Epidemiological, Clinico-haemato-biochemical and Molecular Characterization of Lumpy Skin Disease in Cattle

Lumpy skin disease (LSD) is responsible for huge economic losses due to reduced milk production, degradation of hide, abortion, temporary or permanent sterility of bulls and cows and death. Indirect losses comprised of costs for laboratory diagnosis, treatment, vaccination, disinfection of premises and restrictions on foreign trades. LSD has recently spread beyond its territory into Europe and Asian countries (Tuppurainen et al., 2015 and Ince et al., 2016). In Asia, India, China and Bangladesh have reported the first LSD outbreaks during 2019 (Sudhakar et al., 2020). Huge mortality has been seen in cattle due to LSD in the year 2022 in India.
       
Lumpy skin disease is highly contagious in nature. LSD virus (LSDV) is highly host specific. LSD is transmitted predominantly by arthropod vectors like mosquito, biting flies and ticks mechanically (Lubinga et al., 2015). However, transmission through common grazing, drinking troughs and contaminated fomites is also observed.
       
The clinical entity, morbidity and mortality of the disease vary considerably, depending on the breed, immune status of the cattle population, virus strain and prevalence of the insect vectors (Tuppurainen et al., 2017) therefore it is very difficult to diagnose the disease in early stages. Hence present investigation was undertaken for epidemiological, clinical and molecular characterization of LSD in cattle.

Study population
 
Total 308 cattle were screened for skin diseases from various organized and unorganized farms for a period of 12 months (October 2021 to September 2022) in Rajasthan state in India. Cattle of different breed, age and either sex were examined. Ten apparently healthy cattle (n=10) were selected to have base line data on basic clinical and haemato-biochemical parameters for the comparison and analysis.
 
Clinical examination
 
Each animal was subjected to thorough physical and clinical examination as per the methods described by Radostitis et al., (2007). Entire skin coat of all the animals was examined thoroughly in the day light for lesions. Visible mucous membranes, eyes etc. were also examined and relevant clinical data was recorded.
 
Collection of skin nodular scab samples and DNA extraction
 
Skin nodular scab samples were collected aseptically and placed in clean sterile tube containing phosphate buffer saline for molecular detection of viral DNA. DNA extraction from skin scab homogenates was done using HiPurA viral DNA Purification Kit (HigenoMB Himedia) following the manufacturer’s instructions. Concentration and purity of DNA were estimated in UV absorbance spectrophotometer (Eppendorf, Hamburg, Germany).
 
PCR Reaction for molecular characterization
 
Convention PCR was carried out to find out the presence of LSDV specific nucleic acid through amplification of 192 bp region in the P³² gene as described by Ireland and Binepal (1998).
Forward primer: 5' -TTTCCTGATTTTTCTTACTAT-3' 
Reverse primer: 5' -AAATTATATACGTAAATAAC-3'
       
The PCR reaction was set up in a final volume of 25 µl containing: 12.5 µl of 2X Master mix (Genetix Biotech Asia Pvt. Ltd., New Delhi, India) 0.75 µl of each 50 pmol/µlprimer concentration 8.5 µl of nuclease free water (Genetix Biotech Asia Pvt. Ltd., New Delhi, India) 2.5 µl of extracted DNA (Template DNA).
       
The PCR reaction was carried out in a Bio-Rad T100, Thermo Cycler system (Bio-Rad Pvt. Ltd., California,U S.A.). The PCR conditions had an initial denaturation step of 94°C for 5 minutes, followed by 34 cycles of denaturation at 94°C for one minute, annealing at 50°C for 30seconds, extension at 72°C for 1minute and a final extension step of 72°C for 5 minutes.
       
The PCR product was subjected to electrophoresis in agarose gel containing ethidium bromide for assessing their integrity. 5 µl of PCR product was mixed with 2 μl of 6X gel loading dye (Thermo Scientific) and was loaded in the wells within agarose gel. To determine the size of the amplified PCR products, 5 µl of Gene Ruler 100 bp DNA ladder (Thermo Scientific) was loaded in first well. Gel electrophoresis was allowed to run for 1 hour at a constant voltage of 70V till the dye migrated 3/4^th distance in the agarose gel. The amplified PCR products were visualized under gel documentation system (Biogen Scientific, Cambridge, U.S.A.).
 
Collection and analysis of blood samples
 
Blood samples were collected from LSD affected cattle as well as control animals to determine haemato-biochemical parameters. Haematological parameters viz. Haemoglobin (Hb), packed cell volume (PCV), total erythrocyte count (TEC), total leukocyte count (TLC), platelet count and differential leukocytes count (DLC) were estimated as per the methods described by Feldman et al., (2000).  Biochemical parameters viz. serum glucose, serum total protein (TP), serum albumin, serum globulin, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALKP), creatinine, blood urea nitrogen (BUN) and total bilirubin were estimated by using automated serum biochemistry analyzer (IDEXX Vet test chemistry analyzer).
 
Statistical analysis
 
The data obtained in the research work were statistically analysed and compared using standard formula given for mean and standard error as per statistical methods described by Snedecor and Cochran (1996).

Overall prevalence
 
Out of total 308 cattle screened under present study; 18 animals were found affected with LSD. The overall prevalence of LSD in cattle was 5.84 per cent. The cases were further confirmed by molecular detection of P³² gene of LSDV. Present finding is in agreement with that of Sudhakar et al., (2020) and Nayakwadi et al., (2021).
       
The variation in prevalence of lumpy skin disease might be attributed to difference in geographical location, climate and managemental practices (Ramesh, 2014 and Jubara et al., 2019).
 
Age group-wise prevalence
 
Age group-wise prevalence of LSD in cattle was 3.27 per cent, 4.9 per cent and 7.8 per cent in cattle below 6 months of age, 6 months to 3 years of age group and cattle above 3 years of age, respectively. Age group-wise percent distribution of LSD was 11.11 per cent in cattle below 6 months of age, 27.78 per cent in age group 6 months to 3 years of age and 61.11 per cent in cattle above 3 years of age. Highest prevalence was observed in cattle above 3 years of age whereas lowest prevalence was observed in calves i.e., below 6 months of age. It was revealed that prevalence of LSD increased with advancement of age in cattle.
       
Similar findings were obtained by Elhaig et al., (2017) and Sahoo (2020). Higher prevalence of LSD seen in adult animals might be due to production stress. It may also be due to less exposure of young calves to disease transmitting vectors like mosquito, biting flies etc.
 
Breed-wise prevalence
 
Breed-wise prevalence of LSD in cattle was 4.3 percent and 8.2 per cent in indigenous and crossbred cattle, respectively. Breed-wise percent distribution of LSD was 44.44 per cent in indigenous breeds (Gir and non-descript) and 55.56 per cent in crossbred cattle. Higher prevalence was observed in crossbred cattle as compared to indigenous breeds i.e., Gir and non-descript. Similar findings were reported by Tageldin et al., (2014) and Sahoo (2020).
 
Sex-wise prevalence
 
Sex-wise prevalence of LSD in cattle was 6.66 and 4.42 per cent in female and male cattle, respectively. Sex-wise percent distribution of LSD was 72.22 and 27.78 per cent in female and male animals, respectively. The prevalence was higher in female cattle as compared to male. Findings of present investigation are in agreement with that of Elhaig et al., (2017) and Sahoo (2020).
       
Higher prevalence rate in female animals might be due to production stress in females.
 
Season-wise prevalence
 
Season-wise prevalence of LSD in cattle was 8.73 and 6.48 per cent in winter and rainy season, respectively. Season-wise per cent distribution of LSD was 61.11 and 38.89 per cent in winter and rainy season, respectively. In present study, no any case of LSD was recorded during summer season. The prevalence was highest in winter season followed by rainy season.
       
The higher prevalence of LSD was recorded during winter season which might be attributed to close confinement of animals during winters leading to easier transmission of disease. Vector population also enhances the prevalence of disease in winter season (Hameed et al., 2017).
 
Clinical signs
 
General signs
 
There was highly significant increase (P<0.01) in the mean values of body temperature and respiration in LSD affected animals as compared to control whereas significant increase (P<0.05) was observed in the mean values of heart rate in LSD affected cattle (Table 2). Similar findings were reported by Sahoo (2020) and Rouby et al., (2021).
       
Fever in LSD affected cattle might be due to viraemia which leads to release of endogenous pyrogens and febrile reaction (Vorster and Mapham, 2008). The increase in heart rate in LSD might be associated with febrile and anaemic condition whereas increased respiration rate could be due to secondary bacterial infection resulting in pneumonia. Respiration rate may also be increased because of fever and brisket oedema.
 
Other clinical signs
 
All the affected cattle (100 per cent) showed presence of nodules over the skin. Enlargement of superficial lymph nodes and elevation of rectal temperature each was recorded in 88.88 per cent animals. Oedema of limbs was observed in 72.22 per cent LSD affected cattle while brisket oedema was found in 38.89 per cent animals. Anorexia was observed in 66.67 per cent cases. Nasal and lacrimal secretions were seen in 61.11 per cent animals. Among all affected cattle, 27.78 per cent exhibited lameness. Pneumonia was recorded in 11.11 per cent cases. Out of total lactating cows affected with LSD, 91.67 per cent cows showed reduced milk production. Kerato-conjunctivitis and corneal opacity each was recorded separately in only 5.55 per cent cows. Abortion was recorded in 14.28 per cent pregnant cattle out of total pregnant cows screened under present investigation (Table 1).
 

       
Presence of nodules over the skin was most consistent clinical finding of LSD in cattle. It was followed by fever and enlarged lymph nodes, oedema of limbs and anorexia. Reduction in milk yield was also commonly observed in lactating cows. However, pneumonia, kerato-conjunctivitis and corneal opacity were recorded with lower occurrence. Occasionally, abortion was also observed. The nodules were distributed all over the body viz. on face, neck, abdomen, legs, udder, perineum and scrotum. In few severe cases, nodules were observed on muzzle, oral and nasal mucosa which later on appeared as ulcerative lesion. The diameter of these nodules varied largely ranging from 0.5 cm to 5.5 cm.
       
Findings of present investigation are in agreement with that of Sahoo (2020) and Gayal et al., (2022).
 
Haematological changes
 
There was highly significant (P<0.01) reduction in the mean value of PCV, Hb, TEC and TLC in LSD affected cattle as compared to control whereas reduction in mean value of platelet count was significant (P<0.05). Further, non-significant variation was observed in the mean values of DLC (Table 2).
 

       
Allam et al., (2021) and Keshta et al., (2020) also found reduced PCV and TEC in LSD affected cattle whereas marked decline in mean Hb and TLC has also been reported by Jalali et al., (2017). El-Mandrawy and Alam (2018) and Xavier et al., (2020) also showed reduction in TLC and platelet count in cattle affected with LSD.
       
Decreased PCV, Hb and TEC amy be the result of anorexia and destruction of RBCs. Increased production of reactive oxygen species has adverse effect on membrane and macromolecules of erythrocytes (Nashwa et al., 2017). Thrombocytopenia may be a result of lower production of platelets in bone marrow or sequestration of platelets. Leukopenia in acute viral infection could be attributed to increased tissue demand and neutrophilic margination.
 
Biochemical changes
 
There was highly significant increase (P<0.01) in the mean value of serum TP, serum globulin, ALT, AST, ALKP, BUN and total bilirubin in LSD affected cattle as compared to control group whereas mean value of serum albumin was significantly decreased (P<0.01). Non-significant variation was observed in the mean values of serum creatinine and serum glucose (Table 2).
       
Similar findings were reported by Jalali et al., (2017), El-Mandrawy and Alam (2018), Keshta et al., (2020) Rouby et al., (2021) and Allam et al., (2021).
       
Lower serum albumin in LSD affected cattle might be due to poor nutritional status as a consequence of anorexia whereas serum globulin level is associated with immune response of infected cattle. Increase in hepatic serum enzymes is attributed to virus induced degenerative changes. The increase in serum ALKP might be due to injuries in cardiac and skeletal muscles as a result of degenerative changes in heart, muscles, lymph nodes and skin (Jalali et al., 2017). The increased level of total bilirubin is attributed to hepatocyte and bile duct damage due to vasculitis and vascular thrombosis (Tageldin et al., 2014). Increased level of BUN could be attributed to the effect of LSDV on renal tissues resulted in renal damage or reduction of glomerular filtration rate and protein catabolism (Neamat-Allah, 2015).
 
Molecular characterization of lumpy skin disease
 
In present study, 18 skin scab samples were aseptically collected from the cattle showing characteristic clinical signs of lumpy skin disease infection. DNA was extracted from the collected samples. All the 18 collected samples were processed through conventional PCR, targeting the viral attachment protein encoding P32 gene of lumpy skin disease virus. The expected amplicon size (192 bp) was found in all examined samples (Plate 1). All LSDV positive samples had a band size of 192bp. All the skin scab samples (100 per cent) were positive for the PCR assay (Plate 1).
 

       
The findings of present investigation are in agreement with that of Awad et al., (2010), Ün et al., (2019) and Allam et al., (2021). They reported 100 per cent positive results in molecular detection of lumpy skin disease virus using PCR assay for skin biopsy samples from clinically infected cows.

LSD was first time reported in Rajasthan, India. There were variations in the prevalence of LSD with age, breed, sex and season. Molecular characterization revealed a band size of 192bp in all LSDV positive samples. Marked alterations were observed in haemato-biochemical parameters in LSD affected cattle. Estimation of clinico-haemato-biochemical parameters will pave way in better understanding the pathogenesis and deciding the suitable therapy.
 

There is no conflict of interest.