**Annex 11. Item 5.1. – Chapter 3.4.7. Bovine viral diarrhoea**

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

3	**Paris, 4–8 September 2023**

4

5	**CHAP T E R  3 . 4 . 7 .**

# 6	B OV I N E VI RA L D I A R R H OE A

7	**SUMMARY**

8 *Cattle of all ages are susceptible to infection with bovine viral diarrhoea viruses*(*BVDV*), *including BVDV*

9 *type 1*(Pestivirus bovis), *type 2*(Pestivirus tauri)*, and Hobi-like pestiviruses*(*type 3*[Pestivirus brazilense])*.*

10 *Distribution is world-wide although some countries have recently eradicated the virus. BVDV infection results*

11 *in a wide variety of clinical manifestations, including enteric and respiratory disease in any class of cattle, or*

12 *reproductive and fetal disease following infection of a susceptible breeding female. Infection may be*

13 *subclinical or extend to severe fatal disease. Animals that survive*in-utero *infection in the first trimester of*

14 *gestation are almost always persistently infected*(*PI*)*. PI animals provide the main reservoir of the virus in a*

15 *population and excrete large amounts of virus in urine, faeces, discharges, milk and semen. Identification of*

16 *such PI cattle is a key element in controlling the infection. It is important to avoid the trade of such animals.*

17 *They may appear clinically healthy, or weak and unthrifty. Many PI animals die before reaching maturity.*

18 *They may infrequently develop mucosal disease with anorexia, gastrointestinal erosions, and profuse*

19 *diarrhoea, invariably leading to death. Mucosal disease can arise only in PI animals. Latent infections*

20 *generally do not occur following recovery from acute infection. However bulls may rarely have a persistent*

21 *testicular infection and excrete virus in semen for prolonged periods.*

22 ***Detection of the agent:****BVDV is a pestivirus in the family*Flaviviridae *and is closely related to classical*

23 *swine fever virus*(Pestivirus suis)*and ovine border disease virus~~es~~*(Pestivirus ovis). *BVD viruses are*

24 *classified into the distinct species*Pestivirus bovis (*commonly known as BVDV type 1*)*,*Pestivirus tauri

25 (*BVDV type 2*) *and*Pestivirus brazilense (*BVDV type 3 or Hobi-like pestivirus*)*.**~~The two genotypes~~*~~(*types 1*~~

26 *~~and 2~~*~~) *are classified as separate species in the genus*Pestivirus*. A third putative genotype, BVDV type 3,*~~

27 *~~has also recently been proposed.~~ Although both cytopathic and non-cytopathic biotypes of BVDV type 1 and*

28 *type 2 exist, non-cytopathic strains are usually encountered in field infections and are the main focus of*

29 *diagnostic virus isolation in cell cultures. PI animals can be readily identified by a variety of methods aimed*

30 *to detect viral antigens or viral RNA directly in blood and tissues. Virus can also be isolated by inoculation*

31 *of specimens onto susceptible cell cultures followed by immune-labelling methods to detect the replication*

32 *of the virus in the cultures. Persistence of virus infection should be confirmed by resampling after an interval*

33 *of at least 3 weeks, when virus will again be detected. PI animals are usually seronegative. Viraemia in acute*

34 *cases is transient and usually difficult to detect. Virus isolation in semen from bulls that are acutely or*

35 *persistently infected requires special attention to specimen transport and testing. RNA detection assays are*

36 *particularly useful because they are rapid, have very high sensitivity and do not depend on the use of cell*

37 *cultures.*

38 ***Serological tests:****Acute infection with BVDV is best confirmed by demonstrating seroconversion using*

39 *sequential paired samples, ideally from several animals in the group. The testing of paired*(*acute and*

40 *convalescent samples*) *should be done a minimum of 21 days apart and samples should be tested*

41 *concurrently in the same assay. Enzyme-linked immunosorbent assays and the virus neutralisation test are*

42 *the most widely used.*

43 ***Requirements for vaccines:****There is no standard vaccine for BVD, but a number of commercial*

44 *preparations are available. An ideal vaccine should be able to prevent transplacental infection in pregnant*

45 *cows. Modified live virus vaccine should not be administered to pregnant cattle*(*or to their sucking calves*)

46 *due to the risk of transplacental infection. Live vaccines that contain cytopathic strains of BVDV present a*

47 *risk of inducing mucosal disease in PI animals. Inactivated viral vaccines are safe and can be given to any*

48 *class of animal but generally require booster vaccinations. BVDV is a particularly important hazard to the*

49 *manufacture of vaccines and biological products for other diseases due to the high frequency of*

50 *contamination of batches of fetal calf serum used as a culture medium supplement.*

51

#### 52 1.	Impact of the disease

## A. INTRODUCTION

53 Cattle of all ages are susceptible to infection with bovine viral diarrhoea viruses (BVDV). Distribution of the virus is world-

54 wide although some countries have recently eradicated the virus. BVDV infection results in a wide variety of clinical

55 manifestations, including enteric and respiratory disease in any class of cattle or reproductive and fetal disease following

56 infection of a susceptible breeding female. Infection may be subclinical or extend to severe fatal disease. Clinical

57 presentations and severity of disease may vary with different strains of virus. BVDV viruses also cause immune

58 suppression, which can render infected animals more susceptible to infection with other viruses and bacteria. The clinical

59 impact may be more apparent in intensively managed livestock. Animals that survive *in-utero*infection in the first trimester

60 of gestation are almost always persistently infected (PI). PI animals provide the main reservoir of the virus in a population

61 and excrete large amounts of virus in urine, faeces, discharges, milk and semen. The virus spreads mainly by close contact

62 between PI animals and other cattle. Virus shedding by acutely infected animals is usually less important. This virus may

63 also persist in the environment for short periods or be transmitted ~~with~~via contaminated reproductive materials. Vertical

64 transmission plays an important role in its epidemiology and pathogenesis.

65 Infections of the breeding female may result in conception failure or embryonic and fetal infection which results in abortions,

66 stillbirths, teratogenic abnormalities or the birth of PI calves. Persistently viraemic animals may be born as weak, unthrifty

67 calves or may appear as normal healthy calves and be unrecognised clinically for a long time. However, PI animals have

68 a markedly reduced life expectancy, with a high proportion dying before reaching maturity. Infrequently, some of these

69 animals may later develop mucosal disease with anorexia, gastrointestinal erosions, and profuse diarrhoea, invariably

70 leading to death. Mucosal disease can arise only in PI animals. It is important to avoid the trade of viraemic animals. It is

71 generally considered that serologically positive, non-viraemic cattle are ‘safe’, providing that they are not pregnant.

72 However, a small proportion of persistently viraemic animals may produce antibodies to some of the viral proteins if they

73 are exposed to another strain of BVDV that is antigenically different to the persisting virus. Consequently, seropositivity

74 cannot be completely equated with ‘safety’. Detection of PI animals must be specifically directed at detection of the virus

75 or its components (RNA or antigens). Latent infections generally do not occur following recovery from acute infection.

76 However, semen collected from bulls during an acute infection is likely to contain virus during the viraemic period and often

77 for a short time afterwards. Although extremely rare, some recovered bulls may have a persistent testicular infection and

78 excrete virus in semen, perhaps indefinitely.

79 While BVDV strains are predominantly pathogens of cattle, interspecies transmission can occur following close contact

80 with sheep, goats or pigs. Infection of pregnant small ruminants or pigs with BVDV can result in reproductive loss and the

81 birth of PI animals. BVDV infections have been reported in both New World and Old World camelids. Additionally, strains

82 of border disease virus (BDV) have infected cattle, resulting in clinical presentations indistinguishable from BVDV infection.

83 The birth of cattle PI with BDV and the subsequent development of mucosal disease have also been described. Whilst

84 BVDV and BDV have been reported as natural infections in pigs, the related virus of classical swine fever does not naturally

85 infect ruminants.

86 Although ubiquitous, control of BVDV can be achieved at the herd level, and even at the national level, as evidenced by

87 the progress towards eradication made in many European countries (Moennig *et al*., 2005; Schweizer *et al.,*2021).

#### 88 2.	The causal agent

89 Bovine viral diarrhoea virus (BVDV) is a single linear positive-stranded RNA virus in the genus *Pestivirus*of the family

90 *Flaviviridae.*The genus contains a number of species including ~~the two genotypes of bovine viral diarrhoea virus (~~BVDV)

91 (types 1 [*Pestivirus bovis*],~~and~~ 2 [*Pestivirus tauri*] and 3 [*Pestivirus brazilense*]) and the~~closely~~ related classical swine

92 fever (*Pestivirus suis*) and ovine border disease viruses (*Pestivirus ovis*). Viruses in these ~~genotypes~~pestivirus species

93 show considerable antigenic difference from each other and, within the ~~type 1 and type 2~~species *Pestivirus bovis*and

94 *tauri*, BVDV isolates exhibit considerable biological and antigenic diversity. Within the ~~two BVDV genotypes~~species

95 *Pestivirus bovis*and *taur**i*, further subdivisions are discernible by genetic analysis (Vilcek *et al*., 2001). The two ~~genotypes~~

96 species may be differentiated from each other, and from other pestiviruses, by monoclonal antibodies (MAbs) directed

97 against the major glycoproteins E2 and ERNS or by genetic analysis. Reverse-transcription polymerase chain reaction (RT-

98 PCR) assays enable virus typing direct from blood samples (Letellier & Kerkhofs, 2003; McGoldrick *et al*., 1999). Type 1

99 viruses are generally more common although the prevalence of type 2 strains can be high in North America. BVDV of both

100 ~~genotypes~~species(*Pestivirus bovis*and *tauri*) may occur in non-cytopathic and cytopathic forms (biotypes), classified

101 according to whether or not microscopically apparent cytopathology is induced during infection of cell cultures. Usually, it

102 is the non-cytopathic biotype that circulates freely in cattle populations. Non-cytopathic strains are most frequently

103 responsible for disease in cattle and are associated with enteric and respiratory disease in any class of cattle or

104 reproductive and fetal disease following infection of a susceptible breeding female. Infection may be subclinical or extend

105 to severe fatal disease (Brownlie, 1985). Cytopathic viruses are encountered in cases of mucosal disease, a clinical

106 syndrome that is relatively uncommon and involves the ‘super-infection’ of an animal that is PI with a non-cytopathic virus

107 by a closely related cytopathic strain. The two virus biotypes found in a mucosal disease case are usually antigenically

108 closely related if not identical. Type 2 viruses are usually non-cytopathic and have been associated with outbreaks of

109 severe acute infection and a haemorrhagic syndrome. However some type 2 viruses have also been associated with a

110 disease indistinguishable from that seen with the more frequently isolated type 1 viruses. Further, some type 1 isolates

111 have been associated with particularly severe and fatal disease outbreaks in adult cattle. Clinically mild and inapparent

112 infections are common following infection of non-pregnant animals with either ~~genotype~~virus species.

113 ~~There is an increasing awareness of an “atypical” or “HoBi-like” pestivirus – a putative BVDV type 3~~*Pestivirus H*strains

114 are also associated with clinical disease in cattle, but they appear mainly restricted to South American and Asian cattle

115 populations~~, in cattle, also associated with clinical disease~~ (Bauermann *et al*., 2013; Chen *et al.,*2021)~~, but its distribution~~

116 is presently unclear. These viruses are readily detected by proven pan-reactive assays such as real-time RT-PCR. Some

117 commercial antigen ELISAs (enzyme-linked immunosorbent assays) have been shown to detect these strains (Bauermann

118 *et al*., 2012); generally virus isolation, etc., follows the same principles as for BVDV 1 (*Pestivirus bovis*) and 2 (*Pestivirus*

119 *tauri*). It should be noted however, that antibody ELISAs vary in their ability to detect antibody to BVDV type 3 (*Pestivirus*

120 *brazilense*) and vaccines designed to protect against BVDV 1 and 2 may not confer full protection against infection with

121 these ~~novel~~pestiviruses (Bauermann *et al.,*2012*;*2013).

#### 122 3.	Pathogenesis

##### 123 3.1.	Acute infections

124 Acute infections with BVDV are encountered more frequently in young animals, and may be clinically inapparent or

125 associated with fever, diarrhoea (Baker 1995), respiratory disease and sometimes sudden death. The severity of disease

126 may vary with virus strain and the involvement of other pathogens (Brownlie, 1990). In particular, outbreaks of a severe

127 form of acute disease with haemorrhagic lesions, thrombocytopenia and high mortality have been reported sporadically

128 from some countries (Baker, 1995; Bolin & Ridpath, 1992). Infection with type 2 viruses (*Pestivirus tauri*) in particular

129 has been demonstrated to cause altered platelet function. During acute infections there is a brief viraemia for 7–10 days

130 and shedding of virus can be detected in nasal and ocular discharges. There may also be a transient leukopenia,

131 thrombocytopenia or temperature response, but these can vary greatly among animals. Affected animals may be

132 predisposed to secondary infections with other viruses and bacteria. Although BVDV may cause a primary respiratory

133 disease on its own, the immunosuppressive effects of the virus exacerbate the impact of this virus. BVDV is one of the

134 major pathogens of the bovine respiratory disease complex in feedlot cattle and in other intensive management systems

135 such as calf raising units.

136 Infection of breeding females immediately prior to ovulation and in the first few days after insemination can result in

137 conception failure and early embryonic loss (McGowan & Kirkland, 1995). Cows may also suffer from infertility,

138 associated with changes in ovarian function and secretions of gonadotropin and progesterone (Fray *et al*., 2002).

139 Bulls may excrete virus in semen for a short period during and immediately after infection and may suffer a temporary

140 reduction of fertility. Although the virus level in this semen is generally low it can result in reduced conception rates

141 and be a potential source of introduction of virus into a naive herd (McGowan & Kirkland, 1995).

142 **3.2.	*In-utero*infections**

143 Infection of a breeding female can result in a range of different outcomes, depending on the stage of gestation at

144 which infection occurred. Before about 25 days of gestation, infection of the developing conceptus will usually result

145 in embryo-fetal death, although abortion may be delayed for a considerable time (McGowan & Kirkland, 1995).

146 Surviving fetuses are normal and uninfected. However, infection of the female between about 30–90 days will

147 invariably result in fetal infection, with all surviving progeny PI and seronegative. Infection at later stages and up to

148 about day 150 can result in a range of congenital defects including hydranencephaly, cerebellar hypoplasia, optic

149 defects, skeletal defects such as arthrogryposis and hypotrichosis. Growth retardation may also occur, perhaps as a

150 result of pituitary dysfunction. Fetal infection can result in abortion, stillbirth or the delivery of weak calves that may

151 die soon after birth (Baker, 1995; Brownlie, 1990; Duffell & Harkness, 1985; Moennig & Liess, 1995). Some PI calves

152 may appear to be normal at birth but fail to ~~grow normally~~thrive. They remain PI for life and are usually seronegative,

153 exceptions may be young calves that ingested colostrum containing antibodies. The onset of the fetal immune

154 response and production of antibodies occurs between approximately day 90–120, with an increasing proportion of

155 infected calves having detectable antibodies while the proportion in which virus may be detected declines rapidly.

156 Infection of the bovine fetus after day 180 usually results in the birth of a normal seropositive calf.

##### 157 3.3.	Persistent infections

158 Persistently viraemic animals are a continual source of infective virus to other cattle and are the main reservoir of

159 BVDV in a population. In a population without a rigorous BVDV control programme, approximately 1–2% of cattle are

160 PI. During outbreaks in a naive herd or breeding group, if exposure has occurred in the first trimester of pregnancy,

161 a very high proportion of surviving calves will be PI. If a PI animal dies, there are no pathognomonic lesions due to

162 BVDV and the pathology is often complicated by secondary infections with other agents. Some PI animals will survive

163 to sexual maturity and may breed successfully but the~~ir~~ progeny of female PI animals will also always be PI. Animals

164 being traded or used for artificial breeding should first be screened to ensure that they are not PI.

##### 165 3.4.	Mucosal disease

166 Persistently viraemic animals may later succumb to mucosal disease (Brownlie, 1985). However, cases are rare. This

167 syndrome has been shown to be the outcome of the infection of a PI animal with an antigenically similar cytopathic

168 virus, which can arise either through superinfection, recombination between non-cytopathic biotypes, or mutation of

169 the persistent biotype (Brownlie, 1990). There is usually little need to specifically confirm that a PI animal has

170 succumbed to mucosal disease as this is largely a scientific curiosity and of little practical significance, other than

171 that the animal is PI with BVDV. However, cases of mucosal disease may be the first indication in a herd that BVDV

172 infection is present, and should lead to more in depth investigation and intervention.

##### 173 3.5.	Semen and embryos

174 Bulls that are PI usually have poor quality, highly infective semen and reduced fertility (McGowan & Kirkland, 1995).

175 All bulls used for natural or artificial insemination should be screened for both acute and persistent BVDV infection.

176 A rare event, possibly brought about by acute infection during pubescence, can result in persistent infection of the

177 testes and thus strongly seropositive bulls that intermittently excrete virus in semen (Voges *et al*., 1998). This

178 phenomenon has also been observed following vaccination with an attenuated virus (Givens *et al*., 2007). Embryo

179 donor cows that are PI with BVDV also ~~re~~present a potential source of infection, particularly as there are extremely

180 high concentrations of BVDV in uterine and vaginal fluids. While oocysts without an intact zona pellucida have been

181 shown to be susceptible to infection *in vitro*, the majority of oocysts remain uninfected with BVDV. Normal uninfected

182 progeny have also been ‘rescued’ from PI animals by the use of extensive washing of embryos and *in-vitro*fertilisation.

183 Female cattle used as embryo recipients should always be screened to confirm that they are not PI, and ideally, are

184 seropositive or were vaccinated at least 4 weeks before first use.

185 Biological materials used for *in-vitro*fertilisation techniques (bovine serum, bovine cell cultures) have a high risk of

186 contamination and should be screened for BVDV. Incidents of apparent introduction of virus via such techniques have

187 highlighted this risk. It is considered essential that serum supplements used in media should be free of contaminants

188 as detailed in Chapter 1.1.9 *Tests for sterility and freedom from contamination of biological materials intended for*

189 *veterinary use,*using techniques described in Section B.~~3~~1.1 of this chapter.

#### 190 4.	Approaches to diagnosis and sample collection

191 The diagnosis of BVDV infection can sometimes be complex because of the delay between infection and clinical

192 expression. While detection of PI animals should be readily accomplished using current diagnostic methods, the recognition

193 of acute infections and detection of BVDV in reproductive materials can be more difficult.

##### 194 4.1.	Acute infections

195 Unlike PI animals, acutely infected animals excrete relatively low levels of virus and for a short period of time (usually

196 about 7–10 days) but the clinical signs may occur during the later stages of viraemia, reducing the time to detect the

197 virus even further. In cases of respiratory or enteric disease, samples should be collected from a number of affected

198 animals, preferentially selecting the most recently affected. Swabs should be collected from the nares and conjunctiva

199 of animals with respiratory disease or from rectum and faeces if there are enteric signs. Lung and spleen are preferred

200 from dead animals. Viral RNA may be detected by real-time RT-PCR assays and have the advantages of high

201 sensitivity and being able to detect genome from non-infectious virus. As the virus levels are very low, it is not usually

202 practical to undertake virus isolation unless there is a need to characterise the strain of BVDV involved. Serology

203 undertaken on paired acute and convalescent sera (collected at least 21 days after the acute sample and from 8–10

204 animals) is worthwhile and gives a high probability of incriminating or excluding BVDV infection.

205 Confirmation that an abortion, stillbirth or perinatal death is caused by BVDV is often difficult to establish because

206 there can be a long delay between initial infection and death or expulsion of the fetus. Sampling should take into

207 consideration the need to detect either viral components or antibodies. Spleen and lung are preferred samples for

208 virus detection while pericardial or pleural fluids are ideal samples for serology. The stomach of newborn calves

209 should be checked to confirm that sucking has not occurred. While virus may be isolated from fetal tissue in some

210 cases, emphasis should be placed on the detection of viral antigen by ELISA or RNA by real-time RT-PCR. For

211 serology, both ELISAs and virus neutralisation test (VNT) are suitable though sample quality and bacterial

212 contamination may compromise the ability to detect antibodies by VNT. Maternal serology, especially on a group of

213 animals, can be of value, with the aim of determining whether there has been recent infection in the group. A high

214 antibody titre (>1/1000) to BVDV in maternal serum is suggestive of fetal infection and is probably due to the fetus

215 providing the dam with an extended exposure to virus.

##### 216 4.2.	Persistent infections

217 In the past, identification of PI animals relied heavily on the use of virus isolation in cell cultures. However, antigen

218 detection ELISAs and real-time RT-PCR assays, each with relatively high sensitivity, are widely used for the detection

219 of viral antigens or RNA in both live and dead animals. Virus isolation aimed at the detection of non-cytopathic BVDV

220 in blood is also used, while in some countries, the virus has been identified by immunohistochemistry (IHC). Skin

221 samples have been collected from live animals while a wide range of tissues from dead animals are suitable. Both

222 virus isolation and IHC are labour intensive and costly and can be technically demanding. Virus isolation from blood

223 can be confounded by the presence of maternal antibody to BVDV in calves less than 4–5 months of age (diagnostic

224 gap). Also for antigen detection ELISAs and flow cytometry from blood or blood leukocytes, there are restrictions that

225 limit when animals that ingested colostrum that contains antibodies against BVDV can be reliably tested. In older

226 animals with persistent viraemia infection, low levels of antibody may be present due to their ability to seroconvert to

227 strains of BVDV (including vaccines) antigenically different to the persisting virus (Brownlie, 1990). Bulk (tank) or

228 individual milk samples have been used to monitor dairy herds for the presence of a PI animal. Antigen ELISA, real-

229 time PCR and virus isolation have all been used. To confirm a diagnosis of persistent infection, animals should be

230 retested after an interval of at least 3 weeks by testing of blood samples for the presence of the virus and for ~~evidence~~

231 absence of seroconversion. Care should be taken with retesting of skin samples as it has been shown that, in some

232 acute cases, viral antigen may persist for many weeks in skin (Cornish *et al*., 2005).

##### 233 4.3.	Mucosal disease

234 Although not undertaken for routine diagnostic purposes, for laboratory confirmation of a diagnosis of mucosal

235 disease it is necessary to isolate the cytopathic virus. This biotype may sometimes be isolated from blood, but it can

236 be recovered more consistently from a variety of other tissues, in particular spleen, intestine and Peyer’s patch tissue.

237 Virus isolation is readily accomplished from spleen which is easy to collect and is seldom toxic for cell culture.

##### 238 4.4.	Reproductive materials

239 Semen donor bulls should be sampled for testing for freedom from BVDV infection prior to collection of semen, in

240 accordance with the *Terrestrial Animal Health Code.*It is necessary to confirm that these bulls are not PI, are not

241 undergoing an acute infection and to establish their serological status. This initial testing should be carried out on

242 whole blood or serum samples. To establish that a seropositive bull does not have a persistent testicular infection

243 (PTI), samples of semen should be collected on at least three separate occasions at intervals of not less than 7 days

244 due to the possibility of intermittent low level virus excretion, especially during the early stages of infection. There is

245 also a need to submit a number of straws from each collection, or an appropriate volume of raw semen. Particular

246 care should be taken to ensure that sample transport recommendations are adhered to and that the laboratory

247 documents the condition of the samples on arrival at the laboratory. Further details of collection, transport and test

248 requirements are provided in sections that follow.

## 249	B. DIAGNOSTIC TECHNIQUES

250	***Table 1.****Test methods available for diagnosis of bovine viral diarrhoea and their purpose*

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

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

253 ELISA = enzyme-linked immunosorbent assay; IHC = immunohistochemistry method; NA = nucleic acid; RT-PCR = reverse-

254 transcription polymerase chain reaction; ~~ISH = *in-situ*hybridisation;~~VN = virus neutralisation.

255 (a)A combination of agent detection methods applied on the same clinical sample is recommended.

#### 256 1.	Detection of the agent

257 To prevent the shipment of either animals or animal derivatives (especially semen and embryos) that are infected with

258 BVDV, it is necessary to test for the presence of the infectious virus (virus isolation), viral antigens (antigen detection

259 ELISA) or RNA (real-time RT-PCR) in the blood of the animal being shipped, or the donor of the germplasm (semen or

260 embryos). The exception is for seropositive bulls where semen must be tested rather than the donor bull. Serology only

261 plays a role for establishing that seronegative animals are not undergoing an acute infection or, to establish the serological

262 status of donor bulls. Due to their variable sensitivity without prior virus amplification, procedures such as IHC or *in-situ*

263 hybridisation (ISH) directly on tissues are not considered to be suitable for certification for freedom from BVDV for

264 international trade purposes. In contrast, immune-staining is an essential component of virus isolation in cell culture to

265 detect the presence of non-cytopathic strains of BVDV which predominate in field infections.

266 All test methods must be extensively validated by testing on known uninfected and infected populations of cattle, including

267 animals with low- and high-titre viraemias. Methods based on polyclonal or MAb-binding assays (ELISA or IHC), immune

268 labelling (VI) or on nucleic acid recognition (PCR) must be shown to detect the full range of antigenic and genetic diversity

269 found among BVD viruses. There are ~~three~~designated WOAH Reference Laboratories for BVDV that can assist with

270 relevant information; the reference laboratories for classical swine fever could also be approached to offer some advice.

##### 271 1.1.	Virus isolation

272 When performed to a high standard, BVDV isolation is very reliable. However, it does have very exacting

273 requirements to ensure that the cell cultures and medium components give a system that is very sensitive and are

274 not compromised by the presence of either low levels of BVDV specific antibody or virus. Virus isolation only has the

275 capacity to detect infectious virus which imposes certain limits on sample quality. Further, to detect low levels of virus

276 that may be present in some samples, particularly semen, it may be necessary to examine larger volumes of specimen

277 than is usual. Some of these limitations can be overcome by the use of antigen detection ELISAs with proven high

278 analytical sensitivity, or the use of real-time RT-PCR.

279 The virus may be isolated in a number of bovine monolayer cell cultures (e.g. kidney, lung, testis or turbinate). In

280 some instances, ovine cells are also suitable. Primary or secondary cultures can be frozen as cell suspensions in

281 liquid nitrogen. These can then be tested over a series of passages, or seeded to other susceptible cells and checked

282 for freedom from contaminants and to evaluate their sensitivity compared to an approved batch of cells before routine

283 use. Such problems may be reduced by the use of continuous cell lines, which can be obtained BVD-free, however,

284 their BVDV-free status and susceptibility must be monitored regularly. Continuous cells should be used under a ‘seed

285 lot’ system where they are only used over a limited passage range, within which they have been shown to have

286 acceptable sensitivity to BVDV infection. Although particular continuous cell lines are considered to be appropriate

287 for use for BVDV isolation, there can be significant variation in batches of cells from different sources due to differing

288 passage histories so their suitability must still be confirmed before routine use.

289 Non-cytopathic BVDV is a common contaminant of bovine tissues and cell cultures must be checked for freedom

290 from adventitious virus by regular testing. Cells must be grown in proven cell culture medium components and a large

291 area of cells must be examined. It is not appropriate to screen a few wells of a 96 well plate – examining all wells of

292 a 96 well plate will be more convincing evidence of freedom. The fetal bovine serum that is selected for use in cell

293 culture must also be free not only from virus, but also and of equal or perhaps even greater importance, from BVDV

294 neutralising antibody. Heat treatment (56°C for 30–45 minutes) is inadequate for the destruction of BVDV in

295 contaminated serum; irradiation with a dose of at least 25 kiloGrays (2.5 Mrad) is more certain. Commercial batches

296 of fetal bovine serum mostly test positive by real-time RT-PCR even after the virus has been inactivated by irradiation.

297 Further, most commercially collected batches of fetal bovine serum contain antibodies to pestiviruses, sometimes at

298 levels that are barely detectable but sufficient to inhibit virus isolation. To overcome this, serum can be obtained from

299 BVD virus and antibody free donor animals and used with confidence. Testing of donors for both virus and antibody

300 occurs on an individual animal basis. Although horse serum has been substituted for bovine fetal serum, it is often

301 found to have poorer cell-growth-promoting characteristics. Further there has sometimes been cross contamination

302 with fetal bovine serum during processing, negating the objective of obtaining a BVDV-free product.

303 Buffy coat cells, whole blood, washed leukocytes or serum are suitable for isolation of the virus from live animals.

304 Maternal antibody may interfere with isolation from serum in young calves. Tissue suspensions from post-mortem

305 cases should be prepared by standard methods. Confirmation that a bull is not PI with BVDV is most readily achieved

306 by testing of a blood sample. However, persistent testicular infections (PTI) have been detected in some bulls that

307 have recovered from acute infection, are no longer viraemic and are now seropositive (Voges *et al*., 1998). Virus may

308 be detected in most but not all collections of semen from these bulls. Although still considered to be uncommon, to

309 exclude the potential for a PTI it is essential to screen semen from all seropositive bulls. To be confident that a bull

310 does not have a PTI, batches of semen collected over several weeks should be screened. Once a series of collections

311 have been screened, further testing of semen from a seropositive bull is not warranted. Raw semen, and occasionally

312 extended semen, is cytotoxic and must be diluted in culture medium. For these reasons, it is important to monitor the

313 health of the cells by microscopic examination at intervals during the incubation. These problems are largely

314 overcome by the use of real-time RT-PCR which has several advantages over virus isolation, including higher

315 sensitivity and the potential to be completed within a few hours rather than weeks for virus isolation.

316 There are many variations of procedure in use for virus isolation. All should be optimised to give maximum sensitivity

317 of detection of a standard virus preparation. All biological components used for cell culture should be screened and

318 shown to be free of both BVDV and antibodies to BVDV. Cell cultures (whether primary or continuous lines) should

319 be regularly checked to confirm that they maintain maximum susceptibility to virus infection. Depending on the

320 specimen type and purpose for testing, virus isolation is likely to require one or more passages in cell cultures. While

321 PI animals can be readily identified by screening blood or serum with one passage, semen should be routinely

322 cultured for three passages and biological products such as fetal bovine serum up to five times (original inoculation

323 plus four passages). Conventional methods for virus isolation are used, with the addition of a final immune-staining

324 step (immunofluorescence or, more frequently, peroxidase staining) to detect growth of non-cytopathic virus. Thus

325 tube cultures should include flying cover-slips, while microplate cultures can be fixed and labelled directly in the plate.

326 Examples are given below. Alternatively, culture supernatant from the final passage can be screened by real-time

327 RT-PCR (see below).

###### 328 1.1.1.	Microplate immunoperoxidase method for mass screening for virus detection in serum

329 **samples (Meyling, 1984)**

330 i)	10–25 μl of the serum sample is placed into each of four wells of a 96-well tissue-culture grade

331 microplate. This is repeated for each sample. Known positive and negative controls are

332 included.

333 ii)	100 μl of a cell suspension at the appropriate concentration (usually about 150,000 cells/ml) in

334 medium without fetal calf serum (FCS) is added to all wells. *Note:*the sample itself acts as the

335 cell-growth supplement. If testing samples other than serum, use medium with 10% FCS that is

336 free of antibodies to ruminant pestiviruses.

337 iii)	The plate is incubated at 37°C for 4 days, either in a 5% CO2 atmosphere or with the plate

338 sealed.

339 iv)	Each well is examined microscopically for evidence of cytopathology (cytopathic effect or CPE),

340 or signs of cytotoxicity.

341 v)	The cultures are frozen briefly at approximately –80°C and 50 μl of the culture supernatant is

342 passaged to new cell cultures, repeating steps ~~3~~1.1.1.i to iv above.

343 vi)	The cells are then fixed and stained by one of two methods:

386 An alternative substrate can be made, consisting of 9 mg diaminobenzidine tetrahydrochloride

387 and 6 mg sodium perborate tetrahydrate dissolved in 15 ml of PBS. Though the staining is not

388 quite so intense, these chemicals have the advantage that they can be shipped by air.

389 k)	The plate is examined microscopically. Virus-positive cells show red-brown cytoplasmic

390 staining.

391 Alternative methods for fixation of the cells may be used and include the use of heat (see Chapter

392 3.8.3 *Classical swine fever*, Section B.2.2.1.viii). These should be first evaluated to ensure that the

393 capacity to detect viral antigen is not compromised.

###### 394 1.1.2.	Tube method for tissue or buffy coat suspensions

395 *Note:*this method can also be conveniently adapted to 24-well plastic dishes. *Note:*a minimum of 2 and

396 preferably 3 passages (including primary inoculation) is required.

397 i)	Tissue samples are ground up and a 10% suspension in culture medium is made. This is then

398 centrifuged to remove the debris.

399 ii)	Test tube cultures with newly confluent or subconfluent monolayers of susceptible bovine cells are

400 inoculated with 0.1 ml of the sample. The culture is left to adsorb for 1 hour at 37°C.

401 iii)	The culture is washed with 1 ml of medium; this is then discarded and 1 ml of culture maintenance

402 medium is added.

403 iv)	The culture is incubated for 4–5 days at 37°C, and examined microscopically for evidence of CPE

404 or signs of cytotoxicity.

405 v)	The culture should then be frozen and thawed for passage to fresh cultures for one or preferably

406 two more passages (including the culture inoculated for the final immunostaining). At the final

407 passage, after freeze–thaw the tissue culture fluid is harvested and passaged on to microtitre plates

408 for culture and staining by the immunoperoxidase method (see section B.~~3~~1.1.1 above) or by the

409 immunofluorescent method. For immunofluorescence, cover-slips are included in the tubes and

410 used to support cultured cells. At the end of the culture period, the cover slips are removed, fixed

411 in 100% acetone and stained with an immunofluorescent conjugate to BVDV. Examine the cover

412 slips under a fluorescent microscope for diffuse, cytoplasmic fluorescence characteristic of

413 pestiviruses. Alternatively, culture supernatant from the final passage can be screened by real-time

414 RT-PCR (see below).

###### 415 1.1.3.	Virus isolation from semen

416 The samples used for the test are, typically, extended bovine semen or occasionally raw semen.

417 Semen samples should be transported to the laboratory in liquid nitrogen, or on dry ice. The samples

418 should be stored in liquid nitrogen or at lower than –70°C (for long-term storage) or 4°C (for short-

419 term storage of not more than 1–2 days). The receiving laboratory should document the condition

420 under which samples are received. Raw semen is generally cytotoxic and should be prediluted (e.g.

421 1/10 in BVDV free bovine serum) before being added to cell cultures. At least 0.1 ml of raw semen

422 should be tested with three passages in cell culture. Toxicity may also be encountered with extended

423 semen. For extended semen, an approximation should be made to ensure that the equivalent of a

424 minimum of 0.1 ml raw semen is examined (e.g. a minimum of 1.0 ml extended semen). If toxicity is

425 encountered, multiple diluted samples may need to be tested to reach a volume equivalent to 0.1 ml

426 raw semen (e.g. 5 × 1 ml of a sample of extended semen that has been diluted 1/5 to reduce toxicity).

427 A suggested method is as follows:

428 i)	Dilute 200 μl fresh semen in 1.8 ml bovine serum containing antibiotics. This can be the same

429 serum as is being used for supplementing the cell cultures, and must be shown to be free from

430 antibodies against BVDV.

431 ii)	Mix vigorously and leave for 30 minutes at room temperature.

432 iii)	Inoculate 1 ml of the semen/serum mixture into a monolayer of susceptible cells (see virus

433 isolation from tissue above) in cell culture tubes or a six-well tissue culture plate.

434 iv)	Incubate the cultures for 1 hour at 37°C.

435 v)	Remove the mixture, wash the monolayer several times with maintenance medium and then

436 add new maintenance medium to the cultures.

437 vi)	Include BVDV negative and positive controls in the test. Special caution must be taken to avoid

438 accidental contamination of test wells by the positive control, for example always handling the

439 positive control last.

440 vii)  Observe plates microscopically to ensure freedom from contamination and cytotoxicity. No

441 cytopathology is expected as a result of BVDV infection but other viruses such as BHV-1 could

442 be inadvertently isolated.

443 viii) After 5–7 days, the cultures are frozen at or below approximately –70°C and thawed, clarified

444 by centrifugation, and the supernatant used to inoculate fresh monolayers.

445 ix)	At the end of the second passage, the supernatant from the freeze-thaw preparation should be

446 passaged onto cultures in a suitable system for immunoperoxidase staining or other antigen

447 detection or by real-time RT-PCR after 5 days of culture. This is most readily achieved in 96

448 well microplates. The sample is considered to be negative, if there is no evidence of viral antigen

449 or BVDV RNA detected.

##### 450 1.2.	Nucleic acid detection

451 Conventional gel based RT-PCR has in the past been used for the detection of BVD viral RNA for diagnostic

452 purposes. A multiplex RT-PCR has been used for the simultaneous amplification and typing of virus from cell culture,

453 or direct from blood samples. However, gel based RT-PCR has the disadvantage that it is relatively labour intensive,

454 expensive and prone to cross contamination. These problems had been markedly reduced following the introduction

455 of probe-based real-time or quantitative RT-PCR methods. Nevertheless, stringent precautions should still be taken

456 to avoid nucleic acid contamination in the test system and general laboratory areas where samples are handled and

457 prepared (see Chapter 1.1.6 *Principles and methods of validation of diagnostic assays for infectious diseases*and

458 Chapter 2.2.3 *Development and optimisation of nucleic acid assays*). These assays have even higher sensitivity than

459 gel based RT-PCR and can be completed in a few hours. They are in widespread use for the diagnosis of infectious

460 diseases, allowing the direct detection of viral RNA from a wide range of specimens including serum, whole blood,

461 tissues, milk and semen. The high analytical sensitivity allows the adoption of strategies to screen pools of individual

462 samples or testing of bulk tank milk. By using this approach the presence of one or more PI animals can be identified

463 in herds containing several hundred cows. However, it is not appropriate to pool blood samples taken from calves

464 between day 7 and 40 of life, when colostrum that contains antibodies against BVDV was ingested. During this time

465 the sensitivity of PCR can be reduced and infected animals escape detection. In contrast, the detection of viral RNA

466 in skin biopsy samples remains unaffected (Fux & Wolf*,*2012). Although slightly more expensive than immunostaining

467 methods, real-time RT-PCR is a quick and reliable method that can also be used to screen culture supernatant from

468 the final passage of cell cultures. While real-time RT-PCR has very high sensitivity and can be applied to the screening

469 of biological materials used for vaccine manufacture, caution is needed in the interpretation of results, as the detection

470 of viral RNA does not imply *per se*that infective virus is present. Real-time RT-PCR assays based on fluorescent-

471 labelled DNA probes can also be used to differentiate pestiviruses (e.g. McGoldrick *et al*., 1999).

472 Primers for the assay should be selected in highly conserved regions of the genome, ideally the 5’-noncoding region,

473 or the NS3 (p80 gene). There are published assays that are broadly reactive across the pestivirus genus, detecting

474 all BVDV types (*Pestivirus bovis, tauri*and *brazilense*), CSFV (*Pestivirus suis*), some strains of BDV (*Pestivirus ovis*)

475 and ~~most of the~~several ‘atypical’ pestiviruses (e.g. Hoffman *et al*., 2006). A sensitive broadly reactive assay is

476 recommended for diagnostic applications because interspecies transfer of different pestiviruses is occasionally

477 encountered. When further identification of the specific virus is required, pestivirus species-specific assays can be

478 applied to further type the virus. It is important to thoroughly optimise all aspects of the real-time RT-PCR assay,

479 including the nucleic acid extraction and purification. Optimal concentrations of Mg2+, primers, probe and polymerase,

480 and the cycling parameters need to be determined. However, fully formulated and optimised ‘ready to use’

481 ‘mastermixes’ are now available commercially and only require addition of optimised concentrations of primers and

482 probe. Optimised cycling conditions are often recommended for a particular mastermix.

483 A variety of commercially available nucleic acid purification systems are available in kit form and several can be semi-

484 automated. Systems based on the capture and purification of RNA using magnetic beads are in widespread use and

485 allow rapid processing of large numbers of samples. Specific products should be evaluated to determine the optimal

486 kit for a particular sample type and whether any preliminary sample processing is required. For whole blood samples,

487 the type of anticoagulant and volume of blood in a specimen tube is important. More problems with inhibitors of the

488 PCR reaction are encountered with samples collected into heparin treated blood than EDTA. These differences are

489 also exacerbated if the tube does not contain the recommended volume of blood, thereby increasing the concentration

490 of anticoagulant in the sample. To identify possible false-negative results, it is recommended to spike an exogenous

491 (‘internal control’) RNA template into the specimen prior to RNA extraction (e.g. Hoffman *et al*., 2006). By the inclusion

492 of PCR primers and probe specific to the exogenous sequence, the efficiency of both the RNA extraction and also

493 the presence of any PCR inhibitors can be monitored. While valuable for all sample types, the inclusion of an internal

494 control is particularly desirable when testing semen and whole blood. When using an internal control, extensive testing

495 is necessary to ensure that PCR amplification of the internal control does not compete with the diagnostic PCR and

496 thus lower the analytical sensitivity (see also chapter 1.1.6).

497 When it is suspected that a sample may contain substances that are adversely affecting either the efficiency of RNA

498 extraction or the real-time RT-PCR assay, modest dilution of the sample in saline, cell culture medium or a buffer

499 solution (e.g. phosphate buffered gelatin saline [PBGS]) will usually overcome the problem. Dilution of a semen

500 sample by 1/4 and whole unclotted blood at 1/10 is usually adequate. As the real-time RT-PCR has extremely high

501 analytical sensitivity, dilution of the sample rarely has a significant impact on the capacity of the assay to detect viral

502 RNA when present.

###### 503 1.2.1.	Real-time polymerase chain reaction for BVDV detection in semen

504 Real-time RT-PCR has been shown to be extremely useful to screen semen samples to demonstrate

505 freedom from BVDV and, apart from speed, often gives superior results to virus isolation in cell

506 culture, especially when low virus levels are present, such as may be found in bulls with a PTI. The

507 real-time RT-PCR described here uses a pair of sequence-specific primers for amplification of target

508 ~~D~~RNA and a 5’-nuclease oligoprobe for the detection of amplified products. The oligoprobe is a

509 single, sequence-specific oligonucleotide, labelled with two different fluorophores. The primers and

510 probe are available commercially and several different fluorophores options are available. This pan-

511 pestivirus real-time RT-PCR assay is designed to detect viral ~~D~~RNA of all strains of BVDV types 1

512 (*Pestivirus bovis*)~~and BVDV~~, 2 (*Pestivirus tauri*) and 3 (*Pestivirus brazilense*) as well as ~~BDV,~~CSFV

513 (*Pestivirus suis*), some strains of BDV (*Pestivirus ovis*) and most atypical pestiviruses. The assay

514 selectively amplifies a 208 base pair sequence of the 5’ non-translated region (5’ NTR) of the

515 pestivirus genome. Details of the primers and probes are given in the protocol outlined below.

516 i)	Sample preparation, equipment and reagents

517 a)	The samples used for the test are, typically, extended bovine semen or occasionally raw

518 semen. If the samples are only being tested by real-time RT-PCR, it is acceptable for them

519 to be submitted chilled but they must still be cold when they reach the laboratory.

520 Otherwise, if a cold chain cannot be assured or if virus isolation is being undertaken, the

521 semen samples should be transported to the laboratory in liquid nitrogen or on dry ice. At

522 the laboratory, the samples should be stored in liquid nitrogen or at lower than –70°C (for

523 long-term storage) or 4°C (for short-term storage of up to 7 days). *Note:*samples for virus

524 isolation should not be stored at 4°C for more than 1–2 days.

525 b)	Due to the very high analytical sensitivity of real-time RT-PCR, much smaller volumes of

526 semen may be used. However, at least three straws (minimum 250 μl each) from each

527 collection batch of semen should be processed. The semen in the three straws should be

528 pooled and mixed thoroughly before taking a sample for nucleic acid extraction.

529 c)	A real-time PCR detection system, and the associated data analysis software, is required

530 to perform the assay. A number of real-time PCR detection systems are available from

531 various manufacturers.~~Other equipment required for the test includes a micro-centrifuge,~~

532 ~~a chilling block, a micro-vortex, and micropipettes.~~As real-time RT-PCR assays are able

533 to detect very small amounts of target nucleic acid molecules, appropriate measures are

534 required to avoid contamination. ~~, including dedicated and physically separated ‘clean’~~

535 ~~areas for reagent preparation (where no samples or materials used for PCR are handled),~~

536 ~~a dedicated sample processing area and an isolated area for the PCR thermocycler and~~

537 ~~associated equipment. Each area should have dedicated reagents and equipment.~~

538 Furthermore, a minimum of one negative sample should be processed in parallel to

539 monitor the possibility of low level contamination. Sources of contamination may include

540 product carry-over from positive samples or, more commonly, from cross contamination

541 by PCR products from earlier work.

542 d)	The real-time RT-PCR assay involves two separate procedures.

543 1)	Firstly, BVDV RNA is extracted from semen using an appropriate validated nucleic

544 acid extraction method. Systems using magnetic beads for the capture and

545 purification of the nucleic acid are recommended. It is also preferable that the beads

546 are handled by a semi-automated magnetic particle handling system.

547 2)	The second procedure is the RT-PCR analysis of the extracted RNA template in a

548 real-time RT-PCR system.

549 ii)	Extraction of RNA

550 RNA or total nucleic acid is extracted from the pooled (three straws collected at the same time

551 from the same animal) semen sample. Use of a commercially available magnetic bead based

552 extraction kit is recommended. However, the preferred kit should be one that has been

553 evaluated to ensure optimal extraction of difficult samples (semen and whole blood). Some

554 systems and kit protocols are sufficiently refined that it is not necessary to remove cells from

555 the semen sample. Prior to extraction dilute the pooled semen sample 1/4 in ~~phosphate buffered~~

556 ~~gelatin saline (~~PBGS) or a similar buffered solution. Complete the RNA extraction by taking 50

557 μl of the diluted, pooled sample and add it to the sample lysis buffer. Some commercial

558 extraction kits may require the use of a larger volume. It has also been found that satisfactory

559 results are obtained by adding 25 μl of undiluted pooled sample to sample lysis buffer. Complete

560 the extraction by following the kit manufacturer’s instructions.

561 iii)	Real-time RT-PCR assay procedure

562 a)	Reaction mixture: There are a number of commercial real-time PCR amplification kits

563 available from various sources and the particular kits selected need to be compatible with

564 the real-time PCR platform selected. The required primers and probes can be synthesised

565 by various commercial companies. The WOAH Reference Laboratories for BVDV can

566 provide information on suitable suppliers.

567 b)	Supply and storage of reagents: The real-time PCR reaction mixture is normally provided

568 as a 2 × concentration ready for use. The manufacturer’s instructions should be followed

569 for application and storage. Working stock solutions for primers and probe are made with

570 nuclease-free water at the concentration of 20 μM and 3 μM, respectively. The stock

571 solutions are stored at –20°C and the probe solution should be kept in the dark. Single-

572 use or limited use aliquots can be prepared to limit freeze–thawing of primers and probes

573 and extend their shelf life.

574 c)	Primers and probe sequences

575 Selection of the primers and probe are outlined in Hoffmann *et al*. (2006) and summarised

576 below.

580	d)	Preparation of reaction mixtures

581	The PCR reaction mixtures are prepared in a separate room that is isolated from other

582	PCR activities and sample handling. For each PCR test, appropriate controls should be

583	included. As a minimum, a no template control (NTC), appropriate negative control (NC)

584	and two positive controls (PC1, PC2) should be included. The positive and negative

585	controls are included in all steps of the assay from extraction onwards while the NTC is

586	added after completion of the extraction. The PCR amplifications are carried out in a

587	volume of 25 μl. The protocol described is based on use of a 96 well microplate based

588	system but other options using microtubes are also suitable. Each well of the PCR plate

589	should contain 20 μl of reaction mix and 5 μl of sample as follows:

598 e)	Selection of controls

599 NTC: usually consists of nuclease free water or tRNA in nuclease free water that is added

600 in place of a sample when the PCR reaction is set up.

601 NC: In practice, many laboratories use PBGS or a similar buffer. Ideally the controls for

602 testing of semen samples should be negative semen, from seronegative bulls. However,

603 as a minimum, the assay in use should have been extensively validated with negative and

604 positive samples to confirm that it gives reliable extraction and amplification with semen.

605 PCs: There are two positive controls (PC1=moderate – [Ct 29-32] and PC2=weak [Ct 32–

606 35] positive). Positive semen from naturally infected bulls is preferable as a positive

607 control. However, this is likely to be difficult to obtain. Further, semen from a PI bull is not

608 considered suitable because the virus loads are usually very high and would not give a

609 reliable indication of any moderate reduction in extraction or assay performance. Negative

610 semen spiked with defined quantities of BVDV virus could be used as an alternative. If

611 other samples are used as a routine PC, as a minimum the entire extraction process and

612 PCR assay in use must have been extensively validated using known positive semen from

613 bulls with a PTI or from bulls undergoing an acute infection. If these samples are not

614 available and spiked samples are used for validation purposes, a number of samples

615 spiked with very low levels of virus should be included. On a day to day basis, the inclusion

616 of an exogenous control with each test sample will largely compensate for not using

617 positive semen as a control and will give additional benefits by monitoring the efficiency of

618 the assay on each individual sample. Positive control samples should be prepared

619 carefully to avoid cross contamination from high titred virus stocks and should be prepared

620 in advance and frozen at a ‘ready to use’ concentration and ideally ‘single use’ volume.

621 f)	Extracted samples are added to the PCR mix in a separate room. The controls should be

622 added last, in a consistent sequence in the following order: NTC, negative and then the

623 two positive controls.

624 g)	Real-time polymerase chain reaction

625 The PCR plate or tubes are placed in the real-time PCR detection system in a separate,

626 designated PCR room. Some mastermixes have uniform reaction conditions that are

627 suitable for many different assays. As an example, the PCR detection system is

628 programmed for the test as follows:

629 1× 48°C 10 minutes

630 1× 95°C 10 minutes

631 45 × (95°C 15 seconds, 60°C 1 minute)

632 h)	Analysis of real-time PCR data

633 The software program is usually set to automatically adjust results by compensating for any

634 background signal and the threshold level is usually set according to the manufacturer’s

635 instructions for the selected analysis software used. In this instance, a threshold is set at 0.05.

636 i)	Interpretation of results

637 a) Test controls – all controls should give the expected results with positive controls (PC1

638 and PC2) falling within the designated range and both the negative control (NC) and no

639 template control (NTC) should have no Ct values.

640 b) Test samples

641 1) Positive result: Any sample that has a cycle threshold (Ct) value less than 40 is

642 regarded as positive.

643 2) Negative result: Any sample that shows no Ct value is regarded as negative. However,

644 before reporting a negative result for a sample, the performance of the exogenous

645 internal control should be checked and shown to give a result within the accepted range

646 for that control (for example, a Ct value no more than 2–3 Ct units higher than the

647 NTC).

##### 648 1.3.	Enzyme-linked immunosorbent assay for antigen detection

649 Antigen detection by ELISA has become a widely adopted method for the detection of individual PI animals. These

650 assays are not intended for the detection of acutely infected animals (though from to time this may be achieved).

651 Importantly, these assays are not designed for screening of semen or biological materials used in assays or vaccine

652 manufacture. Several methods for the ELISA for antigen detection have been published and a number of commercial

653 kits are available. Most are based on the sandwich ELISA principle, with a capture antibody bound to the solid phase,

654 and a detector antibody conjugated to a signal system, such as peroxidase. Amplification steps such as the use of

655 biotin and streptavidin in the detection system are sometimes used to increase assay sensitivity. Both monoclonal-

656 and polyclonal-based systems are described. The test measures BVD antigen (NS2-3 or ERNS) in lysates of peripheral

657 blood leukocytes; the new generation of antigen-capture ELISAs (ERNS capture ELISAs) are able to detect BVD

658 antigen in blood as well as in plasma or serum samples. The best of the methods gives a sensitivity similar to virus

659 isolation, and may be preferred in those rare cases where persistent infection is combined with seropositivity. Due to

660 transient viraemia, the antigen ELISA is less useful for virus detection in acute BVD infections.

661 The ~~NS2-3~~antigen detection ELISAs may be less effective in young calves that have had colostrum due to the

662 presence of BVDV maternal antibodies, especially when blood samples or blood leucocytes are tested (Fux & Wolf,

663 2012). Blood or blood leucocytes should not be tested in the first month (ERNS capture ELISA) or the first 3 months

664 (NS2-3 ELISA) of life due to the inhibitory effect of maternal antibodies. The real-time RT-PCR is probably the most

665 sensitive detection method for this circumstance, but the ERNS ELISA has also been shown to be a sensitive and

666 reliable test, ~~particularly~~when used with skin biopsy (ear-notch) samples (Cornish *et al*., 2005).

##### 667 1.4.	Immunohistochemistry

668 Enzyme-labelled methods are useful to detect BVDV antigen in tissue sections, particularly where suitable MAbs are

669 available. However, these assays are not appropriate to certify animals for international trade and use should be

670 limited to diagnostic investigations. It is important that the reagents and procedures used be fully validated, and that

671 nonspecific reactivity be eliminated. For PI cattle almost any tissue can be used, but particularly good success has

672 been found with lymph nodes, thyroid gland, skin, brain, abomasum and placenta. Skin biopsies, such as ear-notch

673 samples, have shown to be useful for *in-vivo*diagnosis of persistent BVDV infection.

#### 674 2.	Serological tests

675 Antibody to BVDV can be detected in cattle sera by a standard VNT or by ELISA, using one of several published methods

676 or with commercial kits (e.g. Edwards, 1990). Serology is used to identify levels of herd immunity, for the detection of the

677 presence of PI animals in a herd, to assist with investigation of reproductive disease and possible involvement of BVDV

678 and to establish the serological status of bulls being used for semen collection and to identify whether there has been a

679 recent infection. ELISA for antibody in bulk milk samples can give a useful indication of the BVD status of a herd (Niskanen,

680 1993). High ELISA values~~(0.8 or more absorbance units)~~in an unvaccinated herd indicates a high probability of the herd

681 having been exposed to BVDV in the recent past, most likely through one or more persistently viraemic animals being

682 present. In contrast, ~~a~~very low or negative values~~(≤0.2)~~ indicate~~s~~ that it is unlikely that persistently viraemic animals are

683 present. However, ELISA values are not always a reliable indicator of the presence of PI animals on farms, due to differing

684 husbandry (Zimmer *et al*., 2002), recent administration of vaccine and also due to the presence of viral antigen in bulk milk,

685 which may interfere with the antibody assay itself. Determination of the antibody status of a small number of young stock

686 (9–18 months) has also been utilised as an indicator of recent transmission of BVDV in the herd (Houe *et al*., 1995), but

687 this approach is also dependent on the degree of contact between different groups of animals in the herd and the potential

688 for exposure from neighbouring herds. VN tests are more frequently used for regulatory purposes (e.g. testing of semen

689 donors) while ELISAs (usually in the form of commercially prepared kits) are commonly used for diagnostic applications.

690 Whether ELISA or VNT, control positive and negative standard sera must be included in every test. These should give

691 results within predetermined limits for the test to be considered valid. In the VNT, a ‘serum control’ to monitor sample

692 toxicity should also be included for each test sample.

##### 693 2.1.	Virus neutralisation test

694 Selection of the virus strain to include in a VNT is very important. No single strain is likely to be ideal for all

695 circumstances, but in practice one should be selected that detects the highest proportion of serological reactions in

696 the local cattle population. Low levels of antibody to BVD type 2 virus (*Pestivirus tauri*) may not be detectable by a

697 neutralisation test that uses type 1 strain of the virus, and vice versa (Fulton *et al*., 1997). It is important that BVD

698 type 1 and BVD type 2 (*Pestivirus bovis*and *tauri*) be used in the test and not just the one that the diagnostician

699 thinks is present, as this can lead to under reporting. Because it makes the test easier to read, most laboratories use

700 highly cytopathic, laboratory-adapted strains of BVDV for VN tests. Two widely used cytopathic strains are ‘Oregon

701 C24V’ and ‘NADL’. However immune-labelling techniques are now available that allow simple detection of the growth

702 or neutralisation of non-cytopathic strains where this is considered desirable, especially to support the inclusion of a

703 locally relevant virus strain. An outline protocol for a microtitre VN test is given below (Edwards, 1990):

###### 704 2.1.1.	Test procedure

705 i)	The test sera are heat-inactivated for 30 minutes at 56°C.

706 ii)	From a starting dilution of 1/4, serial twofold dilutions of the test sera are made in a cell-culture

707 grade flat-bottomed 96-well microtitre plate, using cell culture medium as diluent. For each

708 sample, three or four wells are used at each dilution depending on the degree of precision

709 required. At each dilution of serum, for each sample one well is left without virus to monitor for

710 evidence of sample toxicity that could mimic viral cytopathology or interfere with virus

711 replication. Control positive and negative sera should also be included in each batch of tests.

712 iii)	An equal volume (e.g. 50 μl) of a stock of cytopathic strain of BVDV containing 100 TCID50 (50%

713 tissue culture infective dose) is added to each well. A back titration of virus stock is also done

714 in some spare wells to check the potency of the virus (acceptance limits 30–300 TCID50).

715 iv)	The plate is incubated for 1 hour at 37°C.

716 v)	A flask of suitable cells (e.g. bovine turbinate, bovine testis) is trypsinised and the cell

717 concentration is adjusted to 1.5 × 105/ml. 100 μl of the cell suspension is added to each well of

718 the microtitre plate.

719 vi)	The plate is incubated at 37°C for 4–5 days, either in a 5% CO2 atmosphere or with the plate

720 sealed.

721 vii)  The wells are examined microscopically for CPE or fixed and stained by immunoperoxidase

722 staining using an appropriate monoclonal antibody. The VN titre for each serum is the dilution

723 at which the virus is neutralised in 50% of the wells. This can be calculated by the Spearman–

724 Kärber or Reed Muench methods. A seronegative animal will show no neutralisation at the

725 lowest dilution (1/4), equivalent to a final dilution of 1/8. For accurate comparison of antibody

726 titres, and particularly to demonstrate significant (more than fourfold) changes in titre, samples

727 should be tested in parallel in the same test.

##### 728 2.2.	Enzyme-linked immunosorbent assay

729 Both indirect and blocking types of test can be used. A number of commercial kits are available. As with the virus

730 neutralisation test, ELISAs configured using antigen from one ~~genotype~~species of BVDV may not efficiently detect

731 antibody induced by another ~~genotype~~virus species. Tests should therefore be selected for their ability to detect

732 antibody to the spectrum of types and strains circulating in the country where the test is to be performed.

733 The chief difficulty in setting up the test lies in the preparation of a viral antigen of sufficient potency. The virus must

734 be grown under optimal culture conditions using a highly permissive cell type. Any serum used in the medium must

735 not inhibit growth of BVDV. The optimal time for harvest should be determined experimentally for the individual culture

736 system. The virus can be concentrated and purified by density gradient centrifugation. Alternatively, a potent antigen

737 can be prepared by treatment of infected cell cultures with detergents, such as Nonidet P40, N-decanoyl-N-

738 methylglucamine (Mega 10), Triton X-100 or 1-octylbeta-D-glucopyranoside (OGP). Some workers have used fixed,

739 infected whole cells as antigen. ~~In the future,~~Increasing use ~~may be~~is made of artificial antigens manufactured by

740 expressing specific viral genes in bacterial or eukaryotic systems. Such systems should be validated by testing sera

741 specific to a wide range of different virus strains. In the future, this technology should enable the production of

742 serological tests complementary to subunit or marker vaccines, thus enabling differentiation between vaccinated and

743 naturally infected cattle. An example outline protocol for an indirect ELISA is given below (Edwards, 1990).

###### 744 2.2.1.	Test procedure

745 i)	Roller cultures of secondary calf testis cells with a high multiplicity of infection (about one), are

746 inoculated with BVDV strain Oregon C24V, overlaid with serum-free medium and incubated for

747 24 hours at 37°C.

748 ii)	The cells are scraped off and pelleted. The supernatant medium is discarded. The pellet is

749 treated with two volumes of 2% OGP in PBS for 15 minutes at 4°C, and centrifuged to remove

750 the cell debris. The supernatant antigen is stored in small aliquots at –70°C, or freeze-dried.

751 Non-infected cells are processed in parallel to make a control antigen.

752 iii)	The antigen is diluted to a predetermined dilution in 0.05 M bicarbonate buffer, pH 9.6. Alternate

753 rows of an ELISA-grade microtitre plate are coated with virus and control antigens overnight at

754 4°C. The plates are then washed in PBS with 0.05% Tween 20 or Tween 80 (PBST) before use

755 in the test.

756 iv)	Test sera are diluted 1/50 in serum diluent (0.5 M NaCl; 0.01 M phosphate buffer; 0.05% Tween

757 20; 0.001 M ethylene diamine tetra-acetic acid; 1% polyvinyl pyrrolidone, pH 7.2) and added to

758 virus- and control-coated wells for 1 hour at 37°C. The plates are then washed five times in

759 PBST.

760 v)	Rabbit anti-bovine IgG peroxidase conjugate is added at a predetermined dilution (in serum

761 diluent) for 1 hour at 37°C, then the plates are again washed five times in PBST.

762 vi)	A suitable enzyme substrate is added, such as hydrogen peroxide/tetramethyl benzidine. After

763 colour development, the reaction is stopped with sulphuric acid and the absorbance is read on

764 an ELISA plate reader. The value obtained with control antigen is subtracted from the test

765 reaction to give a net absorbance value for each serum.

766 vii)  It is recommended to convert net absorbance values to sample:positive ratio (or percentage

767 positivity) by dividing net absorbance by the net absorbance on that test of a standard positive

768 serum that has a net absorbance of about 1.0. This normalisation procedure leads to more

769 consistent and reproducible results.

770

771

#### 1.	Background

## C. REQUIREMENTS FOR VACCINES

772 BVDV vaccines are used primarily for disease control purposes. Although they can convey production advantages

773 especially in intensively managed cattle such as in feedlots. In some countries where BVDV eradication is being

774 undertaken, PI animals are removed and remaining cattle are vaccinated to maintain a high level of ~~infection~~antibody

775 positivity and prevent the generation of further PI animals. Vaccination to control BVDV infections can be challenging due

776 in part to the antigenic variability of the virus and the occurrence of persistent infections that arise as a result of fetal

777 infection. Ongoing maintenance of the virus in nature is predominantly sustained by PI animals that are the product of *in-*

778 *utero*infection. The goal for a vaccine should be to prevent systemic viraemia and the virus crossing the placenta. If this

779 is successfully achieved it is likely that the vaccine will prevent the wide range of other clinical manifestations, including

780 reproductive, respiratory and enteric diseases and immunosuppression with its secondary sequelae. There are many

781 different vaccines available in different countries. Traditionally, BVD vaccines fall into two classes: modified live virus or

782 inactivated vaccines. Experimental recombinant subunit vaccines based on BVD viral glycoprotein E2 expressed with

783 baculovirus,~~or~~ transgenic plants or heterologous viruses and BVDV E2 DNA vaccines have been described but few, if

784 any, are in commercial production. They offer a future prospect of ‘marker vaccines’ when used in connection with a

785 complementary serological test.

##### 786 1.1.	Characteristics of a target product profile

787 Traditionally, BVD vaccines fall into two classes: modified live or inactivated virus vaccines. The essential requirement

788 for both types is to ~~afford~~provide a high level of fetal ~~infection~~protection. Many of the live vaccines have been based

789 on a cytopathic strain of the virus which is considered to be unable to cross the placenta. However, it is important to

790 ensure that the vaccine virus does not cause fetal infection. In general, vaccination of breeding animals should be

791 completed well before insemination to ensure optimal protection and avoid any risk of fetal infection. Live virus vaccine

792 may also be immunosuppressive and precipitate other infections. On the other hand, modified live virus vaccines may

793 only require a single dose. Use of a live product containing a cytopathic strain of BVDV may precipitate mucosal

794 disease by superinfection of persistently viraemic animals. Properly formulated inactivated vaccines are very safe to

795 use but, to obtain satisfactory levels of immunity, they usually require booster vaccinations, which may be

796 inconvenient. A combined vaccination protocol using inactivated followed by live vaccine may reduce the risk of

797 adverse reaction to the live strain. Whether live or inactivated, because of the propensity for antigenic variability, the

798 vaccine should contain strains of BVDV that are closely matched to viruses found in the area in which they are used.

799 For example, in countries where strains of BVDV type 2 (*Pestivirus tauri*) are found, it is important for the vaccine to

800 contain a suitable type 2 strain. For optimal immunity against type 1 strains (*Pestivirus bovis*), antigens from the

801 dominant subtypes (e.g. 1a and 1b) should be included. Due to the need to customise vaccines for the most commonly

802 encountered strains within a country or region, it is not feasible to produce a vaccine antigen bank that can be drawn

803 upon globally.

804 Guidance for the production of veterinary vaccines is given in Chapter 1.1.8 *Principles of veterinary vaccine*

805 *production*. The guidelines given here and in chapter 1.1.8 are intended to be general in nature and may be

806 supplemented by national and regional requirements.

#### 807 2.	Outline of production and minimum requirements for vaccines

##### 808 2.1.	Characteristics of the seed

809 For optimal efficacy, it is considered that there should be a close antigenic match between viruses included in a

810 vaccine and those circulating in the target population. BVDV type 2 strains (*Pestivirus tauri*) should be included as

811 appropriate. Due to the regional variations in ~~genotypes~~species and subtypes of BVDV, many vaccines contain more

812 than one strain of BVDV to give acceptable protection. A good appreciation of the antigenic characteristics of

813 individual strains can be obtained by screening with panels of MAbs (Paton *et al*., 1995).

###### 814 2.1.1.	Biological characteristics of the master seed

815 Isolates of cytopathic virus are often mixed with the noncytopathic biotype. The separation and

816 purification of the two biotypes from an initial mixed culture is important to maintain the expected

817 characteristics of the ~~seen~~seed and depends on several cycles of a limiting dilution technique for the

818 noncytopathic virus, or plaque selection for the cytopathic virus. Purity of the cytopathic virus should

819 be confirmed by at least one additional passage at limiting dilution. When isolates have been cloned,

820 their identity and key antigenic characteristics should be confirmed. The identity of the seed virus

821 should be confirmed by sequencing. Where there are multiple isolates included in the vaccine, each

822 has to be prepared separately.

823 While retaining the desirable antigenic characteristics, the strains selected for the seed should not

824 show any signs of disease when susceptible animals are vaccinated. Live attenuated vaccines should

825 not be transmissible to unvaccinated ‘in-contact’ animals and should not be able to infect the fetus.

826 Ideally seeds prepared for the production of inactivated vaccines should grow to high titre to minimise

827 the need to concentrate the antigens and there should be a minimal amount of protein from the cell

828 cultures incorporated into the final product. Master stocks for either live or inactivated vaccines should

829 be prepared under a seed lot system involving master and working stocks that can be used for

830 production in such a manner that the number of passages can be limited and minimise antigenic drift.

831 While there are no absolute criteria for this purpose, as a general guide, the seed used for production

832 should not be passaged more than 20 times beyond the master seed and the master seed should be

833 of the lowest passage from the original isolate as is practical.

###### 834 2.1.2.	Quality criteria (sterility, purity, freedom from extraneous agents)

835 It is crucial to ensure that all materials used in the preparation of the bulk antigens have been

836 extensively screened to ensure freedom from extraneous agents. This should include master and

837 working seeds, the cell cultures and all medium supplements such as bovine serum. It is particularly

838 important to ensure that any serum used that is of bovine origin is free of both adventitious BVDV of

839 all ~~geno~~types and antibodies against BVDV strains because low levels of either virus or antibody can

840 mask the presence of the other. Materials and vaccine seeds should be tested for sterility and

841 freedom from contamination with other agents, especially viruses as described in the chapter 1.1.8

842 and chapter 1.1.9.

###### 843 2.1.3.	Validation as a vaccine strain

844 All vaccines should pass standard tests for efficacy. Tests should include as a minimum the

845 demonstration of a neutralising antibody response following vaccination, a reduction in virus shedding

846 after challenge in vaccinated cattle and ideally a prevention of viraemia. Efficacy tests of BVD

847 vaccines by assessing clinical parameters in non-pregnant cattle can be limited by the difficulty of

848 consistently establishing clinical signs but, when employed, clinical parameters such as a reduction

849 in the rectal temperature response and leukopenia should be monitored. Although it can be difficult

850 by using virus isolation in cell culture to consistently demonstrate the low levels of viraemia associated

851 with an acute infection, real-time PCR could be considered as an alternative method to establish the

852 levels of circulating virus.

853 If a vaccine passes basic tests, the efficacy of vaccination should ultimately be measured by the

854 capacity to prevent transplacental transmission. If there is a substantial reduction and ideally

855 complete prevention of fetal infection, a vaccine would be expected to be highly effective in other

856 situations (for example prevention of respiratory disease). A suitable challenge system can be

857 established by intranasal inoculation of noncytopathic virus into pregnant cows between 60 and 90

858 days of gestation (Brownlie *et al*., 1995). Usually this system will reliably produce persistently viraemic

859 offspring in non-immune cows. In countries where BVDV type 2 viruses (*Pestivirus tauri*) are

860 commonly encountered, efficacy in protecting against BVDV type 2 infections should be measured.

##### 861 2.2.	Method of manufacture

###### 862 2.2.1.	Procedure

863 Both cytopathic and noncytopathic biotypes will grow in a variety of cell cultures of bovine origin.

864 Standard procedures may be used, with the expectation for harvesting noncytopathic virus on days

865 4–7 and cytopathic virus on days 2–4. The optimal yield of infectious virus will depend on several

866 factors, including the cell culture, isolate used and the initial seeding rate of virus. These factors

867 should be taken into consideration and virus replication kinetics investigated to establish the optimal

868 conditions for large scale virus production. Whether a live or inactivated vaccine, the essential aim

869 will be to produce a high-titred virus stock. This bulk antigen preparation can subsequently be

870 prepared according to the type of vaccine being considered.

###### 871 2.2.2.	Requirements for ingredients

872 Most BVDV vaccines are grown in cell cultures of bovine origin that are frequently supplemented with

873 medium components of animal origin. The material of greatest concern is bovine serum due to the

874 potential for contamination with BVD viruses and antibodies to these viruses. These adventitious

875 contaminants not only affect the efficiency of production but also may mask the presence of low levels

876 of infectious BVDV that may have undesirable characteristics. In addition to the virus seeds, all

877 materials should be tested for sterility and freedom from contamination with other agents, especially

878 viruses as described in chapters 1.1.8 and 1.1.9. Further, materials of bovine or ovine origin should

879 originate from a country with negligible risk for transmissible spongiform encephalopathies [TSEs]

880 (see chapter 1.1.9).

###### 881 2.2.3.	In-process controls

882 In-process controls are part of the manufacturing process. Cultures should be inspected regularly to

883 ensure that they remain free from contamination, and to monitor the health of the cells and the

884 development or absence of CPE, as appropriate. While the basic requirement for efficacy is the

885 capacity to induce an acceptable neutralising antibody response, during production, target

886 concentrations of antigen required to achieve an acceptable response may be monitored indirectly

887 by assessment of the quantity of infectious virus or antigen mass that is produced. Rapid diagnostic

888 assays such as the ELISA are useful to monitor BVDV antigen production. Alternatively, the quality

889 of a batch of antigen may be determined by titration of the quantity of infectious virus present,

890 although this may underestimate the quantity of antigen. For inactivated vaccines, infectivity is

891 evaluated before inactivation. For inactivated vaccines the inactivation kinetics should be established

892 so that a suitable safety margin can be determined and incorporated into the routine production

893 processes. At the end of production, *in-vitro*cell culture assays should be undertaken to confirm that

894 inactivation has been complete. These innocuity tests should include a sufficient number of passages

895 and volume of inoculum to ensure that very low levels of infectious virus would be detected if present.

###### 896 2.2.4.	Final product batch tests

897 i)  Sterility

898 Tests for sterility and freedom from contamination of biological materials intended for veterinary use

899 may be found in Chapter 1.1.9.

900 ii) Identity

901 Identity tests should demonstrate that no other strain of BVDV is present when several strains are

902 propagated in a facility producing multivalent vaccines.

903 iii) Safety

904 Safety tests shall consist of detecting any abnormal local or systemic adverse reactions to the vaccine

905 by all vaccination route(s). Batch-to-batch safety tests are required unless safety of the product is

906 demonstrated and APPROVED in the registration dossier and production is consistent with that

907 described in chapter 1.1.8.

908 The safety test is different to the inocuity test (see above).

909 Live vaccines must either be demonstrated to be safe in pregnant cattle (i.e. no transmission to the

910 fetus), or should be licensed with a warning not to use them in pregnant animals. Live vaccines

911 containing cytopathic strains should have an appropriate warning of the risk of inducing mucosal

912 disease in PI cattle.

913 iv) Batch potency

914 BVD vaccines must be demonstrated to produce adequate immune responses, when used in their

915 final formulation according to the manufacturer’s published instructions. The minimum quantity of

916 infectious virus and/or antigen required to produce an acceptable immune response should be

917 determined. *In-vitro*assays should be used to monitor individual batches during production.

##### 918 2.3.	Requirements for authorisation/registration/licensing

###### 919 2.3.1.	Manufacturing process

920 For registration of a vaccine, all relevant details concerning manufacture of the vaccine and quality

921 control testing should be submitted to the relevant authorities. Unless otherwise specified by the

922 authorities, information should be provided from three consecutive vaccine batches with a volume

923 not less than 1/3 of the typical industrial batch volume.

924 There is no standard method for the manufacture of a BVD vaccine, but conventional laboratory

925 techniques with stationary, rolled or suspension (micro-carriers) cell cultures may be used.

926 Inactivated vaccines can be prepared by conventional methods, such as binary ethylenimine or beta-

927 propiolactone inactivation (Park & Bolin, 1987). A variety of adjuvants may be used.

###### 928 2.3.2.	Safety requirements

929 *In-vivo*tests should be undertaken using a single dose, overdose (for live vaccines only) and repeat

930 doses (taking into account the maximum number of doses for primary vaccination and, if appropriate,

931 the first revaccination/booster vaccination) and contain the maximum permitted antigen load and,

932 depending on the formulation of the vaccine, the maximum number of vaccine strains.

933 i)  Target and non-target animal safety

934 The safety of the final product formulation of both live and inactivated vaccines should be assessed

935 in susceptible young calves that are free of maternally derived antibodies and in pregnant cattle. They

936 should be checked for any local reactions following administration, and, in pregnant cattle, for any

937 effects on the unborn calf. Live attenuated vaccines may contribute to immunosuppression that might

938 increase mortality. It may also contribute to the development of mucosal disease in PI animals that

939 is an animal welfare concern. Therefore vaccination of PI animals with live attenuated vaccines

940 containing cytopathic BVDV should be avoided. Live attenuated vaccines must not be capable of

941 being transmitted to other unvaccinated animals that are in close contact.

942 ii) Reversion-to-virulence for attenuated/live vaccines and environmental considerations

943 Virus seeds that have been passaged at least up to and preferably beyond the passage limit specified

944 for the seed should be inoculated into young calves to confirm that there is no evidence of disease.

945 If a live attenuated vaccine has been registered for use in pregnant animals, reversion to virulence

946 tests should also include pregnant animals. Live attenuated vaccines should not be transmissible to

947 unvaccinated ‘in-contact’ animals.

948 iii) Precautions (hazards)

949 BVDV is not considered to be a human health hazard. Standard good microbiological practice should

950 be adequate for handling the virus in the laboratory. A live virus vaccine should be identified as

951 harmless for people administering the product. However adjuvants included in either live or

952 inactivated vaccines may cause injury to people. Manufacturers should provide adequate warnings

953 that medical advice should be sought in the case of self-injection (including for adjuvants, oil-emulsion

954 vaccine, preservatives, etc.) with warnings included on the product label/leaflet so that the vaccinator

955 is aware of any danger.

###### 956 2.3.3.	Efficacy requirements

957 The potency of the vaccine should be determined by inoculation into seronegative and virus negative

958 calves, followed by monitoring of the antibody response. Antigen content can be assayed by ELISA

959 and adjusted as required to a standard level for the particular vaccine. Standardised assay protocols

960 applicable to all vaccines do not exist. Live vaccine batches may be assayed by infectivity titration.

961 Each production batch of vaccine should undergo potency and safety testing as batch release criteria.

962 BVD vaccines must be demonstrated to produce adequate immune responses, as outlined above,

963 when used in their final formulation according to the manufacturer’s published instructions.