Characterization of infectious laryngotracheitis virus (ILTV) isolates from commercial poultry by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP)

Infectious laryngotracheitis (ILT) is a highly contagious, acute respiratory disease of chickens, of worldwide distribution, that affects growth and egg production and leads to significant economic losses during periodic outbreaks of the disease. Live attenuated vaccines (chicken embryo origin [CEO] and tissue-culture origin [TCO]) have been widely used to control the disease in the United States. It is believed that most of the outbreaks in the United States are caused by vaccine-related isolates that persist in the field and spill over into na?ve poultry populations. The objective of this study was to utilize the previously developed polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis to genotype recent ILT virus (ILTV) isolates from commercial poultry. Forty-six samples were collected during January 2006 to April 2007 from five poultry production regions of the United States and were characterized within PCR-RFLP groups III-VI. Sixty-three percent of the samples analyzed were categorized as closely related to the vaccine strains (groups III-V), whereas 33% were categorized as group VI viruses that differed in six and nine PCR-RFLP patterns from the CEO and TCO vaccines; a mixture of group IV and V viruses was detected in two samples (4%). In general, groups V and VI were the most prevalent viruses, found in 52% and 33% of the samples tested respectively. Both types of viruses were detected in vaccinated and nonvaccinated flocks. Although genetically different, both viruses produced severe disease in the field.     

Analysis of infectious laryngotracheitis virus isolates from Ontario and New Brunswick by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify DNA of infectious laryngotracheitis virus (ILTV) isolates obtained from field specimens. The examined 47 samples included 37 isolates representing 35 cases of infectious laryngotracheitis from Ontario and 10 isolates originating from 10 field cases in New Brunswick. The viruses were grown in either embryonated chicken eggs or cell culture, the DNA extracted and amplified using primers designed from the sequence information of a 1.1 kb BamHI fragment of the Ontario 1598 ILTV strain. Thirty-four of the Ontario isolates and all of the New Brunswick isolates were amplified successfully. This suggests that the selected primers would be useful for the majority of the isolates encountered in outbreaks of ILTV.     

Efficacy of live virus vaccines against infectious laryngotracheitis assessed by polymerase chain reaction-restriction fragment length polymorphism

The efficacy of four different commercial live vaccines (vaccines A, B, C, and D) against the infectious laryngotracheitis virus (ILTV) was assessed in specific-pathogen-free (SPF) chickens. SPF chickens were vaccinated intraocularly at 6 wk old with ILTV live vaccines and were challenged intratracheally with the N91B01 strain of virulent Korean ILTV 2 wk after vaccination. The immunity against ILTV live vaccines was assessed by the incidence of latent infection by the challenge virus in the chickens' tracheas and trigeminal ganglia, the reisolation rate of the challenge virus, and the clinical signs in the chickens challenged with the N91B01 strain of ILTV. The latent infection in chickens was assessed by nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Our data showed that the clinical signs and challenge virus isolation were negative in all chickens receiving four difference commercial ILTV live vaccines. The viral DNA of the vaccine strain, but not that of the challenge virus, was detected in chickens vaccinated with vaccine A by nested PCR-RFLP. The viral DNAs of both the vaccine and challenge strains were detected from chickens vaccinated with vaccines B, C, and D. This study showed that only vaccine A can protect chickens from latent infection with the field virulent ILTV. We speculate that the efficacy of infectious laryngotracheitis live vaccines to protect chickens from latent infection with virulent ILTVs can be assessed by nested PCR-RFLP analysis.     

Differentiation of vaccine strains and Georgia field isolates of infectious laryngotracheitis virus by their restriction endonuclease fragment patterns

Seven restriction endonucleases (REs) were used to cleave the DNA from seven vaccine strains of infectious laryngotracheitis (ILT) virus and from six Georgia field isolates of ILT virus. After electrophoresis of the resulting RE fragments, the patterns were compared in order to differentiate strains of ILT virus. The six chicken-embryo-origin (CEO) vaccines were identical with each RE, but the tissue-culture-origin (TCO) vaccine strain differed from the CEO vaccines using five of the REs. Four of the six field isolates were identical by each RE, but two field isolates differed from each other and from the four identical field isolates on the basis of patterns produced by some but not all of the REs. The four identical field isolates could not be differentiated from the CEO vaccine strains by any RE, but the other two field isolates were not identical to either strain of vaccine virus. This work demonstrates that differentiable strains of ILT virus exist in the United States and that viruses other than vaccine viruses are involved in field outbreaks of ILT.     

Differentiation by polymerase chain reaction - restriction fragment length polymorphism of some Oestridae larvae causing myiasis

The cytochrome oxidase I (COI) gene of the most wide-spread Italian species of Oestridae larvae causing myiasis (Gasterophilus spp., Hypoderma bovis, Hypoderma lineatum, Oestrus ovis and Przhevalskiana silenus) was amplified by polymerase chain reaction (PCR) using conserved primers. Restriction fragment length polymorphism (RFLP) of amplicons was also carried out and their restriction profiles compared. A clear genetic difference between the Oestridae larvae examined was demonstrated by using Taq I, Hinf I, Rsa I and Hpa II enzymes. No intra-specific variation in RFLPs was detected between the two species of Hypoderma. The results highlight the taxonomic and phylogenetic relationships among larvae belonging to the different subfamilies, and thus offer additional diagnostic and epidemiological instruments.     

Characterization of western European field isolates and vaccine strains of avian infectious laryngotracheitis virus by restriction fragment length polymorphism and sequence analysis

Infectious laryngotracheitis is a dramatic disease of the upper respiratory tract in poultry caused by a herpesvirus. In this study we investigated the characteristics of western European field isolates of infectious laryngotracheitis virus (ILTV) to gain more information on their diversity. The examined 104 isolates, collected from acute outbreaks during the last 35 years, originated from eight different countries: Switzerland (48), Germany (21), Sweden (14), the United Kingdom (9), Italy (5), Belgium (4), Austria (2), and Norway (1). Two vaccines, a chicken embryo origin product and a tissue culture origin product, were included in the survey. Polymerase chain reaction (PCR) was performed to amplify a 2.1-kb DNA fragment of ILTV using primers generated for the thymidine kinase (TK) gene. After digestion of the resulting PCR products by restriction endonuclease HaeIII, restriction fragment length polymorphism analysis was carried out. PCR amplicons of three field isolates and both vaccine strains were selected for sequencing. Here 98 field isolates showed the same cleavage pattern and were identical to both vaccine strains (clone 1). They differed from five Swiss isolates with identical cleavage pattern (clone 2) and one Swedish isolate (clone 3). The present study demonstrated that at least three clones of ILTV have been circulating in western Europe during the last 35 years. The 104 isolates analyzed showed a high genetic similarity regarding the TK gene, and a large majority of the field isolates (98/104) were genetically related to the vaccine strains.     

鸡传染性喉气管炎病毒PCR诊断方法的建立与应用

根据GenBank登录的传染性喉气管炎病毒(ILTV)的TK基因序列设计并合成1对特异性引物,以ILTV疫苗株DNA为模板,建立了检测ILTV TK基因的PCR方法。应用该方法能从临床分离毒株和疫苗株中扩增到长为427 bp的目的片段;但不能从新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)、禽呼肠孤病毒(ARV)、减蛋综合征病毒(EDSV)、H9亚型禽流感病毒(H9-AIV)、传染性支气管炎病毒(IBV)、大肠杆菌以及金黄色葡萄球菌等病原中扩增出阳性条带;敏感性试验表明其DNA最小检出量为4.9 ng;应用该方法和病毒分离法对2份临床病例和人工感染鸡的检测,两者符合率为100%。上述结果表明该PCR方法具有良好的特异性和敏感性,可用于传染性喉气管炎病毒鉴定和临床诊断。     

Analysis of Korean strains of infectious laryngotracheitis virus by nucleotide sequences and restriction fragment length polymorphism

Korean field strains of infectious laryngotracheitis virus (ILTV) were analyzed by comparison of nucleotide sequences of thymidine kinase (TK) and glycoprotein G (gG) genes and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns. Main differences among TK gene sequence were found in both amino acid at 252 and mRNA polyadenylation signals. In virulent strains, amino acid 252 of TK gene was methionine but was threonine in low virulence and vaccine strains. The mRNA polyadenylation signals of TK gene were identified at 24bp downstream from the stop codon in virulent strains, but not in low virulence and vaccine strains. The gG gene of all virulent strains showed the same nucleotide sequence except for N87278 which had a gG gene sequence identical to that of vaccine strains. The virulent ILTV strains differed from low virulence and vaccine strains in PCR-RFLP patterns of TK and gG genes. The RFLP patterns of TK and gG genes of low virulence ILTV strains were identical to those of vaccine strains. In the case of N87278, the PCR-RFLP patterns of TK and gG genes were identical to those of virulent and vaccine strains of ILTV, respectively. From these results, ILTV field strains were classified into three groups according to sequences of TK and gG genes and PCR-RFLP, and the virulent ILTV strains could be discriminated from low virulence and vaccine strains by PCR-RFLP of TK gene. And it was suspected that N87278 might be produced by in vivo recombination between virulent and vaccine strains of ILTV.     

Epidemic of infectious laryngotracheitis in Italy: characterization of virus isolates by PCR-restriction fragment length polymorphism and sequence analysis

Between May 2007 and October 2008, 34 outbreaks of mild to moderate forms of infectious laryngotracheitis (ILT) occurred in commercial broiler flocks in Italy. Affected birds showed watery eyes, conjunctivitis, nasal discharge, reduction of feed and water consumption, and gasping with expectoration of blood-stained mucus. The mortality rate was < 10%. Gross lesions consisted of conjunctivitis, excess of mucus, blood, or presence of diphtheritic membranes in trachea. A real-time PCR assay was performed to confirm the presence of ILT virus (ILTV) DNA in tracheal tissue homogenates. Twenty-three ILTV isolates were propagated on the chorion-allantoic membrane of embryonated chicken eggs showing typical plaques. PCR combined with restriction fragment length polymorphism and gene sequencing of isolates showed a high genetic correlation between field strains and chicken embryo origin vaccines.     

Differentiation of infectious laryngotracheitis virus strains using restriction endonucleases

Strains of infectious laryngotracheitis virus (ILTV) were examined using an indirect immunofluorescent test (IIF) and with restriction endonucleases for detecting intratypic differences. Electrophoretic analysis of ILTV DNA fragments cleaved with restriction endonuclease Hind 111 clearly distinguished between strains. The IIF test did not discriminate between strains. A molecular weight estimate of ILTV DNA was made by summation of restriction endonuclease fragments cleaved with BamH1 (102.1 X 10(6)) and Hind111 (97.35 X 10(6)). Differences between the estimates may indicate the presence of submolar fragments.     

Detection of infectious laryngotracheitis virus in formalin-fixed, paraffin-embedded tissues by nested polymerase chain reaction

Infectious laryngotracheitis virus (ILTV) is routinely diagnosed by histopathologic examination of trachea, eyelid, and lung tissues. Lesions consistent with infectious laryngotracheitis (ILT) infection include syncytial cell formation with intranuclear inclusion bodies. These changes are present during the acute phase of infection. To increase the sensitivity of detecting ILT, a nested polymerase chain reaction (PCR) was developed for detection of ILTV DNA. Nested PCR assay was specific for the amplification of ILTV DNA and did not amplify a variety of other avian pathogens. To further validate the ability of this assay to detect ILT, nested PCR was performed in formalin-fixed, paraffin-embedded tissues from 35 cases of respiratory disease. Of the 35 cases, 12 were considered ILT suspects on the basis of initial clinical observation. Eleven of the 12 ILT-suspect cases were diagnosed as ILT, and the remaining 24 were diagnosed as nonspecific tracheitis (NST) by histopathologic examination. Histopathologically positive samples were confirmed by direct fluorescent antibody test and virus isolation. Of the 11 ILT-positive cases, 10 were positive by nested PCR. In addition, ILTV DNA was detected in 7 of the 24 samples diagnosed as NST upon histopathologic examination. Therefore, by nested PCR, ILTV DNA was detected in tissues independently of the presence of syncytial cells, intranuclear inclusions, or both. ILT nested PCR is a specific and sensitive assay capable of detecting ILT at different stages of infection and can be utilized in combination with histopathological examination to accelerate the diagnosis of ILT infection.     

Detection and identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay

The polymerase chain reaction (PCR) with primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. A primer pair designed for the detection of human and rodent mycoplasmal species was examined for its ability to detect the most important avian mycoplasmas. After testing the respective reference strains, we found that Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamHI and RsaI). For the detection of Mycoplasma gallisepticum by PCR, we needed species-specific primers. The results of the PCR- and RFLP-based identification procedures of 17 different field isolates agreed with those obtained by conventional methods.     

Differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their ORFs 6 and 7 genes.

Three distinct antigenic profiles were identified by comparing the reactivities of 15 Canadian field isolates, the attenuated U.S. vaccine (Ingelvac MLV) strain and 2 European reference strains (Lelystad and Weybridge) of the porcine reproductive and respiratory syndrome virus (PRRSV) by indirect immunofluorescence with a set of 4 monoclonal antibodies to the nucleocapsid (N) protein and 2 other to the matrix (M) protein. In the present study, 9 Canadian isolates for which the sequences were determined appeared closely related to 2 U.S. reference strains (ATCC VR-2332 and ATCC VR-2385) with amino acid identities varying between 90 to 98% for the M and N proteins; substitutions in the nucleotide sequences were distributed randomly throughout the ORFs 6 and 7 genes, and most were 3rd base silent mutations. In comparison, more than 30% divergence was demonstrated with the Lelystad virus. Furthermore, differentiation between North American and European isolates, and between field isolates and the MLV strain could be achieved by cutting PCR-amplified products encompassing both ORFs 6 and 7 genes with 4 restriction endonucleases. When taken individually, BsaJI and AluI were the more appropriate restriction enzymes for distinguishing the vaccine strain from field isolates. The results obtained suggest that the restriction fragment length polymorphism of the genomic region covering the ORFs 6 and 7 genes may be a valuable tool to differentiate among PRRSV isolates.     

Identification of trichomonadid protozoa from the bovine preputial cavity by polymerase chain reaction and restriction fragment length polymorphism typing.

Accurate identification of the bovine pathogen Tritrichomonas foetus is sometimes complicated by the presence of other trichomonadid protozoa in clinical samples. A highly specific and reproducible approach for differentiating 3 common types of bovine trichomonadid protozoa found in the bovine preputial cavity, T. foetus, Pentatrichomonas hominis, and a Tetratrichomonas species, was developed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Universal trichomonadid protozoa primers, TFR1 and TFR2, were used to amplify the 5.85 rRNA gene and internal transcribed spacer regions (ITSRs), and the products were digested with the restriction enzyme HpyCH4IV. Restriction fragment length polymorphism analysis was performed on 55 trichomonad isolates from bovine preputial washing and scraping samples. The RFLP results correlated 100% with 5.85 rRNA gene and ITSR sequence resultsand PCR results with primers specific for T. foetus. The results of this study demonstrate that PCR and RFLP analysis can be used in lieu of DNA sequencing to identify the specific trichomonadid protozoa isolated from the bovine preputial cavity.     

Differentiation between vaccine strain and field isolates of classical swine fever virus using polymerase chain reaction and restriction test.

The CS vaccine strain of Classical Swine Fever Virus is a derivative from the LK parental strain that has been used in Russia for more than 30 years. A 10697 nucleotide fragment of the CS strain's genome has been sequenced. Sixteen unique restriction markers have been found in the CS genome comparing to the following strains: Alfort187, Alfort Tubingen, Brescia, CAP, Glentorf, ALD, GPE-, Chinese, C-strain, Riems, P97. Fourteen of these sites (AflII, AvaI, CfoI, Eco47II, HaeII, KpnI, MunI, NspI, PstI, ScaI, SmaI, SpeI, StyI, VspI) are only present in the CS strain genome. The 2 sites (BgII, NdeI) are present in all other genomes except for the genome of vaccine strain. A PCR/restriction test has been developed based on these findings in order to distinguish the vaccine strain from field isolates. Two pairs of nested primers and a criteria of analysis have been designed for each restriction marker site. The tests have been conducted first on the reference strains resulting in predicted restriction patterns. Finally, the tests have been applied to a number of field isolates obtained at different locations in Russia in different years. These results give further evidence that PCR/restriction tests can identify the LK and CS vaccines helping to avoid confusion with field strains.     

Nucleotide sequence and polymerase chain reaction/restriction fragment length polymorphism analyses of Aleutian disease virus in ferrets in Japan.

Two ferrets with spontaneous Aleutian disease (AD) were found in Japan. The diagnosis was verified by polymerase chain reaction (PCR) amplification of part of the capsid gene specific to AD virus (ADV). The nucleotide sequences (365 bp in length) of the amplified fragments from the 2 ferrets differed by a single nucleotide, producing an amino acid alteration. Compared with other types of ADV, these isolates had 96% sequence similarity to a published ferret ADV (FADV) in contrast to <91% homology to various types of mink ADV (MADV). The phylogenetic tree of ADVs indicates that these 2 isolates and the published FADV belong to the same genetic group and definitely are divergent from MADVs. The predicted amino acid sequence of the hypervariable segment in the capsid gene was conserved among the 3 types of FADV. These results indicated that the 2 isolates found in Japan were new DNA types of FADV and could have been derived from FADV(s). A restriction fragment length polymorphism (RFLP) method to distinguish the ferret types of ADV from the mink types of ADV was developed on the basis of differences in their nucleotide sequences. Digestion of the PCR products with Afal or ScaI provided different cleavage patterns for FADV and MADV. This PCR/RFLP analysis of the ADV capsid gene will be a valuable asset for diagnosis of this virus infection in ferrets.