Evaluation of the protection achieved at different ages with a pox-vectored infectious laryngotracheitis vaccine in layers reared on a farm and challenged in the laboratory

Vectorized vaccines against Infectious Laryngotracheitis (ILT) are the product of genetic engineering, been developed by ion of genes from the LT virus in a viral vector, which in this case is the avian smallpox virus. The objective for the development of a vaccine with these features was, among others, to achieve a sufficient level immunity so that vaccinated poultry were able to resist the challenge posed by the ILTv. In addition, its development was also aimed at avoiding post-vaccine reactions that are common with live virus, but one of the most relevant facts is that a recombinant vaccine against ILT avoids the latency of the virus, in vaccine-, or field-form, when it stays in the trigeminal nerve, causing the disease when coming out of this latent state.

Vaccination against ILT using vectorized vaccines began in Mexico in 2006 and, although several trials have been made to demonstrate its effectiveness, these have been done mainly in the United States (Davison et al., 2006; Godoy et al., 2008). Little has been studied in Mexico in this respect and all research have been conducted on mast chicken (Etcharren, personal communication;) (Lechuga, personal communication).

The objectives of this study were to assess the protection conferred by a vectorized vaccine against ILT in light breeding hens against the LTv challenge, and to assess the level of immunity achieved at different ages of the productive life of poultry.

Materials & Methods

Poultry
We used 30 light breeding Hy Line hens from commercial farms: 10 30-week old hens, 10 40-week hens and 10 55-week hens, vaccinated with an ILT vectorized vaccine smallpox-ILT at 10 weeks of age by wing-crease puncture at full dosage. Five SPF birds (Birds Free from Specific Pathogens, S. A. de C.V., Tehuacán Pue.) were used as a control group. The birds had access to water and balanced food ad libitum during the entire period of the test.

Challenge
The challenge was performed using a strain of the ILT virus isolated in the area of Tehuacan, Puebla, from a clinical case in commercial poultry, without any reference name. Each bird received 0.2 ml inoculation via intranasal, intraocular and intratracheal application of the LTv, containing a title of 10^4.0 DIE_50%ml.

Experimental Design
The birds were divided into three groups, by age, in isolation units with air filtering at negative pressure of Horsfall-Bauer type. In addition, together with the 30-week hens, five SPF birds were placed as control group for the entire test. In each group, the total number of birds was bled at the beginning and the end in order to perform the enzyme immunoassay (ELISA) LT test, using the CIVTEST-AVI-ILT (Hipra) commercial kit. After the challenge, birds were observed daily for 14 days, reporting weather or not clinical signs of LT were present (swollen heads, blefaroconjuntivitis, nasal exudate and shortness of breath). On day 14 after the challenge, birds were slaughtered and examined in autopsy, assessing the presence of lesions suggestive of LT (mucus, caseose exudate or bleeding in larynx and trachea). From all birds, the trachea was extracted for histopathology purposes. The main protection criterion was the absence of clinical signs and injuries at autopsy. The protection criterion for this parameter, according to the Code of Federal Regulations (9CFR) Animals and products; part 113.328 (2008), is the presence or absence of signs and lesions characteristic of LT (Blefaroconjuntivitis, tears, nasal exudate and coughing in a 14-day period (9CFR 113.328). At autopsy, exudates in trachea and bleeding, in such a way that the protection percentage of the treated group must be equal to or greater than 80%, and the challenged control group should show lesions in 90% of the birds.

Table 1 summarizes the results of the clinical observation. In the SPF control group one bird died during the observation period. However, this was not related to the LT.

Table 1. Presence of clinical signs of LT performed in two stages of the test, 1 to 6 days after challenge and 7-14 days after challenge. The table shows the positive birds / tested birds

In relation to the number of birds showing trachea lesions and the percentage of protection after infection with the Infectious Laryngotracheitis, we observed that protection in the control group was 0 (zero), in the groups of 30 and 55 weeks it was 80% and in the group of 40 weeks protection reached 100%. All the birds showed macroscopic lesions with moderate mucosal to mucosal-purulent exudate.

Histological analysis was performed on all the birds of the different groups in the test. In the control group, 2 birds showed significant changes: Metaplasia areas with loss of cilia. On the lamina propia, they showed moderate Lymphocytic infiltrate and epithelial cells were detected in the lumen. The group of 30-week hens presented metaplasia with loss of cilia and moderate epithelial hyperplasia, and  moderate lymphocytic infiltrate in two birds. The morphological diagnosis was moderate, diffuse lymphocytic tracheitis. In the 40-week Group, none of the birds showed significant lesions. On the other hand, in the 55-week group 3 birds had microscopic lesions, metaplasia with loss of cilia. Moderate lymphocyte infiltration was observed in the lamina propia, and only one of the birds presented areas of bleeding and detachment of epithelial cells. The morphological diagnosis for this case was moderate, diffuse lymphocytic tracheitis. Table 2 summarizes the serology results

Table 2. Serology results obtained before and after the challenge, marked as initial (day 1 of the test) and final (day 14 post-challenge-PD-). The two first columns mark the SP index (Sample positive) and the two remaining columns mark the geometric mean of the ELISA title  and, in parentheses, the variation coefficient

According to 9CFR 113.328 of the United States Department of Agriculture (USDA), the result of the challenge study was considered satisfactory, since none of the birds included in the groups of 30, 40 or 55 weeks of age showed signs of LT. On the macroscopic lesions, birds of 30 and 55 weeks showed 80% protection, according to 9CFR, which is still considered acceptable. The best level of protection in terms of signs and lesions was achieved in the 40-week group, which, combined with the histopathology and serology tests, leads to infer that the consolidation of the immune response is a process that is generated from the application of the vaccine on and reaches its peak at 40 weeks of age, or 30 weeks post-vaccination, showing a decline at 55 weeks of age, while maintaining the level of protection against the ILTv challenge.

The protection generated by the LTv glycoproteins have been widely studied, and among these, the gB glycoprotein stands out, because it has a determining participation in the protection against the herpes-virus (Riviere et al., 2002, Tong et al., 2001), but it has also been shown that immunity considerably improves when you add another glycoprotein, such as UL-32, among others (Lee et al, 2003, Tong et al., 2001), providing a level of immunity that ranges from 80 to 100 per cent (Godoy et al, 2008; Davison et al., 2006). The evaluated vaccine contains genes that encode the gB and UL-32 glycoproteins , and the results obtained in our challenge test against the LTv coincide with those obtained in similar studies, with the addition of the protection factor in terms of time, as a sufficient level of immunity against the challenge was demonstrated from 30 weeks and up to 55 weeks of age, equivalent to 20 and 35 weeks post-vaccination.

Histological lesions have not been a parameter to measure in the challenge tests against the ILTv; nevertheless, they were included in this study in order to confirm if they bear any relationship with the macroscopic lesions and serology results. According to our results, there is no direct correlation with the presence of clinical signs, since all birds of the control group showed signs of ILT but only three showed significant microscopic injuries. On the other hand, microscopic lesions were related to macroscopic lesions and serology results, according to our criteria (see tables). For this test, we wish to emphasize that the 40-week group did not show any kind of lesion.

Similarly to histology tests, serology tests have not been used as parameters to measure the protection in ILTv challenge tests, and this is even a point of view which extends to the commercial poultry production level, by clarifying that the tests have been used are Virus neutralization and Western-Blot. In addition to this, it has been shown that protection against the ILTv is more likely due to the cellular response of type T-cooperators (Chan et al., 1985). However, has also been demonstrated that the ILTv glycoproteins stimulate the immune response, both cellular and humoral (York & Faher, 1991). The antibody response to LT does not guarantee protection, however, is related to the presence or absence of the antigen (Chan et al., 1985) and its behavior in terms of replication in the trachea, where a smaller presence of the antigen will generate a smaller serologic response and, therefore, prevention of the clinical form of LT (York & Faher)(1991). The response of the control group to the presence of antigen was clear, since 75% of the birds tested positive after the challenge. This also reflects this effect in 30- to 55-week groups, but the of 40-week group showed a very different behavior. That is, if index SP is the largest of the three experimental groups, but after the challenge not all the birds become positive, this indicates that, probably, the virus did not replicate as efficiently or as freely as in the other groups. The effect observed in the SP index is repeated in a similar way in the ELISA title; the 40-week group title is the highest of all groups before the challenge, and later, it shows the lowest seroconversion.

Based on the results, we come to the conclusion that the immunity achieved by means of the vectorized vaccine against the ILT was sufficient for resisting the ILTv challenge. Therefore, its use at commercial, light breeding level is feasible.

Chan WL, Lu-King ML, Liew FY. 1985. Helper T cells induced by an inmunopirified herpes simplex virus type 1 (HSV-1) 115 kilodalton glycoprotein (gB) protect mice against HSV-1 infection. J. Exp. Med. 162:1304-1318.

Code Federal of Regulations. 2008. Animals and animal products, Chapter I. Animal and plant health inspection service, department of agriculture.

Davison S, Gingerich E, Casavant S, Croade E. 2006. Evaluation of the efficacy of a live Fowl Pox-vectored infectious laryngotracheitis/avian encaphalomyelitis vaccine against ILT viral challenge. Avian. Dis. 50:50-54.

Godoy A, Flegg P, Oldoni I, García MC, Dorsey KM. 2008. Efficacy of a Vectormune Fowl Pox-laryngotracheitis vaccine and sequence comparison of recent ILTV field isolates. In the American Association of Avian Pathologist Meeting, New Orleans, LA.

Lee LF, Witter RL, Reddy SM, Wu P, Yanagida N, Yoshida S. 2003. Protection and synergism by recombinant Fowl Pox vaccine expressing multiple genes from Marek's Disease virus. Avian Dis. 47:549-588.

Riviere M, Tartaglia J, Norton EK Bongermino CM, Lacoste F, Duert F. 1991. Protection of mice and swine from pseudorabies virus conferred by vaccine virus-based recombinants. J. Virol. 66:3424:3434.

Tong GZ, Zhang SJ, Meng SS, Wang L, Qiu HJ, Wang YF, Yu L, Wang M. 2001. Protection of chickens from Infectious Laryngotrachitis with a recombinant Fowl Pox virus expressing glycoprotein B of Infectious Laryngotracheitis virus. Avian Pathol. 30:143-148.

York JJ & Faher KJ. 1991. Vaccination with affinity-purified glycoproteins protects against Laryngotracheitis herpesvirus. Avian Pathol. 20:693-704