3.5 Detection of LSDV by TaqManTM probe:
The TaqMan™ probe real time PCR assay detected LSDV in 62 skin biopsy
samples (94%), 2/66 blood samples and 3/5 ocular swab samples, which
proved that this assay could be very sensitive and rapid in the
detection of LSDV from field outbreaks (Table 4) (Fig. 2C) .
Average threshold cycle (Ct) values were 15.2±11.2 in tissue biopsy
samples, 29.4±11.6 in blood and 25.6±10.9 in ocular swabs.
Discussion
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 infectionviz., 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 was 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).
Conclusion
This study was primarily focused on the prevalence of LSDV infection in
small and organized farms, its pathological features and molecular
detection using gel-based and real-time PCR assays. LSDV causes
significant economic losses to dairy cattle and there is an urgent need
to develop diagnostic assays and homologous vaccine for the control of
the disease in the Indian subcontinent.