Guidelines for evaluating the efficacy and safety of live anticoccidial vaccines, and obtaining approval for their use in chickens and turkeys

Abstract

These guidelines are intended to aid those engaged in poultry research in the design, implementation and interpretation of laboratory, floor-pen and field studies for the assessment of the efficacy and safety of live anticoccidial vaccines for immunization of chickens and turkeys against Eimeria species. In addition to efficacy and safety requirements, manufacture, quality control and licensing considerations are discussed. The guidelines do not address subunit vaccines comprising non-viable material, but many of the principles described will be relevant to such vaccines if they are developed in the future. Guidelines are available in some countries for avian vaccines of bacterial or viral origin but specific standards for anticoccidial vaccines in poultry have not, as far as we know, been produced. Information is provided on general requirements of registration authorities (based upon regulations applicable in the European Union and the USA) for obtaining marketing authorizations for vaccines. These guidelines may assist poultry specialists in providing specific information for administrators involved in the decision-making process leading to registration of new vaccines, and are intended to facilitate the worldwide adoption of consistent, standard procedures.

Lignes directrices pour évaluer l'efficacité et l'innocuité des vaccins anticoccidiens vivants et obtenir l'approbation de leur utilisation chez le poulet et la dinde

Ces lignes directrices sont destinées à aider ceux qui sont engagés dans la recherche aviaire, dans le but de réaliser et interpréter les études en laboratoire, en parquets et sur le terrain pour l'évaluation de l'efficacité et de l'innocuité des vaccins anticoccidiens vivants pour l'immunisation des poulets et des dindes contre Eimeria species. En plus des exigences relatives à l'efficacité et à l'innocuité, des considérations concernant la fabrication, le contrôle de qualité, et l'autorisation sont discutées. Les lignes directrices ne s'adressent pas aux vaccins sous unitaires, comprenant des matériaux non viables, mais nombre de principes décrits seront pertinents pour de tels vaccins s'ils sont développés dans le futur. Les lignes directrices sont disponibles dans quelques pays pour les vaccins aviaires d'origine virale ou bactérienne, mais à notre connaissance, les standards spécifiques pour les vaccins anticoccidiens destinés aux volailles n'ont pas été élaborés. Il est fourni une information sur les exigences générales d'enregistrement auprès des autorités (basées sur les réglementations applicables au niveau de l'Union Européenne et des USA) pour l'obtention des autorisations de mise sur le marché des vaccins. Ces lignes directrices peuvent aider les spécialistes aviaires en fournissant des informations spécifiques pour les administrations impliquées dans le processus décisionnel conduisant à l'enregistrement de nouveaux vaccins, et sont destinés à faciliter au niveau mondial l'adoption de procédures standard.

Richtlinien zur Evaluierung der Wirksamkeit und Sicherheit von antikokzidiellen Lebendvakzinen und zur Erreichung der Zulassung ihrer Anwendung bei Hühnern und Puten

Diese Richtlinien wurden verfasst, um denjenigen zu helfen, die in der Geflügelforschung mit Entwurf, Durchführung und Interpretation von Labor-, Stall- und Feldstudien zur Bestimmung der Wirksamkeit und Sicherheit von antikokzidiellen Lebendvakzinen zur Immunisierung von Hühnern und Puten gegen Eimeria-Spezies beschäftigt sind. Zusätzlich zu den Wirksamkeit- und Sicherheitsanforderungen werden Gesichtspunkte der Herstellung, Qualitätskontrolle und Lizensierung diskutiert. Die Richtlinien sind nicht für Subunitvakzine mit nicht-vermehrungsfähigem Material gedacht, aber viele der beschriebenen Prinzipien werden auch für diese Vakzinen relevant sein, wenn sie in der Zukunft entwickelt werden. In einigen Ländern stehen Richtlinien für aviäre Impfstoffe bakterieller oder viraler Herkunft zur Verfügung, aber spezifische Standards für antikokzidielle Geflügelvakzine sind, soweit uns bekannt, bislang nicht erstellt worden. Informationen über allgemeine Anforderungen von Registierungsbehörden (basierend auf den in der Europäischen Union und den USA anzuwendenden Regularien) für die Erlangung von Vermarktungsrechten für Impfstoffe werden erwähnt. Diese Richtlinien können auch Geflügelspezialisten bei der Bereitstellung spezieller Informationen für Sachbearbeiter, die in den Entscheidungsprozess für die Registrierung neuer Vakzinen involviert sind, unterstützen und beabsichtigen außerdem, die weltweite Einführung von gleichbleibenden Standardprozeduren zu erleichtern.

Guía para la evaluación de la eficacia y la seguridad de vacunas vivas frente a coccidios y para obtener la aprobación para ser utilizadas en pollos y pavos

Esta guía pretende ayudar a todos aquéllos involucrados en la investigación en el área de avicultura en el diseño, implementación e interpretación de estudios tanto de laboratorio como de campo encaminados a la evaluación de la eficacia y seguridad de vacunas vivas frente a coccidios para inmunizar pollos y pavos frente a diferentes especies de Eimeria. Además de los requerimientos en cuanto a seguridad y eficacia, se discute también la fabricación, el control de calidad y algunas consideraciones del registro. La guía no concierne las vacunas de subunidades que contienen material no viable, pero varios de los principios descritos serán relevantes para estas vacunas si se desarrollan en un futuro. Existen ya, en algunos países, pautas para realizar vacunas aviares de origen bacteriano o vírico, pero los estándares específicos para vacunas frente a coccidios en aves, no se han establecido, hasta el momento. Se proporciona información sobre los requerimientos generales de las autoridades de registro (basada en las regulaciones aplicables en la Unión europea y en USA) para obtener autorización o licencia para comercializar las vacunas. Esta guía puede ayudar a los especialistas en aves de producción en proporcionar información a los administradores involucrados en el proceso de toma de decisiones que conlleva al registro de nuevas vacunas, y también pretende facilitar la adopción de procedimientos estándares y consistentes.

Introduction

Background

It has long been known that parasites of the apicomplexan genus Eimeria can induce a strong immune response in the fowl. Early experiments on immunization of chickens against coccidiosis (the disease caused by Eimeria) were undertaken in the USA in the late 1920s but it was not until the 1950s that the first commercial vaccines for chickens, comprising live oocysts of several Eimeria species, were introduced. Nevertheless, since the discovery of the therapeutic effects of sulphonamides, coccidiosis in poultry has been controlled by incorporating drugs in the feed. Until recently, anticoccidial vaccines for chickens have mainly been used for replacement pullets and broiler breeder stock, and vaccines for turkeys have been used for meat-production birds. Although use of drugs has largely been successful and continues to this day, in the European Union (EU) the desirability of including chemicals in animal feed has been questioned and several anticoccidial drugs have been banned, with more of them likely to be withdrawn in the future. Furthermore, it is well known that drug resistance of coccidia to all classes of drugs has become widespread. This makes the need for alternative methods of coccidiosis control, especially in broilers, more urgent.

Current and future vaccines

Currently, almost all commercially available anticoccidial vaccines contain live oocysts of attenuated or non-attenuated lines that are produced by propagation in the natural host. One vaccine, however, is dependent upon the injection of hens with non-viable antigens from Eimeria maxima gametocytes that stimulate production of maternal antibodies (Wallach et al., 1995), and has been introduced in some countries (Michael et al., 2003). An advantage of live vaccines is that they stimulate a range of immune responses that occur naturally when birds are infected with Eimeria species. Nevertheless, such vaccines are costly to produce and it is desirable to seek alternative means of immunization that are intrinsically safer and efficacious, and that do not depend upon in vivo propagation. Considerable effort has therefore been made to develop new vaccines harnessing recent advances in biotechnology. Much research has been carried out on the selective presentation to the host of specific molecules that can induce protective immunity (Lillehoj & Trout, 1993; Allen & Fetterer, 2002) but, despite early optimism, this approach has not yet resulted in a commercial vaccine. Non-viable subunit vaccines are not addressed in these guidelines. However, many of the basic principles described here for determining the efficacy and safety of live anticoccidial vaccines will apply to subunit vaccines comprising non-viable material when they become a practical proposition.

Commercial and technical significance of vaccines

Many poultry producers now recognize that vaccination with live parasites (exclusively oocysts to date) is economically viable and are investigating it as an alternative to chemotherapy for control of coccidiosis. Technical developments (attenuation of parasites to reduce their pathogenicity, and better methods of vaccine delivery) have made vaccination more acceptable than in the past and several new vaccines have been introduced recently in various countries, with others under development. Information on basic and applied aspects of our knowledge of live anticoccidial vaccines in chickens, including descriptions of currently available commercial vaccines, their biological and immunological characteristics and efficacy, has been provided in several reviews (Shirley & Bedrník, 1997; Chapman, 2000; Chapman et al., 2002; Williams, 2002a b 2003a). Government agencies and academic groups have developed standards for evaluation of anticoccidial drugs (for references, see Holdsworth et al., 2004) and recommendations for evaluating live vaccines in chickens were made by Bedrník et al. (1995) in the publication Guidelines on Techniques in Coccidiosis Research prepared by an EU-funded cooperative group of scientists and edited by Eckert et al. (1995). The present guidelines are intended to encourage the worldwide adoption of uniform standards for the design of experiments to evaluate the efficacy and safety of live (oocyst) anticoccidial vaccines in chickens and turkeys.

General considerations

The immune response and vaccination

The objective of vaccination is to induce an immune response sufficient to enable birds to resist challenge with virulent, heterologous infections. The nature of acquired intestinal immunity to Eimeria species has been extensively reviewed (for example, Rose, 1996; Lillehoj & Lillehoj, 2000). Protective immunity is manifest as a reduction in clinical signs of disease and is readily accomplished by oral inoculation of live sporulated oocysts. A single infection of an immunocompetent bird will induce some degree of immunity to reinfection in all species of Eimeria, although the extent will vary depending upon the species involved (Rose & Long, 1962). Thus, in the chicken, immunity to the highly immunogenic species E. maxima can develop following a single inoculum of 250 oocysts; but for less immunogenic species such as Eimeria tenella, repeated or larger inocula are necessary for protection against challenge (Rose, 1974). The degree of acquired immunity depends not only upon the number of oocysts received by a bird, but upon the timing of exposure to infection, since better immunity is conferred by repeated low doses than by a single dose comprising the same total number of oocysts (Joyner & Norton, 1973 1976). Currently, live oocyst vaccines are administered only once, and auto-reinfection, whether by vaccinal oocysts passed in the faeces or by non-vaccinal oocysts present in the litter, contributes to the achievement of overall protective immunity. This relationship between vaccinal oocysts and the probable presence of background populations has implications for the design of experiments to demonstrate acquisition of immunity.

Characteristics of vaccines

Live anticoccidial vaccines for poultry (including any adjuvants, preservatives or suspending agents) should, ideally, in each type of bird to be immunized:

1. induce protective immunity against economically important species of Eimeria;

2. be safe for the target host species, non-target animals and humans;

3. not represent an environmental hazard;

4. comprise parasites of normal or low virulence, the latter trait being demonstrably stable during propagations through the host;

5. comprise parasites that remain viable during storage under optimal conditions for a reasonable period of time;

6. protect against field strains from different geographical areas, including those where the vaccine is intended to be used;

7. be administered by a commercially practical method to ensure that as many birds as possible receive a similar dose;

8. have no adverse effects upon final performance or other production criteria;

9. be compatible with other poultry vaccines;

10. be free from viral, bacterial, mycoplasmal, fungal, and chemical contaminants;

11. be cost-effective when compared with other methods of coccidiosis control;

12. include drug-sensitive lines, so that they may interbreed with drug-resistant field populations, and thus reduce the overall local resistance.

Although not essential, a method for distinguishing vaccinal lines from field populations is highly desirable. This may be achievable with polymerase chain reaction technology (Schnitzler et al., 1998).

Age of birds when vaccinated

Early immunization (particularly of broilers) is essential to allow immunity to develop well before maximal natural challenge, which most frequently occurs when birds are aged 3 to 5 weeks. Young chicks are less able to respond immunologically to infections than are older birds, and appear to be poorly susceptible to coccidial infections, perhaps because of inefficiencies in oocyst excystation; however, induction of immunity following vaccination of newly hatched chicks is clearly demonstrable (see Williams, 2002b). It is also possible to immunize chicks by inoculation of 18-day-old embryonating eggs (Weber et al., 2004). For broilers, economic and management considerations dictate that vaccination is preferably carried out at the hatchery.

Species of Eimeria and infraspecific characteristics

Terminology for populations of Eimeria species

In this document, the terms “isolate”, “strain”, “line”, “clone” and “stabilate” are defined as follows (based upon Joyner et al. [1978], where guidelines for their designation are also presented):

1. An “isolate”, which may be monospecific or multispecific, comprises viable organisms obtained after introduction of a “sample” (part of a coccidial population collected on a unique occasion from a specific location) into an animal host, avian embryo or cultured cells. One or more “stocks” may be propagated from an isolate by serial passage in vivo or in vitro. Isolates and stocks are not genetically homogeneous and represent the genetic diversity within the Eimeria population at specific and infraspecific levels in the original sample.

2. A “strain” is a monospecific collection of oocysts, derived from an isolate or stock, maintained in the laboratory where it was established by serial passage, and having regularly reproducible characters. Strains are more genetically homogeneous than isolates but there may be some remaining genetic variability among life cycle stages such as the oocysts.

3. A “line” is a laboratory derivative of a strain, maintained under defined conditions different from the parent strain, or at a different location. Thus, parasites of any species selected from strains for use in a live vaccine are properly termed “lines”.

4. A “clone” is a monospecific collection of oocysts, which may be derived from an isolate, stock, strain or line, obtained by inoculation of a single sporozoite (see Shirley & Millard, 1976; Chapman & Rose, 1986), sporocyst (see Shirley & Harvey, 1996) or merozoite (see Haberkorn, 1970) into birds.

5. A “stabilate” is a population of coccidia viably preserved on a unique occasion. Thus, a vaccine master seed frozen in liquid nitrogen is a stabilate. A stabilate may, in fact, be prepared from an isolate, stock, strain, line or clone, but a master seed would in practice be derived only from a line or clone.

For experimental purposes, the passaging of these various populations of coccidia may be carried out in conventional birds reared free from infection. However, the establishment of commercial master seeds and the subsequent passaging of working seeds must be carried out in specific pathogen free (SPF) birds.

Chicken vaccines

Seven Eimeria species host-specific to chickens are recognized: Eimeria acervulina, Eimeria brunetti, E. maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox and E. tenella. The taxonomic validity of Eimeria mivati and Eimeria hagani, incorporated in two vaccines, is controversial (for example, Shirley et al., 1983; Barta et al., 1997; Shirley, 1997). Because immunity to Eimeria is strongly species specific, it is desirable to vaccinate birds against all species that a given type of commercial chicken may encounter. Although all seven species of Eimeria have been identified wherever they have carefully been sought (Williams et al., 1996; Williams, 1998), national regulatory bodies will almost certainly require evidence that all the Eimeria species included in a live vaccine are endemic in countries in which registration is applied for. Regarding types of chicken, birds reared to sexual maturity, such as broiler breeders and layer replacements, are more likely to encounter all of the coccidial species during their lives, so a vaccine intended for such birds should ideally contain all seven species (Shirley & Millard, 1986; Williams, 1998). Shorter-lived broilers probably do not usually require protection against less common species such as E. brunetti or E. necatrix that appear in older flocks, or against the more immunogenic but less pathogenic E. praecox.

Turkey vaccines

In turkeys, there are also seven host-specific Eimeria species (Eimeria adenoeides, Eimeria dispersa, Eimeria gallopavonis, Eimeria innocua, Eimeria meleagridis, Eimeria meleagrimitis and Eimeria subrotunda). However, much less is known about the global distribution of turkey Eimeria species than that of chicken Eimeria species, and there may be important differences in the species found on different continents (Catchpole & Maes, 1996). Anticoccidial vaccines for turkeys have so far been developed only in Canada and the USA, but hardly any information has been published regarding their efficacy. Current vaccines contain some or all of the species E. adenoeides, E. dispersa, E. gallopavonis and E. meleagrimitis (Williams, 2003a). In Europe, the predominant species in the field appear to be E. adenoeides, E. dispersa and E. meleagrimitis (Catchpole & Maes, 1996). Regulatory bodies will require assurance of the endemicity of any turkey coccidia to be included in a live vaccine.

Vaccine characteristics

Cross protection

Some vaccines comprise lines derived from strains that have been maintained in laboratories for many years, whereas others are derived from strains obtained recently from the field. Regardless of origin, the ability of a vaccine to protect birds against homologous (virulent) parental strains and heterologous strains of recent field origin from different geographical locations must be determined. Regulatory authorities usually require information on origin (date and place of isolation), history of subsequent propagation, and biological characteristics of vaccinal lines. Furthermore, information on whether the lines provide protection against antigenically variable field strains of the same species might also be expected. Certain Eimeria species may exhibit immunological variation infraspecifically (for example, Martin et al., 1997; Danforth, 1998). The degree of heterologous protection by a given line may be addressed by obtaining local samples from where the vaccine is intended for use and carrying out cross-protection studies with the candidate vaccine line(s). Such work has justified the inclusion of two lines of E. maxima in some commercial vaccines.

Drug sensitivity

It is important to assess the sensitivity to anticoccidial drugs of lines included in a vaccine. This is an important consideration for producers who integrate vaccination and chemotherapy programmes and wish to introduce drug-sensitive parasites onto farms (Chapman et al., 2002). Although drug-resistant lines have been used to immunize chickens (Danforth et al., 1997), and are generally considered to be widespread in poultry farms, it is undesirable to introduce them to farms where they were previously absent, and therefore to compromise the subsequent use of anticoccidial drugs. Live vaccines containing drug-resistant parasites have been developed for administration to birds given feed containing particular anticoccidial drugs (Schetters et al., 1999; Li et al., 2004 2005); such vaccines are believed to combine the advantages of medication and immunization. In situations where drug resistance is not a major concern, use of such a vaccine may be acceptable.

Vaccine administration

Vaccination methods

Live vaccines may be: sprayed onto chicks in the hatchery or on-farm immediately before placement; included in an edible gel provided at the hatchery or farm; sprayed onto the first feed that placed chicks receive; injected into amniotic fluid surrounding an embryo at a late stage in development; injected into the yolk sac of a hatchling chick; administered by the ocular route; or given in the drinking water, using bell-type or pipeline nipple drinkers. A sensitive oocyst counting technique may be used to monitor vaccine delivery at very low concentrations in drinking water (Williams et al., 2001). The method of vaccination should be precisely documented and, if carried out in a hatchery, carefully supervised. Care should be taken to ensure that vaccinated birds are clearly identified and separated from non-vaccinated control birds in the hatchery and during transit to the test facility.

Uniformity of uptake

Efficacy trials should employ the method of vaccination recommended for particular types of bird in the field. Ideally, vaccination should result in all birds receiving the intended dose but most methods do not achieve complete success (Chapman et al., 2002; Williams, 2002b). Although immunity may eventually develop in some birds that receive no vaccine or an incorrect dose, others might be exposed to large numbers of virulent wild type or vaccinal oocysts before immunity is acquired, and hence may suffer clinical coccidiosis. Uniformity of vaccine uptake can be assessed by transferring samples of vaccinated birds from floor-pens into individual cages and examining their faeces during the patent period of the primary vaccinal infection for small, medium, and large oocysts (for example, Chapman & Cherry, 1997); by this method the presence of at least three species is indicated.

Experimental procedures

General requirements

Requirements common to all evaluative procedures for potential anticoccidial products, including references to published guidelines, have been described (Holdsworth et al., 2004). They include: good laboratory practice, good clinical practice, animal husbandry, diet formulation, study conditions, documentation, data collection, statistical considerations, laboratory procedures, and criteria for evaluating eimerian infections. Laboratory methods for the isolation, identification, propagation, and freeze preservation of Eimeria species have also been provided (for example, Shirley, 1986 1995; Eckert et al., 1995; Chapman & Shirley, 2003). Studies must be conducted objectively and rigorously, according to sound scientific principles. Comprehensive protocols must be prepared describing: the objectives of each experiment; personnel involved and their qualifications; design, materials, and experimental methods; timing and sequence of events; data recording methods; and statistical methods. Approval must be obtained from appropriate authorities before commencement of investigations, and upon study completion a report must be prepared that accurately documents events and results, including unanticipated findings and deviations from the protocol.

In general, good laboratory practice compliance is not legally required for efficacy and safety studies, but it is to the advantage of a sponsor to adopt the principle, since data will then be more acceptable to regulatory authorities. In the EU, however, it is mandatory that laboratory safety studies be carried out in compliance with good laboratory practice (Directive 2001/82/EC).

Criteria for protection and efficacy

The criteria used to determine the effects of a challenge infection in immunized birds are similar to those used to evaluate drug efficacy (see Chapman, 1998; Holdsworth et al., 2004). They may include measurements of weight gain, feed conversion efficiency, oocyst production, the presence of lesions in the intestines, and various biochemical assessments. A “protective index” combining four separate criteria has recently been described (Allen et al., 2004).

Weight gain is a measure of the effects of coccidiosis upon growth; lesion scoring is a subjective assessment of the macroscopic pathology and physical damage caused by the parasite; and oocyst production is a measurement of the parasite's ability to multiply in the host. Advantages and disadvantages of these criteria for assessing vaccine efficacy, and their degree of correlation, have been reviewed (Williams & Catchpole, 2000). Some registration authorities may express a preference for inclusion or exclusion of specific criteria. Most of the common criteria have been used to evaluate vaccine efficacy, but the most useful is undoubtedly measurement of the weight gain of vaccinated birds during the acute phase of infection following separate virulent challenges with each species of Eimeria in the vaccine.

For assessment of weight gain and lesions, the size of the inoculum to be given must be established before the actual experiment. A dose is required that will depress weight gain and cause moderate to severe lesions without killing birds.

Lesion scoring

Whereas measurement of weight gain is straightforward, recording lesions using the method of Johnson & Reid (1970) requires considerable skill, particularly with less pathogenic, non-haemorrhagic species. The absence of lesions in vaccinated birds, following challenge with a dose of oocysts capable of inducing substantial pathology in non-vaccinates, can provide useful information on protective immunity. However, sometimes, lesion scores do not correlate well with weight gain and, in partially or completely immune birds, lesions may be present even though weight gain is not depressed (Long et al., 1980; Williams, 1997b). Furthermore, such lesions may often contain few or no parasites (Williams & Andrews, 2001; Williams, 2003b). In such cases, lesions may actually represent localized or generalized inflammation associated with an active immune response. In our opinion, therefore, lesion scoring is reliable only when a fully susceptible bird receives a primary infection. Although recording lesions has been widely used for evaluation of drug efficacy, and is required by some registration authorities for drug approval, it is proposed that lesion scoring should not be mandatory for vaccine evaluation. Reliance on lesion scoring alone for the determination of the ability of a vaccine to protect birds against a challenge infection is inadvisable; although the absence of lesions in a challenged bird may indicate protection, their presence does not necessarily indicate lack of protection (see Williams, 1997b). If lesion scores are recorded, they should not be evaluated in isolation, but should rather be correlated with other criteria, especially growth rate, used to measure morbidity.

Oocyst production

Measurement of oocyst production by birds in cages cannot always be used to evaluate a vaccine's ability to protect birds against a virulent challenge, since the numbers of oocysts produced do not necessarily correlate with the magnitude of the inoculum (Williams, 2001), particularly in challenge infections using large inocula in naïve birds. However, provided that a small inoculum is given (100 to 1000 oocysts per bird), the number of oocysts in the faeces may be useful for assessing immunity (see Rose, 1974; Smith et al., 1994a b; Wallach et al., 1995). Furthermore, small inocula probably reflect more closely the potential challenge that birds might experience under field conditions. Such data may supplement results obtained from experiments employing severe challenge doses.

Sampling litter at regular intervals during floor-pen or field trials reveals time-related patterns of oocyst accumulation during the life of birds, providing valuable information on vaccine uptake, development of immunity and epidemiology on a flock basis (for example, Williams et al., 2000).

Performance evaluation

In addition to measuring effects during the acute phase of infection following virulent challenge, it is desirable to measure growth and performance of vaccinated birds during their whole lifespan. As in the case of anticoccidial drugs, this is best achieved by recording production criteria such as mortality, final body weight, feed consumption and feed conversion in floor-pen and field trials.

Animals

Experimental studies can be undertaken with healthy, vigorous chicks or poults purchased from a local hatchery. They do not need to be SPF but must be of the commercial type for which marketing authorization is sought. They should not have received an anticoccidial vaccine (unless vaccination in the hatchery is the method under investigation) and care should be taken to ensure that control birds have not been vaccinated. Birds that have been subjected to approved hatchery practices (e.g. toe and beak trimming) are acceptable. Enough birds should be obtained to permit exclusion of any that are small, unthrifty or deformed. Any vaccinations against other diseases, administered before starting the study, should be recorded. Certain pure strains of bird, and broiler and layer hybrids, have different inherent susceptibilities to coccidioses (Long, 1968; Williams & Catchpole, 2000). Therefore, valid comparisons can only be made between trials in which birds of the same strain (and preferably from the same hatchery) have been used in each trial.

Safety studies

Safety must be demonstrated when the vaccine is administered to naïve birds by the recommended method and at the recommended age, but at a dose equivalent to at least 10 times the highest concentration of a commercial vaccine batch. Detailed requirements specified for the EU are given in the registration section. Acute tests may be carried out in battery cages (for example, Evans et al., 1989) and whole-life studies in floor-pens (for example, Williams, 1994) or field trials.

Other safety aspects should include the compatibility of an anticoccidial vaccine with other commonly used poultry vaccines. To establish that interactions between currently licensed vaccines for other organisms and a putative live anticoccidial vaccine do not occur, comparison can be made between birds from a single hatchery that have, or have not, received neonatal or in ovo vaccination for other agents. Also, compatibility with any colourants and suspension agents that might be used with the vaccine, either in laboratory tests (for example, Andrews et al., 2003) or in field trials, must be established.

Floor-pen challenge studies

Type of study

The protective effects of live vaccines are normally assessed in floor-pens. While this is feasible for broilers, it may present husbandry problems for breeder or layer birds. Vaccinated birds should be challenged separately with each species for which a claim is to be made (Williams & Catchpole, 2000; Crouch et al., 2003). Birds reared in cages with wire-mesh floors have limited exposure to faecal material (and hence little opportunity for auto-reinfection) and do not develop satisfactory immunity (for example, McDonald & Ballingall, 1983; Chapman & Cherry, 1997). Battery experiments, widely used for the evaluation of anticoccidial drugs, are therefore of little value for determining live vaccine efficacy. Floor-pens facilitate auto-reinfection and also provide replication for statistical analysis.

Appropriate controls

The design of floor-pen experiments must include appropriate controls, since both the presence of wild-type coccidial infections and spread of vaccinal oocysts can result in immunity developing in birds that have not been vaccinated. One way of keeping challenge controls coccidia-free is to rear them in floor-pens alongside vaccinated birds, but to include a drug in their feed that is able to suppress completely any infection (for example, Long & Millard, 1977; Shirley & Millard, 1986); drugs such as robenidine, clopidol, and nicarbazin have been used for this purpose. In such cases, medicated feed must be withdrawn from control birds at least 2 or 3 days before challenge to avoid the possibility of any residual drug activity. A disadvantage of this procedure is the limited choice of an appropriate drug, because of widespread development of drug resistance by coccidia. Furthermore, certain drugs are excreted largely unmetabolized and so effective concentrations may be ingested by coprophagy before and during the challenge phase of an experiment (for example, Williams et al., 1995).

Perhaps a better solution is to rear same-age controls in a separate coccidia-free facility and to transfer them to the floor-pen facility just before challenge (Chapman & Cherry, 1997). Alternatively, control birds can be reared in laboratory cages with solid floors on which litter is spread to simulate a floor-pen; wire-floored cages can be used for this purpose, by covering the mesh with a board or thick waxed paper covered with litter. Cages can be maintained in isolated rooms (Williams & Catchpole, 2000; Williams & Andrews, 2001) concomitantly with the vaccinated birds in floor-pens. It is essential during the rearing phase that control birds should be managed in a similar manner, maintained at the same stocking density and provided with the same feed, as the vaccinated birds, in order to ensure that the environmental conditions differ as little as possible.

Where appropriate, controls must be included to assess the effect of the inclusion of any feed additives such as antibiotic growth promoters.

Homologous and heterologous challenge infections

Initial experiments should demonstrate protection by challenging birds separately with pure strains of each species included in the vaccine for which a claim is to be made (Williams & Catchpole, 2000; Crouch et al., 2003). For vaccines based upon attenuated lines, the parental (homologous, virulent) strain is acceptable. It is also important to demonstrate protection against heterologous strains recently isolated from the field. Such strains should be obtained from at least three different geographical locations. However, field samples rarely comprise a single species; hence the presence of multiple species in a challenge inoculum can confound conclusions regarding protection, especially in the case of less pathogenic species. Ideally, pure strains of each species, each derived from a single sporocyst or oocyst, should be prepared from field samples. Methods for separating species from multispecific stocks have been described (Shirley, 1995; Williams, 1997a), but this requires specialist expertise. It is proposed that at least 90% of a challenge inoculum should comprise oocysts of the species intended and, if possible, any other species present should be identified.

Experimental design

The experiment should comprise two phases: vaccination of birds and their subsequent challenge. The vaccination phase includes two treatments: birds given the test vaccine in floor-pens and unvaccinated birds from the same hatch maintained coccidia-free in floor-pens or solid-floored cages. The birds of these two treatments should be reared in separate facilities, serviced by different personnel.

For the challenge phase, vaccinated birds and their coccidia-free controls should be randomly selected from their originating pens or cages and transferred to clean, solid-floored cages in a third facility. Before placement, each bird should be identified with a unique numbered tag and weighed. The design should include at least four replicate cages for each of the following treatments, which are repeated for each challenge species: (1) unvaccinated unchallenged control birds, (2) unvaccinated challenged control birds, and (3) vaccinated challenged birds.

The unvaccinated unchallenged control birds may be common to all species in the test. The challenge procedure will need to be repeated at several times after vaccination if it is desired to establish the time of onset of protective immunity.

The entire experiment should be repeated three times using different batches of vaccine. A statistician should be consulted prior to commencement of the study to ensure appropriate analysis of the data. This could involve a mixed-model analysis of variance using the vaccine batch, cage and animal as random-effect factors, and sex and treatment as fixed-effect factors.

Animal husbandry

For the vaccination phase, one-day-old chicks that have received no vaccinations should be obtained from a reputable hatchery and placed in disinfected floor-pens at a commercial stocking density on commercial poultry litter. If necessary, there should be enough chicks for challenges at several time points. If both sexes are included in the experimental design, they should be grouped separately. The vaccinated and control chicks should be cared for in the same way. Heating, lighting and drinking water should be provided according to industry standards. Feed should be formulated according to commercial practice, but excluding any drugs with anticoccidial activity. A non-ionophorous antibiotic growth promoter may be included in the feed of vaccinated and control birds if it is legally used under the commercial conditions pertaining in the country where a trial is carried out. Faecal samples from controls should be examined for oocysts twice weekly to verify freedom from coccidial infection. Any birds that die should be examined to determine cause of death.

Test vaccine material

The test material must be vaccine produced either from a production plant or made under similar conditions and should be identical to that for which registration is proposed. The method of vaccinating birds must be identical to that proposed in the field. Vaccine uptake can be confirmed by counting oocysts present in faecal samples from randomly selected birds 7 days after vaccination. Further samples should be taken after 14 days to confirm cycling of the parasites in the floor-pens.

Experimental material must be of the lowest potency expected of a commercial batch at the conclusion of its stated shelf-life, and at least three batches must be tested in successive trials under similar management conditions. Once efficacy has been satisfactorily demonstrated at minimum potency, trials must be conducted to determine the storage characteristics of the vaccine and the period during which vaccine of a given initial potency will retain a level of potency above the minimum that has been shown to be efficacious. This information will lead to setting the approved shelf-life of the product in compliance with regulatory authority requirements.

Challenge procedures

Birds should be challenged at appropriate intervals after vaccination, using virulent strains of the species represented in the vaccine. Each species should previously have been titrated in susceptible birds of the same breed and age to determine the number of sporulated oocysts necessary to produce a statistically significant reduction in weight gain without killing the birds. The challenge inoculum may be given by gavage in water to each of the vaccinated challenged birds and unvaccinated challenged control birds, and a sham inoculum of water to the unvaccinated unchallenged control birds. Seven days later, all the birds should be individually weighed and their mean gain in weight calculated. Feed consumption should be recorded and the feed conversion calculated for each cage. Other criteria for evaluating efficacy are discussed in experimental procedures.

Floor-pen performance trials

The effects of vaccination upon feed consumption, growth, and mortality are best determined in floor-pens in which birds are reared to market weight. Such trials are intended to simulate commercial conditions, while providing sufficient replication for statistical analysis (for example, Williams, 1994). General considerations for the design, conduct, and analysis of floor-pen trials are similar to those for floor-pen challenge studies and field trials.

Field trials

General considerations

Field trials under commercial conditions must be carried out according to good clinical practice standards with all types of commercial bird for which a vaccine is intended, and using each route of administration to be recommended. They are necessary to establish safety and any beneficial effects of vaccination upon bird performance, and should support results obtained from laboratory and floor-pen studies. Birds vaccinated with test material must be compared with equivalent unvaccinated controls or, if this is not practicable, with a registered anticoccidial drug or another anticoccidial vaccine. It is not possible to conclude that vaccination is solely responsible for immunity, since wild-type infections are very likely to be present. Therefore, in field trials it is not appropriate to challenge birds with virulent Eimeria species.

Design and site selection

Large-scale trials on farms are the most difficult type of experiment to perform because so many variables can confound the results. Success requires careful planning, site selection and supervision, with the elimination of as many sources of variability as possible. When selecting sites, it must be considered whether previously used methods of coccidiosis control, either with drugs or vaccines, might bias the results. The two treatments are assigned randomly to different houses on the same farm (for example, Govoni et al., 1987; Shirley et al., 1995; Williams et al., 1999). Care must be taken to prevent the spread of vaccinal oocysts from a test vaccine or a control vaccine to birds of the other treatment by restricting access, and servicing the control houses first, since it is almost never possible to consign each house to the care of different personnel. Alternatively, if farms have only one or two houses, the two treatments may be assigned to different farms, each with a similar history of disease and anticoccidial protection methods, and managed contemporaneously in an identical fashion (for example, Williams & Gobbi, 2002). Whatever treatment control birds may receive, a feed mill capable of providing high-quality feed with or without anticoccidial drugs should be identified; samples of all batches of feed must be taken and the presence or absence of anticoccidial drugs utilized by the mill (such as ionophores) confirmed for both treatments. If non-ionophorous antibiotic growth promoters are routinely included in the commercial diet, such a compound must be fed to birds in both treatments. It should be shown that the test vaccine is at least as effective as the control treatment. Field trials carried out at different times of year and at three, or preferably more, geographically separate locations (reflecting the geographic areas for which licensing is pursued) will be necessary, to allow for variable climatic and environmental conditions, different management practices, possible strain and species differences in local coccidial populations, and so on.

Registration

Knowledge of any legal requirements for the registration of new vaccines is essential for those involved in the planning and design of appropriate experiments and field trials. As no regulations specifically dealing with anticoccidial vaccines are so far available; such vaccines can only be regulated within the existing framework of general specifications intended for vaccines of quite different sorts. Where national authorities have published guidelines and documented procedures necessary to obtain product licences for vaccines in domestic livestock, these should be consulted by those desiring approval for such products. Although most countries have their own administrative procedures for product licensing, few have issued detailed technical specifications for the requirements needed to support licence applications. In such cases, countries may be willing to accept dossiers of data constructed to meet either the EU or the US format. As far as can be determined, specific requirements for anticoccidial vaccines have not been produced by any regulatory authority. A description of procedures for vaccines in general, based upon requirements specified by the EU and USA, together with suggested specific requirements for avian anticoccidial vaccines, is provided in the following.

European Union

Legislation

Registration of a medicinal product for animals or humans within two or more Member States (countries) of the EU may be achieved by one of two procedures. The Centralized procedure, operated through the European Agency for Evaluation of Medicinal Products (EMEA) in London, leads to a Community marketing authorization valid in each of the 25 Member States. The Decentralized procedure relies on an initial registration by one Member State, which is submitted to as many other Member States as the applicant requires, for mutual recognition. The procedure to use is determined by the nature of the product. All products based on genetic engineering must be registered through the Centralized procedure but the EMEA will also consider products containing a new active agent, or which use novel technology, for registration via this route. All conventional products and varieties of existing products are registered via the Decentralized procedure.

The requirements for registration are codified in a series of Directives, further supported by guidelines published as “The Rules Governing Medicinal Products in the European Union”. For veterinary medicinal products, the requirements are consolidated mainly in Directive 2001/82/EC and in volume 6 of “The Rules”. In addition, quality standards of general application and for specific products are published in the European Pharmacopoeia (EP). Manufacture of medicinal products for sale must comply with the standards of Good Manufacturing Practice (GMP) that, for veterinary products, are described in Directive 91/412/EEC. Any application for a marketing authorization must be accompanied by a certificate of GMP compliance issued by the competent authority of a Member State. Compliance is validated by periodic inspections of manufacturing facilities, conducted by the Member State within which the facility is located, and these inspections are mutually recognized by all other Member States. Such mutual recognitions also exist between the EU and some non-EU countries (e.g. Australia and New Zealand).

Within this framework, there are specific requirements regulating the registration of veterinary immunological products. The EP monograph “Vaccines for Veterinary Use” describes basic standards for quality applicable to all veterinary vaccines, which are modified as appropriate by monographs for individual vaccines. EU rules mirror pharmacopoeial requirements and are further defined by guidelines addressing vaccines of different types (live or inactivated, viral or bacterial) and vaccines for each major target species group. Hence, there are guidelines for avian live vaccines of bacterial or viral origin and there are pharmacopoeial monographs for the majority of avian vaccines. However, anticoccidial vaccines fall outside the scope of those guidelines and currently there are no relevant specific pharmacopoeial monographs. Therefore, although the principles embodied in the avian vaccine guidelines and monographs should be followed, there are no EU standards for regulatory compliance designed specifically for anticoccidial vaccines.

General requirements

Data provided for registration of an anticoccidial vaccine should address: safety for the target species and the environment; efficacy in compliance with the claims made for the product; quality in terms of purity, manufacturing consistency, potency, safety, and stability; and labelling. The data must provide validated evidence that supports claims made for the vaccine and product literature. All conclusions and any departures from any normally expected standards should be fully justified.

Presentation of documentation (the “dossier”) should follow the format prescribed in “The Rules Governing Medicinal Products in the European Union—Notice to Applicants, Veterinary Medicinal Products, Vol 6A”. Documentation should be presented in four parts (Parts I to IV). Part I requires details of the manufacturing facility and evidence of GMP compliance, draft product literature including a “Summary of Product Characteristics” that gives all essential information needed by a person using the product, and three “Expert Reports” (on manufacture and control, on safety, and on efficacy) written by independent experts as critical appraisals. Issues concerned with manufacture and quality should be dealt with in Part II of the dossier, and issues relating to safety in Part III. Evidence that the vaccine meets the claims made for it when used as recommended should be incorporated in Part IV of the dossier.

Specific requirements

Because there are no guidelines or monographs directly addressing anticoccidial vaccines for any target species, it will be necessary to generate data for avian anticoccidial vaccines that comply with the basic requirements for all veterinary immunological agents; and also to address the particular requirements for avian bacterial and viral vaccines, although only in so far as they are relevant to anticoccidial vaccines. All departures from the established requirements expected for bacterial and viral vaccines should be justified, and any specific procedures unique to anticoccidial vaccines validated.

Manufacture

The quantitative formulation of the vaccine should be specified, including all active components and excipients, together with packaging details, normally using materials compliant with EP specifications. A manufacturing flow chart with explanations of all procedures should be provided. Full details of the origin and quality of each component used during manufacture should be given and categorized either as EP compliant or as non-compliant but of biological or non-biological origin. Ingredients of biological origin, including master seeds, and the living substrate in which the organisms (oocysts) are propagated demand most attention.

Current vaccine guidelines are primarily divided into rules for live vaccines and rules for inactivated vaccines. This poses an immediate problem with live anticoccidial vaccines that, although formulated with viable oocysts, should have undergone rigorous physical and chemical treatment to free them from any contaminating microorganisms. Chemical treatment in particular means that, in certain respects concerning the risk of adventitious contamination, live anticoccidial vaccines are more akin to inactivated bacterial or viral vaccines. Therefore, some quality control aspects of anticoccidial vaccine manufacture should bear closer resemblance to those specified for inactivated vaccines than for live vaccines.

Quantification of bacteria and viruses relies on indirect measurement using a titration procedure and calculating back to derive a measure of “infectious units” that may or may not be equivalent to individual organisms. Eimeria oocysts are large enough to be easily observed microscopically and individually counted, so that a fairly accurate direct estimate of oocyst concentration can be obtained. However, enumeration of bacteria and viruses additionally provides a measure of viability, whereas counting of oocysts does not necessarily distinguish between live and dead individuals, even if sporulated. For a live vaccine, it is the quantification of infectious units rather than total units that is important. Quality control procedures for anticoccidial vaccines need to acknowledge these differences between organisms and should be designed accordingly.

Efficient oocyst production requires in vivo propagation in the bird. EU guidelines discourage the use of live animals or primary cell cultures as substrates for vaccine production, preferring established and validated cell lines or inert media from validated sources wherever possible. This minimizes the risk of contamination with adventitious pathogens, with obvious environmental and epizootic consequences, a risk repeated with every new production lot. However, the guidelines do allow the use of live animals if they are SPF. Fortunately, SPF chickens are available and can be used for the manufacture of chicken anticoccidial vaccines (requirements for flocks to be certified as SPF are described in the EP). Unfortunately, compliant SPF turkeys are not readily available. Exhaustive testing of substrate materials and of every manufactured batch of product for freedom from avian pathogens is the only way to provide assurance that vaccines propagated in uncertified stock are safe.

All vaccine manufacture should be based on a seed lot system, whereby defined seed lots that have been fully characterized are stored under stable conditions and used to initiate production batches (termed serials in the USA). Such seed should be a pure culture. For Eimeria species it is technically practicable to derive seeds from single sporozoites, sporocysts or oocysts, which eliminates the risk of cross-contamination with other Eimeria species and, coupled with conventional chemical treatment to render cultures sterile, ensures seed purity. All subsequent handling should be GMP compliant, and limitations are imposed on the number of consecutive in vivo passages permitted between master seeds and final product.

Quality control

At appropriate stages of manufacture, the vaccine must pass a pharmacopoeial sterility test, a test for freedom from Mycoplasma species, and appropriate tests for freedom from extraneous viral agents. Despite the faecal origin of oocysts propagated in birds, extensive washing and dilution procedures and chemical treatments applied to purified suspensions should ensure that compliance with those tests can be routinely and reliably achieved. If such procedures are satisfactorily validated, showing that they eliminate the risk of contamination with specific microorganisms, then the relevant manufacturing in-process testing may, with prior approval of the regulatory authority, be amended or even discontinued.

Every commercial batch of vaccine must be shown to be safe in the target species and meet the potency standard established in efficacy studies. The potency test applied to the vaccine must certify that the batch in question will be as efficacious, when used as recommended, as that demonstrated in the controlled challenge studies detailed in the registration dossier. For anticoccidial vaccines, measurement of specific infectivity or viability, entailing titration in birds, is not practicable routinely and is virtually impossible with a multivalent preparation. Microscopic observation cannot assess viability, and reliable differential counting of oocysts in a multivalent formulation is not possible. As immune responses to Eimeria do not necessarily correlate with protection, there remains little option when verifying potency but to challenge vaccinated birds with each of the Eimeria species in the vaccine.

In the absence of guidelines for potency testing of anticoccidial vaccines, it is open to a licence applicant to devise and validate a test for consideration by regulatory authorities and to demonstrate its correlation with efficacy. The criteria for acceptance of such a test are that it must measure the potency of each active component, must be reproducible and robust (i.e. give similar results in the hands of different operatives), and must show a satisfactory degree of precision. It must be able to detect a subpotent batch of a monovalent vaccine, or a batch of a multivalent vaccine containing a subpotent component. Provided that these requirements are satisfied, there are no restrictions on the design of the challenge test or the criteria for assessing potency.

Safety

In establishing safety in compliance with EU requirements, anticoccidial vaccines should follow the procedures laid down for live vaccines. Assessment of safety in the target species should be based on clinical reaction and weight gain compared with unvaccinated controls. Only if adverse clinical reactions occur will it be necessary to investigate vaccine effects in terms of gross pathology or histopathology.

Safety studies should be conducted at the highest potency at which the vaccine will be manufactured, the assumption being that if this is shown to be safe then any vaccine of lower potency will also be safe. Interpretation of this requirement is the subject of a draft EMEA guideline “EU Requirements for batches with maximum and minimum titre or batch potency for developmental safety and efficacy studies (2002)”. Safety tests should be conducted in the “most sensitive” category of the target species, a description usually interpreted as meaning birds of the youngest age at which the vaccine is to be used. The different inherent susceptibilities to coccidioses exhibited by pure strains of bird, and broiler and layer hybrids, should also be considered. Safety should be assessed during 21 days after administration of the recommended dose, after a 10-fold overdose, and after at least one repeated dosing. The possibility of any adverse effects on reproductive performance and of any possible interference with immunological functions caused by the vaccine should also be considered.

Safety studies must examine the potential for vaccinal lines to spread to non-vaccinated individuals of the target species. It is an EU requirement for live vaccines that, in appropriate target species, faeces, urine, milk, eggs, oral, nasal and other secretions should be tested for the presence of the organism. However, the transmission stage (oocyst) of the Eimeria parasite in chickens and turkeys is excreted in the faeces, and no evidence has been obtained that the parasite may be shed by any other route.

Where attenuated vaccinal lines are used, freedom from reversion to virulence should be demonstrated by conducting at least six consecutive in vivo passages without selection pressure in the target host. The possibility of, and potential consequences arising from, recombination or genomic re-assortment with field strains should be considered.

As in the case of pharmaceuticals administered to food-producing animals, the possibility must be considered of any chemical residues from the vaccine formulation accumulating in edible tissues for human consumption. Such problems can normally be avoided by restricting the choice of non-vaccinal components used in the formulation to substances recognized as being harmless; or by demonstrating that any substances employed during processing have been removed or diluted to insignificant levels by the time the final product is obtained. An assessment of possible harmful effects upon the environment must be considered. Finally, the laboratory-derived safety evidence should be supported by field trial results.

Efficacy

Efficacy studies should be conducted with the lowest potency at which the vaccine will be manufactured, the assumption being that if such material is shown to be effective then any material of higher potency must also be effective. The draft EMEA guideline may be consulted for interpretation of this requirement. Laboratory challenge trials must be carried out, incorporating unvaccinated controls, including each category of bird for which the vaccine is recommended, and must demonstrate the efficacy of each active component in the vaccine by each recommended route of administration. Studies should also justify any booster recommendations and should establish the period to onset of protective immunity after vaccination, and the duration of immunity.

Each individual Eimeria component of the vaccine must be shown to be efficacious in controlled challenge tests that may be conducted in floor-pens. The criteria for assessing efficacy should be appropriate to the Eimeria species concerned; clinical observations, weight gain, and oocyst shedding in birds given appropriate numbers of sporulated oocysts are more likely to provide an objective assessment of efficacy than lesion scores.

It should be recognized that the effectiveness of the vaccine, particularly in terms of duration of immunity, will be influenced by the conditions under which the vaccinated birds are reared. Hence, studies conducted in wire-floored cages, where the opportunity for recycling of vaccinal oocysts through birds is minimized, are unlikely to demonstrate immunity of similar duration to that obtained in floor-pens.

Under EU requirements, experimentally derived safety and efficacy data should be supported by field trial results. Trials in commercial poultry houses should confirm that the vaccine is safe when used on a large scale in accordance with the manufacturer's recommendations. Demonstration of efficacy in field trials, however, will frequently be incomplete because natural challenge is unpredictable and is unlikely to arise against all the Eimeria species represented in the vaccine, even if trials are conducted on multiple sites. It may also be difficult to include absolute controls in field trials, as commercial growers will not want to put any birds at risk. Comparison with birds fed a medicated diet or given another anticoccidial vaccine will, however, provide acceptable data. The repeatedly successful rearing of commercial flocks of birds in a variety of locations should provide sufficient evidence of the efficacy of the vaccine.

Field trials designed to evaluate the economic benefits of vaccination may be of considerable commercial value, but in the EU they are not required for registration. Data submitted in support of applications for product licences should be confined to proving that the vaccine is efficacious and safe.

United States of America

Legislation

In the USA, the Center for Veterinary Biologics is the branch of government responsible for regulating veterinary biological products and ensuring that they are pure, safe, potent, and effective. The agency develops appropriate standards and procedures; issues licences and permits; monitors and inspects products and facilities; and publishes a series of memoranda that cover basic and applied aspects of the production and use of vaccines. Producers of vaccines must have a Veterinary Biological Product License and an Establishment License (veterinary services memorandum number 800.50) for which separate documentation is required. Imported vaccines require a Product Permit for distribution and sale. General licensing considerations regarding study practices, documentation, field safety studies, and so on, are described in memoranda numbers 800.200 and 800.204, and apply to vaccines used to immunize birds against coccidiosis.

The Code of Federal Regulations (9CFR) specifies general requirements for live bacterial and viral vaccines; necessary tests to ascertain purity of master seeds (freedom from extraneous agents); and identity and safety of seed cell cultures. Presentation of the documentation should follow the format prescribed in 9CFR Part 102. Although the Center for Veterinary Biologics provides specific information for several avian viral vaccines (e.g. influenza, infectious bursal disease, anaemia virus, reticuloendotheliosis), none is available for coccidiosis. Thus, establishing appropriate experimental designs for evaluating the efficacy and safety of anticoccidial vaccines requires discussion between individual licence applicants and the agency.

Requirements for conventional live vaccines

Information required for approval of live conventional vaccines includes details of the origin and history of the vaccine lines, virulence of the master seed, and its purity, potency, and efficacy. The potency test is necessary to validate the dose, sensitivity, specificity, and reproducibility of the effects claimed, and to establish whether they correlate with host animal protection. In the USA, regulatory authorities prefer a lesion-score-based potency test or a test that correlates with lesion score reduction. Supporting data should include details of technical procedures used to prepare the product, including each step of preparation, concentration, and assembly of components. Dose determination studies, information on master seed immunogenicity, onset and duration of immunity, and any immunological interference with other products are also required.

Manufacture

Every commercial batch of vaccine must be subjected to tests for potency, purity, and safety. In addition, samples of the vaccine must be submitted to the Center for Veterinary Biologics prior to licensure for confirmatory testing. The guidelines for safety testing for products manufactured in the USA are similar to those described for the EU. Unvaccinated source flocks used to propagate vaccine lines should be free from signs of infectious disease and serologically tested to ensure the absence of the following viruses: avian adenovirus, avian encephalomyelitis, avian influenza, reovirus, infectious bronchitis, infectious bursal disease, laryngotracheitis, lymphoid leucosis, Marek's disease, Newcastle disease, and reticuloendotheliosis. They must also be free from Mycoplasma gallisepticum, Mycoplasma synoviae, Salmonella gallinarum and Salmonella pullorum. The methods and frequency of source flock testing must be described (veterinary services memorandum number 800.65).

Translations of the abstract in French, German and Spanish are available on the Avian Patholgy website.

Acknowledgments

The authors thank John Barta, Graham Knight and Janis McMillen for their very helpful and constructive comments.