Virological, Molecular and Immuno-Biochemical Studies of Lumpy Skin Disease in Naturally Infected Cattle

This study aimed to diagnose Lumpy skin desease by isolation and identification of LSD from suspected naturally infected cattle and to study its effect on immuno-biochemical parameters, inflammatory and oxidative stress markers. The study was conducted on 25 cattle, 1-3 years age from two cities in the Sharkia governorate. Ten clinically healthy cattle represented the control group and fifteen suspected diseased cases by LSD represented the infected group. The clinical signs of suspected cases showed anorexia, skin nodules all over the body, fever above 40 ̊C, edema in forelimbs and dewlap, enlarged superficial lymph nodes, retarded milk production, and abortion in some cases. The virological examination of LSDV from the samples of skin nodules was done. Isolation from skin nodules was done on MDBK cell line and embryonated chiken egg through chorioallantoic membrane route followed by identification using fluorescence technique . Molecular studies including phylogenetic analysis for three positive isolates showed similarity to previously locally isolated strains in Egypt and close relation to strains of SPPV. Immunobiochemical results revealed a significant increase in aspartate aminotransferase , alanine aminotransferase , alkaline phosphatase , Malondialdehyde , Tumor necrosis factor , Interleukin4,immunoglobulin M and immunoglobulin G . Meanwhile, a significant decrease in total protein, albumin, globulin, Glutathione peroxidase, phagocytic assay, calcium, inorganic phosphorous, sodium, and potassium with a nonsignificant change in magnesium was seen when compared to control ones. It could be concluded that the infection with LSD in cattle induce severe effects on immuno-biochemical, antioxidant parameters, and inflammatory markers causing great economic loss ـــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
Lumpy skin disease virus (LSDV), sheep pox and goat poxviruses are members of the genus Capripoxvirus, within the chordo-poxvirinae subfamily of Poxviridae. The genomic sequence of LSDV is about 151-kbp in length.It consists of a central coding region bounded by identical 2.4 kbp-inverted terminal repeats It contains 156 genes. Genomic comparisons revealed that LSDV is closely related to other members of the Chordopoxvirinae, it, however, contains a unique genes responsible for viral host range and virulence (Tulman et al., 2001). LSDV is the etiologic agent of an economically important disease of cattle in the Middle East and Africa (Fenner, 1996).
Lumpy skin desease (LSD) is an acute, subacute, or unapparent viral disease of cattle and occasionally buffaloes. The acute disease is characterized by pyrexia, generalized skin and internal pox lesions, and generalized lymphadenopathy, orchitis, and mastitis (Hamouda et al., 2002, Brenner et al., 2006and Tuppurainen and Oura, 2012. It causes high economic losses, characteristic skin nodules, fever, and emaciation (Ebtsam et al., 2012). The disease considered a ( list A) diseases by the office international des Epizooties (OIE) due to its potential for rapid spread and ability to cause severe significant economic losses, as to hide damage, Loss of milk production, mastitis, infertility, and death (Younis and Aboul souad, 2005 and OIE 2006). In Egypt, LSDV was first isolated and identified from cattle during two outbreaks in Suez and Ismalia governorates during 1989 (House et al 1990 andDavies, 1991).
LSDV exists in skin nodules, crusts of skin lesions, blood, saliva, nasal discharge, semen, and milk of the affected cattle (Babiuk et al., 2008). LSD is primarily spread among animals by biting insects such as mosquitoes and biting flies (lubinga et al., 2014). Less commonly, the virus may be spread by direct contact to the skin lesion, saliva, nasal discharge, milk, or semen of infected animals (Coetzer, 2004).
In Sharkia province during the summer of 2016 , cases of LSD have been observed in cattle previously vaccinated with the Romanian sheep poxvirus (SPPV) vaccine. Phylogenetic analysis revealed that the LSDV isolates were clustered together with other African and European LSDV strains. Also, the LSDV isolates have a unique signature of LSDVs (A11, T12, T34, S99, and P199). ( Fatma et al., 2018).
LSD control through vaccination may be endangered by improper use of vaccines and partial protection of some LSD vaccines (Gelaye et al., 2015) . So the need for genetic characterization of LSDV during outbreaks is important to understand the genetic variation of field isolates(Sylvester ochwo et al., 2020) .On the other hand, abused vaccines bringing may lead to co-infection and recombination of vaccine strains with virulent strains , resulting in virulent reversal of vaccine strains, which may lead to more outbreaks (Sprygin et al., 2018).
This study aimed to diagnose Lumpy skin disease at the molecular level and to study its effect on some immuno-biochemical, oxidative stress parameters, and inflammatory markers in naturally infected cattle.

1.Animal
Twenty five cattle from Belbes & Dyarb Ngm cities in Sharkia governorate at Arab Republic of Egypt(ARE)was used. All cattles aged between 1-3 years old not vaccinated against LSD and were free from external, internal, and blood parasites. The animals were divided into ten clinically healthy cattle without LSD reports used as a control group. And Fifteen suspected infected cattle showing clinical signs were used as an infected group.

Samples
Fifteen skin nodules were collected from suspected infected cases with LSDV from farms during the period between 2018-2019 for virus isolation and identification. Whole blood samples with and without anticoagulants were taken from the jugular vein of healthy and ill cattle for serum separation for immunobiochemical study and phagocytic activity .

Isolation and identification 3.1. Skin nodules preparation
The nodules were incised using a sterile scalpel and the lesions were ground with sterile sand and PBS containing 1000IU/ml penicillin;1mg /ml streptomycin and 100IU\ml Mycostatin in 10%suspension.

Virus isolation 3.2.1.Tissue culture
Samples from Skin nodules prepared in 10% suspension of maintenance media(Dulbecco' s Modified Eagle' s Medium (DMEM)) supplemented with antibiotics ) were inoculated in tissue culture flask 25ml of MDBK cell line. The media used for the growth of MDBK cell line was Dulbecco' s Modified Eagle' s Medium (DMEM) supplemented with antibiotics and 10% foetal calf serum.
The flask is examined daily for 7-14 days for evidence of cytopathic effect (CPE). Contaminated flasks should be discarded. If no CPE is apparent by day 14, the culture should be freeze-thawed three times, and clarified supernatant inoculated on to fresh culture. (OIE terrestrial manual 2010).

Chorioallantoic membrane(CAM)
Skin nodules sample suspension was inoculated in fertile egg via chorioallantoic membrane according to (Van Rooyen et al., 1969). After incubation CAM was harvested, washed in PBS and examined for pock lesion. The positive sample was stored in freeze to be used in Indirect florescent antibodies technique LSDV antiserum: locally prepared in rabbit.Antisera was prepared from previously isolated identified virus.Inactivated virus mixture with incomplete freunds adjuvant was inoculated into rabbits at 0,15,30,45,60 and 90 days respectively according to the protocol of ( Hussein., 2004)

Indirect fluorescent antibody technique
MDBK was grown in Leighton tube containing coverslip. After 24h, the media was discarded and 50µl was inoculated on the coverslip. The inoculated tube was incubated for 1h at 37°c for adsorption time. The maintenance media was added and incubation for farther 24-36h at Co2 incubator (nuaire). The coverslip was taken, air-dried, and fixed in cold acetone for 1h. Also frozen sections of the pock lesion of the CAM were cut using a cryostat, then fixed on a slide using cold acetone. The samples were treated with lumpy skin virus hyperimmune serum prepared in a rabbit and incubate at 37°c for 1h,then wash 3 times with PBS and leave to dry. Add the second anti-rabbit serum conjugate. And further incubation at 37°c for 1h followed by washing 3 times with PBS and leave to dry. Then all samples including negative control were examined using a fluorescent microscope (

. PCR amplification
Primers were utilized in a 25-µl reaction containing 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20pmol concentration, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an applied bio-system 2720 thermal cycler. The primary denaturation step was done at 95 OC for 5 min, followed by 35 cycles of 94⁰C for 30 sec., (annealing 52C⁰ for 40 sec. ) and extension on 72C⁰ for 40 sec. A final extension step was done at 72⁰C for 10 min.

4.2.2Analysis of the PCR Products
The PCR products were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 15 µl of the products were loaded in each gel slot. A gel pilot 100 bp DNA ladder (Qiagen, Germany, GmbH) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

Nucleotide sequencing
The sequencing of the product was done by genetic analyzer 3500(life technology)using a big dye terminator V3.1sequencing kit. The analysis is done by Bioedit and CLC's main workbench 6 software. Inflammatory cytokines TNF -α, IL-4 was measured by using commercial ELISA kits used by method described in standard kits from bio source and ALPCO diagnosis. Further more immunoglobulins titer of both IgG & IgM measured by sandwich ELISA (Erhard et al.,  1992).

Statistical analysis
The obtained data were statistically analyzed by using the T-test (Tamhan and Dunlop, 2000).

RESULTS
The cattle affected by Lumpy skin disease show signs of anorexia, skin nodules, fever above 40˚C, edema in forelimbs, and dewlap with a decrease in the milk production, lacrimation, nasal discharge, and enlarged superficial lymph node (Figs. 1-4). Tissue culture MDBK Infected cells develop a characteristic cytopathic effect CPE consisting of retraction of the cell membrane from surrounding cells, and eventually rounding of cells and margination of the nuclear chromatin . At first, only small areas of CPE can be seen, sometimes as soon as 2 days after infection; over the following 4-6 days, these expand to involve the whole-cell sheet . CPE appeared on inoculated MDBK cell line after 72 h of inoculation(figs.5-6) . Isolated LSDV on tissue culture and frozen sections from positive CAM( fig.9) were identified by the fluorescent antibody technique (Figs.7-8-10-11). Molecular studies included PCR . Analysis of PCR products in the 2% gel as shown in Figure 12 .Results were visualized under ultraviolet (UV) light and images documented using a gel documentation system. Phylogenetic analysis for three positive isolates (Figs.13-14) showed similarity to previously locally isolated strains in Egypt and close relation to strains of SPPV.
who stated that LSD infected cattle characterized by fever, nodules on the skin, edema, and enlargement of superficial lymph nodes. LSDV contains 156 ORFs which have been annotated here as putative genes. These genes represent a 95% coding density and encode proteins of 53 to 2,025 amino acids (Tulman et al., 2001).
The analysis gave a good image to illustrate that our newly isolated strains were similar to previously locally isolated strains in Egypt (accession number MK342936.1 and MK342935.1).Those two previously mentioned registered isolates at gene bank were isolated in 2018 from sheep.
The great similarity between the LSDV isolates and the field SPV isolates indicates that field skin isolate of SPV may be LSDV that changed its host to infect sheep. (El-Kenawy and El-Tholoth 2010). Their results agree with an earlier report (Tulman, et al., 2002) that found sheep and goat poxvirus is probably derived from a common ancestor LSDV, This point needs further study on the whole virus genome.
Although there is some strong evidence suggesting that these viruses emerged thousands of years ago, their genome has evolved through the gain and loss of genes, especially through gene duplication and horizontal gene transfer (  Many of the genes present in the poxvirus genome are not essential to viral replication in cell culture but are important to the modulation of the host antiviral response, and thus are considered virulence genes (McFadden, 2005, and Haller, et al., 2014). The evolution of virulence genes for a certain host is probably influenced by the effectiveness and route of virus transmission, the immune status of the host species, the availability of additional reservoir hosts, intra-and inter-species competition with other viruses. And positive selection for hosts that are more resistant to virus infection (Hughes, et al., 2010;Ebtisam et al., 2012 andHaller, et al., 2014) and according to that our study may give a prediction for the identification of lumpy skin disease virus in Egypt. The significant decrease in T.P and albumin in the infected group may be due to anorexia, off food, and damage of the liver by the virus as the synthesis of albumin occurs in the liver. These results agree with (Hassan et al., 2011) who reported that Hypoproteinemia and hypoalbuminemia result in LSD cattle may be due to decreased synthesis and higher catabolic rate of protein as well as damaged of liver parenchyma.
The infected LSD cattle showed a significant decrease in serum calcium, inorganic phosphorous, sodium, and potassium. This adverse effect may be from decrease food intake and malnutrition and fever with subsequent disturbance of all metabolic processes (Rosby et al., 1991, Ahmed 2007and Hassan et al., 2011. Hypocalcemia could be attributed to hypoproteinemia which result in a decrease of protein bounded calcium (Stockholm and Scott, 2008). Furthermore mentioned that changes in the trace elements may be related to a decrease in food consumption or to hypoproteinaemia which hinders its absorption (Ahmed, 2007 ). This results agree with Helal et al., (2019) who stated that serum calcium and sodium concentrations were significantly decreased in LSD infected cattle. Another explanation of significant decrease in mineral concentration could be attributed to two main factors, decreased synthesis and higher catabolic rate as well as damaged liver parenchyma. Moreover, the infection was considered as a sort of stress on animals and is associated with the increased level of disturbed oxidant/antioxidant status in the body (Ahmed 2007 and Essayed et al., 2016).
The effect of LSD on both antioxidant and inflammatory markers in cattle revealed a significant increase in MDA with a significant decrease in GPx in infected animals compared to the control group. These changes may be attributed to increasing the oxidative stress due to free radical production and lipid peroxidation with the exhaustion of antioxidants in the blood resulting from tissue injury Nashwa et al., there was a significant increase in IL4and TNFα concentration and this may be due to the stimulation of macrophages and lymphocytes to release cytokines that initiate inflammation in different tissue during the viraemic stage of the disease(Shefaa and Rasha 2018).
Our immunological results revealed a significant increase in IgG and IgM with a significant decrease in phagocytic % and phagocytic index in infected cattle compared to the control group. The increase in immunoglobulin may be due to the immune response of the body following viral infection. These . On the other hand,Serological assessment of antibodies to a capripox virus may sometimes be difficult due to the cross-reactivity encountered with other poxviruses as well as to the low antibody titers elicited in some animals following mild infection or vaccination Kitching and Hammond, (1992).
Also there was significant decrease in phagocytic % and index in this study may be due to the stress of infection that decreases cellular immunity as mentioned by Neamat-Allah (2015). Also due to viral infection inhibits macrophage phagocytosis as lowering the mediator's factors such as opsonins and cytokines, which attract and activate leukocytes. The degree of phagocytic activity of macrophages and neutrophils is impaired led to decrease phagocytic uptake of particles, their phagocytic rates, and intracellular killing activities (Murphy et al., 1999).

CONCLUSION
It can be concluded that, lumpy skin disease in cattle induces adverse effects on immuno-biochemical, antioxidant parameters, and inflammatory markers. That causes great economic loss in the animal industry.

Declaration of Competing interest
On behalf of all authors, I hereby declare that no conflict of interest may interfere with the publication of the manuscript.