A five-year surveillance study of vaccination schedules using viral-vectored vaccines against infectious laryngotracheitis in a high-density layer region / Cinco anos de monitoramento de esquemas vacinais com vacinas virais vetorizadas contra laringotraqueíte infecciosa em uma região altamente povoada por galinhas poedeiras

The effectiveness of vectored recombinant vaccines to control infectious laryngotracheitis (ILT) in chickens from a region (State of Minas Gerais, Brazil) with ~10 million layers was evaluated under field conditions from 2014-2018. During this period, only recombinant turkey herpesvirus (rHVT) or fowl poxvirus (rFPV) vaccines that express antigens of infectious laryngotracheitis virus (Gallid herpesvirus-1; GaHV-1) were used. Layer chickens (n=1,283), from eight different egg-producing companies, were individually sampled and examined (active surveillance), and in instances when government poultry health veterinarians were notified due to respiratory disease (passive surveillance). Clinical, macroscopic, and histopathology examinations were performed to diagnose ILT as well as molecular techniques for the detection and characterization of the GaHV-1 DNA from the trachea and trigeminal ganglia (TG). The layer hens sampled and examined belonged to flocks and farms that used different vaccination protocols (non-vaccinated, single dose vaccination, and prime/ boost vaccination). This is the first long-term field study of the effectiveness of ILT vectored vaccines in a high-density multiple age layer hen region. Using various diagnostic methods, the occurrence of GaHV-1 infection and ILT clinical disease in layer hens vaccinated with vectored recombinant vaccines in one quarantined region of Brazil were investigated. The number of ILTV positive chickens by PCR and ILT clinical disease cases was lower in farms when all chickens were vaccinated with at least one vaccine. However, the difference in the detection rates of GaHV-1 infection was significant only when compared farms with prime/ boost and farms using single dose of HTV-LT.

A efetividade das vacinas recombinantes vetorizadas para o controle da laringotraqueíte infecciosa (LTI) nas aves de uma região (Minas Gerais, Brasil) com aproximadamente 10 milhões de poedeiras foi avaliada em condições de campo, no período de 2014 a 2018. Durante este período, somente as vacinas recombinantes "turkey herpesvirus" (rHVT) ou "fowl poxvirus" (rFPV), que expressam antígenos do vírus da laringotraqueíte (Gallid herpesvirus-1; GaHV-1) foram utilizadas. Galinhas poedeiras (n=1.283), de oito diferentes granjas produtoras de ovos, foram individualmente amostradas e examinadas por monitoramento ativo e, na ocorrência de notificação de doença respiratória aos veterinários do serviço oficial, por monitoramento passivo. Exames clínicos, macroscópicos e histopatológicos foram realizados para o diagnóstico de LTI, bem como técnicas moleculares para a detecção e caracterização do DNA de GaHV-1 da traqueia e gânglio trigêmeo. As galinhas poedeiras pertenciam a lotes e granjas que usavam diferentes protocolos de vacinação (não vacinadas, uma dose ou tipo de vacina e duas doses ou tipos de vacina). Este é o primeiro longo estudo a campo sobre a efetividade das vacinas vetorizadas em uma região com população elevada de poedeiras de múltiplas idades. Utilizando vários métodos de diagnóstico, a ocorrência da infecção por GaHV-1 e a LTI clínica em poedeiras de uma região interditada do Brasil foi investigada. O número de galinhas positivas para o vírus GaHV-1 e para casos clínicos de LTI nas granjas foi menor quando todas as aves estavam vacinadas com, pelo menos, um tipo ou dose de vacina. Entretanto, a diferença na taxa de detecção da infecção por GaHV-1 foi significativa somente quando a comparação foi realizada entre granjas com aves vacinadas com duas doses e aves de granjas vacinadas com uma única dose de HVT-LT.

Development of a qPCR for the detection of infectious laryngotracheitis virus (ILTV) based on the gE gene.

1. Infectious laryngotracheitis is a respiratory disease that affects the poultry industry worldwide. It is common in flocks with high-bird density, causing major economic losses. 2. In this study, a SYBR® FAST polymerase chain reaction (PCR) double-strand DNA intercalating agent assay was performed for the detection of infectious laryngotracheitis virus (ILTV) in clinical samples in comparison with a conventional nested-PCR, both based on the glycoprotein E encoding gene. This assay amplified 56 bp and was capable of detecting 19 to 1 copies of virus. 3. In total, 164 clinical samples were obtained from birds with respiratory problems from the period of 2009-2016. In the nested-PCR, there were 45.12% positive samples and 54.88% negative samples, while in the real-time PCR (qPCR), there were 81.1% positive samples and 18.9% negative samples. 4. In conclusion, qPCR from the DNA double-strand intercalating agent SYBR® GREEN FAST was useful for the diagnosis of ILTV because it detected samples that were negative in nested-PCR. This assay has advantages, such as a shortened processing-time, and no need for post-amplification processing (electrophoresis) with additional reagents, such as MgCl2 and agarose. Hence, qPCR proved to be useful, rapid and low cost for use with clinical samples.

Molecular characterization of infectious laryngotracheitis virus in naturally infected egg layer chickens in a multi-age flock in Brazil.

The virus responsible for an outbreak of infectious laryngotracheitis (ILT) in a multi-age flock of egg layer chickens under quarantine in Brazil was characterized. Layer chickens from this area with circulating gallid herpesvirus 1 (GaHV 1) were evaluated using histopathology and molecular characterization techniques based on sequences of infected-cell polypeptide 4 (ICP4) and thymidine kinase (TK) genes. The infected chickens that were analyzed were PCR-positive for GaHV-1 in the trachea and negative in most trigeminal ganglia. The lack of ILT lesions in the conjunctiva and respiratory tissues, combined with detection of viral DNA in the trachea, was found to be associated with latent infection. The sequences from five farms obtained in the present study were identical, and there were no deletions within the 272- to 283-bp region of the ICP4 gene, as observed in the sequences of vaccine strains (CEO and TCO). The lack of a deletion in the ICP4 fragment analyzed in this study indicates that the chickens were infected with a field virus. The absence of the T252M mutation in a fragment of the TK gene, in addition to the low mortality rate observed, suggests that the outbreak in the state of Minas Gerais was not caused by a highly virulent strain but rather by a field virus of lower virulence. In addition, using phylogenetic reconstructions, it was found that this field strain was grouped together in a separate branch, apart from the previously characterized Brazilian strains. The introduction of vectored vaccines apparently has been effective in reducing clinical disease and lesions, and preventing new outbreaks of disease.

Outbreak of infectious laryngotracheitis in large multi-age egg layer chicken flocks in Minas Gerais, Brazil / Surto de laringotraqueíte infecciosa em granjas de galinhas poedeiras de múltiplas idades em Minas Gerais, Brasil

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 São 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).(AU)

Um surto recente (Novembro de 2010) de laringotraqueite infecciosa (LTI) em granjas de postura de múltiplas idades em Minas Gerais, Brasil, é descrito. Um surto de LTI em galinhas de postura havia sido previamente relatado apenas no Estado de São Paulo em 2002. No surto aqui descrito, a população afetada foi de aproximadamente oito milhões de galinhas, com lotes variando de 100.000 a 2.900.000 galinhas. A mortalidade média variou de 1 a 6% e a morbidade atingiu cerca de 90% (a maioria das 27 granjas foram positivas para o virus da LTI). Três granjas com aves de múltiplas idades pertencentes a uma empresa foram selecionadas para o presente relato. Os sinais clinicos incluíram prostração, dispneia, conjuntivite, edema ocasional dos seios paranasais e secreção nasal mucosa e/ou sanguinolenta. As aves severamente afetadas apresentaram acentuada dispneia, aparente engasgo e tornaram-se cianóticas antes da morte. Nestas aves, exsudato fibrinoso denso obstruindo o lúmen da laringe e parte cranial da traqueia foi observado na necropsia. Havia também, conjuntivite com hiperemia intensa e edema, além de sinusite com exsudato caseoso. Na histopatologia, observaram-se necrose e descamação acentuada do epitélio respiratório e da conjuntiva com formação de numerosos sincícios e exsudato fibrinoso. Além disso, infiltrado inflamatório mononuclear (especialmente linfócitos e plasmócitos) moderado a acentuado na lâmina própria foi observado. Corpúsculos de inclusão intranucleares nas células sinciciais foram observados em 16 das 20 aves examinadas. Resultados positivos pela PCR para o virus da LTI foram obtidos de DNA extraído das laringes e traqueias utilizando amostras fixadas em formol e incluidas na parafina. O produto amplificado de uma região pequena do gen ICP4 foi submetido ao sequenciamento e quando comparado com outras sequências depositadas no Genbank mostrou os seguintes resultados: 100% de identidade com uma estirpe do virus de LTI dos Estados Unidos (JN596963.1), 99% de identidade com uma estirpe Australiana e 91% com a estirpe vacinal CEO (JN580316.1) e TCO (JN580315.1).(AU)

Outbreak of infectious laryngotracheitis in large multi-age egg layer chicken flocks in Minas Gerais, Brazil / Surto de laringotraqueíte infecciosa em granjas de galinhas poedeiras de múltiplas idades em Minas Gerais, Brasil

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 São 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).

Um surto recente (Novembro de 2010) de laringotraqueite infecciosa (LTI) em granjas de postura de múltiplas idades em Minas Gerais, Brasil, é descrito. Um surto de LTI em galinhas de postura havia sido previamente relatado apenas no Estado de São Paulo em 2002. No surto aqui descrito, a população afetada foi de aproximadamente oito milhões de galinhas, com lotes variando de 100.000 a 2.900.000 galinhas. A mortalidade média variou de 1 a 6% e a morbidade atingiu cerca de 90% (a maioria das 27 granjas foram positivas para o virus da LTI). Três granjas com aves de múltiplas idades pertencentes a uma empresa foram selecionadas para o presente relato. Os sinais clinicos incluíram prostração, dispneia, conjuntivite, edema ocasional dos seios paranasais e secreção nasal mucosa e/ou sanguinolenta. As aves severamente afetadas apresentaram acentuada dispneia, aparente engasgo e tornaram-se cianóticas antes da morte. Nestas aves, exsudato fibrinoso denso obstruindo o lúmen da laringe e parte cranial da traqueia foi observado na necropsia. Havia também, conjuntivite com hiperemia intensa e edema, além de sinusite com exsudato caseoso. Na histopatologia, observaram-se necrose e descamação acentuada do epitélio respiratório e da conjuntiva com formação de numerosos sincícios e exsudato fibrinoso. Além disso, infiltrado inflamatório mononuclear (especialmente linfócitos e plasmócitos) moderado a acentuado na lâmina própria foi observado. Corpúsculos de inclusão intranucleares nas células sinciciais foram observados em 16 das 20 aves examinadas. Resultados positivos pela PCR para o virus da LTI foram obtidos de DNA extraído das laringes e traqueias utilizando amostras fixadas em formol e incluidas na parafina. O produto amplificado de uma região pequena do gen ICP4 foi submetido ao sequenciamento e quando comparado com outras sequências depositadas no Genbank mostrou os seguintes resultados: 100% de identidade com uma estirpe do virus de LTI dos Estados Unidos (JN596963.1), 99% de identidade com uma estirpe Australiana e 91% com a estirpe vacinal CEO (JN580316.1) e TCO (JN580315.1).

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.

Differentiation of field isolates and vaccine strains of infectious laryngotracheitis virus by DNA sequencing.

Two different regions of the infected cell protein 4 (ICP4) gene of infectious laryngotracheitis virus (ILTV) were amplified and sequenced for characterization of field isolates and tissue culture-origin (TCO) and chicken embryo-origin (CEO) vaccine strains. Phylogenetic analysis of the two regions showed differences in nucleotide and amino acid sequences between field isolates and attenuated vaccines. The PCR-RFLP results were identical to those obtained by DNA sequencing and validated their use to differentiate ILTV strains. The approach using the sequencing of the two fragments of the ICP4 gene showed to be an efficient and practical procedure to differentiate between field isolates and vaccine strains of ILTV.

Natural infection of turkeys by infectious laryngotracheitis virus.

The infectious laryngotracheitis virus (ILTV) is an important respiratory pathogen of chickens that also infects pheasants and peafowl. Epidemiologically non-related commercial turkey flocks with clinical signs such as tracheitis, swollen sinuses, conjunctivitis and expectoration of bloody mucus were examined for the presence of the virus. Laboratory ILTV detection was performed by virus isolation in embryonated eggs and cell cultures, PCR and sequencing of amplification products, histopathology, indirect immunofluorescence and electron microscopy. One ILTV turkey isolate was also experimentally inoculated into susceptible chickens and turkeys, reproducing a mild respiratory disease. This is the first description of natural infections with ILTV in turkeys.

Detecção do vírus da laringotraqueíte das galinhas no Brasil / Detection of infectious laryngotracheitis virus in chickens in Brazil

O propósito deste estudo foi detectar a presença do vírus da laringotraqueíte infecciosa (VLTI) das galinhas em algumas granjas do Brasil. Tecidos da traquéia e suabes foram coletados de 10 lotes de frangos de corte e galinhas de postura com sinais respiratórios. O material foi inoculado em ovos embrionados e as membranas corioalantóides examinadas por histopatologia. Além disso, as amostras foram submetidas à reação em cadeia da polimerase (PCR). Três lotes foram positivos para VLTI por isolamento viral e PCR. Os resultados confirmam a presença do VLTI nas galinhas no Brasil. (AU)

A study was carried out in search for evidences of infectious laryngotracheitis virus (ILTV) infections in some Brazilian chicken flocks. Tracheal tissues and swabs were collected from 10 different flocks of layers and broilers displaying respiratory signs of disease. Samples were processes for virus isolation in embryonated eggs and the membranes examined by histopathology. In addition, specimens were examined by polymerase chain reaction (PCR). Three flocks had ILTV positive chickens by virus isolation and PCR. These results confirm the occurrence of ILTV in chickens in Brazil. (AU)