Occurrence of Infectious Laryngotracheitis Virus ( Iltv ) in 2009-2013 in the State of São Paulo – Brazil

Infectious laryngotracheitis is a very important respiratory disease because it causes significant economic losses in the poultry industry. The target of ILTV infections is the respiratory system, and the main organ in which the virus remains latent is the trigeminal ganglia. However, the virus has demonstrated tropism for other organs as well. The present study was conducted to determine the presence of Infectious Laryngotracheitis Virus (ILTV) in the state of São Paulo. Samples submitted to LABORUSP during the last four years (20092013) analyzed by a nested/PCR technique. Out of the 682 samples from layers tested for LTIV, 12.46 % were positive, and derived from in both traditional (trachea and trigeminal ganglion) and untraditional (cecal tonsils, digestive tract and kidneys) organs utilized for ILTV diagnosis. The present work showed that ILTV is circulating in commercial layer flocks in São Paulo State, and that the LTIV is present in other organs in addition to the respiratory tract and trigeminal ganglion; however, it was not determined if the circulating virus is a vaccinal or field strain.


INTRODUCTION
Infectious laryngotracheitis (ILT) of birds is a highly contagious disease that primarily affects chickens, pheasants, and partridges, with hens as the primary host.Starlings, sparrows, crows, pigeons, and ducks seem to be resistant to the virus (Guy & Garcıa, 2008).The causative agent is a pneumotropic virus of the family Herpesviridae, genus Iltovirus.Taxonomically, this virus is classified as a Gallid herpesvirus 1 (Zhao et al., 2013).This disease is included in the list of mandatory notification of terrestrial and aquatic animal diseases of the OIE.Its notification to the Brazilian Official Service is also mandatory (http://www.oie.int/en/animal-health-in-the-world/oie-listed-diseases-2014/).
Infectious laryngotracheitis was first described in 1925 (May & Tittsler, 1925), and since then it has been reported in many countries, where it is endemic, especially in regions with intensive poultry production with large concentrations of birds and multi-age farms, such as in North America, China, Europe (especially in Poland), Australia, Africa, southwest Asia, New Zealand, and South America (Chacón & Ferreira, 2009;Hidalgo, 2003).Viral transmission occurs via horizontal transfer, and the primary replication sites are in the tracheal mucosa and conjunctiva, where it can causes inflammation, mucoid or serous discharge, cough, and dyspnea.Poor egg production and weight gain are also observed (Coppo et al., 2013b).
The virus invades the trigeminal nerve during the lytic phase of infection, resulting in a latent infection that may be present during the entire life of the animal.Some stressors, such as the onset of lay and placement with other birds, may reactivate virus replication and shedding (Coppo et al., 2013a;Hughes et al., 1991;Hughes et al., 1989;Williams et al., 1992).Recent experimental studies indicate that the virus can also be detected in other organs, such as the heart, liver, spleen, lung, kidney, tongue, thymus, proventriculus, pancreas, duodenum, small intestine, large intestine, cecum, cecal tonsils, bursa, and brain (Oldoni et al., 2009;Wang et al., 2013;Zhao et al., 2013).
Molecular techniques, such as polymerase chain reaction (PCR), have been successfully used for the detection of ILTV in respiratory and other organs (Chacón et al., 2007;Oldoni et al., 2009;Rodríguez Avila et al., 2007).Polymerase chain reaction presents high sensitivity and specificity for the detection of ILTV.It is able to detect viral DNA when other tests, such as histopathology and immunofluorescence, yield negative results (Crespo et al., 2007).Therefore, PCR provides a rapid diagnostic test that can aid in the differentiation between field and vaccinal strains (Clavijo & Nagy, 1997).
The aim of this study was to evaluate the occurrence of the Infectious Laryngotracheitis Virus in samples submitted to the Laboratory of Avian Diseases of the School of Veterinary Medicine of the University of São Paulo between 2009-2013.

Sampling
A total number of 682 samples from layer flocks from several cities of the state of São Paulo (Bastos, Iacri, Tupã, Guapiaçu, Rancharia, Parapuã, Ibiúna) was analyzed.Out of the total number of samples, 337 derived from the trachea, 308 from the trigeminal ganglia, 11 from the lungs, six from the cecal tonsils, seven from the digestive tract, two from the liver, two from the spleen, one from the pancreas, and eight from the kidneys (Table 2).

DNA extraction
Total DNA was extracted according to the method of described by Chomczynski (1993).Briefly, samples were homogenized in sufficient 0.01 M phosphate buffered saline (PBS; pH 7.4) to yield a 10% (w/v) suspension, and clarified at 3000 x g for 20 min at 4°C.The supernatant was separated and an aliquot (200 µL) incubated for 5 min at 37°C with 1000 µL of phenol/guanidine thiocyanate solution.Chloroform (200 µL) was then added to the solution, the mixture was centrifuged (12,000 x g for 15 min at 4°C), propanol (750 µL) was added, and the whole mixture was cooled at -20°C for at least 2 h.Precipitated DNA was collected by centrifugation (12,000 x g for 20 min at 4°C).Any DNA that remained adhered to the wall of the tube was rinsed off with 70% ethanol and the solvent was allowed to evaporate.The total DNA sample was dissolved in 30 µL of Tris-EDTA buffer.

Viral Detection
Viral detection was performed by a nested-PCR technique, which was oriented to the amplification of the gene that encodes protein E, as described by Chacón & Ferreira (2008).
In a DNAse-free fresh microtube, 1 X PCR Buffer, 1.25 mM of each dNTP, 0.5 pmol of each of the forward (GE1S) and reverse (GE2AS) primers (as described in table 1), 1.25 U of Platinum® Taq polymerase (Invitrogen by Life Technologies, Carlsbad, CA, USA), and 2.5 µL of extracted DNA were added.DNA free ultrapure water was included to bring the volume to 25 µL.Amplification was performed in a Mastercycler® Nexus X1Eppendorf (Eppendorf AG, Hamburg, Germany).Thermal cycling consisted of an initial denaturation step of 3 min at 94 °C, followed by 45 cycles of denaturing at 94 °C for 1 min, annealing at 58 °C for 30 s, and extension at 72 °C for 45 s.The end cycle was followed by an extension step at 72 °C for 10 min.The second round of amplification (nested-PCR) was performed in a similar manner, although a second set of primers (GE3S forward and GE4AS reverse) was employed, as described in Table 1.The PCR products were visualized after separation by electrophoresis in an agarose gel (1.5%) using Blue Green Dye (LGC, Sao Paulo, Brazil) to stain the DNA.The size of the amplified product was estimated using the 100 base pair DNA Ladder molecular size marker (Invitrogen).

DISCUSSION AND CONCLUSION
Infectious laryngotracheitis (ILT) is a disease that causes singnificant economic losses due to increased mortality and reduced growth rates and egg production (Guy & Garcıa, 2008).The target of ILTV infections is the respiratory system, specifically the epithelium of the trachea and larynx, although the sinuses and lungs may also be infected.The target site of infection largely depends on the route of inoculation (Bagust & Johnson, 1995).In the present study, ILTV was detected in seven (63.6%) lung samples and 57 (16.6%) tracheal samples, showing a wide viral distribution and strong tropism for the respiratory organs, despite its detection in the intestines, cecal tonsils, and trigeminal ganglia.
The main organ in which ILTV is latent is the trigeminal ganglia, possible because the trigeminal nerve is the main enervator of the upper respiratory tract, tongue and eyes, and the distal part is involved in the innervation of the trachea (Bagust & Johnson, 1995;Bagust, 1986;Williams et al., 1992).The presence of ILTV was detected in 19 of the evaluated trigeminal ganglia samples, showing that the virus became latent in a few chickens.
Our study indicates that viral detection was detected in higher numbers in the lungs, trachea, and trigeminal ganglia.This is consistent with previously reported results, indicating that greater viral replication was detected in the respiratory organs (Oldoni et al., 2009;Rodríguez Avila et al., 2007).However, other organs, such as the lungs, cecal tonsils, digestive tract, and kidneys were also positive.These findings are in agreement with other studies (Wang et al., 2013;Zhao et al., 2013), in which viral DNA was detected and quantified in the heart, liver, spleen, kidneys, tongue, thymus, proventriculus, duodenum, pancreas, small intestine, large intestine, cecum, cecal tonsils, bursa, and brain.These results indicate that circulating ILTV strains may show tropism for other organs in addition to the respiratory system.Further studies, using ILTVspecific immuno-histochemical techniques should be performed in order to confirm such finding.
Five hundred ninety-seven (597) out of 682 (87.5%) samples were negative by the aforementioned molecular tests, implying partial success of the vaccination program regarding reducing viral activity.Indeed, the positive samples (12.46%) revealed viral presence in healthy chickens.In addition, viral presence could also mean that a field strain is circulating among layer flocks.This is suggested by the detection of ILTV in uncommon organs and indicates that the pathogenesis of the disease is not well understood.Several studies using molecular techniques, such as PCR for detection of ILTV (Chacón et al., 2007;Chacón & Ferreira, 2008;Clavijo & Nagy, 1997;Crespo et al., 2007), prove that these techniques are very sensitive and specific.In the present study, a reaction of nested-PCR, oriented to the amplification of part of the gene encoding glycoprotein E, was successfully used to investigate the presence and tissue location of ILTV in layer chickens.
The results of this study demonstrate that ILTV is circulating in laying flocks reared in São Paulo state; however, it is unknown if the circulating virus is a vaccinal or a field-derived strain.Further studies should focus on differentiating the nature of these strains.Additionally, it must be noted that, at the time of diagnosis, organs other than those of the respiratory system presented ILTV infection.

Figure 1 -
Figure 1 -Nested-PCR product (219 bp) of the partial amplification of ILTV glycoprotein E gene.Lanes A, B, E, G, H: tested samples.Lane C: positive control.Lane F: negative control, and Lane D: Molecular size marker (100 bp).

Table 1 -
Primers, nucleotide sequences, amplified products (in bp), and references used in the nested-PCR test.

Table 3 -
ILTV distribution in the 682 analyzed samples from different organs of layers in São Paulo State.