Development and Maturation of the Immune Response
Ontogeny studies of the immune response revealed that the fetus produces a specific antibody response to T cell-dependent antigens (bacteriophage ØX-174), ovine erythrocytes, and Brucella canis. Fetal T cells of the spleen, lymph nodes, and thymus respond to the mitogen phytohemagglutinin (PHA). These studies showed that the fetus possesses a functional lymphocyte (B and T cells) system able to generate humoral and cellular immune responses against several antigens, suggesting that newborns are immunocompetent close to, or at birth [36,37,38,72].
Furthermore, some studies confirmed that colostrum-deprived one-day-old puppies are able to mount humoral immune responses to some antigens [35,84]. Besides, it was demonstrated that puppies with different ages develop lymphoproliferative responses to several types of mitogen [72,74,85], contradicting the idea of antigen neonatal tolerance.
However, MDA inhibits the development of endogenous neonatal immune response and constitutes the main obstacle to successful vaccination [35,38,53]. In an attempt to determine the optimal age to initiate vaccination, the specific humoral immune response elicited by several vaccine preparations was studied in puppies with different ages and from distinct breeds [54,74,86]. It seems that antibody response to vaccination is specific to each animal and depends on the age of the dog, protective antibody titer, and vaccine type [86]. Thus, it is assumed that around 6–12 weeks, MDA no longer interferes with the development of an adequate immune response, and puppies are considered immunocompetent ( C) [38]. Accordingly, international guidelines recommend starting vaccination at 6–8 weeks of age, then re-vaccinate each 2–4 weeks until 16 weeks of age or older, to ensure that at least one vaccine dose induces immunity [27].
In puppies, MHCII is constitutively expressed by APCs [87]. Thus, the phagocytic activity of peripheral blood leukocytes, (PMN and monocytes) at birth and in two-month-old puppies seems not to be compromised [74]. Studies realized in mice and humans have demonstrated some differences between immune responses in adults and neonates. Newborn APCs have a reduced capacity to express CD86 and CD40, and the respective ligands (CD28 and CD40L, respectively) on lymphocytes are also reduced. Binding of antigen to BCR does not induce the hyper-expression of MHC class II molecules, increase the expression of B7.2 (CD86) costimulatory molecule, or the upregulation of CD40 and CD40L. Thus, the defective interaction between B and T cells results in T cell anergy, deviation towards Th2 response, hampers specific B cell response and B cell switch to different B cell classes and subclasses [53,88,89]. Although it is unknown if the same happens in the dog, some studies point towards a Th2 polarization of the neonatal immune response. As the dog grows, the immune system undergoes an educational process provided by exposure to Th1 antigens, achieving a balanced Th1-Th2 immune response [53,90,91] ( D). However, the improvement of dog’s living conditions, good nutrition, vaccination, and deworming programs probably decreases the exposition to Th1 stimulus. Vaccination may also have a profound and long-lasting effect in driving the immune system to a Th1 or Th2 response, that was not yet investigated [90].
Increased life span allowed the recognition of age-related higher susceptibility to infectious, inflammatory, autoimmune, and neoplastic diseases [92]. The gradual deterioration of the immune system function associated with aging is called immunosenescense [93,94,95]. This phenomenon can result from an intrinsic ageing process related to thymus involution and decreased output of naïve cells from bone marrow and thymus ( E). However, the depletion of the reservoir of naïve cells over time by contact with pathogens and their conversion into memory cells during adaptive immune responses may also contribute to the decline of the immune function [96].
Despite contradictory results, some findings were consistently associated with canine elderly, namely the reduction of blood CD4+ T cells, expansion of the CD8+ cell subset with a subsequent reduction of CD4:CD8 ratio, and a decrease of naïve lymphocytes [97,98,99,100,101,102,103].
Age-related changes include impairment of the cell-mediated immune response, as demonstrated by the reduction of proliferative response of blood lymphocytes to mitogens and the reduction of cutaneous delayed type hypersensitivity. Moreover, there is a decline in the humoral immune response probably related to the decreased functionality of Th cells. The ability to mount humoral immune responses seems to prevail, as demonstrated by the persistence of protective vaccine antibody titers, and respond to booster vaccination with elevation in titer [94]. Although the currently adopted triennial re-vaccination program, instead of the prior annual re-vaccination, offers adequate protection to young and adult dogs [104], this vaccination scheme may not confer protection to geriatric dogs [105].
Older dogs commonly present an impairment of immune responses to novel antigenic challenges, such as infections and vaccines, which probably is related to the reduction of the peripheral pool of naïve T cells and low diversity of the repertoire of T cell receptors [95]. Indeed, first-time rabies vaccination of older dogs revealed a significant decrease in antibody titers and an increase of vaccination failure, suggesting that the elderly can compromise the primary response to vaccination [106].
Few investigations have studied the effect of cumulative antigenic exposure and the onset of late-life inflammatory disease (inflammageing) in this species. Available data suggest that elderly dogs exhibit an elevation of serum concentration of oxidative damage biomarker, indicating that they have a reduced ability to respond to oxidative stress. The implementation of strategies to slow the pro-inflammatory state, such as supplementation with antioxidants and the optimization of vaccination protocols, seems to be essential to promote a long and healthy elderly [107].
Development and Maturation of Lymphoid Organs
The ontogeny of the canine immune organs was reviewed in a few publications [36,37,59]. Hematopoietic and immune cells arise from a common bone marrow stem cell. Thereafter, B cells undergo maturation in the fetal liver and bone marrow, which represent successive primary lymphoid organs. B cells maturation involves the acquisition of BCR and selection to ensure that only B cells that express functional BCR (positive selection) and do not ligate self-antigens (negative selection) survive. On the other hand, immature T cells are exported to the thymus for final maturation [1,60,61]. The thymus generates a diverse repertoire of T cells that undergo positive and negative selection, ensuring that autoreactive cells are eliminated before reach peripheral organs [1,62]. Monocytes and granulocytes (neutrophils, eosinophils, and basophils) mature in the bone marrow and are released into the bloodstream [1].
Maturation of the immune system occurs from birth to approximately six months old. Although the puppy was considered immunocompetent between 6–12 weeks of age, it is not possible to predict accurately the onset of immunocompetence, since it depends on the presence of MDA [38].
In growing animals, hematopoietic bone marrow is located inside the long and flat bones, but as the animal ages the medullary cavity is replaced by fatty tissue and active bone marrow is confined to the trabecular cavities of flat bones, and epiphyses and metaphysis of long bones [63]. The proportion of T helper cells (CD4+), cytotoxic T cells (CD8+), and ‘unconventional’ γδ-T cells increases with age. Mature T cells (CD3+) reaches more than 60% in adult dogs ( ). With increasing age, bone marrow plays a dual role of primary and secondary lymphatic organ, maintaining a pool of effector and memory lymphocytes [64].
Thymus rapidly grows in dogs, reaching maximum size at six months of age. Then, when the dog reaches sexual maturity (between 6 and 23 months), the organ suffers involution, characterized by reduction of thymic parenchyma, which is replaced by adipose, connective tissue, and prominent epithelial structures (cords, tubules, cysts) [24,65,66,67]. In newborn puppies, the organ comprises approximately 12% of CD4+ and 3% of CD8+ T cells, of which 69% were double-positive (CD4+CD8+) and 13% double negative (CD4−CD8−) cells [68]. The CD4+CD8+ double positive phenotype characterizes immature T cells during thymic development [69]. Approximately 5% of the cells recovered by thymus tissue teasing were CD34+ progenitor cells [70].
Secondary lymphoid tissues include the encapsulated organs (lymph nodes and spleen) and mucosal associated lymphoid tissue (MALT). Secondary lymphoid tissues facilitate interactions between naïve lymphocytes and APC, leading to productive adaptive immune responses [71].
The distribution of lymphocyte subsets in secondary lymphoid tissue of one-day-old puppies and adult dogs was studied. The percentage of T cells (CD3+) was lower in the spleen compared with lymph nodes. Conversely, B cells (CD21+) predominate in the spleen relative to other compartments ( ). The highest proportion of B cells observed in the spleen of puppies may be related to its function as primary B-lymphopoietic organ. The subsequent reduction of B cell population in adult dogs may result from the peripheral negative selection in the spleen and recirculation of these cells to the induction sites, namely lymph nodes. A relatively high number of γδ-T cells were found in the spleen, although very few cells were present in lymph nodes [72]. These cells retain cytotoxic activity and constitute a first line of defense of epidermal and mucosal epithelial linings [14,73].
MALT comprises non-encapsulated lymphoid tissue that is continuously exposed to antigens against which it is necessary to mount an immune response or maintain immune tolerance [76,77,78,79]. The presence of nasal associated lymphoid tissue was documented in dogs [67], but bronchus associated lymphoid tissue seems not to be a constitutive structure [77]. Gut associated lymphoid tissue (GALT) is characterized by agglomerates of lymphocytes, organized into discrete follicles denominated Peyer’s patches within the mucosa, as well as lymphocytes scattered throughout the lamina propria [67]. Dogs have two distinct types of Peyer’s patches: duodenal and jejunal (proximal units), and single ileal Peyer’s patches. The proximal units contribute to the mucosal immune response, while the ileal Peyer’s patches present features of a primary lymphoid organ, participating in the early development of the B cell system, and show involution when dogs reach sexual maturity [80,81]. There is a remarkable increase in the weight of mesenteric lymph nodes after weaning, which reflects the importance played by lymph nodes in fighting exogenous antigens [65].
Regarding circulating leukocyte population, polymorphonuclear neutrophils (PMN) predominates in the first day of life and were almost three times higher than lymphocyte count. During the first week, there is a decrease in PMN and a transient predominance of lymphocytes, which might reflect immune system activation through contact with foreign antigens [74]. Reference values of blood cells determined for puppies with age ranging between 16 and 60 days old are presented in .
| 16–24 Days | 28–46 Days | 46–60 Days | Adult (Reference Range) | |
|---|---|---|---|---|
| WBC (×109/L) | 11.0 (2.9) | 13.1 (2.7) | 13.7 (3.8) | 6–18 |
| Neutrophils (×109/L) | 5.5 (2.4) | 7.0 (1.8) | 7.1 (2.1) | 3.6–13.0 |
| Lymphocytes (×109/L) | 4.2 (1.4) | 4.6 (1.1) | 5.0 (2.1) | 0.8–5.8 |
| Monocytes (×109/L) | 0.6 (0.18) | 0.9 (0.2) | 0.9 (0.3) | 0–1.6 |
| Eosinophils (×109/L) | 0.5 (0.26) | 0.4 (0.25) | 0.5 (0.39) | 0–1.8 |
The phenotype of the circulating lymphocyte subpopulation in neonatal dogs differs significantly from that of adult dogs. Indeed, peripheral blood of a one-day-old puppy contains a lower proportion of T cells, with a very low rate of CD8+ T cells, and a high amount of B cells [72,74]. Although the percentage of blood CD4+ T cells remains relatively stable from birth to adulthood, the amount of CD8+ T cells increases with age. The high levels of circulating B cells observed in newborn puppies decreased progressively with age. Moreover, it was also reported a nonsignificant age-related decrease of γδ-T lymphocytes [74,75] ( ).
Thus, newborn puppies present a developing immune system, which is different from adults. The fetus lives in the uterus, which is a sterile environment, ensuring that T and B cells of the newborn not met its cognate antigen in the periphery (naïve cells) and that activated, and memory cells were absent [83]. After birth, the newborn is exposed to microbial-rich surroundings. The exposure to external antigens stimulates the immune system, inducing the massive activation and redistribution of peripheral lymphocytes, which change the size and structure of lymphoid organs and promote the appearance of lymphocytes in previously empty spaces [72]. The maturation of the immune system progresses from birth to approximately six months old [38].
What form of vitamin C is best for dogs?
You may be familiar with vitamin C as ‘ascorbic acid’. However, this is a form that the body struggles to use to best effect. Experts agree that ‘sodium ascorbate’ is the easiest to digest and also lasts longest in the body. Also, choose sodium ascorbate products marked as ‘USP Pure’.