Lumpy skin disease (LSD) is a viral disease caused by LSD virus and is one of the most economically significant transboundary and emerging diseases of cattle. LSD causes considerable economic losses due to emaciation, damage to hides, infertility, and loss of milk production. In Ethiopia, the disease is distributed almost in all regions and is regarded as one of the most economically important livestock diseases in the country. An outbreak investigation of the disease was monitored from October 2016 to April 2017 in southern pastoral areas of Bale Zone, Oromia, Ethiopia. In December 2016, LSD outbreak occurred in Sawena district of Bale Zone, from which necessary biopsy samples were collected from actively infected animals for the purpose of virus isolation, and characterization using different molecular techniques at National Animal Health and Diagnostic Investigation Center (NAHDIC) of Sebeta, Ethiopia. In addition, clinical examination of infected and in-contact animals was carried out together with a questionnaire survey. Based on the clinical manifestations, LSD was recorded in 18% (94/522) of examined cattle, whereas biopsy samples from 20 clinically positive animals were collected for further laboratory process. The morbidity rate was higher in animals less than two years 28.97% (31/107) than other ages and showed a statistically significant difference with
In the Greater Horn of Africa (GHA), pastoralists occupy large parts of arid and semiarid lands of Ethiopia, Kenya, Somali, Djibouti, Eritrea, Sudan, Uganda, and Tanzania [
This livestock sector has contributed considerable portion to the economy of the country and is still promising to rally round the economic development of the country. Livestock production remains crucial and represents a major asset among resource-poor small holder farmers by providing milk, meat, skin, and manure and traction force [
In the highlands, livestock are kept under settled or transhumant systems utilizing common pastures, many of which have a high clover content and crop residues. Such livestock include some 9.3 million oxen providing draught power for the mixed farming system that prevails. In the arid and semiarid extensive grazing areas of the eastern, western, and southern lowlands, cattle, sheep, goats, and camels are managed in migratory pastoral production systems [
The Bale pastoralists’ livelihoods depend predominantly on livestock and their products. They practice a transhumance nomadic system, which had been their traditional and primary survival strategy. However, physical infrastructure is poorly developed in areas where pastoralists live. Poor health and productivity of animal due to disease has considerably become the major stumbling block to the potential of livestock industry [
LSD is one of the most economically significant transboundary, emerging viral diseases. It is currently endemic in most Africa countries and expanded to the Middle East region [
The disease is now the problem of almost all the regions and agroecological zones of Ethiopia. A major outbreak of LSD has occurred in different regions of Ethiopia like Amhara and W/Oromia regions in 2000/2001, Oromia and Southern nations’ nationalities and people (SNNP) regions in 2003/2004, and Tigray, Amhara, and Benishangul regions in 2006/2007 [
Hence, it is important to generate information on isolation of LSD from outbreak reported and its molecular characterization. Therefore, the objectives of this study are to investigate the occurrence of LSD outbreak in pastoral areas and to isolate the infectious LSDV from nasal swab and skin lesion samples in Vero cell cultures in the study area.
The outbreak investigation was conducted from November 2016 to April 2017 in Bale Administrative Zone of Oromia Regional State, which is located 430 km southeast from the capital city, Addis Ababa. The town of the Robe Zone is geographically found at 7°7′N 40°0′E with an elevation of 2,492 meters above sea level. Bale Zone of Oromia Regional State was purposively selected for the study based on LSD outbreak report to Yabello Regional Veterinary Laboratory and Zonal pastoral office.
The altitude of the study area ranges from 850 to 2800 m.a.s.l, where the lowland area predominates with a narrow strip of high land area in the northern part of Sawena district (Figure
Map of the study area.
The active outbreak investigation was conducted in extensive management system in local cattle. Cattle that showed clinical signs of pox like skin lesion were targeted for this study. All age and sex groups which are reared under two different production and management systems (small number of animals especially milking cows and calves that are kept around the home and the majority of animals which are driven long distances in search of good pasture and surface water) were involved in the outbreak investigation.
The study areas were selected purposively based on the reports of LSD outbreaks to Yabello Regional Veterinary Laboratory. Active outbreaks were assessed together with veterinary professionals who are working in the regional veterinary laboratories, zonal veterinary office, and district veterinary clinics. Clinical and epidemiological data were recorded and samples were collected for virus isolation and identification that were conducted at Animal Health and Diagnostic Investigation Center (NAHDIC) at Sebeta, Ethiopia.
Additionally, semistructured questionnaires were used to interview 20 pastoralists on LSD occurrence and its associated impacts. The cattle owners expressed their views and shared their practical knowledge about the prevailing situations regarding LSD using their native language (Afan Oromo); among the questions were awareness about clinical signs of LSD, age affected, sex affected, seasons of LSD outbreak, livestock movement, milk reduction, abortion in pregnant cows, presence of death due to the disease, status of vaccination, and water sources for the livestock. The pastoralist called the local name of the diseases “Itesa.” Relevant data were gathered by observing clinically sick animals and interviewing cattle owners and animal health workers working at the field. Information was carefully recorded in a designed format.
During the study period, the total number of animals examined was 522. A field investigation was conducted purposively at the specific site of the outbreak within Bale Zone. Cattle with clear signs and symptoms and suspected to be diseased with LSD were selected to be sampled. Skin nodules from cattle which were with severe clinical signs of the disease were taken aseptically by washing and cleaning the area and removing the hairs with the help of sterile scalpel blade. Nasal swabs were collected with sterile swabs from infected animals.
According to the procedures of OIE [
Skin nodules were taken aseptically by washing and cleaning the area and removing the hairs with the help of sterile scalpel blade and nasal swabs were taken aseptically by cleaning the external part of noses. Tissue samples were placed in the sterilized universal bottle containing virus transport medium and nasal swabs were placed in the sterilized cryovial tubes containing virus transport medium kept at −20°C until transported to National Animal Health Diagnostic and Investigation Center, Sebeta, Ethiopia.
The skin biopsy samples were thawed at room temperature and washed three times in sterile phosphate-buffered saline (PBS, pH 7.2). Approximately 1 g washed tissue sample was mixed with 9 ml sterile PBS and grounded using a sterile mortar and pestle. The tissue suspension was centrifuged at 4000 rpm for 15 min. And the supernatant was filtered through a membrane of pore size 0.45
Approximately 1 ml filtered supernatant was inoculated onto a monolayer of Vero cells in 25 cm2 tissue culture flasks, incubated at 37°C for an hour for adsorption, and then 9 ml Glasgow minimum essential medium (GMEM, Sigma-Aldrich), containing 0.1% gentamicin and 2% fetal calf serum (Sigma-Aldrich), was added. The inoculated flasks were incubated at 37°C in a humidified incubator with 5% CO2. Cells were monitored daily for 14 days, using an inverted microscope, for evidence of virus-induced cytopathic effects (CPEs); finally, cells were frozen at –80°C [
DNA was extracted by Qiagen kit, according to the manufacturer’s instructions. The tissue sample was cut into pieces and grinded with sterile sand by adding PBS buffer and after centrifugation at 2000 rpm for 2 minutes, the supernatant was collected into new microcentrifuge tubes; after that, 200
Real-time polymerase chain reaction (RT-PCR) assay was used to detect the virus with
Forward: 5″-GGTGTAGTACGTATAAGATTATCGTATAGAAACAAGCCTTTA-3″; reverse: 5″-AATTTCT-TTCTCTGTTCCATTTG-3″
DNA was amplified in a final volume of 20
After amplification of the DNA template, the positive samples were noted by amplification fluorescence curves, melting curves (at 73°C), and cycle threshold (Ct) values from the assay which were used to describe the positive samples: Ct value higher than 40 was indicated as negative suggesting absence of the virus from the tissue specimens and nasal swabs [
The amplified DNA from extracted specimens was analyzed by agarose gel electrophoresis as described by [
The collected data were coded, entered, and stored into Microsoft Excel spreadsheet 2010. The data were thoroughly screened and properly coded before subjecting to statistical analysis. The data were imported from Microsoft Excel and analyzed using Statistical Package for Social Sciences (SPSS) software version 20. The morbidity and mortality were estimated in accordance with sex and different age categories.
Confidence interval was also used to describe morbidity and mortality across different variables. Chi-square (
Sampling from animals was carried out according to the experimental practice and standard approved by the Animal Welfare and Research Ethics Committee at Addis Ababa University College of Veterinary Medicine and Agriculture, Bishoftu Campus, which is in accordance with the International Guidelines for Animal Welfare, with verification number VM/ERC/25/06/09/2017.
Out of 522 cattle examined, clinical manifestation relevant to LSD was recorded in 18% (94/522) of cattle. The common clinical signs observed in cattle affected by LSD were fever, development of different sizes of circumscribed nodules on the skin, necrotic nodules, deep scab formation, swelling of dewlap, and enlargement of superficial lymph nodes. Lacrimation (Figure
An inflammation and whiteness of the eye of cattle infected with LSD. Encircled area shows lacrimation probably because of secondary bacterial complication.
Characteristic signs of LSD with generalized circumscribed skin nodules covering the entire body. Encircled area shows developed circumscribed nodules on the skin. Pictures were taken during field investigation.
According to the result of study where outbreak of LSD was investigated, there were 18% morbidity, 1.34% mortality, and 7.44 case fatality rates observed in study area. In this study, a slightly higher morbidity rate was observed in Arda Galma kebele, but the mortality and case fatality are relatively lower (Table
Morbidity rate, mortality rate, and case fatality rates of LSD outbreaks in the study area.
Name of kebeles | No. of susceptible cattle | No. of affected cattle | No. of deaths | Morbidity rate (%) | Mortality rate (%) | Case fatality |
---|---|---|---|---|---|---|
Arda Galma | 186 | 39 | 2 | 20.96 | 1.08 | 5.13 |
Kore Korme | 336 | 55 | 5 | 16.36 | 1.49 | 9.09 |
Total | 522 | 94 | 7 | 18.00 | 1.34 | 7.44 |
LSD morbidity rate in different age and sex groups.
Risk factors | No. of cattle at risk | No. of cattle affected | Morbidity rate (%) | ||
---|---|---|---|---|---|
Age | |||||
<2 | 107 | 31 | 29.71 | 14.562 | 0.001 |
≥2–4 | 314 | 41 | 13.05 | ||
>4 | 101 | 22 | 21.78 | ||
Total | 522 | 94 | 18.00 | ||
Sex | |||||
F | 369 | 76 | 20.59 | 11.554 | 0.003 |
M | 153 | 18 | 11.76 | ||
Total | 522 | 94 | 18.00 |
The mortality observed among the different age groups was significant (
Mortality rate in association with risk factors of age and sex.
Risk factors | No. of cattle at risk | No. of cattle affected | No. of deaths | Mortality rate (%) | Case fatality | ||
---|---|---|---|---|---|---|---|
Age | |||||||
<2 | 107 | 31 | 5 | 4.67 | 11.55 | 0.003 | 16.12 |
>2–4 | 314 | 41 | 1 | 0.31 | 7.14 | ||
>4 | 101 | 22 | 1 | 0.99 | 4.54 | ||
Total | 522 | 94 | 7 | 1.34 | 7.44 | ||
Sex | |||||||
F | 369 | 76 | 4 | 1.08 | 0.62 | 0.428 | 5.2 |
M | 153 | 18 | 3 | 1.96 | 16.66 | ||
Total | 522 | 94 | 7 | 1.34 | 7.44 |
Out of 20 samples, 7 skin biopsy samples and 13 nasal swabs were inoculated in Vero culture and the virus was passaged three times. Although it was not be able to isolate all samples, the characteristic LSDV CPE was perceived in most infected cell cultures (Figure
LSD growth on Vero cell culture. (a) Uninoculated Vero monolayer cell after 24 hours. (b) Infected Vero cells showing typical CPE at 11th day.
The extracted DNA of 20 specimens was amplified using
Classical PCR gel picture from skin nodule and nasal swabs of LSDV infected cattle. Lane M: DNA ladder (100bp, Fermentas); lanes 1–10 represent positive samples from Sawena district of Bale; N: negative control without template; P1: positive control for LSD; P2: positive control for SPP.
Conventional PCR was run targeting the RPO30 gene of the collected samples. Amplicons were analyzed by 3% agarose gel electrophoresis. The specific primers set amplified a DNA fragment of 172 bp equal to the expected amplification product size from LSDV. The result showed that the reference strain of the LSDV and the local isolate from skin nodules and nasal swabs had the same size of RPO30 gene fragment 172 bp.
Melting curves are generated from the DNA of skin biopsy and nasal swabs sample with respect to known LSDV controls (Figure
Melting curve analysis of field isolates of LSDV.
According to the pastoralist community, the local name for LSD was mentioned as “Itesa.” About 17 of the 20 respondents said that the LSD outbreak reoccurs in the frequency of three years, while 3 individuals said the disease occurs at an interval of two years. They also reported that the incidence of the disease increased during the rainy season (Figure
Picture taken during questionnaire survey in the study areas.
The survey enabled an estimation of the direct economic losses resulting from animals dying from LSD. Production losses were estimated from the weighted average price of each animal that died. An average cost of a single ox dying from this disease is 7,000 Ethiopian birr. For the one active outbreak investigated in two villages, the total economic losses from the deaths of 7 animals were 50,000 ETB (US$2174). The estimated total expense incurred for the treatment of LSD was also assessed. An average of 21 birr/animal was incurred for treatment of LSD with a frequency of treatment of once per month. A high economic loss was incurred for supportive veterinary treatment of LSD during outbreak, estimated as 2286 ETB (US$99.42).
As a pastoralist said, 0.6% (3/74) of pregnant cows have aborted due to the diseases. Further, animals that recovered were no longer fit for export purposes and were therefore sold at local markets at a lower price. Lastly, the survey found that animals that had recovered from LSD produced less milk and suffered a loss in draught power (Figure
The present study reported an outbreak of lumpy skin disease that occurred at the end of November 2016. As far as the objective is to characterize LSDV from an outbreak cases, the occurrence of LSD was examined using clinical diagnosis, PCR, and virus isolation. Accordingly, out of 20 typical clinical cases sampled and tested in the present study, 10 were confirmed as positive for LSD using PCR.
The clinical manifestations observed in the present finding, such as fever, circumscribed nodules on the skin, necrotic nodules, enlargement of superficial lymph nodes, and lacrimation, are in agreement with those documented by [
In the present study, the observed morbidity rate (18%) is slightly higher than that reported in [
With regard to mortality rate, the present finding report (1.34%) is slightly lower than that reported in [
In the present study, LSDV was isolated from samples collected from naturally infected cattle by inoculation on Vero cell. Characteristic pock lesions were observed after 1st passage and become clear after 3rd passage; this finding agrees with [
The CPEs characterized by rounding of cells, aggregation of dead cells, and destruction of monolayers are in line with the reports made by [
In agreement with the present finding, the authors in [
PCR was the test of choice for rapid detection and identification of the LSD outbreak causative agent. The PCR assay used in this work showed high specificity as a unique band of the expected size (172 bp) was obtained for DNA samples derived from skin biopsies, nasal swabs, and Neethling reference strain of LSDV.
The present study outbreak was reported at the end of November 2016, which is after the end of the main rainy season in most parts of the lowland and some highland agroecological zones. The seasonality of the outbreaks was also substantiated by questionnaire respondents who provided information on active disease surveillance. This agrees with the report of [
Other environmental risk factors associated with spread of LSD were found to be warm humid agroclimate, communal grazing/watering, and introduction of new animals in a herd. The incidence of LSD occurrence is high during wet seasons when biting-fly populations are abundant and it decreases or ceases during the dry season [
The findings associated with LSD mortality and veterinary expenses for treating sick animals suggest heavy losses in the sector. In line with current findings, various studies showed that lumpy skin disease causes severe economic losses as a result of the prolonged debilitating clinical course of the illness, reduced weight gain, temporary or permanent loss of milk production, infertility problems or even sterility in bulls, abortions in pregnant cows, and permanent damage to hides [
LSD was found to be the major cattle health problem that causes severe economic loss because of permanent damage to hides, a prolonged debilitating clinical course, reduced weight gain, temporary or permanent loss of milk production, temporary or permanent infertility or even sterility in bulls, and abortion of pregnant cows. In the present study, LSD causes significant effect on morbidity and mortality of animals that leads to economic loss because of abortion of pregnant cows, milk yield reduction, cost of dead animals, and cost of treatment. From the outbreak investigated in the present study, all age groups of animals were infected with significantly higher morbidity and mortality rate in young animals than other groups. Extracted DNA from skin biopsies and nasal swabs tested by using PCR revealed that all tested samples were LSDV. This implies failure of LSD vaccines, because a studied group of animals was annually vaccinated against the disease. Lumpy skin disease is considered as transboundary and trade band disease which has significant impediment on livestock market and animal products. Based on the above conclusions, the following recommendations are forwarded: Increasing appropriate handling facilities of vaccine besides providing quality vaccine and good administration skills should be the major considerations because vaccination is the only effective method to control the LSD in endemic countries like Ethiopia The government should establish strategic policies for effective control and eradication of the disease, i.e., strategic vaccination program and restriction of livestock movement Availability of a simple diagnostic test that can help confirm the cases at the field level is important to take control measures early during its occurrence More investigations should be carried out on the economic impact of LSD and the methods of spread, particularly the involvement of vectors Detailed molecular analysis of different isolates within the country needs to be carried out for further confirmation
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest.
Shubisa Abera is highly grateful to Yabello Regional Veterinary Laboratory and Oromia Pastoral Area Development Commission for having allowed him to join MSc class and supporting him with logistic and financial during his field works. He also expresses his heartfelt gratitude to his staff members Ato Huka Liban and Ato Boru Bule for their support in field work. An appreciation goes to the National Animal Health Diagnostic Center (NAHDIC) that allowed Shubisa to use the laboratory facilities. He is well pleased to gratify Mr. Melaku Sombo, Mr. Abdi Aliye, and Mrs. Tsion Bilat for their cooperativeness and unreserved technical support in all his laboratory works. His sincere thanks go to Bale Zone Pastoral Office and in particular to Dr. Bekele Dinsa and Dr. Elias Araga. He is very much grateful to Dr. Bekele Dinsa working at Sawena District Pastoral Office. Lots of thanks are due to Arda Galma and Kore Korme cattle owners and animal attendants because they allowed Shubisa to take samples from diseased animals.