A sensitive nested-Polymerase Chain Reaction protocol to detect infectious laryngotracheitis virus
Beltrão, NilzaneEgochega, Raul FloresFurian, Thales QuediRodenbusch, Carla RosaneFallavena, Luiz Cesar BelloPasquali, GiancarloCanal, Cláudio Wageck
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.
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