**Annex 12. Item 5.1 – Chapter 3.4.12 Lumpy Skin Disease**

###### 2	MEETING OF THE WOAH BIOLOGICAL STANDARDS COMMISSION

3	**Paris, 4–8 September 2023**

4

5	**CHAP T E R  3 . 4 . 1 2 .**

# 6	L U M PY SK I N D I SEASE 

7	**SUMMARY**

8 ***Description of the disease:****Lumpy skin disease*(*LSD*) *is a poxvirus disease of cattle characterised by*

9 *fever, nodules on the skin, mucous membranes and internal organs, emaciation, enlarged lymph nodes,*

10 *oedema of the skin, and sometimes death. The disease is of economic importance as it can cause a*

11 *temporary reduction in milk production, temporary or permanent sterility in bulls, damage to hides and,*

12 *occasionally, death*. *Various strains of capripoxvirus are responsible for the disease. These are antigenically*

13 *indistinguishable from strains causing sheep pox and goat pox yet distinct at the genetic level. LSD has a*

14 *partially different geographical distribution from sheep and goat pox, suggesting that cattle strains of*

15 *capripoxvirus do not infect and transmit between sheep and goats. Transmission of LSD virus*(*LSDV*) *is*

16 *thought to be predominantly by arthropods, natural contact transmission in the absence of vectors being*

17 *inefficient. Lumpy skin disease is endemic in most~~many~~African and Middle Eastern countries. Between*

18 *2012 and 2022, LSD spread into south-east Europe, the Balkans, Russia and Asia as part of the Eurasian*

19 *LSD epidemic.*

20 ***Pathology:****the nodules are firm, and may extend to the underlying subcutis and muscle. Acute histological*

21 *key lesions consist of epidermal vacuolar changes with intracytoplasmic inclusion bodies and dermal*

22 *vasculitis. Chronic key histological lesions consist of fibrosis and necrotic sequestrae.*

23 ***Detection of the agent:****Laboratory confirmation of LSD is most rapid using a real-time or conventional*

24 *polymerase chain reaction*(*PCR*) *method specific for capripoxviruses in combination with a clinical history*

25 *of a generalised nodular skin disease and enlarged superficial lymph nodes in cattle. Ultrastructurally,*

26 *capripoxvirus virions are distinct from those of parapoxvirus, which causes bovine papular stomatitis and*

27 *pseudocowpox, but cannot be distinguished morphologically from orthopoxvirus virions, including cowpox*

28 *and vaccinia viruses, both of which can cause disease in cattle, although neither causes generalised*

29 *infection and both are uncommon in cattle. LSDV will grow in tissue culture of bovine, ovine or caprine origin.*

30 *In cell culture, LSDV causes a characteristic cytopathic effect and intracytoplasmic inclusion bodies that is*

31 *distinct from infection with Bovine herpesvirus 2, which causes pseudo-lumpy skin disease and produces*

32 *syncytia and intranuclear inclusion bodies in cell culture. Capripoxvirus antigens can be demonstrated in*

33 *tissue culture using immunoperoxidase or immunofluorescent staining and the virus can be neutralised using*

34 *specific antisera.*

35 *A variety of conventional and real-time PCR tests as well as isothermal amplification tests using*

36 *capripoxvirus-specific primers have been published for use on a variety of samples.*

37 ***Serological tests:****The virus neutralisation test*(*VNT*) *and enzyme-linked immunosorbent assays*(*ELISAs*)

38 *are widely used and have been validated. The agar gel immunodiffusion test and indirect immunofluorescent*

39 *antibody test are less specific than the VNT due to cross-reactions with antibody to other poxviruses.*

40 *Western blotting using the reaction between the P32 antigen of LSDV with test sera is both sensitive and*

41 *specific, but is difficult and expensive to carry out.*

42 ***Requirements for vaccines:****All strains of capripoxvirus examined so far, whether derived from cattle,*

43 *sheep or goats, are antigenically similar. Attenuated cattle strains, and strains derived from sheep and goats*

44 *have been used as live vaccines against LSDV.*

## 45	A. INTRODUCTION

46 Lumpy skin disease (LSD) was first seen in Zambia in 1929, spreading into Botswana by 1943 (Haig, 1957), and then into

47 South Africa the same year, where it affected over eight million cattle causing major economic loss. In 1957 it entered

48 Kenya, ~~at the same time as~~associated with an outbreak of sheep pox (Weiss, 1968). In 1970 LSD spread north into the

49 Sudan, by 1974 it had spread west as far as Nigeria, and in 1977 was reported from Mauritania, Mali, Ghana and Liberia.

50 Another epizootic of LSD between 1981 and 1986 affected Tanzania, Kenya, Zimbabwe, Somalia and the Cameroon, with

51 reported mortality rates in affected cattle of 20%. The occurrence of LSD north of the Sahara desert and outside the African

52 continent was confirmed for the first time in Egypt and Israel between 1988 and 1989, and was reported again in 2006

53 (Brenner *et al.,*2006). In the past decade, LSD occurrences have been reported in the Middle Eastern, European and

54 Asian regions (for up-to-date information, consult WOAH WAHIS interface 40). Lumpy skin disease outbreaks tend to be

55 sporadic, depending upon animal movements, immune status, and wind and rainfall patterns affecting vector populations.

56 The principal method of transmission is thought to be mechanical by various arthropod vectors (Tuppurainen *et al*., 2015).

57 Lumpy skin disease virus (LSDV) belongs to the family *Poxviridae*, subfamily *~~Chordopoxvirinae~~Chordopoxviridae*, and

58 genus *Capripoxvirus*. In common with other poxviruses LSDV replicates in the cytoplasm of an infected cell, forming distinct

59 perinuclear viral factories. The LSD virion is large and brick-shaped measuring 293–299nm (length) and 262–273nm

60 (width). The LSDV genome structure is also similar to other poxviruses, consisting of double-stranded linear DNA that is

61 25% GC-rich, approximately 150,000 bp in length, and encodes around 156 open reading frames (ORFs). An inverted

62 terminal repeat sequence of 2200–2300 bp is found at each end of the linear genome. The linear ends of the genome are

63 joined with a hairpin loop. The central region of the LSDV genome contains ORFs predicted to encode proteins required

64 for virus replication and morphogenesis and exhibit a high degree of similarity with genomes of other mammalian

65 poxviruses. The ORFs in the outer regions of the LSDV genome have lower similarity and likely encode proteins involved

66 in viral virulence and host range determinants.

67 Phylogenetic analysis shows the majority of LSDV strains group into two monophyletic clusters (cluster 1.1 and 1.2)

68 (Biswas *et al.,*2020; Van Schalkwyk *et al.,*2021). Cluster 1.1 consists of LSDV Neethling vaccine strains that are based

69 on the LSDV/Neethling/LW-1959 vaccine strain (Kara *et al.,*2003; Van Rooyen *et al.,*1959; van Schalkwyk *et al.,*2020)

70 and historic wild-type strains from South Africa. Cluster 1.2 consists of wild-type strains from southern Africa, Kenya, the

71 northern hemisphere, and the Kenyan KSGP O-240 commercial vaccine. In addition to these two clusters, there have

72 recently been recombinant LSDV strains isolated from clinical cases of LSD in the field in Russia and central Asia (Flannery

73 *et al.,*2021; Sprygin *et al.,*2018; 2020; Wang *et al.,*2021). These recombinant viruses show unique patterns of accessory

74 gene alleles, consisting of sections of both wild-type and “vaccine” LSDV strains.

75 The severity of the clinical signs of LSD is highly variable and depends on a number of factors, including the strain of

76 capripoxvirus the age of the host, immunological status and breed. *Bos taurus*is generally more susceptible to clinical disease

77 than *Bos indicus;*the Asian buffalo (*Bubalus spp.*) has also been reported to be susceptible. Within *Bos taurus,*the fine-

78 skinned Channel Island breeds develop more severe disease, with lactating cows appearing to be the most at risk. However,

79 even among groups of cattle of the same breed kept together under the same conditions, there is a large variation in the

80 clinical signs presented, ranging from subclinical infection to death (Carn & Kitching, 1995). There may be failure of the virus

81 to infect the whole group, probably depending on the virulence of the virus isolate, immunological status of the host, host

82 genotype, and vector prevalence. Seroprevalence studies, experimental infections and case reports have provided indications

83 that several wildlife species (e.g. springbok, impala, giraffe, camel, banteng) are susceptible to LSDV infection (Dao *et al.,*

84 2022; Hedger & Hamblin, 1983; Kumar *et al.,*2023; Porco *et al.,*2023). The scarcity of documented outbreaks in wildlife and

85 the fact that available studies remain limited in number and mostly involve only a few animals, make it difficult to determine

86 the role of wildlife in LSDV epidemiology. This topic deserves further study, especially given the current spread of LSDV in

87 new geographical areas where large numbers of naïve, potentially susceptible wild bovines and other ruminants are present.

88 The incubation period under field conditions has not been reported, but following experimental inoculation is 6–9 days until

89 the onset of fever. In the acutely infected animal, there is an initial pyrexia, which may exceed 41°C and persist for 1 week.

90 All the superficial lymph nodes become enlarged. In lactating cattle there is a marked reduction in milk yield. Lesions

91 develop over the body, particularly on the head, neck, udder, scrotum, vulva and perineum between 7 and 19 days after

92 virus inoculation (Coetzer, 2004). The characteristic integumentary lesions are multiple, well circumscribed to coalescing,

93 0.5–5 cm in diameter, firm, flat-topped papules and nodules. The nodules involve the dermis and epidermis, and may

1 https://www.woah.org/en/what-we-do/animal-health-and-welfare/disease-data-collection/

94 extend to the underlying subcutis and occasionally to the adjacent striated muscle. These nodules have a creamy grey to

95 white colour on cut section, which may initially exude serum, but over the ensuing 2 weeks a cone-shaped central core or

96 sequestrum of necrotic material/necrotic plug (“sit-fast”) may appear within the nodule. The acute histological lesions

97 consist of epidermal vacuolar changes with intracytoplasmic inclusion bodies and dermal vasculitis. The inclusion bodies

98 are numerous, intracytoplasmic, eosinophilic, homogenous to occasionally granular and they may occur in endothelial

99 cells, fibroblasts, macrophages, pericytes, and keratinocytes. The dermal lesions include vasculitis with fibrinoid necrosis,

100 oedema, thrombosis, lymphangitis, dermal-epidermal separation, and mixed inflammatory infiltrate. The chronic lesions

101 are characterised by an infarcted tissue with a sequestered necrotic core, often rimmed by granulation tissue gradually

102 replaced by mature fibrosis. At the appearance of the nodules, the discharge from the eyes and nose becomes

103 mucopurulent, and keratitis may develop. Nodules may also develop in the mucous membranes of the mouth and

104 alimentary tract, particularly the abomasum and in the trachea and the lungs, resulting in primary and secondary

105 pneumonia. The nodules on the mucous membranes of the eyes, nose, mouth, rectum, udder and genitalia quickly

106 ulcerate, and by then all secretions, ocular and nasal discharge and saliva contain LSD virus (LSDV). The limbs may be

107 oedematous and the animal is reluctant to move. Pregnant cattle may abort, and there is a report of intrauterine

108 transmission (Rouby & Aboulsoudb, 2016). Bulls may become permanently or temporarily infertile and the virus can be

109 excreted in the semen for prolonged periods (Irons *et al*., 2005). Recovery from severe infection is slow; the animal is

110 emaciated, may have pneumonia and mastitis, and the necrotic plugs of skin, which may have been subject to fly strike,

111 are shed leaving deep holes in the hide (Prozesky & Barnard, 1982).

112 The main differential diagnosis is pseudo-LSD caused by bovine herpesvirus 2 (BoHV-2). This is usually a milder clinical

113 condition, characterised by superficial nodules, resembling only the early stage of LSD. Intra-nuclear inclusion bodies and

114 viral syncytia are histopathological characteristics of BoHV-2 infection not seen in LSD. Other differential diagnoses (for

115 integumentary lesions) include: dermatophilosis, dermatophytosis, bovine farcy, photosensitisation, actinomycosis,

116 actinobacillosis, urticaria, insect bites, besnoitiosis, nocardiosis, demodicosis, onchocerciasis, pseudo-cowpox, and

117 cowpox. Differential diagnoses for mucosal lesions include: foot and mouth disease, bluetongue, mucosal disease,

118 malignant catarrhal fever, infectious bovine rhinotracheitis, and bovine papular stomatitis.

119 LSDV is not transmissible to humans. However, all laboratory manipulations must be performed at an appropriate

120 containment level determined using biorisk analysis (see Chapter 1.1.4 *Biosafety and biosecurity: Standard for managing*

121 *biological risk in the veterinary laboratory and animal facilities*)*.*

## 122	B. DIAGNOSTIC TECHNIQUES

123	***Table 1.****Test methods available for the diagnosis of LSD and their purpose*

124	Key: +++ = recommended for this purpose; ++ recommended but has limitations;

125	+ = suitable in very limited circumstances; – = not appropriate for this purpose.

126 PCR = polymerase chain reaction; TEM = Transmission electron microscopy; VNT = virus neutralisation test;

127 IFAT = indirect fluorescent antibody test; ELISA = enzyme-linked immunosorbent assay.

#### 128 1.	Detection of the agent

##### 129 1.1.	Specimen collection, submission and preparation

130 Material for virus isolation and antigen detection should be collected as biopsies or from skin nodules at post-mortem

131 examination. Samples for virus isolation should preferably be collected within the first week of the occurrence of

132 clinical signs, before the development of neutralising antibodies (Davies, 1991; Davies *et al*., 1971), however virus

133 can be isolated from skin nodules for at least 3–4 weeks thereafter. Samples for genome detection using conventional

134 or real-time polymerase chain reaction (PCR) may be collected when neutralising antibody is present. Following the

135 first appearance of the skin lesions, the virus can be isolated for up to 35 days and viral nucleic acid can be

136 demonstrated via PCR for up to 3 months (Tuppurainen *et al*., 2005; Weiss, 1968). Buffy coat from blood collected

137 into heparin or EDTA (ethylene diamine tetra-acetic acid) during the viraemic stage of LSD (before generalisation of

138 lesions or within 4 days of generalisation), can also be used for virus isolation. Samples for histology should include

139 the lesion and tissue from the surrounding (non-lesion) area, be a maximum size of 2 cm3, and be placed immediately

140 following collection into ten times the sample volume of 10% neutral buffered formal saline.

141 Tissues in formalin have no special transportation requirements in regard to biorisks. Blood samples with

142 anticoagulant for virus isolation from the buffy coat should be placed immediately on ice after gentle mixing and

143 processed as soon as possible. In practice, the samples may be kept at 4°C for up to 2 days prior to processing, but

144 should not be frozen or kept at ambient temperatures. Tissues for virus isolation and antigen detection should be kept

145 at 4°C, on ice or at –20°C. If it is necessary to transport samples over long distances without refrigeration, the medium

146 should contain 10% glycerol; the samples should be of sufficient size (e.g. 1 g in 10 ml) that the transport medium

147 does not penetrate the central part of the biopsy, which should be used for virus isolation.

148 ~~Samples for histology should include the lesion and tissue from the surrounding (non-lesion) area, be a maximum~~

149 ~~size of 2 cm~~3~~, and be placed immediately following collection into ten times the sample volume of 10% neutral buffered~~

150 ~~formaldehyde. Tissues in formalin have no special transportation requirements in regard to biorisks.~~Material for

151 histology should be prepared using standard techniques and stained with haematoxylin and eosin (H&E) (Burdin,

152 1959). Lesion material for virus isolation and antigen detection is minced using a sterile scalpel blade and forceps

153 and then macerated in a sterile steel ball-bearing mixer mill, or ground with a pestle in a sterile mortar with sterile

154 sand and an equal volume of sterile phosphate buffered saline (PBS) or serum-free modified Eagle’s medium

155 containing sodium penicillin (1000 international units [IU]/ml), streptomycin sulphate (1 mg/ml), mycostatin

156 (100 IU/ml) or fungizone (amphotericin, 2.5 μg/ml) and neomycin (200 IU/ml). The suspension is freeze–thawed three

157 times and then partially clarified using a bench centrifuge at 600 ***g***for 10 minutes. In cases where bacterial

158 contamination of the sample is expected (such as when virus is isolated from skin samples), the supernatant can be

159 filtered through a 0.45 μm pore size filter after the centrifugation step. Buffy coats may be prepared from unclotted

160 blood using centrifugation at 600 ***g***for 15 minutes, and the buffy coat carefully removed into 5 ml of cold double-

161 distilled water using a sterile Pasteur pipette. After 30 seconds, 5 ml of cold double-strength growth medium is added

162 and mixed. The mixture is centrifuged at 600 ***g***for 15 minutes, the supernatant is discarded and the cell pellet is

163 suspended in 5 ml growth medium, such as Glasgow’s modified Eagle’s medium (GMEM). After centrifugation at

164 600 ***g***for a further 15 minutes, the resulting pellet is suspended in 5 ml of fresh GMEM. Alternatively, the buffy coat

165 may be separated from a heparinised sample by using a Ficoll gradient.

##### 166 1.2.	Virus isolation on cell culture

167 LSDV will grow in tissue culture of bovine, ovine or caprine origin. MDBK (Madin–Darby bovine kidney) cells are often

168 used, as they support good growth of the virus and are well characterised (Fay *et al.,*2020). Primary cells, such as

169 lamb testis (LT) cells also support viral growth, but care needs to be taken to ensure they are not contaminated with

170 viruses such as bovine viral diarrhoea virus. One ml of clarified supernatant or buffy coat is inoculated onto a confluent

171 monolayer in a 25 cm2 culture flask at 37°C and allowed to adsorb for 1 hour. The culture is then washed with warm

172 PBS and covered with 10 ml of a suitable medium, such as GMEM, containing antibiotics and 2% fetal calf serum. If

173 available, tissue culture tubes containing appropriate cells and a flying cover-slip, or tissue culture microscope slides,

174 are also infected.

175 The flasks/tissue culture tubes are examined daily for 7–14 days for evidence of cytopathic effects (CPE). Infected

176 cells develop a characteristic CPE consisting of retraction of the cell membrane from surrounding cells, and eventually

177 rounding of cells and margination of the nuclear chromatin. At first only small areas of CPE can be seen, sometimes

178 as soon as 2 days after infection; over the following 4–6 days these expand to involve the whole cell monolayer~~sheet~~.

179 If no CPE is apparent by day 14, the culture should be freeze–thawed three times, and clarified supernatant inoculated

180 on to a fresh cell monolayer. At the first sign of CPE in the flasks, or earlier if a number of infected cover-slips are

181 being used, a cover-slip should be removed, fixed in acetone and stained using H&E. Eosinophilic intracytoplasmic

182 inclusion bodies, which are variable in size but up to half the size of the nucleus and surrounded by a clear halo, are

183 diagnostic for poxvirus infection. PCR may be used as an alternative to H&E for confirmation of the diagnosis. The

184 CPE can be prevented or delayed by adding specific anti-LSDV serum to the medium. In contrast, the herpesvirus

185 that causes pseudo-LSD produces a Cowdry type A intranuclear inclusion body. It also forms syncytia.

186 An ovine testis cell line (OA3.T~~s~~) has been evaluated for the propagation of capripoxvirus isolates (Babiuk *et al.*,

187 2007), however this cell line has been found to be contaminated with pestivirus and should be used with caution.

##### 188 1.3.	Polymerase chain reaction (PCR)

189 The conventional gel-based PCR method described below is a simple, fast and sensitive method for the detection of

190 capripoxvirus genome in EDTA blood, semen or tissue culture samples (Tuppurainen *et al*., 2005).

###### 191 1.3.1.	Test procedure

192 The extraction method described below can be replaced using commercially available DNA extraction

193 kits.

194 i)	Freeze and thaw 200 μl of blood in EDTA, semen or tissue culture supernatant and suspend in

195 100 μl of lysis buffer containing 5 M guanidine thiocyanate, 50 mM potassium chloride, 10 mM

196 Tris/HCl (pH 8); and 0.5 ml Tween 20.

197 ii)	Cut skin and other tissue samples into fine pieces using a sterile scalpel blade and forceps.

198 Grind with a pestle in a mortar. Suspend the tissue samples in 800 μl of the above mentioned

199 lysis buffer.

200 iii)	Add 2 μl of proteinase K (20 mg/ml) to blood samples and 10 μl of proteinase K (20 mg/ml) to

201 tissue samples. Incubate at 56°C for 2 hours or overnight, followed by heating at 100°C for

202 10 minutes. Add phenol:chloroform:isoamylalcohol (25:24:1 [v/v]) to the samples in a 1:1 ratio.

203 Vortex and incubate at room temperature for 10 minutes. Centrifuge the samples at 16,060 ***g***

204 for 15 minutes at 4°C. Carefully collect the upper, aqueous phase (up to 200 μl) and transfer

205 into a clean 2.0 ml tube. Add two volumes of ice cold ethanol (100%) and 1/10 volume of 3 M

206 sodium acetate (pH 5.3). Place the samples at –20°C for 1 hour. Centrifuge again at 16,060 ***g***

207 for 15 minutes at 4°C and discard the supernatant. Wash the pellets with ice cold 70% ethanol

208 (100 μl) and centrifuge at 16,060 ***g***for 1 minute at 4°C. Discard the supernatant and dry the

209 pellets thoroughly. Suspend the pellets in 30 μl of nuclease-free water and store immediately at

210 –20°C (Tuppurainen *et al*., 2005). Alternatively a column-based extraction kit may be used.

211 iv)	The primers for this PCR assay were developed from the gene encoding the viral attachment

212 protein. The size of the expected amplicon is 192 bp (Ireland & Binepal, 1998). The primers

213 have the following gene sequences:

214 Forward primer 5’-TCC-GAG-CTC-TTT-CCT-GAT-TTT-TCT-TAC-TAT-3’

215 Reverse primer 5’-TAT-GGT-ACC-TAA-ATT-ATA-TAC-GTA-AAT-AAC-3’.

216 v)	DNA amplification is carried out in a final volume of 50 μl containing: 5 μl of 10 × PCR buffer,

217 1.5 μl of MgCl2 (50 mM), 1 μl of dNTP (10 mM), 1 μl of forward primer, 1 μl of reverse primer,

218 1 μl of DNA template (~10 ng), 0.5 μl of *Taq*DNA polymerase and 39 μl of nuclease-free water.

219 The volume of DNA template required may vary and the volume of nuclease-free water must

220 be adjusted to the final volume of 50 μl.

221 vi)  Run the samples in a thermal cycler as follows: 2 minutes at 95°C; then 45 seconds at 95°C,

222 50 seconds at 50°C and 1 minute at 72°C (34 cycles); 2 minutes at 72°C and hold at 4°C until

223 analysis.

224 vii) Mix 10 μl of each sample with loading dye and load onto a 1.5% agarose gel in TAE buffer

225 (Tris/acetate buffer containing EDTA). Load a parallel lane with a 100 bp DNA-marker ladder.

226 Electrophoretically separate the products using approximately 8–10 V/cm for 40–60 minutes

227 and visualise with a suitable DNA stain and transilluminator.

228 Quantitative real-time PCR methods have been described that are reported to be faster and have

229 higher sensitivity than conventional PCRs (Balinsky *et al*., 2008; Bowden *et al*., 2008). A real-time

230 PCR method that differentiates between LSDV, sheep pox virus and goat pox virus has been

231 published (Lamien *et al.,*2011).

232 Quantitative real-time PCR assays have been designed to differentiate between Neethling-based

233 LSDV strains, which are often used for vaccination, and wild-type LSDV strains from cluster 1.2

234 (Agianniotaki *et al.,*2017; Pestova *et al.,*2018; Vidanovic *et al.,*2016). These “DIVA” assays (DIVA:

235 differentiation of infected from vaccinated animals) enable, for example, differentiation of “Neethling

236 response” caused by vaccination with a LSDV Neethling vaccine strain from disease caused by

237 infection with a cluster 1.2 wild-type virus. However these DIVA PCR assays cannot distinguish

238 between a LSDV Neethling vaccine strain and the novel recombinant LSDV strains ~~recently~~isolated

239 from disease outbreaks in Asia (~~Byadovskaya *et al.,*2021;~~ Flannery *et al.,*2021). These DIVA assays

240 are also not capable of discriminating between LSDV Neethling vaccine strains and recently

241 characterised (historic) wild-type viruses from South Africa belonging within cluster 1.1 (Van

242 Schalkwyk *et al.,*2020; 2021). Consequently, in regions where recombinant strains (currently Asia

243 and possibly elsewhere) or wild-type cluster 1.1 strains are circulating (currently South Africa and

244 possibly elsewhere), these DIVA assays are not suitable for distinguishing vaccine and wild-type

245 virus. Thus, in order to overcome these constraints, whole genome sequencing is recommended.

##### 246 1.4.	Transmission electron microscopy

247 The characteristic poxvirus virion can be visualised using a negative staining preparation technique followed by

248 examination with an electron microscope. There are many different negative staining protocols, an example of which

249 is given below.

###### 250 1.4.1.	Test procedure

251 Before centrifugation, material from the original biopsy suspension is prepared for examination under

252 the transmission electron microscope by floating a 400-mesh hexagonal electron microscope grid,

253 with pioloform-carbon substrate activated by glow discharge in pentylamine vapour, onto a drop of

254 the suspension placed on parafilm or a wax plate. After 1 minute, the grid is transferred to a drop of

255 Tris/EDTA buffer, pH 7.8, for 20 seconds and then to a drop of 1% phosphotungstic acid, pH 7.2, for

256 10 seconds. The grid is drained using filter paper, air-dried and placed in the electron microscope.

257 The capripox virion is brick shaped, covered in short tubular elements and measures approximately

258 290 × 270 nm. A host-cell-derived membrane may surround some of the virions, and as many as

259 possible should be examined to confirm their appearance (Kitching & Smale, 1986).

260 The ~~capripox~~virions of capripoxvirus are indistinguishable from those of orthopoxvirus, but, apart

261 from vaccinia virus and cowpox virus, which are both uncommon in cattle and do not cause

262 generalised infection, no other orthopoxvirus is known to cause lesions in cattle. However, vaccinia

263 virus may cause generalised infection in young immunocompromised calves. In contrast,

264 orthopoxviruses are a common cause of skin disease in domestic buffalo (*Bubalus bubalis*) causing

265 buffalo pox, a disease that usually manifests as pock lesions on the teats, but may cause skin lesions

266 at other sites, such as the perineum, the medial aspects of the thighs and the head. Orthopoxviruses

267 that cause buffalo pox cannot be readily distinguished from capripoxvirus by electron microscopy.

268 The virions of parapoxvirus ~~virions~~that cause bovine papular stomatitis and pseudocowpox are

269 smaller, oval in shape and each is covered in a single continuous tubular element that appears as

270 striations over the virion. Capripoxvirus virions are also distinct from the herpesvirus that causes

271 pseudo-LSD (also known as “Allerton” or bovine herpes mammillitis).

##### 272 1.5.	Fluorescent antibody tests

273 Capripoxvirus antigen can be identified on infected cover-slips or tissue culture slides using fluorescent antibody

274 tests. Cover-slips or slides should be washed and air-dried and fixed in cold acetone for 10 minutes. The indirect test

275 using immune cattle sera is subject to high background colour and nonspecific reactions. However, a direct conjugate

276 can be prepared from sera from convalescent cattle (or from sheep or goats convalescing from capripox) or from

277 rabbits hyperimmunised with purified capripoxvirus. Uninfected tissue culture should be included as a negative control

278 as cross-reactions can cause problems due to antibodies to cellular components (pre-absorption of these from the

279 immune serum helps solve this issue).

##### 280 1.6.	Immunohistochemistry

281 Immunohistochemistry using F80G5 monoclonal antibody specific for capripoxvirus ORF 057 has been described for

282 detection of LSDV antigen in the skin of experimentally infected cattle (Babiuk *et al.,*2008).

##### 283 1.7.	Isothermal genome amplification

284 Molecular tests using loop-mediated isothermal amplification to detect capripoxvirus genomes are reported to provide

285 sensitivity and specificity similar to real-time PCR with a simpler method and lower cost (Das *et al.,*2012; Murray *et*

286 *al.,*2013). Field validation of the Das *et al.*method was reported by Omoga *et al.*(2016).

#### 287 2.	Serological tests

288 All the viruses in the genus *Capripoxvirus*share a common major antigen for neutralising antibodies and it is thus not

289 possible to distinguish strains of capripoxvirus from cattle, sheep or goats using serological techniques.

##### 290 2.1.	Virus neutralisation

291 A test serum can either be titrated against a constant titre of capripoxvirus (100 TCID50 [50% tissue culture infective

292 dose]) or a standard virus strain can be titrated against a constant dilution of test serum in order to calculate a

293 neutralisation index. Because of the variable sensitivity of tissue culture to capripoxvirus, and the consequent difficulty

294 of ensuring the accurate and repeatable seeding of 100 TCID50/well, the neutralisation index is the preferred method

295 in most laboratories, although it does require a larger volume of test sera. The test is described using 96-well flat-

296 bottomed tissue-culture grade microtitre plates, but it can be performed equally well in tissue culture tubes with the

297 appropriate changes to the volumes used, although it is more difficult to read an end-point in tubes.

###### 298 2.1.1.	Test procedure

299 i)	Test sera, including a negative and a positive control, are diluted 1/5 in Eagle’s/HEPES (N-2-

300 hydroxyethylpiperazine, N-2-ethanesulphonic acid) buffer and inactivated at 56°C for

301 30 minutes.

302 ii)	Next, 50 μl of the first inactivated serum is added to columns 1 and 2, rows A to H of the

303 microtitre plate. The second serum is placed in columns 3 and 4, the third in columns 5 and 6,

304 the positive control serum is placed in columns 7 and 8, the negative control serum is placed in

305 columns 9 and 10, and 50 μl of Eagle’s/HEPES buffer (without serum) is placed in columns 11

306 and 12, and to all wells in row H.

307 iii)	A reference strain of capripoxvirus, usually a vaccine strain known to grow well in tissue culture,

308 with a titre of over log10 6 TCID50 per ml is diluted in Eagle’s/HEPES in bijoux bottles to give a

309 log dilution series of log10 5.0, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 TCID50 per ml (equivalent to log10 3.7,

310 2.7, 2.2, 1.7, 1.2, 0.7, 0.2 TCID50 per 50 μl).

311 iv)	Starting with row G and the most diluted virus preparation, 50 μl of virus is added to each well

312 in that row. This is repeated with each virus dilution, the highest titre virus dilution being placed

313 in row A.

314 v)	The plates are covered and incubated for 1 hour at 37°C.

315 vi)	An appropriate cell suspension (such as MDBK cells) is prepared from pregrown monolayers

316 as a suspension of 105 cells/ml in Eagle’s medium containing antibiotics and 2% fetal calf serum.

317 Following incubation of the microtitre plates, 100 μl of cell suspension is added to all the wells,

318 except wells H11 and H12, which serve as control wells for the medium. The remaining wells of

319 row H are cell and serum controls.

320 vii)  The microtitre plates are covered and incubated at 37°C for 9 days.

321 viii) Using an inverted microscope, the monolayers are examined daily from day 4 for evidence of

322 CPE. There should be no CPE in the cells of row H. Using the 0240 KSGP vaccine strain of

323 capripoxvirus, by way of example, the final reading is taken on day 9, and the titre of virus in

324 each duplicate titration is calculated using the Kärber method. If left longer, there is invariably a

325 ‘breakthrough’ of virus in which virus that was at first neutralised appears to disassociate from

326 the antibody.

327 ix)	*Interpretation of the results:*The neutralisation index is the log titre difference between the titre

328 of the virus in the negative serum and in the test serum. An index of ≥1.5 is positive. The test

329 can be made more sensitive if serum from the same animal is examined before and after

330 infection. Because the immunity to capripoxviruses is predominantly cell mediated, a negative

331 result, particularly following vaccination, after which the antibody response may be low, does

332 not imply that the animal from which the serum was taken is not protected.

333 x)	Antibodies to capripoxvirus can be detected from 1 to 2 days after the onset of clinical signs.

334 These remain detectable for about 7 months.

##### 335 2.2.	Enzyme-linked immunosorbent assay

336 Enzyme-linked immunosorbent assays (ELISAs) for the detection of capripoxviral antibodies are widely used and are

337 available in commercial kit form (Milovanovic *et al.,*2019; Samojlovic *et al.,*2019).

##### 338 2.3.	Indirect fluorescent antibody test

339 Capripoxvirus-infected tissue culture grown on cover-slips or tissue culture microscope slides can be used for the

340 indirect fluorescent antibody test. Uninfected tissue culture control, and positive and negative control sera, should be

341 included in the test. The infected and control cultures are fixed in acetone at –20°C for 10 minutes and stored at 4°C.

342 Dilutions of test sera are made in PBS, starting at 1/20 or 1/40, and positive samples are identified using an anti-

343 bovine gamma-globulin conjugated with fluorescein isothiocyanate. Antibody titres may exceed 1/1000 after infection.

344 Sera may be screened at 1/50 and 1/500. Cross-reactions can occur with orf virus (contagious pustular dermatitis

345 virus of sheep), bovine papular stomatitis virus and perhaps other poxviruses.

##### 346 2.4.	Western blot analysis

347 Western blotting of test sera against capripoxvirus-infected cell lysate provides a sensitive and specific system for

348 the detection of antibody to capripoxvirus structural proteins, although the test is expensive and difficult to carry out.

349 Capripoxvirus-infected LT cells should be harvested when 90% CPE is observed, freeze–thawed three times, and

350 the cellular debris pelleted using centrifugation. The supernatant should be decanted, and the proteins should be

351 separated using SDS/PAGE (sodium dodecyl sulphate/polyacrylamide gel electrophoresis). A vertical discontinuous

352 gel system, using a stacking gel made up of acrylamide (5%) in Tris (125 mM), pH 6.8, and SDS (0.1%), and a

353 resolving gel made up of acrylamide (10–12.5%) in Tris (560 mM), pH 8.7, and SDS (0.1%), is recommended for use

354 with a glycine running buffer containing Tris (250 mM), glycine (2 M), and SDS (0.1%). Samples of supernatant should

355 be prepared by boiling for 5 minutes with an appropriate lysis buffer prior to loading. Alternatively, purified virus or

356 recombinant antigens may replace tissue-culture-derived antigen.

357 Molecular weight markers should be run concurrently with the protein samples. The separated proteins in the

358 SDS/PAGE gel should be transferred electrophoretically to a nitrocellulose membrane (NCM). After transfer, the NCM

359 is rinsed thoroughly in PBS and blocked in 3% bovine serum albumin (BSA) in PBS, or 5% skimmed milk powder in

360 PBS, on a rotating shaker at 4C overnight. The NCM can then be separated into strips by employing a commercial

361 apparatus to allow the concurrent testing of multiple serum samples, or may be cut into strips and each strip incubated

362 separately thereafter. The NCM is washed thoroughly with five changes of PBS for 5 minutes on a rotating shaker,

363 and then incubated at room temperature on the shaker for 1.5 hours, with the appropriate serum at a dilution of 1/50

364 in blocking buffer (3% BSA and 0.05% Tween 20 in PBS; or 5% milk powder and 0.05% Tween 20 in PBS). The

365 membrane is again thoroughly washed and incubated (in blocking buffer) with anti-species immunoglobulin

366 horseradish-peroxidase-conjugated immunoglobulins at a dilution determined using titration. After further incubation

367 at room temperature for 1.5 hours, the membrane is washed and a solution of diaminobenzidine tetrahydrochloride

368 (10 mg in 50 ml of 50 ~~mM~~mm Tris/HCl, pH 7.5, and 20 μl of 30% [v/v] hydrogen peroxide) is added. Incubation is

369 then undertaken for approximately 3–7 minutes at room temperature on a shaker with constant observation, and the

370 reaction is stopped by washing the NCM in PBS before excessive background colour is seen. A positive and negative

371 control serum should be used on each occasion.

372 Positive test samples and the positive control will produce a pattern consistent with reaction to proteins of molecular

373 weights 67, 32, 26, 19 and 17 kDa – the major structural proteins of capripoxvirus – whereas negative serum samples

374 will not react with all these proteins. Hyperimmune serum prepared against parapoxvirus (bovine papular stomatitis

375 or pseudocowpox virus) will react with some of the capripoxvirus proteins, but not the 32 kDa protein that is specific

376 for capripoxvirus.

## 377	C. REQUIREMENTS FOR VACCINES

#### 378 1.	Background: rationale and intended use of the product

379 Live attenuated strains of capripoxvirus have been used as vaccines specifically for the control of LSD (Brenner *et al*.,

380 2006; Capstick & Coakley, 1961; Carn, 1993). Capripoxviruses are cross-reactive within the genus. Consequently, it is

381 possible to protect cattle against LSD using strains of capripoxvirus derived from sheep or goats (Coakley & Capstick,

382 1961). However, it is recommended to carry out controlled trials, using the most susceptible breeds, prior to introducing a

383 ~~vaccine strain not usually used in cattle. The duration of protection provided by LSD vaccination is unknown.~~

384 Capripoxvirus vaccine strains can produce a large local reaction at the site of inoculation in *Bos taurus*breeds (Davies,

385 1991), which some stock owners find unacceptable. This has discouraged the use of vaccine, even though the

386 consequences of an outbreak of LSD are invariably more severe. Risk–benefit of vaccination should be assessed following

387 ~~stakeholder discussion.~~

388 Vaccines are a key tool to control LSD. Different types of LSD vaccines have been developed and several are commercially

389 available (Tuppurainen *et al.,*2021).

390 Live attenuated vaccines (LAV) based on the Neethling LSDV strain (homologous LAV vaccines) have been shown to offer

391 high levels of protection against LSD under experimental conditions (Haegeman *et al.,*2021) and have been used

392 successfully to control the disease in the field, through systematic vaccination of the entire country’s cattle population for

393 a number of consecutive years (Klement *et al.,*2020). Homologous vaccines may induce fever, produce a local reaction

394 at the site of inoculation, cause a temporary reduction in milk production and on rare occasions induce a ‘Neethling’

395 response (Ben-Gera *et al.,*2015; Davies, 1991; Haegeman *et al.,*2021). Such adverse effects, however, usually resolve

396 within a few days and are largely outweighed by the overall benefits of vaccination with homologous vaccines. The duration

397 of immunity induced by good quality live attenuated LSDV vaccines was shown to be at least 18 months (Haegeman *et*

398 *al.,*2023).

399 As capripox viruses provide cross-reactive protection within the genus, heterologous LAVs comprising sheep pox virus or

400 goat pox virus strains have also been tested and used to protect cattle against LSD. Sheep pox virus-based heterologous

401 vaccines usually contain higher doses of virus than when administered to sheep. Although safe, their effectiveness in

402 protecting cattle against LSD is inferior compared to homologous vaccines (Ben-Gera *et al*., 2015; Zhugunissov *et al*.,

403 2020). Heterologous vaccines containing goat pox virus strains for use in cattle against LSD have been developed more

404 recently. One such vaccine based on the Gorgan strain provided protection under experimental conditions comparable to

405 homologous vaccines (Gari *et al*., 2015). On the other hand, a goat pox vaccine based on an attenuated Uttarkashi goat

406 pox virus strain performed suboptimally under field conditions in India (Naveem *et al*., 2023), indicating that further research

407 is warranted before asserting that all goat pox virus-based vaccines induce protection equal to homologous vaccines in

408 cattle against LSD.

409 In addition, homologous inactivated vaccines against LSD have been developed and tested (Haegeman *et al*., 2023; Hamdi

410 *et al*., 2020; Wolff *et al*., 2022). These vaccines are reported to be safe and efficacious. They however require a booster

411 vaccination one month after primo-vaccination and then every 6 months thereafter, based on the fact that the duration of

412 immunity is shorter than 1 year (Haegeman *et al*., 2023).

413 None of the commercial vaccines currently available has practical DIVA capacity. This problem may be resolved in the

414 future by introducing new types of vaccines (e.g. vector-vaccines, subunit vaccines, mRNA vaccines) that are at various

415 stages of development and evaluation.

#### 416 2.	Outline of production of LSD vaccines~~and minimum requirements for conventional~~

417 **~~vaccines~~**

418 ~~General requirements set for the facilities used for the production of vaccines and for the documentation and record keeping~~

419 ~~throughout the whole manufacturing process are described in Chapter 1.1.8 *Principles of veterinary vaccine production*.~~

420 ~~The documentation should include standard operating procedures (SOP) for the method of manufacture and each step for~~

421 ~~the testing of cells and reagents used in the process, each batch and the final product.~~

422 The production of vaccines, including LSD vaccines, starts within research and development (R&D) facilities where vaccine

423 candidates are produced and tested in preclinical studies to demonstrate the quality, safety and efficacy of the product.

424 Minimum requirements for different production stages of veterinary vaccines are available in different chapters of the

425 *Terrestrial Manual.*These are intended to be used in combination with country-specific regulatory requirements for vaccine

426 production and release. Here we outline the most important requirements for the production of live and inactivated LSD

427 vaccines. Full requirements are available in Chapter 1.1.8 *Principles of veterinary vaccine production,*Chapter 2.3.3

428 *Minimum requirements for the organisation and management of a vaccine manufacturing facility*and Chapter 2.3.4

429 *Minimum requirements for the production and quality control of vaccine,*and other regulatory documentation*.*

##### 430 2.1.	Quality assurance

431 Facilities for manufacturing LSD vaccines should operate in line with the concepts of good laboratory practice (GLP)

432 and good manufacturing practice (GMP) to produce high quality products. Quality risk management and quality

433 control with adequate documentation management, as an integral part of the production process, have to be in place.

434 In case some activities of the production process are outsourced, those should also be appropriately defined,

435 recorded and controlled.

436 The vaccine production process (Outline of Production) should be documented in a series of standard operating

437 procedures (SOPs), or other documents describing the manufacturing of each batch and the final product (including

438 starting materials to be used, manufacturing steps, in-process controls and controls on the final product). Detailed

439 requirements for documentation management in the process of vaccine production are available in Chapter 2.3.3.

440 A completed Outline of Production is to be enclosed in a vaccine candidate dossier and used for the evaluation of

441 the production process and product by regulatory bodies.

##### 442 2.2.	Process validation

443 The dossier with the enclosed Outline of Production for the vaccine candidate has to be submitted for regulatory

444 approval, so it can be assessed and authorised by the competent authority to ensure compliance with local regulatory

445 requirements. Among others, data on quality, safety, and efficacy will be assessed. The procedures necessary to

446 obtain these data are described in the subsequent sections.

447 National regulatory authorities might also require official control authority re-testing (check testing) of final products

448 and batches in government laboratories or an independent batch quality control by a third party.

449 **3.	Requirements for LSD vaccine candidates and batch production**

##### 450 3.1.	Requirements for starting materials

451 Live attenuated vaccines (LAV) and inactivated vaccines (IV) for LSD are produced using the system of limited and

452 controlled passages of master seed and working seed virus and cell banks with a specified maximum. This approach

453 aims to prevent possible and unwanted drift of properties of seed virus and cells that might arise from repeated

454 passaging.

###### 455 3.1.1.	Characteristics of the seed virus

456 ~~Each seed strain of capripoxvirus used for vaccine production must be accompanied by records~~

457 ~~clearly and accurately describing its origin, isolation and tissue culture or animal passage history.~~

458 ~~Preferably, the species and strain of capripoxvirus are characterised using PCR or DNA sequencing~~

459 ~~techniques.~~

460 ~~A quantity of master seed vaccine virus should be prepared, frozen or desiccated and stored at low~~

461 ~~temperatures such as –80°C and used to produce a consistent working seed for regular vaccine~~

462 ~~production.~~

463 ~~Each master seed strain must be non-transmissible, remain attenuated after further tissue culture~~

464 ~~passage, and provide complete protection against challenge with virulent field strains for a minimum~~

465 ~~of 1 year. It must produce a minimal clinical reaction in cattle when given via the recommended route.~~

466 ~~The necessary safety and potency tests are described in Section C.2.2.4 *Final product batch tests*.~~

###### 467 ~~2.1.2.	Quality criteria (sterility, purity, freedom from extraneous agents)~~

468 ~~Each master seed must be tested to ensure its identity and shown to be free from adventitious~~

469 ~~viruses, in particular pestiviruses, such as border disease and bovine viral diarrhoea virus, and free~~

470 ~~from contamination with bacteria, fungi or mycoplasmas.~~

471 ~~The general procedures for sterility or purity tests are described in Chapter 1.1.9 *Tests for sterility*~~

472 *~~and freedom from contamination of biological materials intended for veterinary use~~*~~.~~

473 Master seed virus is a quantity of virus of uniform composition derived from an original isolate,

474 passaged for a documented number of times and distributed into containers at one time and stored

475 adequately to ensure stability (via freezing or lyophilisation). Selection of master seed viruses (MSVs)

476 should ideally be based on their ease of growth in cell culture, virus yield, and in accordance with the

477 regional epidemiological importance. Also, measures to minimise transmissible spongiform

478 encephalopathies (TSE) contamination should be taken into account (see Section C.3.5.1 *Purity*

479 *tests*).

480 For each seed strain selected for LSD vaccine production, the following information should be

481 provided:

482 -	Historical record: geographical origin, animal species from which the virus was recovered,

483 isolation procedure, tissue culture or animal passage history

484 -	Identity: species and strain identification using DNA sequencing

485 -	Purity: the absence of bacteria, fungi, mycoplasma, and other viruses (see Chapter 1.1.9 *Tests*

486 *for sterility and freedom from contamination of biological materials intended for veterinary use*)

487 -	Safety (overdose, one/repeated dose tests, and reversion to virulence tests) (see Section C.3.3

488 *Vaccine safety*)

489 -	Efficacy data, linked to a specified (protective) dose (see Section C.3.4 *Vaccine efficacy*)

490 -	Stability

491 Each master seed strain selected for production of live attenuated LSD vaccines must remain

492 attenuated after further passage in animals (see Section C.3.3. *Vaccine safety*), produce minimal

493 clinical reaction when given via the recommended route, provide complete protection against

494 challenge with virulent field strains, and is ideally not transmissible.

495 A quantity of master seed virus should be prepared and stored to be further used for the preparation

496 of working seeds and production seeds. Working seed viruses may be expanded in one or more (but,

497 limited) cell culture passages from the master seed stock and used to produce vaccine batches. This

498 approach and limitation of seed virus passaging will assist in maintaining uniformity and consistency

499 in production.

###### 500 3.1.2.	Master cell stocks

501 The production process of LSD vaccines ideally employs an established master cell stock (MCS)

502 system with defined lowest and highest cell passage to be used to grow the vaccine virus. Primary

503 cells derived from normal tissues can be used in the production process, but the use of primary cells

504 has an inherently higher risk of introducing extraneous agents compared with the use of established

505 (well characterised) cell lines and should be avoided where alternative methods of producing effective

506 vaccines exist. For each MCS, manufacturers should demonstrate:

507 -	MCS identity

508 -	genetic stability by subculturing from the lowest to the highest passage used for production

509 -	stable MCS karyotype with a low level of polyploidy

510 -	freedom from oncogenicity or tumorigenicity by using *in-vivo*studies using the highest cell

511 passage that may be used for production

512 -	purity of MCSs from extraneous bacteria, fungi, mycoplasma, and viruses

513 -	implemented measures to lower TSE contamination risk (see Section C.3.5.1 *Purity tests*).

##### 514 3.2.	Method of vaccine manufacturing

515 ~~The method of manufacture should be documented as the Outline of Production.~~

###### 516 ~~2.2.1.	Procedure~~

517 **3.2.1.	LSD vaccine batch production**

518 ~~Vaccine batches are produced on an appropriate cell line such as MDBK.~~As already mentioned in

519 the first paragraphs of Section C, all steps undertaken in the production of vaccine batches should

520 be described and documented in the Outline of Production. The production of LAV and IV against

521 LSD starts with the inoculation of the required number of working vials of seed virus ~~is~~reconstituted

522 ~~with GMEM or other~~in appropriate medium ~~and inoculated~~onto a suitable primary or continuous cell

523 line grown in suspension or monolayer~~. Cells should be harvested after 4–8 days when they exhibit~~

524 ~~50–70% CPE for maximum~~in the exponential growth phase. At the time highest viral~~infectivity, or~~

525 ~~earlier if CPE is extensive and cells appear ready to detach. Techniques such as~~ loads are present,

526 sonication or repeated freeze–thawing ~~are~~is used to release the intracellular virus from the

527 cytoplasm. The lysate may then be clarified using centrifugation to remove cellular debris~~(for~~

528 ~~example by use of centrifugation at 600 ***g***for 20 minutes~~, with retention of the supernatant). ~~A second~~

529 ~~passage of the virus may be required to produce sufficient virus for a production batch.~~

530 An aliquot of the virus suspension is titrated to check the virus titre. For LAV, the virus-containing

531 suspension is diluted to attain the dose at which the vaccine candidate will be evaluated or to at least

532 the determined protective dose for approved vaccines and is then mixed with a suitable protectant

533 ~~such as an equal volume of sterile, chilled 5% lactalbumin hydrolysate and 10% sucrose (dissolved~~

534 in double-distilled water or appropriate balanced salt solution), and transferred to individually

535 ~~numbered~~labelled bottles or bags for storage at low temperatures such as –80°C, or for freeze–

536 drying. A written record of all the procedures followed must be kept for all vaccine batches.

###### 537 ~~2.2.2.	Requirements for substrates and media~~

538 ~~The specification and source of all ingredients used in the manufacturing procedure should be~~

539 ~~documented and the freedom of extraneous agents (bacteria, fungi, mycoplasma and viruses) should~~

540 ~~be tested. The detailed testing procedure is described in Chapter 1.1.9. The use of antibiotics must~~

541 ~~meet the requirements of the licensing authority.~~

###### 542 2~~.2.3.	In-process control~~

543 ~~i)	Cells~~

544 ~~ii)	Records of the source of the master cell stocks should be maintained. The highest and lowest~~

545 passage numbers of the cells that can be used for vaccine production must be indicated in the

546 Outline of the Production. The use of a continuous cell line (such as MDBK, etc.) is strongly

547 recommended, unless the virus strain only grows on primary cells. The key advantage of

548 continuous over primary cell lines is that there is less risk of introduction of extraneous agents.

549 ~~iii)	Serum~~

550 ~~iv)	Serum used in the growth or maintenance medium must be free from antibodies to capripoxvirus~~

551 and free from contamination with pestivirus or other viruses, extraneous bacteria, mycoplasma

552 ~~or fungi.~~

553 ~~v)	Medium~~

554 ~~vi)	Media must be sterile before use.~~

555 ~~vii)  Virus~~

556 ~~viii) Seed virus and final vaccine must be titrated and pass the minimum release titre set by the~~

557 manufacturer. For example, the minimum recommended field dose of the South African

558 Neethling strain vaccines (Mathijs *et al.,*2016) is log10 3.5 TCID50, although the minimum

559 protective dose is log10 2.0 TCID50. Capripoxvirus is highly susceptible to inactivation by sunlight

560 ~~and allowance should be made for loss of activity in the field.~~

561 ~~ix)	The recommended field dose of the Romanian sheep pox vaccine for cattle is log10 2.5 sheep~~

562 infective doses (SID50), and the recommended dose for cattle of the RM65-adapted strain of

563 ~~Romanian sheep pox vaccine is log10 3 TCID50 (Coakley & Capstick, 1961).~~

###### 564 3.2.2.	Inactivation process for inactivated LSD vaccines

565 Unlike LAV, inactivated LSD vaccines contain inactivated antigens in combination with adjuvants to

566 strengthen the induced immune response after administration. The vaccine evaluation process

567 described below needs to show the amount of antigen necessary to elicit a protective immune

568 response. Currently, literature data indicate that an inactivated vaccine originating from an LSDV

569 virus stock with titre 104 cell culture infectious dose50 (CCID50)/ml before inactivation can be sufficient

570 to induce an efficient immune response to prevent clinical disease, viremia and virus shedding after

571 challenge of young cattle (Wolf *et al.,*2022)

572 To monitor the inactivation process and the level of antigen inactivation, samples are taken at regular

573 intervals during inactivation and titrated. Inactivation conditions and the length of initial and repeated

574 exposure should be documented in detail since one or more factors during the process could

575 influence the outcomes. The inactivation kinetics should reach a predefined target e.g. one remaining

576 infectious unit per million doses (1 × 10–6 infectious units/dose) as suggested by APHIS (2013). The

577 confirmatory testing of inactivation is performed on each vaccine lot and represents an important part

578 of the inactivation process monitoring. In addition to all the procedures mentioned above, the

579 inactivation procedure and tests demonstrating that antigen inactivation is complete and consistent

580 must additionally be documented in the Outline of Production.

##### 581 3.3.	Vaccine safety

582 During the vaccine development process, vaccine safety must be evaluated in the target animal (target animal batch

583 safety test –TABST) to demonstrate the safety of the dose intended for registration. The animals used in the safety

584 testing should be representative (species, age and category [calves, heifers, bulls, cows.]) for all the animals for which

585 the vaccine is intended. Vaccinated and control groups are appropriately acclimatised, housed and managed in line

586 with animal welfare standards. Animal suffering has to be eliminated or reduced and euthanasia is recommended in

587 moribund animals.

588 Essential parameters to be evaluated in safety studies are local and systemic reactions to vaccination, including local

589 reactions at the site of administration, fever, effect on milk production, and induction of a ‘Neethling’ response. The

590 effect of the vaccine on reproduction needs to be evaluated where applicable.

591 A part of the safety evaluation of LAV and IV can be performed during the efficacy trials (see Section C.3.4 *Vaccine*

592 *efficacy*) by measuring local and systemic responses following vaccination and before challenge.

593 Guidelines for safety evaluation are provided by the European Medicine Agency (EMEA) in VICH GL44: TABST for

594 LAV and IV (EMEA, 2009). Safety aspects of LAV and IV against LSD to be evaluated are:

###### 595 3.3.1.	Overdose test for LAV

596 Local and systemic responses should be measured following an overdose test whereby 10× the

597 maximum vaccine titre is administered. If the maximum vaccine titre is not specified, 10× the minimum

598 vaccine titre can be applied in multiple injection sites. Ideally, the 10× dose is dissolved in the 1×

599 dose volume of the adjuvants or diluent. Generally, eight animals per group should be used (EMEA,

600 2009).

###### 601 3.3.2.	One dose and repeat dose test

602 This aims to test the safety of the vaccine dose applied in the vaccination regime intended for

603 registration. LAV LSD vaccines require one dose per year, while inactivated LSD vaccines require a

604 booster dose in addition to the primary dose. The minimal recommended interval between

605 administrations is 14 days.

606 Generally, eight animals per group should be used unless otherwise justified (EMEA, 2009). For each

607 target species, the most sensitive breed, age and sex proposed on the label should be used.

608 Seronegative animals should be used. In cases where seronegative animals are not reasonably

609 available, alternatives should be justified.

###### 610 3.3.3.	Reversion to virulence tests

611 Live attenuated vaccines inherently carry the risk of vaccine virus reverting to virulence when

612 repeated passages in a host species could occur due to shedding and transmission from vaccinated

613 animals to contact animals. LAV LSD vaccines should therefore be tested for non-reversion to

614 virulence by means of passage studies. Vaccine virus (MSV, not the finished vaccine) is inoculated

615 in a group of target animals of susceptible age via the natural route of infection or the route that is

616 most likely to result in infection. The vaccine virus is subsequently recovered from tissues or

617 excretions and is used directly to inoculate a further group of animals. After not less than four

618 passages (see chapter 1.1.8), i.e. use of a total of five groups of animals, the re-isolate must be fully

619 characterised, using the same procedures used to characterise the master seed virus.

###### 620 3.3.4.	Environmental consideration

621 This includes the evaluation of the ability of LAV LSD vaccines to be shed, to spread and to infect

622 contact target and non-target animals, and to persist in the environment.

###### 623 ~~2.2.4.	Final product batch tests~~

624 ~~i)	Sterility/purity~~

625 ~~ii)	Vaccine samples must be tested for sterility/purity. *Tests for sterility and freedom from*~~

626 *~~contamination of biological materials intended for veterinary use~~*~~may be found in Chapter 1.1.9.~~

627 ~~iii)	Safety and efficacy~~

628 ~~iv)	The efficacy and safety studies should be demonstrated using statistically valid vaccination–~~

629 challenge studies using seronegative young LSDV susceptible dairy cattle breeds. The group

630 numbers recommended here can be varied if statistically justified. Fifteen cattle are placed in a

631 high containment level large animal unit and serum samples are collected. Five randomly

632 chosen vials of the freeze-dried vaccine are reconstituted in sterile PBS and pooled. Two cattle

633 are inoculated with 10 times the recommended field dose of the vaccine, and eight cattle are

634 inoculated with the recommended field dose. The remaining five cattle are unvaccinated control

635 animals. The animals are clinically examined daily and rectal temperatures are recorded. On

636 day 21 after vaccination, the animals are again serum sampled and challenged with a known

637 virulent capripoxvirus strain. The challenge virus solution should also be tested free from

638 extraneous viruses. The clinical response is recorded during the following 14 days. Animals in

639 the unvaccinated control group should develop the typical clinical signs of LSD, whereas there

640 should be no local or systemic reaction in the vaccinates other than a raised area in the skin at

641 the site of vaccination, which should disappear after 4 days. Serum samples are again collected

642 on day 30 after vaccination. The day 21 serum samples are examined for seroconversion to

643 selected viral diseases that could have contaminated the vaccine, and the days 0 and

644 30 samples are compared to confirm the absence of antibody to pestivirus. Because of the

645 variable response in cattle to LSD challenge, generalised disease may not be seen in all of the

646 ~~unvaccinated control animals, although there should be a large local reaction.~~

647 ~~v)	Once the efficacy of the particular strain being used for vaccine production has been determined~~

648 in terms of minimum dose required to provide immunity, it is not necessary to repeat this on the

649 ~~final product of each batch, provided the titre of virus present has been ascertained.~~

650 ~~vi)	Batch potency~~

651 ~~vii)  Potency tests in cattle must be undertaken for vaccine strains of capripoxvirus if the minimum~~

652 immunising dose is not known. This is usually carried out by comparing the titre of a virulent

653 challenge virus on the flanks of vaccinated and control animals. Following vaccination, the

654 flanks of at least three animals and three controls are shaved of hair. Log10 dilutions of the

655 challenge virus are prepared in sterile PBS and six dilutions are inoculated intradermally (0.1 ml

656 per inoculum) along the length of the flank; four replicates of each dilution are inoculated down

657 the flank. An oedematous swelling will develop at possibly all 24 inoculation sites on the control

658 animals, although preferably there will be little or no reaction at the four sites of the most dilute

659 inocula. The vaccinated animals may develop an initial hypersensitivity reaction at sites of

660 inoculation within 24 hours, which should quickly subside. Small areas of necrosis may develop

661 at the inoculation site of the most concentrated challenge virus. The titre of the challenge virus

662 is calculated for the vaccinated and control animals; a difference in titre >log10 2.5 is taken as

663 ~~evidence of protection.~~

##### 664 3.4.	Vaccine efficacy

665 Data enclosed in the vaccine candidate dossier should support the efficacy of the vaccine in each animal species for

666 each vaccination regimen that is described in the product label recommendation. This includes studies regarding the

667 onset of protection when claims for onset are made and for the duration of immunity. Efficacy studies should be

668 conducted with the vaccine candidate that has been produced at the highest passage level permitted for vaccine

669 production as specified in the Outline of Production.

670 Efficacy (and safety) should be demonstrated in vaccination–challenge studies using representative (by species, age

671 and category) seronegative healthy animals for which the vaccine is intended and which are tested negative for

672 standard viral pathogens.

673 An example of a vaccination-challenge test set-up is outlined here. The group numbers mentioned can be varied if

674 statistically justified. Thirteen animals are placed in a high containment large animal unit and are divided into two

675 groups:

676 -	single/repeated dose test group (*n*=8) – animals inoculated with the vaccine dose and route intended for

677 registration (in case of an IV against LSD, a booster dose should follow primary vaccination after minimum 14

678 days).

679 -	control group (*n*=5) – non-vaccinated animals

680 Throughout the *in-vivo*study, all animals are clinically examined and rectal temperatures recorded. Blood, serum and

681 swab samples are regularly collected and subjected to laboratory testing. On day 21 after the vaccination with a LAV

682 or after the booster vaccination for an IV, the animals in both groups are challenged with a known virulent LSDV

683 strain. The challenge virus solution should be of known titre and tested free from extraneous viruses. Experience

684 obtained from previous animal experiments indicates that a dose of challenge virus between 104.0 and 106.5 TCID50

685 produces clinical disease in about half of the susceptible experimental cattle (;.Tuppurainen *et al*., 2021).

686 The clinical response following challenge is recorded over a period of 14 days. No clinical signs should occur in the

687 vaccinates, other than a local reaction at the site of inoculation. At least 1 animal in the unvaccinated control group

688 should develop the typical clinical signs of LSD. Although a generalised disease with skin nodules may not be seen

689 in all the unvaccinated control animals based on the knowledge that the outcome of a LSDV infection can range from

690 inapparent to severe, at the very least a large local reaction is to be expected.

691 Clinical and laboratory results will enable assessment of the safety and efficacy of the LSD vaccine candidate and

692 the induced immune responses. Serum samples collected at different time points during the trial can be examined to

693 study seroconversion against selected viral diseases that could have contaminated the vaccine.

##### 694 ~~2.3.	Requirements for regulatory approval~~

###### 695 ~~2.3.1.	Safety requirements~~

696 ~~i)	Target and non-target animal safety~~

697 ii)	The vaccine must be safe to use in all breeds of cattle for which it is intended, including young

698 ~~and pregnant animals. It must also be non-transmissible and remain attenuated after further~~

699 ~~tissue culture passage.~~

700 iii)	Safety tests should be carried out on the final product of each batch as described in Section

701	~~C.2.2.4.~~

702 ~~iv)	Reversion-to-virulence for attenuated/live vaccines~~

703 v)	The selected final vaccine should not revert to virulence during further passages in target

704 ~~animals.~~

705 ~~vi)	Environmental consideration~~

706 vii)  Attenuated vaccine should not be able to perpetuate autonomously in a cattle population.

707 ~~Strains of LSDV are not a hazard to human health.~~

###### 708 ~~2.3.2.	Efficacy requirements~~

709 ~~i)	For animal production~~

710 The efficacy of the vaccine must be demonstrated in statistically valid vaccination challenge

711 experiments under laboratory conditions. The group numbers recommended here can be varied if

712 statistically justified. Fifteen cattle are placed in a high containment level large animal unit and serum

713 samples are collected. Five randomly chosen vials of the freeze-dried vaccine are reconstituted in

714 sterile PBS and pooled. Two cattle are inoculated with 10 times the field dose of the vaccine, eight

715 cattle are inoculated with the recommended field dose. The remaining five cattle are unvaccinated

716 control animals. The animals are clinically examined daily and rectal temperatures are recorded. On

717 day 21 after vaccination, the animals are again serum sampled and challenged with a known virulent

718 capripoxvirus strain using intravenous and intradermal inoculation (the challenge virus solution

719 should also be tested and shown to be free from extraneous viruses). The clinical response is

720 recorded during the following 14 days. Animals in the unvaccinated control group should develop the

721 typical clinical signs of LSD, whereas there should be no local or systemic reaction in the vaccinates

722 other than a raised area in the skin at the site of vaccination which should disappear after 4 days.

723 Serum samples are again collected on day 30 after vaccination. The day 21 serum samples are

724 examined for seroconversion to selected viral diseases that could have contaminated the vaccine,

725 and the days 0 and 30 samples are compared to confirm the absence of antibody to pestivirus.

726 Because of the variable response in cattle to challenge with LSDV, generalised disease may not be

727 ~~seen in all of the unvaccinated control animals, although there should be a large local reaction.~~

728 Once the potency of the particular strain being used for vaccine production has been determined in

729 terms of minimum dose required to provide immunity, it is not necessary to repeat this on the final

730 ~~product of each batch, provided the titre of virus present has been ascertained.~~

731 ~~ii)	For control and eradication~~

732 ~~Vaccination is the only effective way to control LSD outbreaks in endemic countries and recent~~

733 ~~experiences of the disease in Eastern Europe and the Balkans suggests this is also true for outbreaks~~

734 in non-endemic countries. Unfortunately, currently no marker vaccines allowing a DIVA strategy are

735 ~~available, although to a limited extent PCR can be used for certain vaccines.~~

736 ~~The duration of immunity produced by LSDV vaccine strains is currently unknown.~~

###### 737 ~~2.3.3.	Stability~~

738 ~~All vaccines are initially given a shelf life of 24 months before expiry. Real-time stability studies are~~

739 ~~then conducted to confirm the appropriateness of the expiry date. Multiple batches of the vaccine~~

740 ~~should be re-titrated periodically throughout the shelf-life period to determine the vaccine stability.~~

741 ~~Properly freeze-dried preparations of LSDV vaccine, particularly those that include a protectant, such~~

742 ~~as sucrose and lactalbumin hydrolysate, are stable for over 25 years when stored at~~

743 ~~–20°C and for 2–4 years when stored at 4°C. There is evidence that they are stable at higher~~

744 ~~temperatures, but no long-term controlled experiments have been reported. No preservatives other~~

745 ~~than a protectant, such as sucrose and lactalbumin hydrolysate, are required for the freeze-dried~~

746 ~~preparation.~~

##### 747 3.5.	Batch/serial tests before release for distribution

748 Quality tests on MSV and safety and efficacy tests on vaccine candidates are performed during the evaluation process

749 for new LSD vaccines. Once vaccines are approved to be used in the field, it remains important to verify the quality

750 of each vaccine batch produced. An independent batch quality control assessment may be warranted or requested

751 by national or international regulatory authorities.

###### 752 3.5.1.	Purity test

753 Purity is defined by the absence of different contaminants (bacteria, fungi, mycoplasma, and other

754 viruses; see full details in chapter 1.1.9) in the vaccine and its associated diluent/adjuvants. Virus

755 isolation and bacterial culture tests can be used to show freedom from live competent replicating

756 microorganisms, but molecular methods are more rapid and sensitive, but positives can be caused

757 by genome fragments and incompetent replicating microorganisms.

758 Besides the contaminants mentioned above, manufacturers should demonstrate implemented

759 measures to minimise the risk of TSE contamination in ingredients of animal origin such as:

760 -	all ingredients of animal origin in production facilities are from countries recognised as having the lowest

761 possible risk of bovine spongiform encephalopathy

762 -	tissues or other substances used are themselves recognised as being of low or nil risk of containing TSE

763 agents

###### 764 3.5.2.	Identity tests

765 In addition to identity tests performed on the MSV, the identity tests on final batches aim to

766 demonstrate the presence of only the selected capripoxvirus species and strain in the vaccine as

767 indicated in the Outline of Production and the absence of other strains or members of the genus and

768 any other viral contaminant that might arise during the production process. Identity testing could be

769 assured by using appropriate tests (e.g. PCRs, sanger sequencing, NGS).

###### 770 3.5.3.	Potency tests

771 Standard requirements for potency tests can be found in CFR Title 9 part 113, in the European

772 Pharmacopoeia, and in this Terrestrial Manual.

773 **3.5.3.1. Live vaccines**

774 The potency of LAV against LSD can be measured by means of virus titration. The virus titre

775 must, as a rule, be sufficiently greater than that shown to be protective in the efficacy test

776 for the vaccine candidate. This will ensure that at any time prior to the expiry date, the titre

777 will be at least equal to the evaluated protective titre. The titres of currently available

778 commercial homologous LSD vaccines range between 103 and 104 infectious units/dose

779 (Tuppurainen *et al*., 2021).

780 **3.5.3.2. Inactivated LSD vaccines**

781 For inactivated LSD vaccines, potency tests are performed using vaccination–challenge

782 efficacy studies in animal hosts (see Section C.3.4. *Vaccine efficacy*).

###### 783 3.5.4.	Safety/efficacy

784 Safety and efficacy testing is undertaken during the evaluation process of the vaccine candidate, and

785 also needs to be performed on a number of vaccine batches until robust data are generated in line

786 with international and national regulations. Afterwards, when using a seed lot system in combination

787 with strict implementation of GMP standards and depending on local regulations, TABST could be

788 waived as described in VICH50 and VICH55, providing the titer has been ascertained using potency

789 testing. Batches or serials are considered satisfactory if local and systemic reactions to vaccination

790 are in line with those described in the dossier of the vaccine candidate and product literature.

791 **3.5.4.1. Field safety/efficacy tests**

792 Field testing of two or more batches should be performed on all animal categories for which

793 the product is indicated before release of the product for general use (see chapter 1.1.8).

794 The aim of these studies is to demonstrate the safety and efficacy of the product under

795 normal field conditions of animal care and use in different geographical locations where

796 different factors may influence product performance. A protocol for safety/efficacy testing in

797 the field has to be developed with defined observation and recording procedures. However,

798 it is generally more difficult to obtain statistically significant data to demonstrate efficacy

799 under field conditions. Even when properly designed, field efficacy studies may be

800 inconclusive due to uncontrollable outside influences.

801 **3.5.4.2. Duration of Immunity**

802 The duration of immunity (DOI) following vaccination should be demonstrated via challenge

803 or the use of a validated serology test. Efficacy testing at the end of the claimed period of

804 protection should be conducted in each species for which the vaccine is indicated or the

805 manufacturer should indicate that the DOI for that species is not known. Likewise, the

806 manufacturer should demonstrate the effectiveness of the recommended booster regime in

807 line with these guidelines, usually by measuring the magnitude and kinetics of the

808 serological response observed.

#### 809 ~~3.	Vaccines based on biotechnology~~

810 ~~A new generation of capripox vaccines is being developed that uses the LSDV as a vector for the expression and delivery~~

811 ~~of immuno-protective proteins of other ruminant pathogens with the potential for providing dual protection (Boshra *et al*.,~~

812 ~~2013; Wallace & Viljoen, 2005), as well as targeting putative immunomodulatory genes for inducing improved immune~~

813 ~~responses (Kara *et al.,*2018).~~

814 **4.	Post-market studies**

##### 815 4.1.	Stability

816 Stability testing shall be carried out as specified in Annex II of Regulation (EU) 2019/6 and in the Ph. Eur. 0062:

817 Vaccines for veterinary use, on not fewer than three representative batches providing this mimics the full-scale

818 production described in the application. At the end of shelf-life, sterility has to be re-evaluated using sterility testing

819 or by showing container closure integrity. Multiple batches of the vaccine should be re-titrated periodically throughout

820 the shelf-life period to determine the vaccine stability.

##### 821 4.2.	Post-marketing surveillance

822 After release of a vaccine, its performance under field conditions should continue to be monitored by competent

823 authorities and by the manufacturer itself. Not all listed adverse effects may show up in the clinical trials performed

824 to assess safety and efficacy of the vaccine candidate due to the limited number of animals used. Post-marketing

825 surveillance studies can also provide information on vaccine efficacy when used in normal practice and husbandry

826 conditions, on duration of induced immunity, on ecotoxicity, etc.

827 First, a reliable reporting system should be in place to collect consumer complaints and notifications of adverse

828 reactions. Secondly, post-marketing surveillance should be established to investigate whether the reported

829 observations are related to the use of the product and to identify, at the earliest stage, any serious problem that may

830 be encountered from its use and that may affect its future uptake. Vaccinovigilance should be an on-going and integral

831 part of all regulatory programmes for LSD vaccines, especially for live vaccines.

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970 like lumpy skin disease virus from flies near the western border of China. *Transbound. Emerg. Dis*., doi:

971 10.1111/tbed.14159

972 ~~WALLACE D.B. & VILJOEN G.J. (2005). Immune responses to recombinants of the South African vaccine strain of lumpy skin~~

973 ~~disease virus generated by using thymidine kinase gene insertion. *Vaccine*, **23**, 3061–3067.~~

974 WEISS K.E. (1968). Lumpy skin disease. *Virol. Monogr*., **3**, 111–131.

975 WOLFF J., BEER M. & HOFFMANN B. (2022). High Efficiency of Low Dose Preparations of an Inactivated Lumpy Skin Disease

976 Virus Vaccine Candidate. *Vaccines*(Basel), **10**, 1029. doi: 10.3390/vaccines10071029.

977 ZHUGUNISSOV K., BULATOV Y., ORYNBAYEV M., KUTUMBETOV L., ABDURAIMOV Y., SHAYAKHMETOV Y., TARANOV D., AMANOVA Z.,

978 MAMBETALIYEV M., ABSATOVA Z., AZANBEKOVA M., KHAIRULLIN B., ZAKARYA K. & TUPPURAINEN E. (2020). Goatpox virus (G20-

979 LKV) vaccine strain elicits a protective response in cattle against lumpy skin disease at challenge with lumpy skin disease

980 virulent field strain in a comparative study. *Vet Microbiol*., **245**, 108695. doi: 10.1016/j.vetmic.2020.108695.

981	*

982	* *

983 **NB:**There are WOAH Reference Laboratories for lumpy skin disease (please consult the WOAH Web site:

984 https://www.woah.org/en/what-we-offer/expertise-network/reference-laboratories/#ui-id-3).

985 Please contact WOAH Reference Laboratories for any further information on

986 diagnostic tests, reagents and vaccines for lumpy skin disease

###### 987 **NB:**FIRST ADOPTED IN 1989. MOST RECENT UPDATES ADOPTED IN 2021.