2006; Bora et al. 2018).
Himalayan goral (Naemorhedus goral), the smallest members of goat-antelopes (subfamily Caprinae), are found in the forests of the Himalayas including Bhutan, Nepal, southern Tibet, Myanmar, and northeastern states of India including Sikkim. They are protected under Schedule III of the Indian Wildlife Protection Act (1972) and are categorized as Near Threatened according to the International Union for Conservation of Nature Red List of Threatened Species (Duckworth and MacKinnon 2008). We report an outbreak of goatpox affecting Himalayan gorals and domestic goat (Capra aegagrus hircus) with evidence of possible transmission of the infection to and from domestic goats and gorals.
The outbreak occurred in Himalayan gorals during April–May, 2018 in the Tawang district (27°35′18″N, 91°51′55″E) of Arunachal Pradesh, a hilly state of India, situated at the northeastern-most corner and sharing international borders with Bhutan in the west, Myanmar in the east, and China in the north. In most cases, carcasses were recovered in a decomposed state inside jungles, making it difficult to ascertain the exact number of deaths, but local villagers gave an estimate of up to 100 deaths in the outbreak. On examination, the carcasses were found to be intact with numerous necrosed, ruptured, and circular pox-like lesions in the skin that measured about 1 cm in diameter over the entire body and corneal opacity with the pasting of eyelids. Pox-like lesions were also seen in the anal region involving the underside of the tail (Fig. 1A, B). A domestic goat in the same area was also suffering from a similar pox-like disease and was in its recovery stage (Fig. 1C, D). Considering the postmortem findings in goral as well as the clinical findings in the affected goat, we suspected a typical poxvirus infection. It was also evident from the findings that both the animals had suffered from the same disease. Clinical signs of goatpox in sheep and goats are well described (Tuppurainen et al. 2017). In earlier studies, we recorded goatpox outbreaks in the domestic goat population of Assam, a neighboring state of Arunachal Pradesh. The most likely manner for CaPVs to enter a new area is by the introduction of infected animals (Bhanuprakash et al. 2006). There is frequent movement of animals and humans between the two states. It is possible that the present outbreak may have been related to the outbreaks in Assam. Also, the domestic goats in the affected areas of Arunachal Pradesh were maintained under unorganized and free-ranging systems, favoring the frequent mixing of domestic and wild animals and serving as a portal of transmission in both directions.
Clinical pictures of goral (Naemorhedus goral) and domestic goat (Capra aegagrus hircus) affected with goatpox in the 2018 outbreak at Arunachal Pradesh, India. (A) Carcass of goral that died in the goatpox outbreak. (B) Pox lesions underneath the tail of a goral carcass. (C) Clinically affected domestic goat in the same area of Arunachal Pradesh, India. (D) Typical pox lesions in the udder of the affected domestic goat.
Clinical pictures of goral (Naemorhedus goral) and domestic goat (Capra aegagrus hircus) affected with goatpox in the 2018 outbreak at Arunachal Pradesh, India. (A) Carcass of goral that died in the goatpox outbreak. (B) Pox lesions underneath the tail of a goral carcass. (C) Clinically affected domestic goat in the same area of Arunachal Pradesh, India. (D) Typical pox lesions in the udder of the affected domestic goat.
We collected skin scabs (n=2) and lung tissue (n=2) from gorals and a skin scab (n=1) from an affected domestic goat for confirmatory diagnosis. The samples were processed to make a suspension (Bora et al. 2018) and subjected to standard virologic and molecular diagnosis by counter immunoelectrophoresis (Sharma et al. 1988), PCR (Ireland and Binepal 1998), and PCR-restriction fragment length polymorphism (PCR-RFLP; Hosamani et al. 2004). Virus was isolated in Vero cells (Bora et al. 2018). Molecular characterization of the virus was done by sequencing and phylogenetic analysis of the P32 gene of CaPV.
The tissue suspension was initially subjected to counter immunoelectrophoresis, performed on an agarose gel slide (1%), and the suspension was run against known goatpox hyperimmune sera followed by electrophoresis. After electrophoresis, clear precipitin bands could be observed between the tissue suspension and goatpox-hyperimmune sera in case of skin scab samples from both goral and the domestic goat, but the test was negative in lung tissue samples collected from goral. Further, PCR amplification of full- or partial-length of the P32 gene yielded a positive result showing the presence of capripox virus. On PCR-RFLP analysis, the P32 gene products of the viruses from both Himalayan goral and domestic goat yielded expected results with two fragments of 327 base pairs and 697 base pairs that were specific to GTPV.
Based on the results, the agent associated with the present outbreak in gorals, as well as in domestic goat, was identified as GTPV (Hosamani et al. 2004). On inoculation of the tissue suspensions in Vero cells, the cytopathic effect in infected cells was characterized by ballooning, increased refractility, formation of syncytia, and detachment of the cells from surface at 6 d postinfection (Bora et al. 2018; Dutta et al. 2019). The virus isolates were recovered after four blind passages in Vero cells and confirmed by PCR-RFLP to be GTPV (Hosamani et al. 2004). The findings were further supported by molecular characterization and phylogenetic analysis of the viruses. Sequence analyses of the full length P32 gene of GTPV isolates from goral (GenBank accession no. MN967026) and domestic goat (GenBank no. MT017655) in our study revealed a homology of 99.4–99.8% with other GTPV isolates reported from India and other parts of the world, both at the nucleotide and amino acid levels. Both the viruses isolated from goral and the domestic goat shared 100% homology on nucleotide and amino acid sequences. The phylogenetic tree constructed based on the nucleotide sequence of the P32 gene of viruses isolated from the present outbreak, in comparison to other CaPV, showed all GTPVs in one separate group were closely clustered to each other and were branched separately from other members such as SPPV and lumpy skin disease virus (Fig. 2). Both of our isolates were clustered close to the earlier GTPV isolate in wild goats (serow, Capricornis rubidus) from northeast India (Dutta et al. 2019), indicating that the outbreaks in gorals and serows may be caused by a similar agent.
Phylogenetic tree constructed based on the nucleotide sequence of the P32 gene of capripox virus. The viruses of the Himalayan goral (Naemorhedus goral) and the domestic goat (Capra aegagrus hircus) from the present study are indicated by triangles.
Phylogenetic tree constructed based on the nucleotide sequence of the P32 gene of capripox virus. The viruses of the Himalayan goral (Naemorhedus goral) and the domestic goat (Capra aegagrus hircus) from the present study are indicated by triangles.
The emergence of GTPV infection in domestic animals of northeastern India extending to wild animals has been reported earlier (Bora et al. 2018; Dutta et al. 2019). Further, cross-species infections of SPPV and GTPV have been reported (Bhanuprakash et al. 2010; Santhamani et al. 2015; Ramakrishnan et al. 2016). Our findings confirm the involvement of GTPV in the present outbreak in affecting both wild gorals and domestic goats with possible cross-transmission. Due to climate change, habitat destruction, continuous increase in international trade, and tourism, new diseases can become established in areas where they were previously unknown. The present outbreak of goatpox in Tawang, Arunachal Pradesh, India involving Himalayan gorals and domestic goats is an example of entry of the viruses to new areas, affecting both wild as well as domestic populations.
We acknowledge the help and cooperation received from the State Forest Department State Veterinary and Animal husbandry Department and other officials, Government of Arunachal Pradesh, World Wildlife Fund, India team, and the local people in collection of the samples from gorals.