Respiratory and GIT tract immune responses of broiler chickens following experimental infection with Newcastle disease’s virus

Newcastle disease is one the most contagious viral diseases which could infect almost all species around the world. Vaccination is a practical and useful way against the endemic cases of Newcastle disease (Usman 2002). Currently, common vaccination programs against Newcastle disease in most countries include administering live attenuated and killed vaccines to control the endemic strains. Vaccines belonging to B1 and LaSota strains are the commonest vaccines used to prevent Newcastle disease in the poultry industry though sometimes facing with vaccine failures due to unknown reasons. I-2 vaccine against Newcastle disease is becoming more popular not only in village chicken but also in industrial broiler’s one. This study was conducted to elaborate some aspects of cell-mediated immunity of this vaccine compared to a commonly used commercial vaccine against Newcastle disease. Several studies proved I-2 vaccine as an appropriate choice with good immunogenicity, low level of vaccine reaction, less expenses with vaccine production in comparison with the common vaccines, and thermostability making the vaccine no need to follow cold chain during the transportation (Bell 2001). Fifty percent embryo infectious dose (EID50) of the virus used for the vaccine was supposed to be 10^6/5 since EID50/bird higher 10⁶ was reported as the immunizing titer in the studies conducted on the protective antibody titers against I-2 virus by other researchers (Nili et al. 2005). If the vaccine was kept in 4–8 °C for a long time, at least a year, it could result in achieving an acceptable antibody titer. I-2 vaccine could be administered via different routes including eye drop, drinking water, feed, and injection. However, most farmers prefer to administer the vaccine through the eye drop route (Bensink and Spradbrow 1999).

The first line of specific immune response to NDV is cellular immunity appearing in 2–3 days after administering ND live vaccines. It is common to assess the effects of Newcastle vaccines with specific antibody titers against the vaccine. Titration of the sera after administrating the vaccines indicates the outcome of the vaccine and whether the resulted titer results in the immunization or not (OIE 2012; Swayne et al. 2013). Ghumman et al. used leukocyte migration inhibition assay to evaluate the cellular immunity in 1976 and indicated that lymphocyte mitogens were occurred 2 days after the primary vaccination. This finding was related to bird’s resistance against the challenged virus. They concluded that the cellular immunity against certain strains of NDV is an integral part of the total immunity together with other immunity parameters such as the humoral and the local (Ghumman and Bankowski 1976; White and Appleton 1953). It was also indicated that although bursectomized chicks received Newcastle vaccines were incapable of producing antibodies up to immunization level, they were resistant to the disease. In addition, Newcastle disease HI titer was not depended on cellular immunity response (Marino and Hanson 1987). However, since the role of immunity is clear in all poultry diseases, it is necessary to study the immunity system more to develop and modify the prevention strategies (Lillehoj and Trout 1993). Cellular immunity is a type of specific adaptive immunity with T lymphocytes and important roles in developing the immunity in vaccinated chicks against the Newcastle disease virus and eliminating it (Sharma 1999). T cells are classified based on the cellular evolution and expression to CD4 and CD8 co-receptors. CD8 is usually expressed on the surface of cytotoxic T cells, while CD4 is found on the surface of T helper cells (Chan et al. 1988). Glycoprotein antigens on the surface of leukocytes are biomarkers or cluster of differentiation (CD) that could be used to differentiate the leukocytes based on the cell type and the maturity stage (Saalmuller et al. 1997; Glick 2000). Also, several studies indicated that measuring T cell-released interferon-γ after in vitro or in vivo stimulation could be also an appropriate marker to evaluate the cellular immunity after the infection or vaccination (Breed et al. 1997; Karaca et al. 1996; Martin et al. 1994; Prowse and Pallister 1989). It became obvious that the chicken’s body also uses several mechanisms in addition to producing Newcastle disease virus specific antibody against the virus (Marino and Hanson 1987). Enzyme-linked immunosorbent assay (ELISA) indicated some reliable and simple methods to evaluate the cytokine release after the activation proving this parameter as an appropriate one to assess the cellular immunity (Mateu de et al. 1998; Whiteside 1994; Rothel et al. 1990; Mateu de et al. 1998). Several cell-mediated immunity (CMI)-associated cytokines have been detected in chickens during the recent years giving the chance to develop a new immunologic assay for the species. Recently, monoclonal antibodies (mAbs) were developed based on specific interferon-γ ELISA test (Lambrecht et al. 2000). The aim of this study was to evaluate the cellular immunity with serum interferon-γ using ELISA assay, to study the changes in T cell subsets, CD4 and CD8, in intestinal and tracheal samples with monoclonal antibodies against the surface or intracellular glycoproteins as the markers using immunohistochemistry assay after vaccinating with I-2 thermostable vaccine and to compare the results of this vaccine with acute virus and B1 vaccine challenges.

Herts 33.56 (ICPI 1.88) was used in this study as an international acute strain. This virus was originally isolated from Hertforshir chicken in England in 1933 (Allan et al. 1978). Herts 33.56 was propagated in 9-day-old to 11-day-old embryonated chicken eggs. Concentration of the virus in infected chick embryo allantoic fluid was 10⁸ EID50/mL. As the dose of challenge virus, determined in previous studies, 1 × 10⁻⁴ EID50/mL was the basic dose of the challenge in this study (Alexander et al. 2006).

Interferon-γ changes were significant in time (p < 0.001). Totally, there was no significant difference between I-2 and B1 groups, but 7 days after primary vaccination and 14 days after the booster, a significant difference was observed. The statistical difference was significant between I-2 group and negative control group, and finally, the differences of this parameter between various groups and on different days were significant (p < 0.001) (Table 2) (Fig. 1).

To improve the vaccination strategies in the field and to evaluate the vaccines, it is necessary to increase our knowledge about immunostimulatory conditions by the vaccine and the challenge virus (Noraup et al. 2011). Several studies have been conducted to evaluate the immune response to infection with NDV. Some of them suggested that innate immunity can play an important role against NDV infection (Bing-guo et al. 2011; Rauw et al. 2009). Recent studies also confirmed detection of CMI reaction to NDV, shortly after vaccination with a live vaccine (Reynolds and Maraqa 2000).

Immunohistochemistry is an accurate and specific immunoassay technique that could indicate the tissue distribution of the selected agents in tissue sections. Several studies have been conducted to evaluate the effects of viruses and various vaccines such as HVT, MDV, IBDV, and so forth on the infiltration of T cell subtypes (CD4+ and CD8+) using IHC method (Kamran et al. 2010; Silke and Christine 2005; Takao et al. 1999). Also, some limited studies have been conducted to evaluate the distribution of T cell subtypes (CD4+ and CD8+) following the vaccination and challenge with Newcastle disease virus (Xiaofei et al. 2007; Awad et al. 2014; Russell et al. 1997). On the other hand, interferon-γ ELISA test indicated a high potential in measuring CMI role in protecting the chickens against the poultry infectious diseases in the future and studying the role of interferon-γ in various immune mechanisms of the chickens (Lambrecht et al. 2004). Interferon-γ (interferon type II) is secreted by a few immune cells (including T helpers, cytotoxic T cells, and natural killer cells) with antiviral and immunostimulatory effects (Staeheli et al. 2001). Overall, this type of immune response could affect on the type of cellular immunity involved in NDV clearance (Agrawal and Reynolds 1991; Reynolds and Maraqa 2000; Seal et al. 2000). However, limited data is available regarding the functions of interferon-γ in the avian immune system, and the knowledge regarding this interferon is not well developed in various avian diseases including the Newcastle disease (Susta et al. 2013).

In a study on the increased of γ interferon levels in the chicken’s splenocytes infected with California acute (CA) strain comparing with a lentogenic Newcastle virus strain, the increment of interferon-γ level in the sera of acute strain-infected chicks was noticed 2 and 3 days PI (Rue et al. 2011). In addition, it was indicated that very virulent Newcastle disease virus (velogenic viscerotropic strain, ZJ1) induced high level of interferon-γ production in the backyard poultry, just prior to death, while the vaccine strain did not have similar effect (Cornax et al. 2012). Cellular immunity response was evaluated by measuring interferon-γ with ELISA method after splenocyte stimulation by mitogens and antigen induction by vaccinating with inactive and live LaSota vaccine. Many vaccinated chicks produced interferon-γ 2 to 4 weeks after stimulation of vaccinating with a live vaccine. In case of the inactive vaccine, half of the chicks responded significantly 4 weeks after the vaccination. This indicated that the sensitivity of ELISA in measuring interferon-γ production by T cells in the response to mitogens and specific antigen induction in in vitro conditions was sufficient (Lambrecht et al. 2004).

The effects of three antibiotics with vaccinating with LaSota live and inactive vaccination in the cellular immunity response were assessed in 20-week SPF chickens by measuring interferon-γ in the cell culture using ELISA. Five days after cell culture inoculation, the group received antibiotics and the vaccine simultaneously showed a significant increment in interferon-γ production compared with the control group and the group that received antibiotics only (Khalifeh et al. 2009). The importance of interferon-γ was indicated using IFN-γ and levamisole as the adjuvant in Newcastle DNA vaccine. Selecting this cytokine as the adjuvant would enhance the protection of the chicks against the acute Newcastle disease virus challenge (Yin et al. 2006). Evaluating the cellular immunity in the birds is cumbersome and hence is not a common practice in this field.

As mentioned above, measuring interferon-γ as a cytokine after the stimulation could be an appropriate evaluation method for the cellular immunity. Interferon-γ changes were significant in the period of this study (p < 0.001), and the levels of its titer were increasing. There was no significant difference between two groups received I-2 and B1 vaccines. However, a significance difference was noticed between I-2 group with negative control and B1 groups. Significant different serum levels of these parameters were also noticed in different days (p < 0.001). The level of interferon-γ started to increase 3 days after the vaccination and peaked 6 days post-challenge in the vaccinated groups. Cellular immunity has an important role in many immune responses against the viruses with various mechanisms such as cytokine production (Kaiser and Staheli 2008). It was indicated in a study that interferon-γ could decrease the effects of Newcastle virus if only its level is high at the beginning of the infection. It was suggested that interferon-γ might induce a delayed and insufficient response against the virus replication. The virus load and its replication might be decreased with its direct anti-viral effect, changes in innate immune response by its immunostimultory effects, or a combination of both (Peiris et al. 2009, 2010).