Characterization by restriction fragment length polymorphism and sequence analysis of field and vaccine strains of infectious laryngotracheitis virus involved in severe outbreaks
TL;DR: This work shows that the severe ILT outbreak was caused by a highly virulent, non-vaccine strain.
Abstract: At the end of 2002 and throughout 2003, there was a severe outbreak of infectious laryngotracheitis (ILT) in an intensive production area of commercial hens in the Sao Paulo State of Brazil. ILT virus was isolated from 28 flocks, and 21 isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using four genes and eight restriction enzymes, and by partial sequencing of the infected cell protein 4 (ICP4) and thymidine kinase (TK) genes. Three groups resulted from the combinations of PCR-RFLP patterns: 19 field isolates formed Group I, and the remaining two isolates together with the chicken embryo origin (CEO) vaccine strains formed Group II. Group III comprised the tissue-culture origin (TCO) vaccine strain by itself. The PCR-RFLP results agreed with the sequencing results of two ICP4 gene fragments. The ICP4 gene sequence analysis showed that the 19 field isolates classified into Group I by RFLP-PCR were identical among themselves, but were different to ...
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TL;DR: Overall, molecular typing data collected worldwide have identified live attenuated vaccine-related isolates as the primary source for outbreaks of infectious laryngotracheitis.
Abstract: Infectious laryngotracheitis (ILT) is an economically important respiratory disease of poultry that affects the poultry industry worldwide. The disease is caused by gallid herpesvirus I (GaHV-1), a member of the genus Iltovirus, family Herpesviridae, subfamily Alphaherpesvirinae. The current incidence of the disease is heavily influenced by live attenuated vaccines, which have been used extensively since their introduction in the mid-twentieth century. The capability of current live attenuated vaccine viruses to revert to virulence and spread from bird to bird has shaped the molecular epidemiology of ILT. Because of the antigenic homogeneity among GaHV-1 strains, differentiation of strains has been achieved by targeting genomic differences between outbreak-related isolates and vaccine strains. Numerous genes and genomic regions have been utilized in the development of DNA-based diagnostic assays to differentiate outbreak-related isolates from vaccine strains in countries where ILT outbreaks have occurred. More recently, full genome sequences have allowed determination of the origin of some of the outbreak-related isolates circulating in some poultry production countries. Overall, molecular typing data collected worldwide have identified live attenuated vaccine-related isolates as the primary source for outbreaks of the disease.
64 citations
Cites background or methods from "Characterization by restriction fra..."
...Differentiation of field isolates and vaccine strains from Brazil was achieved using PCR-RFLP of the TK, ICP4, UL47/gG, and gE genes combined with sequencing of the TK and ICP4 genes (Chacon & Ferreira, 2009; Chacon et al., 2010)....
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...…these from non-vaccine-related isolates, particularly in North America, Europe, South America and East Asia (Chang et al., 1997; Han & Kim, 2001; Kirkpatrick et al., 2006b; Ojkic et al., 2006; Oldoni & Garcia, 2007; Oldoni et al., 2008; Neff et al., 2008; Chacon et al., 2010; Moreno et al., 2010)....
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...Typing methods that distinguish between GaHV-1 isolates and vaccine strains of the USA, Australia, Europe and Asia (Chang et al., 1997; Ojkic et al., 2006; Oldoni & Garcia, 2007; Chacon et al., 2010; Moreno et al., 2010) utilized varied combinations of genes (multilocus genotyping) (Table 3)....
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...In South America the ICP4 gene has been sequenced (Chacon & Ferreira, 2009; Chacon et al., 2010), and in the UK the TK and ICP4 genes are the targets (Creelan et al., 2006)....
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TL;DR: Next-generation vaccines, along with more appropriate immunological screening strategies, are identified as particularly promising options to enhance ILT control in the future.
Abstract: Over the past 80 years, biosecurity measures and vaccines have been used to prevent the occurrence of outbreaks of infectious laryngotracheitis (ILT). Despite these control strategies, ILT continues to have an impact on intensive poultry industries. Attenuated vaccines, particularly those derived by passage in chicken embryos, have been associated with a number of side effects, including residual virulence, transmission to naive birds, establishment of latent infections with subsequent reactivation and shedding of virus, and reversion to virulence after in vivo passage. Most recently, recombination between attenuated ILT vaccines in the field has been shown to be responsible for the emergence of new virulent viruses that have caused widespread disease. To address some of these issues, new-generation virally vectored recombinant vaccines have been developed and recently released in some countries. In addition, recombinant deletion mutants of ILT virus have been proposed as vaccine candidates. In this review, recent advances in the understanding of the epidemiology of traditionally attenuated ILT vaccines as well as in the development and use of new generation vaccines are examined. Next-generation vaccines, along with more appropriate immunological screening strategies, are identified as particularly promising options to enhance ILT control in the future.
51 citations
Cites background from "Characterization by restriction fra..."
...Following the occurrence of these outbreaks, the poultry industries of Brazil and Peru elected to utilize CEO ILT vaccines (Brazil) or recombinant ILT vaccines (Peru) to control disease outbreaks (Chacón & Ferreira, 2009; Chacón et al., 2010)....
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TL;DR: Results suggest that CEO laryngotracheitis vaccine viruses could increase in virulence after bird-to-bird passages causing severe outbreaks in susceptible birds.
Abstract: 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.
23 citations
TL;DR: Data on genomic variation among Italian ILTV strains is generated revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and may be involved in ILTV virulence/attenuation.
Abstract: Infectious laryngotracheitis (ILT) is an acute and highly contagious respiratory disease of chickens caused by an alphaherpesvirus, infectious laryngotracheitis virus (ILTV). Recently, full genome sequences of wild-type and vaccine strains have been determined worldwide, but none was from Europe. The aim of this study was to determine and analyse the complete genome sequences of five ILTV strains. Sequences were also compared to reveal the similarity of strains across time and to discriminate between wild-type and vaccine strains. Genomes of three ILTV field isolates from outbreaks occurred in Italy in 1980, 2007 and 2011, and two commercial chicken embryo origin (CEO) vaccines were sequenced using the 454 Life Sciences technology. The comparison with the Serva genome showed that 35 open reading frames (ORFs) differed across the five genomes. Overall, 54 single nucleotide polymorphisms (SNPs) and 27 amino acid differences in 19 ORFs and two insertions in the UL52 and ORFC genes were identified. Similarity among the field strains and between the field and the vaccine strains ranged from 99.96% to 99.99%. Phylogenetic analysis revealed a close relationship among them, as well. This study generated data on genomic variation among Italian ILTV strains revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and may be involved in ILTV virulence/attenuation. The results of this study can contribute to the understanding of the molecular bases of ILTV pathogenicity and provide genetic markers to differentiate between wild-type and vaccine strains.
23 citations
TL;DR: An outbreak of infectious laryngotracheitis in a multi-age laying hen facility in Minas Gerais state, Brazil, is described and marked necrosis and desquamation of respiratory ephitelium and conjunctiva with numerous syncytial cells formation and fibrinous exudate was observed.
Abstract: A recent (November 2010) outbreak of infectious laryngotracheitis (ILT) in a multi-age laying hen facility in Minas Gerais state, Brazil, is described. Previous ILT outbreak in laying hens was only notified in Sao Paulo state, Brazil, in 2002. In the outbreak described here, the affected population was approximately eight million hens, with flock sizes ranging from 100,000 to 2,900,000 chickens. The average mortality ranged from 1 to 6%, and morbidity was around 90% (most of the twenty seven farms of the area were positive for ILT virus). Three multi-age laying farms from one company were selected for this report. Clinical signs included prostration, dyspnea, conjunctivitis, occasional swelling of the paranasal sinuses and bloody mucous nasal discharge. Severely affected chickens presented with dyspnea, gasping and became cyanotic before death. At necropsy, these chickens had fibrinous exudate blocking the larynx and the lumen of cranial part of the trachea. In addition, conjunctivitis with intense hyperemia, edema and sinuses with caseous exudate were present. On histopathology, there were marked necrosis and desquamation of respiratory ephitelium and conjunctiva with numerous syncytial cells formation and fibrinous exudate. Moderate to marked non suppurative (especially lymphocytes and plasma cells) infiltration in the lamina propria also was observed. Sixteen out of 20 examined chickens, eosinophilic intranuclear inclusion bodies were observed in the syncytial cells. The DNA extracted from larynx and trachea produced positive PCR results for ILT virus (ILTV) DNA using formalin-fixed, paraffin embedded (FFPE) samples. Amplicons from a small region of ICP4 gene were submitted to sequencing and showed 100% identity with ILTV EU104910.1 (USA strain), 99% with ILTV JN596963.1 (Australian strain) and 91% with ILTV JN580316.1 (Gallid herpesvirus 1 CEO vaccine strain) and JN580315.1 (Gallid herpesvirus 1 TCO vaccine strain).
20 citations
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TL;DR: MEGA2 vastly extends the capabilities of MEGA version 1 by facilitating analyses of large datasets, enabling creation and analyses of groups of sequences, and expanding the repertoire of statistical methods for molecular evolutionary studies.
Abstract: Summary: We have developed a new software package, Molecular Evolutionary Genetics Analysis version 2 (MEGA2), for exploring and analyzing aligned DNA or protein sequences from an evolutionary perspective. MEGA2 vastly extends the capabilities of MEGA version 1 by: (1) facilitating analyses of large datasets; (2) enabling creation and analyses of groups of sequences; (3) enabling specification of domains and genes; (4) expanding the repertoire of statistical methods for molecular evolutionary studies; and (5) adding new modules for visual representation of input data and output results on the Microsoft Windows platform. Availability: http://www.megasoftware.net.
6,184 citations
"Characterization by restriction fra..." refers methods in this paper
...Phylogenetic trees were generated by the neighbour-joining methods with 1000 bootstrap replicates using the MEGA software program version 3.1 (Kumar et al., 2001)....
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Journal Article•
TL;DR: The complete recovery of DNA from samples used for the RNA and protein isolation makes it possible to normalize the results of gene expression studies based on DNA content instead of on the more variable total RNA, protein content or tissue weight.
Abstract: This report describes a new method for simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. The method is based on the use of a reagent containing phenol and guanidine thiocyanate. A biological sample is homogenized in the reagent and the simultaneous isolation of RNA, DNA and proteins is accomplished in a single step by a liquid-phase separation. The isolation of RNA can be completed in about 1 h, and DNA and proteins in about 3 h. The simultaneously isolated RNA, DNA and proteins are ready for Northern, Southern and Western blotting. The complete recovery of DNA from samples used for the RNA and protein isolation makes it possible to normalize the results of gene expression studies based on DNA content instead of on the more variable total RNA, protein content or tissue weight.
2,807 citations
"Characterization by restriction fra..." refers methods in this paper
...DNA from field samples, homogenized CAM plaques and commercial vaccine strains was extracted according to Chomczynski (1993) and Chacón & Ferreira (2008)....
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TL;DR: Modified-live infectious laryngotracheitis vaccine viruses, both tissue-culture-origin and chicken-embryo-origin, were passaged 20 times in specific-pathogen-free chickens to suggest that ML ILT vaccine viruses may increase in virulence after bird-to-bird passage.
Abstract: Modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, both tissue-culture-origin (TCO) and chicken-embryo-origin (CEO), were passaged 20 times in specific-pathogen-free chickens. After serial bird-to-bird passage, increased virulence was observed for CEO virus but not TCO virus. Increased mortality and increased severity and duration of respiratory disease were observed in chickens inoculated with chicken-passaged CEO viruses; only mild respiratory disease (no mortality) occurred in chickens inoculated with chicken-passaged TCO viruses. These findings suggest that ML ILT vaccine viruses may increase in virulence after bird-to-bird passage.
170 citations
01 Jan 2016
TL;DR: In this paper, modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, both tissue-culture-origin (TCO) and chicken-embryo origin (CEO), were passaged 20 times in specific-pathogen-free chickens.
Abstract: SUMMARY. Modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, both tissue-culture-origin (TCO) and chicken-embryo-origin (CEO), were passaged 20 times in specific-pathogen-free chickens. After serial bird-to-bird passage, increased virulence was observed for CEO virus but not TCO virus. Increased mortality and increased severity and duration of respiratory disease were observed in chickens inoculated with chicken-passaged CEO viruses; only mild respiratory disease (no mortality) occurred in chickens inoculated with chicken-passaged TCO viruses. These findings suggest that ML ILT vaccine viruses may increase in virulence after bird-to-bird passage.
166 citations
TL;DR: It is concluded that the trigeminal ganglion is the main site of latency of ILT virus in laying chickens, as it did not detect viral DNA in any of the other tissues sampled from clinically recovered birds.
Abstract: Mature laying chickens were inoculated intratracheally with a field strain of infectious laryngotracheitis (ILT) virus. Tracheal swabs were collected regularly from all birds for virus culture. At various times post-inoculation, pairs of birds were killed and tissues removed for detection of virus products using conventional tissue homogenization and culture, organ culture, indirect immunofluorescence (IF) and also the polymerase chain reaction (PCR). The latter was used to detect a DNA sequence from the ILT virus thymidine kinase gene. Following inoculation the birds developed mild respiratory disease with clinical signs characteristic of ILT from 3 to 10 days post-inoculation. Trachea and turbinate tissues were virus-positive as determined by virus isolation, organ culture, IF and PCR on day 4 post-inoculation. After recovery from the acute phase, virus shedding initially ceased, then intermittent, low level shedding was recorded for five of the six remaining birds. In an attempt to locate sites of latency, pairs of birds were sampled at 31, 46 and 61 days post-inoculation. Virus was not detected in upper respiratory tract or ocular tissues by conventional techniques, or in the trigeminal, proximal and distal ganglia. All tissues were also negative by PCR, except for the trigeminal ganglia of five of the six birds. All PCR-positive birds had previously shed ILT virus intermittently between days 19 and 59 post-inoculation. As we did not detect viral DNA in any of the other tissues sampled from clinically recovered birds, we conclude that the trigeminal ganglion is the main site of latency of ILT virus.
130 citations
"Characterization by restriction fra..." refers background in this paper
...The capacity of ILTV strains to maintain a state of latency in the chickens, in particular within the trigeminal ganglion, is a distinct characteristic of GaHV-1 that makes the disease difficult to control (Williams et al., 1992)....
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