**Annex 10. Item 5.1. – Chapter 3.4.1. Bovine anaplasmosis**

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

3	**Paris, 4–8 September 2023**

4

## 5	SEC T I O N  3. 4. 

6	**BO V I NA E**

# 7	C H A P T E R 3 . 4 . 1 .

8	**B OV I N E A N AP L A S M OS IS**

9	**SUMMARY**

10 ***Definition of the disease:****Bovine anaplasmosis results from infection with*Anaplasma marginale*. A second*

11 *species,*A. centrale*, has long been recognised and usually causes benign infections.*Anaplasma marginale

12 *is responsible for almost all outbreaks of clinical disease.*Anaplasma phagocytophilum *and*A. bovis, *which*

13 *infect cattle, ~~have been recently~~are also included within the genus~~but they are not reported to~~.*Anaplasma

14 phagocytophilum *can**cause ~~clinical~~self-limiting disease in cattle. There are no reports of disease associated*

15 *with*A. bovis *infection**. The organism is classified in the genus*Anaplasma *belonging to the family*

16 Anaplasmataceae *of the order*Rickettsiales*.*

17 ***Description of the disease:****Anaemia, jaundice in acute, severe cases and ~~sudden~~unexpected death are*

18 *characteristic signs of bovine anaplasmosis. Other signs include rapid loss of milk production and weight,*

19 *but the clinical disease can only be confirmed by identifying the organism. Once infected, cattle may remain*

20 *carriers for life, and identification of these animals depends on the detection of specific antibodies using*

21 *serological tests, or of rickettsial DNA using molecular amplification techniques. The disease is typically*

22 *transmitted by tick vectors, but mechanical transmission by biting insects or by needle can occur.*

23 ***Detection~~Identification~~of the agent:****Microscopic examination of blood or organ smears stained with*

24 *Giemsa stain is the most common method of identifying*Anaplasma *in clinically affected animals. In these*

25 *smears,*A. marginale *organisms appear as dense, rounded, intraerythrocytic bodies approximately 0.3–*

26 *1.0 μm in diameter situated on or near the margin of the erythrocyte.*Anaplasma centrale *is similar in*

27 *appearance, but most of the organisms are situated toward the centre of the erythrocyte. It can be difficult*

28 *to differentiate*A. marginale *from*A. centrale *in a stained smear, particularly with low levels of rickettsaemia.*

29 *Commercial stains that give very rapid staining of*Anaplasma spp.*are available in some countries.*

30 Anaplasma phagocytophilum *can only be observed in infected granulocytes, mainly neutrophils and*A. bovis

31 *can only be observed in infected monocytes~~infecting granulocytes, mainly neutrophils~~.*

32 *It is important that smears be well prepared and free from foreign matter. Smears from live cattle should*

33 *preferably be prepared from blood drawn from the jugular vein or another large vessel. For post-mortem*

34 *diagnosis, smears should be prepared from internal organs*(*including liver, kidney, heart and lungs*) *and*

35 *from blood retained in peripheral vessels. The latter are particularly ~~desirable~~useful if post-mortem*

36 *decomposition is advanced.*

37 ***Serological tests:****A competitive enzyme-linked immunosorbent assay*(*C-ELISA*) *has ~~been demonstrated~~*

38 *~~to have~~good sensitivity in detecting carrier animals. Card agglutination is the next most frequently used*

39 *assay. The complement fixation test*(*CFT*) *is no longer considered a reliable test ~~for disease certification of~~*

40 *individual animals due to variable sensitivity. Cross reactivity between*Anaplasma *spp. can complicate*

41 *interpretation of serological tests. In general, the C-ELISA has the best specificity, with cross-reactivity*

42 *described between*A. marginale, A. centrale, A. phagocytophilum *and*Ehrlichia *spp. Alternatively, an*

43 *indirect ELISA ~~using the CFT with modifications~~*(*I-ELISA*)*is a reliable test used in many laboratories and*

44 *can be prepared in-house for routine diagnosis of anaplasmosis. Finally, a displacement double-antigen*

45 *sandwich ELISA has been developed to differentiate between*A. marginale *and*A. centrale *antibodies.*

46 ***Nucleic-acid-based tests****~~have been used~~are often used in diagnostic laboratories and experimentally,*

47 *and are capable of detecting the presence of low-level infection in carrier cattle and tick vectors. A nested*

48 *conventional polymerase chain reaction (PCR*)*~~reaction is necessary~~has been used to identify low-level*

49 *carriers~~using conventional polymerase chain reaction~~*~~(*PCR*)~~, *~~and~~although nonspecific amplification can*

50 *occur. ~~Recently,~~Real-time PCR assays ~~with~~have analytical sensitivity equivalent to nested conventional*

51 *PCR ~~have been described~~and are preferable in a diagnostic setting to reduce the risk of amplicon*

52 *contamination.*

53 ***Requirements for vaccines:****Live vaccines are used in several countries to protect cattle against*

54 ~~A. marginale *infection*~~*bovine anaplasmosis. A vaccine consisting of live*A. centrale *is most widely used and*

55 *gives partial protection against challenge with virulent*A. marginale*. Vaccination with*A. centrale *leads to*

56 *infection and long-term persistence in many cattle. Vaccinated cattle are typically protected from disease*

57 *caused by*A. marginale*, but not infection.*

58 Anaplasma centrale *vaccine is provided in chilled or frozen forms. Quality control is very important as other*

59 *blood-borne agents that may be present in donor cattle can contaminate vaccines and be disseminated*

60 *broadly. For this reason, frozen vaccine is recommended as it allows thorough post-production quality control,*

61 *which limits the risk of contamination with other pathogens.*

62 Anaplasma centrale *vaccine is not entirely safe. A practical recommendation is to restrict its use, as far as*

63 *possible, to calves, as nonspecific immunity will minimise the risk of some vaccine reactions that may require*

64 *treatment with tetracycline or imidocarb. Partial immunity develops in 6–8 weeks and lasts for several years*

65 *after a single vaccination. In countries where*A. centrale *is exotic, it cannot be used as a vaccine against*A.

66 marginale.

## 67	A. INTRODUCTION

68 Outbreaks of bovine anaplasmosis are due to infection with *Anaplasma marginale. Anaplasma centrale*is capable of

69 ~~producing~~can produce a moderate degree of anaemia, but clinical outbreaks in the field are extremely rare. ~~New species~~

70 ~~of *Anaplasma*,~~Other members of the family Anaplasmataceae that infect cattle include*A. phagocytophilum*and *A. bovis*

71 (Dumler *et al*., 2001)*~~,~~*~~with a primary reservoir~~. *Anaplasma phagocytophilum*has a broad host range and causes the

72 diseases human granulocytic anaplasmosis (HGE), equine granulocytic anaplasmosis (EGA), and canine granulocytic

73 anaplasmosis (CGA), in humans, horses, and dogs, respectively (Matei *et al.,*2019). In northern Europe~~in rodents~~, *A.*

74 *phagocytophilum*causes tick-borne fever, primarily affecting lambs. In cattle, *A. phagocytophilum*infections have been

75 reported ~~to infect cattle, but do not cause~~from many geographical regions, however the association with disease is less

76 commonly reported. Naturally occurring clinical disease as reported in Germany was characterised by fever (39.5–41.7°

77 C), sudden reduction in milk production, lower limb oedema, and stiffness with leukopenia, erythropenia, neutropenia,

78 lymphocytopenia and monocytopenia. The affected animals recovered without antibiotic treatment (~~Dreher *et al.,*2005;~~

79 ~~Hofmann-Lehmann *et al.*, 2004~~Silaghi *et al*., 2018).

80 The most marked clinical signs of bovine anaplasmosis are anaemia and jaundice, the latter occurring in acute severe,

81 cases or late in the disease. Haemoglobinaemia and haemoglobinuria are not present, and this may assist in the differential

82 diagnosis of bovine anaplasmosis from babesiosis, which is often endemic in the same regions. The disease can only be

83 confirmed, however, by identification of the organism in erythrocytes from the affected animal. Caution must be exercised

84 if using nucleic acid techniques alone to diagnose *A. marginale*in anaemic cattle. Persistent, low-level infection can be

85 detected by these techniques and may lead to a misdiagnosis of bovine anaplasmosis. Visualisation of *A. marginale*bodies

86 in erythrocytes is therefore required for confirmation.

87 *Anaplasma marginale*occurs in most tropical and subtropical countries and is widely distributed in ~~some more~~temperate

88 regions. *Anaplasma centrale*was first described from South Africa. The organism has since been imported by other

89 countries – including Australia and some countries in South America, South-East Asia and the Middle East – for use as a

90 vaccine against *A. marginale.*

91 *Anaplasma*species~~were~~, though originally ~~regarded~~described as protozoan parasites, ~~but further research showed they~~

92 ~~had no significant attributes to justify this description. Since the last major accepted revision of the~~are obligate intracellular

93 Gram-negative bacteria. Based on taxonomy established in 2001 (Dumler *et al.*, 2001), the Family *Anaplasmataceae*

94 (Order *Rickettsiales*) is ~~now~~ composed of ~~four~~five genera, *Anaplasma*, *Ehrlichia, Neorickettsia,*~~and~~*Wolbachia*~~. The genus~~

95 and*Aegyptianella*~~is retained within the Family *Anaplasmataceae*as genus *incertae sedis*. The revised genus~~. The genus

96 *Anaplasma*~~now~~ contains *Anaplasma marginale*as the type species, *A. phagocytophilum*the agent of human granulocytic

97 ehrlichiosis (formerly *Ehrlichia phagocytophila*and *E. equi*), *A. platys,*and *A. bovis*(formerly *E. bovis*)*.~~Haemobartonella~~*

98 ~~and *Eperythrozoon*are now considered most closely related to the mycoplasmas.~~

99 *Anaplasma*species are transmitted either mechanically or biologically by arthropod vectors. ~~Reviews based on careful~~

100 ~~study~~Detection of ~~reported transmission experiments list up~~pathogen DNA within a tick is insufficient to ~~19 different ticks~~

101 ~~as capable of~~determine the ability of a particular tick species to transmit a pathogen. Studies demonstrating transmission

102 of the pathogen are critical in determining the potential role of a particular tick species in pathogen transmission~~transmitting~~

103 *~~A. marginale~~*~~(Kocan *et al.*, 2004). These are: *Argas persicus, Ornithodoros lahorensis,*~~. Many studies have demonstrated

104 the	transmission	ability	of*Dermcentor	~~albipictus,	D.~~andersoni*,	*~~D. hunteri,	D. occidentalis,~~D. variabilis*,

105 *~~Hyalomma excavatum, H. rufipes, Ixodes ricinus, I. scapularis,~~*and *D. albipictus*. Additionally, transmission by multiple

106 *Rhipicephalus*species is well recognised including *R.**annulatus*~~(formerly *Boophilus annulatus),*~~*R. bursa*, *R. calcaratus,*

107 *R. decoloratus, R. evertsi, R. microplus~~, R. sanguineus~~*~~and *R. simus.*However, the classification of several ticks in these~~

108 ~~reports has been questioned.~~and *R. sanguineous*. Other species of *Rhipicephalus*also likely serve as biological vectors

109 of *A. marginale*. *Anaplasma marginale*DNA has been widely reported in *Hyalomma*species, and transmission has been

110 demonstrated with *H. excavatum*. It is likely that multiple *Hyalomma*species also serve as vectors of *A. marginale*(Shkap

111 *et al*., 2009).

112 Intrastadial or transstadial transmission ~~is the usual mode~~can occur, even in the one-host, *Rhipicephalus*species. Male

113 ticks may be particularly important as vectors, as they ~~can become persistently infected and serve as a reservoir~~are most

114 likely to move between cattle searching for ~~infection~~female ticks. Experimental demonstration of vector competence does

115 not necessarily imply a role in transmission in the field. However, *Rhipicephalus*species are clearly important vectors of

116 anaplasmosis in ~~countries such as~~ Australia~~and countries in~~, many regions of Africa, and Latin America~~, and some species~~

117 ~~of~~. *Dermacentor*spp. are efficient vectors in the United States of America (USA).

118 Various other biting arthropods have been implicated as mechanical vectors, particularly in the USA. Experimental

119 transmission has been demonstrated with a number of species of *Tabanus*(horseflies), and with mosquitoes of the genus

120 *Psorophora*~~(Kocan *et al.*, 2004)~~. The importance of biting insects in the natural transmission of anaplasmosis appears to

121 vary greatly from region to region. *Anaplasma marginale*also can be readily transmitted during vaccination against other

122 diseases unless a fresh or sterilised needle is used for injecting each animal. Similar transmission by means of unsterilised

123 surgical instruments has been described (Reinbold *et al.*, 2010a).

124 The ~~main~~only known biological vector~~s~~ of *A. centrale*~~appear to be multihost ticks~~is *R. simus*, endemic in Africa~~, including~~

125 *~~R. simus.~~*~~The~~*.*Though multiple transmission studies have been done, there is no evidence that the common cattle tick

126 (*R. microplus*) ~~has not been shown to be~~can serve as a vector for *A. centrale*. This is ~~of relevance~~relevant where

127 *A. centrale*is used as a vaccine in *R. microplus*-infested regions.

128 *Anaplasma marginale*infection has not been reported in humans. ~~Thus,~~There is ~~no~~minimal risk of field or laboratory

129 transmission to workers ~~and~~from laboratories working with *A. marginale*~~may operate at the lowest biosafety level,~~

130 ~~equivalent to BSL1~~. Nevertheless the agent should be handled with appropriate biosafety and containment procedures as

131 determined by biorisk analysis (see Chapter 1.1.4 *Biosafety and biosecurity: Standard for managing biological risk in the*

132 *veterinary laboratory and animal facilities*).

## 133	B. DIAGNOSTIC TECHNIQUES

134 ***Table 1.****Test methods available for the diagnosis of bovine anaplasmosis and their purpose*

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

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

137 Agent id. = agent identification; CAT = card agglutination test; ~~CFT = complement fixation test;~~

138 C-ELISA = competitive enzyme-linked immunosorbent assay; ddasELISA = displacement double-antigen, sandwich ELISA;

139 IFAT = indirect fluorescent antibody test; PCR = polymerase chain reaction.

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

141 (b)These tests do not distinguish infected from vaccinated animals.

#### 142 1.	Detection of the agent

##### 143 1.1.	Microscopic examination

144 Samples from live cattle should include thin blood smears and blood collected into an anticoagulant. Air-dried thin

145 blood smears can be kept satisfactorily at room temperature for at least 1 week. The blood sample in anticoagulant

146 should be held and transferred at 4°C, unless it can reach the laboratory within a few hours. This sample is useful for

147 preparing fresh smears if those submitted are not satisfactory. In addition, a low packed cell volume ~~and/~~or erythrocyte

148 count can help to substantiate the involvement of *A. marginale*when only small numbers of the parasites are detected

149 in smears, ~~for example~~particularly during the recovery stage of the disease.

150 In contrast to *Babesia bovis, A. marginale-*~~does~~infected erythrocytes do not accumulate in capillaries, so blood drawn

151 from the jugular or other large vessel is satisfactory. *Anaplasma marginale*replicate in the erythrocytes to form small

152 membrane-bound colonies, also termed inclusion bodies or initial bodies.~~Because of the rather indistinctive~~

153 ~~morphology of *Anaplasma*~~**These initial bodies can be visualised on a blood smear, but are small and easily confused

154 with debris or stain precipitate (see Figure 1)*.*Thus it is essential that smears are well prepared~~and~~, including

155 ensuring slides are free ~~from foreign matter, as specks~~of debris ~~can confuse diagnosis~~and stain is recently filtered

156 (Watman #1 filter paper). Thick blood films as are used sometimes for the diagnosis of babesiosis are not appropriate

157 for the diagnosis of anaplasmosis, as *~~Anaplasma~~A. marginale*are difficult to identify once they become dissociated

158 from erythrocytes.

159

160 ***Figure. 1.****Anaplasma marginale initial bodies. A Diff-Quick stained blood smear from a bovine experimentally infected*

161 *with A. marginale. Arrows point to the A. marginale initial bodies. Photo from S. Noh.*

162 Samples from dead animals should include air-dried thin smears from the liver, kidney, heart and lungs and from a

163 peripheral blood vessel. The latter is particularly recommended should there be a significant delay before post-

164 mortem examination because, under these circumstances, bacterial contamination of organ smears often makes

165 identification of *~~Anaplasma~~A. marginale*equivocal. Brain smears, which are useful for the diagnosis of some forms

166 of babesiosis, are of no direct value for diagnosing anaplasmosis, but should be included for differential diagnosis

167 where appropriate.

168 Blood from organs, rather than organ tissues *per se*, is required for smear preparation, as the aim is to be able to

169 examine microscopically intact erythrocytes for the presence of *~~Anaplasma~~A.marginalecolonies.*Organ-derived

170 blood smears can be stored satisfactorily at room temperature for several days.

171 Both blood and organ smears can be stained in 10% Giemsa stain for approximately 30 minutes after fixation in

172 absolute methanol for 1 minute. After staining, the smears are rinsed three or four times with tap water to remove

173 excess stain and are then air-dried. Conditions for Giemsa staining vary from laboratory to laboratory, but distilled

174 water is not recommended for dilution of Giemsa stock. Water should be pH 7.2–7.4 to attain best resolution with

175 Giemsa stain. Commercial stains that give very rapid staining of *~~Anaplasma~~A.marginale*are available in some

176 countries. Smears ~~are~~must be examined under oil immersion at a magnification of ×700–1000.

177 *Anaplasma marginale*~~appear as dense,~~initial bodies are round~~ed and~~ deeply stained~~intraerythrocytic bodies,~~and

178 approximately 0.3–1.0 μm in diameter. Most of these bodies are located on or near the margin of the erythrocyte.

179 This feature distinguishes *A. marginale*from *A. centrale,*as in the latter most of the organisms have a more central

180 location in the erythrocyte. However, particularly at low levels of rickettsaemia, differentiation of these two species in

181 smears can be difficult. Appendages associated with the *Anaplasma*~~body~~initial body have been described in some

182 isolates of *A. marginale*~~(Kreier & Ristic, 1963; Stich *et al*., 2004)~~.

183 The percentage of infected erythrocytes varies with the stage and severity of the disease. Maximum rickettsaemias

184 in excess of 50% may occur with *A. marginale.*Multiple infections of individual erythrocytes are common during

185 periods of high rickettsaemias.

186 The infection becomes visible microscopically 2–6 weeks following transmission. During the course of clinical

187 disease, the rickettsaemia approximately doubles each day for up to about 10 days, and then decreases at a similar

188 rate. Severe anaemia may persist for some weeks after the parasites have become virtually undetectable in blood

189 smears. Following recovery from initial infection, cattle remain latently infected for life.

##### 190 1.2.	Polymerase chain reaction

191 Nucleic-acid-based tests to detect *A. marginale*~~infection~~ in ~~carrier~~infected cattle have been developed although not

192 ~~yet~~fully validated. The analytical sensitivity of polymerase chain reaction (PCR)-based methods has been estimated

193 at 0.0001% infected erythrocytes, but at this level, only a proportion of carrier cattle would be detected. A nested PCR

194 has been used to identify *A. marginale*carrier cattle with a capability of identifying as few as 30 infected erythrocytes

195 per ml of blood, well below the lowest levels in carriers. However, nested PCR is time consuming as it requires two

196 full PCR reactions, and poses significant quality control ~~and specificity~~problems for routine use (Torioni De Echaide

197 *et al.*, 1998). Real-time PCR assays are reported to achieve a level of analytical sensitivity equivalent to nested PCR

198 ~~has also been described for identification of *A. marginale*~~**and should be considered instead of the nested PCR (Carelli

199 *et al.*, 2007; Decaro *et al.*, 2008~~; Reinbold *et al.*, 2010b~~). ~~Two~~Advantages of ~~this technique~~the real-time PCR, which

200 uses a single closed tube for amplification and analysis, are reduced opportunity for amplicon contamination and a

201 semi-quantitative assay result. Equipment and reagents needed for real-time PCR ~~is~~are expensive~~, requires~~

202 ~~preventive maintenance,~~ and may be beyond the capabilities of some laboratories. ~~Real-time PCR assays may target~~

203 ~~one of several genes (Carelli *et al.*, 2007; Decaro *et al.*, 2008), or 16S rRNA (Reinbold *et al.*, 2010b), and are reported~~

204 ~~to achieve a level of analytical sensitivity equivalent to nested conventional PCR (Carelli *et al.*, 2007; Decaro *et al.*,~~

205 ~~2008; Reinbold *et al.*, 2010b).~~

206 The most widely cited assays for the detection *A. marginale*in individual animals use a probe for increased specificity

207 and are designed to detect *msp1b*(Carelli *et al.*, 2007) or *msp5*(Futse *et al.,*2003) in genomic DNA extracted from

208 whole blood. The assay based on detection of *msp1b*has been partially validated to detect the pathogen in individual

209 animals and was used to define samples for the validation of a C-ELISA (Carelli *et al.*, 2007; Chung *et al.,*2014). The

210 analytical test performance of this assay is robust, and exclusivity testing confirmed other bacterial and protozoal tick-

211 borne pathogens of cattle were not detected. The assay, evaluated using 51 blood samples from 18 cattle herds in

212 three regions of southern Italy, had 100% concordance with nested PCR.

213 *Msp1b*is a multigene family. Based on the annotation of the St. Maries strain of *A. marginale*, the designed primers

214 and probe will amplify multiple members of this gene family, including *msp1b-1*, *msp1b-2*, and *msp1-pg3*). This may

215 help increase diagnostic sensitivity, but may pose challenges if quantification of the pathogen is desired. Additionally,

216 some *A. marginale*strains have single nucleotide polymorphisms in *msp1b*within the primer and probe binding

217 regions. Thus, if *msp1b*is used as a diagnostic target, primer and probe design should consider local *A. marginale*

218 strains. *Msp1b*has the advantage as a target in that orthologs of this gene family are absent in the related *A.*

219 *phagocytophilum*and *Ehrlichia*spp., including *E. ruminantium*, thus helping ensure specificity of the test.

220 *Msp5*has also been used as a target to detect *A. marginale*in cattle in field samples and more frequently in

221 experimental samples (Futse *et al.,*2003). *Msp5*is highly conserved among *A. marginale*strains and is a single copy

222 gene, thus providing some advantages as a target for ensuring detection of widely variant strains of *A. marginale*.

223 However, the related *Anaplasma*spp. and *Ehrlichia*spp. all have *msp5*orthologs with 50% identity to an *E.*

224 *ruminantium*gene (NCBI accession: L07385.1), thus specificity must be determined in laboratory and field samples.

225 Additionally, little work has been done to validate an *msp5*-based real-time PCR test for diagnostic purposes.

226 A third primer–probe set is designed to detect *A. marginale*using real-time, reverse transcriptase PCR. The primers

227 amplify a 16sRNA gene segment from *A. marginale*and *A. phagocytophilum*, while the probe differentiates between

228 the two species (Reinbold *et al.*, 2010b). The analytical performance of this assay is robust. However, the diagnostic

229 sensitivity, specificity, and of particular importance with 16sRNA sequence-based tests, exclusivity for other tick-

230 borne pathogens of cattle have not been evaluated. Additionally, this assay is designed for use following RNA

231 extraction and reverse transcription, which is more laborious and expensive than DNA extraction. Bacterial RNA is

232 rapidly degraded, and this may ultimately reduce diagnostic sensitivity of this assay.

233 In regions that use *A. centrale*as a vaccine, it may be useful to differentiate between *A. marginale*and *A. centrale*

234 infected/vaccinated animals. PCR is best suited for this task. The real-time PCR assay developed by Carelli *et al.*

235 can also be used in a duplex reaction to detect and differentiate between *A. centrale*and *A. marginale*(Decaro *et al.*,

236 2008). Primers and probe have been designed to specifically amplify a region of *A. centrale groEL*, but not *A.*

237 *marginale*groEL, despite 97% sequence identity between the two genes. The *A. marginale-*specific primers and

238 probes perform similarly in the single and duplex PCR (Carelli *et al.*, 2007). Using the same 51 field samples from

239 cattle in Italy, the *A. centrale*assay had less analytical sensitivity compared with nested PCR and discordance in 4

240 of 51 samples between an *A. centrale*reverse line blot test and the duplex PCR assay.

241 ***Table 1.****Oligonucleotides used in PCR assays to detect*A. marginale *and*A. centrale

242 (a)*Am*denotes *A. marginale*, *Ac*denotes *A. centrale*, Pb denotes probe sequence.

243 (b)Fluorophores and quenchers not included in probe sequences.

244 (c)Amplifies *A. phagocytophilum*and *A. marginale*16S rRNA gene.

245 (d)Probe is specific for *A. marginale*16S rRNA gene.

246 (e)Can be used as a duplex PCR with *msp1b*primers and probe based on Carelli *et al.,*2007.

247 (f)Primers and probe amplify *A. centrale groEL*.

#### 248 2.	Serological tests

249 In general, unless animals have been treated or are at a very early stage of infection (<14 days), serology using the

250 competitive enzyme-linked immunosorbent assay (C-ELISA), indirect ELISA (I-ELISA) or card agglutination test (CAT)

251 (see below) may be the preferred methods of identifying infected animals in most laboratories. *Anaplasma marginale*

252 infections usually persist for the life of the animal. However, except for occasional small recrudescences, *~~Anaplasma~~*

253 *A. marginale initial bodies*cannot readily be detected in blood smears after acute rickettsaemia and, ~~even~~end-point PCR

254 may not detect the presence of *~~Anaplasma~~*the pathogen in blood samples from asymptomatic carriers. Thus, a number of

255 serological tests have been developed with the aim of detecting persistently infected animals.

256 A feature of the serological diagnosis of anaplasmosis is the highly variable results with regard to both sensitivity and

257 specificity reported for many of the tests from different laboratories. This is due at least in part to inadequate ~~evaluation~~

258 validation of the tests using significant numbers of known positive and negative animals. ~~Importantly, the capacity of severa~~l

259 ~~assays to detect known infections of long-standing duration has been inadequately addressed.~~ An exception is a C-ELISA

260 (see below), which ~~has been~~was initially validated using true positive and negative animals defined by nested PCR (Torioni

261 De Echaide *et al.*, 1998)~~, and the card agglutination assay, for which relative sensitivity and specificity in comparison with~~

262 ~~the C-ELISA has been evaluated (Molloy *et al.*, 1999).~~And updated in 2014 (Chung *et al*., 2014). Therefore, while most of

263 the tests described in this section are useful for obtaining broad-based epidemiological data, caution is advised on their

264 use for disease certification. The C-ELISA, I-ELISA and CAT are described in detail below.

265 It should be noted that there is a high degree of cross-reactivity between *A. marginale*and *A. centrale*, as well as cross-

266 reactivity with both *A. phagocytophilum*and *Ehrlichia*spp. in serological tests (Al-Adhami *et al.*, 2011; Dreher *et al*., 2005).

267 While the infecting species can sometimes be identified using antigens from homologous and heterologous species,

268 equivocal results are obtained on many occasions. Efforts have been made to develop tests that differentiate between

269 naturally acquired immunity to *A. marginale*and vaccine acquired immunity due to immunisation with *A. centrale*(Bellezze

270 *et al*., 2023; Sarli *et al*., 2020).

##### 271 2.1.	Competitive enzyme-linked immunosorbent assay

272 ~~A C-ELISA using a recombinant antigen termed~~Major surface protein 5 (MSP5) is an immunodominant protein

273 expressed by A. *marginale*, *A. ovis*, and *A. centrale*. In *A. marginale*the gene is highly conserved making it a useful

274 target across broad geographical regions with high *A. marginale*strain diversity (Knowles *et al.*, 1996; Torioni De

275 Echaide *et al.*, 1998). Thus, a C-ELISA based on recombinantly expressed (rMSP5~~and MSP5-~~) in combination with

276 an MSP5-specific monoclonal antibody (mAb) has proven very sensitive and specific for detection of *Anaplasma-*

277 infected animals (~~Hofmann-Lehmann *et al.*, 2004~~Molloy *et al*., 1999; Reinbold *et al.*, 2010b; Strik *et al.*, 2007). ~~Al~~l

278 *~~A. marginale~~*~~strains tested, along with~~Additionally, A. *ovis*and *A. centrale*, express ~~the~~MSP5 ~~antigen~~and ~~induce~~

279 infected animals produce antibodies against the immunodominant epitope recognised by the MSP5-specific ~~mAb. A~~

280 ~~recent report~~mAb used in the C-ELISA. This C-ELISA was updated in 2014 to improve performance by using

281 glutathione S-transferase (GST) instead of maltose binding protein (MBP) as the tag on the rMSP5 (Chung *et al*.,

282 2014). This assay no longer requires adsorption to remove the antibodies directed against MBP, thus it is faster and

283 easier than the previous version of the C-ELISA. The diagnostic sensitivity is 100% and the diagnostic specificity is

284 99.7% using a cut-off of 30% inhibition as determined by receiver operating characteristic (ROC) plot (Chung *et al*.,

285 2014). For this validation, 385 sera defined as negative were from dairy cattle maintained in tick-free facilities from

286 farms with no clinical history of bovine anaplasmosis. The 135 positive sera were from cattle positive for *A. marginale*

287 using nested PCR and serology.

288 One study suggested that antibodies from cattle experimentally infected with *A. phagocytophilum*will test positive in

289 the C-ELISA (Dreher *et al.*, 2005). However, in another study no cross-reactivity could be demonstrated, and the mAb

290 used in the assay did not react with *A. phagocytophilum*MSP5 in direct binding assays (Strik *et al.*, 2007). Cross

291 reactivity has been demonstrated between *~~A. marginale~~*~~and *Ehrlichia*spp, in naturally and experimentally infected~~

292 ~~cattle (Al-Adhami *et al*, 2011). Earlier studies had shown that the C-ELISA was 100% specific using 261 known~~

293 ~~negative sera from a non-endemic region, detecting acutely infected cattle as early as 16 days after experimental tick~~

294 ~~or blood inoculation, and was demonstrated to detect cattle that have been experimentally infected as long as 6 years~~

295 ~~previously (Knowles *et al.*, 1996). In detecting persistently infected cattle from an anaplasmosis-endemic region that~~

296 ~~were defined as true positive or negative using a nested PCR procedure, the rMSP5 C-ELISA had a sensitivity of~~

297 ~~96% and a specificity of 95% (Torioni De Echaide *et al.*, 1998)~~*A. marginale*and *Ehrlichia*sp. BOV2010 isolated in

298 Canada, in naturally and experimentally infected cattle (Al-Adhami *et al*, 2011).

299 Test results using the rMSP5 C-ELISA are available in less than 2~~.5~~hours. A test kit is available commercially that

300 contains specific instructions. Users should follow the manufacturer’s instructions. I~~n general, however, it is conducted~~

301 ~~as follows.~~

###### 302 ~~2.1.1.	Kit reagents~~

303 ~~A 96-well microtitre plate coated with rMSP5 antigen,~~

304 ~~A 96-well coated adsorption/transfer plate for serum adsorption to reduce background binding,~~

305 ~~100×Mab-peroxidase conjugate,~~

306 ~~10× wash solution and ready-to-use conjugate-diluting buffer,~~

307 ~~Ready-to-use substrate and stop solutions,~~

308 ~~Positive and negative controls~~

###### 309 ~~2.1.2.	Test procedure~~

310 i)	Add 70 μl of undiluted serum sample to the coated adsorption/transfer plate and incubate at

311 ~~room temperature for 30 minutes.~~

312 ~~ii)	Transfer 50 μl per well of the adsorbed undiluted serum to the rMSP5-coated plate and incubate~~

313 ~~at room temperature for 60 minutes.~~

314 ~~iii)	Discard the serum and wash the plate twice using diluted wash solution.~~

315 iv)	Add 50 μl per well of the 1× diluted MAb-peroxidase conjugate to the rMSP5-coated plate wells,

316 ~~and incubate at room temperature for 20 minutes.~~

317 ~~v)	Discard the 1×diluted MAb-peroxidase conjugate and wash the plate four times using diluted~~

318 ~~wash solution.~~

319 ~~vi)	Add 50 μl per well of the substrate solution, cover the plate with foil, and incubate for 20 minutes~~

320 ~~at room temperature.~~

321 ~~vii)  Add 50 μl per well of stop solution to the substrate solution already in the wells and gently tap~~

322 ~~the sides of the plate to mix the wells.~~

323 ~~viii) Immediately read the plate in the plate reader at 620, 630 or 650 nm.~~

###### 324 ~~2.1.3.	Test validation~~

325 ~~The mean average optical density (OD) of the negative control must range from 0.40 to 2.10. The average per~~

326 ~~cent inhibition of the positive control must be ≥30%.~~

###### 327 ~~2.1.4.	Interpretation of the results~~

328 ~~The % inhibition is calculated as follows:~~

329 ~~% inhibition = 100[1 – (Sample OD ÷ Negative Control OD)]~~

330 ~~Samples with <30% inhibition are negative. Samples with ≥30% inhibition are positive.~~

331 ~~Specificity of the MSP5 C-ELISA may be increased by using a higher percentage inhibition cut-off value~~

332 ~~(Bradway *et al*., 2001); however the effect of this change on sensitivity has not been thoroughly evaluated.~~

333 ~~Recently, an improved MSP5 C-ELISA was developed by replacing rMBP-MSP5 with rGST-MSP5 in addition~~

334 ~~to an improvement in the antigen-coating method by using a specific catcher system. The new rMSP5-GST~~

335 ~~C-ELISA was faster, simpler, had a higher specificity and an improved resolution compared with the rMSP5-~~

336 ~~MBP C-ELISA with MBP adsorption (Chung *et al*., 2014).~~

##### 337 2.2.	Indirect enzyme-linked immunosorbent assay

338 An I-ELISA was first developed using the CAT antigen, which is a crude *A. marginale*lysate (see below). ~~and it~~The

339 test can be implemented where the commercial C-ELISA is not available. Unlike the C-ELISA, most reagents, such

340 as buffers and ready-to dissolve substrates, are available commercially in many countries. Any laboratory can prepare

341 the antigen using local strains of *A. marginale*, though standardised methods have not been developed. I-ELISA uses

342 small amounts of serum and antigen that~~and the sensitivity and specificity of the test standardised with true positive~~

343 ~~and negative sera is as good as for the C-ELISA. As it~~ can be prepared in each laboratory.~~Only the general procedure~~

344 is described here (Barry *et al.*, 1986). For commercial kits, the manufacturer’s instructions should be followed. In the

345 ~~case of in-house I-ELISA~~The sensitivity and specificity of the test was 87.3% and 98.4–99.6% respectively, though

346 this varied by laboratory (Nielsen *et al*., 1996). For general methods, refer to Barry *et al.*(1986). ~~Initial bodies and~~

347 ~~membranes are obtained as for the complement fixation test (Rogers *et al.,*1964). This antigen is treated with 0.1%~~

348 ~~sodium dodecyl sulphate for 30 minutes prior to fixing the antigen to the microtitre plate.~~For each laboratory, the

349 specific amount of antigen ~~has to~~must be ~~adjusted~~optimised to obtain the best reading and the least expenditure.

350 Alternatively, rMSP5 can be used as the antigen in this test. This eliminates the need for preparation and

351 standardisation of antigen derived from splenectomised, *A. marginale*infected animals (Silva *et al*., 2006). In a

352 comparison between I-ELISA using the CAT antigen and rMSP with a histidine tag (rMSP5-HIS), these two I-ELISAs

353 performed identically. In this comparison, IFAT was used as the gold standard test (Silva *et al*., 2006).

354 Test results using the I-ELISA are available in about 4 to 5 hours. It is generally conducted as follows:

###### 362 2.2.2.	Test procedure (this test is run in triplicate)

363 i)	Plates can be prepared ahead of time and kept under airtight conditions at –20°C.

364 ii)	Carefully remove the plastic packaging before using plates, being careful not to touch the bottom

365 of them as this can distort the optical density reading.

366 iii)	Remove the lid and deposit 200 l PBST20 solution in each well and incubate for 5 minutes at

367 room temperature (RT).

368 iv)	For one plate, dissolve 1.1 g of skim milk (blocking agent) in 22 ml of PBST20.

369 v)	Remove the plate contents and deposit in each well 200 μl of blocking solution, put the lid on

370 and incubate for 60 minutes at 37°C.

371 vi)	Wash the plate three times for 5 minutes with PBST20.

372 vii)  Dilute all serum samples including controls 1/100 in PBST20 solution.

373 viii) Remove the contents of the plate and deposit 200 μl of diluted serum in each of the three wells

374 for each dilution, starting with the positive and negative and blank controls.

375 ix)	Incubate plate at 37°C covered for 60 minutes.

376 x)	Wash three times as described in subsection vi.

377 xi)	Dilute 1/1000 anti-IgG bovine alkaline phosphatase conjugate in PBST20 solution. Add 200 μl

378 of the diluted conjugate per well. Incubate the covered plate at 37°C for 60 minutes.

379 xii)  Remove the lid and wash three times as described in point vi above~~make three washes with~~

380 ~~PBST20~~.

381 xiii) Remove the contents of the plate and deposit 195 μl of 0.075% *p*-Nitrophenyl phosphate

382 disodium hexahydrate in Tris buffer in each well and incubate for 60 minutes at 37°C.

383 xiv) The reaction is quantified by a microplate reader spectrophotometer, adjusted to 405 nm

384 wavelength. The data are expressed in optical density (OD).

###### 385 2.2.3.	Data analysis

386 Analysis of results should take into account the following parameters.

387 i)	The mean value of the blank wells.

388 ii)	The mean value of the positive wells with their respective standard deviations.

389 iii)	The mean value of negative wells with their respective standard deviations.

390 iv)	The mean value of the blank wells is subtracted from the mean of all the other samples if not

391 automatically subtracted by the ELISA reader.

392 v)	Control sera are titrated to give optical density values ranging from 0.90 to 1.50 for the positive

393 and, 0.15 to 0.30 for the negative control.

394 Positive values are those above the cut-off calculated value which is the sum of the average of the

395 negative and two times the standard deviation.

396 ~~For purposes of assessing the consistency of the test operator, the error “E” must alsoo be estimated;~~

397 ~~this is calculated by determining the percentage represented by the standard deviation of any against~~

398 ~~their mean serum.~~

399 As with all diagnostic tests, it is important to measure reproducibility. For more details see Chapter

400 2.2.4 *Measurement uncertainty*.

##### 401 2.3.	Displacement double-antigen sandwich ELISA to differentiate between *A. marginale*and

402 ***A. centrale*antibodies**

403 In regions where vaccination with *A. centrale*is used to control bovine anaplasmosis, differentiation between

404 *A. centrale*-vaccinated and *A. marginale*-infected animals may be useful. Because there is often high amino acid

405 identity between *A. marginale*and *A. centrale*surface proteins, identifying unique targets for serological assays for

406 this purpose is difficult. Epitopes from MSP5 (aa28-210, without the transmembrane region) that are not shared

407 between *A. marginale*and *A. centrale*were used to develop a displacement double-antigen sandwich ELISA

408 (ddasELISA) (Bellezze *et al*., 2023; Sarli *et al*., 2020). The recombinant MSP5 epitopes from *A. marginale*or *A.*

409 *centrale*are expressed in *E. coli*with a histidine tag and purified. The ELISA plates are then coated with either the

410 recombinant *A. marginale*MSP5 epitope, or the *A. centrale*MSP5 epitope and blocked. Serum is added to the wells

411 and allowed to incubate. Following washing, a combination of biotinylated and non-biotinylated recombinant proteins

412 are added to improve specificity of the reaction (see below for specifics). The protein–biotin binding to the serum

413 antibody is detected with a peroxidase-streptavidin based detection system. The optical density for the *A. marginale*

414 MSP5-coated well (ODAm) and the OD for the *A. centrale*MSP5 (ODAc) coated well for each animal is measured. If

415 the OD for either target is <0.2, the sample is excluded from the analysis. For the remaining samples, the ratio

416 between the OD values (ODAm/ODAc) is calculated. If the ratio is >0.38 the sample is considered positive for anti-

417 *A. marginale*antibodies, and a ratio ≤ 0.38 is classified as vaccinated with *A. centrale*.

418 For the detection of *A. marginale*the test has a diagnostic specificity of 98% and a diagnostic sensitivity of 98.9%.

419 For 702 field samples evaluated, 131 (19%) had an OD <0.2 in the ddasELISA and thus were excluded from the

420 analysis. Of those animals, 52% were nested PCR positive for *A. marginale*, 23% were nested PCR positive for *A.*

421 *centrale*, 4.6% were nested PCR positive for *A. marginale*and *A. centrale*, 20% were nested PCR negative for both,

422 suggesting the ddasELISA may lack sensitivity.

423 Of the 571 ddasELISA positive field samples, the agreement between the ddasELISA and nested PCR was 84% and

424 the kappa coefficient was 0.70 (95% CI: 0.635–0.754), indicating substantial agreement between tests. There was

425 agreement between the ddasELISA and nested PCR for 93% of the *A. marginale*ddasELISA positive samples and

426 86% of the *A. centrale*ddasELISA positive samples. Additionally, 36 nested PCR negative samples tested positive

427 for antibodies against *A. marginale*(*n*=28) or *A. centrale*(*n*=8) by ddasELISA. This test could not identify animals

428 with co-infections, meaning animals vaccinated with *A. centrale*that are then infected with *A. marginale*, which is not

429 uncommon.

430 Test results using the ddasELISA are available in 5–6 hours. It is conducted as outlined below, see Bellezze *et al.*,

431 2023 for more details.

###### 432 2.3.1.	Test reagents

433 i)	A 96-well microtitre plate coated with either *A. marginale*or A. centrale recombinant protein

434 ii)	PBS/Tween buffer (PBS (50mM sodium phosphate, 150 mM NaCL, pH 7.2) with 0.05% Tween-

435	20)

436 iii)	Blocking reagent (PBS with 10% commercial dried skim milk)

437 iv)	Purified recombinant *A. marginale*MSP5 epitopes and *A. centrale*epitopes

438 v)	Biotinylated recombinant *A. marginale*MSP5 epitopes and *A. centrale*epitopes

439 vi)	Streptavidin-horse radish peroxidase (HRP) detection system

440 vii)  Chromogenic  substrate  (1  mM  2,2’-Azinobis  [3-ethylbenzothiazoline-6-sulfonic  acid]-

441 diammonium salt in0.05 M sodium citrate, pH 4.5, 0.0025% V/V H2O2 (100 μl/well).

442 viii) ELISA plate reader (405 nm reading)

443 ix)	Positive and negative control sera for *A. marginale*and *A. centrale*

###### 444 2.3.2.	Test procedure

445 i)	Plates are coated overnight.

446 ii)	Block with blocking buffer for 1 hour at room temperature and wash three times with PBS/Tween

447 buffer.

448 iii)	Add undiluted serum 100 ul/well and incubate for 1 hour at 25°C at 100 rpm.

449 iv)	Wash three times with PBS/Tween buffer.

450 v)	Add 100 μl of *A. marginale*MSP5-biotin (1 μg/ml) plus *A. centrale*MSP5 (10 μg/ml) to

451 *A. marginale*test wells. Add *A. centrale*MSP5-biotin (1 μg/ml) plus *A. marginale*MSP5

452 (10 μg/ml) in PBS/Tween buffer + 10% fat-free dried milk to *A. centrale*test wells.

453 vi)	Incubate 1 hour at 25°C, 100 rpm and wash the plate five times with PBS/Tween buffer.

454 vii)  To detect the bound protein–biotin complex, add streptavidin-HRP diluted in 1/500 in

455 PBS/Tween buffer with 10% dried milk for 1 hour at 25°C, 100 rpm.

456 viii) Wash five times with PBS/Tween buffer.

457 ix)	Add chromogenic substrate based on manufacturer’s instructions.

458 x)	The reaction is measured by microplate reader spectrophotometer at 405 nm wavelength. The

459 data are expressed in optical density (OD).

460 xi)	OD405nm <0.2 is considered negative.

461 xii)  Results are expressed as the ratio between antibodies specific for *A. marginale*MSP5 and for

462 *A. centrale*MSP5 (ODAm/ODAc). If the ratio is >0.38 the sample is considered positive for anti-

463 *A. marginale*antibodies, and a ratio ≤ 0.38 is classified as vaccinated with *A. centrale*.

##### 464 2.4.	Card agglutination test

465 ~~The advantages of the CAT are that it is sensitive~~The sensitivity of the CAT is from 84% to 98% (Gonzalez *et al*.,

466 1978; Molloy *et al*., 1999) and the specificity is 98.6% (Molloy *et al*., 1999). Though sometimes giving variable results,

467 the CAT can be useful under certain circumstances, as it may be undertaken either in the laboratory or in the field,

468 and it gives a result within a few minutes. Nonspecific reactions may be a problem, and subjectivity in interpreting

469 assay reactions can result in variability in test interpretation. In addition, the CAT antigen, which is a ~~suspension~~

470 lysate of *A. marginale*~~particles~~ isolated from erythrocytes, can be difficult to prepare and can vary from batch to batch

471 and laboratory to laboratory. To obtain the antigen, splenectomised calves are infected by intravenous inoculation

472 with blood containing *~~Anaplasma~~A. marginale*-infected erythrocytes. When the rickettsaemia exceeds 50%, the

473 animal is exsanguinated, the infected erythrocytes are washed, lysed, and the erythrocyte ghosts and *~~Anaplasma~~*

474 ~~particles~~*A. marginale*are pelleted. The pellets are sonicated, washed, and then resuspended in a stain solution to

475 produce the antigen suspension.

476 A test procedure that has been slightly modified from that originally described (Amerault & Roby, 1968; Amerault *et*

477 *al.*, 1972) is as follows, and is based on controlled conditions in a laboratory setting:

###### 478 2.4.1.	Test procedure

479 i)	Ensure all test components are at a temperature of 25–26°C before use (this constant

480 temperature is critical for the test).

481 ii)	On each circle of the test card (a clear perspex/plastic or glass plate marked with circles that

482 are 18 mm in diameter), place next to each other, but not touching, 10 μl of bovine serum factor

483 (BSF), 10 μl of test serum, and 5 μl of CAT antigen 39. Negative and low positive control sera

484 must be tested on each card.

485 iii)	BSF is serum from a selected animal with high known conglutinin level. If the conglutinin level

486 is unknown, fresh serum from a healthy animal known to be free from *Anaplasma*can be used.

487 The BSF must be stored at –70°C in small aliquots, a fresh aliquot being used each time the

488 tests are performed. The inclusion of BSF improves the sensitivity of the test.

489 iv)	Mix well with a glass stirrer. After mixing each test, wipe the stirrer with clean tissue to prevent

490 cross-contamination.

491 v)	Place the test card in a humid chamber and rock at 100–110 rpm for 7 minutes.

492 vi)	Read immediately against a backlight. Characteristic clumping of the antigen (graded from +1

493 to +3) is considered to be a positive result. The test is considered to give a negative result when

494 there is no characteristic clumping.

495 A latex card agglutination test, a relatively simple and rapid test platform, has been partially validated.

496 This test uses rMSP5-HIS rather than *A. marginale*lysate and does not require BSF. The

497 performance of this test was compared with that of the I-ELISA using rMSP5-HIS as the antigen. The

498 relative sensitivity was 95.2% and relative specificity was 91.86% (Ramos *et al.,*2014).

1 The test as conducted in the USA and Mexico uses larger volumes of reagents: antigen (15 μl), serum (30 μl), and bovine serum factor (30 μl), and a 4-minute reaction time (see step iv).

##### 499 ~~2.4.	Complement fixation test~~

500 ~~The complement fixation (CF) test has been used extensively for many years; however, it shows variable sensitivity~~

501 ~~(ranging from 20 to 60%), possibly reflecting differences in techniques for antigen production, and poor reproducibility.~~

502 ~~In addition, it has been demonstrated that the CF assay fails to detect a significant proportion of carrier cattle (Bradway~~

503 *~~et al.~~*~~, 2001). It is also uncertain as to whether or not the CF test can identify antibodies in acutely infected animals~~

504 ~~prior to other assays (Coetzee *et al.*, 2007; Molloy *et al.*, 1999). Therefore, the CF test is no longer recommended as~~

505 ~~a reliable assay for detecting infected animals.~~

##### 506 2.5.	Indirect fluorescent antibody test

507 Because of the limitations on the number of indirect fluorescent antibody (IFA) tests that can be performed daily by

508 one operator, other serological tests are generally preferred to the IFA test. The IFA test is performed as described

509 for bovine babesiosis in chapter 3.4.2, except that *A. marginaIe*infected blood is used for the preparation of antigen

510 smears. A serious problem encountered with the test is nonspecific fluorescence. The reported sensitivity is 97.6%

511 and specificity 89.6% (Gonzalez *et al*., 1978). Antigen made from blood collected as soon as adequate rickettsaemia

512 (5–10%) occurs is most likely to be suitable. Nonspecific fluorescence due to antibodies adhering to infected

513 erythrocytes can be reduced by washing the erythrocytes in an acidic glycine buffer before antigen smears are

514 prepared. Infected erythrocytes are washed twice in 0.1 M glycine buffer (pH 3.0, centrifuged at 1000 ***g***for 15 minutes

515 at 4°C) and then once in PBS, pH 7.4. Recently published data show that the IFA, like the C-ELISA, can cross react

516 with other members of the *Anaplasmataceae*family, and specifically an *Ehrlichia*spp. identified as BOV2010 (Al-

517 Adhami *et al.,*2011).

##### 518 2.6.	Complement fixation test

519 The complement fixation test (CFT) was used extensively for many years; however, it has variable sensitivity (ranging

520 from 20 to 60%), possibly reflecting differences in techniques for antigen production, and poor reproducibility. In

521 addition, the CF assay fails to detect a significant proportion of carrier cattle (Bradway *et al.*, 2001). It is also uncertain

522 as to whether or not the CF test can identify antibodies in acutely infected animals prior to other assays (Coetzee *et*

523 *al.*, 2007; Molloy *et al.*, 1999). Therefore, the CF test is no longer recommended as a reliable assay for detecting

524 infected animals.

525

526

#### 1.	Background

## C. REQUIREMENTS FOR VACCINES

527 Several immunisation methods have been used to protect cattle against anaplasmosis in countries where the disease is

528 endemic, but none is ideal to date~~(McHardy, 1984)~~. A review of *A. marginale*vaccines and antigens has been published

529 (Kocan *et al.*, ~~2003~~2010; Noh *et al.,*2012). Use of the less pathogenic *A. centrale,*which gives partial cross-protection

530 against *A. marginale,*is the most widely accepted method, although not used in many countries~~where the disease is~~

531 ~~exotic~~, including north America.

532 In this section, the production of live *A. centrale*vaccine is described. It involves infection of a susceptible, splenectomised

533 calf and the use of its blood as a vaccine. Detailed accounts of the production procedure are available and reference should

534 be made to these publications for details of the procedures outlined here (Bock *et al.*, 2004; de Vos & Jorgensen, 1992;

535 Pipano, 1995).

536 Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8 *Principles of veterinary vaccine production*.

537 The guidelines given here and in Chapter 1.1.8 are intended to be general in nature and may be supplemented by national

538 and regional requirements.

539 *Anaplasma centrale*vaccine can be provided in either frozen or chilled~~form~~ depending on demand, transport networks,

540 and the availability of liquid nitrogen or dry ice supplies. Frozen vaccine is recommended in most instances, as it allows

541 for thorough post-production quality control of each batch. It is, however, more costly to produce and more difficult to

542 transport than chilled vaccine. The risk of contamination makes post-production control essential, but may be prohibitively

543 expensive.

#### 544 2.	Outline of production and minimum requirements for conventional vaccines

##### 545 2.1.	Characteristics of the seed

###### 546 2.1.1.	Biological characteristics

547 *Anaplasma centrale*was isolated in 1911 in South Africa and has been used as a vaccine in South

548 America, Australia, Africa, the Middle East, and South-East Asia. It affords only partial, but adequate,

549 protection in regions where the ~~challenging~~circulating strains are of moderate virulence (e.g.

550 Australia) (Bock & de Vos, 2001). In the humid tropics where *A. marginale*~~appears to~~may be ~~a very~~

551 more virulent~~rickettsia~~, the protection afforded by *A. centrale*may be inadequate to prevent disease

552 in some animals.

553 *Anaplasma centrale*usually causes benign infections, especially if used in calves under 9 months of

554 age. Severe reactions following vaccination have been reported when adult cattle are inoculated. The

555 suitability of an isolate of *A. centrale*as a vaccine can be determined by inoculating susceptible cattle,

556 monitoring the subsequent reactions, and then challenging the animals and susceptible controls with

557 a virulent local strain of *A. marginale.*Both safety and efficacy can be judged by monitoring

558 rickettsaemias in stained blood films and the depression of packed cell volumes of inoculated cattle

559 during the vaccination and challenge reaction periods.

560 Infective material for preparing the vaccine is readily stored as frozen stabilates of infected blood in

561 liquid nitrogen or dry ice. Dimethyl sulphoxide (DMSO) ~~and~~or polyvinylpyrrolidone M.W. 40,000

562 (Bock *et al.*, 2004) are the recommended cryopreservatives, as they allow for intravenous

563 administration after thawing of the stabilate. A detailed account of the freezing technique using DMSO

564 is reported elsewhere (Mellors *et al.,*1982), but briefly involves the following: infected blood is

565 collected, chilled to 4°C, and cold cryoprotectant (4 M DMSO in PBS) is added slowly with stirring to

566 a final blood:protectant ratio of 1:1, to give a final concentration of 2 M DMSO. The entire dilution

567 procedure is carried out in an ice bath and the diluted blood is dispensed into suitable containers

568 (e.g. 5 mI cryovials), and frozen, as soon as possible, in the vapour phase of a liquid nitrogen

569 container.

###### 570 2.1.2.	Quality criteria

571 Evidence of purity of the *A. centrale*isolate can be determined by serological testing of paired sera

572 from the cattle used in the safety test for possible ~~contaminants~~pathogens that may be present (Bock

573 *et al.*, 2004; Pipano, 1997). Donor calves used to expand the seed for vaccine production should be

574 examined for all blood-borne infections prevalent in the vaccine-producing country, including

575 *Babesia*, *Anaplasma*, *Ehrlichia*, *Theileria*and *Trypanosoma*. This can be done by routine examination

576 of stained blood films after splenectomy, PCR, and preferably also by serology. Any calves showing

577 evidence of natural infections of any of these agents should be rejected. The absence of other

578 infective agents should also be confirmed. These may include the agents of enzootic bovine leukosis,

579 mucosal disease, infectious bovine rhinotracheitis, ephemeral fever, Akabane disease, bluetongue,

580 and foot and mouth disease~~, and rinderpest~~. The testing procedures will depend on the diseases

581 prevalent in the country and the availability of tests but should involve serology of paired sera at the

582 very least and, in some cases, virus isolation, antigen, or DNA/RNA detection (Bock *et al.*, 2004;

583 Pipano, 1981; 1997).

##### 584 2.2.	Method of manufacture

###### 585 2.2.1.	Procedure

586 i)  Production of frozen vaccine

587 Quantities of the frozen stabilate (5–10 ml) are thawed by immersing the vials in water preheated to

588 40°C. The thawed material is kept on ice and used as soon as possible (within 30 minutes) to infect

589 a susceptible, splenectomised calf by intravenous inoculation.

590 The rickettsaemia of ~~the~~this donor calf is monitored daily by examining stained films of jugular blood,

591 and the blood is collected for vaccine production when suitable rickettsaemias are reached. A

592 rickettsaemia of 1 × 108/ml (approximately 2% rickettsaemia in jugular blood) is the minimum required

593 for production of vaccine as this is the dose to vaccinate a bovine. If a suitable rickettsaemia is not

594 obtained, passage of the strain by subinoculation of 100–200 ml of blood to a second splenectomised

595 calf may be necessary.

596 Blood from the donor is collected by aseptic jugular or carotid cannulation using heparin as an

597 anticoagulant (5 International Units [IU] heparin/ml blood). The use of blood collection units for human

598 use are also suitable and guarantee sterility and obviate the need to prepare glass flasks that make

599 the procedure more cumbersome.

600 In the laboratory, the infective blood is mixed in equal volumes with 3 M glycerol in PBS supplemented

601 with 5 mM glucose at 37°C (final concentration of glycerol 1.5 M). The mixture is then equilibrated at

602 37°C for 30 minutes and dispensed into suitable containers (e.g. 5 ml cryovials). The vials are cooled

603 at approximately 10°C/minute in the vapour phase of liquid nitrogen and, when frozen, stored in the

604 liquid phase (Bock *et al.,*2004).

605 DMSO can be used as a cryoprotectant in the place of glycerol. This is done in the same way as

606 outlined for the preparation of seed stabilate (Mellors *et al.,*1982; Pipano, 1981).

607 If glycerolised vaccine is to be diluted, the diluent should consist of PBS with 1.5 M glycerol and 5 mM

608 glucose (Jorgensen *et al.,*1989). Vaccine cryopreserved with DMSO should be diluted with diluent

609 containing the same concentration of DMSO as in the original cryopreserved blood (Pipano *et al.,*

610 1986).

611 ii) Production of chilled vaccine

612 Infective material for chilled vaccine is prepared in the same way as for frozen vaccine, but it must

613 be issued and used as soon as possible after collection. The infective blood can be diluted to provide

614 1 × 107 parasites per dose of vaccine. A suitable diluent is 10% sterile bovine serum in a

615 glucose/balanced salt solution containing the following quantities per litre: NaCI (7.00 g), MgCI2.6H2O

616 (0.34 g), glucose (1.00 g), Na2HPO4(2.52 g), KH2PO4(0.90 g), and NaHCO3(0.52 g).

617 If diluent is not available, acid citrate dextrose (20% [v/v]) or citrate phosphate dextrose (20% [v/v])

618 should be used as anticoagulant to provide the glucose necessary for survival of the organisms.

619 iii) Use of vaccine

620 In the case of frozen vaccine, vials should be thawed by immersion in water, preheated to 37°C to

621 40°C, and the contents mixed with suitable diluent to the required dilution. If glycerolised vaccine is

622 prepared, it should be kept cool and used within 8 hours (Bock *et al.,*2004). If DMSO is used as a

623 cryoprotectant, the prepared vaccine should be kept on ice and used within 15–30 minutes (Pipano,

624 1981). The vaccine is most commonly administered subcutaneously.

625 iv) Chilled vaccine should be kept refrigerated and used within 4–7 days of preparation.

626 The strain of *A. centrale*used in the vaccine is of reduced virulence, but is not entirely safe. A practical

627 recommendation is, therefore, to limit the use of vaccine to calves, where nonspecific immunity will

628 minimise the risk of vaccine reactions. When older animals have to be vaccinated, there is a risk of

629 severe reactions. These reactions occur infrequently, but valuable breeding stock or pregnant

630 animals obviously warrant close attention,	and should be observed daily for 3 weeks post-

631 vaccination. Clinically sick animals should be treated with oxytetracycline or imidocarb at dosages

632 recommended by the manufacturers. Protective immunity develops in 6–8 weeks and usually lasts

633 for several years.

634 Anaplasmosis and babesiosis vaccines are often used concurrently, but it is not advisable to use any

635 other vaccines at the same time (Bock *et al.,*2004).

###### 636 2.2.2.	Requirements for substrates and media

637 *Anaplasma centrale*~~cannot~~can be cultured in ~~vitro~~*Rhipicephalus appendiculatus*and *Dermacentor variabilis*

638 cells lines, though antigen expression and immunogenicity of the cultured *A. centrale*need to be tested (Bell-

639 Sakyi *et al*., 2015). No substrates or media other than buffers and diluents are used in vaccine production.

640 DMSO or glycerol should be purchased from reputable companies.

###### 641 2.2.3.	In-process controls

642 i)  Source and maintenance of vaccine donors

643 A source of calves free from natural infections of *~~Anaplasma~~A. marginale*and other tick-borne

644 diseases should be identified. If a suitable source is not available, it may be necessary to breed the

645 calves under tick-free conditions specifically for the purpose of vaccine production.

646 The calves should be maintained under conditions that will prevent exposure to infectious diseases

647 and to ticks and biting insects. In the absence of suitable facilities, the risk of contamination with the

648 agents of infectious diseases present in the country involved should be estimated, and the benefits

649 of local production of vaccine weighed against the possible adverse consequences of spreading

650 disease (Bock *et al.*, 2004).

651 ii) Surgery

652 Donor calves should be splenectomised to allow maximum yield of organisms for production of

653 vaccine. This is best carried out in young calves and under general anaesthesia.

654 iii) Screening of vaccine donors before inoculation

655 As for preparation of seed stabilate, donor calves for vaccine production should be examined for all

656 blood-borne infections prevalent in the vaccine-producing country, including *Babesia*, *Anaplasma*,

657 *Ehrlichia*, *Theileria*and *Trypanosoma*. This can be done by routine examination of stained blood films

658 after splenectomy, and preferably also by serology. Any calves showing evidence of natural infections

659 of any of these agents should be rejected. The absence of other infective agents should also be

660 confirmed. These may include the agents of enzootic bovine leukosis, bovine viral diarrhoea,

661 infectious bovine rhinotracheitis, ephemeral fever, Akabane disease, bluetongue, and foot and mouth

662 disease. The testing procedures will depend on the diseases prevalent in the country and the

663 availability of tests, but should involve serology of paired sera at the very least and, in some cases,

664 virus isolation, antigen, or DNA/RNA detection (Bock *et al.*, 2004; Pipano, 1981; 1997).

665 iv) Monitoring of rickettsaemias following inoculation

666 It is necessary to determine the concentration of rickettsia in blood being collected for vaccine. The

667 rickettsial concentration can be estimated from the erythrocyte count and the rickettsaemia

668 (percentage of infected erythrocytes).

669 v) Collection of blood for vaccine

670 All equipment should be sterilised before use (e.g. by autoclaving). Once the required rickettsaemia

671 is reached, the blood is collected in heparin using strict aseptic techniques. This is best done if the

672 calf is sedated and with the use of a closed-circuit collection system.

673 Up to 3 litres of heavily infected blood can be collected from a 6-month-old calf. If the calf is to live,

674 the transfusion of a similar amount of blood from a suitable donor is indicated. Alternatively, the calf

675 should be killed immediately after collection of the blood.

676 vi) Dispensing of vaccine

677 All procedures are performed in a suitable environment, such as a laminar flow cabinet, using

678 standard sterile techniques. Use of a mechanical or magnetic stirrer will ensure thorough mixing of

679 blood and diluent throughout the dispensing process. Penicillin (500,000 lU/litre) and streptomycin

680 (370,000 μg/litre) are added to the vaccine at the time of dispensing.

###### 681 2.2.4.	Final product batch tests

682 The potency, safety and sterility of vaccine batches cannot be determined in the case of chilled vaccine, and

683 specifications for frozen vaccine depend on the country involved. The following are the specifications for frozen

684 vaccine produced in Australia.

685 i)  Sterility and purity

686 Standard tests for sterility are employed for each batch of vaccine and diluent (see Chapter 1.1.9

687 *Tests for sterility and freedom from contamination of biological materials intended for veterinary use*).

688 The absence of contaminants is determined by doing appropriate serological testing of donor cattle,

689 by inoculating donor lymphocytes into sheep and then monitoring them for evidence of viral infection,

690 and by inoculating cattle and monitoring them serologically for infectious agents that could potentially

691 contaminate the vaccine. Cattle inoculated during the test for potency (see Section C.2.2.4.iii) are

692 suitable for the purpose. Depending on the country of origin of the vaccine, these agents include the

693 causative organisms of enzootic bovine leukosis, infectious bovine rhinotracheitis, bovine viral

694 diarrhoea, ephemeral fever, Akabane disease, Aino virus, bluetongue, parainfluenza, foot and mouth

695 disease, lumpy skin disease, rabies, Rift Valley fever, contagious bovine pleuropneumonia,

696 Jembrana disease, heartwater, pathogenic *Theileria*and *Trypanosoma spp*., *Brucella abortus*,

697 *Coxiella*, and *Leptospira*(Bock *et al.*, 2004; Pipano, 1981; 1997). Other pathogens to consider include

698 the causal agents of bovine tuberculosis and brucellosis as they may spread through contaminated

699 blood used for vaccine production. Most of these agents can be tested by means of specific PCR and

700 there are many publications describing primers, and assay conditions for any particular disease.

701 ii) Safety

702 Vaccine reactions of the cattle inoculated in the test for potency (see Chapter 1.1.8 *Principles of*

703 *veterinary vaccine production*) are monitored by measuring rickettsaemia and depression of packed

704 cell volume. Only batches with pathogenicity levels equal to or lower than a predetermined standard

705 are released for use.

706 iii) Potency

707 Vaccine is thawed and diluted 1/5 with a suitable diluent (Bock *et al.*, 2004). The diluted vaccine is

708 then incubated for 8 hours at 4°C, and five cattle are inoculated subcutaneously with 2 ml doses. The

709 inoculated cattle are monitored for the presence of infections by examination of stained blood smears.

710 All should become infected for a batch to be accepted. A batch proving to be infective is

711 recommended for use at a dilution of 1/5 with isotonic diluent.

##### 712 2.3.	Requirements for authorisation

###### 713 2.3.1.	Safety

714 The strain of *A. centrale*used in vaccine is of reduced virulence but is not entirely safe. A practical

715 recommendation is, therefore, to limit the use of vaccine to calves, where nonspecific immunity will

716 minimise the risk of vaccine reactions. When older animals have to be vaccinated, there is a risk of

717 severe reactions. These reactions occur infrequently, but valuable breeding stock or pregnant

718 animals obviously warrant close attention, and should be observed daily for 3 weeks post-

719 vaccination. Clinically sick animals should be treated with oxytetracycline or imidocarb at dosages

720 recommended by the manufacturers.

721 *Anaplasma centrale*is not infective to other species, and the vaccine is not considered to have other

722 adverse environmental effects. The vaccine is not infective for humans. When the product is stored

723 in liquid nitrogen, the usual precautions pertaining to the storage, transportation and handling of deep-

724 frozen material applies.

###### 725 2.3.2.	Efficacy requirements

726 ~~Partial but long-lasting immunity results from one inoculation. There is no evidence that repeated~~

727 ~~vaccination will have a boosting effect.~~Immunisation with live *A. centrale*results in long-term infection

728 of the vaccinee, thus repeated vaccination is unnecessary. Infection with *A. centrale*does not prevent

729 subsequent infection with *A. marginale*, but does at least result in protection from disease (Shkap *et*

730 *al*., 2009). The vaccine is used for control of clinical anaplasmosis in endemic areas. It will not provide

731 sterile immunity, and should not be used for eradication of *A. marginale*.

###### 732 2.3.3.	Stability

733 The vaccine can be kept for 5 years when stored in liquid nitrogen. Once thawed, it rapidly loses its

734 potency. Thawed vaccine cannot be refrozen.

#### 735 3.	Vaccines based on biotechnology

736 There are no vaccines based on biotechnology available for anaplasmosis.

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833 ~~Fractionation of the complement-fixing antigen. *J. Bacteriol*., **88**, 81–86.~~

834 SARLI M., THOMPSON C.S., NOVOA M., VALENTINI B.S., MASTROPAOLO M., ECHAIDE I.E., DE ECHAIDE S. T. & PRIMO M.E. (2020).

835 Development and evaluation of a double-antigen sandwich ELISA to identify *Anaplasma marginale*-infected and

836 *A. centrale*-vaccinated cattle. *J. Vet. Diagn. Invest.*, **32**, 70–76.

837 SHKAP V., KOCAN K., MOLAD T., MAZUZ M., LEIBOVICH B., KRIGEL Y., MICHOYTCHENKO A., BLOUIN E., DE LA FUENTE J., SAMISH

838 M., MTSHALI M., ZWEYGARTH E., FLEIDEROVICH E. L. & FISH L. (2009). Experimental transmission of field *Anaplasma marginale*

839 and the *A. centrale*vaccine strain by *Hyalomma excavatum, Rhipicephalus sanguineus*and *Rhipicephalus*(*Boophilus*)

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844 ~~STICH R.W., OLAH G.A., BRAYTON K.A., BROWN W.C., FECHHEIMER M., GREEN-CHURCH K., JITTAPALAPONG S., KOCAN K.M.,~~

845 ~~MCGUIRE T.C., RURANGIRWA F.R. & PALMER G.H. (2004).Identification of a novel *Anaplasma marginale*appendage-~~

846 ~~associated protein that localizes with actin filaments during intraerythrocytic infection. *Infect Immun.,***72**, 7257–7264.~~

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848 C.A., BACANELLI G. & TORRES R.A. (2006). Comparison between indirect enzyme-linked immunosorbent assays for

849 *Anaplasma marginale*antibodies with recombinant Major Surface Protein 5 and initial body antigens. *Mem. Inst. Oswaldo*

850 *Cruz.,***101**, 511–516.

851 STRIK N.I., ALLEMAN A.R., BARBET A.F., SORENSON H.L., ~~WANSLEY~~WAMSLEY H.L., GASCHEN F.P., LUCKSCHANDER N., WONG S.,

852 CHU F., FOLEY J.E., BJOERSDORFF A., STUEN S. & KNOWLES D.P. (2007). Characterization of *Anaplasma phagocytophilum*

853 major surface protein 5 and the extent of its cross-reactivity with *A. marginale*. *Clin. Vaccine Immunol.*, **14**, 262–268.

854 TORIONI DE ECHAIDE S., KNOWLES D.P., MCGUIRE T.C., PALMER G.H., SUAREZ C.E. & MCELWAIN T.F. (1998). Detection of

855 cattle naturally infected with *Anaplasma marginale*in a region of endemicity by nested PCR and a competitive enzyme-

856 linked immunosorbent assay using recombinant major surface protein 5. *J. Clin. Microbiol.,***36**, 777–782.

857	*

858	* *

859 **NB:**There is a WOAH Reference Laboratory for anaplasmosis (please consult the WOAH Web site:

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

861 Please contact the WOAH Reference Laboratory for any further information on

862 diagnostic tests, reagents and vaccines for bovine anaplasmosis

863 **NB:**FIRST ADOPTED IN 1991. MOST RECENT UPDATES ADOPTED IN 2015.

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