Overview of the Immune Dynamics of the Digestive System

The digestive tract of the chicken is a major site of pathogen exposure. Although the bird has a multifaceted set of tools to prevent or resist infection, any activation of the immune system can divert nutrients away from production. Therefore, prevention of pathogenic exposure is preferred. However, it is unlikely that the bird can escape exposure to all pathogens during its life, thus the ability to respond to immunologic challenges is essential. The immune system of birds is similar to that of mammals in terms of structure and function, although some differences do exist, particularly in regulatory aspects. The innate immune system responds nonspecifically to foreign molecules and is essential for the induction of the specific (acquired) immune response. Cells of the innate immune system include macrophages, dendritic cells, heterophils, and natural killer cells. The acquired immune response involves recognition of a specific antigen and response by lymphocytes. Cytotoxic T lymphocytes are especially effective at inducing cells infected with intracellular pathogens to undergo apoptosis. Helper T lymphocytes increase the effectiveness of innate immune cells in combating extracellular pathogens and are also essential for activating B lymphocytes, which produce antibodies specific to the invading pathogen. All aspects of the immune system function together, although one aspect will often dominate, depending on the type and severity of the infection. This paper reviews the basics of avian immune function in general and discusses the immune system in the digestive tract in particular in birds. The consequences of activation of the immune system are presented.Currently, growth-promoting antibiotics are not used in poultry in many countries; the North American industry may be moving in that direction as well, either through legislation or consumer pressure. Several nonantibiotic means of manipulating the immune system to prevent the health- and performance-suppressing effects of immune system activation are presented here.     

Systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin.

Serpulina hyodysenteriae infection of pigs, swine dysentery, causes a mucohemorrhagic diarrhoea resulting in significant economic losses to swine producers. The pathogenesis of this disease is poorly understood. Regardless, commercial vaccines have been developed and are in use. Thus, the present study was designed to examine cellular immune responses induced by parenteral S. hyodysenteriae vaccination. Significant antigen-specific interferon-gamma (IFN-gamma) and blastogenic responses were detected from peripheral blood lymphocytes isolated from vaccinated pigs. However, poor IFN-gamma responses were detected from colonic lymph node lymphocytes from these same pigs despite significant antigen-specific blastogenic responses. In addition, peripheral blood IFN-gamma responses were diminished by either in vitro depletion of CD4 expressing cells or by in vitro treatment with porcine IL-10. Colonic lymph node IFN-gamma responses were not inhibited by treatment with porcine IL-10. Vaccination also resulted in increased percentages of both mucosal and peripheral blood CD8 single positive cells with concurrent decreases in percentages of CD4 single positive cells as compared to percentages of these same populations from non-vaccinated pigs. In conclusion, these studies show that parenteral S. hyodysenteriae vaccination results in cellular immune responses detectable both peripherally (systemic immunity) as well as at the site of infection (mucosal immunity). However, it appears that regulatory mechanisms affecting IFN-gamma production in response to S. hyodysenteriae antigen differ between peripheral and colonic compartments.     

猪免疫口蹄疫灭活疫苗后外周血细胞亚群变化的研究

为了解动物在注射口蹄疫灭活疫苗后机体免疫状态的变化情况,本试验选取口蹄疫阴性猪进行疫苗免疫,利用流式细胞术检测方法来测定外周血中免疫细胞亚群的变化。结果显示,树突状细胞在免疫后能够快速产生应答,在24 h内其含量出现明显升高;B细胞和Th细胞含量在免疫后第10天都出现了明显升高,且两者之间的变化存在着高度相关性,而抗体水平的变化情况与这两种细胞含量的变化也高度相关;细胞毒性T细胞含量在免疫后也出现升高。这说明灭活疫苗免疫动物后不仅可以激活机体的体液免疫应答反应,产生高水平抗体,发挥免疫保护作用,更重要的是能够在短时间内激发机体的固有免疫应答,同时能够激活细胞免疫应答,全方位发挥免疫保护作用。     

Study on Change of Lymphocyte Subpopulations in Swine after Immunization with Inactivated Vaccine

In order to investigate the change of lymphocyte subpopulations in the peripheral blood after injection of foot-and-mouth disease inactivated vaccine, blood samples were collected after vaccination and assayed with flow cytometry.The results showed that the proportion of dendritic cells (DCs) increased significantly in a short time after vaccination, after which the number of B cells, helper T (Th) cells and cytotoxic lymphocytes (CTLs) also raised with significance.The changing trend of B cells proportion was highly relevant with Th cells and a same correlation also appeared in the antibody titers.This indicated that the innate immune system could be activated rapidly after vaccination with the inactivated vaccine, and the adaptive immune response including cellular immune response and humoral immune response could be invoked subsequently.The high level of neutralizing antibodies was of great importance in evading FMDV and the cellular immune response also exerted a crucial role.     

Ovine and murine T cell epitopes from the non-structural protein 1 (NS1) of bluetongue virus serotype 8 (BTV-8) are shared among viral serotypes

Bluetongue virus (BTV) is a non-enveloped dsRNA virus that causes a haemorrhagic disease mainly in sheep. It is an economically important Orbivirus of the Reoviridae family. In order to estimate the importance of T cell responses during BTV infection, it is essential to identify the epitopes targeted by the immune system. In the present work, we selected potential T cell epitopes (3 MHC-class II-binding and 8 MHC-class I binding peptides) for the C57BL/6 mouse strain from the BTV-8 non-structural protein NS1, using H2^b-binding predictive algorithms. Peptide binding assays confirmed all MHC-class I predicted peptides bound MHC-class I molecules. The immunogenicity of these 11 predicted peptides was then determined using splenocytes from BTV-8-inoculated C57BL/6 mice. Four MHC-class I binding peptides elicited specific IFN-γ production and generated cytotoxic T lymphocytes (CTL) in BTV-8 infected mice. CTL specific for 2 of these peptides were also able to recognise target cells infected with different BTV serotypes. Similarly, using a combination of IFN-γ ELISPOT, intracellular cytokine staining and proliferation assays, two MHC-class II peptides were identified as CD4⁺ T cell epitopes in BTV-8 infected mice. Importantly, two peptides were also consistently immunogenic in sheep infected with BTV-8 using IFN-γ ELISPOT assays. Both of these peptides stimulated CD4⁺ T cells that cross-reacted with other BTV serotypes. The characterisation of these T cell epitopes can help develop vaccines protecting against a broad spectrum of BTV serotypes and differentiate infected from vaccinated animals.     

ISVD‐1 Highlights of the skin immune system

Similar to intestine and pulmonary tissue, skin is in contact with the environment and hence, requires efficient immune surveillance. The concept of skin‐associated lymphoid tissue (SALT) was first introduced in 1978. The skin immune system (SIS) with its cellular and humoral components and the associated draining lymph nodes fulfills the three major functions of the innate and adaptive immune system: (1) induction of the primary immune response; (2) expression of immunity to previously encountered antigens (memory); and (3) avoidance of deleterious immune responses to nonthreatening antigens (tolerance). The cellular components of the SIS include dendritic antigen‐presenting cells (Langerhans cells and dermal dendritic cells), skin‐homing αβ‐T lymphocytes and γδ‐T lymphocytes, keratinocytes and their environment, the microvascular unit (endothelial cells of the dermal postcapillary venules), neural cells and dendritic neural processes, and the draining lymph node with its high endothelial venules. Humoral components of the SIS are defensins, complement components, immunoglobulins, cytokines, chemokines, fibrinolysin, eicosanoids and neuropeptides. Important characteristics and functions of these components and their interactions with each other will be discussed with regard to normal skin versus recruitment phase, retention phase and resolution phase associated with inflammatory conditions. The discussion will primarily focus on cutaneous dendritic antigen‐presenting cells, cutaneous lymphocytes and their homing into the skin, and keratinocytes. Moreover, the importance of the microvascular unit in relation to leukocyte recruitment and migration will be discussed.     

BCG vaccination of neonatal calves: potential roles for innate immune cells in the induction of protective immunity

Bovine tuberculosis is a disease of increasing incidence in the UK causing major economic losses and with significant impact on bovine and, potentially human health: the causative agent Mycobacterium bovis is a zoonotic pathogen. Neonatal vaccination with the attenuated M. bovis Bacille Calmette Guerin (BCG) vaccine confers a significant degree of protection in cattle, and is a widely used control strategy for human TB. The adaptive immune system is relatively immature in neonates and increased numbers of innate effector cells present in young animals and human infants may compensate for this, enabling effective immune responses to vaccination. Natural killer cells and subsets of γδ TCR+ T lymphocytes secrete high levels of interferon gamma and can interact with antigen presenting cells to promote both innate and adaptive immune responses. These cell populations may be pivotal in determining immune bias following neonatal vaccination with BCG.     

Antigen presentation in vaccine development

A variety of microorganisms, nutrients or toxins are generally intrude our body through mucosal tissues or skin, where equipment for both preventing their invasions and catching their information to activate internal immune systems for adapting surroundings is arranged. Among the equipment, cells in charge of innate immunity, particularly dendritic cells (DCs), having an excellent capacity for prompt recognition of invaded pathogens via toll-like receptors (TLRs) to alert B and T cells for establishing aquired/adaptive immunity by presenting their processed antigenic fragments, have been paid great attention. These TLR-activated, antigen captured DCs are divided into two groups; one is pathogen-retaining unit and the other is pathogen-controlling unit. The latter DCs present processed antigenic molecules from the pathogens to competent β T cells together with special containers, such as class I, class II MHC and CD1 to generate specific cellular immunity. The former two MHC molecules can present processed peptide antigens, whereas the last CD1 molecule can present glycolipid/lipid antigens. In contrast, B lymphocytes that captured antigens via their specific immunoglobulin (Ig) receptors present digested peptide fragments with their class II MHC to stimulate suitable CD4⁺ helper T cells which in turn secrete various cytokines to efficiently expand and maintain antibody production from that partner B cells to establish humoral immunity. These β T cells and antibodies, recognize either processed antigenic peptide or glycolipid fragments, and thus, identification of these epitopes enables us to generate artificial pathogen-specific vaccines. Based on the recent findings about precise mechanisms of antigen processing and presentation orchestrated at the surface compartment, future development of vaccines against various pathogens are discussed.     

新城疫病毒样颗粒研究进展

新城疫(Newcastle disease,ND)是由新城疫病毒(Newcastle disease virus,NDV)引起的一种急性、高度接触性禽类烈性传染病,以呼吸道、消化道及神经系统症状为主要特征,在易感家禽中死亡率高达100%,给中国及世界养禽业和贸易往来带来了严重的经济损失。病毒样颗粒(virus-like particles,VLPs)是由病毒蛋白在感染过程中体外表达或自发组装而形成的不含病毒基因组、不能复制、不具有感染能力的病毒样蛋白颗粒。VLPs适宜的大小及独特的表面结构可被天然免疫细胞和分子有效识别,诱导良好的先天性免疫应答和适应性免疫应答,且VLPs不含病毒的感染性核酸,安全性较高,近年来成为疫苗领域中的研究热点。树突状细胞(dendritic cells,DCs)作为专职抗原递呈细胞,在连接天然免疫与适应性免疫之间具有独特功能,是目前抗原递呈能力最强的专职免疫细胞。成熟DCs (mature DCs,mDCs)迁移至次级淋巴组织能刺激初始型T细胞活化和增殖,被认为是特异性免疫应答的始动者,体外培养的不成熟DCs (immature DCs,imDCs) 也可辅助增强抗原递呈能力。NDV-VLPs主要是以NDV-M蛋白为骨架,来装配HN、F和NP蛋白,可表现出与活的NDV颗粒相似的外观和免疫原性。可见通过NDV-VLPs制成的疫苗同样具备安全、稳定、可刺激体液和细胞免疫应答、作为载体表达其他抗原及可设计成区分自然感染与免疫接种等诸多优点。文章主要就NDV-VLPs的相关内容展开探讨,以期为后续研究提供参考。     

A comparison of DNA vaccines expressing the 45W, 18k and 16k host-protective antigens of Taenia ovis in mice and sheep

The immunogenicity of DNA vaccines encoding three different Taenia ovis host-protective antigens was compared in mice and sheep. DNA vaccines encoding the 45W, 18k and 16k antigens of T. ovis were constructed. The ability of DNA vaccines encoding the 45W and 18k genes to express antigen was confirmed by Western blotting of transfected Cos-7 cells. BALB/c mice were vaccinated intramuscularly with 45W, 18k or 16k DNA vaccines and the humoral immune response analysed by ELISA. DNA vaccines expressing 45W, 18k or 16k antigen were immunogenic in mice and generated significant titres of antigen-specific antibody. Intramuscular vaccination of outbred sheep with the T. ovis DNA vaccines generated significantly lower titres of 45W-specific antibody and failed to generate 18k or 16k-specific antibody. The findings of this study show that each of the three T. ovis host-protective antigens are amenable to delivery via DNA vaccines, and that the parameters governing the efficacy of DNA vaccines in sheep require further investigation.     

Investigation of antigen specific lymphocyte responses in healthy horses vaccinated with an inactivated West Nile virus vaccine

West Nile virus (WNV) is a single-stranded, enveloped RNA virus capable of causing encephalitic disease in horses. Unvaccinated horses are at risk for developing WNV disease in endemic geographic regions. Effective vaccination reduces disease frequency and diminishes disease severity in vaccinated individuals that become infected with WNV. Recent data indicate CD4+ lymphocytes are required for effective protection against disease; in particular, cross talk between CD4+ and CD8+ lymphocytes must be functional. The objective of this project was to investigate immune responses in horses throughout a series of three vaccinations using a commercial inactivated vaccine under natural conditions. Immune responses to vaccination were determined by neutralizing antibody titers with plaque reduction neutralization test (PRNT), IgM titer (capture ELISA), WNV specific antibody Ig subclass responses, WNV lymphocyte proliferative responses and intracellular cytokine expression. Horses were vaccinated with a series of three vaccines at 3-week intervals using an inactivated product. An initial measure of immune activation following vaccination was determined by evaluating changes in lymphocyte cytokine expression. Interferon (IFN) gamma and interleukin (IL)-4 expressing CD4+ lymphocytes significantly increased 14 days following initial vaccination compared to unvaccinated horses (P<0.05). IFN-gamma expressing CD8+ lymphocytes also increased and remained elevated for 110 days. Antigen specific lymphocyte proliferative responses were significantly increased up to 90 days following the third vaccination (P<0.05). As expected, vaccinated horses produced increased neutralizing antibody based on PRNT data and WNV antigen-specific Ig subclass responses compared with unvaccinated horses (P<0.05). Our data indicate that WNV vaccination with an inactivated product effectively induced an antigen-specific antibody responses, as well as CD4+ and CD8+ lymphocyte activation.     

Overview of the avian immune system.

The avian immune system operates on the same general principles as the mammalian immune system. Antigenic stimulation initiates an immune response that involves cellular cooperation most notably between macrophages, B lymphocytes and T lymphocytes. Macrophages process the antigen and present the antigen to the lymphocytes. B lymphocytes, the principal cells that mediate humoral immunity, transform into plasma cells and produce antibodies. T lymphocytes, most important for cellular immunity, differentiate into functionally diverse subpopulations. The subpopulations of avian T cells have been identified with monoclonal reagents and appear to be similar to those of mammalian T cells. Lymphokines, the soluble products secreted by immune cells, mediate the functions of these cells. Studies on avian lymphokines have lagged behind those on mammalian lymphokines because the genes coding for avian lymphokines have not been cloned. The avian lymphokines studied thus far appear to function along the same lines as the mammalian lymphokines. The immune response in birds is highly regulated and breakdown in regulation often results in immunodepression.     

Canine leishmaniosis. Modulation of macrophage/lymphocyte interactions by L. infantum

Canine leishmaniosis, caused by Leishmania infantum, is a systemic disease with variable clinical signs and a progressive evolution. This disease is characterized by impaired T cell-mediated immune response, which has been associated with disease chronicity and high mortality. Protective immunity against leishmaniosis is thought to be mediated by T cell and cytokine production. The T cell activation requires a primary signal delivered by the major histocompatibility complex (MHC) molecules present on the surface of antigen presenting cells, and a non-specific signal generated by co-stimulatory molecules. To characterize canine immune responses in the presence of L. infantum parasites or their antigens, in vitro cell cultures of canine macrophages and lymphocytes were established, and the macrophages presenting MHC class II molecules were evaluated as well as the expression of IL-12 and CD80-86 co-stimulatory molecules and nitric oxide production. The results showed for the first time the up-regulation of MHC class II molecules on the surface in canine peripheral blood monocyte-derived macrophages during L. infantum infection in the presence of lymphocytes. In addition, a lack of co-stimulatory expression and a reduced release of nitric oxide were observed, suggesting a loss of T cell function and consequently an inactivation of the macrophage oxidative burst which, in turn, favors the survival of Leishmania. These results constitute a new contribution for the understanding of the interactions between L. infantum and the canine immune system.     

Characterization of the immune response to Mycoplasma bovis lung infection

To better understand the interaction between Mycoplasma bovis and its bovine host, we have characterized the immune response generated during an experimental lung infection with M. bovis. Proliferation ([3H]-thymidine blastogenesis) and Th1/Th2 cytokine production were used to monitor peripheral cellular immune responses. Flow cytometry analysis was used to determine T-cell subset activity by CD25 expression. Humoral immune response was monitored by the identification of antigen-specific IgG1 and IgG2 isotypes over time. Herein, we show that M. bovis antigen stimulates activation of CD4+ and CD8+ cells in vitro in a manner consistent with memory, and that gammadelta-T cells are activated by antigen in a manner consistent with innate immunity. In addition, the percentage of cells producing IFN-gamma during recall response is equal to that of IL-4 producing cells. Serological analysis shows M. bovis stimulates increased production of antigen-specific IgG1 while very little IgG2 is produced. We therefore submit that experimental lung infection of cattle with M. bovis results in a Th2-skewed immune response.     

Immunoprophylaxis of bovine dermatophytosis.

The literature on immunoprophylaxis as control method for ringworm in cattle is reviewed. Scientific papers on immune response to dermatophyte antigens and vaccination against ringworm were obtained from personal files and computerized search in 4 relevant databases. Vaccines with antigens of Trichophyton verrucosum stimulate a humoral and cellular immune response. In animals vaccinated with inactivated vaccines, some protection is observed after challenge. However, the protective immunity is inadequate in most cases. Vaccination with live vaccines elicits an immune response that prevents the development of clinical disease. The protective immunity is based mainly on the cellular branch of the immune system. The efficacy and safety of live dermatophyte vaccines have been demonstrated in both challenge experiments and field trials from different countries. Effective control of ringworm in cattle has been achieved in regions implementing systematic vaccination.     

Functional expression of a bovine major histocompatibility complex class I gene in transgenic mice

Major histocompatibility complex (MHC) class I restricted cellular immune responses play an important role in immunity to intracellular pathogens. By binding antigenic peptides and presenting them to T cells, class I molecules impose significant selection on the targets of immune responses. Candidate vaccine antigens for cellular immune responses should therefore be analysed in the context of MHC class I antigen presentation. Transgenic mice expressing human MHC (HLA) genes provide a useful model for the identification of potential cytotoxic T lymphocyte (CTL) antigens. To facilitate the analysis of candidate CTL vaccines in cattle, we have produced transgenic mice expressing a common bovine MHC (BoLA) class I allele.The functional BoLA-A11 gene, carried on a 7 kb genomic DNA fragment, was used to make transgenic mice by pronuclear microinjection. Three transgenic mouse lines carrying the BoLA-A11 gene were established. Expression of the BoLA-A11 gene was found in RNA and the A11 product could be detected on the surface of spleen and blood cells. Functional analysis of the A11 transgene product, and its ability to act as an antigen presenting molecules in the mouse host will be discussed.     

Lentivirus-induced immune dysregulation

FIV/HIV infections are associated with an early robust humoral and cellular anti-viral immune response followed by a progressive immune suppression that eventually results in AIDS. Several mechanisms responsible for this immune dysfunction have been proposed including cytokine dysregulation, immunologic anergy and apoptosis, and inappropriate activation of immune regulatory cells. Studies on FIV infection provide evidence for all three. Cytokine alterations include decreases in IL2 and IL12 production and increases in IFNγ and IL10 in FIV⁺ cats compared to normal cats. The elevated IL10:IL12 ratio is associated with the inability of FIV⁺ cats to mount a successful immune response to secondary pathogens. Additionally, chronic antigenic (FIV) stimulation results in an increase in the percent of activated T cells expressing B7 and CTLA4 co-stimulatory molecules in infected cats. The expression of these molecules is associated with T cells that are undergoing apoptosis in the lymph nodes. As ligation of CTLA4 by B7 transduces a signal for induction of anergy, one can speculate that the activated T cells are capable of T cell–T cell interactions resulting in anergy and apoptosis. The inability of CD4⁺ cells from FIV⁺ cats to produce IL2 in response to recall antigens and the gradual loss of CD4⁺ cell numbers could be due to B7–CTLA4 interactions. The chronic antigenemia may also lead to activation of CD4⁺CD25⁺ T regulatory cells. Treg cells from FIV⁺ cats are chronically activated and inhibit the mitogen-induced proliferative response of CD4⁺CD25⁻ by down-regulating IL2 production. Although Treg cell activation can be antigen-specific, the suppressor function is not, and thus activated Treg cells would suppress responses to secondary pathogens as well as to FIV. Concomitant with the well-known virus-induced immune suppression is a progressive immune hyper-activation. Evidence for immune hyper-activation includes polyclonal B cell responses, gradual replacement of naïve CD4⁺ and CD8⁺ T cell phenotypes with activation phenotypes (CD62L⁻, B7⁺, CTLA4⁺), and the chronic activation of CD4⁺CD25⁺ Treg cells. Thus lentivirus infections lead to severe immune dysregulation manifested as both chronic immune suppression and chronic immune activation. FIV infection of cats provides a number of advantages over other lentivirus infections as a model to study this immune dysregulation. It is a natural infection that has existed in balance with the cat's immune system for thousands of years. As such, the natural history and pathogenesis provides an excellent model to study the long-term relationships between AIDS lentivirus and host immune system function/dysregulation.     

Bovine viral diarrhea virus type 1- and type 2-specific bovine T lymphocyte-subset responses following modified-live virus vaccination

Expression of CD25 (interleukin-2 receptor alpha chain) was used to monitor antigen-specific activation of T lymphocyte subsets (CD4+, CD8+, and gamma delta T cells) from cattle immunized with modified-live virus (MLV) bovine viral diarrhea virus (BVDV) vaccines. Two groups of 15 animals each were vaccinated with one dose of either BVDV genotype 1 (BVDV-1) or BVDV-1 and BVDV genotype 2 (BVDV-1/2). Six animals negative for both BVDV antibody and BVDV virus were used as negative controls. Three animals vaccinated 7 and 5 weeks before the start of the experiment with MLV BVDV-1 vaccine served as positive controls. Blood samples were taken from the negative control group, the positive control group, and the BVDV-1/2 group 0, 21, 35, 60, and 90 days after vaccination. Blood samples were taken from the BVDV-1 group 0, 21, and 90 days after vaccination. Isolated peripheral blood lymphocytes from immunized and control animals were incubated for 5 days with and without BVDV-1 or BVDV-2. Compared with nonvaccinated animals, a significant (P <.05) increase in expression of CD25 by CD4+ (60 days), CD8+, and gammadelta T (35 to 90 days) lymphocytes from the group given BVDV-1/2 was detected following in vitro exposure to BVDV-1 or BVDV-2 after vaccination. The CD8+ and gammadelta T cells from the group vaccinated with BVDV-1 had significantly (P <.05) increased expression of CD25 compared with nonvaccinates following postvaccination exposure to in vitro BVDV-1 but not to BVDV-2. There was no significant difference between the two vaccinated groups in CD25 expression on any of the T cell subsets in response to BVDV-1 or BVDV-2 exposure. A single administration of MLV BVDV vaccine may be more effective at stimulating CD8+ and gammadelta T cell-specific immune responses to the homologous genotype than to the heterologous genotype.     

Comparison of in ovo and post-hatch vaccination with particular reference to infectious bursal disease. A review

In ovo vaccination is an alternative approach to post-hatch vaccination of chickens, particularly in broilers. Vaccination at embryonation day 18 helps to 'close the window' of susceptibility i.e. the time between vaccination and early exposure to infectious agents compared with post-hatch vaccination. Attempts on embryonal vaccination as a mode of vaccine delivery were approached from the observation that chickens already develop certain immunologic functions before hatching. The immune system in birds begins to develop early during embryogenesis and various immune reactions have been induced in the late stage chicken embryos. Compared with post-hatch vaccination, in ovo vaccination stimulates both the innate and adaptive immune responses with the advantage that because of the prenatal immunization, in ovo vaccinated chicks have developed an appreciable degree of protection by the time of hatch. Effects of maternal antibodies on vaccines to be used for in ovo vaccination can be prevented by developing vaccines that are insensitive to maternal antibodies. It has been described that vaccination of chicken embryos at embryonation day 18 did not significantly affect the immune competence of hatched chickens. The apparent absence of tolerance in chicks hatched from embryos exposed to an antigen at the late stage of embryonation implies the feasibility of in ovo vaccination. Investigations on in ovo vaccination to produce safe and efficient vaccines are still in progress. Currently a large number of vaccines are under investigation for viral, bacterial and protozoal diseases.