This study mainly described the overall morbidity, pathomorphological features, molecular detection and phylogenetic analysis of LSD in cattle of organized and backyard dairy cattle in the west coastal region of India. The overall morbidity of LSDV infection in cattle was 4.48% with no mortality. It was observed that the backyard farms were more affected due to lack of adequate floor space to keep the affected animals isolated, lack of timely diagnosis and lack of awareness about the disease, presence of stray cattle around and grazing practice. Our findings were similar to the outbreak report from Europe, where higher morbidity has been reported in cattle in small farms with fewer animals than in medium and large farms (EFSA, 2017; Sevik & Dogan, 2017).
The pathology investigation in present study revealed that the animals exhibited characteristic lesions of LSD such as fever, cutaneous nodules, lymphadenopathy, pustules on various body parts, lameness, etc., which were described earlier also (Prozesky, & Barnard1982; Abdallah, et al., 2018; Sanz-Bernardo et al., 2020; Sudhakar et al., 2020; Kumar et al., 2021). This study observed morbidity rate of 4.48%, which is in the range of the LSD morbidity rate reported earlier (Sudhakar et al., 2020; Kumar et al., 2021). Higher morbidity rate of 40–75% is also reported by some workers (Babiuk et al., 2008; Tuppurainen&Oura, 2012). The characteristic microscopic lesions of LSD were vasculitis, perivascular infiltration of inflammatory cells around the dermal blood vessels, spongiosis and necrosis (Tageldin et al., 2014; Abdallah et al., 2018; Vaskovic, et al., 2019; Prozesky and Barnard,1982). The characteristic lesions of pox viral infection viz.,vacuolar degeneration of keratinocytes, spongiosis, and intracytoplasmic eosinophilic inclusion bodies were also observed in our study (Body, et al., 2012; Tageldin, et al., 2014; Sanz-Bernardo et al., 2020).
Three major genes namely, capri pox specific -P32 and LSD specific fusion protein gene (F) and RPO30 were targeted for PCR amplification to detect the LSDV nucleic acid. The P32 gene encodes envelope protein which is homologous to vaccinia virus H3L gene located on the membrane surface of a mature virion (Tulman et al., 2001). Several studies demonstrated that the P32 gene is highly conserved among capripox viruses and therefore it has been used as a diagnostic tool for the detection of LSDV (Tuppurainen et al. 2005; El-Kholy et al. 2008; Shooshtari et al., 2009), SPPV and GTPV (Shooshtari et al., 2009). Hosamani et al. (2006) carried out the differentiation of SPPV, GTPV and LSDV by sequence analysis of the P32 gene. Sudhakar et al (2020) reported PCR based diagnosis of LSD targeting P32, F and RPO30 genes of LSDV in DNA isolated from blood and skin biopsies of cattle.
LSD is an emerging transboundary viral disease for India as neighboring countries such as China and Bangladesh have also evidenced its impact during the same time and there is always a risk of spread of diseases across the borders due to uncontrolled movement of animals, animal products and presence of porous boundaries (EFSA 2020; Sudhakar et al., 2020). After the report of the first outbreak of LSD in Odisha state in the west coast region of India in August 2019, many states situated in the southern region of India witnessed large outbreaks. Although LSDV, SPPV and GTPV have high antigenic similarity and genetic identity, genome sequence analyses have shown that, they are phylogenetically distinct (Le Goff et al., 2009; Lamien, et al., 2011). In this study, LSDV isolate LSDV/CCARI-Goa1/India/2021 (Acce.No.MW590715) was distinctly different from SPPV and GTPV isolates from India and showed highest similarity with Kenyan, Bangladesh and other Indian LSD isolates. Phylogenetic analysis of RNA polymerase subunit (RPO30) gene showed clustering of LSDV, GPTV & SPPV into three different clusters and our isolate exhibited close relation with LSDV isolates of India, Kenya, China and Bangladesh. In comparison with other Indian isolates, our isolate showed similarity with the isolates of Odisha and Haryana states. RPO30 gene have also been used to differentiate lumpy skin disease virus and sheep poxvirus field and vaccinal strains (Rouby, 2018).
In addition to the diagnostic application of real time PCR in LSDV infection, it has been highly recommended for the differentiation of wild-type LSDV and vaccine virus strains (Vidanovic et al., 2016; Pestova et al., 2018). Real time PCR is significantly recognized as a sensitive technique than traditional gel-based PCR in contrast with detection and quantification of the LSD viral gene (Zeynalova et al., 2016; Pestova et al., 2018). In this study, gel-based PCR could not detect LSDV genome in blood samples, however, few blood samples were positive in TaqManTM Probe-based real time PCR assay. As per earlier studies, samples collected during active viraemic phase of infection were positive for viral genome either by gel PCR or Real time PCR (Babiuk et al., 2008; Sudhakar et al., 2020).