**Annex 9. Item 5.1. – Chapter 3.3.6. Avian tuberculosis**

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

3	**Paris, 4–8 September 2023**

4

5	**CHAP T E R  3 . 3 . 6 .**

# 6	A V I A N T U B ER C U L OS I S

7	**SUMMARY**

8 ***Description of the disease:****Avian tuberculosis, or avian mycobacteriosis, is ~~an important~~a significant*

9 *disease that affects companion, captive exotic, wild, and domestic birds and mammals. The disease is most*

10 *often caused by*Mycobacterium avium *subsp.*avium (M. a. avium), a member of the M. avium complex.

11 *However, more than ten other mycobacterial species have been reported to infect birds. The most significant*

12 *cause of poultry disease is M*. *a*. *avium.*

13 *Clinical signs of the disease vary depending on the organs involved. The classical presentation is*

14 *characterised by chronic and progressive wasting and weakness. Diarrhoea is common and joint swelling*

15 *are standard features in infected flocks. Some birds may show respiratory signs, and occasionally, sudden*

16 *death occurs. Some birds may develop granulomatous ocular lesions.*

17 Mycobacterium tuberculosis*, the agent that most commonly causes human tuberculosis*(*gene IS*61101)*is*

18 *less commonly rarely the cause of infection in birds, and it is often ~~as a~~the result of transmission from pet*

19 *bird owners.*

20 *Members of*M. avium *complex:*M. a. avium (*serotypes 1–3; containing gene segments IS*901 *and IS*1245),

21 M. avium *subsp.*hominissuis (*serotypes 4–6, 8–11, and 21; lacking gene segment IS*901 *and containing*

22 *segment IS*1245) *and*M. intracellulare (*serotypes 7, 12–20, and 22–28; lacking both IS*901 *and IS*1245) *can*

23 *also infect an extensive range of mammals such as swine, cattle, deer, sheep, goats, horses, cats, dogs,*

24 *and exotic species. In humans, all members of the*M. avium *complex and*M. genavense *~~are capable of~~*

25 *inducing can induce a progressive disease that is refractory to treatment, ~~mostly~~mainly  in*

26 *immunocompromised patients.*

27 *~~All manipulations involving~~Due to the contagious nature of this group of organisms, handling ~~of~~open live*

28 *cultures or ~~of~~material from infected birds must only be carried out ~~with~~after an appropriate ~~biorisk~~*

29 *~~management~~risk assessment and the implementation of biosafety measures designed to avoid infection.*

30 *Diagnosis of avian tuberculosis in birds depends on the demonstration of*~~the above-mentioned~~a

31 mycobacterial species *in live or dead birds or the detection of an immune response, cellular or humoral,*

32 *culture examination, or gene segments~~IS~~*~~6110*, IS*901 *and IS*1245~~*by polymerase chain reaction*(*PCR*) *in*

33 *the excretions or secretions of live birds.*

34 ***Detection of the agent:****Where clinical signs of avian tuberculosis are seen in the flock, or typical*

35 *tuberculous lesions are present in birds at necropsy, the demonstration of acid-fast bacilli in smears or*

36 *sections made from affected organs is sufficient for a quick positive diagnosis. If acid-fast bacilli are not*

37 *found but typical tuberculous signs or lesions are present in the birds, a culture of the organism or PCR must*

38 *be attempted~~. PCR could also be carried out directly~~ on tissue samples. Any acid-fast organism isolated*

39 *should be identified by nucleic-acid-based tests or chromatographical*(*e.g. high-performance liquid*

40 *chromatography*[*HPLC*]) *criteria; serotyping of isolates of*M. avium *complex members or PCR for 16S*r*RNA*

41 g*ene followed by sequencing, or the presence of an amplicon for the insertion sequences**IS*6110*, IS*901,

42 *and IS*1245 *could also be performed.*

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

100

43 ***Tuberculin ~~test~~and serological tests:****These tests are ~~normally~~typically used to determine ~~the~~disease*

44 *prevalence ~~of disease~~in a flock or ~~to~~detect infected birds. When used to detect the presence of avian*

45 *tuberculosis in a flock, they should be supported by the necropsy of any birds that give positive reactions.*

46 *In domestic fowl, the tuberculin test in the wattle is the test of choice. This test is less useful in other species*

47 *of bird. ~~A better test, especially in waterfowl, is~~The whole blood stained-antigen agglutination test is better,*

48 *especially in waterfowl. It is more reliable and ~~has the advantage that it will~~can give a result within a few*

49 *minutes while the bird is still being held.*

50 ***Requirements for vaccines and diagnostic biologicals:****No vaccines are available for use in birds. Avian*

51 *tuberculin purified protein derivative*~~(*PPD*)~~*is the standard preparation for use in the tuberculin test of*

52 *domestic poultry. Avian PPD is also used as a component in the comparative intradermal tuberculin test in*

53 *cattle*(*see Chapter 3.1.13*Mammalian tuberculosis [infection with *Mycobacterium tuberculosis*complex])*.*

## 54	A. INTRODUCTION

55 Several mycobacterial species can be involved in the aetiology of avian tuberculosis~~and~~, also known as avian

56 mycobacteriosis. Avian tuberculosis is most commonly ~~produced~~caused by infection with *Mycobacterium avium*subsp.

57 *avium*(serotypes 1, 2, and 3: containing specific gene segment IS*901*and nonspecific segment IS*1245*) and less frequently

58 by *M. genavense*(Guerrero *et al.*, 1995; Pavlik *et al.*, 2000; Salamatian *et al.,*2020; Sattar *et al.,*2021; Tell *et al*., 2001).

59 Avian mycobacteriosis is also caused by other two members of the*M. avium*complex: *M. avium*subsp. *hominissuis*

60 (serotypes 4–6, 8–11, and 21: lacking gene segment IS*901*and containing segment IS*1245*and mainly infecting humans

61 and pigs) and *M. intracellulare*(serotypes 7, 12–20, and 22–28: lacking both gene segments IS*901*and IS*1245*) and by

62 *~~M. intracellulare~~*~~, *M. scrofulaceum*, *M. fortuitum,*and~~ other potentially pathogenic mycobacterial species including

63 *M. scrofulaceum*and *M. fortuitum*. Under some circumstances, an extensive range of mammalian species, such as swine,

64 cattle, deer, sheep, goats, horses, cats, dogs, and exotic animals, can be infected by these mycobacterium species

65 (Dvorska *et al.*, 2004; Kunze *et al.,*1992; Mijs *et al.*, 2002; Shitaye *et al.*, 2009; Tell *et al*., 2001; Thorel *et al*., 1997; 2001).

66 *Mycobacterium tuberculosis*and *M. bovis*are ~~less common as causal~~rarely the causative agents of tuberculosis in birds

67 (Hoop, 2002; Lanteri *et al.,*2011; Peters *et al.,*2007; Schmidt *et al.,*2022; Tell *et al*., 2001).

68 *Mycobacterium avium*species with standing in nomenclature as of 202335 (Arahal *et al.,*2023) consists of ~~four~~three

69 subspecies: *M. avium*subsp. *avium*, *~~M. avium~~*~~subsp. *hominissuis*,~~*M. avium*subsp. *silvaticum*, and *M. avium*subsp.

70 *paratuberculosis*(Mijs *et al.*, 2002; Thorel *et al*., 1990). The latter is the causal agent of Johne’s disease, or

71 paratuberculosis, in ruminants and other mammalian species (see Chapter 3.1.16 *Paratuberculosis*[*Johne’s disease*]).

72 *Mycobacterium a. silvaticum,*which like *M. avium*subsp. *paratuberculosis*grows *in-vitro*only on media with Mycobactin,

73 which can cause avian tuberculosis in wood pigeons (Thorel *et al.*, 1990). With the widespread use of whole genome

74 sequencing (WGS) and bioinformatics, some studies have investigated the classification of species belonging to the genus

75 *Mycobacterium*and have proposed that *M. avium*be three subspecies *M. avium*subsp. *avium*, *M. avium*subsp.

76 *paratuberculosis*, and *M. avium*subsp. *lepraemurium*. Further subdividing *M. avium*subsp. *avium*into three variants *M.*

77 *avium*subsp. *avium*var. *avium*, *M. avium*subsp. *avium*var. *silvaticum*, and *M. avium*subsp. *avium*var. *hominissuis*(Riojas

78 *et al.,*2021; Tortoli *et al.,*2019).

79 ~~All *M. a avium*isolates from birds and mammals, including humans, have a multiple repetitive sequence IS*901*in their~~

80 ~~genome and produce a characteristic three-band pattern in IS*1245*restriction fragment length polymorphism (RFLP) as~~

81 ~~described and standardised previously (Dvorska *et al.*, 2003; Ritacco *et al*., 1998). This repetitive sequence is also present~~

82 in *M. a. silvaticum*and RFLP analysis can help with identification. IS*901*has only been detected in *M. avium*strains with

83 ~~serotypes 1, 2 and 3 (Pavlik *et al*., 2000; Ritacco *et al*., 1998) that are apparently more pathogenic to birds than other~~

84 ~~serotypes (Tell *et al*., 2001). On the basis of genetic and phenotypic differences it has recently been proposed to~~

85 ~~differentiate *M. a. avium*into two subspecies based on the target organism: *M. a. hominissuis*for human and porcine~~

86 isolates and *M. a. avium*for bird-type isolates (Mijs *et al*., 2002). *Mycobacterium a. hominissuis*has polymorphic multiband

87 ~~IS*1245*RFLP patterns and is able to grow between 24 and 45°C (Mijs *et al*., 2002; Van Soolingen *et al.*, 1998). It is worth~~

88 ~~noting that the typical features of bird-isolates, the three-band pattern in IS*1245*RFLP and presence of IS*901*, have also~~

89 ~~been found in cervine and bovine isolates of *M. a. avium*.~~

90 Avian tuberculosis in birds is most prevalent in gallinaceous poultry and in wild birds raised in captivity. Turkeys are ~~quite~~

91 susceptible, but ducks, geese, and other water birds are comparatively resistant. The practices of allowing poultry to roam

92 at large on the farm (free range) and of keeping the breeders for several years are conducive to the spread of the causal

93 agent of avian tuberculosis among them. Infected individuals and contaminated environments (water and soil) are the ~~main~~

94 primary sources of infection. The above-mentioned mycobacterial species causing avian tuberculosis can survive for

95 several months in the environment (Dvorska *et al.*, 2007; Kazda *et al.*, 2009; Shitaye *et al.*, 2008; Tell *et al*., 2001).

1 https://lpsn.dsmz.de/species/mycobacterium-avium

96 ~~In most cases,~~Infected birds usually show no clinical signs but ~~they~~may eventually become lethargic and emaciated.

97 Many affected birds show diarrhoea and swollen joints, and comb and wattles may regress and become pale. Affected

98 birds, especially gallinaceous poultry, are usually older than 1 year. Some show respiratory signs~~and~~, including sudden

99 death may occur, dyspnoea is less common, and granulomatous ocular lesions (Pocknell *et al*., 1996) ~~as well as~~and skin

100 lesions have been reported. Under intensive husbandry conditions, sudden death may occur, often associated with severe

101 lesions in the liver; such lesions are easily observed at post-mortem examination (Salamatian *et al.,*2020; Tell *et al*., 2001).

102 The primary lesions of avian tuberculosis in ~~birds~~poultry (chickens and turkeys) are nearly always in the intestinal tract.

103 Such lesions take the form of deep ulcers filled with caseous material containing many mycobacterial cells, and these are

104 discharged into the lumen and appear in the faeces. Before the intestinal tract is opened, the ulcerated areas appear as

105 tumour-like masses attached to the gut wall~~, but~~. Still, when the intestine is opened, the true nature of the mass becomes

106 evident. Typical caseous lesions are nearly always found in the liver and spleen~~, and~~; these organs are usually~~are~~ greatly

107 enlarged because of the formation of new tuberculous tissue. The lungs and other tissues are ordinarily free from lesions

108 even in advanced cases (Salamatian *et al.,*2020; Tell *et al.*, 2001; Thorel *et al.*, 1997).

109 ~~Among domestic animals (mammals), domestic pigs (*Sus scrofa*f. *domesticus*) are the most susceptible to avian~~

110 ~~tuberculosis. Usually, no clinical manifestations are observed in such animals. Avian tuberculosis is suspected when~~

111 ~~tuberculous lesions are found in the head and mesenteric lymph nodes on meat inspection after slaughter. Findings of~~

112 ~~tuberculous lesions that involve other organs (liver, spleen, lungs, etc.) are rare, usually occurring at the advanced stage~~

113 ~~of the disease. *Mycobacterium a. avium*accounted for up to 35% of the *Mycobacteria*isolated from such tuberculous~~

114 lesions (Dvorska *et al.,*1999; Pavlik *et al.,*2003, 2005; Shitaye *et al.,*2006). Unlike the other species mentioned previously,

115 ~~cattle are highly resistant to the causative agent of avian tuberculosis, and tuberculous lesions are detected in head lymph~~

116 ~~nodes, or occasionally in liver lymph nodes, only on meat inspection. *Mycobacterium a. avium*can be successfully isolated~~

117 ~~from tuberculous lesions in mesenteric lymph nodes from juvenile cattle: the isolation rate from cattle under 2 years of age~~

118 ~~was 34.4% in contrast to 13.0% from cattle over 2 years of age (Dvorska *et al.,*2004).~~

119 Pet and wild birds with avian mycobacteriosis have clinical presentations often exacerbated by age and viral and fungal

120 co-infections (Schmidt *et al.,*2022; Schmitz *et al.,*2018b). The presence of nonspecific clinical signs and the absence of

121 gross finds during necropsy in psittacine and passeriform birds may confound diagnosis. Furthermore, differences in body

122 condition and gross pathology are observed, where psittacines have more severe lesions than passeriform birds. These

123 differences could also be attributed to the fact that they are often more likely infected with *M. genavense*than *M*. *avium*

124 (Schmitz *et al.,*2018a). The advent of more affordable WGS has allowed the study of *M*. *avium*and *M*. *genavense*and

125 their epidemiology in a large captive population of birds belonging to multiple taxa for over 22 years. In this large bird

126 population, 68% of all birds at necropsy had isolates that were infected with *M. avium*or *M. genavense*. The WGS study

127 of these mycobacterium isolates demonstrated strong evidence of disease clustering among those birds infected with *M.*

128 *avium*but not among those harbouring *M. genavense*(Witte *et al.,*2021). This works sheds light on the epidemiology of

129 mycobacterium among captive birds, and future studies are necessary to understand these pathogens’ epidemiology better

130 and to help identify its reservoirs.

131 It is essential to bear in mind that all members of *M. avium*~~complex~~ and *M. genavense*are capable of giving rise to a

132 progressive disease in humans that is refractory to treatment, especially in immunocompromised individuals (Narsana *et*

133 *al.,*2023; Pavlik *et al.*, 2000; Tell *et al*., 2001). ~~Members of *Mycobacterium avium*complex are classed in Risk Group 2 for~~

134 ~~human infection and should be handled with appropriate measures~~All *Mycobacterium*species can cause infection in

135 people (Cowman *et al.,*2019). Caution should be exercised by those working with birds in environments infected with

136 *Mycobacterium*, especially those immunosuppressed. All laboratory manipulations with live cultures or potentially

137 infected/contaminated material must be performed at an appropriate biosafety and containment level determined by

138 conducting a thorough risk assessment as described in Chapter 1.1.4 *Biosafety and biosecurity: Standard for managing*

139 *biological risk in the veterinary laboratory and animal facilities*. ~~Biocontainment measures should be determined by risk~~

140 ~~analysis as described in Chapter 1.1.4.~~The CDC’s online Manual for Biosafety in Microbiological and Biomedical

141 Laboratories is also a good reference 36.

2 https://www.cdc.gov/labs/pdf/SF 19_308133-A_BMBL6_00-BOOK-WEB-final-3.pdf

## 142	B. DIAGNOSTIC TECHNIQUES

143	***Table 1.****Test methods available for the diagnosis of avian tuberculosis and their purpose*

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

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

146 PCR = polymerase chain reaction.

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

#### 148 1.	Identification of the agent

149 If there is a characteristic history of avian tuberculosis in a flock and typical lesions are found in birds at post-mortem, the

150 detection of acid-fast bacilli (AFB) in smears or sections from affected organs, stained by the Ziehl–Neelsen method usually

151 is ~~normally~~sufficient to establish a diagnosis. Confirmation of *M. avium*subspecies should be carried out by PCR or other

152 molecular techniques (Kaevska *et al.,*2010; Slana *et al.,*2010). Occasionally a case will occur, presumably ~~as a result of~~

153 due to large infecting doses giving rise to acute overwhelming disease, in which affected organs, most obviously the liver,

154 have a ‘morocco leather’ appearance with fine greyish or yellowish mottling. ~~In such cases~~AFB may not be found in such

155 cases, but careful inspection will reveal parallel bundles of brownish refractile bacilli. Prolongation of the hot carbol-fuchsin

156 stage of Ziehl–Neelsen staining to 10 minutes will usually reveal that these are indeed AFB, with unusually high resistance

157 to penetration of the stain. ~~Recently,~~ DNA probes~~and~~, polymerase chain reaction (PCR), and WGS techniques have been

158 used to identify the agent at the species and subspecies level specifically. Traditionally, *M. a. avium*is separated from

159 common nonchromogenic slow-growing organisms by their ability to grow at 42°C (*M. a. avium*). *Mycobacterium*

160 *genavense*is particularly fastidious and has ~~special~~unique requirements for growth and identification (Shitaye *et al.,*2010).

##### 161 1.1.	Culture

162 If there is a characteristic flock history and suggestive lesions are found at necropsy, but no AFB are seen in smears

163 or sections, an attempt must be made to isolate the causative organism from the necropsy material. The liver or

164 spleen is usually the best organ to use, but if the carcass is decomposed, bone marrow may prove more satisfactory

165 as it could be less contaminated. As with the culture of *M. bovis*, non-sterile specimens need to be processed with

166 detergent, alkali, or acid to eliminate rapidly growing microorganisms before culture (see Chapter 3.1.13 *Mammalian*

167 *tuberculosis*[*infection with*Mycobacterium tuberculosis *complex*]). *Mycobacterium a. avium*grows best on media

168 such as Lowenstein–Jensen, Herrold’s medium, Middlebrook 7H10~~and~~, 7H11, or Coletsos, with 1% sodium pyruvate

169 added. It ~~may occasionally be~~is necessary to incorporate mycobactin J, as it is used ~~for the isolation of~~to isolate

170 *M. ~~a~~*~~. *paratuberculosis*~~*genavense*and *M. a. silvaticum.*~~Growth may be confined to the edge of the condensation~~

171 ~~water.~~ Cultures should be incubated for at least ~~8~~12 weeks, less if using liquid media. Typically, *M. a*. *avium*produces

172 ‘smooth’ colonies within 2–4 weeks; rough variants ~~do~~occur. Shorter incubation times can be achieved using the

173 liquid culture BACTEC system or the automated fluorescent MGIT 960 culture system. *Mycobacterium a. avium*can

174 also be detected in ~~massively~~ infected tissue by a conventional PCR, which also ~~allows acceleration of the~~accelerates

175 pathogen detection and identification (Moravkova *et al.,*2008). ~~Currently,~~Direct detection and quantification of

176 *M. a. avium*using IS*901*quantitative real-time PCR can be considered ~~as~~the ~~best~~fast and inexpensive method

177 ~~(despite its rather high cost per test)~~(Kaevska *et al.,*2010; Slana *et al.,*2010).

178 For *M. genavense*, the optimal medium is liquid media supplemented with Mycobactin J (an iron chelator) and then

179 plated onto a solid medium ~~is~~such as Middlebrook 7H11~~medium acidified to pH 6 and supplemented with blood and~~

180 ~~charcoal (Realini *et al*., 1999)~~. The incubation period at 37°C with 5–7% CO2 should be extended for at least ~~6 months~~

181 42 days. If samples are directly plated onto solid media, plates should be held for at least 12 weeks. Bacterial growth

182 should be prepared in a smear and stained using an acid-fast stain. All acid-fast organisms should be identified using

183 MALDI-TOF (matrix assisted laser desorption ionisation–time of flight [mass spectrometry]) or PCR (Buckwalter *et*

184 *al.,*2016; Hall *et al.,*2003; Shitaye *et al.*, 2010).

185 Typing of mycobacteria to the species and subspecies level requires a specialised laboratory. Conventional

186 biochemical tests for species identification are lengthy and fail to distinguish between *M. avium*and *M. intracellulare*.

187 Thus, a miscellaneous group of mycobacteria that includes both species is usually classified under the ~~denomination~~

188 ~~of~~*M. avium*complex denomination. Seroagglutination, ~~which is~~based on the sugar residue specificity of surface

189 glycopeptidolipids, allows ~~classification~~the parsing of *M. avium*complex organisms into 28 serovars (Wolinsky &

190 Schaefer, 1973). More sophisticated typing methods directed at cell-wall-specific targets are currently available, such

191 as enzyme-linked immunosorbent assays with monoclonal antibodies to major serovars, ~~and~~ high-performance liquid

192 chromatography~~(HPLC)~~, and WGS. Based on DNA–rRNA hybridisation serovars 1 to 6, 8 to 11, and 21 ~~are currently~~

193 have been ascribed to *M. a. avium*and *M. a. hominissuis*, and serovars 7, 12 to 20, and 25 to *M. intracellulare*.

194 However, no consensus was achieved on other serovars, and some isolates cannot be serotyped (Inderlied *et al*.,

195 1993). For final species and subspecies identification, the current methods are WGS and bioinformatic analysis of

196 isolates obtained from sick birds. Avian tuberculosis in birds is commonly caused by *M. a. avium*types 1, 2, or 3. If

197 the isolate is not one of these three serotypes, further molecular identification tests ~~(IS*901*PCR)~~must be ~~carried out~~

198 conducted in a specialised laboratory. However, it should be ~~borne in mind~~noted that ~~superficial~~tuberculous lesions

199 in ~~caged~~pet birds, especially psittacines, may be caused by *M. tuberculosis,*and ~~IS*6110*PCR should be used for~~

200 precise identification should always be attempted (Hoop, 2002; Lanteri *et al.*, 2011; Peters *et al.,*2007; Schmidt *et*

201 *al.,*2008; Tell *et al.,*2001).

##### 202 1.2.	Nucleic acid recognition methods

203 Specific and reliable genetic tests for speciation ~~are currently~~have been available (Saito *et al*., 1990). , including

204 commercial nucleic ~~acid~~ hybridisation probes ~~have become a ‘gold standard’~~reference method for ~~distinction between~~

205 distinguishing*M. avium,*~~and~~*M. intracellulare*~~cultures.~~and *M. genavense*~~can also be distinguished with these tests~~.

206 A further probe that covers the whole *M. avium*complex was also developed, as genuine *M. avium*complex strains

207 have been described that fail to react with specific *M. avium*and *M. intracellular*probes (Soini *et al*., 1996).

208 Nevertheless, identification errors were reported due to the cross-reactivity, which may have serious consequences

209 (van Ingen *et al.,*2009). Various in-house molecular methods have been reported ~~for the identification of~~to identify

210 mycobacterial cultures, including ~~MAC.~~members of the *Mycobacterium avium*complex. The following gene segments

211 could be used to identify *Mycobacterium*isolates as *M. avium*in one multiplex PCR reaction: IS*900*, IS*901*, IS*1245*.

212 The isolates of *M. a. avium/M. a. silvaticum*are IS900−, IS901+, IS1245+, the isolates of *M. a. hominissuis*are

213 IS900−, IS901−, IS1245+, and the isolates of *M. a. paratuberculosis*are IS900+, IS901−, IS1245− (Kaevska *et al.,*

214 2010; Moravkova *et al.,*2008). A ~~multiplex~~16S rRNA PCR and sequencing method for differentiating *M. avium*from

215 *M. intracellulare*and *M. tuberculosis*complex ~~has some advantages (Cousins *et al*., 1996). 16S rRNA~~is currently

216 commercially available. Similarly, many veterinary diagnostic laboratories commonly perform in-house PCR and

217 sequencing (Kirschner *et al*., 1993)~~may also be used~~. Culture-independent in-house molecular tests have been

218 developed ~~for the detection~~to detect and identification of identify species belonging to the *M. avium*complex directly

219 from samples (Hall *et al.,*2003; Kaevska *et al.,*2010). WGS of isolates has recently become the go-to molecular

220 method to identify mycobacterium isolates from birds with great accuracy. It enables, with the use of bioinformatic

221 tools, not only an accurate identification of species and subspecies, but also helps to determine the organism

222 relatedness within a flock or environment (Witte *et al.,*2021). In recent years, veterinary diagnostic laboratories have

223 extensively adopted real-time PCR methods to detect *M. a. avium*directly from different specimens (faeces, tissues,

224 formalin-fixed tissues, and environmental samples). The technique rapidly detects fastidious and slow-growing

225 microorganisms, such as *M. a. avium*(Tell *et al.,*2003a; 2003b).

226 Several commercial diagnostic PCR tests for detecting *M. a. avium*are available. Still, users should consider the skill

227 set and equipment necessary to perform such tests. Furthermore, it is important to determine the fitness for the

228 purpose of these tests before implementation. The interpretation of the results of these molecular tests also requires

229 veterinary expertise.

230 *~~Mycobacterium a. avium,~~*~~the causative agent of avian tuberculosis (Thorel *et al.,*1990), previously designated as~~

231 *~~M. avium~~*~~species only, is assigned to serotypes 1 to 3 within the *M. avium*complex of 28 serotypes (Wolinsky &~~

232 ~~Schaefer, 1973). As revealed by molecular biological studies, the detected insertion sequence IS*901*(Kunze *et al.,*~~

233 ~~1992) is possessed not only by the isolates of the above-named serotypes, but also by isolates, virulent for birds, that~~

234 ~~could not be typed because agglutination occurred (Pavlik *et al.,*2000). In epidemiological studies, a standardised~~

235 ~~IS*901*RFLP methods replaced serotyping (Dvorska *et al.,*2003).~~

#### 236 2.	Immunological methods

237 Tests used for export depend on the importing requirement of individual countries. In the main, the tuberculin test or the

238 haemagglutination (stained antigen) test are most frequently used for export testing of poultry.

##### 239 2.1.	Tuberculin test

240 The tuberculin test is the most widely used test ~~in~~for domestic fowl and the only test for which an international

241 standard for the reagent exists. Tuberculin is the standard avian purified protein derivative (PPD). Birds are tested by

242 intradermal inoculation in the wattle with 0.05 ml or 0.1 ml of tuberculin (containing approximately 2000 International

243 Units [IU]), using a ~~very~~ fine needle of approximately 26 gauge, 10 mm long~~× 0.5 mm~~. The test is read after 48 hours

244 ~~and~~. A positive reaction is any swelling at the site, from a small firm nodule approximately 5 mm in diameter to gross

245 oedema extending into the other wattle and down the neck. ~~With practice,~~ Even very small wattles on immature birds

246 can be inoculated successfully. However, ~~in immature birds~~the comb may be used in immature birds, although the

247 results are not ~~so~~as reliable. Tuberculin testing of the wattle in turkeys is much less ~~reliable~~consistent than in ~~the~~

248 domestic ~~fowl~~chickens. Inoculation in the wing web has been recommended as ~~being~~more efficient, but this is still

249 not as good as ~~for domestic fowl~~in chickens. Other birds may also be tested in the wing web, but results are not

250 generally satisfactory. The bare ornamental skin areas on Muscovy ducks and some ~~species of~~pheasant species

251 can be used, but ~~reliability~~dependability is doubtful, and interpretation is difficult. Testing in the foot web of waterfowl

252 has also been described; the test is not very sensitive and is often complicated by infections of the inoculation site.

253 In the common pheasant, the tuberculin test can be performed in either of two ways. In the first, 0.05 ml or 0.1 ml of

254 tuberculin is injected into the skin of the lower eyelid. A positive result is indicated by marked swelling at the injection

255 site after 48 hours. Alternatively, 0.25 ml of tuberculin is injected into the thoracic muscles, and the birds are observed

256 for 6–10 hours. Infected birds will show signs of depression and keep aside from the flock, and there may be cases

257 of sudden death. No clinical signs will be provoked in uninfected birds.

##### 258 2.2.	Stained antigen test

259 The stained-antigen agglutination test has been used with good results, especially in domestic and ornamental

260 waterfowl. A drop (0.05–0.1 ml) of the antigen is mixed with the same volume of fresh whole blood, obtained by

261 venipuncture, on a white porcelain or enamel tile. The mixture is rocked for 2 minutes and examined for agglutination.

262 The agglutination may be coarse, in which case it is obvious, or quite fine, in which case it may be most clearly seen

263 as an accumulation of the malachite-green-stained antigen around the edge of the drop, leaving the centre a normal

264 blood-red colour. This test is especially useful for screening large flocks for immediate culling and therefore has

265 advantages over the tuberculin test for controlling the disease, even in domestic fowl. It has also been claimed that it

266 is more reliable in domestic poultry than the tuberculin test.

###### 267 2.2.1.	Preparation of the antigen

268 An antigen stained with 1% malachite green is used for the rapid whole blood plate agglutination test

269 (Rozanska, 1965). The strain used to prepare the stained antigen must be smooth and not auto-

270 agglutinate in saline suspension. It must conform to the characteristics of *M. a. avium*, preferably

271 obtained from a culture collection, to guarantee its authenticity.

272 A strain that will detect infection with any serotype is recommended instead of the specific serotype

273 most likely to be encountered (in Europe, serotype 2 for domestic fowl, serotype 1 for waterfowl, and

274 birds and swine in the USA). Using a highly specific strain for the serotype is recommended. The

275 specificity of strains can be determined only by testing them as antigens, although, in general, a

276 serotype 2 antigen will always detect serotype 3 infection and vice versa. Serotype 1 strains detect a

277 wide  spectrum  of  infections  and  frequently  detect  infections  with  mycobactin-dependent

278 mycobacteria or *M. a. silvaticum*. There is no reason not to use a culture containing more than one

279 strain of *M. a. avium*if it shows the desired properties of sensitivity and specificity. Consistency of

280 results between batches will be easier using pure cultures.

281 The organism should be grown in a suitable liquid medium, such as Middlebrook 7H9 containing 1%

282 sodium pyruvate. Good growth should be obtained in approximately 7 days. The liquid culture is used

283 as a seed for bulk antigen preparation.

284 Antigen for agglutination tests is best obtained on a solid medium, such as Löwenstein–Jensen or

285 7H11, containing 1% sodium pyruvate instead of glycerol, using Roux flasks or large bottles. Using

286 a solid medium maximizes the chance of detecting contamination, and antigens grown in some liquid

287 media are not agglutinated by specific antibodies. Liquid seed culture should be diluted (based on

288 experience) to give discrete colonies on the solid medium. This will usually give the best yield

289 increasing the chance of detecting contamination. About 10 ml of inoculum will usually allow it to

290 wash over the whole surface and provide sufficient moisture to keep the air in the bottle near 100%

291 humidity.

292 The bottles are incubated at 37°C, and good growth should be obtained in 14–21 days with most

293 strains. The antigen is harvested by adding sterile glass beads and twice the volume of sterile normal

294 saline (containing 0.3% formalin) as was used to inoculate the bottle. The bottle is then shaken gently

295 to wash off all the growth, and the washing is collected into a sterile bottle and re-incubated at 37°C

296 for 7 days. The killed bacilli are washed twice in sterile normal saline with 0.2% formalin by

297 centrifugation and re-suspension. This sequence is safer than the original method in which the

298 washing was carried out before the incubation that kills the organisms. Finally, bacilli are again

299 centrifuged and re-suspended in sterile normal saline containing 0.2% formalin and 0.4% sodium

300 citrate to a concentration of about 1010 bacteria per ml. This corresponds to a concentration ten times

301 that which matches tube No. 4 on McFarland’s scale.

302 Cultures for antigen should be inspected for contamination daily for the first 5 days of incubation. The

303 suspension made from the culture washings is also re-examined microscopically (for likely

304 contaminants such as yeasts) and rechecked by culture to ensure that the formalin has killed the

305 mycobacteria.

###### 306 2.2.2.	Validation of the antigen

307 Cultures should be checked by Gram staining for contamination by organisms other than

308 mycobacteria.

309 One or more batches for agglutinating antigen must be tested for efficacy in using serum from

310 naturally or artificially infected tuberculous birds by comparison with a standard preparation of known

311 potency. When using animals for research or reagent testing, approval of the procedures and the use

312 of animals by the institution’s ethics committee should be sought before any testing occurs. The

313 potency relative to that of the standard preparation must not differ significantly from that declared on

314 the label. Each bottle of antigen must be tested with normal chicken serum (to detect

315 autoagglutination) and *M. a. avium*positive chicken serum of low and high antibody content. This

316 should be done, where possible, alongside a previous batch of stained antigens. Those bottles that

317 give satisfactory agglutination reactions with the antisera can now be pooled and the antigen stained.

318 This is done by adding 3 ml of 1% malachite green solution per 100 ml of suspension. The stained

319 antigen should be checked using whole blood, just as the unstained antigen was tested with serum.

320 The agglutinating antigen should stay in the refrigerator for at least 6 months at 4°C and much longer

321 if frozen at –20°C or below. If a batch has not been used for several weeks, it should be rechecked,

322 especially for autoagglutination.

323 It is critical to perform a safety test of the unwashed antigen by culture and incubation to ensure that

324 all the bacilli are dead.

#### 325 Note on limitation of use

326 Neither the tuberculin test with avian tuberculin nor the stained-antigen agglutination test is likely to be of any value in

327 cases of *M. tuberculosis*infection in ~~caged~~pet birds.

## 328	C. REQUIREMENTS FOR DIAGNOSTIC BIOLOGICALS

#### 329 1.	Background

330 No vaccines are available.

331 Avian tuberculin is a preparation of purified protein derivatives (PPD-A) made from the heat-treated products of growth of

332 *M. a. avium*. It is used by intradermal injection to reveal delayed hypersensitivity ~~as a means of identifying~~to identify birds

333 infected with or sensitised to the same species of ~~tubercle bacillus~~*Mycobacterium*. Importantly it is also used ~~as an~~to aid

334 ~~to~~ differential diagnosis in the comparative intradermal tuberculin test for bovine tuberculosis (see Chapter 3.1.13). An

335 international standard preparation of PPD-A is being developed by WOAH to replace the former WHO Standard 37.

336 The general principles ~~as given~~in Chapter 1.1.8 *Principles of veterinary vaccine production,*should be followed for

337 injectable diagnostic biologicals such as tuberculin. The standards ~~set out~~here and in chapter 1.1.8 are intended to be

338 general~~in nature~~ and may be supplemented by national and regional requirements.

#### 339 2.	Outline of production and minimum requirements for tuberculin production

##### 340 2.1.	Characteristics of the seed

###### 341 2.1.1.	Biological characteristics of the master seed

342 Strains of *M. a. avium*used to prepare seed cultures should be purchased from a culture collection

343 and identified as ~~to~~species by appropriate tests. Several strains are recommended ~~by~~for this

344 purpose in different countries. For example, in the European Union~~(EU), for example, are~~, D4ER

345 and TB56~~. Reference may also be made to~~are recommended. The relevant national

346 recommendations should be followed. Globally there are commercial sources for PPD-A.

###### 347 2.1.2.	Quality criteria (sterility, purity, freedom from extraneous agents)

348 Seed cultures should be shown to be free from contaminating organisms and to be capable of

349 producing tuberculin ~~with~~of sufficient potency. The necessary tests are described below.

##### 350 2.2.	Method of manufacture

###### 351 2.2.1.	Procedure

352 The seed material is kept as a stock of freeze-dried cultures. If the cultures have been grown on solid

353 media, it will be necessary to adapt the organism to grow as a floating culture. This is most easily

354 accomplished by incorporating a piece of potato in the flasks of liquid medium (e.g. Watson Reid’s

355 medium). When the culture has been adapted to a liquid medium, it can be maintained by a passage

356 at 2–4-week intervals (Angus, 1978; Haagsma & Angus, 1995).

357 The organism is cultivated in modified Dorset-Henley’s synthetic medium, then killed by heating in

358 flowing steam and filtered to remove cells. The protein in the filtrate is precipitated chemically

359 (ammonium sulphate or trichloroacetic acid~~[TCA]~~ are used), washed, and resuspended. An

360 antimicrobial preservative that does not give rise to false-positive reactions, such as phenol (not more

361 than 0.5% [w/v]), may be added. Mercurial derivatives should not be used. Glycerol (not more than

362 10% [w/v]) or glucose (2.2% [w/v]) may be added as a stabiliser. The product is dispensed aseptically

363 into sterile neutral glass containers,~~which are~~ then sealed to prevent contamination. The product

364 may be freeze-dried.

###### 365 2.2.2.	Requirements for ingredients

366 The production culture substrate must be shown to ~~be capable of producing~~produce a product that

367 conforms to the ~~standards of the~~European Pharmacopoeia (~~2000~~202438 ) standards or other

368 international standards such the WHO (WHO, 1987). It must be free from ingredients known to cause

369 toxic or allergic reactions.

###### 370 2.2.3.	In-process controls

371 The production flasks, inoculated from suitable seed cultures, are incubated for the appropriate time

372 period. Any flasks showing contamination or grossly abnormal growth should be discarded after

373 autoclaving. As incubation proceeds, the surface growth of many cultures becomes moist and may

374 sink into the medium or to the bottom of the flask. In PPD-A tuberculin, the pH of the dissolved

375 precipitate (the so-called concentrated tuberculin) should be pH 6.6–6.7. The Kjeldahl method

376 determines the protein level (total organic nitrogen) of the PPD-A concentrate~~is determined by the~~

377 ~~Kjeldahl method~~. Total nitrogen and trichloroacetic acid precipitable nitrogen are usually compared.

###### 378 2.2.4.	Final product batch tests

3  PPD of *M. avium*tuberculin, WHO (1955) Technical Report Series, no.96, 11.

4 https://www.edqm.eu/en/d/234640?p_l_back_url=%2Fen%2Fsearch%3Fq%3Dpurified%2Bprotein%2Bderivative

379 i)	Sterility

380 Sterility testing is generally performed according to the European Pharmacopoeia (~~2000~~2024) or

381 other guidelines (see~~also~~ Chapter 1.1.9 *Tests for sterility and freedom from contamination of*

382 *biological materials intended for veterinary use*).

383 ii)	Identity

384 One or more batches of tuberculin may be tested for specificity together with a standard preparation

385 of bovine tuberculin by comparing the reactions produced in guinea-pigs sensitised with *M. bovis*

386 using a procedure similar to that described in Section C.2.2.4.iv. ~~In guinea-pigs sensitised with~~

387 *~~M. bovis~~*~~,~~ The potency of the preparation of avian tuberculin must be shown to be not more than 10%

388 of the potency of the standard preparation of bovine tuberculin used in the potency test. The use of

389 animals for this purpose should be reviewed and approved by your institution's ethical committee.

390 iii)	Safety

391 Tuberculin PPD-A can be examined for freedom from living mycobacteria using the culture method

392 described previously. This culture method, which does not require the use of animals, is used in many

393 laboratories, and its use is encouraged over the use of animals for this purpose. The following is the

394 previously described method, using experimental animals to evaluate the safety of PPD. The use of

395 animals for this purpose should be reviewed and approved by the institution’s ethics committee. Two

396 guinea-pigs, each weighing not less than 250 g and ~~that have~~not ~~been treated~~previously treated

397 with any material that will interfere with the test, are injected subcutaneously with 0.5 ml of the

398 tuberculin under test. No abnormal effects should occur within 7 days.

399 Tests on tuberculin for living mycobacteria may be performed either on the tuberculin immediately

400 before it is dispensed into final containers or on samples taken from the final containers themselves.

401 A sample of at least 10 ml must be taken and ~~this must be~~ injected intraperitoneally or subcutaneously

402 into at least two guinea-pigs, dividing the volume to be tested equally between the guinea-pigs. It is

403 desirable to take a larger sample, 50 ml, and to concentrate any residual mycobacteria by

404 centrifugation or membrane filtration. The guinea-pigs are observed for at least 42 days and are

405 examined macroscopically at post-mortem. Any lesions found are examined microscopically and by

406 culture. Each filled container must be inspected before it is labelled, and any showing abnormalities

407 must be discarded.

408 A test for the absence of toxic or irritant properties must be ~~carried out~~conducted according to the

409 ~~specifications of the~~European Pharmacopoeia (~~2000~~2024) specifications or the equivalent

410 regulatory documents for each country or region.

411 To test for lack of sensitising effect, three guinea-pigs that have not previously been treated with any

412 material that could interfere with the test are each injected intradermally on ~~each of~~three occasions

413 with the equivalent of 500 ~~IU~~International units – one IU is equal to the biological activity 0.02 μg of

414 PPD – of the preparation under test in a 0.1 ml volume. In the USA and Canada, the potency of the

415 tuberculin is expressed as tuberculin unit (TU) rather than IU. One TU is also defined as 0.02 μgs of

416 PPD. Each guinea-pig, together with ~~each of~~the three control guinea-pigs that have not been injected

417 previously, is injected intradermally 15–21 days after the third injection with the same dose of ~~the~~

418 ~~same~~tuberculin. The reactions of the two groups of guinea-pigs should not be significantly different

419 when measured 24–28 hours later.

420 iv)	Batch potency

421 The potency of avian tuberculin is determined in guinea-pigs sensitised with *M. a. avium*~~, by~~

422 ~~comparison~~compared with a standard preparation calibrated in IU or TU.

423 Use no fewer than nine albino guinea-pigs, each weighing 400–600 g. Sensitise the guinea-pigs by

424 administering ~~to each, by deep intramuscular injection,~~a suitable dose of inactivated or live

425 *M. a. avium*to each by deep intramuscular injection. The test is performed between 4 and 6 weeks

426 later~~as follows: Shave~~. Briefly, have the guinea-pigs’ flanks~~so as~~ to provide space for three-to-four

427 injections on each side. Prepare at least three dilutions of the tuberculin under test and at least three

428 dilutions of the standard preparation in an isotonic buffer solution containing 0.0005% (w/v)

429 polysorbate 80 (Tween 80). Choose the dilutions so that the reactions produced have diameters of

430 not less than 8 mm and not more than 25 mm. Allocate the dilutions to the injection sites randomly

431 ~~according to~~using a Latin square design. The dilutions correspond to 0.001, 0.0002, and 0.00004

432 mg of protein in a final dose of 0.2 ml, injected intradermally.

433 At 24 hours, the reactions’ diameters ~~of the reactions~~are measured, and the results are calculated

434 using standard statistical methods, taking the diameters to be directly proportional to the logarithms

435 of the concentrations of the tuberculin~~s~~. The estimated potency must be not less than 75% and not

436 more than 133% of the potency stated on the label. The test is not valid unless the fiducial limits of

437 error (*p*= 0.95) are not less than 50% and not more than 200% of the estimated potency. If the batch

438 fails a potency test, the test may be repeated one or more times, provided that the final estimate of

439 potency and~~of~~ fiducial limits is based on the combined results of all the tests.

440 It is recommended that avian tuberculin should contain the equivalent of at least 25,000 IU/ml or

441 approximately 0.5 mg protein per ml, giving a dose for practical use of 2500 IU/0.1 ml.

#### 442 3.	Requirements for authorisation/registration/licensing

##### 443 3.1.	Manufacturing process

444 The manufacturing process should follow the requirements of European Pharmacopoeia (~~2000~~2024) or other

445 international standards.

##### 446 3.2.	Safety requirements

###### 447 3.2.1.	Target and non-target animal safety

448 Antimicrobial preservatives or other substances that may be added to a tuberculin must have been shown not

449 to impair the safety and effectiveness of the product. The maximum permitted concentration~~s~~ for phenol is

450 0.5% (w/v), and for glycerol, it is 10% (v/v). The pH should be between 6.5 and 7.5.

###### 451 3.2.2.	Precautions (hazards)

452 Experience~~both~~ in humans and animals led to the observation that appropriately diluted tuberculin injected

453 intradermally results in a localised reaction at the injection site without generalised manifestations. Even in

454 very sensitive persons, severe, generalised reactions are extremely rare and limited.

##### 455 3.3.	Stability

456 During storage, liquid avian tuberculin should be protected from the light and held at a temperature of 5°C (±3°C).

457 Freeze-dried preparations may be stored at higher temperatures (~~but~~not exceeding 25°C) and protected from ~~the~~

458 light. During use, periods of exposure to higher temperatures or to direct sunlight should be kept at a minimum.

459 ~~Provided the tuberculins are~~Following accepted practice, tuberculin should be stored at a temperature of between

460 2°C and 8°C and protected from light; they may be used up to the end of the following periods ~~subsequent to~~after

461 the last satisfactory potency test: Liquid PPD tuberculin~~s~~: 2 years; lyophilised PPD-A tuberculin~~s~~: 8 years; HCSM

462 (heat-concentrated synthetic-medium) tuberculin~~s~~ diluted: 2 years. Recent research on the temperature stability of

463 human, bovine, and avian tuberculin solutions has shown that they are stable for a year at 37°C. This should be

464 further explored as these products are used in the field in remote areas of the world where maintaining temperature

465 control is very difficult (Maes *et al.,*2011).

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610	*

611	*  *

612	**NB:**There is currently (2024) no WOAH Reference Laboratory for avian tuberculosis

613	(please consult the WOAH Web site for the current list:

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

615	**NB:**FIRST ADOPTED IN 1989 AS TUBERCULOSIS IN BIRDS. MOST RECENT UPDATES ADOPTED IN 2014.