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Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study
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Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study J. Ben-Gera a , E. Klement a,∗ , E. Khinich b , Y. Stram b , N.Y. Shpigel a a Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel b Kimron Veterinary Institute, Beit Dagan, Israel
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Article history: Received 16 June 2015 Received in revised form 20 July 2015 Accepted 22 July 2015 Available online xxx Keywords: Lumpy skin disease Vaccine efficacy Randomized controlled trial Sheep pox virus Epidemic Capripox
a b s t r a c t Lumpy skin disease (LSD) is a viral disease of cattle and buffalo, caused by a Capripox virus. A field study was performed during an LSD epidemic which occurred in 2012–2013 in Israel, in order to assess the efficacy of two commercial vaccines for protection against LSD. Fifteen dairy herds, vaccinated 2–5 months prior to study onset with a single dose of 102.5 TCID50 of RM65 attenuated sheep-pox vaccine, and not affected previously, were enrolled in the study. 4694 cows were randomized to be either vaccinated with a 103.5 TCID50 /dose of RM65 vaccine (x10RM65) or with a same dose of an attenuated Neethling LSD virus vaccine. A case of LSD was defined as the appearance of at least 5 lesions typical to LSD and a severe case was defined if this sign was accompanied by either fever (>39.5 ◦ C) or/and a 20% reduction in milk production. Deep lesion biopsies and blood samples were collected from 64.5% of the cases in an attempt to detect DNA of LSD virus by PCR and to differentiate between the wild strain and the vaccine Neethling strain. Seventy-six cows were affected by LSD in 8 herds with an incidence of 0.3–5.7%. Mantel–Haenszel relative risk (RRMH ) for LSD morbidity at least 15 days after vaccination in x10RM65 vs. Neethling was 2.635 (CI95% = 1.44–4.82) and 11.2 (2.3–54.7) for severe morbidity. RRMH for laboratory confirmed cases was 4.28 (1.59–11.53). An incidence of 0.38% (9/2356) of Neethling associated disease was observed among Neethling vaccinated cows while no such disease occurred in x10RM65 vaccinated cows. We conclude that the Neethling vaccine is significantly more effective than x10RM65 in preventing LSD morbidity, though it might cause a low incidence of Neethling associated disease. No transmission of the Neethling strain to non-Neethling vaccinated cows was observed in this study. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lumpy skin disease (LSD) is a re-emerging economically significant disease of cattle, caused by the lumpy skin disease virus (LSDV), of the genus Capripoxvirus [1]. The virus is believed to be transmitted mechanically by blood feeding flying insects such as Stomoxys sp. and Aedes sp. [2] and potentially by ticks [3–8]. In Israel the disease was first recorded in 1989 [9] and afterwards in 2006–2007 [10]. In both epidemics it was suspected that the virus arrived from Egypt by wind borne arthropod vectors [11]. In 1989 all livestock in the infected village were culled, including sheep and
∗ Corresponding author. Tel.: +972 8 9489560; fax: +972 8 9489634. E-mail address: [email protected] (E. Klement).
goats. These control measures were followed by ring vaccination of the surrounding herds. In the epidemics which occurred during 2006–2007 only clinically infected animals were slaughtered and quarantine was applied to all affected herds. Along with this the entire southern region of Israel was vaccinated, using subcutaneous injection of 1 ml of an attenuated sheep pox vaccine of the RM65 strain (Abic® , Israel) (102.5 TCID50 /ml). On late July, 2012 first suspected cases of LSD were reported from several beef herds located in north-eastern Israel, near the borders of Israel with Syria and Lebanon. The epidemic kept advancing southward despite the implementation of movement restrictions and emergency vaccination with an attenuated sheep pox vaccine of the RM-65 strain (JovivacTM , Jovac® , Jordan) (102.5 TCID50 /ml). On October, 2012 first cases began to appear among intensive zero-grazing high producing Holstein dairy cattle herds
http://dx.doi.org/10.1016/j.vaccine.2015.07.071 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ben-Gera J, et al. Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.071
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onset. (Fig. 1, Table 1). These herds accommodated a total of 8480 animals (cows, heifers, suckling calves). All participating herds held proper restriction facilities, which enabled animal identification and proper vaccination technique. 2.2. Prior vaccination As a part of the initial attempts made by the veterinary services to mitigate the outbreak, all herds were vaccinated subcutaneously 2–5 months prior to the study with 1 ml of an attenuated sheep pox vaccine of the strain RM65 (JovivacTM , Jovac® , Jordan) (102.5 TCID50 /ml) (Table 1). 2.3. Study design
Fig. 1. Spread of lumpy skin disease among dairy and beef cattle in Israel during 2012–2013. Black stars represent the 15 dairy herds participating in the efficacy study during 2013 (affected herds are circled in red. Non-affected herds are circled in black).
and reached its peak at the spring of 2013. During April alone 70 new herds were affected, including more than 40 new dairy herds. At this stage the disease has spread among the herds in the entire northern part of Israel, with one isolated foci in the center of Israel more than 100 km south, probably due to unauthorized movement of sick cattle (the affected farms apart of this isolated foci are depicted in Fig. 1). The failure of controlling the outbreak by using the attenuated RM65 sheep-pox vaccine, in its original concentration (102.5 TCID50 /ml) led the Israeli Veterinary Services to take a decision, on March the 18th 2013, to vaccinate all cattle in Israel against LSD, using the attenuated RM65 sheep-pox vaccine in a 10× dose (x10RM65) and the attenuated Neethling lumpy skin disease vaccine (OBP® , Republic of South-Africa). Roughly, almost all dairy herds were vaccinated within 3 months with the Neethling vaccine while most of the feedlots and beef cattle herds were vaccinated with the x10RM65 vaccine. The current study was planned, parallel to this campaign, aiming to evaluate the field efficacy and safety of the two vaccines during the ongoing LSD epidemic which occurred in Israel. Both vaccines were used for the first time in Israel, and to the best of our knowledge their efficacy was never compared previously under field conditions. 2. Materials and methods 2.1. Study population The study was conducted in 15 commercial dairy herds in the north of Israel in which no LSD was documented prior to the study
All the cattle in the 15 participating herds were vaccinated according to the following scheme with the exception of 29 animals that could not be restrained. Cows were allocated to two vaccination groups: Cows with an even brand number were vaccinated subcutaneously with 2 ml of an attenuated LSDV Neethling strain vaccine. In addition, in 7 herds (herds 1, 4, 5, 6, 9, 11 and 14, Table 1) all animals under 24 months of age (suckling calves, heifers) were vaccinated with 2 ml (as recommended by the manufacturer) of the Neethling strain vaccine (Lumpy Skin Disease Vaccine for Cattle, 103.5 TCID50 /dose (according to data provided by the manufacturer in the registration file of the vaccine), OBP® , Republic of SouthAfrica, Lot numbers: 435, 8655). Cows with an odd brand numbers were vaccinated with the x10RM65 vaccine, which was prepared by diluting the RM65 vaccine (JovivacTM , Sheep pox virus strain RM-65, 5 × 102.5 TCID50 /ml = 103.5 TCID50 /dose, Jovac® , Jordan, Lot numbers: 20DO310, 23D112) with fifth of the volume recommended by the manufacturer and injecting twice the recommended dose (i.e. 2 ml instead of 1 ml). In 8 herds the same preparation was administered to all animals aged under 24 months (suckling calves, heifers) (herds 2, 3, 7, 8, 10, 12, 13 and 15, Table 1). Both vaccines were injected subcutaneously in the neck region. All vaccinations were performed by trained veterinarians, using syringes and needles that were disposed after a single use. 2.4. Data collection Age of all vaccinated animals and daily milk yield of all vaccinated milking cows were retrieved from the herd management software (NOATM , Israel Cattle Breeders Association). After vaccination, all cattle in the herds were monitored daily for any signs of LSD or other illness by the herdsmen and twice weekly by the attending veterinarian. When illness was suspected its date of identification was recorded and notified to the study veterinarian (JBG). The study veterinarian arrived at the farm to conduct physical examination and to collect blood samples and biopsies from some of the suspected animals, usually within a day from notification. Blood was sampled from the coccygeal or jugular vein (5 ml, EDTA tube). Full skin deep lesion biopsies were taken under local anesthesia. Samples were collected from 49 (64.5%) out of 76 clinically affected cows. 2.5. Case definition Any animal with at least five typical LSD skin nodules was considered a positive case. Any cow, calve or heifer in which the appearance of typical skin nodules was accompanied by fever (>39.5 ◦ C) was defined as a severe case. In milking cows severe case definition included also a decrease of over 20% in daily milk yield compared to average milk yield in the two preceding days. Such cases were defined as severe even when fever was not recorded.
Please cite this article in press as: Ben-Gera J, et al. Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.071
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Table 1 Total number of cows vaccinated and time from vaccination to lumpy skin disease morbidity in 15 Israeli dairy herds participating in the vaccine efficacy study during 2013. Herd# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Total
# Vaccinated x10RM65
# Vaccinated Neethling
131 160 267 52 308 208 149 265 150 58 15 155 129 139 152
146 124 239 248 276 214 154 136 164 22 24 135 161 164 149
2338
2356
Date vaccinated RM65
Date vaccinated study (x10RM65\Neethling)
Date of first clinical case
Date of last clinical case
8.1.2013 15.10.2012 2.10.2012 1.1.2013 11.1.2013 8.1.2013 27.9.2012 10.1.2013 1.8.2012 25.12.2012 25.12.2012 23.1.2013 21.9.2012 29.1.2013 28.1.2013
13.3.2013 17.3.2013 17.3.2013 21.3.2013 21.3.2013 19.3.2013 23.3.2013 12.3.2013 12.3.2013 19.3.2013 19.3.2013 14.3.2013 19.3.2013 19.3.2013 17.3.2013
26.3.2013 27.5.2013 27.3.2013 31.3.2013 2.4.2013 3.4.2013 27.3.2013 13.5.2013 – – – – – – –
2.4.2013 9.7.2013 25.4.2013 14.4.2013 21.5.2013 9.4.2013 4.4.2013 4.7.2013 – – – – – – –
2.6. Virus identification Biopsies and blood samples collected from positive cases were delivered in 4 ◦ C to the Kimron Veterinary Institute for virus DNA detection by PCR and virus strain identification. PCR was first performed on the skin lesion biopsies whenever these were obtained. If a skin lesion biopsy was not available or if the PCR performed on this tissue was negative, a whole blood PCR was performed. Viral DNA from blood and skin were extracted with the DNeasy Blood and Tissue Kit (Qiagene, Hilden, Germany), according to the manufacturer’s instructions. PCR, Nested PCR and restriction enzyme digestion were performed as was previously described [12]. 2.7. Statistical analysis Incidence of disease (all cases and severe cases) was calculated separately for each vaccination group. An adjusted relative risk (RRMH ) for all herds was then calculated by the Mantel–Haenszel method and statistical significance was calculated using the Mantel–Haenszel test. Heterogeneity between the herds was tested by the heterogeneity Chi-Square test. When the p-value for this test was <0.05, the adjusted RRMH was calculated using a Randomeffects model (DerSimonian-Laird procedure). Since only cows were randomized, the entire procedure was calculated twice; Once for all cattle in the herds (data not shown) and once just for cows. These RRMH s were calculated for the following definitions of morbidity: (1) Any case. (2) Severe case. (3) Any case occurring at least 14 days after vaccination. (4) Severe cases occurring at least 14 days after vaccination. (5) Clinical cases in which a wild strain LSDV was identified by PCR. These analyses were performed
by the COMPARE2 module in the WinPepi statistical package [13,14]. Time of case and severe case occurrence for each vaccination group was compared visually by drawing Kaplan–Meyer ‘one minus survival’ curves. These were performed for all cattle participating in the study. 3. Results LSD was detected and confirmed in 8 herds out of the 15 farms participating in the study. Morbidity in the affected farms ranged from 0.3% to 5.7%. Eighty-nine animals developed clinical signs of LSD (an overall incidence of 1.6%). Of these 51 were x10RM65 vaccinated (incidence of 1.85%), 31 were Neethling vaccinated (incidence of 1.11%) and 7 were unvaccinated calves located in one herd, which were born after vaccine administration (7 of 59 calves borne in this herd after vaccination (an incidence of 11.8%)). Up to 50% of the clinical cases were classified as severe (Table 2). Mortality of 7 animals was recorded in 3 herds, 3 of which were unvaccinated calves. Seventy six cases out of the 89 cases occurred in cows (incidence of 2.47%), compared to only six cases which occurred among 2440 vaccinated calves and heifers in these herds (RR = 9.52, CI95% = 4.14–21.85). Since there was better randomization among cows, and only very low morbidity among calves and heifers, most of the analysis is presented for cows only. An incidence of 46/1540 (2.99%) was observed among x10RM65 vaccinated cows, while it reached 30/1537, (1.95%) in Neethling vaccinated cows. In 5 sick herds the incidence among x10RM65 vaccinated cows was higher than in Neethling vaccinated cows while in 2 herds the morbidity among the latter was higher, in one herd the morbidity was
Table 2 Relative risk (RR) and Mantel–Haenszel relative risk (RRMH ) for lumpy skin disease morbidity among cows vaccinated with x10RM65 and Neethling vaccines in 8 affected herds in Israel. Herd# 1 2 3 4 5 6 7 8 Total Total RRMH (CI95% )
x10RM65 vaccinated
Neethling vaccinated
131 160 267 52 308 208 149 265
146 124 239 248 276 214 154 136
1540
1537
x10RM65 morbidity (%)
Neethling morbidity (%)
RR (CI95% )
x10RM65 severe morbidity (%)
Neethling severe morbidity (%)
RR (CI95% )
7 (5.34%) 17 (10.6%) 1 (0.37%) 2 (3.84%) 4 (1.3%) 3 (1.44%) 0 (0%) 12 (4.52%)
2 (1.37%) 4 (3.22%) 10 (4.2%) 6 (2.41%) 3 (1.1%) 1 (0.46%) 2 (1.3%) 2 (1.47%)
3.9 3.3 0.088 1.6 1.18 3.13 0 3.1
0 (0%) 15 (9.37%) 1 (0.37%) 2 (3.8%) 2 (0.65%) 1 (0.48%) 0 (0%) 8 (3%)
0 (0%) 0 (0%) 4 (1.67%) 1 (0.4%) 1 (0.36%) 0 (0%) 1 (0.65%) 0 (0%)
0 – 0.22 9.5 1.8 – 0 –
46 (3%)
30 (2%)
1.5 1.49 (0.92–2.41)
29 (1.88%)
7 (0.45%)
4.18 3.65 (1.59–8.35)
Please cite this article in press as: Ben-Gera J, et al. Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.071
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Fig. 2. Accumulation of lumpy skin disease cases (a) and severe cases (b) among cattle vaccinated with Neethling vaccine (green) and RM65 vaccine (blue). Cattle diagnosed with non-severe lumpy skin disease are censored. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
approximately the same (Table 2). The RRMH for morbidity among x10RM65 vaccinated cows compared to the Neethling vaccinated cows was 1.49 (0.92–2.41). The RRMH for severe cases was 3.65 (1.59–8.35). Observation of the dynamics of case accumulation revealed an interaction between the time elapsed from vaccination and incidence of all cases or severe cases (Fig. 2). The incidence among x10RM65 vaccinated cows was significantly lower during the first two weeks after vaccination (RRMH = 0.315, 0.11–0.91) (Table 3) while it was significantly higher two weeks after vaccination both for the occurrence of all cases (RRMH = 2.635, 1.44–4.82) and for the occurrence of severe cases (RRMH = 11.2, 2.3–54.7) (Table 3). Field strain of LSDV was identified in all the affected farms. Field strain viruses were identified from skin lesion biopsies and whole blood samples obtained from 17 and five x10RM65 vaccinated cows, respectively and from skin lesion biopsies and a whole blood sample obtained from five and one Neethling vaccinated cows, respectively. Adjusted relative risk in x10RM65 vs. Neethling vaccinated cows for morbidity caused by LSDV wild strain was RRMH = 4.28 (CI95% = 1.59–11.53) (Table 4). Neethling strain was identified from a total of 9 Neethling vaccinated cows in 4 farms. All were identified from skin lesion biopsies. Among these cases, one was classified as a severe case. These results indicate an overall incidence of Neethling associated disease of 0.38% (9/2356) and 0.04% (1/2356) of Neethling associated severe morbidity (Table 4). In all of these cases signs of illness appeared within 14 days from vaccination. 4. Discussion The RM65 strain based vaccine was used to prevent LSD in Israel after outbreaks which occurred in 1989, 2006 and 2007. However, the incidence in some herds vaccinated with this vaccine, at least once and up to 3 times prior to the outbreak of 2007 reached up to 44% [15]. This prior experience combined with the observation of high LSD incidence in RM65 vaccinated beef herds during the outbreak of 2012 indicated that the effectiveness of the RM65 vaccine was very limited and motivated the evaluation of the efficacy of other vaccines. In addition to the RM65 vaccine, which was used in Israel and other countries in the Middle-East, two other vaccines were used for prevention of lumpy skin disease; The Neethling LSDV attenuated vaccine used in southern Africa [16] and the O-240 capripox
vaccine, which was recently shown to actually be a strain of Lumpy skin disease virus [17], and was primarily used in Central Africa [18]. Prior vaccination of dairy herds with O-240 capripox vaccine was associated with the appearance of skin lesions similar to LSD [19]. The Israeli veterinary service, therefore refrained from using this vaccine and decided to use the Neethling attenuated vaccine and a 10× dose of the RM65 vaccine along with comparison of their efficacy. A recent study found a Gorgan goat pox based vaccine to provide protection from challenge by LSDV. However, extensive field experience with this vaccine is still lacking [20]. In this study the efficacy of the x10RM65 and the Neethling lumpy skin disease virus live attenuated vaccines were compared. Both were administered subcutaneously in an approximate virus dose of 103.5 TCID50 . The results of this study demonstrate a significantly higher efficacy of the attenuated Neethling vaccine for LSD prevention: The risk for a confirmed wild strain infection was more than 4 folds higher among x10RM65 vaccinated cows, compared to Neethling vaccinated cows (Table 4). During the first two weeks after vaccination, incidence among Neethling vaccinated cows seems higher. However, these cases can be mostly attributed either to infection by the vaccine strain (Table 4) or to infection by a wild strain prior to vaccination or prior to reaching full protection by the vaccine (as incubation time averages approximately 10 days [21]). The risk for LSD cases and severe cases appearing at least two weeks after vaccination was 2.635 and 11.2 folds higher, respectively among x10RM65 compared to Neethling vaccinated cows (Table 3). These results are opposed to the results of a recent study in which a Neethling based vaccine failed to protect vaccinated calves against challenge with LSDV. However, the vaccine in that study was manufactured by a different manufacturer (The Ethiopian National Veterinary Institute) and the authors suggest that its poor efficacy might be attributed to its poor immunogenicity, which might occurred due to over-attenuation [20]. The main event observed after Neethling vaccination was the development of a disease similar to LSD. This is opposed to the potential adverse effect described by the vaccine manufacturer, which is characterized by a swelling in vaccine injection site, appearing at least 4 days after vaccination (Lumpy Skin Disease Vaccine for Cattle, OBP® , vaccine label). In this study 0.38% (9/2356) of the Neethling vaccinated cows showed a confirmed Neethling associated disease, but only one cow showed severe illness (0.04%). All confirmed cases of Neethling associated disease appeared within a period of 14 days from vaccination. It should
Please cite this article in press as: Ben-Gera J, et al. Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.071
≤14 days from vaccination 1 2 3 4 5 6 7 8 Total Total RRMH (CI95% )
Total Total RRMH (CI95% )
Neethling vaccinated
x10RM65 morbidity (%)
131 160 267 52 308 208 149 265
146 124 239 248 276 214 154 136
1 (0.76%) 0 1 (0.37%) 0 2 (0.65%) 0 0 0
1540
1537
4 (0.26%)
131 160 267 52 308 208 149 265
146 124 239 248 276 214 154 136
6 (3.9%) 17 (10.6%) 0 2 (3.8%) 2 (0.64%) 3 (1.44%) 0 12 (4.5%)
1540
1537
42 (2.7%)
Neethling morbidity (%) 0 0 7 (3%) 6 (2.4%) 2 (0.75%) 0 2 (1.3%) 0 17 (1.1%)
2 (0.92%) 4 (3.2%) 3 (1.25%) 0 1 (0.36%) 1 (0.45%) 0 2 (1.47%) 13 (0.84%)
RR (CI95% )
x10RM65 severe morbidity (%)
Neethling severe morbidity (%)
RR (CI95% )
– – 1.12 – 0.86
0 0 1 (0.37%) 0 1 (0.32%) 0 0 0
0 0 4 (1.67%) 1 (0.24%) 0 0 1 (0.65%) 0
– – 0.22 – –
0.23 0.315 (0.11–91)
2 (0.13%)
6 (0.4%)
0.32 0.34 (0.06–1.79)
4.23 3.3 – – 1.78 3.2 – 3.06
0 8 (5%) 0 2 (3.8%) 1 (0.32%) 1 (0.49%) 0 8 (3%)
0 0 0 0 1 (0.36%) 0 0 0
– – – – 0.88 – – –
3.2 2.635 (1.44–4.82)
20 (1.3%)
1 (0.06%)
21.67 11.2 (2.3–54.7)
Table 4 Relative risk (RR) and Mantel–Haenszel relative risk (RRMH ) for lumpy skin disease morbidity caused by wild strain virus among cows vaccinated with x10RM65 and Neethling vaccines in 8 affected herds in Israel. Vaccine herd#
x10RM65
Attempted virus DNA detectiona /sick 1 2 3 4 5 6 7 8
6/7 6/17 1/1 2/2 3/4 2/3 0/0 9/12
Total Total RRMH (CI95% )
30/46
a b c
Neethling
Wild strain virus identificationb /vaccinated (% incidence) 4 (3%) 5 (3.1%) 1 (0.37%) 2 (3.8%) 1 (0.32%) 2 (1%) 0 7 (2.64%) 22 (1.43%)
RR (CI95% ) for wild strain identificationb
Attempted virus DNA detectiona /sick
Wild strain virus identificationb /vaccinated (% incidence)
Neethling virus identificationb /vaccinated (% incidence)
2/2 1/4 5/10 6/6 3/3 0/1 2/2 0/2
1 (0.7%) 1 (0.8%) 0 2 (0.8%) 1 (0.36%) 0 1 (0.65%) 0
0 0 3 (1.25%) 3 (1.2%) 2 (0.72%) 0 1 (0.65%) 0
4.28 3.87 – 4.75 0.88 – – –
19/30
6 (0.4%)
9 (0.38%c )
3.57 4.28 (1.59–11.53)
Number of samples obtained for virus DNA detection by PCR (i.e. attempted virus DNA detection) and for further virus identification out of the total number of cases appearing in each vaccinated group. Identification was performed as described by Menasherow et al. [16]. Calculated out of all the Neethling vaccinated cows (n = 2356).
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>14 days from vaccination 1 2 3 4 5 6 7 8
x10RM65 vaccinated
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Please cite this article in press as: Ben-Gera J, et al. Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.071
Table 3 Relative risk (RR) and Mantel–Haenszel relative risk (RRMH ) for lumpy skin disease morbidity among cows vaccinated with x10RM65 and Neethling vaccines in 8 affected herds in Israel, within 14 days post vaccine and 15 days onwards.
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be mentioned, however, that only 19/30 (63%) of the cases among Neethling vaccinated cows were sampled (Table 4). Thus, actual incidence of Neethling related disease could reach 0.6%. No case of Neethling associated disease appeared in cows which were not vaccinated with the Neethling vaccine, indicating that transmission of the vaccine strain between animals was not observed during this study. Considering these findings it seems that adverse effects after vaccination with the attenuated Neethling strain are rare and its use is therefore relatively safe. In the current study no adverse effects were observed after vaccinating with the x10RM65 vaccine. The main strengths of the current study stem from its design. This is a prospective randomized study and therefore confounding and information biases are minimized. Attempt for virus DNA detection by PCR in 64.5% of the clinical cases further supports the results of the study and increases the specificity of case identification. Despite this, lack of sensitivity due to lower awareness of some of the herdsmen and the veterinarians might always exist. Such a bias could be the cause for higher disease incidence observed in cows when compared to calves and heifers. As milked cows may have been under better surveillance the sensitivity for detection of disease among this population might have been higher. Reduced sensitivity can also be the result of the conservative case definition we chose (i.e. at least five skin lesions as a minimal definition for a clinical disease). A relatively specific case definition was prefered because estimation of vaccine efficacy was the main goal of this study and under conditions of rare morbidity (as occurred in this study), specificity is more important than sensitivity in determining bias in the estimate of relative risk (and the resultant vaccine efficacy) [22]. Another explanation can be that vaccinated calves and heifers are less susceptible to disease, compared to high producing cows, which may be in higher stress. This finding is in agreement with the finding of Magori-Cohen et al. [21] who found a marginally statistically significant association between lactation and higher risk of LSD morbidity during an LSD outbreak in a dairy farm (i.e. p = 0.053). As opposed to these findings, the results of a retrospective study performed in Ethiopia found higher susceptibility of calves to LSD [18]. Another potential limitation of the study is the fact that all herds were vaccinated 3–5 months prior to the study with an RM-65 vaccine in a regular dose. Though prior experience indicates that this vaccine, administered in a dose of 102.5 TCID is not effective for prevention of LSD, its prior administration could have resulted in a booster effect to vaccination by the x10RM65 and the Neethling vaccines. While the results of this study do not support nor refute the effectiveness of the x10RM65 vaccine for disease prevention they clearly show that the Neethling vaccine is highly effective in protection from LSD. As during the 2012–2013 Israeli LSD epidemic almost all of the dairy herds were vaccinated with the Neethling vaccine, the results of this study may explain the successful control of LSD in these herds in Israel (last confirmed case of LSD in Israel to date was documented on August 29th, 2013). Beef herds, which were almost entirely vaccinated by the x10RM65 vaccine were already highly affected when vaccinated. Thus, it is more difficult to assess the contribution of the x10RM65 vaccine to the control of the disease in Israel. 5. Conclusion Though the current study supports the use of the Neethling vaccine for protection against lumpy skin disease, its use should be considered against the potential adverse effects associated with its
administration and the risk of inserting an actual disease agent into a potentially naïve country. Choosing the right vaccine should be based on consideration of the advantages and disadvantages of each vaccine and tailored to the specific situation in each country. Acknowledgments This study was supported by a grant from the Israeli Veterinary Services. Conflict of interests: The authors do not have any conflict of interest to declare. References [1] Tuppurainen ES, Oura CA. Lumpy skin disease: an emerging threat to Europe, the Middle East and Asia (Review). Transbound Emerg Dis 2011. [2] Chihota CM, Rennie LF, Kitching RP, Mellor PS. Mechanical transmission of lumpy skin disease virus by Aedes aegypti (Diptera: Culicidae). Epidemiol Infect 2001;126:317–21. [3] Lubinga JC, Tuppurainen ES, Stoltsz WH, Ebersohn K, Coetzer JA, Venter EH. Detection of lumpy skin disease virus in saliva of ticks fed on lumpy skin disease virus-infected cattle. Exp Appl Acarol 2013;61:129–38. [4] Tuppurainen ES, Lubinga JC, Stoltsz WH, Troskie M, Carpenter ST, Coetzer JA, et al. Evidence of vertical transmission of lumpy skin disease virus in Rhipicephalus decoloratus ticks. Ticks Tick Borne Dis 2013;4:329–33. [5] Tuppurainen ES, Lubinga JC, Stoltsz WH, Troskie M, Carpenter ST, Coetzer JA, et al. Mechanical transmission of lumpy skin disease virus by Rhipicephalus appendiculatus male ticks. Epidemiol Infect 2013;141:425–30. [6] Lubinga JC, Clift SJ, Tuppurainen ES, Stoltsz WH, Babiuk S, Coetzer JA, et al. Demonstration of lumpy skin disease virus infection in Amblyomma hebraeum and Rhipicephalus appendiculatus ticks using immunohistochemistry. Ticks Tick Borne Dis 2014;5:113–20. [7] Lubinga JC, Tuppurainen ES, Coetzer JA, Stoltsz WH, Venter EH. Evidence of lumpy skin disease virus over-wintering by transstadial persistence in Amblyomma hebraeum and transovarial persistence in Rhipicephalus decoloratus ticks. Exp Appl Acarol 2014;62:77–90. [8] Lubinga JC, Tuppurainen ES, Mahlare R, Coetzer JA, Stoltsz WH, Venter EH. Evidence of transstadial and mechanical transmission of lumpy skin disease virus by Amblyomma hebraeum ticks. Transbound Emerg Dis 2015;62:174–82. [9] Yeruham I, Nir O, Braverman Y, Davidson M, Grinstein H, Haymovitch M, et al. Spread of lumpy skin disease in Israeli dairy herds. Vet Rec 1995;137:91–3. [10] Brenner J, Bellaiche M, Gross E, Elad D, Oved Z, Haimovitz M, et al. Appearance of skin lesions in cattle populations vaccinated against lumpy skin disease: statutory challenge. Vaccine 2009;27:1500–3. [11] Klausner Z, Fattal E, Klement E. Using synoptic systems’ typical wind trajectories for the analysis of potential atmospheric long distance dispersal of lumpy skin disease virus. Transbound Emerg Dis 2015. [12] Menasherow S, Rubinstein-Giuni M, Kovtunenko A, Eyngor Y, Fridgut O, Rotenberg D, et al. Development of an assay to differentiate between virulent and vaccine strains of lumpy skin disease virus (LSDV). J Virol Methods 2014;199:95–101. [13] Abramson JH. WINPEPI (PEPI-for-Windows): computer programs for epidemiologists. Epidemiol Perspect Innov 2004;1:6. [14] Abramson JH. WINPEPI updated: computer programs for epidemiologists, and their teaching potential. Epidemiol Perspect Innov 2011;8:1. [15] (AHAW) EPoAHaW. Scientific Opinion on lumpy skin disease. EFSA J 2015;13:3986. [16] Hunter P, Wallace D. Lumpy skin disease in southern Africa: a review of the disease and aspects of control. J S Afr Vet Assoc 2001;72:68–71. [17] Tuppurainen ES, Pearson CR, Bachanek-Bankowska K, Knowles NJ, Amareen S, Frost L, et al. Characterization of sheep pox virus vaccine for cattle against lumpy skin disease virus. Antiviral Res 2014;109:1–6. [18] Ayelet G, Haftu R, Jemberie S, Belay A, Gelaye E, Sibhat B, et al. Lumpy skin disease in cattle in central Ethiopia: outbreak investigation and isolation and molecular detection of the virus. Rev Sci Tech 2014;33:877–87. [19] Yeruham I, Perl S, Nyska A, Abraham A, Davidson M, Haymovitch M, et al. Adverse reactions in cattle to a capripox vaccine. Vet Rec 1994;135:330–2. [20] Gari G, Abie G, Gizaw D, Wubete A, Kidane M, Asgedom H, et al. Evaluation of the safety, immunogenicity and efficacy of three capripoxvirus vaccine strains against lumpy skin disease virus. Vaccine 2015;33:3256–61. [21] Magori-Cohen R, Louzoun Y, Herziger Y, Oron E, Arazi A, Tuppurainen E, et al. Mathematical modelling and evaluation of the different routes of transmission of lumpy skin disease virus. Vet Res 2012;43:1. [22] Thrusfield M. Observational studies. Veterinary epidemiology. 3rd ed. UK: Blackwell Science; 2005. p. 266–88.
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Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study J. Ben-Gera a , E. Klement a,∗ , E. Khinich b , Y. Stram b , N.Y. Shpigel a a Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel b Kimron Veterinary Institute, Beit Dagan, Israel
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Article history: Received 16 June 2015 Received in revised form 20 July 2015 Accepted 22 July 2015 Available online xxx Keywords: Lumpy skin disease Vaccine efficacy Randomized controlled trial Sheep pox virus Epidemic Capripox
a b s t r a c t Lumpy skin disease (LSD) is a viral disease of cattle and buffalo, caused by a Capripox virus. A field study was performed during an LSD epidemic which occurred in 2012–2013 in Israel, in order to assess the efficacy of two commercial vaccines for protection against LSD. Fifteen dairy herds, vaccinated 2–5 months prior to study onset with a single dose of 102.5 TCID50 of RM65 attenuated sheep-pox vaccine, and not affected previously, were enrolled in the study. 4694 cows were randomized to be either vaccinated with a 103.5 TCID50 /dose of RM65 vaccine (x10RM65) or with a same dose of an attenuated Neethling LSD virus vaccine. A case of LSD was defined as the appearance of at least 5 lesions typical to LSD and a severe case was defined if this sign was accompanied by either fever (>39.5 ◦ C) or/and a 20% reduction in milk production. Deep lesion biopsies and blood samples were collected from 64.5% of the cases in an attempt to detect DNA of LSD virus by PCR and to differentiate between the wild strain and the vaccine Neethling strain. Seventy-six cows were affected by LSD in 8 herds with an incidence of 0.3–5.7%. Mantel–Haenszel relative risk (RRMH ) for LSD morbidity at least 15 days after vaccination in x10RM65 vs. Neethling was 2.635 (CI95% = 1.44–4.82) and 11.2 (2.3–54.7) for severe morbidity. RRMH for laboratory confirmed cases was 4.28 (1.59–11.53). An incidence of 0.38% (9/2356) of Neethling associated disease was observed among Neethling vaccinated cows while no such disease occurred in x10RM65 vaccinated cows. We conclude that the Neethling vaccine is significantly more effective than x10RM65 in preventing LSD morbidity, though it might cause a low incidence of Neethling associated disease. No transmission of the Neethling strain to non-Neethling vaccinated cows was observed in this study. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Lumpy skin disease (LSD) is a re-emerging economically significant disease of cattle, caused by the lumpy skin disease virus (LSDV), of the genus Capripoxvirus [1]. The virus is believed to be transmitted mechanically by blood feeding flying insects such as Stomoxys sp. and Aedes sp. [2] and potentially by ticks [3–8]. In Israel the disease was first recorded in 1989 [9] and afterwards in 2006–2007 [10]. In both epidemics it was suspected that the virus arrived from Egypt by wind borne arthropod vectors [11]. In 1989 all livestock in the infected village were culled, including sheep and
∗ Corresponding author. Tel.: +972 8 9489560; fax: +972 8 9489634. E-mail address: [email protected] (E. Klement).
goats. These control measures were followed by ring vaccination of the surrounding herds. In the epidemics which occurred during 2006–2007 only clinically infected animals were slaughtered and quarantine was applied to all affected herds. Along with this the entire southern region of Israel was vaccinated, using subcutaneous injection of 1 ml of an attenuated sheep pox vaccine of the RM65 strain (Abic® , Israel) (102.5 TCID50 /ml). On late July, 2012 first suspected cases of LSD were reported from several beef herds located in north-eastern Israel, near the borders of Israel with Syria and Lebanon. The epidemic kept advancing southward despite the implementation of movement restrictions and emergency vaccination with an attenuated sheep pox vaccine of the RM-65 strain (JovivacTM , Jovac® , Jordan) (102.5 TCID50 /ml). On October, 2012 first cases began to appear among intensive zero-grazing high producing Holstein dairy cattle herds
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onset. (Fig. 1, Table 1). These herds accommodated a total of 8480 animals (cows, heifers, suckling calves). All participating herds held proper restriction facilities, which enabled animal identification and proper vaccination technique. 2.2. Prior vaccination As a part of the initial attempts made by the veterinary services to mitigate the outbreak, all herds were vaccinated subcutaneously 2–5 months prior to the study with 1 ml of an attenuated sheep pox vaccine of the strain RM65 (JovivacTM , Jovac® , Jordan) (102.5 TCID50 /ml) (Table 1). 2.3. Study design
Fig. 1. Spread of lumpy skin disease among dairy and beef cattle in Israel during 2012–2013. Black stars represent the 15 dairy herds participating in the efficacy study during 2013 (affected herds are circled in red. Non-affected herds are circled in black).
and reached its peak at the spring of 2013. During April alone 70 new herds were affected, including more than 40 new dairy herds. At this stage the disease has spread among the herds in the entire northern part of Israel, with one isolated foci in the center of Israel more than 100 km south, probably due to unauthorized movement of sick cattle (the affected farms apart of this isolated foci are depicted in Fig. 1). The failure of controlling the outbreak by using the attenuated RM65 sheep-pox vaccine, in its original concentration (102.5 TCID50 /ml) led the Israeli Veterinary Services to take a decision, on March the 18th 2013, to vaccinate all cattle in Israel against LSD, using the attenuated RM65 sheep-pox vaccine in a 10× dose (x10RM65) and the attenuated Neethling lumpy skin disease vaccine (OBP® , Republic of South-Africa). Roughly, almost all dairy herds were vaccinated within 3 months with the Neethling vaccine while most of the feedlots and beef cattle herds were vaccinated with the x10RM65 vaccine. The current study was planned, parallel to this campaign, aiming to evaluate the field efficacy and safety of the two vaccines during the ongoing LSD epidemic which occurred in Israel. Both vaccines were used for the first time in Israel, and to the best of our knowledge their efficacy was never compared previously under field conditions. 2. Materials and methods 2.1. Study population The study was conducted in 15 commercial dairy herds in the north of Israel in which no LSD was documented prior to the study
All the cattle in the 15 participating herds were vaccinated according to the following scheme with the exception of 29 animals that could not be restrained. Cows were allocated to two vaccination groups: Cows with an even brand number were vaccinated subcutaneously with 2 ml of an attenuated LSDV Neethling strain vaccine. In addition, in 7 herds (herds 1, 4, 5, 6, 9, 11 and 14, Table 1) all animals under 24 months of age (suckling calves, heifers) were vaccinated with 2 ml (as recommended by the manufacturer) of the Neethling strain vaccine (Lumpy Skin Disease Vaccine for Cattle, 103.5 TCID50 /dose (according to data provided by the manufacturer in the registration file of the vaccine), OBP® , Republic of SouthAfrica, Lot numbers: 435, 8655). Cows with an odd brand numbers were vaccinated with the x10RM65 vaccine, which was prepared by diluting the RM65 vaccine (JovivacTM , Sheep pox virus strain RM-65, 5 × 102.5 TCID50 /ml = 103.5 TCID50 /dose, Jovac® , Jordan, Lot numbers: 20DO310, 23D112) with fifth of the volume recommended by the manufacturer and injecting twice the recommended dose (i.e. 2 ml instead of 1 ml). In 8 herds the same preparation was administered to all animals aged under 24 months (suckling calves, heifers) (herds 2, 3, 7, 8, 10, 12, 13 and 15, Table 1). Both vaccines were injected subcutaneously in the neck region. All vaccinations were performed by trained veterinarians, using syringes and needles that were disposed after a single use. 2.4. Data collection Age of all vaccinated animals and daily milk yield of all vaccinated milking cows were retrieved from the herd management software (NOATM , Israel Cattle Breeders Association). After vaccination, all cattle in the herds were monitored daily for any signs of LSD or other illness by the herdsmen and twice weekly by the attending veterinarian. When illness was suspected its date of identification was recorded and notified to the study veterinarian (JBG). The study veterinarian arrived at the farm to conduct physical examination and to collect blood samples and biopsies from some of the suspected animals, usually within a day from notification. Blood was sampled from the coccygeal or jugular vein (5 ml, EDTA tube). Full skin deep lesion biopsies were taken under local anesthesia. Samples were collected from 49 (64.5%) out of 76 clinically affected cows. 2.5. Case definition Any animal with at least five typical LSD skin nodules was considered a positive case. Any cow, calve or heifer in which the appearance of typical skin nodules was accompanied by fever (>39.5 ◦ C) was defined as a severe case. In milking cows severe case definition included also a decrease of over 20% in daily milk yield compared to average milk yield in the two preceding days. Such cases were defined as severe even when fever was not recorded.
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Table 1 Total number of cows vaccinated and time from vaccination to lumpy skin disease morbidity in 15 Israeli dairy herds participating in the vaccine efficacy study during 2013. Herd# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Total
# Vaccinated x10RM65
# Vaccinated Neethling
131 160 267 52 308 208 149 265 150 58 15 155 129 139 152
146 124 239 248 276 214 154 136 164 22 24 135 161 164 149
2338
2356
Date vaccinated RM65
Date vaccinated study (x10RM65\Neethling)
Date of first clinical case
Date of last clinical case
8.1.2013 15.10.2012 2.10.2012 1.1.2013 11.1.2013 8.1.2013 27.9.2012 10.1.2013 1.8.2012 25.12.2012 25.12.2012 23.1.2013 21.9.2012 29.1.2013 28.1.2013
13.3.2013 17.3.2013 17.3.2013 21.3.2013 21.3.2013 19.3.2013 23.3.2013 12.3.2013 12.3.2013 19.3.2013 19.3.2013 14.3.2013 19.3.2013 19.3.2013 17.3.2013
26.3.2013 27.5.2013 27.3.2013 31.3.2013 2.4.2013 3.4.2013 27.3.2013 13.5.2013 – – – – – – –
2.4.2013 9.7.2013 25.4.2013 14.4.2013 21.5.2013 9.4.2013 4.4.2013 4.7.2013 – – – – – – –
2.6. Virus identification Biopsies and blood samples collected from positive cases were delivered in 4 ◦ C to the Kimron Veterinary Institute for virus DNA detection by PCR and virus strain identification. PCR was first performed on the skin lesion biopsies whenever these were obtained. If a skin lesion biopsy was not available or if the PCR performed on this tissue was negative, a whole blood PCR was performed. Viral DNA from blood and skin were extracted with the DNeasy Blood and Tissue Kit (Qiagene, Hilden, Germany), according to the manufacturer’s instructions. PCR, Nested PCR and restriction enzyme digestion were performed as was previously described [12]. 2.7. Statistical analysis Incidence of disease (all cases and severe cases) was calculated separately for each vaccination group. An adjusted relative risk (RRMH ) for all herds was then calculated by the Mantel–Haenszel method and statistical significance was calculated using the Mantel–Haenszel test. Heterogeneity between the herds was tested by the heterogeneity Chi-Square test. When the p-value for this test was <0.05, the adjusted RRMH was calculated using a Randomeffects model (DerSimonian-Laird procedure). Since only cows were randomized, the entire procedure was calculated twice; Once for all cattle in the herds (data not shown) and once just for cows. These RRMH s were calculated for the following definitions of morbidity: (1) Any case. (2) Severe case. (3) Any case occurring at least 14 days after vaccination. (4) Severe cases occurring at least 14 days after vaccination. (5) Clinical cases in which a wild strain LSDV was identified by PCR. These analyses were performed
by the COMPARE2 module in the WinPepi statistical package [13,14]. Time of case and severe case occurrence for each vaccination group was compared visually by drawing Kaplan–Meyer ‘one minus survival’ curves. These were performed for all cattle participating in the study. 3. Results LSD was detected and confirmed in 8 herds out of the 15 farms participating in the study. Morbidity in the affected farms ranged from 0.3% to 5.7%. Eighty-nine animals developed clinical signs of LSD (an overall incidence of 1.6%). Of these 51 were x10RM65 vaccinated (incidence of 1.85%), 31 were Neethling vaccinated (incidence of 1.11%) and 7 were unvaccinated calves located in one herd, which were born after vaccine administration (7 of 59 calves borne in this herd after vaccination (an incidence of 11.8%)). Up to 50% of the clinical cases were classified as severe (Table 2). Mortality of 7 animals was recorded in 3 herds, 3 of which were unvaccinated calves. Seventy six cases out of the 89 cases occurred in cows (incidence of 2.47%), compared to only six cases which occurred among 2440 vaccinated calves and heifers in these herds (RR = 9.52, CI95% = 4.14–21.85). Since there was better randomization among cows, and only very low morbidity among calves and heifers, most of the analysis is presented for cows only. An incidence of 46/1540 (2.99%) was observed among x10RM65 vaccinated cows, while it reached 30/1537, (1.95%) in Neethling vaccinated cows. In 5 sick herds the incidence among x10RM65 vaccinated cows was higher than in Neethling vaccinated cows while in 2 herds the morbidity among the latter was higher, in one herd the morbidity was
Table 2 Relative risk (RR) and Mantel–Haenszel relative risk (RRMH ) for lumpy skin disease morbidity among cows vaccinated with x10RM65 and Neethling vaccines in 8 affected herds in Israel. Herd# 1 2 3 4 5 6 7 8 Total Total RRMH (CI95% )
x10RM65 vaccinated
Neethling vaccinated
131 160 267 52 308 208 149 265
146 124 239 248 276 214 154 136
1540
1537
x10RM65 morbidity (%)
Neethling morbidity (%)
RR (CI95% )
x10RM65 severe morbidity (%)
Neethling severe morbidity (%)
RR (CI95% )
7 (5.34%) 17 (10.6%) 1 (0.37%) 2 (3.84%) 4 (1.3%) 3 (1.44%) 0 (0%) 12 (4.52%)
2 (1.37%) 4 (3.22%) 10 (4.2%) 6 (2.41%) 3 (1.1%) 1 (0.46%) 2 (1.3%) 2 (1.47%)
3.9 3.3 0.088 1.6 1.18 3.13 0 3.1
0 (0%) 15 (9.37%) 1 (0.37%) 2 (3.8%) 2 (0.65%) 1 (0.48%) 0 (0%) 8 (3%)
0 (0%) 0 (0%) 4 (1.67%) 1 (0.4%) 1 (0.36%) 0 (0%) 1 (0.65%) 0 (0%)
0 – 0.22 9.5 1.8 – 0 –
46 (3%)
30 (2%)
1.5 1.49 (0.92–2.41)
29 (1.88%)
7 (0.45%)
4.18 3.65 (1.59–8.35)
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Fig. 2. Accumulation of lumpy skin disease cases (a) and severe cases (b) among cattle vaccinated with Neethling vaccine (green) and RM65 vaccine (blue). Cattle diagnosed with non-severe lumpy skin disease are censored. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
approximately the same (Table 2). The RRMH for morbidity among x10RM65 vaccinated cows compared to the Neethling vaccinated cows was 1.49 (0.92–2.41). The RRMH for severe cases was 3.65 (1.59–8.35). Observation of the dynamics of case accumulation revealed an interaction between the time elapsed from vaccination and incidence of all cases or severe cases (Fig. 2). The incidence among x10RM65 vaccinated cows was significantly lower during the first two weeks after vaccination (RRMH = 0.315, 0.11–0.91) (Table 3) while it was significantly higher two weeks after vaccination both for the occurrence of all cases (RRMH = 2.635, 1.44–4.82) and for the occurrence of severe cases (RRMH = 11.2, 2.3–54.7) (Table 3). Field strain of LSDV was identified in all the affected farms. Field strain viruses were identified from skin lesion biopsies and whole blood samples obtained from 17 and five x10RM65 vaccinated cows, respectively and from skin lesion biopsies and a whole blood sample obtained from five and one Neethling vaccinated cows, respectively. Adjusted relative risk in x10RM65 vs. Neethling vaccinated cows for morbidity caused by LSDV wild strain was RRMH = 4.28 (CI95% = 1.59–11.53) (Table 4). Neethling strain was identified from a total of 9 Neethling vaccinated cows in 4 farms. All were identified from skin lesion biopsies. Among these cases, one was classified as a severe case. These results indicate an overall incidence of Neethling associated disease of 0.38% (9/2356) and 0.04% (1/2356) of Neethling associated severe morbidity (Table 4). In all of these cases signs of illness appeared within 14 days from vaccination. 4. Discussion The RM65 strain based vaccine was used to prevent LSD in Israel after outbreaks which occurred in 1989, 2006 and 2007. However, the incidence in some herds vaccinated with this vaccine, at least once and up to 3 times prior to the outbreak of 2007 reached up to 44% [15]. This prior experience combined with the observation of high LSD incidence in RM65 vaccinated beef herds during the outbreak of 2012 indicated that the effectiveness of the RM65 vaccine was very limited and motivated the evaluation of the efficacy of other vaccines. In addition to the RM65 vaccine, which was used in Israel and other countries in the Middle-East, two other vaccines were used for prevention of lumpy skin disease; The Neethling LSDV attenuated vaccine used in southern Africa [16] and the O-240 capripox
vaccine, which was recently shown to actually be a strain of Lumpy skin disease virus [17], and was primarily used in Central Africa [18]. Prior vaccination of dairy herds with O-240 capripox vaccine was associated with the appearance of skin lesions similar to LSD [19]. The Israeli veterinary service, therefore refrained from using this vaccine and decided to use the Neethling attenuated vaccine and a 10× dose of the RM65 vaccine along with comparison of their efficacy. A recent study found a Gorgan goat pox based vaccine to provide protection from challenge by LSDV. However, extensive field experience with this vaccine is still lacking [20]. In this study the efficacy of the x10RM65 and the Neethling lumpy skin disease virus live attenuated vaccines were compared. Both were administered subcutaneously in an approximate virus dose of 103.5 TCID50 . The results of this study demonstrate a significantly higher efficacy of the attenuated Neethling vaccine for LSD prevention: The risk for a confirmed wild strain infection was more than 4 folds higher among x10RM65 vaccinated cows, compared to Neethling vaccinated cows (Table 4). During the first two weeks after vaccination, incidence among Neethling vaccinated cows seems higher. However, these cases can be mostly attributed either to infection by the vaccine strain (Table 4) or to infection by a wild strain prior to vaccination or prior to reaching full protection by the vaccine (as incubation time averages approximately 10 days [21]). The risk for LSD cases and severe cases appearing at least two weeks after vaccination was 2.635 and 11.2 folds higher, respectively among x10RM65 compared to Neethling vaccinated cows (Table 3). These results are opposed to the results of a recent study in which a Neethling based vaccine failed to protect vaccinated calves against challenge with LSDV. However, the vaccine in that study was manufactured by a different manufacturer (The Ethiopian National Veterinary Institute) and the authors suggest that its poor efficacy might be attributed to its poor immunogenicity, which might occurred due to over-attenuation [20]. The main event observed after Neethling vaccination was the development of a disease similar to LSD. This is opposed to the potential adverse effect described by the vaccine manufacturer, which is characterized by a swelling in vaccine injection site, appearing at least 4 days after vaccination (Lumpy Skin Disease Vaccine for Cattle, OBP® , vaccine label). In this study 0.38% (9/2356) of the Neethling vaccinated cows showed a confirmed Neethling associated disease, but only one cow showed severe illness (0.04%). All confirmed cases of Neethling associated disease appeared within a period of 14 days from vaccination. It should
Please cite this article in press as: Ben-Gera J, et al. Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.071
≤14 days from vaccination 1 2 3 4 5 6 7 8 Total Total RRMH (CI95% )
Total Total RRMH (CI95% )
Neethling vaccinated
x10RM65 morbidity (%)
131 160 267 52 308 208 149 265
146 124 239 248 276 214 154 136
1 (0.76%) 0 1 (0.37%) 0 2 (0.65%) 0 0 0
1540
1537
4 (0.26%)
131 160 267 52 308 208 149 265
146 124 239 248 276 214 154 136
6 (3.9%) 17 (10.6%) 0 2 (3.8%) 2 (0.64%) 3 (1.44%) 0 12 (4.5%)
1540
1537
42 (2.7%)
Neethling morbidity (%) 0 0 7 (3%) 6 (2.4%) 2 (0.75%) 0 2 (1.3%) 0 17 (1.1%)
2 (0.92%) 4 (3.2%) 3 (1.25%) 0 1 (0.36%) 1 (0.45%) 0 2 (1.47%) 13 (0.84%)
RR (CI95% )
x10RM65 severe morbidity (%)
Neethling severe morbidity (%)
RR (CI95% )
– – 1.12 – 0.86
0 0 1 (0.37%) 0 1 (0.32%) 0 0 0
0 0 4 (1.67%) 1 (0.24%) 0 0 1 (0.65%) 0
– – 0.22 – –
0.23 0.315 (0.11–91)
2 (0.13%)
6 (0.4%)
0.32 0.34 (0.06–1.79)
4.23 3.3 – – 1.78 3.2 – 3.06
0 8 (5%) 0 2 (3.8%) 1 (0.32%) 1 (0.49%) 0 8 (3%)
0 0 0 0 1 (0.36%) 0 0 0
– – – – 0.88 – – –
3.2 2.635 (1.44–4.82)
20 (1.3%)
1 (0.06%)
21.67 11.2 (2.3–54.7)
Table 4 Relative risk (RR) and Mantel–Haenszel relative risk (RRMH ) for lumpy skin disease morbidity caused by wild strain virus among cows vaccinated with x10RM65 and Neethling vaccines in 8 affected herds in Israel. Vaccine herd#
x10RM65
Attempted virus DNA detectiona /sick 1 2 3 4 5 6 7 8
6/7 6/17 1/1 2/2 3/4 2/3 0/0 9/12
Total Total RRMH (CI95% )
30/46
a b c
Neethling
Wild strain virus identificationb /vaccinated (% incidence) 4 (3%) 5 (3.1%) 1 (0.37%) 2 (3.8%) 1 (0.32%) 2 (1%) 0 7 (2.64%) 22 (1.43%)
RR (CI95% ) for wild strain identificationb
Attempted virus DNA detectiona /sick
Wild strain virus identificationb /vaccinated (% incidence)
Neethling virus identificationb /vaccinated (% incidence)
2/2 1/4 5/10 6/6 3/3 0/1 2/2 0/2
1 (0.7%) 1 (0.8%) 0 2 (0.8%) 1 (0.36%) 0 1 (0.65%) 0
0 0 3 (1.25%) 3 (1.2%) 2 (0.72%) 0 1 (0.65%) 0
4.28 3.87 – 4.75 0.88 – – –
19/30
6 (0.4%)
9 (0.38%c )
3.57 4.28 (1.59–11.53)
Number of samples obtained for virus DNA detection by PCR (i.e. attempted virus DNA detection) and for further virus identification out of the total number of cases appearing in each vaccinated group. Identification was performed as described by Menasherow et al. [16]. Calculated out of all the Neethling vaccinated cows (n = 2356).
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>14 days from vaccination 1 2 3 4 5 6 7 8
x10RM65 vaccinated
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Please cite this article in press as: Ben-Gera J, et al. Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.071
Table 3 Relative risk (RR) and Mantel–Haenszel relative risk (RRMH ) for lumpy skin disease morbidity among cows vaccinated with x10RM65 and Neethling vaccines in 8 affected herds in Israel, within 14 days post vaccine and 15 days onwards.
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be mentioned, however, that only 19/30 (63%) of the cases among Neethling vaccinated cows were sampled (Table 4). Thus, actual incidence of Neethling related disease could reach 0.6%. No case of Neethling associated disease appeared in cows which were not vaccinated with the Neethling vaccine, indicating that transmission of the vaccine strain between animals was not observed during this study. Considering these findings it seems that adverse effects after vaccination with the attenuated Neethling strain are rare and its use is therefore relatively safe. In the current study no adverse effects were observed after vaccinating with the x10RM65 vaccine. The main strengths of the current study stem from its design. This is a prospective randomized study and therefore confounding and information biases are minimized. Attempt for virus DNA detection by PCR in 64.5% of the clinical cases further supports the results of the study and increases the specificity of case identification. Despite this, lack of sensitivity due to lower awareness of some of the herdsmen and the veterinarians might always exist. Such a bias could be the cause for higher disease incidence observed in cows when compared to calves and heifers. As milked cows may have been under better surveillance the sensitivity for detection of disease among this population might have been higher. Reduced sensitivity can also be the result of the conservative case definition we chose (i.e. at least five skin lesions as a minimal definition for a clinical disease). A relatively specific case definition was prefered because estimation of vaccine efficacy was the main goal of this study and under conditions of rare morbidity (as occurred in this study), specificity is more important than sensitivity in determining bias in the estimate of relative risk (and the resultant vaccine efficacy) [22]. Another explanation can be that vaccinated calves and heifers are less susceptible to disease, compared to high producing cows, which may be in higher stress. This finding is in agreement with the finding of Magori-Cohen et al. [21] who found a marginally statistically significant association between lactation and higher risk of LSD morbidity during an LSD outbreak in a dairy farm (i.e. p = 0.053). As opposed to these findings, the results of a retrospective study performed in Ethiopia found higher susceptibility of calves to LSD [18]. Another potential limitation of the study is the fact that all herds were vaccinated 3–5 months prior to the study with an RM-65 vaccine in a regular dose. Though prior experience indicates that this vaccine, administered in a dose of 102.5 TCID is not effective for prevention of LSD, its prior administration could have resulted in a booster effect to vaccination by the x10RM65 and the Neethling vaccines. While the results of this study do not support nor refute the effectiveness of the x10RM65 vaccine for disease prevention they clearly show that the Neethling vaccine is highly effective in protection from LSD. As during the 2012–2013 Israeli LSD epidemic almost all of the dairy herds were vaccinated with the Neethling vaccine, the results of this study may explain the successful control of LSD in these herds in Israel (last confirmed case of LSD in Israel to date was documented on August 29th, 2013). Beef herds, which were almost entirely vaccinated by the x10RM65 vaccine were already highly affected when vaccinated. Thus, it is more difficult to assess the contribution of the x10RM65 vaccine to the control of the disease in Israel. 5. Conclusion Though the current study supports the use of the Neethling vaccine for protection against lumpy skin disease, its use should be considered against the potential adverse effects associated with its
administration and the risk of inserting an actual disease agent into a potentially naïve country. Choosing the right vaccine should be based on consideration of the advantages and disadvantages of each vaccine and tailored to the specific situation in each country. Acknowledgments This study was supported by a grant from the Israeli Veterinary Services. Conflict of interests: The authors do not have any conflict of interest to declare. References [1] Tuppurainen ES, Oura CA. Lumpy skin disease: an emerging threat to Europe, the Middle East and Asia (Review). Transbound Emerg Dis 2011. [2] Chihota CM, Rennie LF, Kitching RP, Mellor PS. Mechanical transmission of lumpy skin disease virus by Aedes aegypti (Diptera: Culicidae). Epidemiol Infect 2001;126:317–21. [3] Lubinga JC, Tuppurainen ES, Stoltsz WH, Ebersohn K, Coetzer JA, Venter EH. Detection of lumpy skin disease virus in saliva of ticks fed on lumpy skin disease virus-infected cattle. Exp Appl Acarol 2013;61:129–38. [4] Tuppurainen ES, Lubinga JC, Stoltsz WH, Troskie M, Carpenter ST, Coetzer JA, et al. Evidence of vertical transmission of lumpy skin disease virus in Rhipicephalus decoloratus ticks. Ticks Tick Borne Dis 2013;4:329–33. [5] Tuppurainen ES, Lubinga JC, Stoltsz WH, Troskie M, Carpenter ST, Coetzer JA, et al. Mechanical transmission of lumpy skin disease virus by Rhipicephalus appendiculatus male ticks. Epidemiol Infect 2013;141:425–30. [6] Lubinga JC, Clift SJ, Tuppurainen ES, Stoltsz WH, Babiuk S, Coetzer JA, et al. Demonstration of lumpy skin disease virus infection in Amblyomma hebraeum and Rhipicephalus appendiculatus ticks using immunohistochemistry. Ticks Tick Borne Dis 2014;5:113–20. [7] Lubinga JC, Tuppurainen ES, Coetzer JA, Stoltsz WH, Venter EH. Evidence of lumpy skin disease virus over-wintering by transstadial persistence in Amblyomma hebraeum and transovarial persistence in Rhipicephalus decoloratus ticks. Exp Appl Acarol 2014;62:77–90. [8] Lubinga JC, Tuppurainen ES, Mahlare R, Coetzer JA, Stoltsz WH, Venter EH. Evidence of transstadial and mechanical transmission of lumpy skin disease virus by Amblyomma hebraeum ticks. Transbound Emerg Dis 2015;62:174–82. [9] Yeruham I, Nir O, Braverman Y, Davidson M, Grinstein H, Haymovitch M, et al. Spread of lumpy skin disease in Israeli dairy herds. Vet Rec 1995;137:91–3. [10] Brenner J, Bellaiche M, Gross E, Elad D, Oved Z, Haimovitz M, et al. Appearance of skin lesions in cattle populations vaccinated against lumpy skin disease: statutory challenge. Vaccine 2009;27:1500–3. [11] Klausner Z, Fattal E, Klement E. Using synoptic systems’ typical wind trajectories for the analysis of potential atmospheric long distance dispersal of lumpy skin disease virus. Transbound Emerg Dis 2015. [12] Menasherow S, Rubinstein-Giuni M, Kovtunenko A, Eyngor Y, Fridgut O, Rotenberg D, et al. Development of an assay to differentiate between virulent and vaccine strains of lumpy skin disease virus (LSDV). J Virol Methods 2014;199:95–101. [13] Abramson JH. WINPEPI (PEPI-for-Windows): computer programs for epidemiologists. Epidemiol Perspect Innov 2004;1:6. [14] Abramson JH. WINPEPI updated: computer programs for epidemiologists, and their teaching potential. Epidemiol Perspect Innov 2011;8:1. [15] (AHAW) EPoAHaW. Scientific Opinion on lumpy skin disease. EFSA J 2015;13:3986. [16] Hunter P, Wallace D. Lumpy skin disease in southern Africa: a review of the disease and aspects of control. J S Afr Vet Assoc 2001;72:68–71. [17] Tuppurainen ES, Pearson CR, Bachanek-Bankowska K, Knowles NJ, Amareen S, Frost L, et al. Characterization of sheep pox virus vaccine for cattle against lumpy skin disease virus. Antiviral Res 2014;109:1–6. [18] Ayelet G, Haftu R, Jemberie S, Belay A, Gelaye E, Sibhat B, et al. Lumpy skin disease in cattle in central Ethiopia: outbreak investigation and isolation and molecular detection of the virus. Rev Sci Tech 2014;33:877–87. [19] Yeruham I, Perl S, Nyska A, Abraham A, Davidson M, Haymovitch M, et al. Adverse reactions in cattle to a capripox vaccine. Vet Rec 1994;135:330–2. [20] Gari G, Abie G, Gizaw D, Wubete A, Kidane M, Asgedom H, et al. Evaluation of the safety, immunogenicity and efficacy of three capripoxvirus vaccine strains against lumpy skin disease virus. Vaccine 2015;33:3256–61. [21] Magori-Cohen R, Louzoun Y, Herziger Y, Oron E, Arazi A, Tuppurainen E, et al. Mathematical modelling and evaluation of the different routes of transmission of lumpy skin disease virus. Vet Res 2012;43:1. [22] Thrusfield M. Observational studies. Veterinary epidemiology. 3rd ed. UK: Blackwell Science; 2005. p. 266–88.
Please cite this article in press as: Ben-Gera J, et al. Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease – The results of a randomized controlled field study. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.07.071