**Annex 17. Item 5.1. – Chapter 3.10.4. Infection with *Campylobacter jejuni*and *C. coli***

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

3	**Paris, 4–8 September 2023**

4

5	**CHAP T E R  3 . 1 0 . 4 .**

# 6	INF E CT ION WIT H

7	***CAMPYLOBACTER JEJUNI*A N D *C. COLI***

8	**SUMMARY**

9 ***Description of the disease:***Campylobacter jejuni (C. jejuni) *and*Campylobacter coli (C. coli) *can colonise*

10 *the intestinal tract of most mammals and birds and are the most frequently isolated*Campylobacter *species*

11 *in humans with gastroenteritis. Although poultry is the main reservoir of*Campylobacter, *transmission to*

12 *humans is only partly through handling and consumption of poultry meat; other transmission routes are also*

13 *considered to be important. This chapter focuses on*C. jejuni *and*C. coli *in primary livestock production with*

14 *regard to food safety.*

15 Campylobacter jejuni *and*C. coli *do not normally cause clinical disease in adult animals except for sporadic*

16 *cases of abortion in ruminants~~and very rare cases of hepatitis in ostriches~~. The faecal contamination of*

17 *meat*(*especially poultry meat*) *during processing is considered to be an important source of human food-*

18 *borne disease. In humans, extraintestinal infections, including bacteraemia, can occur and some sequelae*

19 *of infection, such as polyneuropathies, though rare, can be serious.*

20 ***Identification of the agent:****In mammals and birds, detection of intestinal colonisation is based on the*

21 *isolation of the organism from faeces, rectal swabs or caecal contents, or the use of polymerase chain*

22 *reaction*(*PCR*)*.*Campylobacter jejuni *and*C. coli *are thermophilic, Gram-negative, highly motile bacteria*

23 *that, for optimal growth, require microaerobic environment and incubation temperatures of 37–42°C. Agar*

24 *media containing selective antibiotics are required to isolate these bacteria from faecal/intestinal samples.*

25 *Alternatively, their high motility can be exploited using filtration techniques for isolation. Enrichment*

26 *techniques to detect intestinal colonisation are not routinely used. Preliminary confirmation of isolates can*

27 *be made by examining the morphology and motility using a light microscope. The organisms in the log growth*

28 *phase are short and S-shaped in appearance, while coccoid forms predominate in older cultures. Under*

29 *phase-contrast microscopy the organisms have a characteristic rapid corkscrew-like motility. Phenotypic*

30 *identification is based on reactions under different growth conditions. Biochemical and molecular tests,*

31 *including PCR and MALDI-TOF*(*matrix assisted laser desorption ionisation–time of flight*) *mass*

32 *spectrometry can be used to identify*Campylobacter *strains at species level. PCR assays can also be used*

33 *for the direct detection of*C. jejuni *and*C. coli*.*

34 ***Serological tests:****serological assays are not routinely in use for the detection of colonisation by*C. jejuni

35 and C. coli*.*

36 ***Requirements for vaccines:****There are no effective vaccines available for the prevention of enteric*

37 Campylobacter *infections in birds or mammals.*

38

39 **1.	Disease**

## A. INTRODUCTION

40 *Campylobacter jejuni*and *C. coli*are generally considered commensals of livestock, domestic pet animals and birds. ~~Large~~

41 High numbers of *Campylobacter*have been isolated from young livestock with enteritis, including piglets, lambs and calves,

42 but the organisms are also found in healthy animals. One specific *C. jejuni*clone has been associated with abortion in

43 sheep (Tang *et al.,*2017).~~Outbreaks of avian hepatitis have been reported, but although *C. jejuni*is associated with the~~

44 ~~disease, it is not the causative agent (Jennings *et al.,*2011). Recently, a new *Campylobacter*was isolated as the causative~~

45 ~~agent of spotty liver disease in layers (Crawshaw *et al.,*2015).~~*Campylobacter jejuni*and *C. coli*are of interest mainly from

46 the point of view food safety.*Campylobacter*is the main cause of human bacterial intestinal disease identified in many

47 industrialised countries (~~Havelaar *et al.,*2013; Scallan *et al.,*2011~~CDC, 2022; EFSA, 2021), and *C. jejuni*and *C. col**i*

48 together account for more than 90% of all human campylobacteriosis cases. Over 80% of cases are caused by *C. jejuni*

49 and about 10% of cases are caused by *C. coli*. In humans, *C. jejuni/C. coli*infection is associated with acute enteritis and

50 abdominal pain lasting for 7 days or more. Although such infections are generally self-limiting, complications can arise and

51 may include bacteraemia, Guillain–Barré syndrome, reactive arthritis, and abortion (WHO, 2013). ~~Attribution~~ Studies have

52 shown~~that~~the majority of campylobacteriosis cases in humans can be attributed to poultry and a smaller fraction to cattle

53 (Mughini-Gras *et al.,*2012)~~is the main reservoir of *Campylobacter*and responsible for between 50 and 80% of the human~~

54 infections. In the European Union (EU), an estimated ~~30~~20–40% of the human infections are associated with handling and

55 consumption of poultry meat while up to 80% of the strains infecting humans have their origin in the poultry reservoir

56 (EFSA, 2010).~~; but~~A considerable proportion of the poultry-derived strains has a non-poultry meat transmission route,

57 e.g. via ~~environmental contamination~~surface water (~~EFSA, 2010b,~~Mulder *et al.,*2020). Contact with pets and livestock,

58 the consumption of contaminated water or raw milk and travelling ~~in high prevalence areas~~ are also considered risks factors

59 in human disease (Domingues *et al.,*2012; Mughini-Gras *et al.,*2021). The control of *Campylobacter*in the food chain has

60 ~~now~~become a major target of agencies responsible for food safety world-wide.

61 Laboratory manipulations should be performed with appropriate biosafety and containment procedures as determined by

62 biorisk analysis (see Chapter 1.1.4 *Biosafety and biosecurity: standard for managing biological risk in the veterinary*

63 *diagnostic laboratory and animal facilities*).

#### 64 2.	Taxonomy

65 There are currently ~~34~~43*Campylobacter*species recognised (July 2023), but with the improved diagnostic techniques and

66 genomic analysis, this number is expected to increase over time (*~~cf~~*List of prokaryotic names with standing in

67 nomenclature: (https://lpsn.dsmz.de/genus/campylobacter~~~~~~http://www.bacterio.net/index.html~~). Members of the genus

68 *Campylobacter*are typically Gram-negative, non-spore-forming, S-shaped or spiral shaped bacteria (0.2–~~0.8~~0.5 μm wide

69 and 0.5–~~5~~8 μm long), with single polar flagella at one or both ends, conferring a characteristic corkscrew-like motility.

70 ~~These bacteria~~*Campylobacter*requires microaerobic conditions, but some strains also grow aerobically or anaerobically.

71 They neither ferment nor oxidise carbohydrates. Some species, particularly *C. jejuni*, *C. coli*and *C. lari*, are thermophilic,

72 growing optimally at 42°C. They can colonise mucosal surfaces, usually the intestinal tract, of most mammalian and avian

73 species tested. The species *C. jejuni*includes two subspecies (*C. jejuni*subsp. *jejuni*and C. *jejuni*subsp. *doylei*) that can

74 be discriminated on the basis of several phenotypic tests, but this subspeciation has no added value for epidemiological

75 or intervention purposes~~(nitrate reduction, selenite reduction, sodium fluoride, and safranine) and growth at 42°C (subsp.~~

76 *~~doylei~~*~~does not grow at 42°C) (Garrity, 2005). Subspecies *jejuni*is much more frequently isolated then subspecies *doylei*~~.

## 77	B. DIAGNOSTIC TECHNIQUES

78 ***Table 1.****Test methods available for the diagnosis of Campylobacter jejuni and C. coli and their purpose*

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

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

81 ~~MALDI-TOF = matrix assisted laser desorption ionisation–time of flight; PCR = polymerase chain reaction.~~

82 (a)Regarding the control of the agent: *Campylobacter jejuni*and *C. coli*are endemic globally and very rarely cause disease. These

83 species are of interest from the point of view of food safety. There is no eradication programme. For broiler flocks there are worldwide

84 efforts to try to prevent colonisation with *C. jejuni*and *C. coli*to prevent contamination of the carcasses during slaughter. Therefore, only

85 the columns ‘population freedom’ (= broiler flock) and prevalence of infection surveillance are filled in where “infection” should be read

86 as “colonisation”.

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

#### 88 1.	Isolation and identification of the agent

89 Two ISO (International Organization for Standardization) procedures for detection of *Campylobacter*exist. ISO 10272

90 describes a horizontal method for detection and enumeration of thermotolerant *Campylobacter*spp. ~~(ISO 10272)~~in food

91 and animal feeding stuffs with 2 parts: (part 1 detection method (ISO 10272-1:2017) and part 2 colony count technique

92 (ISO 10272-2:2017). ~~Both parts of the ISO are under revision and will be published in 2017. The revised standard will~~

93 include methods for the isolation of *Campylobacter*from live animals, and a procedure for ISO 17995 concerns water

94 quality, with detection and enumeration of thermotolerant *Campylobacter*spp*.*from water (ISO, 2005 – last reviewed in

95 ~~2014).~~

##### 96 1.1.	Collection of specimens

###### 97 1.1.1.	Poultry at the farm

98 Poultry is frequently colonised with *C. jejuni*(65–95%), less often with *C. coli*and rarely with other

99 *Campylobacter*species (~~Newell & Wagenaar*,*2000~~Wagenaar *et al*., 2023). Colonisation rates in

100 broiler chickens are age-related. Most flocks are negative until 2 weeks of age. Once *Campylobacter*

101 colonisation occurs in a broiler flock, transmission, via exposure to faecal contamination, is extremely

102 rapid and up to 100% of birds within a flock can become colonised within a few days. Samples from

103 live birds, destined for the food chain, should therefore be taken as close to slaughter as possible

104 (~~Newell & Wagenaar*,*2000~~Wagenaar *et al*., 2023). The majority of birds shed large numbers of

105 organisms (>106 colony-forming units/g faeces). Campylobacters can be isolated from fresh

106 faeces/caecal droppings or cloacal swabs. For reliable detection of *Campylobacter*by culture, freshly

107 voided faeces (preferably without traces of urine) should be collected. **Such samples must be**

108 **prevented from drying out before culturing.**When swabs are used, a transport medium such as

109 Cary Blair, Amies, or Stuart must be used. Sampling strategy in primary poultry has been reviewed

110 (Vidal *et al.,*2013) and is normally based on boot-swab samples, faecal/caecal droppings or cloacal

111 swabs.

###### 112 1.1.2.	Cattle, sheep and pigs at the farm

113 Campylobacters are frequent colonisers of the intestine of livestock such as cattle, sheep and pigs;

114 data have been reviewed by Newell *et al.,*(~~in press~~2017). Cattle and sheep are found to be colonised

115 mainly with *C. jejuni*, *C. coli*, *C. hyointestinalis*, and *C. fetus*, whereas pigs are predominantly

116 colonised by *C. coli*. In young animals, the numbers are higher than in older animals. In older animals,

117 the organisms can be intermittently detected in faeces, probably due to low numbers or due to

Report of the Meeting of the Biological Standards Commission / September 2023

216

118 intermittent shedding. Fresh samples have to be taken (rectal samples if possible) and **they should**

119 **be prevented from drying out**. When swabs are used, a transport medium (like Cary Blair, Amies,

120 or Stuart) must be used.

###### 121 1.1.3.	At slaughter

122 In poultry, the caecal contents are usually used for the detection of *Campylobacter*. ~~They~~Caeca can

123 be cut with sterile scissors from the remaining part of the intestines and submitted intact to the

124 laboratory in a suitable container.

125 Samples from cattle, sheep and pigs are collected from the intestines by aseptically opening the gut

126 wall or by taking guarded rectal swabs.

127 At all stages from collecting the samples until they are processed in the laboratory, utmost attention

128 should be given to make sure that campylobacters do not die. Follow the instructions for

129 transportation and shipment carefully.

##### 130 1.2.	Transportation and treatment of specimens

###### 131 1.2.1.	Transport

132 Campylobacters are sensitive to environmental conditions, including dehydration, atmospheric

133 oxygen, sunlight and elevated temperature. Transport to the laboratory and subsequent processing

134 should therefore be as rapid as possible preferably the same day.~~, but~~It is recommended to process

135 the samples within 72 hours, but if not possible, storage of samples is accepted up to 96 hours (Tast-

136 Lahti *et al*., 2022)~~within at least 3 days~~. The samples must be protected from light, extreme

137 temperatures and desiccation.

138 No recommendation on the ideal temperature for transportation can be made, but it is clear that

139 freezing or high temperatures can reduce viability. If possible, samples should be maintained at a

140 temperature of 4°C (±2°C).~~High temperatures (>20°C), low temperatures (<0°C) and fluctuations in~~

141 ~~temperature must be avoided. When the time between sampling and processing is longer than 48~~

142 ~~hours, storage at 4°C (±2°C) is advised.~~

###### 143 1.2.2.	Transport media

144 *Swabs:*When samples are collected on boot-swabs or rectal swabs, the use of commercially

145 available transport tubes, containing a medium, such as Cary Blair or Amies, is recommended. This

146 medium may be plain agar or charcoal-based. The function of the medium is not for growth of

147 *Campylobacter*spp., but to protect the swab contents from drying and the toxic effects of oxygen.

148 When only small amounts of faecal/caecal samples can be collected and transport tubes are not

149 available, shipment of the specimen in transport medium is recommended. Several transport media

150 have been described: Amies, Cary-Blair, modified Cary-Blair, modified Stuart medium, Campy-

151 thioglycolate medium, alkaline peptone water and semisolid motility test medium. ~~Good recovery~~

152 ~~results have been reported using Cary-Blair (Luechtefeld *et al*., 1981; Sjogren *et al*., 1987).~~

###### 153 1.2.3.	Maintenance of samples

154 On arrival at the laboratory, samples should be processed as soon as possible, preferably on the day

155 of arrival. It is recommended to process the samples within 72 hours, but if not possible, storage of

156 samples is accepted up to 96 hours, whereby *C. coli*is more sensitive for long storage times than *C.*

157 *jejuni*~~but no longer than 3 days after collecting the samples~~(Tast-Lahti *et al*., 2022). To avoid

158 temperature variation, samples should only be refrigerated when they cannot be processed on the

159 same day, otherwise they should be kept at room temperature when processed the same day. When

160 samples are submitted or kept in the laboratory at 4°C, they should be allowed to equilibrate to room

161 temperature before processing to avoid temperature shock.

162 **1.3.	Isolation of *Campylobacter***

163 For the isolation of *Campylobacter*from faecal/caecal or intestinal samples, no pre-treatment is needed;

164 samples can be plated on selective medium or the filtration method on non-selective agar can be used. In the

165 case of caecal samples, caeca are aseptically opened by cutting the end with a sterile scissors and squeezing

166 out the material to be processed. Enrichment ~~is recommended~~can be considered to enhance the culture

167 sensitivity ~~of potentially environmentally stressed organisms or in the case~~ of low levels of organisms in faeces

168 (ISO, 2017), for example from cattle, sheep or pigs. However, enrichment of faecal samples is usually subject

169 to overgrowth by competing bacteria and is not carried out routinely. There is no need to use enrichment

170 media to isolate *Campylobacter*from poultry caeca.

###### 171 1.3.1.	Selective media for isolation

172 Many media can be used in the recovery of *Campylobacter*spp. The selective medium modified

173 charcoal, cefoperazone, desoxycholate agar (mCCDA), is the most commonly recommended

174 ~~medium~~and is prescribed in the ISO standard, although alternative media may be used (ISO, 2017).

175 A detailed description on *Campylobacter*detection by culture and the variety of existing media is

176 given by Corry *et al.*(1995; 2003). The selective media can be divided into two main groups: blood-

177 based media and charcoal-based media. Blood components and charcoal serve to remove toxic

178 oxygen derivatives. Most media are commercially available. The selectivity of the media is

179 determined by the antibiotics used. Cefalosporins (generally cefoperazone) are used, sometimes in

180 combination with other antibiotics (e.g. vancomycin, trimethoprim). ~~Cycloheximide (actidione) and~~

181 ~~more recently~~Amphotericin B or cycloheximide are used to inhibit yeasts and molds (Martin *et al*.,

182 2002). The main difference between the media is the degree of inhibition of contaminating flora. All

183 the selective agents allow the growth of both *C. jejuni*and *C. coli*. There is no medium available that

184 allows growth of *C. jejuni*and inhibits *C. coli*or vice versa. To some extent, other *Campylobacter*

185 species (e.g. *C. lari*, *C. upsaliensis*, *C. helveticus, C. fetus*and *C. hyointestinalis*) will grow on most

186 media, especially at the less selective temperature of 37°C.

187 Examples of selective blood-containing solid media:

188 i)  Preston agar

189 ii) Skirrow agar

190 iii) Butzler agar

191 iv) Campy-cefex

192 Examples of charcoal-based solid media:

193 i)  mCCDA (modified charcoal cefoperazone deoxycholate agar), slightly modified version of the

194 originally described CCDA) (Bolton *et al*., 1984; 1988)

195 ii) Karmali agar or CSM (charcoal-selective medium) (Karmali *et al*., 1986)

196 iii) CAT agar (cefoperazone, amphotericin and teicoplanin), facilitating growth of *C. upsaliensis*

197 (Aspinall *et al*., 1993).

###### 198 1.3.2	Enrichment

199 The ISO standard describes the isolation of *Campylobacter*from samples with low numbers of

200 *Campylobacter*and high numbers of background flora by using Preston enrichment medium (ISO,

201 2017). This can be considered for samples from pigs, cattle and sheep. Samples are added to

202 Preston broth with a 1 in 10 dilution (e.g. 10 g faecal sample with 90 ml broth) and incubated under

203 microaerobic conditions for 24 hours at 41.5°C.

204 After enrichment, campylobacters can be isolated on selective media as described before with plating

205 one loop (10 μl) to solid media.

###### 206 1.3.3.	Passive filtration

207 Passive filtration, a method developed by Steele & McDermott (1984) obviates the need for selective

208 media; thus it is very useful for the isolation of antimicrobial-sensitive *Campylobacter*species. As the

209 method does not use expensive selective media, it may be used in laboratories with fewer resources.

210 For passive filtration, faeces are mixed with PBS (approximately 1/10 dilution) to produce a

211 suspension. Approximately 10–15 drops~~100 μ~~l of this suspension are then carefully layered on to a

212 ~~0.45 or~~0.65 μm sterile cellulose acetate filter, which has been previously placed on top of a non-

213 selective blood agar plate. Care must be taken not to allow the inoculum to spill over the edge of the

214 filter. The bacteria are allowed to migrate through the filter for 30–45 minutes at 37°C ~~or room~~

215 ~~temperature~~(microaerobic conditions are not required) and the filter is then removed. The plate is

216 incubated microaerobically at 37°C or 42°C.

Report of the Meeting of the Biological Standards Commission / September 2023

218

###### 217 1.3.4.	Incubation

218 i)  Atmosphere

219 Microaerobic atmospheres of 5–10% oxygen, 5–10% carbon dioxide are required for optimal growth

220 (Corry *et al*., 2003~~; Vandamme, 2000~~). Appropriate atmospheric conditions may be produced by a

221 variety of methods. In some laboratories, (repeated) gas jar evacuations followed by atmosphere

222 replacement with bottled gasses are used. Gas generator kits are available from commercial sources.

223 Variable atmosphere incubators are more suitable if large numbers of cultures are undertaken.

224 ii) Temperature

225 Media may be incubated at 37°C or 42°C, but it is common practice to incubate at 42°C to minimise

226 growth of contaminants and to select for optimal growth of *C. jejuni*and *C. coli*. The fungistatic agents

227 ~~cycloheximide or~~amphothericin B or cycloheximide are added in order to prevent growth of yeasts

228 and mould at 37°C (Bolton *et al*., 1988). In some laboratories, incubation takes place at 41.5°C to

229 harmonise with *Salmonella*and *Escherichia coli*O157 isolation protocols~~(ISO, 2006)~~.

230 iii) Time

231 *Campylobacter jejuni*and *C. coli*usually show growth on solid media within 24–48 hours at 37–42°C.

232 As the additional number of positive samples obtained by prolonged incubation is very low, 48 hours

233 of incubation is recommended for routine diagnosis (Bolton *et al*., 1988).

##### 234 1.4.	Confirmation

235 A pure culture is required for confirmatory tests, but a preliminary confirmation can be obtained by direct microscopic

236 examination of suspect colony material.

###### 237 1.4.1.	Identification on solid medium

238 On Skirrow or other blood-containing agars, characteristic *Campylobacter*colonies are slightly pink,

239 round, convex, smooth and shiny, with a regular edge. On charcoal-based media such as mCCDA,

240 the characteristic colonies are greyish, flat and moistened, with a tendency to spread, and may have

241 a metal sheen.

###### 242 1.4.2.	Microscopic examination of morphology and motility

243 Material from a suspect colony is suspended in saline and evaluated, preferably by a phase-contrast

244 microscope, for characteristic, spiral or curved slender rods with a corkscrew-like motility. Older

245 cultures show less motile coccoïd forms.

###### 246 1.4.3.	Detection of oxidase

247 Take material from a suspect colony and place it on to a filter paper moistened with oxidase reagent.

248 The appearance of a violet or deep blue colour within 10 seconds is a positive reaction. If a

249 commercially available oxidase test kit is used, follow the manufacturer’s instructions.

###### 250 1.4.4.	Aerobic growth at 25°C

251 Inoculate the pure culture on to a non-selective blood agar plate and incubate at 25C in an aerobic

252 atmosphere for 48 hours.

###### 253 1.4.5.	Latex agglutination tests

254 *Latex agglutination tests*for confirmation of pure cultures of *C. jejuni*and *C. coli*(often also including

255 *C. lari*) are commercially available.

256 **1.5. Biochemical identification of *Campylobacter*to the species level**

257 Among the *Campylobacter*spp. growing at 42°C, the most frequently encountered species from samples of animal

258 origin are *C. jejuni*and *C. coli*. However, low frequencies of other species, including *Helicobacter*species, have been

259 described. Generally, *C. jejuni*can be differentiated from other *Campylobacter*species on the basis of the hydrolysis

260 of hippurate as this is the only hippurate-positive species isolated from veterinary or food samples. The presence of

261 hippurate-negative *C. jejuni*strains has been reported (Steinhauserova *et al*., 2001). Table 2 gives some basic

262 classical phenotypic characteristics of the most important thermophilic *Campylobacter*species (ISO, ~~2006~~2017).

263 More extensive speciation schemes have been described in the literature (~~On, 1996; Vandamme, 2000~~). Speciation

264 results should be confirmed using defined positive and negative controls.

265 Biochemical speciation may be supplemented or replaced with MALDI-TOF mass spectrometry. MALDI-TOF can be

266 used to identify *Campylobacter*isolates rapidly and efficiently at the genus and species level (Bessede *et al.*, 2011).

267 ***Table 2.****Basic phenotypic characteristics of selected thermophilic*Campylobacter *species*

268 The confirmatory tests for the presence of thermophilic campylobacters and the interpretation (ISO, ~~2006~~2017) are

269 given in Table 3. Confirm results of confirmation tests using positive and negative controls.

270 ***Table 3.****Confirmatory tests for thermophilic*Campylobacter

###### 271 1.5.1.	Detection of hippurate hydrolysis

272 Suspend a loopful of growth from a suspect colony in 400 μl of a 1% sodium hippurate solution (care

273 should be taken not to incorporate agar). Incubate aerobically at 37°C for 2 hours, then slowly add

274 200 μl 3.5% ninhydrin solution to the side of the tube to form an overlay. Re-incubate at 37°C for ~~10~~

275 15–30 minutes, and read the reaction. Positive reaction: dark purple/blue. Negative reaction: clear or

276 grey. If commercially available hippurate hydrolysis test disks are used, follow the manufacturer’s

277 instructions. The hippurate hydrolysis test is not very robust and the test is often replaced by

278 molecular tests (see below).

###### 279 1.5.2.	Detection of indoxyl acetate hydrolysis

280 Place a suspect colony on an indoxyl acetate disk and add a drop of sterile distilled water. If indoxyl

281 acetate is hydrolysed a colour change to dark blue occurs within 5–10 minutes. No colour change

282 indicates hydrolysis has not taken place. If commercially available indoxyl acetate hydrolysis test

283 disks are used, follow the manufacturer’s instructions.

284 Biochemical speciation may be supplemented or replaced with molecular methods or MALDI-TOF mass

285 spectrometry. MALDI-TOF can be used to identify *Campylobacter*isolates rapidly and efficiently at the genus and

286 species level (Bessede *et al.*, 2011). A variety of DNA probes and polymerase chain reaction (PCR)-based

287 identification assays has been described for the identification of *Campylobacter*species (On, 1996; Vandamme,

288 2000). On & Jordan (2003) evaluated the specificity of 11 PCR-based assays for *C. jejuni*and *C. coli*identification*.*

289 A fast method to differentiate *C. jejuni*and *C. coli*strains is a duplex real-time PCR, targeting gene *mapA*for *C. jejuni*

290 identification and gene *CeuE*for *C. coli*identification (Best *et al*., 2003). Another real-time PCR method commonly

291 used to identify and differentiate between *C. jejuni, coli*and *lari*is described by Mayr *et al.*(2010). A gel-based method

292 that is commonly used differentiates between *C. jejuni, C. coli, C. lari and C. upsaliensis*(Wang *et al.,*2002).

293 *Campylobacter*isolates can also be molecular identified at species level with 16S rRNA sequencing (Gorkiewicz *et*

294 *~~al.,~~*~~2003).~~

295 **1.6.	Molecular detection and identification of *Campylobacter***

296 Multiple PCR-based methods for the detection of *Campylobacter*in animal faecal samples and enriched meat

297 samples have been ~~extensively~~described in the literature~~(Bang *et al*., 2001 Lund *et al*., 2003; Olsen *et al*., 1995)~~.

298 Lund *et al*. describe a real-time PCR method to detect *Campylobacter*spp. in chicken faecal samples using magnetic

299 bead DNA isolation followed by a real-time PCR targeting the 16S rRNA gene (Lund *et al*., 2003; 2004). For food

300 samples, a combined method is described of Bolton broth enrichment and multiplex real-time PCR targeting gene

301 *mapA*for *C. jejuni*, gene *ceuE*for *C. coli*and a ATP-binding protein for both *C. jejuni*and *C. coli*(Lanzl *et al.,*2022).

302 ~~Many molecular tests are available to identify *Campylobacter*species, but there is not a specific recommended one.~~

303 *~~Campylobacter~~*~~isolates can be identified at species level with 16S rRNA sequencing (Gorkiewicz *et al.,*2003).~~

304 Inclusion of positive and negative reference strains and process controls to detect inhibition of the PCR reaction by

305 the sample matrix are required for all molecular *Campylobacter*detection methods.

306 A variety of DNA probes and PCR-based identification assays has been described for the identification of

307 *Campylobacter*species (Ferrari *et al.,*2023; Jribi *et al.,*2017). On & Jordan (2003) evaluated the specificity of

308 11 PCR-based assays for *C. jejuni*and *C. coli*identification*.*A fast method to differentiate *C. jejuni*and *C. coli*strains

309 is a duplex real-time PCR, targeting gene *mapA*for *C. jejuni*identification and gene *ceuE*for *C. coli*identification

310 (Best *et al*., 2003). Another real-time PCR method commonly used to identify and differentiate between *C. jejuni, C.*

311 *coli*and C. *lari*is described by Mayr *et al.*(2010). *Campylobacter*isolates can also be identified at species level with

312 16S rRNA sequencing (Gorkiewicz *et al.,*2003).

##### 313 1.7.	Antigen-capture-based tests

314 Enzyme immunoassays are available for the detection of *Campylobacter*in human and animal stool samples (Ricke

315 *et al*., 2019). Some are of the lateral flow format. While antigen tests are convenient to use, in an evaluation study

316 where human stool samples were tested with four commercial *Campylobacter*antigen tests, it was shown that no

317 stool antigen test offered the necessary combination of high sensitivity, high specificity, and moderate to high positive

318 predictive value needed in a standalone diagnostic test (Fitzgerald *et al.,*2016). By using antigen-capture-based

319 tests, the sensitivity and specificity should be critically evaluated through an in-house validation.

#### 320 2.	Serological tests

321 There are no serological assays in routine use for the detection of colonisation of *C. jejuni*or*/C. coli*in livestock.

## 322	C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

323 There are no vaccines specifically developed for *C. jejuni*or *C. coli*in animals or birds.

### 324 REFERENCES

325 ASPINALL S.T., WAREING D.R.A., HAYWARD P.G. & HUTCHINSON D.N. (1993). Selective medium for thermophilic

326 campylobacters including *Campylobacter upsaliensis. J. Clin. Pathol*., **46**, 829–831.

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453 http://www.who.int/iris/handle/10665/80751

454	*

455	* *

456	**NB:**There is a WOAH Reference Laboratory for campylobacteriosis

457	(please consult the WOAH Web site for the most up-to-date list:

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

459	Please contact the WOAH Reference Laboratories for any further information on

460	diagnostic tests and reagents for campylobacteriosis

461	**NB**: FIRST ADOPTED IN 2004. MOST RECENT UPDATES ADOPTED IN 2017.