Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus.

A previous sequence analysis of a US5 gene fragment of infectious laryngotracheitis virus (ILTV) performed in an Argentinian epidemiological study allowed to differentiate between wild and vaccine strains. This analysis also defined five ILTV haplotypes with specific variations at positions 461, 484, 832, 878 and 894 of the US5 gene. This characterization of viral strains may also be accomplished using the High-Resolution Melting Analysis (HRMA), which has been described as an effective, fast and sensitive method to detect mutations in PCR products. In the present study, an HRM protocol was developed with the aim of characterizing the circulating ILTV strains in Argentina. The specificity of this tool was confirmed in different DNA diluents, without interference from heterologous DNA or other cellular metabolites. Additionally, the salt concentration in the elution buffer used for DNA extraction did not alter the curve profiles. Higher concentrations of DNA (Ct≅26.0) displayed well-defined curve profiles, whereas lower concentrations (Ct≅32.5) exhibited more heterogeneous curves. The HRMA showed 97.49% concordance with the reference technique, i.e., sequencing. The HRM protocol has the capability to perform DNA amplification prior to its characterization. Thus, eventually this technique may be used simultaneously as a diagnostic tool. This advantage implies a significant reduction in the time and effort involved in sample processing.

A Review of Eight High-Priority, Economically Important Viral Pathogens of Poultry within the Caribbean Region.

Viral pathogens cause devastating economic losses in poultry industries worldwide. The Caribbean region, which boasts some of the highest rates of poultry consumption in the world, is no exception. This review summarizes evidence for the circulation and spread of eight high-priority, economically important poultry viruses across the Caribbean region. Avian influenza virus (AIV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV), fowl adenovirus group 1 (FADV Gp1), and egg drop syndrome virus (EDSV) were selected for review. This review of serological, molecular, and phylogenetic studies across Caribbean countries reveals evidence for sporadic outbreaks of respiratory disease caused by notifiable viral pathogens (AIV, IBV, NDV, and ILTV), as well as outbreaks of diseases caused by immunosuppressive viral pathogens (IBDV and FADV Gp1). This review highlights the need to strengthen current levels of surveillance and reporting for poultry diseases in domestic and wild bird populations across the Caribbean, as well as the need to strengthen the diagnostic capacity and capability of Caribbean national veterinary diagnostic laboratories.

Phylogenetic Evidence of a Close Relationship between the Peruvian Strain Vfar-043 and Two U.S. Origin Iltv Field Strains.

Infectious laryngotracheitis virus (ILTV) is the causative agent of an acute respiratory avian disease known as infectious laryngotracheitis (ILT), which has been associated with economic losses in poultry. The presence of ILTV has been widely reported in South American countries; however, only one full genomic sequence (VFAR-043 strain) has been recently published, from an outbreak in Peru. The aim of this study was to determine the genetic relationship of the Peruvian strain with other ILTV strains from different geographic regions. The phylogenetic analyses revealed a close relationship between VFAR-043 and two U.S. origin strains (1874C5 and J2) using only the whole genome, Unique Long (UL), and Unique Short (US) genomic regions. Then these three genomic sequences were compared to evaluate their genetic variations using the USDAref as a reference strain. Genetic variations such as synonymous and nonsynonymous single-nucleotide polymorphisms, insertions, deletions, and nucleotide-codon variations were identified among these three strains. Moreover, the phylogenetic tree analysis using gene sequences of the US5 and ICP4 coding regions from South American isolates showed that VFAR-043 does not have a close relationship with either the Argentinian (US5) or Brazilian (ICP4) reported sequences. However, a close relationship was observed between VFAR-043 and another Peruvian isolate (USP-81) when the ICP4 gene sequence was analyzed. All these results suggest that VFAR-043 together with 1874C5 and J2 are closely related. These findings contribute to our understanding of the epidemiology of ILTV in South America.

Evidencia filogenética de una relación genética cercana entre la cepa peruana del virus de la laringotraqueítis infecciosa aviar VFAR-043 y dos cepas de campo con origen en los Estados Unidos. El virus de laringotraqueítis infecciosa aviar es el agente causal de una enfermedad aviar respiratoria aguda conocida como laringotraqueítis infecciosa que está asociada con pérdidas económicas en la industria avícola. La presencia del virus de la laringotraqueítis infecciosa ha sido ampliamente reportada en países de América del Sur; sin embargo, solamente una secuencia genómica completa (cepa VFAR-043) ha sido publicada recientemente y obtenida a partir de un brote en Perú. El objetivo de este estudio fue determinar la relación genética de la cepa peruana con otras cepas de diferentes regiones geográficas. El análisis filogenético reveló una cercana relación entre el virus VFAR-043 y dos cepas de los Estados Unidos (18746C5 y J2) usando el genoma completo y las regiones genómicas única larga (UL) y la región genómica única corta (UC). Posteriormente, estas tres secuencias genómicas fueron comparadas con la cepa de referencia del Departamento de Agricultura de los Estados Unidos (USDAref) para evaluar sus variaciones genéticas. Variaciones genéticas como polimorfismo de nucleótido único (con las siglas en inglés SNP) tanto de tipo sinónimo y no-sinónimo, inserciones, deleciones y variación de nucleótido en un codón fueron identificadas entre estas tres cepas (VFAR-043, 18746C5 y J2). Además, el análisis de los árboles filogenéticos usando secuencias genéticas de la región codificadora de US5 e ICP4 de aislamiento sudamericanos reveló que el virus VFAR-043 no mostró relación genética cercana con secuencias argentinas (US5) ni secuencias brasileras (ICP4) que están reportadas. No obstante, se observó una relación cercana entre el virus VFAR-043 y otro aislamiento peruano (USP-81) cuando se analizó la secuencia genética del gen ICP4. Todos estos resultados sugieren que los virus VFAR-043, 1874C5 y J2 están genéticamente relacionados. Estos hallazgos contribuyen al conocimiento de la epidemiologia del virus de la laringotraqueítis infecciosa aviar en América del Sur.

Molecular Characterization and Cluster Analysis of Field Isolates of Avian Infectious Laryngotracheitis Virus from Argentina.

Avian infectious laryngotracheitis (ILT) is a worldwide infectious disease that causes important economic losses in the poultry industry. Although it is known that ILT virus (ILTV) is present in Argentina, there is no information about the circulating strains. With the aim to characterize them, seven different genomic regions (thymidine kinase, glycoproteins D, G, B, C, and J, and infected cell polypeptide 4) were partially sequenced and compared between field samples. The gJ sequence resulted to be the most informative segment, it allowed the differentiation among field sample strains, and also, between wild and vaccine viruses. Specific changes in selected nucleotidic positions led to the definition of five distinct haplotypes. Tests for detection of clustering were run to test the null hypothesis that ILTV haplotypes were randomly distributed in time in Argentina and in space in the most densely populated poultry region of this country, Entre Rios. From this study, it was possible to identify a 46 km radius cluster in which higher proportions of haplotypes 4 and 5 were observed, next to a provincial route in Entre Rios and a significant decline of haplotype 5 between 2009 and 2011. Results here provide an update on the molecular epidemiology of ILT in Argentina, including data on specific genome segments that may be used for rapid characterization of the virus in the field. Ultimately, results will contribute to the surveillance of ILT in the country.

Persistence of the tissue culture origin vaccine for infectious laryngotracheitis virus in commercial chicken flocks in Brazil.

Infectious laryngotracheitis (ILT) is a respiratory disease of great importance that causes serious economic losses in the poultry industry. Its control is based on biosecurity procedures and vaccination programs that use live attenuated vaccines such as tissue culture origin (TCO), chicken embryo origin (CEO), and vectored vaccines. However, problems have been reported, such as the reversion of virulence, virus latency, and field virus outbreaks. Several molecular techniques have been developed to differentiate between the field and vaccine strains. This study was conducted to determine the presence of infectious laryngotracheitis virus (ILTV) in Brazil from 2012 to 2014. PCR-RFLP (restriction fragment length polymorphism) was used to detect and differentiate ILTV strains; DNA sequencing and predictive RFLP analysis were also used for this purpose. Molecular analysis detected the presence of ILTV in 15 samples that were characterized as strains of TCO vaccine origin. This study showed that the ILTV TCO vaccine strain has been circulating in commercial chicken flocks in Brazil since its introduction during the 2002 outbreak.

A sensitive nested-Polymerase Chain Reaction protocol to detect infectious laryngotracheitis virus

Background: Infectious laryngotracheitis virus (ILTV) is a member of the family Herpesviridae that has a worldwide distribution, although it is well controlled in areas of intensive production in which periodic outbreaks of the disease occur. ILTV is an important respiratory pathogen of chickens that may cause severe or mild disease in layers and broilers. Severe disease is characterized by respiratory depression, gasping, expectoration of bloody exudate and high mortality. Mild diseased chickens exhibit milder clinical signs and low mortality, and laboratory techniques are mandatory for a final diagnosis. Several techniques have been described for the detection of ILTV, however they have disadvantages that constrains their use in routine diagnosis. Viral multiplication is limited to respiratory tissue, which makes the trachea the ideal site to look for the virus. The purpose of the present study was to develop a sensitive and specific nested Polymerase Chain Reaction (PCR) protocol to detect ILTV DNA directly from tracheal swabs of naturally or experimentally infected chickens. Materials, Methods & Results: The nested-PCR was carried out with two sets of primers selected from a portion of the ILTV thymidine kinase gene. PCR sensitivity was determined by using five-fold serial dilutions of a commercial laryngotracheitis vaccine. PCR was specific as determined by testing related respiratory viruses (pathogens of chickens), ILTV strain, and field isolates. Nested-PCR was 250 times more sensitive than virus isolation (VI). To further validate the ability of this assay to detect ILTV from tracheal swabs, experimentally infected chickens and ILTV suspect cases were examined by VI, PCR, and histopathology. VI and nested-PCR both detected virus in tracheas during all the experimental period. With one exception, all positive samples by VI were also positive by the nested-PCR. However, nested-PCR detected 5 additional positive samples in the end of the experimental period. Through direct histopathology, typical syncytia and inclusion bodies were found in only two samples. In the clinical cases of respiratory illness, VI detected ILTV positive samples in 4 out of the 8 flocks with respiratory illness and histopathological analyses detected 3 flocks but the nested-PCR detected 5 positive fl ocks including those positive by VI and histopathology. Discussion: In the experimental infection, VI and PCR both detected ILTV in the majority of the samples but PCR detected some additional positive samples close to the end of the experimental period. By comparison of the PCR with VI sensitivity, it can be concluded that the protocol here described has a greater advantage over the previously described protocols that afford a direct comparison. Histopathology was the least sensitive test, since viral inclusion bodies and syncytial cells were only observed in two tracheal sections and a possible explanation for this result may be that necrosis and desquamation destroy infected epithelium. In the detection of the virus from clinical cases, the nested-PCR also detected a greater number of positive samples than VI and histopathology. Comparison of the nested-PCR with VI in experimentally infected broilers indicates that the two diagnostic tests are very efficient to detect ILTV in the early time of infection. However, VI tests in late infection may be not as sensitive as the nested-PCR if majority of the ILTV have been inactivated or become latent. Two distinctive sequences were obtained from the positive controls and field isolates. The sequences were specific to ILTV since they had a minimum of 99.5% similarity with previously described strains. The assay described in the present study indicates that the nested-PCR protocol is more sensitive than previously described tests and can replace histopathology and virus isolation with advantage.

A sensitive nested-polymerase chain reaction protocol to detect infectious Laryngotracheitis virus / A sensitive nested-polymerase chain reaction protocol to detect infectious Laryngotracheitis virus

Background: Infectious laryngotracheitis virus (ILTV) is a member of the family Herpesviridae that has a worldwide distribution, although it is well controlled in areas of intensive production in which periodic outbreaks of the disease occur. ILTV is an important respiratory pathogen of chickens that may cause severe or mild disease in layers and broilers. Severe disease is characterized by respiratory depression, gasping, expectoration of bloody exudate and high mortality. Mild diseased chickens exhibit milder clinical signs and low mortality, and laboratory techniques are mandatory for a fi nal diagnosis. Several techniques have been described for the detection of ILTV, however they have disadvantages that constrains their use in routine diagnosis. Viral multiplication is limited to respiratory tissue, which makes the trachea the ideal site to look for the virus. The purpose of the present study was to develop a sensitive and specifi c nested Polymerase Chain Reaction (PCR) protocol to detect ILTV DNA directly from tracheal swabs of naturally or experimentally infected chickens.Materials, Methods & Results: The nested-PCR was carried out with two sets of primers selected from a portion of the ILTV thymidine kinase gene. PCR sensitivity was determined by using fi ve-fold serial dilutions of a commercial laryngotracheitis vaccine. PCR was specifi c as determined by testing

Background: Infectious laryngotracheitis virus (ILTV) is a member of the family Herpesviridae that has a worldwide distribution, although it is well controlled in areas of intensive production in which periodic outbreaks of the disease occur. ILTV is an important respiratory pathogen of chickens that may cause severe or mild disease in layers and broilers. Severe disease is characterized by respiratory depression, gasping, expectoration of bloody exudate and high mortality. Mild diseased chickens exhibit milder clinical signs and low mortality, and laboratory techniques are mandatory for a fi nal diagnosis. Several techniques have been described for the detection of ILTV, however they have disadvantages that constrains their use in routine diagnosis. Viral multiplication is limited to respiratory tissue, which makes the trachea the ideal site to look for the virus. The purpose of the present study was to develop a sensitive and specifi c nested Polymerase Chain Reaction (PCR) protocol to detect ILTV DNA directly from tracheal swabs of naturally or experimentally infected chickens.Materials, Methods & Results: The nested-PCR was carried out with two sets of primers selected from a portion of the ILTV thymidine kinase gene. PCR sensitivity was determined by using fi ve-fold serial dilutions of a commercial laryngotracheitis vaccine. PCR was specifi c as determined by testing

A sensitive nested-polymerase chain reaction protocol to detect infectious Laryngotracheitis virus / A sensitive nested-polymerase chain reaction protocol to detect infectious Laryngotracheitis virus

Background: Infectious laryngotracheitis virus (ILTV) is a member of the family Herpesviridae that has a worldwide distribution, although it is well controlled in areas of intensive production in which periodic outbreaks of the disease occur. ILTV is an important respiratory pathogen of chickens that may cause severe or mild disease in layers and broilers. Severe disease is characterized by respiratory depression, gasping, expectoration of bloody exudate and high mortality. Mild diseased chickens exhibit milder clinical signs and low mortality, and laboratory techniques are mandatory for a fi nal diagnosis. Several techniques have been described for the detection of ILTV, however they have disadvantages that constrains their use in routine diagnosis. Viral multiplication is limited to respiratory tissue, which makes the trachea the ideal site to look for the virus. The purpose of the present study was to develop a sensitive and specifi c nested Polymerase Chain Reaction (PCR) protocol to detect ILTV DNA directly from tracheal swabs of naturally or experimentally infected chickens.Materials, Methods & Results: The nested-PCR was carried out with two sets of primers selected from a portion of the ILTV thymidine kinase gene. PCR sensitivity was determined by using fi ve-fold serial dilutions of a commercial laryngotracheitis vaccine. PCR was specifi c as determined by testing

Background: Infectious laryngotracheitis virus (ILTV) is a member of the family Herpesviridae that has a worldwide distribution, although it is well controlled in areas of intensive production in which periodic outbreaks of the disease occur. ILTV is an important respiratory pathogen of chickens that may cause severe or mild disease in layers and broilers. Severe disease is characterized by respiratory depression, gasping, expectoration of bloody exudate and high mortality. Mild diseased chickens exhibit milder clinical signs and low mortality, and laboratory techniques are mandatory for a fi nal diagnosis. Several techniques have been described for the detection of ILTV, however they have disadvantages that constrains their use in routine diagnosis. Viral multiplication is limited to respiratory tissue, which makes the trachea the ideal site to look for the virus. The purpose of the present study was to develop a sensitive and specifi c nested Polymerase Chain Reaction (PCR) protocol to detect ILTV DNA directly from tracheal swabs of naturally or experimentally infected chickens.Materials, Methods & Results: The nested-PCR was carried out with two sets of primers selected from a portion of the ILTV thymidine kinase gene. PCR sensitivity was determined by using fi ve-fold serial dilutions of a commercial laryngotracheitis vaccine. PCR was specifi c as determined by testing

Diverse uses of feathers with emphasis on diagnosis of avian viral infections and vaccine virus monitoring

The large amounts of feathers produced by the poultry industry, that is considered as a waste was explored for possible uses in various industries, such as meals for animals, biofuels, biodegradable plastic materials, combating water pollution and more. That review mentions these uses, but concentrate on the utilization of feathers for the diagnosis of viral infections and for monitoring vaccine viruses in chickens after vaccination. The viral diseases in which diagnosis using nucleic acids extracted from the feather shafts was described are, Marek's disease virus, circoviruses, chicken anemia virus, fowlpox virus, avian retroviruses, avian influenza virus and infectious laryngotracheitis virus. In two cases, of Marek's disease virus and of infectious laryngotracheitis virus, the differentiation of vaccine and wild-type viruses from feather shafts was made possible, thus allowing for monitoring the vaccination efficacy. The present review demonstrates also the stability of DNA viruses in feather shafts, and the possible evaluation of environmental dissemination of pathogens. When viruses are transmitted vertically, like in the cases of the retrovirus REV, a teratogenic effect on the development of feathers of the day-old newly hatched chick might occur in the case of avian influenza and the chicken anemia virus, which might indicate on a viral infection.

Diverse uses of feathers with emphasis on diagnosis of avian viral infections and vaccine virus monitoring

The large amounts of feathers produced by the poultry industry, that is considered as a waste was explored for possible uses in various industries, such as meals for animals, biofuels, biodegradable plastic materials, combating water pollution and more. That review mentions these uses, but concentrate on the utilization of feathers for the diagnosis of viral infections and for monitoring vaccine viruses in chickens after vaccination. The viral diseases in which diagnosis using nucleic acids extracted from the feather shafts was described are, Marek's disease virus, circoviruses, chicken anemia virus, fowlpox virus, avian retroviruses, avian influenza virus and infectious laryngotracheitis virus. In two cases, of Marek's disease virus and of infectious laryngotracheitis virus, the differentiation of vaccine and wild-type viruses from feather shafts was made possible, thus allowing for monitoring the vaccination efficacy. The present review demonstrates also the stability of DNA viruses in feather shafts, and the possible evaluation of environmental dissemination of pathogens. When viruses are transmitted vertically, like in the cases of the retrovirus REV, a teratogenic effect on the development of feathers of the day-old newly hatched chick might occur in the case of avian influenza and the chicken anemia virus, which might indicate on a viral infection.

Laryngotracheitis: reproducibility of the disease and comparison of diagnostic methods

Infectious laryngotracheitis virus (ILTV) cause mild to severe respiratory disease in chickens, the purpose of our study being to use Brazilian isolate of ILTV to reproduce ILTV disease in chickens by experimental infection and to compare three diagnostic methods (nested polymerase chain reaction (PCR), virus isolation, histopathology) for detection of ILTV. Forty-eight chickens intratracheally inoculated with ILTV and a further 48 with PBS, showing mild respiratory signs 48 hours post infection (PI) but no signs of infection after day 10 PI. Every 2 days PI, six birds were arbitrarily selected from the control and infected groups, sacrificed and the trachea collected. Both the nested PCR and virus isolation detected the virus from day 2 until day 12 PI. However, at day 12 PI, PCR detected ILTV DNA in 100% of the samples while the virus isolation method detected ILTV in only 33% of the samples. Tracheal histopathology showed intranuclear inclusion bodies on days 8 and 10 PI. The results indicate that the field-isolate of ILTV studied by us is of low pathogenicity and that our nested PCR protocol was able to detect positive samples over a longer infection period than many ILTV diagnostic test already described.

O vírus da laringotraqueíte (VLT) causa de leve a severa doença respiratório em galinhas, o propósito do nosso estudo foi usar um isolado brasileiro de VLT para reproduzir a doença em frangos através da infecção experimental e comparar três métodos de diagnóstico (nested PCR, isolamento viral e histopatologia) para detectar o VLT. Quarenta e oito frangos inoculados intratraquealmente com VLT e outros 48 com PBS, apresentaram sinais respiratórios leves 48 horas após a infecção (PI), mas nenhum sinal após o dia 10 PI. A cada dois dias, seis aves foram selecionados arbitrariamente do controle e do grupo infectado, sacrificados e as traquéias coletadas. Ambos nested PCR e isolamento viral detectaram o vírus do dia 2 até o dia 12 PI. No entanto, no dia 12 PI, a PCR detectou o DNA viral em 100% das amostras enquanto o isolamento viral detectou em somente 33% das amostras. Histopatologia da traquéia revelou corpúsculos de inclusão intranuclear nos dias 8 e 10 PI. Os resultados indicam que o VLT isolado de campo estudado é de baixa patogenicidade e que o protocolo de Nested PCR foi capaz de detectar amostras positivas por um período mais longo da infecção do que muitos testes de diagnósticos descritos.

Laryngotracheitis: reproducibility of the disease and comparison of diagnostic methods

Infectious laryngotracheitis virus (ILTV) cause mild to severe respiratory disease in chickens, the purpose of our study being to use Brazilian isolate of ILTV to reproduce ILTV disease in chickens by experimental infection and to compare three diagnostic methods (nested polymerase chain reaction (PCR), virus isolation, histopathology) for detection of ILTV. Forty-eight chickens intratracheally inoculated with ILTV and a further 48 with PBS, showing mild respiratory signs 48 hours post infection (PI) but no signs of infection after day 10 PI. Every 2 days PI, six birds were arbitrarily selected from the control and infected groups, sacrificed and the trachea collected. Both the nested PCR and virus isolation detected the virus from day 2 until day 12 PI. However, at day 12 PI, PCR detected ILTV DNA in 100% of the samples while the virus isolation method detected ILTV in only 33% of the samples. Tracheal histopathology showed intranuclear inclusion bodies on days 8 and 10 PI. The results indicate that the field-isolate of ILTV studied by us is of low pathogenicity and that our nested PCR protocol was able to detect positive samples over a longer infection period than many ILTV diagnostic test already described.

O vírus da laringotraqueíte (VLT) causa de leve a severa doença respiratório em galinhas, o propósito do nosso estudo foi usar um isolado brasileiro de VLT para reproduzir a doença em frangos através da infecção experimental e comparar três métodos de diagnóstico (nested PCR, isolamento viral e histopatologia) para detectar o VLT. Quarenta e oito frangos inoculados intratraquealmente com VLT e outros 48 com PBS, apresentaram sinais respiratórios leves 48 horas após a infecção (PI), mas nenhum sinal após o dia 10 PI. A cada dois dias, seis aves foram selecionados arbitrariamente do controle e do grupo infectado, sacrificados e as traquéias coletadas. Ambos nested PCR e isolamento viral detectaram o vírus do dia 2 até o dia 12 PI. No entanto, no dia 12 PI, a PCR detectou o DNA viral em 100% das amostras enquanto o isolamento viral detectou em somente 33% das amostras. Histopatologia da traquéia revelou corpúsculos de inclusão intranuclear nos dias 8 e 10 PI. Os resultados indicam que o VLT isolado de campo estudado é de baixa patogenicidade e que o protocolo de Nested PCR foi capaz de detectar amostras positivas por um período mais longo da infecção do que muitos testes de diagnósticos descritos.

Laryngotracheitis: reproducibility of the disease and comparison of diagnostic methods

Infectious laryngotracheitis virus (ILTV) cause mild to severe respiratory disease in chickens, the purpose of our study being to use Brazilian isolate of ILTV to reproduce ILTV disease in chickens by experimental infection and to compare three diagnostic methods (nested polymerase chain reaction (PCR), virus isolation, histopathology) for detection of ILTV. Forty-eight chickens intratracheally inoculated with ILTV and a further 48 with PBS, showing mild respiratory signs 48 hours post infection (PI) but no signs of infection after day 10 PI. Every 2 days PI, six birds were arbitrarily selected from the control and infected groups, sacrificed and the trachea collected. Both the nested PCR and virus isolation detected the virus from day 2 until day 12 PI. However, at day 12 PI, PCR detected ILTV DNA in 100% of the samples while the virus isolation method detected ILTV in only 33% of the samples. Tracheal histopathology showed intranuclear inclusion bodies on days 8 and 10 PI. The results indicate that the field-isolate of ILTV studied by us is of low pathogenicity and that our nested PCR protocol was able to detect positive samples over a longer infection period than many ILTV diagnostic test already described.

O vírus da laringotraqueíte (VLT) causa de leve a severa doença respiratório em galinhas, o propósito do nosso estudo foi usar um isolado brasileiro de VLT para reproduzir a doença em frangos através da infecção experimental e comparar três métodos de diagnóstico (nested PCR, isolamento viral e histopatologia) para detectar o VLT. Quarenta e oito frangos inoculados intratraquealmente com VLT e outros 48 com PBS, apresentaram sinais respiratórios leves 48 horas após a infecção (PI), mas nenhum sinal após o dia 10 PI. A cada dois dias, seis aves foram selecionados arbitrariamente do controle e do grupo infectado, sacrificados e as traquéias coletadas. Ambos nested PCR e isolamento viral detectaram o vírus do dia 2 até o dia 12 PI. No entanto, no dia 12 PI, a PCR detectou o DNA viral em 100% das amostras enquanto o isolamento viral detectou em somente 33% das amostras. Histopatologia da traquéia revelou corpúsculos de inclusão intranuclear nos dias 8 e 10 PI. Os resultados indicam que o VLT isolado de campo estudado é de baixa patogenicidade e que o protocolo de Nested PCR foi capaz de detectar amostras positivas por um período mais longo da infecção do que muitos testes de diagnósticos descritos.