Rev. sci. tech. Off. int. Epiz., 2007, 26 (2), 351-372 Marker vaccines and the impact of their use on diagnosis and prophylactic measures P. Vannier (1) , I. Capua (2) , M.F. Le Potier (1) , D.K.J. Mackay (3)* , B. Muylkens (4)** , S. Parida (5) , D.J. Paton (5) & E. Thiry (4) (1) Agence française de sécurité sanitaire des aliments (AFSSA), BP 53, 22440 Ploufragan, France (2) Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy (3) European Medicines Agency (EMEA), London, E14 4HB, United Kingdom (4) Virology, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium (5) Institute for Animal Health, Pirbright, GU24 0NF, United Kingdom After the first author, who coordinated the composition, the authors’ names are listed in alphabetical order. The different authors wrote on the following topics: I. Capua on avian influenza; S. Parida, D.J. Paton and D. Mackay on foot and mouth disease; M.F. Le Potier on classical swine fever; B. Muylkens and E. Thiry on infectious bovine rhinotracheitis and P. Vannier on Aujeszky’s disease * The views expressed in this article are the personal views of the author and should not be taken to represent the position of the EMEA, the Committee for Medicinal Products for Veterinary Use or the European Commission ** Research fellow at the Fonds de la Recherche Scientifique – FNRS in Belgium Summary Molecular biology and technical advances in DNA recombination have ushered in a new era in vaccinology. This article examines the recent development of specific marker vaccines and examines the impact of their use on the diagnosis and prevention of major infectious diseases. Gene-deleted vaccines, DIVA strategies (differentiating infected from vaccinated animals) and similar methods have been successfully applied in the control and eradication of Aujeszky’s disease, infectious bovine rhinotracheitis, classical swine fever, foot and mouth disease and, recently, avian influenza. The efficacy and performance of existing marker vaccines and their companion diagnostic tools (which should be assesed by an independent body) are discussed, as are the ways in which these tools are deployed by competent authorities. The limits and the advantages of the use of marker vaccines are carefully analysed in the light of practical experiences. Although these vaccines can limit the speed and the extent of virus dissemination and thus reduce the number of animals slaughtered, marker vaccines are no substitute for sanitary measures. Early detection and warning systems and the quick implementation of sanitary measures, including stamping out, remain key issues in the control of highly contagious diseases. Keywords Aujeszky’s disease – Avian influenza – Classical swine fever – Companion diagnostic tool – Control – DIVA (Differentiating infected from vaccinated animals) strategy – Eradication – Foot and mouth disease – Latent infection – Marker vaccine – Sanitary measure – Vaccination – Viral excretion – Virus carrier state.
IntroductionMolecular biology and technological advances indeoxyribonucleic acid (DNA) recombination have usheredin a new era in vaccinology. In particular, ‘deleted’vaccines, used in conjunction with an appropriatediagnostic kit, have emerged over the past ten years,enabling infection-specific antibodies to be recognisedregardless of an animal’s vaccination status. The first suchvaccines were used to protect pigs against Aujeszky’sdisease (AD). The same principles were subsequentlyapplied to the development of vaccines against infectiousbovine rhinotracheitis (IBR). For classical swine fever(CSF), subunit proteins were obtained from baculovirusrecombinant and the resulting vaccine obtained aEuropean marketing authorisation, re-launching thedebate on whether or not to use sanitary measures ormedical prophylactic treatments. The same principle is alsoat work when the detection of antibodies to non-structuralproteins (NSP) is used to identify animals infected withfoot and mouth disease virus (FMDV), whether or not theyhave also been vaccinated. Furthermore, more recently,recombinant vaccines have been used to protect birdsagainst avian influenza (AI).
An historic example: marker vaccines used againstAujeszky’s diseaseAujeszky’s disease virus (ADV) belongs to the subfamilyAlphaherpesviridae of the family Herpesviridae, which infectthe central nervous system and other organs (such as therespiratory tract) in virtually all mammals, except humansand the tailless apes. It is associated primarily with pigs,the natural host, which remain latently infected followingclinical recovery. After primary infection, most pigsdevelop clinical signs, depending on their age. In naïvepiglets, nervous signs are observed and the mortality canbe very high. In sows, reproductive disorders are inducedafter infection. In fattening pigs, general clinical signs, suchas fever and loss of appetite associated with respiratorydisorders of varying severity, are observed. Silent infectioncan also occur.
Currently available Aujeszky’s disease marker vaccines
Advances in molecular biology have contributed to betterknowledge of the genome of existing vaccine strains. Bystudying conventional vaccine strains, it was found thatcertain coding sequences of the single-sequence shortsection of the Bartha strain of the ADV had been deleted.
These sequences, situated in enzymatic restrictionfragment BamHI no. 7, code for two structuralglycoproteins: gE and gI. Accordingly, the Bartha strain,when isolated under natural conditions, does not expressgE, which makes it possible to distinguish vaccinated pigsfrom infected pigs, provided, of course, that thecorresponding enzyme-linked immunosorbent assay(ELISA) kits are used. ELISA kits make it possible to detectanti-gE antibodies in the serum of pigs, by usingmonoclonal antibodies that are very specific to certainantigenic determinants of gE, as described by van Oirschotet al. (129, 131).
Subsequently, knowledge about the molecular biology ofmutants of the ADV led to a better understanding of thefunctions of the viral glycoproteins. The first factor ofvirulence that was identified in the herpes virus was thethymidine-kinase enzyme, which allows the virus toreplicate itself in the central nervous system. Later, thevirulence of strains of the ADV not expressing theglycoprotein membrane gE was seen to have diminishedconsiderably compared with that of field viruses (9). ThisgE would therefore appear to play a major role in thespread of the virus within the nervous system, with theinfection spreading both through the olfactory tract andtrigeminal cavity (82). This knowledge has made itpossible to develop new vaccines by means of geneticrecombination, which modifies the genome of the vaccinestrains in order to excise, remove or delete certainsequences that code for glycoproteins and prevents theirexpression. These proteins do not induce antibodies invaccinated animals and so are used as serological markersfor infection by wild-type viruses. The functions of thesesame proteins are often partially responsible for thevirulence of field strains (such as gE); their non-expressionhelps to reduce or eliminate the pathogenicity of thesevaccine strains, which always express the majorglycoproteins (gB, gC, gD), thus inducing protectiveimmune responses in vaccinated or infected pigs.
Another generation of vaccines, not yet on the market, hasappeared, which uses live vaccine strains of the geneticallymodified ADV as an expression vector of the gene codingfor the immunogenic proteins of other viruses, such as CSF(135). These ‘hybrid’ viruses protect the vaccinated animalagainst both AD and CSF. Moreover, in-depth knowledgeof the molecular biology of the ADV has led to the creationof recombinants that cannot be shed by the vaccinatedanimal in an infectious form. Such recombinants can,however, spread from one cell to another in the inoculatedorganism, as do conventional live vaccine strains, but inrather limited sites (53).
Finally, one should not overlook the considerable progressthat has been made with immunological adjuvanttechnology, even though this is not directly linked withmolecular biology. We have seen the emergence of vaccines
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against AD that are produced by tank-mixing a liveattenuated strain with an adjuvant comprised of mineraloils. At the same time, the nature of the oils used inadjuvant composition has evolved, as has emulsiontechnology, making the vaccines increasinglyimmunogenic and considerably reducing local reactions atthe site of injection.
ELISA kits which are available commercially use indirect orcompetitive techniques for measuring antibody levels.When marker deleted vaccines are used, several types ofkits can be employed to detect specifically the infectedvaccinated animals; the ELISA tests that detect gE antibodies are the most commonly used in the field inEurope, whereas tests that detect gG (NSP) or gC antibodies are used more frequently in the UnitedStates of America (USA) (89, 144). A combination ofseveral kits is often used in the framework of controlprogrammes against AD, such as gE kits associated withkits that detect gB antibodies or antibodies against thewhole viral proteins. The latter two kits are used mainly inunvaccinated herds or regions where vaccination is notcarried out; but they can also be used to interpret the herdstatus in regard to infection (gE+; gB+) or vaccination (gB+;gE–). As well as testing sera, the ELISA can be adapted totest filter paper disks that have been moistened with a small quantity of blood obtained by puncturing asuperficial vein (144). This technique is convenient fortesting large numbers of pigs. The disks are air-dried beforeshipment to the laboratory. Moreover, muscular exudatescan also be used as alternative biological samples as thesekits have been evaluated taking serum samples as thereference; the individual sensitivity of the test was 93.2%and the individual specificity was 98.3% (64).
Requirements for the detection of gE, gB or global viralantigen have been defined by several competentauthorities. When comparing different serological tests thatdetect AD antibodies, the sensitivity of ELISA tests appearsvery good and the blocking ones often appear a little moresensitive than indirect ones, allowing earlier detection ofmore than 95% of infected pigs (88).
Marker vaccines used against bovine herpesvirus-1Bovine herpesvirus-1 (BoHV-1), classified as analphaherpesvirus, is a major pathogen of cattle. Primaryinfection is accompanied by various clinical manifestationssuch as rhinotracheitis, pustular vulvovaginitis, abortion,and systemic infection in neonates. Following clinicalrecovery, a life-long latent infection is established in thenervous sensory ganglia of infected animals.
BoHV-1 is a pathogen that is found throughout the worldand which displays significant differences in regionalincidence and prevalence depending on geographicallocation and breeding management (1). BoHV-1 isresponsible for significant financial losses incurred throughdisease and trading restrictions within the cattle industry,prompting the development of control programmes inNorth American herds. Based on serological surveys,several studies have aimed at identifying the risk factors forBoHV-1 seropositivity. Some of them are well characterised,e.g. age, sex (males are more frequently positive thanfemales) and herd size (11, 107). Direct animal contacts,such as purchase of cattle and participation in cattle showswere also found to be important risk factors for theintroduction of BoHV-1 (132, 133, 134). Other factorssuch as farm density or cattle density may increase the riskof BoHV-1 introduction (139). As reported for otherdiseases caused by herpesviruses in man and animals, thelatency-reactivation cycle has a deep epidemiologicalimpact since it is responsible for the maintenance of BoHV-1 in a cattle population. BoHV-1 infection of newgeneration cattle by latent carriers submitted toreactivation/re-excretion stimulus can occur at severaldifferent times, e.g. at birth (118), at mating, duringtransport (117) or following the introduction of heifersinto a group of dairy cows. Therefore, the detection ofBoHV-1 latent carriers is the main concern in the setting upof BoHV-1 control programmes. Moreover, sanitarymeasures must be taken to prevent the introduction ofseropositive animals or even animals originating from aseropositive herd in order to improve vaccine programmeefficacy.
Depending on the seroprevalence of BoHV-1, eradicationprogrammes are based either on the detection and theculling of seropositive animals, or on the repeatedvaccination of infected herds. The use of vaccines tocontrol BoHV-1 infections has evolved over the last fewdecades: previously they were used simply as an effectivemeans of reducing the clinical impact of the disease,whereas nowadays vaccination programmes areimplemented with the additional intent of preventingtransmission, although this is not as readily achievable as areduction in clinical signs. Indeed, BoHV-1 vaccines arenot efficacious at preventing BoHV-1 infection andestablishment of latency. Moreover, vaccine schemes mustbe accompanied by strict management measures to preventthe reintroduction of BoHV-1 into the cattle herd.Therefore, BoHV-1 control programmes may take a longtime to eliminate this well-adapted virus infection of cattle.
Currently available and future bovineherpesvirus-1 marker vaccines
In Europe, several countries have initiated controlprogrammes aimed at BoHV-1 elimination and some
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countries are already BoHV-1-free (Finland, Sweden,Denmark, Switzerland and Austria). In this context, theability to differentiate infected from vaccinated animals(known as the DIVA strategy) was critical for preventingtrading restrictions in Europe. To set up the DIVA strategy,marker vaccines and reliable companion tests were developed.
On the one hand, the marker vaccines must include BoHV-1 antigens able to induce a protective immuneresponse. On the other hand, they consist either of subunitvaccines or of BoHV-1 strains from which a gene encodinga non-essential glycoprotein has been deleted. The deletedglycoprotein must be expressed by all BoHV-1 field strainsand induce a detectable immune response. It should alsobe a virulence factor of BoHV-1 in order to ensure a furtherattenuation of the marker vaccine strain by the deletion ofits gene.
Non-essential BoHV-1 glycoproteins gC, gE, gI, gG and gMmay be deleted to construct BoHV-1 marker vaccines. Fourcandidate deletion mutants (BoHV-1 gC-, gM-, gI- and gG-null mutants) do not correspond to the above-mentionedrequirements. BoHV-1 gC-null mutants retain virulence inthe natural host (56) and BoHV-1 gM-null mutants havenever been tested in vivo (61). BoHV-1 gI-null mutants arenot sufficiently immunogenic (56). BoHV-1 gG-nullmutants are easily reactivated from latency (56) and gG-specific antibody tests are not available.
Evidence of consistent results led to the selection of theBoHV-1 gE-null mutant as a candidate BoHV-1 deletedmutant for use as a marker vaccine. First, it was shown tobe immunogenic (56) and to possess very little residualvirulence (127). Moreover, BoHV-1 gE was shown to beexpressed in a very large subset of BoHV-1 field strains(101) and a gE-specific antibody test has been developedas a companion test for the differentiation of vaccinatedfrom infected animals (66, 130). The development of aBoHV-1 marker vaccine took advantage of the knowledgegained from ADV control and marker vaccines based onADV gE-null mutants have successfully been developed forthe control of ADV (137).
Several studies have been conducted, and others are still inprogress, to produce new generation vaccines againstBoHV-1. Ideal marker vaccines should combine high levelsof safety and efficacy. Several subunit vaccines have beentested. They consist mainly of glycoproteins B, C or Dexpressed in different systems such as transfected cellcultures (126), recombinant baculoviruses (125),recombinant adenoviruses (42, 43, 100, 148), orrecombinant tobacco mosaïc viruses (96). The gD-basedsubunit vaccines are the most efficacious at reducingclinical disease and virus excretion when they areformulated with effective adjuvants. For example,chitosans (42) and CpG oligodeoxynucleotides (54, 86)
are new adjuvants that significantly enhance the protectiveimmune response, as evidenced by increased neutralisingantibody titres and reduced clinical disease and viralshedding following challenge. The latest vaccineapproaches consist of plasmid DNA vaccines containingsequences encoding for the three major immunodominantBoHV-1 glycoproteins gC (50), gB (71) or gD (87, 120,121). These constructs could potentially be administeredby needle-free delivery methods which would help toprevent losses due to the tissue damage that classicalvaccine delivery methods can cause (54, 124).
The biological properties
of glycoprotein E (gE)-deleted bovine
herpesvirus-1 marker vaccines
As for the majority of the BoHV-1 vaccines, markervaccines are very effective at preventing clinical signs afterchallenge with highly virulent strains (55). However, noneare able to fully prevent infection by the challenge strain,which establishes a latent infection, and might be re-excreted following a reactivation stimulus. New vaccineformulations and protocols have therefore been developedin order to improve the viral protection. Equivocal resultswere obtained when the two forms (inactivated and liveattenuated) of the same marker vaccine were tested. Whenit was administered twice to seronegative cattle, theattenuated marker vaccine induced a better viral protectionthan the inactivated marker vaccine after challenge (12).However, the inactivated vaccine was more efficacious atreducing virus excretion after reactivation of latentlyinfected calves than the live attenuated vaccine (13). Aninteresting approach was to combine the use of theattenuated vaccine as the priming dose and the inactivatedvaccine as a booster injection to complete the primarycourse of vaccination. This kind of protocol was shown tobe the most efficacious at reducing virus excretionfollowing challenge (44, 58). The immune status of aBoHV-1 latent carrier is the key factor controlling viral re-excretion following a reactivation stimulus. Therefore,latent carriers must be repeatedly vaccinated at regular 6-month intervals in order to maximally decrease the riskof re-excretion (34).
The efficacy of the DIVA vaccines was demonstrated in twofield trials. In the first trial, a significant decrease in thenumber of gE seroconversions was observed in herdswhere the gE-deleted vaccines were used (77). The secondstudy demonstrated that repeated vaccination using eitherinactivated or live attenuated gE-deleted BoHV-1 vaccinesis efficacious at reducing the incidence of gE seroconversion in dairy cattle and consequently theherd prevalence of gE-positive animals (34). This studyshowed the superior efficacy of a protocol whereby all ofthe herd is vaccinated together at regular 6-month intervals
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compared to protocols where all of the herd is vaccinated,but not all at the same time (34).
Three safety concerns about the live attenuated gE-deletedvaccine have to be addressed. First, gE-null BoHV-1 wasdemonstrated to establish a latent infection (67, 128) andto be re-excreted following experimental stimuli (67, 105)and in field conditions (35). However, there is so far noevidence that this deletion mutant can persist in the cattlepopulation (78).
A second concern rose from the production of seronegativeBoHV-1 latent carriers following the use of BoHV-1 markervaccines in passively immunised calves. Indeed, passivelyacquired colostral immunity interferes with an activeantibody response following infection. It has beenexperimentally demonstrated that gE-negative BoHV-1vaccine, when used in passively immunised calves, givesrise to seronegative vaccine virus carriers (67).
The third concern is the potential for BoHV-1recombinants to arise after co-infection of animals with areplicative gE-deleted BoHV-1 strain and a virulent BoHV-1 field strain (115, 116). One field observation and twoexperimental findings underlie this concern:
a) the isolation of a gE-deleted BoHV-1 vaccine strain incows vaccinated eight months before (35)
b) the frequent appearance of BoHV-1 recombinants in co-infected calves (105)
c) the experimental isolation of a virulent gE-deletedBoHV-1 recombinant (84, 85).
Properties of companion diagnostic tools
Even if the DIVA strategy has been demonstrated to beefficacious, it presents some weaknesses (7). Indeed, thestrength of the tool is fully dependent on the capacity ofthe diagnostic test to detect BoHV-1 gE-specific antibody.But the sensitivity of the only available gE-specific ELISA isaround 70% (62, 97). This rather low level of sensitivityhas a 30% false-negative rate in individual tests but itremains sufficiently high for use at infected-herd level. Theproblem of the low sensitivity of this test is compoundedby the weak level of the delayed immune response raisedagainst BoHV-1 gE, which means that it can be as many as42 days after infection before gE antibodies can be detected(7). The specificity of the gE-specific ELISA test is 92%(62). Although it is an acceptable level in the first steps ofa control programme, this lack of specificity will beresponsible for several misleading false positive results inherds where BoHV-1 has been eradicated. In these herds, aserial combination of serological tests should beperformed. The Danish test system (consisting of ablocking and an indirect ELISA), which was used in the
BoHV-1 eradication programme in the Netherlands, has avery high sensitivity (> 99.0%) and a very high specificity(> 99.9%) (29). Finally, a useful approach for dairy cattleherd monitoring is the regular serological testing of milktank samples (140). A positive result is obtained when15% of the dairy cows are seropositive towards BoHV-1.This level of seropositivity is rapidly obtained in cases ofBoHV-1 introduction into a previously negative herd.
Marker vaccines used against classical swine feverClassical swine fever, previously known as hog cholera, isstill a serious threat for the domestic pig population as it isa highly contagious viral disease of worldwide importance.Pigs and wild boars are the only natural reservoir of CSFvirus (CSFV). CSFV, bovine viral diarrhoea virus (BVDV),and border disease belong to the genus Pestivirus of theFlaviviridae family. These are small, enveloped, positivesingle-stranded ribonucleic acid (RNA) viruses. The pigletsdevelop more evident clinical signs than the adults. Theusual clinical sign is hyperthermia, which is usually higherthan 40°C in piglets (which pile together in the corner) butin adults it can be lower (39.5°C). The first usual signs of theacute form are anorexia, lethargy, conjunctivitis, respiratorysigns and constipation followed by diarrhoea. The chronicform of the disease is generally fatal. Often the infected pigspresent jaundice and cyanosis before death (65).
Currently available classical swine fever vaccines
Two live attenuated vaccines have been used successfullyfor many years. The live vaccines include the Chinesestrain also known as C strain vaccine, which wasattenuated by serial passages in rabbits and later adapted tocell cultures (136), and the Thiverval strain derived fromthe virulent Alfort strain through more than 170 serialpassages at 29°C to 30°C (4, 63). These traditional livevaccines induce a high level of protection against thedevelopment of clinical signs and neutralising antibodies attwo weeks post challenge. Dewulf et al. (32) demonstratedthat no virus transmission was detectable even when thepigs were challenged on the same day as the vaccination.The vaccinal protection lasts at least six to ten monthswhatever the immunisation route used: intramuscular ororonasal (57, 114). The main problem of using these livevaccines is that it is impossible to distinguish antibodiesthat are the result of vaccination from those that are due tonatural infection.
To clear up this difficulty, different teams have worked onthe development of marker vaccines. CSFV envelope
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glycoprotein E2 is the major protective immunogenicprotein responsible for eliciting neutralising antibodies andconferring protective immunity against the virus and it hasbeen demonstrated to be a highly conformationaldependent immunogenic protein (70, 149). Different typesof subunit or marker vaccines have been developed as non-replicative ADV expressing the E2 of CSFV (94), or livevectors such as porcine adenovirus. DNA vaccines havealso been developed (3) but as yet these do not induce anyreal protection in pigs. Different vaccine schemes orcombinations have been used such as: a prime-booststrategy (DNA-adenovirus) (52) or a co-administrationwith some interleukin (IL) recombinant protein such as IL-3, 12 or 18 or via a DNA vector (2, 141).
At the present time, only two E2 recombinant proteinsubunit vaccines produced in the baculovirus expressionsystem have been licensed for market use. The efficacy ofthese two commercially available E2 marker vaccines hasbeen extensively assessed in different vaccination-challenge and transmission trials. The results of theseexperiments were rather variable. A single vaccination witha vaccine dose of 32 µg of E2 in a water-oil-water adjuvantprevented clinical signs and mortality following a CSFVchallenge three weeks after vaccination (15). At least 14days were needed to obtain clinical protection in growingpigs vaccinated with a single dose (14, 123), but in the caseof earlier challenge, no protection against the disease andno reduction of virus shedding has been demonstrated(123). The ability of the two marker vaccines to preventtransplacental transmission of CSFV has also beenevaluated. The results showed that with a doublevaccination, virus spreading by transplacental infectionunder the conditions of emergency vaccination could notbe prevented in most of the vaccinated animals and couldlead to the carrier sow syndrome and, consequently, to thelate onset form of CSF (30). Based on the results of usingthe double vaccination protocol on pregnant giltschallenged 46 days after the second immunisation, it wasconcluded that double vaccination with an E2 subunitmarker vaccine protects pregnant gilts from the clinicalcourse of the disease but does not prevent horizontal norvertical spread of the CSFV (31). Despite the fact that theseresults indicated that the efficacy of these vaccines was notideal, their use in an emergency vaccination protocol hasnot been banned by the European Commission (EC).
Properties of companion diagnostic tools and their sensitivity and specificity limits
Discriminatory companion ELISA tests are based on thedetection of antibodies to the Erns protein. In 1999, sixteennational swine fever laboratories participated in testing thediscriminatory ELISAs. The two available kits were testedfor sensitivity, specificity, reproducibility and practicability.Reference sera (CSFV and BVDV antibody positive) and
field sera were used as well as sera from the weaner andsow experiments carried out during the marker vaccinetrial. Both discriminatory ELISAs were less sensitive thanconventional CSF antibody ELISAs, although there wasconsiderable variation between them. Neitherdiscriminatory ELISA consistently detected the marker-vaccinated, CSF-challenged weaner pigs correctly as ‘CSFpositive’, although CSF-challenged pregnant sows wereidentified correctly. The limitations of these discriminatoryELISAs would prevent the use of the two licensed markervaccines under emergency field conditions (40).
In 2003, the EC supported another large-scale inter-laboratory trial to assess the performance of a new versionof a companion Erns ELISA test. It was concluded that evenif the specificity and the sensitivity of the test was betterthan the previous kits tested in 1999 (40), there was still aneed for more reliable tests to be sure that a vaccinated pighas not been infected and is not a virus carrier.
Marker vaccines used againstfoot and mouth diseaseFoot and mouth disease is a highly contagious disease ofdomestic and wild cloven-hoofed animals including cattle,sheep, goats and pigs. It is caused by a virus (FMDV) of thegenus Aphthovirus, family Picornaviridae and exists asmultiple serotypes and subtypes; it causes severe economiclosses through decreased livestock productivity and traderestrictions. The virus is widely distributed and the diseaseis completely absent only in the European Union (EU) andin the Australasian and North American continents.
Current vaccines and their biological propertiesin the framework of eradication and control
In areas with endemic FMD, vaccination is commonly usedin conjunction with zoosanitary measures to minimiselosses and reduce virus circulation. In FMD-free countries,such as those of the EU and North America, the policy forcontrolling outbreaks has been primarily based upon‘stamping out’, i.e. slaughtering of infected and contactanimals together with restrictions on the movement ofanimals and animal products. Nevertheless, provision isretained to vaccinate under emergency circumstanceswhere an outbreak is or threatens to become extensive.During and after the 2001 FMD outbreaks in Europe therewas a growing desire to place more emphasis on a‘vaccinate-to-live’ policy to reduce reliance upon large-scale slaughter of herds at risk of becoming infected.According to such a policy, the stamping out of infectedpremises and the imposition of movement restrictionswould be accompanied by a limited period of emergency
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vaccination of surrounding herds, followed byserosurveillance to detect and eliminate infected animalsnot identified on the basis of clinical disease orepidemiological tracing. In support of this policy, newregulations have been approved by the World Organisationfor Animal Health (OIE) and the EU so that countries usingthis approach can now regain their FMD-free status sixmonths after the last infection has been reported ratherthan one year later as was previously the case (39, 145).
Current FMD vaccines are produced by infectingsusceptible cell cultures (most frequently baby-hamsterkidney cell lines) with virulent FMDV, followed bychemical inactivation with binary ethyleneimine andpurification by ultra filtration. At formulation, antigen ismixed with either an aluminium hydroxide and saponinadjuvant to make an aqueous vaccine for use in ruminantsor is emulsified in oil to make a vaccine for theimmunisation of pigs and ruminants. These vaccines havebeen used successfully for decades to control FMD andregular mass vaccination, mainly of cattle, has helped inthe eradication of the disease in some regions such asEurope and South America. Emergency vaccine banks havebeen established by national and international agencies,holding reserves of concentrated unformulated antigenfrozen over liquid nitrogen (36). These may be formulatedat higher doses than would be used for prophylacticvaccination so as to induce a rapid onset of immunity afteradministration as a single dose.
Nevertheless, there are a number of concerns andlimitations with the use of conventional vaccines inemergency control programmes. Their production requiresthe growth to high titre and subsequent completeinactivation of virulent strains of FMDV. Although this isconducted within high containment facilities there is stillthe potential for escape of live virus from these facilities orfor inadequate inactivation of virus and these concernshave led some FMD-free countries to prohibit vaccinemanufacture on their territory. Conventional FMD vaccinesare more difficult to standardise than vaccines produced bysynthetic or recombinant techniques and final producttesting for safety and efficacy still requires in vivo testing inanimals. As mentioned earlier, most vaccines are preparedfrom concentrated cell culture supernatants from FMDVinfected cells and therefore contain variable amounts ofviral NSP. Induction of antibody to NSP by vaccines‘contaminated’ with residual amounts of these proteinsmakes it difficult to identify infection in vaccinatedpopulations by the use of NSP antibody tests (see ahead).In addition, a consistent cold chain is required in the fieldfor the vaccine to remain efficacious. Althoughconventional vaccines can prevent clinical signs and spreadof the disease in vaccinated animals, they do not induce asterile immunity and therefore may not prevent virus-exposed animals from becoming acutely infected, and aproportion of such animals will become persistently
infected virus carriers (25, 90, 91) whose presencejeopardises recovery of FMD-free status. Moreover, fullprotection takes time to develop and is short-lived withoutrepeated booster doses (27, 37). Even when emergencyvaccine was administered with a ten times greater antigenpayload than the normal dose it could not fully protectvaccinated cattle from a severe challenge at ten days postvaccination (Cox and Barnett, unpublished results).
Possible future vaccines
Much work has been done on the development ofalternative vaccines, including subunit vaccines based onhighly immunogenic FMDV proteins or peptides and DNAvaccines (10, 26, 33, 41, 59, 60, 69, 99, 109), but theirimmunogenicity has been found to be much lower thanthat of conventional FMD vaccines.
Efforts to produce attenuated FMD vaccines by theadaptation and further passage of FMDV in non-susceptible hosts have been unsuccessful due to thereversion of the attenuated viruses to virulent forms (76).Targeted deletion of the Lpro gene, which is not essential tovirus replication, produced a vaccine that induced a goodFMD-specific neutralising antibody response, but couldnot protect fully (23, 79). Although a recombinant virus inwhich the RGD receptor binding site was deleted inducedprotection in natural hosts (74), with such a virus there ispotential for selection of virus variants that could entercells by utilising other receptors (5, 6).
Another approach has been to produce a vaccine whichexpresses the entire virus capsid, and therefore all of theimmunogenic sites present on intact virus, but lacks theinfectious nucleic acids (8, 48, 49, 68, 102). Using thisstrategy, the Plum Island Animal Disease Center in the USA(47, 80, 83, 147) inserted the complete capsid codingsequences of FMDV into a live replication-defective humanadenovirus vector, along with the FMDV 3C proteaseneeded for capsid assembly. One parenteral inoculationwith this vaccine induced antibody and clinical protectionwithin seven days. The same adenovirus vector expressingthe cytokine porcine interferon-alpha could protectanimals from FMD for three to five days within one day ofvaccination (22, 47).
Properties of companion diagnostic tools and their sensitivity and specificity limits
Conventional FMD vaccines are mainly comprised of viralstructural proteins (SP) plus RNA and contain only traceamounts of viral NSP synthesised during viral replication.Furthermore, the amount of residual NSP present invaccines can be reduced by additional purification stepsduring antigen preparation. Therefore, conventionalvaccines elicit a mainly anti-SP antibody response, whereas
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infection with replicating virus elicits antibodies to both SPand NSP. Consequently, serological methods which detectantibodies to FMDV SP such as the liquid phase blockingELISA (51), the solid phase competition ELISA (73) or thevirus neutralisation test (45) cannot distinguish betweeninfection and vaccination with conventional vaccines.However, ELISAs that measure antibodies to different NSP(3ABC, 3AB, 3A, 3B, 2A, 2B and 2C) can be used asmarker tests to detect infection in conventionallyvaccinated animals (17, 26, 28, 75, 98, 103, 106, 108).Incomplete purification of vaccine antigen as well asmultiple vaccination increases the likelihood of inducingNSP antibodies, but the latter will not arise after emergencyvaccination of previously naïve animals.
To date, the most promising NSP tests have been those thatdetect antibody to NSP 3ABC, and the OIE index methodis an indirect 3ABC screening ELISA (first developed inSouth America [NCPanaftosa]) supported by aconfirmatory immunoblotting test against NSP 3A, 3B, 2C,3D and 3ABC (143). A number of commercial 3ABCELISA test kits have recently become available and theirsensitivity and specificity were compared to one anotherand to the index screening method at a workshop inBrescia (Italy) in 2004 (16). The specificity of the testsranged between 97% and 98%, including when used totest cattle that had been given a single dose of Europeanvaccine. All of the tests were highly sensitive for detectionof infection in unvaccinated cattle, whereas the sensitivityof the tests to detect viral carriers in vaccinated andsubsequently infected cattle ranged from 68% to 94%depending on the test used. The workshop concluded thattwo tests performed comparably to the OIE index method(Ceditest® FMDV-NS, Cedi Diagnostics B.V. and 3ABCtrapping-ELISA, IZS-Brescia) of which the Ceditest® is theonly one available as a commercial kit.
Guidance on how to carry out post-outbreakserosurveillance for FMD in vaccinated populations isprovided by the OIE Terrestrial Animal Health Code(Terrestrial Code) (145) and by the European Directive onFMD control (39). The Terrestrial Code requires that herdscontaining seroreactors must be followed up to determinewhether these contain infected animals or not; findingevidence of infection at any stage automatically invalidatesfreedom from infection status. The European Directive onFMD control specifies that serosurveillance should becarried out at least one month after an outbreak hasfinished or one month after the last use of vaccine,whichever is the later. Further, it states that the entirevaccinated population should be sampled and tested orenough should be sampled and tested to give 95%confidence to detect a within-herd prevalence of infectionof 5%.
The problem of some vaccinated animals becomingcarriers without seroconverting to NSP can be overcome by
interpreting NSP test results on a herd basis (104),although this still leaves a lack of certainty over freedomfrom infection in small herds (46, 92), since a test with80% sensitivity at the individual level requires at least twoinfected animals in the herd to be sampled to have 95%confidence of detecting at least one of them (93). Imperfecttest specificity can be partly overcome by retesting positivesamples. For example, with the Ceditest® at the Bresciaworkshop, discounting the positive results that were notconfirmed on Cedi retest increased the specificity to 99.2%and decreased the sensitivity from 86.4% to 85.1% (93). Afurther increase in test specificity could be achieved by asecond retesting of Ceditest-confirmed positive samplesusing another non-covariant, commercially available NSPassay (SVANOVIR™ FMDV 3ABC-Ab ELISA, Svanova,Upsala, Sweden), resulting in an overall specificity andsensitivity of 99.98% and 71.2% respectively (93).
Foot and mouth disease is therefore a good example ofwhere advances in vaccine production technology toreduce contamination of vaccines with NSP antigens,together with advances in diagnostic techniques to detectantibody to these antigens, have resulted in thedevelopment of marker vaccines and companiondiagnostic tests that are sufficiently robust that they haveresulted in amendments to FMD control policy such that apolicy of ‘vaccinate to live’ is now supported in appropriatecircumstances. Improved FMD marker vaccines and testsmay be available in the future. For example, theexperimental adenovirus vectored vaccines describedabove express recombinant viral capsids that are devoid ofNSP 3D, which is a protein that elicits a strong antibodyresponse but cannot be eliminated from conventionalvaccines by purification (81). This would enable use of acompanion marker test for infection based on detection ofanti-3D antibody (47). Testing of saliva for FMDV-specificIgA is also a promising tool for detection of infection in animals given conventional vaccine by the parenteralroute (90).
Marker vaccines used
against avian influenzaAvian influenza viruses all belong to the InfluenzavirusA genus of the Orthomyxoviridae family. They areenveloped, negative-stranded RNA viruses with asegmented genome, consisting of 8 genes (PB1, PB2, PA,HA, NP, NA, MA and NS). AI viruses may be classified onthe basis of the severity of the clinical signs they cause insusceptible birds. Low pathogenicity AI (LPAI), may becaused by viruses belonging to all 15 haemagglutinin types(H1-H15) and produce a mild disease in susceptiblepoultry characterised by respiratory and enteric signs thatare often associated in breeders and laying hens withreproductive disorders. Some LPAI viruses are termed
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mildly pathogenic AI viruses. Highly pathogenic AI (HPAI)is, in contrast, a systemic viral disease of poultry withmortality that approaches 100% in many gallinaceousbirds. The clinical disease HPAI is caused only by virusesof the H5 and H7 subtypes that contain multiple basicamino acids at the deduced sequence of the cleavage site ofthe precursor of the haemagglutinin molecule. The mainclinical signs presented by HPAI-infected poultry areanorexia, depression, cessation of egg-laying, followed bycomplete reluctance to move and tremors of the head,paralysis of the wings and incoordination of the legmovements (19).
Marker vaccines and companion
diagnostic tests for notifiable avian influenza
Increased knowledge of AI occurrence and epidemiologyhas driven a revision process of the definition of AI forinternational trade regulations laid down by the OIE. Therevised Chapter on AI now reads ‘For the purposes of thisTerrestrial Code, avian influenza in its notifiable form (NAI)is defined as an infection of poultry caused by anyinfluenza A virus of the H5 or H7 subtypes or by any AIvirus with an intravenous pathogenicity index (IVPI)greater than 1.2 (or as an alternative at least 75% mortality)as described below. NAI viruses can be divided into highlypathogenic notifiable AI (HPNAI) and low pathogenicitynotifiable AI (LPNAI)’ (146). The aim of this section is toreview currently available marker vaccines and companiondiagnostic tests for NAI viruses of the H5 and H7 subtypes.
Research in AI vaccinology has only recently become afield of interest for pharmaceutical companies and forresearch institutions, and for this reason the selection ofproducts and performance of companion diagnostic testsare not adequate to cover fully all the complex fieldsituations this infection may cause.
Antigenic cross-reactivity between strains of the same Hsubtype is believed to occur even between strainsbelonging to different lineages. However, how cross-protection will be influenced by immunological pressuregenerated by the variety of seed strains is currentlyunknown (112). Similarly, the occurrence and extent ofantigenic drift in this situation is impossible to predict, butcould become a significant issue in the future.
Inactivated conventional vaccines
and companion diagnostic tests
or systems to reveal field exposure
These vaccines are based on a preparation containinginactivated virus grown in embryonated eggs. The seedstrains that are currently being used are field isolates
collected from natural outbreaks, selected without definedcriteria. For this reason they may contain seed viruses of either high or low pathogenicity, although the OIE guidelines indicate that LPNAI strains should be used (146).
In order to be efficacious, inactivated conventionalvaccines must contain a seed virus of the same H subtypeas the field strain against which vaccination is directed,while the subtype of the other surface antigen, theneuraminidase protein (N), is virtually irrelevant withregard to protection (111). Thus, vaccines may contain aseed virus of the same subtype as the field strain (H5N1vaccine to combat an H5N1 field challenge) or may be ofthe same H subtype but of a different N (H5N9 vaccine tocombat an H5N1 field challenge). The latter is known asheterologous vaccination.
Currently there are two methods of detecting fieldexposure with this type of vaccine that have been evaluatedin the field with satisfactory results.
The introduction of unvaccinated sentinels into the shedhas been used as a method of identifying field exposurewithin a vaccinated population, regardless of the strainused in the vaccine. This system requires the identificationof unvaccinated birds and regular clinical inspections inconjunction with serological testing to detect LPNAI,HPNAI being clearly visible as clinical pathology in thesentinels. The system is deemed to be valid, but requirespreparatory work and is more time consuming, especiallywhen the number of herds to be vaccinated is very high,and particularly when birds that are not confined to cagesneed to be identified within the flock. Furthermore, therisk that sentinel birds could be substituted with naïvebirds in order to escape restriction policies exists.
In the case of heterologous vaccination, it is possible to usethe diversity between the N antigen contained in thevaccine and the one in the field as a natural marker system.This DIVA system was developed in Italy in 2001, and hasbeen successfully used to combat multiple introductions ofNAI (18). The system is based on the detection ofantibodies to the N protein of the field virus, whichrepresent evidence of field infection. Currently, only anindirect immunofluorescence antibody test has been fullyvalidated (20, 21).
An encouraging system that can be used as a companiontest to vaccines containing homologous or heterologousseed strains, is based on the detection of anti-NS1antibodies (122). This system is based on the fact that theNS1 protein is synthesised only during active viralreplication and is therefore not present in significantamounts when inactivated vaccines, that do not replicatein the bird, are used. Birds that are vaccinated with such
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vaccines will develop antibodies to the NS1 only followingfield exposure (38). Full and field validation of this systemunder different circumstances is still in progress (H. Chen,personal communication) and should be made availablebefore this system is recommended. No diagnostic kit hasbeen validated to date.
Engineered vaccines and companion tests
Engineered vaccines include all vaccines that are notnatural isolates or natural reassortants. These include:recombinant live vectored vaccines and vaccines based onreverse genetics and recombinant proteins.
Recombinant live vectored vaccines are based on insertingan H5 or H7 haemagglutinin gene in a suitable, replicatingvector which will, during its replication, induce theproduction of antibodies to the haemagglutinin of theinfluenza gene. Fowlpox-based recombinant viruses haveobtained marketing authorisations and are being usedcurrently in several countries. Newcastle disease virus(NDV)-based recombinants have been developed byseveral groups (H. Chen, personal communication; 113, 138), with a product developed by Chineseresearchers being used currently in the field in the People’sRepublic of China. An infectious laryngotracheitis-basedproduct has also been described (72).
All these preparations, with the exception of the onedeveloped by Swayne (113), offer protection from clinicalsigns and reduce shedding levels. However, for most ofthese products, their efficacy in the presence of pre-existing antibodies due to natural infection with a fieldstrain of the vector virus, e.g. NDV or fowlpox virusremains to be established (111, 113).
The greatest advantage to the use of these products is thatcompanion diagnostic tests directed to detect antibodies toany viral proteins other than the haemagglutinin may beused to identify field-exposed flocks. Thus, the agar gelimmunodiffusion or ELISA tests directed to the detectionof antibodies to the NP or M proteins, can be successfullyused, and enable the detection of field challenge caused byany influenza A virus. Tests directed to the detection ofantibodies to the N protein, identify field exposure only toviruses of known N subtype.
Inactivated vaccines generated by reverse genetics haverecently been developed in the USA and in the People’sRepublic of China (119). The seed strain contained in thevaccine is basically a synthetic virus, completelyengineered in the laboratory. These viruses contain abackbone derived from a virus that has high replicationcapacities (A/Puerto Rico/8/XX) with the two genesencoding for surface antigens (H and N) derived fromcontemporary viruses. This combination of genes allows
on the one hand an excellent replicative efficiency – whichensures high titred, consistent virus yields duringproduction – and on the other, suitable surface antigens.Since these vaccines have the same properties asconventional inactivated vaccines, the same companiontests apply.
Recombinant protein-based vaccines are synthesised in thelaboratory by expressing the haemagglutinin in a suitablesystem, for example baculoviruses, plants and yeasts (110).Several prototypes have been generated and have beentested in the laboratory with encouraging results, however,probably due to the cost of production, they have notattracted any commercial interest to date. The use of any of these ‘engineered’ vaccines in the field would haveseveral advantages, including the possibility of using avariety of companion diagnostic tests, as is the case withrecombinant vectored vaccines – and of course, of updating the haemagglutinin component should this be required.
In summary, to date, only conventional inactivated(containing natural or synthetic strains) and recombinantlive vectored vaccines are available for use and can becoupled with a suitable companion diagnostic test. Bothcategories have some advantages and limitations in theirapplication in the field, and certainly, considering thecomplexities of these vaccines and the need to extend theiruse, more research is needed to optimise products andcompanion tests in order to tackle the current limitationsto their use in the field.
Limitations and advantages of the use of marker vaccinesIn spite of the major progress that has been made as aresult of the development of marker vaccines, it would bea mistake to consider that their use could simply replacesanitary prophylactic measures. Indeed, past experience isvery useful for assessing the limits and the advantages ofthe use of these marker vaccines, which could be apowerful tool in a set of measures to control and eradicatea contagious disease. However, the use of such vaccineshas to be adapted to the epidemiological situation, thecontagiousness of the disease concerned and to thepresence or absence of conditions with the capacity toinfluence the spread of infection. To control a disease, thekey point is to detect clinically inapparent infected animals(healthy carriers) which can infect in-contact susceptibleanimals. When vaccination is used, the critical stage ofalert induced by the appearance of clinical signs isremoved or suppressed. For this reason, such vaccineshave to be as efficient as possible, not only to protectvaccinated animals against clinical signs, but also to
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prevent, as far as possible, the excretion of the virus byvaccinated and subsequently infected animals. Moreover,the sensitivity of the diagnostic kits should be as high aspossible to reduce to the greatest possible extent, theprobability of false negative results; indeed, in such astrategy, the epidemiological consequences of false positiveresults are less significant than false negative results, aspositive results are generally confirmed (in a second,complimentary phase) by a reference laboratory usinganother diagnosis tool.
The longest experience of using marker vaccines has beenaccumulated in relation to the control and eradication ofAD. In this case, the use of deleted marker vaccines hasrepresented a considerable advance in programmes tocontrol the disease in several countries.
First, these vaccines have made mass vaccination possible,whilst retaining the means for serological detection ofinfection. This has enabled vaccinated herds whichsubsequently become infected to be pinpointed so that thenecessary measures can be applied to prevent the fieldvirus from spreading further.
Second, it has become possible to implement sanitarymeasures in a gradual manner in vaccinated, infectedherds, by culling the infected sows at varying speeds, asrequired. These infected sows are detected throughserological screening using the ELISA technique, whichenables vaccinated pigs to be distinguished from those thathave been vaccinated and then subsequently infected.
This means that vaccination has a combined effect whichallows a programme of prophylactic treatment to be carriedout in total safety. Mass vaccination, conducted severalyears in succession, limits the quantity of virus shed intothe air by the infected pigs, thereby considerably reducingthe probability and scale of the air-borne spread ofcontagion between herds (95, 137). Furthermore,systematic vaccination avoids economic losses due to apoorly controlled infection. Consequently, after severalyears of vaccination in a country or region, and theintroduction of sanitation measures into the infected herds
and the continual culling of the oldest infected sows, theprevalence of infection gradually diminishes; in addition,the incidence of infection remains very low and is keptunder control. However, the cost of vaccination must betaken into account when calculating the total cost of aprophylactic treatment.
Authors compared the cumulative costs, over ten years, ofvarious measures for controlling AD in northern Germany,following the introduction of prophylactic treatment (Table I). Of the five possible strategies, the mosteconomical is based on systematic vaccination, followed byscreening of infected herds and the slaughter of sowspresenting infection-specific antibodies.
Of course, this is a cumulative cost which takes all costsinto account: those of the State, those of tradeorganisations and those of breeders. The authors note thatthe prevalence of infection diminishes during the first twoyears, but that vaccination alone is not enough to eliminateinfection; during the final years of the programme the ADVpersisted in a small number of herds. After 42 months ofvaccination, few herds still harboured infected breedinganimals. The detection and elimination of these breedinganimals lead to a sharp drop in the prevalence of infectionin breeding herds, whilst the risk of infection in fatteningfarms (or of fattened animals in other farms) becomes zero (142).
As a result of past and present experience, it has becomepossible to develop a strategy for using vaccines to controlAD. In countries that have sufficient economic resources toenvisage eradication of the infection, there are two possibleoptions:
– where the prevalence of infection in a given territory ishigh, or there is a high density of pig herds, massvaccination with effective deleted vaccines is the onlymeans of reducing prevalence; however, although thesemeasures are necessary, they are not in themselvessufficient to eradicate the infection. Identification,screening and culling of the infected breeding animalsappear to be essential to successful eradication whilst
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Table I
Total costs of potential control strategies for Aujeszky’s disease in Germany over the ten years following initiation of prophylactic
treatment (147)
Strategy Cumulated cost over ten years (in thousands of euros)
Vaccination of sows twice per year 18,085
Vaccination of sows three times per year 18,143
Vaccination of sows three times per year, and of pigs for consumption once per year 13,534
Vaccination of sows three times per year, and of pigs for consumption once per year. Serological
controls and slaughter of pigs presenting infectious antibodies (where prevalence is < 10%) 9,907
Control and slaughter of pigs possessing infectious antibodies 19,342
continuing to systematically vaccinate the animals at leasttwo years after elimination of the last infected pig. In thelatter case, it is advisable to control the movements ofpiglets, pigs for consumption and breeding animals asmuch as possible;
– by contrast, in regions with a low herd density and lowprevalence of AD, serological screening and the culling ofinfected breeders or total slaughter of certain herds, appearto be the most effective, and in some cases the mosteconomical, measures for achieving eradication. Suchmeasures have been successfully introduced, for example,in Denmark, the United Kingdom, Sweden and severalregions of France.
However, independently of the performances of thevaccines and the companion kits (which are key issues indetermining the use of such tools), other examples showthat the use of marker vaccines would not have had asignificant impact on the control of the situation. When aserious CSF epizootic hit several European countries in1997, many people believed that the use of these newgeneration serological marker vaccines could prevent afurther animal health catastrophe. However, an analysis ofthe situation that existed when the first CSF outbreaksappeared in the Netherlands revealed that more than 22herds were already infected when the primary outbreakwas identified in the region of Venhorst on 4 February1997. The situation rapidly became dramatic for the regionbecause farmers had already sold piglets before theveterinary administration could isolate the infected zone.This led to a rapid spread of the infection in the south ofthe country.
Under such circumstances, the use of a serological markervaccine would not radically alter the basic nature of theproblem, as it does not obviate the need to identifypotentially infected animals and to take a sample of serumbefore any animals are transported, in other words, tostrictly control the movement of pigs. Indeed, at the startof an epizootic, the success of control measures dependson their being rapidly implemented after the appearance ofthe first outbreak and before extensive, undetected spreadhas occurred. Vaccination is no substitute for basicmeasures to control contagious diseases.
So, as a general rule, as long as CSF has not beeneradicated in the world, there is still a great risk ofreintroduction of the virus in free areas. Farmers andveterinarians are in the best position to detect a newintroduction of CSFV, so they should receive training in thedetection of clinical signs and remain extremely vigilant. Anon-vaccination policy is logical in disease-free states butemergency vaccination may be considered in contingencyplans to avoid destroying millions of pigs. When outbreaksoccur, the use of the traditional live C strain vaccine is as
effective at preventing the spread of virus as culling theneighbouring herds. This strategy can be used after thestart of an epizootic when there are too many outbreaksoccurring at the same time, but this means that the pigs areseropositive and leads to their destruction. For this reason,the development of efficacious marker vaccines andreliable discriminatory tests should be encouraged. As ithas been demonstrated that 14 days are necessary toinduce good protection with the available subunit E2vaccines, their use could be envisaged when severaloutbreaks occur at the same time and the use of strictsanitary prophylactic measures alone may not be enoughto control the disease: vaccinating pigs in the zone aroundthe outbreaks would allow the movement of pigs andprevent mass culling. As these E2 subunit vaccines do notprevent vertical transmission, their use must be limited togrowing pigs. Simulation models will also be useful toolsfor choosing the best control measures to apply, dependingon the epidemiological situation.
At the start of an epizootic, in regions with a high densityof pig herds, ring or zonal vaccination can also beenvisaged in order to prevent the virus from replicating toorapidly and to limit the cost of preventive slaughter.However, in this case, transmission of the virus must belimited and control measures must be properly applied andeffective. Such an approach is particularly pertinent forhighly contagious diseases such as FMD in thosecircumstances under which the air-borne transmission isone of the main epidemiological factors in the spread of thevirus. So, if the first outbreaks appear in an area with ahigh density of susceptible herds and underepidemiological conditions that favour air-borne spread,ring vaccination, implemented on the basis of the results ofmodels and assessment to determine the risks anddirections of spreading, could be useful in limiting thespeed and the extent of the virus dissemination (24).However, due to the ability of vaccination to mask theappearance of clinical signs without preventing infection,vaccinated herds, even with a serological monitoringprogramme, represent a greater risk for undetected spreadthan unvaccinated herds, where monitoring can be basedon clinical inspection alone.
In the case of AI, the use of marker vaccines has provedvery effective in controlling LPNAI infection in turkeys andpoultry (18). Nevertheless, the difficulty of implementingsuch a strategy will depend on the species in which thevaccines are intended to be used and, consequently, on theefficiency of one type of vaccine in regard to one particularspecies. The efficiency of a vaccine and the performance ofa companion diagnostic kit cannot be extrapolated fromone avian species to another, so the use of marker vaccineswill depend on the availability of validated data on theperformance of the vaccine and companion diagnostic kitin the avian species that is to be vaccinated.
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In regard to the HPNAI strains, some Asian countries haveimplemented a massive vaccination campaign due to thehigh prevalence of infection in the domestic flocks. Insome countries, such as Vietnam, this strategy seems tohave given good results, but the lack of validated dataprevents drawing definitive conclusions on the beststrategies to use in the face of such complex and difficultanimal health crises.
ConclusionThe power of the combination of a marker vaccine and acompanion diagnostic kit for the control of an infectiousdisease depends not only on the performances of thevaccine and the kit themselves but also on the way inwhich these tools are deployed by the competent authorityfor use in the field. Ideally, both the vaccine and thediagnostic kit should be scientifically assessed by anindependent body following submission of a dossier ofdata in support of an official application for a marketingauthorisation. With respect to tests, at least for those usedin the context of official control and eradicationcampaigns, national or international ReferenceLaboratories should control the quality of each batchreleased onto the market. If several diagnosis laboratoriesare involved in the diagnosis and the surveillance of theinfection and the disease, a Reference Laboratory shouldorganise proficiency tests, the results of which should beused to deliver an agreement to the diagnosis laboratoriesto allow them to carry out the diagnostic tests. Regulatory
systems already exist in most regions of the world (e.g. EU,USA, Japan) to control the quality of vaccines released ontothe market. The situation is more varied with respect tocompanion diagnostic tests as not all regions or countrieshave legislation governing their marketing. Adequatevalidation of tests as ‘fit for purpose’ and appropriatequality control, either by the manufacturer themselves orby official Reference Laboratories, are necessary to ensurethat the test performs as claimed and that all batchesreaching the market are of consistent, high quality. Inaddition, where testing is carried out by multiplelaboratories, experience has shown a clear need for qualityassurance of the testing performed in all participatinglaboratories. This can be achieved by a combination oflaboratories working to recognised quality standards,backed up by external accreditation, and participation inregular proficiency tests conducted by an appropriateReference Laboratory. Only those laboratories reaching therequired standards should be certified as competent to beinvolved in national disease eradication campaigns.
A successful programme can be based on vaccination, butshould also include sanitary measures. Furthermore, whenvaccination is part of a control programme, it should beimplemented only for a certain period of time. Most of thetime, when the prevalence of the infection decreasessignificantly and when the epidemiological unit is correctlyprotected from outside introduction of the agent,vaccination should be replaced by sanitary measures.
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Les vaccins à marqueurs et les conséquences
de leur utilisation sur le diagnostic et la prophylaxie
P. Vannier, I. Capua, M.F. Le Potier, D.K.J. Mackay, B. Muylkens, S. Parida, D.J. Paton & E. Thiry
Résumé
La biologie moléculaire et les avancées techniques liées à la recombinaison de
l’ADN marquent le début d’une nouvelle ère en vaccinologie. Les auteurs
examinent le développement récent de vaccins à marqueurs spécifiques ainsi
que les conséquences de leur utilisation sur le diagnostic et la prévention des
principales maladies infectieuses. Les vaccins obtenus par délétion de gène, les
stratégies DIVA (visant à différencier les animaux infectés des animaux
vaccinés) et d’autres méthodes similaires ont été appliqués avec succès pour
P. Vannier, I. Capua, M.F. Le Potier, D.K.J. Mackay, B. Muylkens, S. Parida, D.J. Paton & E. Thiry
Resumen
La biología molecular y, en particular, los adelantos registrados en la técnica de
recombinación de ADN, han marcado el comienzo de una nueva era para la
vacunación. En este artículo se examinan las vacunas marcadoras específicas,
de reciente desarrollo, y las repercusiones de su administración en el
diagnóstico y la prevención de importantes enfermedades infecciosas. Se han
obtenido resultados satisfactorios en materia de control y erradicación de la
enfermedad de Aujeszky, la rinotraqueítis infecciosa bovina, la peste porcina
clásica, la fiebre aftosa y, más recientemente, la influenza aviar con vacunas
producidas mediante deleción de genes, estrategias para diferenciar entre
animales vacunados e infectados (estrategias DIVA) y otros métodos similares.
Los autores también analizan la eficacia y los resultados obtenidos con las
vacunas marcadoras existentes y los equipos de diagnóstico asociados (que
deberán ser evaluados por un organismo independiente), así como la forma en
que las autoridades competentes las utilizarán. Asimismo, examinan
detalladamente las limitaciones y las ventajas de la administración de vacunas
marcadoras a la luz de la experiencia adquirida en la práctica. Pese a que
pueden limitar la velocidad y la importancia de la diseminación del virus y, por
consiguiente, reducir el número de animales sacrificados, las vacunas
Rev. sci. tech. Off. int. Epiz., 26 (2)364
contrôler et éradiquer la maladie d’Aujeszky, la rhinotrachéite infectieuse
bovine, la peste porcine classique, la fièvre aphteuse et plus récemment
l’influenza aviaire. Les auteurs examinent l’efficacité et les performances des
vaccins à marqueurs existants ainsi que des outils diagnostiques compagnons
(dont l’évaluation devrait être conduite par un organisme indépendant) ; ils
analysent également la manière dont ces outils sont utilisés par les autorités
compétentes. Ils étudient ensuite en détail les avantages et les limites de
l’utilisation des vaccins à marqueurs, à la lumière des expériences pratiques en
la matière. Bien que l’utilisation de ces vaccins ait pour effets de freiner et de
restreindre la dissémination virale, et partant de réduire le nombre d’animaux
abattus, elle n’a pas vocation à remplacer les mesures sanitaires. Les systèmes
de détection et d’alerte précoces et la mise en œuvre rapide de mesures
sanitaires, y compris l’abattage sanitaire, demeurent des mesures
incontournables de la lutte contre les maladies très contagieuses.
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