Resultados para el objetivo específico definir un sistema de competencias

The successful application of DIVA vaccination in eradication programmes for AD in pigs and AI in birds instigated the development of marker vaccines and DIVA systems for a number of other important notifiable diseases that inflict economical and ethical strain on the meat and poultry industries (Pasick, 2004).

The majority of developments carried out on DIVA-compatible vaccines and diagnostic tests initially focused on four of the most economically important trans-boundary diseases in Europe: AD, AI, FMD and CSF (Van Oirschot et al., 1996; Babiuk, 1999; Van Oirschot, 1999; Clavijo et al., 2004; Pasick, 2004; Bouma, 2005; Suarez, 2005; 2012; Beer et al., 2007; Vannie et al., 2007; Uttenthal et al., 2010). There have since been a vast number of studies and approaches to marker/DIVA vaccine development for many other diseases.

Although the purpose of applying DIVA vaccine strategies for many of these diseases is similar, the technological approaches that have been applied are extremely diverse and differ depending on the biology of the RNA or DNA virus, as well as the characteristics of the disease that they inflict and antibody response they provoke (Table 1.1 & 1.2). For example, the groundwork undertaken on PrV using molecular biological approaches enabled mapping of, and functional roles to be assigned to, the various structural proteins of the virus prior to development of the marker vaccine and companion diagnostic test (Van Oirschot et al., 1986;

1996; Van Oirschot, 1999). Subsequently, the introduction of DIVA vaccination was largely attributed to collated knowledge of the herpesvirus glycoproteins of PrV (Mettenleiter, 2002).

As conventionally attenuated PrV vaccines harbour a deletion within their genomes encoding for an immunogenic glycoprotein (Van Oirschot et al., 1990) marker vaccine approaches

initially focused towards several of these characterised envelope glycoproteins. Glycoprotein I or E (gI/gE) (Van Oirschot et al., 1996), was one of these targets (Van Oirschot et al., 1988;

1990; 1991). This envelope protein had proved an effective marker antigen as antibodies to this glycoprotein persist for >2 years in infected/exposed animals and it is expressed by at least the majority of field strains (Van Oirschot et al., 1990).

Different DIVA strategies have been developed since the origin of marker vaccines, some of which require the use of appropriate vaccines and specific companion serologic discriminatory tests (Avellaneda et al, 2010). However, many marker vaccines have been developed through more conventional routes without using DNA recombinant technology to engineer the marker vaccine. Conventional inactivated vaccines have been applied successfully for DIVA approaches with companion diagnostic tests targeting proteins involved in virus replication (Mackay et al., 1998; Chung et al., 2002; Suarez, 2005; 2012;

Lambrecht et al., 2007; Barros et al., 2009; Hemmatzadeh et al., 2013). Recent advances in immunology, microbiology, molecular biology, proteomics, genetics, genomics and microbial pathogenesis have led to a wide variety of biotechnological approaches based on DNA mediated vaccine development. Vaccines engineered with gene deletions and additions, live vectored vaccines, chimeric vaccines, peptide and subunit vaccines have all been utilised to induce differential antibody responses (Babiuk, 1999; Henderson, 2005; Meeusen et al., 2007). Importantly, many marker vaccines retain the essential properties to: (1) reduce clinical signs after infection; (2) reduce wild-type virus replication after infection; (3) reduce transmission of the virus in the laboratory and in the field (Pensaert et al., 1990; Vannie et al., 1991; Swayne et al., 2000; Uttenthal et al., 2010). Many of the approaches to develop marker/DIVA vaccines have utilised virus structural and non-structural proteins, depending on family-specific aspects of the virion particle, and their role in virus pathogenesis and host-pathogen interactions. However, regardless of the approach taken, the essential properties

required to fulfil the DIVA principle is the ability to specifically detect antibodies of infected animals to the marker antigen with a sensitive ‘marker assay’ (Beer et al., 2007). Exploiting various biotechnological tools and expression systems have therefore also contributed to minimised production costs and time lag for development and analysis of diagnostic serological tests (Clavijo et al., 2004; Perkins et al., 2007a; b; Hema et al., 2007; Gómez-Sebastián et al., 2008). Furthermore, different expression systems have be used for maximised purity of immunogenic proteins both for development of the vaccine and companion diagnostic test (Van Drunen Little-van den Hurk et al., 1997; Wang et al., 2002;

Clavijo et al., 2004; Sørensen et al., 2005; Huang et al., 2006; Choi et al., 2013).

A prerequisite for DIVA vaccination is that all field strains express the marker antigen and that infected animals always elicit antibodies to that protein after infection (Van Oirschot et al., 1996; Van Drunen Little-van den Hurk et al., 2006). A number of requirements for the DIVA diagnostic test were proposed by Van Oirschot et al. (1996):

1. Antibodies must be detectable within three weeks after infection 2. Antibodies must persist for a long period after infection

3. Vaccinated and subsequently infected animals elicit antibodies if wild-type virus replicates within the host

4. Repeatedly vaccinated animals must score negative to the marker 5. A high sensitivity, specificity and reproducibility must be obtained

There is often a lag time before detectable antibody responses are produced to the marker, not only following vaccination, but also following infection, which can vary depending on the disease and antigen used for serological screening (Van Oirschot et al., 1996; Van Rijn et al., 1996; Bouma et al., 1999; De Smit et al., 2001; Beer et al., 2007). Temperature may also represent an issue with the approach in fish as antibody responses are temperature dependent

for poikliotherms (Bly and Clem, 1992). Therefore, direct virus detection, of either antigen or nucleic acid, is usually necessary, especially if screening individual animals, to confirm their infection status. Furthermore, serology is only currently utilised for detecting suspect cases, but confirmation requires direct pathogen detection methods (OIE, 2012).

Few bacterial DIVA vaccine approaches have been conducted, e.g. a subunit and negative marker vaccine for Actinobacillus pleuropneumoniae, the causative agent of Porcine pleuropneumonia, by deletion of the Apx2A gene which expresses Apx2 toxins (Goethe et al., 2001; Tonpitak et al., 2002; Mass et al., 2006). However, copious studies have been conducted for marker vaccines and DIVA approaches for RNA and DNA viral diseases of mammals and birds (Table 1.1 & 1.2). These DIVA approaches have been achieved by taking advantage of the properties of serum immunoglobulin specificity and affinity, particularly immunoglobulin G (IgG) in mammals and IgY in birds.

DIVA approaches have varied considerably depending on the virus type. The approach to developing a DIVA vaccine requires either (1) construction of vaccines that exhibit different immunogenic properties to the wild-type strain or (2) exploit immunogenic variations that exist between vaccine and wild-type strain.

Since no DIVA approach has been applied for aquatic viruses, developments undertaken for mammalian and avian viruses and their success in the field may provide useful models for aquatic DIVA developments.

Table 1.1 Animal RNA virus DIVA vaccine approaches

(5) Live attenuated ,

(1) Live G_L neg. live vaccine (1) Synthetic G_L peptide ELISA Castillo-Olivares et al., 2003

Blutongue virus

(1) Inactivated vaccine (1) E.coli expressed rec. NS3 indirect ELISA

(1) E^RNS GP competitive ELISA with baculovirus rec. E^RNS

Susceptible hosts are listed in italics under viruses; Env. = enveloped; Nak. = naked; ss = single stranded; kbp = kilo base pairs; seg = segments;

ORF = open reading frame; rec. = recombinant; EITB = Enzyme-linked immunoelectrotransfer blot; ELISA = enzyme-linked immunosorbent assay; IFAT = indirect fluorescent antibody technique

Table 1.2 Animal DNA virus DIVA vaccine approaches

(1) Competitive gE blocking ELISA Delhon et al., 2003;

Anziliero et al., 2011

(1) Rec. gG deleted vaccine (1) Rec. baculovirus/E.coli expressed gG ELISA

Fuchs et al.., 2007;

Lee et al., 2011;

Shil et al., 2012

Susceptible hosts are listed in italics under viruses; Env. = enveloped; Nak. = naked; ss = single stranded; kbp = kilo base pairs; seg = segments;

ORF = open reading frame; rec. = recombinant; ELISA = enzyme-linked immunosorbent assay; IFAT = indirect fluorescent antibody technique