RFLP analysis of recent Northern Ireland isolates of infectious laryngotracheitis virus: Comparison with vaccine virus and field isolates from England, Scotland and the Republic of Ireland

Abstract

Restriction fragment length polymorphism (RFLP) analysis was used to assist epidemiological investigations following the recent introduction of infectious laryngotracheitis virus (ILTV) to commercial poultry flocks in Northern Ireland (NI). A 4.9 kbp PCR product of the ILTV ICP4 gene was generated from each of 16 field isolates of ILTV originating from England, Scotland, NI and the Republic of Ireland (RoI) and of the single vaccine strain currently licenced for use within the United Kingdom. With the exception of isolate PV6/94 from RoI, all field isolates generated RFLP patterns, following digestion with HaeIII, similar to that obtained using the vaccinal strain. Following MspI digestion, NI isolates were indistinguishable from the vaccinal strain and recent English isolates. However, one English and one Scottish isolate, both made prior to the introduction of vaccination, and two isolates from RoI generated a second pattern following digestion with MspI.

Rapid detection and characterization from field cases of infectious laryngotracheitis virus by real-time polymerase chain reaction and restriction fragment length polymorphism

“…Identification of a ''wild-type'' 724-bp fragment within the ICP4 gene Msp I digest of the 4.9-kbp ICP4 PCR product from the ILTV PRC isolate (see Table 1) was electrophoresed on a 3% NuSieve GTG low melting point gel, and a 724-bp band that is unique to the ''wild-type'' strain (Graham et al ., 2000) was excised and purified using the QIAQuick gel extraction kit system (Qiagen). The purified DNA fragment was labelled using the ECL direct nucleic acid labelling and detection system (Amersham).…”

“…A 4.9-kbp fragment of the ICP4 gene was amplified from all field cases and isolates presented in Table 1 using the Elongase Amplification System (Invitrogen) and primers as previously described (Chang et al ., 1997) in a GeneAmp PCR System 9700 thermal cycler. The PCR products were purified using a QIAQuick PCR purification system (Qiagen), digested using Msp I as previously described (Graham et al ., 2000) and electrophoresed on a 1.5% agarose gel.…”

“…However, although a low virulent phenotype was identified through analysis of the TK gene, no differences were identified in the TK region between field isolates, older ''wild-type'' ILTV isolates and vaccinal strains, necessitating the development of a PCR assay based on another region of the viral genome. Assays targeting the ICP4 gene, which enables differentiation between vaccinal and non-vaccinal strains, have been previously described (Chang et al ., 1997;Graham et al ., 2000;OIE, 2004), and therefore this gene was selected as a region likely to be suitable for development for a real-time PCR assay.…”

“…The disease is of economic importance as virulent strains can cause high levels of mortality and reduced productivity, while recovery from the disease can result in birds with a latent carrier status (Hughes et al ., 1987(Hughes et al ., , 1991b. Although strains may vary considerably in their virulence, there is recent evidence that vaccine-derived strains have become established in the field (Guy et al ., 1989;Graham et al ., 2000). This ability of ILT vaccine strains to recirculate may also be responsible for some outbreaks in susceptible birds, as passage in birds has been reported to result in increasing virulence (Guy et al ., 1991), while stress in latently infected birds has also been demonstrated to be responsible for the reexcretion of ILTV (Hughes et al ., 1987;Bagust et al ., 2000).…”

“…These methods have included the detection of field strains using polymerase chain reaction (PCR) coupled with non-radioactive probes (Abbas et al ., 1996), while differentiation between field and vaccine strains has been demonstrated through the use of PCR coupled with restriction fragment length polymorphism (RFLP) (Chang et al ., 1997;Clavijo & Nagy, 1997;Graham et al ., 2000;Han & Kim, 2001a). Differentiation between field isolates of high and low virulence has also been achieved through the use of PCR, RFLP and sequence analysis (Han & Kim, 2001b), while multiplex PCR has been used for ILT diagnosis (Pang et al ., 2002).…”