Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.

No abstract

Infectious laryngotracheitis virus (ILTV; Gallid herpesvirus 1) is an alphaherpesvirus that causes acute respiratory disease in chickens. The role of glycoprotein G (gG) in vitro has been investigated in a number of alphaherpesviruses, but the relevance of gG in vivo in the pathogenicity of ILTV or in other alphaherpesviruses is unknown. In this study, gG-deficient mutants of ILTV were generated and inoculated into specific-pathogen-free chickens to assess the role of gG in pathogenicity. In chickens, gG-deficient ILTV reached a similar titre to wild-type (wt) ILTV but was significantly attenuated with respect to induction of clinical signs, effect on weight gain and bird mortality. In addition, an increased tracheal mucosal thickness, reflecting increased inflammatory cell infiltration at the site of infection, was detected in birds inoculated with gG-deficient ILTV compared with birds inoculated with wt ILTV. The reinsertion of gG into gG-deficient ILTV restored the in vivo phenotype of the mutant to that of wt ILTV. Quantitative PCR analysis of the expression of the genes adjacent to gG demonstrated that they were not affected by the deletion of gG and investigations in vitro confirmed that the phenotype of gG-deficient ILTV was consistent with unaltered expression of these adjacent genes. This is the first reported study to demonstrate definitively that gG is a virulence factor in ILTV and that deletion of gG from this alphaherpesvirus genome causes marked attenuation of the virus in its natural host.

The efficacy and safety of transplanting autologous mesenchymal stem cells (MSCs), from granulocyte-colony-stimulating factor (G-CSF)-mobilised peripheral blood, was investigated in diabetic patients with critical limb ischaemia (CLI). After 3 months, the transplanted group of patients (n=7) showed a significant improvement in ischaemia manifestations, including pain and neurological signs, wound healing and the rate of lower-limb amputation, compared to the control group of patients (n=14). Pain was significantly reduced in the transplanted group compared to controls (P=0.014). The ankle-brachial index (ABI) and the pulse strength within ischaemic tissues of the transplanted group were significantly improved (P=0.035 and P=0.01, respectively). Importantly, 50% of the control group (7/14 patients) faced major amputation of a limb at the study's conclusion, compared to none of 7 patients in the transplanted group (P=0.047). The safety of transplantation was confirmed by observing no adverse reactions among the transplanted group, including infection and immunological rejection. Hence, this study provides further evidence that transplantation of autologous peripheral blood MSCs, mobilised by G-CSF, induces angiogenesis and improves the wound healing process in diabetic patients with CLI.

Previous studies in our laboratory using a combination of polymerase chain reaction and restriction fragment length polymorphism have identified at least five different genotypes of infectious laryngotracheitis virus (ILTV). However, the virulence of these classes of ILTV was not investigated. In this study, five groups (16 birds each) of 3-week-old specific pathogen free chickens were inoculated via the intratracheal route with 10 3 median embryo infected dose of five different strains of ILTV. Three further groups of chickens were inoculated similarly with the vaccine strains SA2 and A20 or with sterile phosphate-buffered saline (PBS) for comparison. Four days post-inoculation, clinical signs were monitored for scoring, and eight chickens from each group were subsequently euthanized, weighed and subjected to pathological and histopathological examinations. The remaining birds were monitored for clinical signs and mortality until 21 days post-inoculation.All groups inoculated with ILTV strains showed moderate to severe clinical signs 4 days after inoculation. The strain Q1-96 caused only minimal breathing symptoms with a median score that was not significantly different to that of the group inoculated with PBS, but was significantly different to those of the groups inoculated with other ILTV strains. The strain Q1-96 caused severe photophobia and conjunctivitis with a median score that was significantly higher than those of all other groups except for the group inoculated with the strain N3-04. All ILTV strains caused a significant reduction in weight gain when compared with the group inoculated with PBS. The strain Q1-96 caused an average weigh loss of 14% that was significantly higher than those of other ILTV strains. The strains S2-04 and Q1-96 induced only minor microscopic tracheal lesions while all the other ILTV strains, including the vaccine strains A20 and SA2, induced moderate to severe microscopic tracheal lesions. Median scores for microscopic tracheal lesions were well correlated with the number of viral genomes detected in trachea.The results revealed that there is considerable variation among ILTV strains in their tropism for trachea or conjunctiva. In addition it was revealed that ILTV strains with high affinity for conjunctiva can severely affect weigh gain. The ILTV numbers and microscopic lesions in trachea were not found to be reliable indicators of virulence since they are not necessarily correlated with mortality rate in ILT. IntroductionInfectious laryngotracheitis (ILT) is a significant respiratory disease of chickens in many countries and can cause reduced egg production and predisposition to other respiratory pathogens (Guy & Bagust, 2003). Outbreaks of mild to moderate forms of ILT are not uncommon in commercial layer flocks worldwide, while sporadic outbreaks of ILT in broiler flocks have also been recognized in recent years as an emerging problem in several countries including Australia (Critchley, 2004; Wells, 2004). Two live attenuated vaccines, SA2 and A20 (Fort Dodge, Australia...