Lumpy skin disease outbreak in cattle population of Chattogram, Bangladesh

Abstract Background Lumpy skin disease (LSD) is an important viral disease causing significant economic losses in commercial livestock production. In mid‐2019, an outbreak of LSD has been reported in cattle population from different parts of Bangladesh including Chattogram division. A cross‐sectional surveillance study was undertaken from August 2019 to December 2019 to investigate the prevalence and associated risk factors of LSD in cattle in Chattogram district. Methods A total of 3,327 cattle from 19 commercial farms were examined for the LSD specific skin lesions and associated risk factors. A total of 120 skin biopsies were collected from the suspected animal for the confirmation of the disease using molecular detection and histopathological examination. Partial genome sequencing and phylogenetic analyses were performed on selected viral isolates. Results The overall clinical prevalence of LSD in the study population was 10% (95% confidence interval [CI]: 9.4%–11%) where the highest farm level outbreak frequency was 63.33% (95% CI: 45.51%–78.13%) and the lowest 4.22% (95% CI: 3.39%–5.25%). Crossbred and female cattle showed a significantly higher prevalence of the disease compared to their counterparts. Introduction of new animals in farms was found to be one of the most significant risk factors in the transmission of the disease. All suspected skin biopsies were positive for LSD virus (LSDV) infection with granulomatous and pyogranulomatous dermatitis was revealed on histopathology. Phylogenetic analysis based on the inverted terminal repeat region of the LSDV gene suggested that the locally circulating strain was closely related to the strains isolated from the Middle East and North African countries. Conclusions The data generated in this study would be beneficial to the field veterinarians and animal health decision makers in the country as well as it will aid in taking appropriate measures to prevent further relapse or outbreak of this disease in future.


| INTRODUC TI ON
Lumpy skin disease (LSD) is a viral disease caused by LSD virus (LSDV) that belongs to the family Poxviridae and genus Capripoxvirus. The disease affects a wide range of domestic animals including cattle, buffalo, sheep and goats (Alkhamis & VanderWaal, 2016;El-Nahas et al., 2011), and the main symptoms are fever and nodular lesions on the skin, mucous membrane of respiratory and digestive tracts (Coetzer & Tuppurainen, 2004). The World Organization for Animal Health (OIE) included the disease in notifiable transboundary disease list due to its substantial economic losses in terms of reduced productivity, poor hide quality, poor growth rate, infertility and even death (Anonymous, 2021;Tuppurainen et al., 2017;Tuppurainen & Oura, 2012). LSDV is believed to be transmitted mainly biting arthropods such as mosquitoes, flies and ticks (Magori-Cohen et al., 2012). Higher incidence of this disease is observed in crossbred young animals with communal grazing and during the wet season when the arthropods vectors are abundant. Introduction of new animals is another important risk factor (Al Rammahi & Jassim, 2015;Alemayehu et al., 2013;Chihota et al., 2001;El-khabaz, 2014;Kiplagat et al., 2020;Ochwo et al., 2019). Zambia is the first country where LSD was first identified in 1929 that was followed by many African and Middle Eastern countries (Kasem et al., 2018). Although many countries have experienced the outbreak of LSD, but which strain or variants will be the perfect match for vaccine production is remained as a debatable issue (Ayelet et al., 2013;Ben-Gera et al., 2015). Some recent articles claimed the potentiality of vaccine candidates for LSD prevention (Klement et al., 2020;Wolff et al., 2020;Zhugunissov et al., 2020). In contrast, vaccine strains were also found during the disease outbreak in Russia which raised further doubts on the vaccine candidate and its efficacy (Kononov et al., 2019).
In Bangladesh, an outbreak of an unknown syndrome with nodular skin lesions was reported by local veterinary services authority in mid-2019 in commercial and backyard cattle population in some locations (Anwara, Karnaphuli and Patiya) of Chattogram district (Anonymous, 2019). Same pattern of clinical onset was reported later in different districts of the country (Giasuddin et al., 2020;Khalil et al., 2021). The outbreak report was preliminary confirmed based on clinical signs and later using the reverse transcription polymerase chain reaction (RT-PCR) test by the Department of Livestock Services (DLS), Bangladesh and notified the disease as LSD to OIE in August, 2019 (Anonymous, 2019). Therefore, a cross-sectional surveillance study was undertaken on clinically suspected LSD cases throughout Chattogram district; the south-eastern part of Bangladesh. The aim of the present study was to confirm the disease occurrence based on clinical, molecular and pathological identification and unveiled the plausible risk factors of LSDV infection in this region. We further analysed the sequence data of the circulating LSDV strains to identify the probable geographical origin of this strain.

| Study design
The study was conducted over a period of 5 months (August to December 2019) in Chattogram District of Bangladesh at the onset of a lumpy skin disease outbreak. A cross-sectional study was designed to collect the samples, and individual animal was considered as the sampling unit. A standard questionnaire was used to collect demographic data such as breed, age, sex and other data (e.g., introduction of new animals, source of water supply in the farm, etc.  . Selection of study areas and animals were based on the suspected cases reported by the local veterinarians and physical visit to the farms. A case was considered positive for LSD when an animal showed two or more of the following signs such as nodular characteristic lesions on the skin, fever, lameness, lymphadenopathy, edema and decreased production (e.g., reduction of milk yield) (Magori-Cohen et al., 2012). A total of 19 commercial farms from Chattogram district (six farms from Pahartali area, three farms from Sitakunda and two farms from each of Chattogram Port, Double Mooring, Hathazari, Panchlaish and Chadgaon area) were selected ( Figure 2). Farms comprising less than 15 cattle were excluded from the present survey . Sample from affected animals was collected from the individual farms using a simple randomization technique. A farm was considered positive for ectoparasites (flies, ticks, lice) when at least one animal was infested by one of these parasites.

| Sample collection and preservation
A total of 19 farms having 3,327 animals were considered consisting of 669 calves, 281 heifers, 2,272 cows and 105 bulls. Data were collected by face to face interview of the animal attendants of the particular farm and physical examination of the cattle. Among the diseased or suspected cattle (Figure 1a,b), a total of 120 skin biopsies from nodular lesions were collected aseptically using punch biopsy techniques (Kasem et al., 2018). Briefly, the biopsy site was shaved by the sterile blades, and a small punch was taken deeply in the skin so that all layers along with the subcutaneous tissue were collected. Half of the skin biopsy specimen was kept in neutral buffered formalin (10%) for histological examination following conventional haematoxylin and eosin (H&E) staining (Fischer et al., 2008).
The rest half of the skin biopsy samples were preserved in −20°C for molecular confirmation of the infection.

| DNA extraction and PCR presence of LSDV
Total genomic DNA was extracted from all suspected skin biopsies using commercially available kits following manufacturer's instruction with some modifications (DNeasy Blood & Tissue Kits®). PCR was then performed to confirm the presence of LSDV using a set of published primers (forward; GTGGAAGCCAATTAAGTAGA and reverse; GTAAGAGGGACATTAGTTCT) targeting the inverted repeat region (ITR) of the genome (Stram et al., 2008). In brief, PCR reactions were set up in 50μl final volumes containing 25μl master mix, 2.5μl forward primer, 2.5μl reverse primer, 5.0μl DNA template and 15μl nuclease free water. The PCR conditions were follows as an initial denaturation step of 95°C for 1 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 49°C for 30 s, extension at 72°C for 70 s and a final extension step at 72°C for 5 min. Then, 5μl of amplified amplicons were taken and stained using 0.05% ethidium bromide (Sigma-Aldrich ® ) and a visualization of the band (1,237 bp) after agarose gel (1%) electrophoresis.

| Nucleotide sequencing and phylogenetic analysis
Four randomly selected LSDV PCR amplicons were gel purified using Wizard® SV Gel and PCR Clean-Up System (Promega) and sequenced by sanger dideoxy sequencing (Macrogen ® ). The sequence read data were then manually cleaned up using the chromatogram software (Geneious Prime version 2020) and deposited in GenBank. NCBI BLAST was performed for each of the sequence reads and the ITR from a diverse range related LSDV and other poxvirus genome sequences (Nt sequence identity 100%-70%) were retrieved (N = 63) and aligned with MAFFT v7.017 using G-INS-i (gap open penalty 1.53; offset value 0.123) alignment algorithm (Katoh et al., 2002). jModelTest program 2.1.3 favoured a generaltime-reversible model with gamma distribution rate variation and a proportion of invariable sites (GTR + I + G4) for the phylogeny (Darriba et al., 2012). Maximum likelihood (ML) phylogenetic trees was reconstructed using the program PhyML v3.1 (Guindon & Gascuel, 2003), and FigTree v1.4 was used to generate the consensus tree (Smith et al., 2009). The proportion of bootstrap support (%) was showed in each branch while multiple taxa showing polytomy, and closely related isolates were collapsed for better visualization.

| Statistical analysis
All data were inserted and coded in Microsoft office Excel 2016 spreadsheet, and both univariable and multivariable analyses were performed using generalized linear mixed models in STATA-IC 13.
Farm was included in the model as random effect. Backward elimination procedure was followed, and a p value ≤ 0.05 was considered significant in both univariable and multivariable analyses. Prevalence map along with location and size of the farms was created using QGIS 3.12.0.

| Clinical prevalence of LSD
The overall clinical prevalence of LSD was 10% (95% CI: 9.4%-11%) in the study farms. The farm level highest frequency was 63.33% in one of the farms located in Chadgaon region, and the lowest was 4.22% in a farm at Sitakunda region of Chattogram District (Figure 2).
The clinical prevalence ranges from 20% to 40% in five farms of

| Risk factors associated with the occurrence of LSD
The clinical prevalence was observed the lowest in bulls (5%) (95% CI: 2%-10%). Univariable analysis showed that odds ratio (OR) of having the disease in calves, cows and heifers were 1.37 (CI: 0.53-3.55), 2.52 (CI: 1.02-6.26) and 3.51(CI: 1.35-9.14) times higher compared to bulls, respectively (Table 1). Females were in higher risk (OR = 2.26, CI: 1.28-4.0) than males. In terms of lactation, with increasing lactation number decrease in prevalence was observed; odds of having the disease in first lactation was 7 times higher compared to fourth lactation. The univariable analysis also showed that local cattle were less susceptible than the crossbred. Besides, introduction of new animals, sources of water supply and floor types (brick or cemented) in the farm act as potential risk factors of the disease (Table 1). In multivariable model, crossbred (p = .0080, OR = 3.58, CI: 1.40-9.17) and female (p ≤ .0001, OR = 3.96, CI: 2.16-7.27) cattle had a significantly higher risk of getting the disease compared to their counterparts (Table 2). support) with sheep pox reference sequence (CAPIS1ITR) (Figure 3).

| Molecular identification of LSDV
On the other hand, Isolate M2 on the other hand showed stronger bootstrap support (80%) towards recent isolates of LSDV from Egypt (KF588352, KR052866 and KF58835). The phylogenetic reconstruction thus reaffirm that the viral isolates from the nodular skin biopsies were LSDV genotypes most closely related to those from Egypt (Figure 3).

| Histological features of the skin biopsies
All cases involved granulomatous and pyogranulomatous dermatitis with multifocal to diffuse deep dermal necrosis and panniculitis ( Figure 4) (Figure 4). Pannicular infarction and subcutaneous vasculitis were present in the samples.  (Gari et al., 2010;Kasem et al., 2018).

| D ISCUSS I ON
Comparatively shorter duration of the actual study period and culling of diseased animals might be a reason for the paucity of mortality. However, clinical form of LSD is generally associated with economic loss in terms of production and treatment expenditure (Babiuk et al., 2008a;2008b).
Risk factors analyses suggests that crossbred cattle were more susceptible to LSD than indigenous cattle which was consistent with the findings of previous studies (Al Rammahi & Jassim, 2015;Kiplagat et al., 2020;Klement et al., 2018). Higher susceptibility of crossbred cattle might be due to lower disease resistance capability in comparison to indigenous breeds (Tageldin et al., 2014).
Further, the higher number of crossbred animals (96.79%) was sampled over local (3.21%) cattle might explain the variation of the results. Heifers were affected largely with LSD in comparison to bulls, calves and cows. In previous studies, a higher morbidity was recorded in younger cattle (<2 years) in Saudi Arabia (Kasem et al., 2018) and calves (0.5-1 year) in Ethiopia (Molla et al., 2018).
This might be due to management system of the farms where heifer was kept in poor hygienic conditions in comparison to other animals (calf, cow or bull). Females were more prone to LSD compared to males which was consistent with previous research (Ayelet et al., 2014;Magori-Cohen et al., 2012;Salib & Osman, 2011). Higher frequency of LSD in female cattle could be due to their exposure to many stress conditions, e.g., pregnancy, parturition and sometimes less amount of feed supplied compared to their actual requipment (Kasem et al., 2018). We observed an inverse relationship with lactation number in the occurrence of LSD in cattle although we were unable to identify the possible reasons. Farm-specific risk factors such as introduction of new animals to the farm demonstrated a significantly higher risk to be infected with the virus or its transmission which was supported by previous findings (Gari et al., 2010;Macpherson, 1994;Munyeme et al., 2008). The PCR-based molecular test targeting ITR region of the LSDV has successfully confirmed all suspected cases of LSD in this study, and the local genotype circulating in Chattogram district was deposited in GenBank as well (Gene bank accession no. MT070969-MT070972). The ML tree reconstructed from the ITR region of all related poxviruses showed that the LSDV strains circulating in Bangladesh are closely related to that in Middle East and North Africa as three out of four sequences had closest phylogenetic relationship with isolates from Egypt. However, the ITR region of the genome used for amplification and sequencing of LSDV is a pseudogene, relatively conserved and homologous to many other poxvirus genomes (Gershoni & Black, 1989). Therefore, the phylogenetic reconstruction had lack of discriminatory resolution, as Further studies should incorporate sequencing at least three different core gene groups along with concatenation or partitioning approach for alignment and subsequent phylogenetic analyses to reconstruct a comprehensive evolutionary tree with better discriminatory power and resolution.  (Khatun et al., 2016).
It is also mentionable that this outbreak was reported just a month after the festival. It is plausible that unregulated and illegal import of live animals without prior health check or quarantine measures have embarked the clinical outbreak of LSD. Unrestricted incountry movements of livestock even after the first reporting might have significantly aggravated the viral transmission (Tuppurainen et al., 2017). However, outbreak of this disease occurred in China and Odisha of India in August 2019 (Anonymous, 2019;Sudhakar et al., 2020), and this could be an unexplored link to this outbreak as cattle movements were speculated as a risk factor (Klausner et al., 2017). Within farm LSDV transmission is further related with the biosecurity measures and other management practices.
We found a positive correlation between the communal water supply as well as the floor made of brick as observed by others (Babiuk et al., 2008a;2008b;Tuppurainen & Oura, 2012 The data generated in this study would be beneficial to the field veterinarians and animal health decision makers in Bangladesh, and also it will aid in taking appropriate measures to prevent further relapse or outbreak of this disease in future.

E TH I C A L A PPROVA L S TATE M E NT
Ethical approval was obtained from the institutional ethical ap-  (13)]. for analyses of the data and review of the manuscript.

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest between the authors.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.524.