Fowl Cholera PDF

N B : V e rsi o n a d opt e d b y th e Wo r ld A s se m bly o f D ele g a te s of t he O I E i n Ma y 20 0 8 1 CHAPTER 2.3.9. 2 FOWL CHOLERA 3...

N B : V e rsi o n a d opt e d b y th e Wo r ld A s se m bly o f D ele g a te s of t he O I E i n Ma y 20 0 8 1 CHAPTER 2.3.9. 2 FOWL CHOLERA 3 SUMMARY 4 Fowl cholera (avian pasteurellosis) is a commonly occurring avian disease that can affect all types 5 of birds and is distributed world-wide. Fowl cholera outbreaks often manifest as acute fatal 6 septicaemia, primarily in adult birds. Chronic and asymptomatic infections also occur. Diagnosis 7 depends on isolation and identification of the causative bacterium, Pasteurella multocida. 8 Presumptive diagnosis may be based on the occurrence of typical signs and lesions and/or on the 9 microscopic demonstration of myriad bacteria in blood smears, or impression smears of tissues 10 such as liver or spleen. Mild or chronic forms of the disease also occur where the disease is 11 endemic, with localised infection primarily of the respiratory and skeletal systems. 12 Identification of the agent: Pasteurella multocida is readily isolated, often in pure culture, from 13 visceral organs such as lung, liver and spleen, bone marrow, gonads or heart blood of birds that 14 succumb to the acute bacteraemic form of the disease, or from the caseous exudate characteristic 15 of chronic fowl cholera lesions. It is a facultative anaerobic bacterium that grows best at 37°C. 16 Primary isolation is usually accomplished using media such as dextrose starch agar, blood agar, 17 and trypticase–soy agar. Isolation may be improved by the addition of 5% heat-inactivated serum. 18 Colonies range from 1 to 3 mm in diameter after 18–24 hours of incubation and are discrete, 19 circular, convex, translucent, and butyraceous. The cells are coccobacillary or short rod-shaped, 20 0.2–0.4 × 0.6–2.5 µm in size, stain Gram negative, and generally occur singly or in pairs. Bipolar 21 staining is evident with Wright or Giemsa stains. 22 Identification of P. multocida is based on the results of biochemical tests, which include 23 carbohydrate fermentation, enzyme production, and selected metabolite production. 24 Serological characterisation of strains of P. multocida includes capsular serogrouping and somatic 25 serotyping. Polymerase chain reaction-based methods also allow capsular and somatic typing. 26 DNA fingerprinting can differentiate among P. multocida having the same capsular serogroup and 27 somatic serotype. These characterisations require a specialised laboratory with appropriate 28 diagnostic reagents. 29 Serological tests: Serological tests are rarely used for diagnosis of fowl cholera. The ease of 30 obtaining a definitive diagnosis through isolation and identification of the causative organism 31 generally precludes the need for serodiagnosis. 32 Requirements for vaccines and diagnostic biologicals: The P. multocida vaccines in general 33 use are bacterins, containing aluminium hydroxide or oil as adjuvant, prepared from multiple 34 serotypes. Two doses of the killed vaccine are typically required. Live culture vaccines tend to 35 impart greater protective immunity, but are used less frequently because of potential post-vaccinal 36 sequelae such as pneumonitis and arthritis. Multivalent vaccines typically incorporate somatic 37 serotypes 1, 3, and 4 as they are among the more commonly isolated avian serotypes. Safety and 38 potency testing of bacterins usually use the host animal. Final containers of live cultures are tested 39 for potency by bacterial counts. 40 A. INTRODUCTION 41 Fowl cholera is a contagious bacterial disease of domesticated and wild avian species caused by infection with 42 Pasteurella multocida. It typically occurs as a fulminating disease with massive bacteraemia and high morbidity 43 and mortality in older birds. Chronic infections also occur with clinical signs and lesions related to localised 44 infections. The pulmonary system and tissues associated with the musculoskeletal system are often the seats of 45 chronic infection. Common synonyms for fowl cholera are avian pasteurellosis and avian haemorrhagic OIE Terrestrial Manual 2015 1  Chapter 2.3.9. – Fowl cholera 46 septicaemia. Fowl cholera is not considered to have zoonotic potential as avian isolates are generally 47 nonpathogenic in mammals exposed by the oral or subcutaneous routes. Other bacterial diseases, including 48 salmonellosis, colibacillosis, and listeriosis in chickens, and pseudotuberculosis, erysipelas, and chlamydiosis in 49 turkeys, may present with clinical signs and lesions similar to fowl cholera. Differentiation is based on isolation 50 and identification, as P. multocida is readily cultured from cases of fowl cholera. 51 B. DIAGNOSTIC TECHNIQUES 52 Fowl cholera (avian pasteurellosis) is a commonly occurring avian disease that can affect all types of birds and is 53 often fatal (Derieux, 1978; Rimler & Glisson, 1997; Glisson et al. 2013 2008). In the peracute form, fowl cholera is 54 one of the most virulent and infectious diseases of poultry. Diagnosis depends on identification of the causative 55 bacterium, P. multocida, following isolation from birds with signs and lesions consistent with this disease. 56 Presumptive diagnosis may be based on the observance of typical signs and lesions and/or on the microscopic 57 demonstration of bacteria showing bipolar staining in smears of tissues, such as blood, liver, or spleen. Mild forms 58 of the disease may occur. 59 All avian species are susceptible to P. multocida, although turkeys may be the most severely affected. Birds older 60 than 16 weeks are primarily affected. Often the first sign of disease is dead birds. Other signs include: fever, 61 anorexia, depression, mucus discharge from the mouth, diarrhoea, ruffled feathers, drop in egg production 62 coupled with smaller eggs, increased respiratory rate, and cyanosis at the time of death. Lesions that are often 63 observed include: congested organs with serosal haemorrhages, enlarged liver and spleen, multiple small 64 necrotic areas in the liver and/or spleen, pneumonia, and mild ascites and pericardial oedema. Birds that survive 65 the acute septicaemic stage or those infected with organisms of low virulence may develop chronic fowl cholera, 66 characterised by localised infections. These infections often involve joints, foot pads, tendon sheaths, sternal 67 bursa, conjunctivae, wattles, pharynx, lungs, air sacs, middle ears, bone marrow, and meninges. Lesions resulting 68 from these infections are usually characterised by bacterial colonisation with necrosis, fibrino-suppurative exudate, 69 and degrees of fibroplasia. 70 Diagnosis depends on isolation and identification of the causative organism. 71 Table 1. Test methods available for the diagnosis of fowl cholera and their purpose Purpose Individual Method Population Immune status in animal Contribute to Confirmation Prevalence of freedom individual animals freedom from eradication of clinical infection – from or populations infection prior policies cases surveillance infection post-vaccination to movement Agent identification1 Culture – – – +++ – – Detection of immune response Serological ELISA – – – – – ++ 72 Key: +++ = recommended method; ++ = suitable method; + = may be used in some situations, but cost, reliability, or other 73 factors severely limits its application; – = not appropriate for this purpose. 74 Although not all of the tests listed as category +++ or ++ have undergone formal standardisation and validation, their routine 75 nature and the fact that they have been used widely without dubious results, makes them acceptable. 76 ELISA = enzyme-linked immunosorbent assay. 77 1. Identification of the agent 78 Pasteurella multocida is a facultative anaerobic bacterium that grows best at 35–37°C. Primary isolation is usually 79 accomplished using media such as blood agar, trypticase–soy agar or dextrose starch agar, and isolation may be 80 improved by supplementing these media with 5% heat-inactivated serum. Maintenance media usually do not 81 require supplemental serum. Colonies range from 1 to 3 mm in diameter after 18–24 hours of incubation. They 1 A combination of agent identification methods applied on the same clinical sample is recommended. 2 OIE Terrestrial Manual 2015  Chapter 2.3.9. – Fowl cholera 82 usually are discrete, circular, convex, translucent, and butyraceous. Capsulated organisms usually produce larger 83 colonies than those of noncapsulated organisms. Watery mucoid colonies, often observed with mammalian 84 respiratory tract isolates, are very rare with avian isolates. The cells are coccobacillary or short rod-shaped, 85 usually 0.2–0.4 × 0.6–2.5 µm in size, stain Gram negative, and generally occur singly or in pairs. Recently 86 isolated organisms or those found in tissue smears show bipolar staining with Wright or Giemsa stains or 87 methylene blue, and are usually encapsulated. 88 Isolation of the organism from visceral organs, such as liver, bone marrow, spleen, or heart blood of birds that 89 succumb to the acute form of the disease, and from exudative lesions of birds with the chronic form of the disease, 90 is generally easily accomplished. Isolation from those chronically affected birds that have no evidence of disease 91 other than emaciation and lethargy is often difficult. In this condition or when host decomposition has occurred, 92 bone marrow is the tissue of choice for isolation attempts. The surface of the tissue to be cultured is seared with a 93 hot spatula and a specimen is obtained by inserting a sterile cotton swab, wire or plastic loop through the heat- 94 sterilised surface. Alternatively the sterilised surface can be cut with sterile scissors/scalpel and the swab or loop 95 inserted into the cut without touching the outer surface. The specimen is inoculated directly on to agar medium or 96 into tryptose or another broth medium, incubated for 2–3 a few hours, transferred to agar medium, and incubated 97 again. 98 Identification is based primarily on the results of biochemical tests. Carbohydrate fermentation reactions are 99 essential. Those carbohydrates that are fermented include: glucose, mannose, galactose, fructose, and sucrose. 100 Those not fermented include: rhamnose, cellobiose, raffinose, inulin, erythritol, adonitol, m-inositol, and salicin. 101 Mannitol is usually fermented. Arabinose, maltose, lactose, and dextrin are usually not fermented. Variable 102 reactions occur with xylose, trehalose, glycerol, and sorbitol. Pasteurella multocida does not cause haemolysis, is 103 not motile and only rarely grows on MacConkey agar. It produces catalase, oxidase, and ornithine decarboxylase, 104 but does not produce urease, lysine decarboxylase, beta-galactosidase, or arginine dihydrolase. Phosphatase 105 production is variable. Nitrate is reduced; indole and hydrogen sulphide are produced, and methyl red and 106 Voges–Proskauer tests are negative. Detection of hydrogen sulphide production may require lead acetate-laden 107 paper strips suspended above a modified H2S liquid medium (Rimler, 1998; Glisson, et al., 2008). Commercial 108 biochemical test kits are available. Polymerase chain reaction (PCR) based methods may enable rapid 109 identification of P. multocida colonies. However, no absolutely specific DNA-based test for the identification of P. 110 multocida has been published (Miflin & Blackall, 2001). 111 Differentiation of P. multocida from other avian Pasteurella spp. and Riemerella (Pasteurella) anatipestifer can 112 usually be accomplished using the tests and results indicated in Table 2. Laboratory experience has shown that 113 P. multocida is most easily identified by its colony morphology and appearance in Gram stains. Positive reactions 114 to indole and ornithine decarboxylase are the most useful biochemical indications. 115 Table 2. Tests used to differentiate Pasteurella multocida from other avian 116 Pasteurella species and Riemerella anatipestifer Test* Pasteurella Riemerella multocida gallinarum anatipestifer Haemolysis on blood agar *  v Growth on MacConkey’s agar    Indole production    Gelatin liquefaction   u Catalase production    Urease production   v Glucose fermentation    Lactose fermentation u   Sucrose fermentation    Maltose fermentation u   Ornithine decarboxylase    117 *Test reaction results:  = no reaction;  = reaction; v = variable reactions; u = usually no reaction; u usually a reaction. 118 Antigenic characterisation of P. multocida is accomplished by capsular serogrouping and somatic serotyping. 119 Capsular serogroups are determined by a passive haemagglutination test (Carter, 1955; 1972). Capsular 120 serogroups, determined by a passive haemaglutination test, are A, B, D, E, and F. All but serogroup E have been 121 isolated from avian hosts. A nonserological disk diffusion test that uses specific mucopolysaccharidases to 122 differentiate serogroups A, D, and F has been developed (Rimler & Glisson, 1997; Rimler, 1994). A specific OIE Terrestrial Manual 2015 3  Chapter 2.3.9. – Fowl cholera 123 multiplex capsular PCR assay has been developed that allows for rapid and specific capsular typing (Townsend 124 et al., 2001). 125 Somatic serotypes are usually determined by an agar gel immunodiffusion (AGID) test (Heddleston, 1962; 126 Heddleston et al., 1972). Serotypes 1 through 16 have been reported; all 16 serotypes have been isolated from 127 avian hosts (Rimler, 1998; Glisson et al. 2013 2008). The most effective characterisation involves determination 128 of both serogroup and serotype. These determinations require a specialised laboratory with appropriate 129 diagnostic reagents. To determine the serotype, the laboratory prepares the unknown bacterial culture as antigen 130 for the AGID test and then must test it against all 16 serotype-specific antisera. Antigens present in a single 131 isolate may react with multiple serotype-specific antisera resulting in bi- or trinomial serotypes, as illustrated by 132 the 3, 4 and 3, 4, 12 strains (Rimler, 1998; Glisson et al. 2013 2008). A highly specific multiplex PCR assay allows 133 for differentiation among the 16 somatic serotypes. It has proven more accurate and less laborious than 134 conventional typing (Harper et al., 2015). 135 1.1. Somatic typing procedure using the gel diffusion precipitin 136 test 137 1.1.1. Test procedure 138 i) Inoculate a dextrose starch agar (DSA) plate (20 × 150 mm containing 70 ml of medium or 139 two 15 × 100 mm plates containing 20 ml of medium per plate) with cells from a pure 140 culture of P. multocida by using a sterile cotton swab. Swab the entire surface of the 141 plate(s). Incubate the plate(s) in a 37°C incubator for 18–24 hours. This procedure is used 142 to produce antigen for positive control purposes or to prepare antigen from diagnostic 143 cultures. 144 ii) Harvest the cells from the plate(s) using 2.5 ml of 0.85% saline with 0.6% formaldehyde 145 and a sterile hockey stick. Place the cells in a tube using a sterile pipette. 146 iii) Autoclave the cells at 100°C for 1 hour. 147 iv) Centrifuge the cell suspension mixture at 13,300 g for 20 minutes. 148 v) Remove the supernatant and place in a sterile tube. 149 vi) Prepare the agar gel for use in the gel diffusion precipitin test (GDPT) by placing 17.0 g of 150 NaCl, 1.8 g of Noble agar, and 200 ml of distilled water into a 500 ml flask. Microwave the 151 contents of the flask with the cap loose for 2.5 minutes. Swirl the contents of the flask and 152 microwave again for 2.5 minutes. Allow the agar to cool slightly for 10–15 minutes. Do not 153 prepare less than 200 ml of agar in a microwave. Dehydration during the microwave 154 process can increase the agar concentration and negatively impact or inhibit diffusion. 155 vii) Place 5 ml of melted agar onto the surface of a 75 × 25 mm plain glass microscope slide. 156 It is important that the slides are level prior to dispensing the agar. Allow the agar to cool 157 (approximately 30 minutes) completely. 158 viii) Cut wells in the agar bed. The wells are 3 mm in diameter and 3 mm apart from edge-to- 159 edge. Frequently an Ouchterlony template is used to create two or three replicates of wells 160 per slide. Each replicate has a centre well and is surrounded by four wells located at 90° 161 angles (from centre). 162 ix) Always place reference antiserum in the centre well (of a replicate). Place antigen from a 163 diagnostic or reference culture in one of the surrounding wells within a replicate. Fill each 164 well to capacity. 165 x) Incubate the slides within a moist chamber in a 37°C incubator for 48 hours. Precipitin 166 lines of a reaction can be best observed with subdued lighting from underneath the slide. 167 When present, reactions should occur between the centre and surrounding well(s) as an 168 arc of precipitin. Sometimes these reactions are close to the edge of a well. Examine the 169 slides carefully. Diagnostic cultures can react to more than one reference somatic 170 antiserum. 171 xi) Use positive controls. Test reference antiserum against reference antigen each time the 172 test is performed. 173 Somatic typing by a multiplex PCR assay is based on the LPS (lipopolysaccharide) genes expressed 174 by the different Heddleston type strains and offers a reliable and fast assay for somatic typing. 175 DNA fingerprinting of P. multocida by restriction endonuclease analysis (REA) has proved valuable in 176 epidemiological investigations of fowl cholera in poultry flocks. Isolates of P. multocida having both 177 capsular serogroup and somatic serotype in common may be distinguished by REA. Ethidium-bromide- 4 OIE Terrestrial Manual 2015  Chapter 2.3.9. – Fowl cholera 178 stained agarose gels are analysed following electrophoresis of DNA digested with either Hhal or Hpall 179 endonuclease (Wilson et al., 1992). 180 2. Serological tests 181 Serological tests for the presence of specific antibodies are not used for diagnosis of fowl cholera. The ease of 182 obtaining a definitive diagnosis by isolation and identification of the causative organism precludes the need for 183 serodiagnosis. Serological tests, such as agglutination, AGID, and passive haemagglutination, have been used 184 experimentally to demonstrate antibody against P. multocida in serum from avian hosts; none were highly 185 sensitive. Determinations of antibody titres using enzyme-linked immunosorbent assays have been used with 186 varying degrees of success in attempts to monitor seroconversion in vaccinated poultry, but not for diagnosis. 187 C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS 188 C1. Inactivated vaccine 189 1. Background 190 1.1. Rationale and intended use of the product 191 Fowl cholera may be caused by any of 16 Heddleston serotypes of P. multocida, although certain 192 serotypes appear to be more often associated with disease. The P. multocida vaccines in general use 193 are inactivated, containing aluminium hydroxide or oil adjuvant, prepared from cells of serotypes 194 selected on the basis of epidemiological information. Commercial vaccines are usually composed of 195 serotypes 1, 3, and 4. Vaccination plays a significant role in the control of this disease. Guidelines for 196 the production of veterinary vaccines are given in Chapter 1.1.6 Principles of veterinary vaccine 197 production. The guidelines given here and in chapter 1.1.6 are intended to be general in nature and 198 may be supplemented by national and regional requirements. 199 Bacterin Inactivated vaccine is normally administered by intramuscular injection in the leg or breast 200 muscles, or subcutaneously at the back of the neck. Two doses are typically administered at 2- to 4- 201 week intervals. As with most killed vaccines, full immunity cannot be expected until approximately 202 2 weeks after the second dose of a primary vaccination course. Live vaccines are typically 203 administered in the drinking water. Vaccination of diseased birds or those in poor nutritional status 204 should be avoided as a satisfactory immune response may not be generated in such circumstances. 205 2. Outline of production and minimum requirements for vaccines 206 2.1. Characteristics of the seed 207 2.1.1. Biological characteristics 208 All strains of P. multocida to be incorporated into a bacterin or vaccine must be well 209 characterised, of known serotype, pure, safe and immunogenic. The culture(s) that is evaluated 210 and characterised is designated by lot number and called a master seed. All cultures used in 211 the production of licensed bacterins or vaccines must be derived from an approved master 212 seed(s) and must be within an accepted number of passages from the master seed lot See 213 chapter 1.1.6 for guidelines on master seeds. 214 2.1.2. Quality criteria (sterility, purity, freedom from extraneous 215 agents) 216 Pasteurella multocida seeds must be pure culture and free from extraneous bacteria and fungi 217 (sse Chapter 1.1.7 Tests for sterility and freedom from contamination of biological materials. 218 2.1.3. Validation as a vaccine strain 219 Suitability as a vaccine strain is demonstrated in efficacy and safety trials. 220 2.1.4. Emergency procedure for provisional acceptance of new master 221 seed virus (MSV) in the case of an epizootic (with pathogens with 222 many serotypes, e.g. bluetongue virus, highly pathogenic avian 223 influenza, FMD, etc.) 224 Individual countries may have provisions to expedite the licensing or authorisation procedure in 225 the event of an animal health emergency where currently available vaccines do not protect. For OIE Terrestrial Manual 2015 5  Chapter 2.3.9. – Fowl cholera 226 example, the United States of America (USA) has the authority to issue a conditional license 227 based on a reasonable expectation of efficacy instead of requiring completion of definitive 228 efficacy trials prior to licensure. All requirements for master seed testing and product safety 229 must be completed prior to conditional licensure. 230 2.2. Method of manufacture 231 2.2.1. Procedure 232 Production cultures of each bacterial isolate to be included in the final product are prepared 233 separately. Pasteurella multocida cultures may be grown in a suitable broth media or initially 234 grown on agar media and scaled up to broth media. Cultures are subpassaged until the desired 235 volume is prepared. Cultures are harvested when they reach a suitable density, frequently 236 measured by spectrophotometry (optical density). 237 Cultures are then inactivated by formaldehyde or other suitable inactivant. The inactivated 238 harvest may be concentrated, typically by centrifugation or filtration, or diluted to reach the 239 proper concentration for blending into completed product. All the standardised component 240 cultures are mixed, and usually blended with an adjuvant, prior to filling sterile final containers. 241 2.2.2. Requirements for ingredients 242 See chapter 1.1.6. 243 2.2.3. In-process controls 244 The purity of the cultures is determined at each stage of production prior to inactivation. This 245 may be achieved by microscopic examination (e.g. phase–contrast microscopy, Gram strain) 246 and/or by culture. Killed cultures are tested for completeness of inactivation. Analytical assays 247 to determine the levels of formaldehyde or other preservatives are done on bulk vaccine and 248 must be within specified limits. During manufacturing, production parameters must be tightly 249 controlled to ensure that all serials (batches) are produced in the same manner as that used to 250 produce the serials used for immunogenicity efficacy studies. 251 2.2.4. Final product batch tests 252 i) Sterility/purity 253 Sterility tests are done on filled vaccine. Each lot must pass sterility requirements, for 254 example those detailed in the 9 CFR Part 113.26 or 113.27 (CFR USDA, 2001 2013). (See 255 also Chapter 1.1.7 Tests for sterility and freedom from contamination of biological 256 materials.) 257 ii) Identity 258 The identity of the antigens in inactivated products is typically ensured through the master 259 seed concept and good manufacturing controls. Separate identity testing on completed 260 product batches is not required in the USA, but procedures may differ in other countries. 261 iii) Safety 262 Safety testing is conducted on each bulk or filled vaccine lot Live vaccines are tested 263 according to the method described in Section C1.2.3.2.i, except that only one 264 representative animal species is required. Bacterins are administered according to label 265 recommendations, and the may be assessed in birds are observed vaccinated for 14 days; 266 at least 18 of 20 birds must show no unfavourable reactions attributable to the bacterin 267 batch potency tests. 268 Certain countries or regions, such as the European Union (EU), also may require testing 269 each batch for endotoxin content. 270 iv) Batch potency 271 Each production lot of bacterin or live vaccine must be tested for potency by a test that is 272 related to, and considered predictive of, efficacy. Potency tests are performed on the 273 product in its final form. 274 Bacterins are In the USA, inactivated vaccines are typically tested for batch potency in a 275 vaccination–challenge trial, such as described in 9 CFR Parts 113.116-118 (USDA, 2013). 6 OIE Terrestrial Manual 2015  Chapter 2.3.9. – Fowl cholera 276 Separate groups of birds (20 vaccinates, 10 controls) must be are challenged with each of 277 the serotypes of P. multocida for which protection is claimed. Bacterins Vaccines are 278 administered according to the dose and route recommended on the label Two doses are 279 administered 3 weeks apart, and all birds are challenged 2 weeks after the second dose. 280 The birds are observed for 14 days after challenge. For a satisfactory test according to 281 9 CFR, at least 14 of 20 vaccinates must survive and at least 8 of 10 controls must die. 282 The mean bacterial count of any vaccine lot at the time of preparation must be sufficiently 283 high to ensure that at any time prior to product expiration, the count is at least twice the 284 immunogenicity standard. (The European Pharmacopoeia requires a count that is at least 285 equal to the immunogenicity standard.) 286 In the EU, a serological test or other validated method may be used for batch potency after 287 a batch of minimum permissible potency is initially tested in a vaccination–challenge trial 288 (European Pharmacopoeia, 2008). 289 v) Formaldehyde content 290 Vaccines inactivated with formaldehyde are tested for residual formaldehyde. 291 2.3. Requirements for authorisation/registration/licensing 292 The following section is based on the requirements for inactivated P. multocida vaccines in the USA. 293 Other countries may have slightly different requirements. 294 2.3.1. Manufacturing process 295 The general method for production of manufacturers should demonstrate that the procedure 296 used to inactivate bacteria is sufficient for complete inactivation. A test should be developed to 297 confirm inactivation of each bacterial culture. 298 P. multocida bacterins is presented here. Production cultures of each bacterial isolate to be 299 included in the final product are prepared. The cultures are typically started in small vessels and 300 subpassaged into progressively larger volumes of media until the desired production volume is 301 achieved. Each production culture is inactivated by formalin or other acceptable means. All of 302 the component cultures are mixed, and usually blended, with an adjuvant prior to filling sterile 303 final containers. 304 The following section is based on the requirements for P. multocida bacterins and vaccines as 305 found in Title 9, United States Code of Federal Regulations (CFR). 306 2.3.2. Safety requirements 307 i) Target and non-target animal safety 308 Inactivated vaccines should pose no hazard to non-target species. Safety in target animals 309 may be evaluated according to harmonised requirements in VICH GL44 (VICH, 2009). The 310 EU and USA recommend vaccinating at least 20 non-immune, unexposed birds according 311 to label recommendations and evaluating daily for adverse reactions. The EU monitors for 312 21 days. In the USA, target animal safety is evaluated during the pre-challenge period of 313 the efficacy study, which is typically 5 weeks. 314 Safety also should be evaluated in a field setting prior to product licensure or registration. 315 This evaluation typically involves multiple geographical locations or husbandry conditions 316 and much larger numbers of birds. 317 ii) Reversion-to-virulence for attenuated/live vaccines and environmental considerations 318 Not applicable. 319 Each of 10 birds is given an equivalent of 10 vaccine doses and observed for 10 days. At 320 least 8 of 10 birds must show no unfavourable reactions attributable to the master seed. 321 Additionally, the master seeds must be tested for reversion to virulence and evaluated for 322 excretion from the host and transmission to other target species. 323 iii) Precautions (hazards) OIE Terrestrial Manual 2015 7  Chapter 2.3.9. – Fowl cholera 324 Vaccines prepared with aluminium-based adjuvants may cause temporary nodules at the 325 site of injection. Operator self-injection poses no immediate problems, but medical advice 326 should be sought as there is a risk of infection via a contaminated needle. 327 Vaccines prepared with oil-based adjuvants may cause more severe reactions at the site 328 of injection, which may manifest as large nodules. Care should be taken to administer 329 these vaccines correctly. Operator self-injection requires immediate medical attention, 330 involving prompt incision and irrigation of the site. 331 2.3.3. Efficacy requirements 332 Products prepared from candidate master seeds should be shown to be effective against 333 challenge infection. Efficacy should be demonstrated in each animal species (e.g., chickens, 334 turkeys) and by each route of administration for which the product will be recommended, and 335 protection must be demonstrated against each challenge serotype for which protection is 336 claimed. Birds used in efficacy studies should be immunologically naïve to fowl cholera and at 337 the minimum age recommended for product use. The lot of product used to demonstrate 338 efficacy should be produced from the highest allowable passage of master seed. 339 For live avian Pasteurella vaccines, Efficacy of bacterins must be demonstrated similarly prior to 340 licensure. However, no immunogenicity standards are derived from the lot that was used to 341 demonstrate initial efficacy; each production lot is satisfactorily tested in a vaccination-challenge 342 trial prior to release for sale and distribution. 343 In the USA and EU, 20 vaccinates and 10 controls are used in each efficacy trial. Birds are 344 challenged not less than 14 (USA) or 21 (EU) days after vaccination and are observed for 345 14 days after challenge. In the USA, mortality is measured, and a satisfactory test requires that 346 at least eight of the controls die and at least 16 of the vaccinates survive (USDA, 2013). In the 347 EU, birds are expected to remain free from severe signs of disease, and a satisfactory test 348 requires at least 70% of the control birds to be affected while at least 70% of the vaccinates 349 remain free from disease (European Pharmacopoeia, 2008). 350 2.3.4. Vaccines permitting a DIVA strategy (detection of infection 351 in vaccinated animals) 352 Not applicable to this disease. 353 2.3.5. Duration of immunity 354 Formal duration of immunity studies are not typically required, although it is important to check 355 the requirements of individual countries. Revaccination recommendations, beyond the primary 356 vaccination series, are more often determined empirically. 357 2.3.6. Stability 358 Vaccine stability should be confirmed by testing the product for potency at periodic intervals 359 through the dating period. In the USA, at least three lots of vaccine are tested and must pass 360 established potency requirements at the end of dating. Vaccines are typically stored at 2–7°C 361 and protected from freezing. Partly used containers should be discarded at the end of a day’s 362 operations. 363 C2. Live vaccine 364 1. Background 365 1.1. Rationale and intended use of the product 366 Live vaccines containing modified P. multocida are not generally used except in North America. Live 367 vaccines are typically administered in the drinking water or wing web. Vaccination of diseased birds or those 368 in poor nutritional status should be avoided as a satisfactory immune response may not be generated in 369 such circumstances. 370 2. Outline of production and minimum requirements for vaccines 371 Guidelines for the production of the veterinary vaccines are given in chapter 1.1.6. 372 2.1. Characteristics of the seed 8 OIE Terrestrial Manual 2015  Chapter 2.3.9. – Fowl cholera 373 2.1.1. Biological characteristics 374 All strains of P. multocida to be incorporated into a vaccine must be well characterised, of known 375 serotype, pure, safe and immunogenic. See chapter 1.1.6 for guidelines on master seeds. 376 2.1.2. Quality criteria (sterility, purity, freedom from extraneous 377 agents) 378 Pasteurella multocida seeds must be pure culture and free from extraneous bacteria and fungi. 379 2.1.3. Validation as a vaccine strain 380 Suitability as a vaccine strain is demonstrated in efficacy and safety trials. In addition, Seeds used in 381 live vaccines must be genetically and phenotypically stable upon repeated in-vivo passage. Ideally, 382 they should not persist in the vaccinated animal and any shedding of the vaccine organism from 383 vaccinated birds should be of limited magnitude and duration. 384 2.1.4. Emergency procedure for provisional acceptance of new master 385 seed virus (MSV) in the case of an epizootic (with pathogens with 386 many serotypes, e.g. bluetongue virus, highly pathogenic avian 387 influenza, FMD, etc.) 388 Many countries have mechanisms for provisional acceptance in the event of an epizootic in 389 which commercially available vaccines are not effective. As inactivated fowl cholera vaccines 390 are typically effective and pose less safety risk, however, it is more likely that an inactivated 391 vaccine would be considered for a fowl cholera epizootic. 392 2.2. Method of manufacture 393 2.2.1. Procedure 394 Production cultures of each bacterial isolate to be included in the final product are prepared separately. 395 Pasteurella multocida cultures may be grown in a suitable broth media or initially grown on agar media 396 and scaled up to broth media. Cultures are subpassaged until the desired volume is prepared. Cultures 397 are harvested when they reach a suitable density, frequently measured by spectrophotometry (optical 398 density). 399 Each component culture may be standardised, by concentration or dilution, to a desired concentration. 400 All of the standardised component cultures are mixed prior to filling sterile final containers. Live 401 vaccines are typically lyophilised, to be reconstituted with sterile diluent immediately prior to use. 402 2.2.2. Requirements for ingredients 403 See chapter 1.1.6 404 2.2.3. In-process controls 405 The purity of the cultures is determined at each stage of production. This may be achieved by 406 microscopic examination (e.g. phase–contrast microscopy, Gram strain) or by culture. During 407 manufacturing, production parameters must be tightly controlled to ensure that all serials 408 (batches) are produced in the same manner as that used to produce the serials used for 409 efficacy studies. 410 2.2.4. Final product batch tests 411 i) Sterility/purity 412 Sterility tests are done on filled vaccine. Each lot must pass sterility requirements, for 413 example those detailed in the 9 CFR Part 113.27 (CFR USDA, 2013). (See also chapter 414 1.1.7.) 415 ii) Purity 416 Each batch shall pass a test for purity carried out using sold media and ignoring the growth 417 of the vaccinal bacterium, for example as detailed in the 9 CFR Part 113.27 (CFR USDA, 418 2013). (See also chapter 1.1.7.). 419 iii) Identity OIE Terrestrial Manual 2015 9  Chapter 2.3.9. – Fowl cholera 420 Each batch of live vaccine in the USA is tested for identity. Requirements of other 421 countries may vary. This is most commonly accomplished by characterising the bacteria in 422 vitro. 423 iv) Safety 424 Live vaccines may be tested according to the method described in Section C1.2.3.2.i, 425 except that frequently only one representative animal species is required. 426 Certain countries (e.g. EU) also may require testing each batch for endotoxin content 427 (European Pharmacopoeia, 2008). 428 v) Batch potency 429 The potency of live vaccine lots is determined by a bacterial count performed on 430 reconstituted lyophilised product in its final container. In the USA, the mean bacterial count 431 of any vaccine lot at the time of preparation must be sufficiently high to ensure that at any 432 time prior to product expiration, the count is at least twice the immunogenicity standard. 433 The EU requires a count that is at least equal to the immunogenicity standard. 434 vi) Moisture content 435 Lyophilised vaccine is tested for moisture content. Harmonised requirements for testing 436 moisture by a gravimetric method are found in VICH GL26 (VICH, 2003). Typically 437 moisture is expected to be less than 5%. 438 2.3. Requirements for authorisation/registration/licensing 439 2.3.1. Manufacturing process 440 See chapter 1.1.6. 441 2.3.2. Safety requirements 442 i) Target and non-target animal safety 443 The safety of master seeds used in the production of live vaccines must be evaluated prior 444 to licensing. Safety must be tested in each animal species (chickens, turkeys, ducks, 445 psittacines) for which the product is recommended. Harmonised VICH GL44 (VICH, 2006) 446 is available for target animal safety. 447 Overdose studies are typically required for live vaccines. For example, each of 10 birds is 448 given an equivalent of 10 vaccine doses and observed for 10 days. If unfavourable 449 reactions are seen, this finding should be included in a risk assessment, and it may be 450 appropriate to designate maximum permissible serial potency requirements. 451 The master seed is also tested in representative non-target species (e.g. rodents or non- 452 target avian species) that may be expected to come into contact with vaccine bacteria 453 shed by vaccinated birds. Master Seed bacteria should be administered to the most 454 sensitive species at the most sensitive age, by the route (e.g. oral) expected to occur in 455 the field. 456 ii) Reversion-to-virulence for attenuated/live vaccines and environmental considerations 457 Master seed bacteria for live vaccines should be evaluated for their stability with repeated 458 passage in vivo. The seed should remain avirulent and genotypically stable after multiple 459 passages. Harmonised requirements for reversion to virulence studies are described in 460 VICH GL40 (VICH, 2006). 461 Seeds for live vaccines also should be tested for their potential to shed from vaccinated 462 animals and persist and spread in the environment. Ideally vaccine organisms should shed 463 no more than briefly and should not persist in the environment. Exceptions from the ideal 464 should be addressed in a risk assessment for the product. 465 iii) Precautions (hazards) 466 Inadvertent human exposure to the vaccine organism should be reported to a physician. 467 2.3.3. Efficacy requirements 10 OIE Terrestrial Manual 2015  Chapter 2.3.9. – Fowl cholera 468 Products prepared from candidate master seeds should be shown to be effective against 469 challenge infection. Efficacy should be demonstrated in each animal species (e.g. chickens, 470 turkeys) and by each route of administration for which the product will be recommended, and 471 protection must be demonstrated against each challenge serotype for which protection is 472 claimed. Birds used in efficacy studies should be immunologically naïve to fowl cholera and at 473 the minimum age recommended for product use. The lot of product used to demonstrate 474 efficacy should be produced from the highest allowable passage of master seed. 475 For live avian Pasteurella vaccines in the USA, 20 vaccinates and 10 controls are used in each 476 efficacy trial. Birds are challenged not less than 14 days after vaccination and are observed for 477 10 days after challenge. A satisfactory test requires that at least eight of the controls die and at 478 least 16 of the vaccinates survive. 479 The arithmetic mean count of colony-forming units in the lot of product that is used to 480 demonstrate efficacy is used as the minimum standard (immunogenicity standard) for all 481 subsequent production lots of vaccine. 482 2.3.4. Vaccines permitting a DIVA strategy (detection of infection 483 in vaccinated animals) 484 Not applicable 485 2.3.5. Duration of immunity 486 Formal duration of immunity studies are not typically required, although it is important to check 487 the requirements of individual countries. Revaccination recommendations, beyond the primary 488 vaccination series, are more often determined empirically. 489 2.3.6. Stability 490 Vaccine stability should be confirmed by testing the product for potency at periodic intervals 491 through the dating period. In the USA, batches of vaccine are tested until a statistically valid 492 stability record is established. Each lot must pass established potency requirements at the end 493 of dating. Live vaccines should be used promptly upon opening. 494 REFERENCES 495 CARTER G.R. (1955). Studies on Pasteurella multocida. I. A hemagglutination test for the identification of 496 serological types. Am. J. Vet. Res., 16, 481–484. 497 CARTER G.R. (1972). Improved hemagglutination test for identifying type A strains of Pasteurella multocida. Appl. 498 Microbiol, 24, 162–163. 499 CODE OF FEDERAL REGULATIONS (CFR) (OF THE UNITED STATES OF AMERICA) (2001). Title 9, Animals and Animal 500 Products. US Government Printing Office, Washington D.C., USA. 501 DERIEUX W.T. (1978). Response of young chickens and turkeys to virulent and avirulent Pasteurella multocida 502 administered by various routes. Avian Dis., 22, 131–39. 503 EUROPEAN PHARMACOPOEIA (2008). Vaccines for Veterinary Use. Fowl Cholera Vaccine Inactivated. 1945. 504 European Directorate for the Quality of Medicines and Health Care (EDQM), Council of Europe, Strasbourg, 505 France. 506 GLISSON J.R., HOFACRE C.L. & CHRISTENSEN J.P. (2008). Pasteurellosis and other respiratory bacterial infections. In: 507 Diseases of Poultry, Twelfth Edition, Saif Y.M., Fadly A.M., Glisson J.R., McDougald L.R., Nolan L.K. & Swayne 508 D.E., eds. Blackwell Publishing Professional, Ames, Iowa, USA, 739–758. 509 GLISSON J.R., HOFACRE C.L. & CHRISTENSEN J.P. (2013). In: Diseases of Poultry; Thirteenth Edition, Swayne D.E., 510 Editor in Chief, Glisson J.R., McDougald L.R., Nolan L.K., Suarez D.L. & Nair V., Associate Editors. Wiley- 511 Blackwell, Ames, Iowa, USA and Oxford, UK, pp 807–823. 512 GLISSON J.R., SANDHU T.S., & HOFACRE C.L. (2008). Pasteurellosis, Avibacteriosis, Gallibacteriosis, Riemerellosis, 513 and Pseudotuberculosis. In: A Laboratory Manual for the Isolation, Identification, and Characterization of Avian 514 Pathogens, Fifth Edition, Dufour-Zavala L., Swayne D.E., Glisson J.R., Pearson J.E., Reed W.M., Jackwood M.W. 515 & Woolcock P.R., eds. American Association of Avian Pathologists, Athens, Georgia, USA, 12–14. OIE Terrestrial Manual 2015 11  Chapter 2.3.9. – Fowl cholera 516 HARPER M., JOHN M., TURNI C., EDMUNDS M., ST MICHAEL F., ADLER B., BLACKALL P.J., COX A.D. & BOYCE J.D. (2015). 517 Development of a rapid multiplex PCR to genotype Pasteurella multocida strains using the lipopolysaccharide 518 outer core biosynthesis locus. J. Clin. Microbiol., 53 (3), 477–485. 519 HEDDLESTON K.L. (1962). Studies on pasteurellosis. V. Two immunogenic types of Pasteurella multocida 520 associated with fowl cholera. Avian Dis., 6, 315–321. 521 HEDDLESTON K.L., GALLAGHER J.E. & REBERS P.A. (1972). Fowl cholera: Gel diffusion precipitin test for serotyping 522 Pasteurella multocida from avian species. Avian Dis., 16, 925–936. 523 International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal 524 Products (VICH) (2003). Guideline 25: Testing of residual formadehyde. 525 http://www.vichsec.org/pdf/03_2003/Gl25_st7.pdf 526 International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal 527 Products (2003). Guideline 26: Testing of residual moisture. http://www.vichsec.org/pdf/03_2003/Gl26_st7.pdf 528 International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal 529 Products (2006). Guideline 40: Target Animal Safety – Examination of Live Veterinary Vaccines in Target Animals 530 for Absence of Reversion to Virulence. http://www.vichsec.org/pdf/0807/GL41-st7.pdf 531 International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal 532 Products (VICH) (2009). Guideline 44: Target Animal Safety – Examination of Live Veterinary Vaccines in Target 533 Animals for Absence of Reversion to Virulence. http://www.vichsec.org/pdf/0807/GL41-st7.pdf 534 MIFLIN J.K. & BLACKALL P.J. (2001). Development of a 23s rRNA-based PCR assay for the identification of 535 Pasteurella multocida. Lett. Appl. Microbiol., 33, (3), 216–221. 536 RIMLER R.B. (1994). Presumptive identification of Pasteurella multocida serogroups A, D, and F by capsule 537 depolymerisation with mucopolysaccharidases. Vet. Rec., 134, 191–192. 538 RIMLER R.B. & GLISSON J.R. (1997). Fowl cholera. In: Diseases of Poultry, Tenth Edition, Calnek B.W., Barnes H.J., 539 Beard C.W., McDougald L.R. & Saif Y.M., eds. Iowa State University Press, Ames, Iowa, USA. 143–159. 540 RIMLER R.B., SANDHU T.S. & GLISSON J.R. (1998). Pasteurellosis, Infectious Serositis, and Pseudotuberculosis. In: 541 A Laboratory Manual for the Isolation and Identification of Avian Pathogens, Fourth Edition, Swayne D.E., Glisson 542 J.R., Jackwood, M.W., Pearson, J.E. & Reed W.M., eds. American Association of Avian Pathologists, Kennett 543 Square, Pennsylvania, USA, 17–28. 544 TOWNSEND K.M, BOYCE J.D., CHUNG J.Y., FROST A.J. & ADLER B. (2001). Genetic organization of Pasteurella 545 multocida cap Loci and development of a multiplex capsular PCR typing system. J. Clin. Microbiol., 39 (3), 924– 546 929. Erratum in: J. Clin. Microbiol., (2001), 39 (6), 2378. 547 UNITED STATES DEPARTMENT OF AGRICULTURE (USDA) (2013). Code of Federal Regulations, Title 9, Animals and 548 Animal Products. Office of the Federal Register, National Archives and Records Administration. US Government 549 Printing Office, Washington D.C., USA. 550 WILSON M.A., RIMLER R.B. & HOFFMAN L.J. (1992). Comparison of DNA fingerprints and somatic serotypes of 551 serogroups B and E Pasteurella multocida isolates. J. Clin. Microbiol., 30, 1518–1524. 552 * 553 * * 12 OIE Terrestrial Manual 2015