Comparison of the safety and protective efficacy of vaccination with glycoprotein-G-deficient infectious laryngotracheitis virus delivered via eye-drop, drinking water or aerosol.

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated virus strains. These vaccines have limitations due to residual pathogenicity and reversion to virulence. To avoid these problems and to better control disease, attention has recently turned towards developing a novel vaccine strain that lacks virulence gene(s). Glycoprotein G (gG) is a virulence factor in ILTV. A gG-deficient strain of ILTV has been shown to be less pathogenic than currently available vaccine strains following intratracheal inoculation of specific pathogen free chickens. Intratracheal inoculation of gG-deficient ILTV has also been shown to induce protection against disease following challenge with virulent virus. Intratracheal inoculation, however, is not suitable for large-scale vaccination of commercial poultry flocks. In this study, inoculation of gG-deficient ILTV via eye-drop, drinking water and aerosol were investigated. Aerosol inoculation resulted in undesirably low levels of safety and protective efficacy. Inoculation via eye-drop and drinking water was safe, and the levels of protective efficacy were comparable with intratracheal inoculation. Thus, gG-deficient ILTV appears to have potential for use in large-scale poultry vaccination programmes when administered via eye-drop or in drinking water.     

Protection of chickens from infectious laryngotracheitis with a recombinant fowlpox virus expressing glycoprotein B of infectious laryngotracheitis virus

Infectious laryngotracheitis (ILT) is an economically important disease of chickens caused by a type I gallid herpesvirus, infectious laryngotracheitis virus (ILTV). The vaccines currently available are modified live viruses, which are effective in preventing disease outbreaks. However, they have often been associated with a variety of adverse effects including spread of vaccine virus to non-vaccinates, inadequate attenuation, production of latently infected carriers, and increased virulence as a result of in vivo passage. In this study, a recombinant fowlpox virus expressing glycoprotein B (gB) of ILTV (rFPV-ILTVgB) was constructed. Protection of specific pathogen free (SPF) and commercial chickens from ILT with the rFPV-ILTVgB and commercial ILTV vaccine (Nobilis ILT) were compared after challenge with a lethal dose of virulent ILTV.Both the rFPV-ILTVgB- and the Nobilis ILT-vaccinated SPF chickens were completely protected from death, while 90% of the unvaccinated chickens died after challenge. The immunized commercial chickens were also 100% protected with rFPV-ILTVgB, compared with 85% protected with Nobilis ILT. The protective efficacy was also measured by the antibody response to ILTV gB, isolation of challenge virus and polymerase chain reaction amplification of the ILTV thymidine kinase gene after challenge. The results showed that rFPV-ILTVgB could be a potential safe vaccine to replace current modified live vaccines for preventing ILT.     

Chicken embryo origin-like strains are responsible for Infectious laryngotracheitis virus outbreaks in Egyptian cross-bred broiler chickens

Infectious laryngotracheitis virus (ILTV) continues to cause respiratory disease in Egypt in spite of vaccination. The currently available modified live ILTV vaccines provide good protection but may also induce latent infections and even clinical disease if they spread extensively from bird-to-bird in the field. Four field ILTV isolates, designated ILT-Behera2007, ILT-Giza2007, ILT-Behera2009, and ILT-Behera2010 were isolated from cross-bred broiler chickens. The pathogenicity based on intratracheal pathogenicity index, tracheal lesion score, and mortality index for chicken embryos revealed that ILT-Behera2007, ILT-Behera2009 and ILT-Behera2010 isolates were highly pathogenic whereas ILT-Giza2007 was non-pathogenic. To study the molecular epidemiology of these field isolates, the infected cell protein 4 gene was amplified and sequenced. Phylogenetic analysis revealed that ILT-Behera2007, ILT-Behera2009, and ILT-Behera2010 are chicken embryo origin (CEO) vaccine-related isolates while ILT-Giza2007 is a tissue culture origin vaccine-related isolate. These results suggest that CEO laryngotracheitis vaccine viruses could increase in virulence after bird-to-bird passages causing severe outbreaks in susceptible birds.     

Pathogenicity and vaccine efficacy of a thymidine kinase gene deleted infectious laryngotracheitis virus expressing the green fluorescent protein gene

Summary.  The deletion of the thymidine kinase (TK) gene of herpesviruses causes a reduction in their virulence. However, the effects of the TK gene in infectious laryngotracheitis virus (ILTV) have not been clearly elucidated. In the present study, we constructed a TK gene-deleted recombinant ILTV expressing the green fluorescent protein (GFP) gene as a marker. The GFP gene was inserted in place of the TK gene in both virulent and low virulence strains of ILTV. The GFP gene in the recombinants was expressed in chicken kidney cells, LMH cells and in the chorioallantoic membrane of embryonated chicken eggs. The recombinants produced cytopathic effects in chicken kidney cells and LMH cells and formed pocks in the chorioallantoic membrane of embryonated chicken eggs. The growth rate of the recombinant in chicken kidney cells was similar to that of wild type viruses. The recombinants showed a reduction of virulence compared to that of parental viruses and induced protection against virulent ILTV in specific pathogen free chickens. The recombinant expressing GFP gene may be a candidate for a genetically engineered vaccine and provide a means to study growth kinetics and mechanism of latent infection and reactivation of ILTV. In this study, we confirmed that the TK gene is directly related to virulence of ILTV. This is the first report to show the evidence that the TK gene is a major gene related to virulence of ILTV. Received May 1, 2001; accepted November 14, 2001     

Genotyping of infectious laryngotracheitis virus using allelic variations from multiple genomic regions

Live attenuated vaccines are extensively used worldwide to control the outbreak of infectious laryngotracheitis. Virulent field strains showing close genetic relationship with the infectious laryngotracheitis virus (ILTV) vaccines of chicken embryo origin have been detected in the poultry industry. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, a reliable molecular epidemiological method, of multiple genomic regions was performed. The PCR-RFLP is a time-consuming method that requires considerable amount of intact viral genomic DNA to amplify genomic regions greater than 4?kb. In this study, six variable genomic regions were selected and amplified for sequencing. The multi-allelic PCR-sequence genotyping showed better discrimination power than that of previous PCR-sequencing schemes using single or two target regions. The allelic variation patterns yielded 16 strains of ILTV classified into 14 different genotypes. Three Korean field strains, 550/05/Ko, 0010/05/Ko and 40032/08/Ko, were found to have the same genotype as the commercial vaccine strain, Laryngo Vac (Zoetis, Florham Park, NJ, USA). Three other Korean field strains, 40798/10/Ko, 12/07/Ko, and 30678/14/Ko, showed recombined allelic patterns. The multi-allelic PCR-sequencing method was proved to be an efficient and practical procedure to classify the different strains of ILTV. The method could serve as an alternate diagnostic and differentiating tool for the classification of ILTV, and contribute to understanding of the epidemiology of the disease at a global level.     

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

A real-time polymerase chain reaction (PCR) assay was developed to specifically amplify infectious laryngotracheitis virus (ILTV) DNA from field samples. The 222-base-pair PCR fragment was amplified using primers located in a conserved region of the infected cell protein 4 gene that was demonstrated in this work to encompass a single nucleotide polymorphism. Subsequent restriction fragment length polymorphism (RFLP) analysis of real-time PCR amplified fragments from a range of ILTV isolates using the restriction endonuclease MspI enabled differentiation between older ILTV isolates that were prevalent in the 1960s prior to the availability of vaccine strains and more recent isolates that predominantly are identical to vaccine strains. The assay, using real-time PCR, RFLP and sequence analysis, was used to characterize two recent field cases of infectious laryngotracheitis from Northern Ireland. One of the field cases was demonstrated to be similar to older "wild-type" isolates, while the other field case was identified to have a concurrent ILTV infection of both "wild-type" and vaccinal type origin. The assay described here using real-time PCR and RFLP provides a rapid, specific method that enables detection and characterization of ILTV directly from field cases.     

Comparison of the replication and transmissibility of an infectious laryngotracheitis virus vaccine delivered via eye-drop or drinking-water

Live attenuated vaccines have been extensively used to control infectious laryngotracheitis (ILT). Most vaccines are registered/recommended for use via eye-drop although vaccination via drinking-water is commonly used in the field. Drinking-water vaccination has been associated with non-uniform protection. Bird-to-bird passage of chick-embryo-origin (CEO) ILT vaccines has been shown to result in reversion to virulence. The purpose of the present study was to examine the replication and transmission of a commercial CEO infectious laryngotracheitis virus (ILTV) vaccine strain following drinking-water or eye-drop inoculation. Two groups of 10 specific-pathogen-free chickens were each vaccinated with Serva ILTV vaccine strain either via eye-drop or drinking-water. Groups of four or five unvaccinated birds were placed in contact with vaccinated birds at regular intervals. Tracheal swabs were collected every 4 days from vaccinated and in-contact birds to assess viral replication and transmission using quantitative polymerase chain reaction. Compared with eye-drop-vaccinated birds, drinking-water-vaccinated birds showed delayed viral replication but had detectable viral DNA for a longer period of time. Transmission to chickens exposed by contact on day 0 of the experiments was similar in both groups. Birds exposed to ILTV by contact with eye-drop vaccinated birds on days 4, 8, 12 and 16 of the experiment had detectable ILTV for up to 8 days post exposure. ILTV was not detected in chickens that were exposed by contact with drinking-water vaccinated birds on day 12 of the experiment or later. Results from this study provide valuable practical information for the use of ILT vaccine.     

Relationship between mortality, clinical signs and tracheal pathology in infectious laryngotracheitis.

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.     

Infectious laryngotracheitis virus, an alpha herpesvirus that does not interact with cell surface heparan sulfate

Among the alpha herpesviruses studied to date, the initial stage of wild-type virus attachment involves an interaction between virally encoded structural envelope glycoproteins (predominantly glycoprotein C) and cell surface heparan sulfate proteoglycans. An analysis of the infectious laryngotracheitis virus (ILTV) glycoprotein C and glycoprotein B sequences suggested that these proteins lacked consensus heparin-binding domains. This indicated that ILTV might attach to its host cell in a heparan-independent manner, distinct from other alpha herpesviruses. The infectivity of two ILTV strains, a tissue-culture-adapted vaccine strain and a virulent field challenge strain, were found to be insensitive to the presence of exogenous heparin or chondroitin. Furthermore, infectivity was retained in chicken embryonic liver cells treated with heparinase. However, 4 degrees C attachment studies and penetration studies in the presence of citrate buffer clearly demonstrated that ILTV attaches stably to and effectively penetrates chicken embryonic liver cells. Consequently, ILTV represents an alpha herpesvirus whose initial attachment step does not involve interactions with heparan or chondroitin sulfate containing proteoglycans.     

Comparative in vivo safety and efficacy of a glycoprotein G-deficient candidate vaccine strain of infectious laryngotracheitis virus delivered via eye drop

Infectious laryngotracheitis (ILT) is an acute respiratory disease in poultry that is commonly controlled by vaccination with conventionally attenuated virus strains. Despite the use of these vaccines, ILT remains a threat to the intensive poultry industry. Our laboratory has developed a novel candidate vaccine strain of infectious laryngotracheitis virus (ILTV) lacking glycoprotein G (ΔgG-ILTV). The aim of the present study was to directly compare this candidate vaccine with three currently available commercial vaccines in vivo. Five groups of specific-pathogen-free chickens were eye-drop inoculated with one of the three commercial vaccine strains (SA2-ILTV, A20-ILTV or Serva-ILTV), or ΔgG-ILTV, or sterile medium. Vaccine safety was assessed by examining clinical signs, weight gain and persistence of virus in the trachea. Vaccine efficacy was assessed by scoring clinical signs and conducting post-mortem analyses following challenge with virulent virus. Following vaccination, birds that received ΔgG-ILTV had the highest weight gain among the vaccinated groups and had clinical scores that were significantly lower than birds vaccinated with SA2-ILTV or A20-ILTV, but not significantly different from those of birds vaccinated with Serva-ILTV. Analysis of clinical scores, weight gain, tracheal pathology and virus replication after challenge revealed a comparable level of efficacy for all vaccines. Findings from this study further demonstrate the suitability of ΔgG-ILTV as a vaccine to control ILT.     

Evaluation of the protection elicited by direct and indirect exposure to live attenuated infectious laryngotracheitis virus vaccines against a recent challenge strain from the United States

In a recent study (Oldoni & García, 2007), some field strains of infectious laryngotracheitis viruses (ILTV) were characterized as genotypically different (group VI) from ILT vaccine strains. The objective of this study was to evaluate the protection elicited by one chicken embryo origin (CEO) and one tissue culture origin (TCO) vaccine against a field isolate from group VI after direct and indirect exposure to ILTV live attenuated vaccines. In phase 1 of the experiment, non-vaccinated chickens were placed into contact with the eye drop vaccinates for a period of four weeks after vaccination. Transmission of the vaccine virus to these in-contact birds was demonstrated by real time PCR and antibody production, although the in-contact birds did not become protected against disease when subsequently challenged in phase 2 of the experiment. This emphasized the importance of uniform vaccination to obtain adequate protection, both to avoid the occurrence of susceptible chickens, and to minimize the potential for reversion to virulence of live-attenuated vaccines. In phase 2, protection against challenge with a group VI field virus was assessed four weeks after vaccination by scoring clinical signs and mortality, and quantifying weight gain. Sentinel birds were added to the groups one day after challenge to assess shedding of challenge virus, using real time PCR and virus isolation, during the period 2 to 12 days post challenge. The results showed that the CEO and TCO eye drop-vaccinated chickens were protected against challenge with the group VI virus, even though it was genetically different from the vaccine strains, and that challenge virus was not transmitted from these protected birds to the sentinels.     

Infectious laryngotracheitis virus in chickens

Infectious laryngotracheitis (ILT) is an important respiratory disease of chickens and annually causes significant economic losses in the poultry industry world-wide. ILT virus (ILTV) belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The transmission of ILTV is via respiratory and ocular routes. Clinical and post-mortem signs of ILT can be separated into two forms according to its virulence. The characteristic of the severe form is bloody mucus in the trachea with high mortality. The mild form causes nasal discharge, conjunctivitis, and reduced weight gain and egg production. Conventional polymerase chain reaction (PCR), nested PCR, real-time PCR, and loop-mediated isothermal amplification were developed to detect ILTV samples from natural or experimentally infected birds. The PCR combined with restriction fragment length polymorphism (RFLP) can separate ILTVs into several genetic groups. These groups can separate vaccine from wild type field viruses. Vaccination is a common method to prevent ILT. However, field isolates and vaccine viruses can establish latent infected carriers. According to PCR-RFLP results, virulent field ILTVs can be derived from modified-live vaccines. Therefore, modified-live vaccine reversion provides a source for ILT outbreaks on chicken farms. Two recently licensed commercial recombinant ILT vaccines are also in use. Other recombinant and gene-deficient vaccine candidates are in the developmental stages. They offer additional hope for the control of this disease. However, in ILT endemic regions, improved biosecurity and management practices are critical for improved ILT control.     

Comparative full genome analysis of four infectious laryngotracheitis virus (Gallid herpesvirus-1) virulent isolates from the United States

Gallid herpesvirus-1 (GaHV-1), commonly named infectious laryngotracheitis (ILT) virus, causes the respiratory disease in chickens known as ILT. The molecular determinants associated with differences in pathogenicity of GaHV-1 strains are not completely understood, and a comparison of genomic sequences of isolates that belong to different genotypes could help identify genes involved in virulence. Dideoxy sequencing, 454 pyrosequencing and Illumina sequencing-by-synthesis were used to determine the nucleotide sequences of four genotypes of virulent strains from GaHV-1 groups I-VI. Three hundred and twenty-five open reading frames (ORFs) were compared with those of the recently sequenced genome of the Serva vaccine strain. Only four ORFs, ORF C, U(L)37, ICP4 and U(S)2 differed in amino acid (aa) lengths among the newly sequenced genomes. Genome sequence alignments were used to identify two regions (5' terminus and the unique short/repeat short junction) that contained deletions. Seventy-eight synonymous and 118 non-synonymous amino acid substitutions were identified with the examined ORFs. Exclusive to the genome of the Serva vaccine strain, seven non-synonymous mutations were identified in the predicted translation products of the genes encoding glycoproteins gB, gE, gL and gM and three non-structural proteins U(L)28 (DNA packaging protein), U(L)5 (helicase-primase) and the immediate early protein ICP4. Furthermore, our comparative sequence analysis of published and newly sequenced GaHV-1 isolates has provided evidence placing the cleavage/packaging site (a-like sequence) within the inverted repeats instead of its placement at the 3' end of the U(L) region as annotated in the GenBank's entries NC006623 and HQ630064.     

Restriction endonuclease analysis of the DNA of Rickettsia prowazekii vaccine strain E and its revertant

The DNA of Rickettsia prowazekii vaccine strain E was analysed by restriction analysis with 17 endonucleases in comparison with its virulent revertant - Evir and the virulent reference strain Breinl. The DNA of cloned and uncloned strains showed identical restriction endonuclease patterns. In spite of stable differences in virulence, strains E and Evir displayed a totally identical DNA cleavage pattern indicating the absence of marked structural differences between their genomes. On the other hand 9 endonucleases showed differences in the restrictograms of the DNA strain Breinl as compared with strains E and Evir.     

A double recombinant herpes virus of turkeys for the protection of chickens against Newcastle,infectious laryngotracheitis and Marek’s diseases

A double recombinant strain of herpes virus of turkeys (HVT) was constructed that contains the fusion (F) gene from Newcastle disease virus (NDV) and the gD plus gI genes from infectious laryngotracheitis virus (ILTV) inserted into a non-essential region of the HVT genome. Expression of the F protein was controlled by a human cytomegalovirus promoter, whereas expression of gD plus gI was driven by an ILTV promoter. The double recombinant vaccine virus (HVT-NDV-ILT) was fully stable genetically and phenotypically following extended passage in cell culture and infection of chickens. Safety of the vaccine virus was confirmed by overdose and backpassage studies in specific-pathogen-free chickens. Chickens vaccinated with a single dose of HVT-NDV-ILT administered by the in ovo route were highly protected from challenge with the velogenic NDV (GB Texas), ILTV (LT 96-3) and Marek’s disease virus (GA 5) strains (97%, 94% and 97%, respectively). Similarly, chickens vaccinated with a single dose by subcutaneous (SC) route at 1 day of age were highly protected from challenge with the same three viruses (100%, 100%, and 88%, respectively). The protection level of a single dose given by in ovo or SC route against challenge with a virulent Marek’s disease virus strain demonstrates that insertion of multiple genes from two different pathogens within the HVT genome had no adverse effect on the capacity of HVT to protect against Marek’s disease. These results demonstrate that HVT-NDV-ILT is a safe and efficacious vaccine for simultaneous control of NDV, ILTV and Marek’s diseases.     

Genome sequence comparison of two United States live attenuated vaccines of infectious laryngotracheitis virus (ILTV)

This study was conducted to identify unique nucleotide differences in two U.S. chicken embryo origin (CEO) vaccines [LT Blen (GenBank accession: JQ083493) designated as vaccine 1; Laryngo-Vac^? (GenBank accession: JQ083494) designated as vaccine 2] of infectious laryngotracheitis virus (ILTV) genomes compared to an Australian Serva vaccine reference ILTV genome sequence [Gallid herpesvirus 1 (GaHV-1); GenBank accession number: HQ630064]. Genomes of the two vaccine ILTV strains were sequenced using Illumina Genome Analyzer 2X of 36 cycles of single-end reads. Results revealed that few nucleotide differences (23 in vaccine 1; 31 in vaccine 2) were found and indicate that the US CEO strains are practically identical to the Australian Serva CEO strain, which is a European-origin vaccine. The sequence differences demonstrated the spectrum of variability among vaccine strains. Only eight amino acid differences were found in ILTV proteins including UL54, UL27, UL28, UL20, UL1, ICP4, and US8 in vaccine 1. Similarly, in vaccine 2, eight amino acid differences were found in UL54, UL27, UL28, UL36, UL1, ICP4, US10, and US8. Further comparison of US CEO vaccines to several ILTV genome sequences revealed that US CEO vaccines are genetically close to both the Serva vaccine and 63140/C/08/BR (GenBank accession: HM188407) and are distinct from the two Australian-origin CEO vaccines, SA2 (GenBank accession: JN596962) and A20 (GenBank accession: JN596963), which showed close similarity to each other. These data demonstrate the potential of high-throughput sequencing technology to yield insight into the sequence variation of different ILTV strains. This information can be used to discriminate between vaccine ILTV strains and further, to identify newly emerging mutant strains of field isolates.     

Occurrence of infectious laryngotracheitis outbreaks in commercial layer hens detected by ELISA

Infectious laryngotracheitis (ILT) is an acute respiratory disease of chickens and a cause of great economic loss in commercial layers. The aims of this study were to investigate the prevalence of ILT in the field outbreaks and to compare the characteristics of ILT-infected and free flocks of commercial layers. A total of 625 blood serum samples were collected from 25 different layer flocks. The presence of antibodies against infectious laryngotracheitis virus (ILTV) in each sample was determined by ELISA. Of the 625 serum samples, 266 (42.56%) were found to be positive for ILTV antibodies. A total of 16 (64%) flocks were detected ILT positive by ELISA method. The mortality of infected flocks was statistically higher (P < 0.05) than uninfected flocks. The egg production of positive flocks was lower than that of the free flocks, but this difference was not statistically significant. The average live weight of hens in infected flocks was lower (P > 0.05) than hens in free flocks. In conclusion, the results of this study indicated a high prevalence of ILT infection in the commercial layer flocks in Konya region, Turkey. In outbreaks, ILT significantly increased the mortality rate and decreased the average live weight in layer hens.     

An in vitro and in vivo evaluation of the virulence of Newcastle disease virus and vaccines for the chicken reproductive tract

The virulence of two vaccine strains and two field strains of Newcastle disease virus (NDV) for the female reproductive tract of chickens was assessed using oviduct organ cultures (OOC) prepared from precociously-induced oviducts in young chicks by oestrogen treatment. Ciliostasis, haemagglutination and virus isolation from infected OOC supernatants, histopathology and immunoperoxidase test results indicated the pathogenic nature of both vaccine and virulent NDVs for the precocious oviducts. The virulent viruses, mesogenic and lentogenic vaccines caused damage in that order of magnitude and the uterus had a higher susceptibility than oviducts. One virulent and the mesogenic strain of NDV were used for in vivo trials. The pathogenicity was assessed in oestrogen-treated infected chickens using histopathology and immunoperoxidase test. The vaccine virus produced transient damage up to 6 days post-infection, while the damage with the virulent isolate persisted for at least 9 days post-infection. This technique could be a pointer to possible variations in virulence of NDV vaccine and field strains, and warrants further investigation. The potential value of OOC from young chickens for testing the possibility of NDV vaccines causing damage by themselves and offering protection against damage of the reproductive tract caused by virulent isolates is emphasized.     

Effects of reticuloendotheliosis virus on the response of chickens to infectious laryngotracheitis virus

Effects of reticuloendotheliosis virus (REV) on the response to infectious laryngotracheitis virus (ILTV) were investigated in young chickens with and without maternally derived antibody (MAb) to REV. In the first experiment a group of 1-day-old chickens without REV MAb were inoculated at 1 day of age with REV whilst another group of similar chickens were left uninoculated. All chickens were vaccinated with ILTV at 7 days of age. There was a significantly higher proportion of infectious laryngotracheitis (ILT) post-vaccinal ophthalmia (p.v.o.) in the group inoculated with REV. In the second experiment chickens with and without MAb to REV were inoculated at 1 day old with REV. These chickens, together with others not inoculated with REV, were vaccinated with ILTV isolate SA-2 8 days later. A virulent ILTV isolate, G, was used to challenge all the chickens 20 days after vaccination. Again the chickens without MAb to REV inoculated with REV showed a higher proportion of ILT p.v.o. and a significantly higher mortality rate due to ILT following vaccination. In the chickens inoculated with REV at 1 day of age and not vaccinated but challenged with ILTV there was a significantly higher mortality and rate of clinical signs due to ILT in those birds without than in those with REV MAb. In both experiments chickens from REV negative parents were found to be free of REV neutralising MAb. However, only 30% of chickens originating from a flock known to be infected with REV had a titre of 1/40 or higher. In spite of this, this group was significantly more resistant than the group without REV MAb to the immunosuppressive effect of inoculation at 1 day old with REV. This was demonstrated by their lower susceptibility (i.e. less p.v.o. and mortality) to the vaccination and challenge with ILTV. Chickens without REV MAb developed neutralising antibodies within 2 weeks of inoculation with REV. Irrespective of the REV MAb status 1-day-old chickens inoculated with REV were viraemic within a week.