African swine fever is a devastating, infectious viral haemorrhagic disease of domestic and wild pigs, which can cause severe disease and high mortality in susceptible hosts. The host range of African swine fever virus (ASFV) is very narrow with suids as sole vertebrate hosts, and soft ticks of the genus Ornithodoros as competent arthropod vector^1,2. Most isolates of ASFV circulating in Europe, the Russian Federation, and Asia cause the acute form of disease, although some reduced-virulence isolates have been obtained from infected wild boar in the Baltic States^3,4,5. However, highly virulent ASFV isolates were found in European wild boars of all ages^5,6.

African swine fever is caused by ASFV belonging to the genus Asfivirus of the family Asfarviridae. ASFV is the only virus listed in that genus⁷. ASFV is an enveloped virus having linear genome of double-stranded DNA of length between 170 kb and 190 kb in size containing 150-167 ORFs⁸. The ASFV contains a central DNA with a thick protein layer called the core shell, an inner lipoprotein envelope surrounding the core and the icosahedral capsid (p72)⁹. On the basis of p72 protein¹⁰, a total of 24 different genotypes of ASFV are identified with only one serotype detectable by antibody test.

The first occurrence of African swine fever (ASF) in domestic pigs in India was recorded from two states of northeast India, namely Arunachal Pradesh and Assam in early 2020. Considering the geographical location and widespread ongoing ASF outbreaks in China since 2018, the possible source of ASF into Arunachal Pradesh and subsequently to Assam state of India was considered as through China¹¹. Subsequently, the outbreak of ASF was reported from Mizoram state in the year 2021¹². The present study is reporting outbreaks of ASF in different districts of Meghalaya during the period from April 2021 to

November, 2021. We have studied the pathology, confirmed the ASF outbreaks by detection of p72 gene of ASFV and identified the circulating strain as the ASFV genotype II.

Out of the 14 pig farms studied, outbreak of ASF was confirmed by PCR in 6 different farms located in five different districts of Meghalaya, namely RiBhoi, East Khasi Hills, West Garo Hills, South West Khasi Hills and North Garo Hills district. Details of the farms visited, epidemiological data and PCR results are given in Table 2. Onset of the outbreaks in all the 6 farms was sudden and characterized by severe depression, lethargy, cutaneous haemorrhages or purplish discolouration of the skin and high fever. Infected pigs died within 3-5 days, after onset of clinical signs and resulted 90-100% mortality in affected farms.

The detailed post mortem examination revealed discolouration of the skin to cutaneous haemorrhages andbloody discharge from the nose. Upon opening of the carcass, all the lymph nodes (deep and superficial) were found to be enlarged, haemorrhagic and dark black in colour. The large intestine showed haemorrhagic colitis with bloody intestinal content. Spleen was severely enlarged with rounded edges and infarcts were also seen (Fig 2). Petechial haemorrhages were observed on the cortical surfaces of the kidney (Fig 3). Liver was dark chocolate colored, enlarged with rounded edges. The gall bladder was distended and haemorrhagic (Fig 4). Hydropericardium with paint brush like haemorrhages on both epicardium and endocardium in heart and haemorrhagic pneumonia was observed in lungs (Fig 5).

Histopathological examination of all the lymph nodes revealed necrosis and lymphoid depletion in the follicles. Interfollicular region showed severe congestion and haemorrhages (Fig 6). Severe congestion, haemorrhages and lymphoid depletion was observed in spleen (Fig 7). In liver, the hepatic cells showed degeneration to coagulative necrosis and dilatation of sinusoidal spaces (Fig 8). Haemorrhages and mononuclear infiltration was observed in portal area. Tissue sections from lungs revealed haemorrhagic pneumonia with severe accumulation of oedematous fluid in the interstitium and bronchioles. The alveolar septae were thickened due to oedema, vascular congestion and haemorrhages and infiltration by mononuclear cells (Fig 9). Hyperplasias of bronchiolar epithelial cells were also observed. Kidneys showed hypercelluiarity of glomerular tuft, infiltration of inflammatory cells in the interstitium and coagulative necrosis of tubular epithelium.

Representative tissue samples collected from these six farms were tested for CSF and PRRS by using reverse transcription-PCR, which have yielded negative results for CSF and PRRS. DNA samples from all the tissue samples were again tested by PCR for PCV2, PPV and ASF. While the results for PCV2 and PPV were negative, the samples from six different pig farms have given positive results for ASF. The fragment of 478 bp region of P72 gene of ASFV was successfully amplified by using PCR, which confirmed the outbreak of ASF in pig population of Meghalaya (Fig 10).

The amplified nucleotide sequence from the 478 bp region of p72 gene was analysed and compared with previously defined 75 reference sequences of major genotypes of ASFV (Table 3), to classify the ASFV from the field outbreaks in Meghalaya, India. The comparison of nucleotide and deduced amino acid sequences of p72 region of ASFV from Meghalaya, India with the reference genotype II sequences from 3 African (AY274455, AY274456 & AF270706), 3 European (JX857521, MK628478, KJ627218), 3 Russian (KJ627212, KJ627208, JX857510) and 24 Asian isolates including isolates from recent outbreaks from India and China revealed 100% homology. Comparison of the genotype II sequences with other genotypes revealed highest sequence homology of 99.01% with the genotype I. Phylogenetic tree based on the same region of p72 gene grouped the sequences from Meghalaya, India along with the 33 reference sequences closely in a single group identifying our sequence in genotype II (Fig 11).

Since time immemorial, pig farming has been a vital part of the lifestyle in North Eastern region of India, which includes a high percentage of tribal people²⁴. Pig farming is a source of income for rural poor from the lowest socio-economic strata, in this region. Pig farming is a significant sub-sector in the state of Meghalaya, with a long history of backyard production and a steadily increasing level of consumption²⁵.

African swine fever is an infectious haemorrhagic disease characterised by high mortality in affected pig population resulting in severe economic losses. The neighbouring countries of India has already faced the catastrophic effects of ASF. The North-East India shares international border with China and Myanmar, which have already witnessed ASF outbreaks in recent years. OIE reported the first outbreak in India on May 21, 2020, from Assam and Arunachal Pradesh states of India¹¹. Later in March 2021, another North-East Indian state, Mizoram also reported its first outbreak of ASF¹². Eventually, in recent months severe mortalities in pig population were recorded from different districts of Meghalaya. The risk factor associated with occurrence of disease in Meghalaya is that it shares border with Assam by which the disease might have spread.

In the present study, 5 districts of Meghalaya were chosen to determine cause of severe mortality in pig population. Out of the 14 pig farms studied, outbreak of ASF was confirmed by PCR in 6 different farms. The clinical signs observed in this study included severe depression, lethargy, cutaneous haemorrhages or purplish discolouration of the skin and high fever with a mortality rate of 90-100%, which are the characteristic features of ASF outbreaks in naïve population^26,27.

ASF affected carcass is characterised by cyanotic skin, cutaneous haemorrhages, bloody discharge from the nose^28,29, as seen during this study. Post mortem examination showed extensive haemorrhages in the visceral organs. Most prominent features included enlarged tarry coloured lymph nodes, enlarged dark spleen, pneumonic lungs, hepatomegaly with distended gall bladder, haemorrhages on the cortical surface of the kidney, hydropericardium and myocarditis. All these findings were very similar to the lesions observed by earlier studies^30,31,32,33.

The histopathological lesions of ASF like, lymphoid depletion, vascular congestion and haemorrhages in lymphoid organs were seen. Microscopic lesions included dilatation of sinusoidal spaces and degenerative changes in liver, haemorrhagic pneumonia in lungs and necrotic changes in kidney parenchyma, which were typical lesions of ASF described earlier^{29,30,32,34,35}.

Tissue samples were also screened for CSF, PRRS, PCV2 and PPV by PCR, to confirm any coinfections. The major capsid protein p72, which is encoded by B646L gene, is the most dominant structural component of the ASF virus³⁶. We have successfully amplified the 478 bp region of P72 gene of ASFV¹⁵ by PCR in tissue samples. Further, the nucleotide and deduced amino acid sequences and phylogenetic analysis of p72 gene confirmed the outbreak of ASF in pig population of Meghalaya and clearly characterized the Meghalaya strain in genotype II (Fig 11).

Early disease detection is the key to maintain a sound animal health and to adopt the most effective strategy to control ASF. A strategic research plan on surveillance and sero-epidemiology of ASF on domestic and wild pig population in the state will help to implement immediate containment measures to prevent further spread of the disease. Studies on the involvement of biological vectors in the perpetuation and transmission of ASFV are also important to estimate the risks more accurately in the Indian scenario.

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