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Immune System Development in the Dog and Cat
ARTICLE IN PRESS J. Comp. Path. 2007,Vol. 137, S10^S15
www.elsevier.com/locate/jcpa
Immune System Development in the Dog and Cat M. J. Day Division of Veterinary Pathology, Infection and Immunity, School of ClinicalVeterinary Science, University of Bristol, Langford BS40 5DU, UK
Summary Routine vaccination of young puppies and kittens takes place within the ¢rst 16 weeks of life, during which time there is considerable change in the immune system of these animals. Newborn pups and kittens must obtain passive immune protection through the ingestion of colostrum within the ¢rst hours of life. The timing of early life vaccination is determined by the period of time required for passively acquired immunoglobulin to degrade, thereby permitting an endogenous immune response to be generated by the neonate. In the absence of inhibitory maternally derived antibody (MDA), pups and kittens are capable of mounting a protective immune response at an early age. New generation molecular vaccines appear able to circumvent the inhibitory e¡ects of MDA. In addition to changes in serum immunoglobulin concentrations, there are alterations in the numbers and proportions of blood and tissue leucocytes (particularly CD4+ and CD8+ Tcells, and B cells) during the ¢rst year of life. The qualitative nature of the newborn immune system may also alter fromTh2 regulation in utero to Th1 regulation in the neonatal period. Immune function is likely to be genetically determined, and in dogs there is evidence for breed e¡ects on immune function which likely relate to the inheritance of particular haplotypes of major histocompatibility complex (MHC) genes. The design of vaccines for young animals of these species must take into account these immunological changes and the potential modulatory e¡ect of vaccines on immune development. r 2007 Elsevier Ltd. All rights reserved. Keywords: dog; cat; immune development; colostrum; vaccine
Introduction The focus of this overview is the in utero development and neonatal maturation (to the age of approximately 3^6 months) of the immune system of the dog and cat. Despite the obvious importance of clear understanding of this area for optimizing neonatal vaccination of these species, there is in reality a sparse evidence-base concerning the development of the immune system in puppies and kittens.
In utero Development As for other domestic animal species, there is an assumption that the newborn pup or kitten has in place the constitutive components of a functional, but yet na|« ve, immune system which has to date been under the regulatory in£uence of the maternal immune milieu Correspondence to: M.J. Day (e-mail: [email protected]). 0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2007.04.005
(Felsburg, 2002). The in utero ontogeny of lymphoid tissue in the dog and cat was de¢ned many years ago, but there is little knowledge of the development of immune cell subsets and soluble proteins in these species. In the dog, development of the thymus commences on day 27 of gestation and is complete by day 45. Lymphocytes appear in the thymus on day 35, in lymph nodes on day 46 (Bryant and Shifrine, 1972) and in the spleen around day 50^55. Canine fetal splenic and thymic lymphocytes can be stimulated by mitogen from day 45 and 50, respectively (Bryant et al., 1973). Lymphocytes are known to appear in the circulation of fetal cats at approximately day 25 of gestation (Tiedemann and van Ooyen, 1978). In the ¢nal 2 weeks of gestation, the proportions of blood lymphocytes in the fetal kitten alters; with a marked elevation inTcells and reduction in ‘null cells’ (Sellon et al.,1996). Few studies have investigated whether antigenic challenge to the developing canine or feline fetus induces premature immune r 2007 Elsevier Ltd. All rights reserved.
ARTICLE IN PRESS Immune Development in Dogs and Cats
responsiveness in utero, but in the dog such responses to a range of antigens are possible in the ¢nal trimester of gestation (Krakowka,1998).
Maternal Immunity The best documented aspect of immune system development in the dog and cat relates to the essential requirement for passive transfer of immunity via the ingestion of colostral immunoglobulin (Ig). These species have endotheliochorial placentation in which there is a relatively impenetrable barrier (comprising maternal endothelium and the chorionic epithelium) to the in utero transfer of maternal immunoglobulin. It is generally accepted that small quantities of IgG may pass through this barrier, such that the newborn pup or kitten has a serum concentration of IgG that approximates to 5 per cent of the adult level. In the ¢rst 24 h after birth, the pup or kitten must ingest immunoglobulin-rich colostrum which provides passively acquired immune protection throughout the neonatal period. In newborn kittens, such absorption does not occur after the ¢rst 16 h of life (Casal et al., 1996). Although not speci¢cally documented in the dog and cat, it is assumed that this transfer is permitted to occur via mechanisms such as low concentration of intestinal proteolytic enzymes and the transient expression of the intestinal FcgRn immunoglobulin receptor allowing absorption of IgG into the neonatal vascular and lymphatic circulations. IgM and IgA are also absorbed, but it is not speci¢cally known whether colostral IgA is signi¢cantly absorbed and re-secretedçor largely remains within the intestinal lumen in these species. Similarly, there is little published information as to whether there might be absorption of other proteins (e.g. cytokines) or maternal leucocytes from this colostral source. There may be considerable variation between littermates in the e⁄ciency of uptake of colostral immunoglobulin; relating
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to the size and strength of the individual newborn and the maternal abilities of the bitch or queen. There may also be variation between the individual bitch/queen in the concentration of speci¢c antibodies within the colostral immunoglobulin. Several studies have examined the immunoglobulin composition of canine and feline colostrum and milk (Table 1). Canine colostrum is rich in both IgG and IgA and both immunoglobulins are present in higher concentration than in the serum of the bitch. By contrast, milk contains signi¢cantly more IgA than IgG and this IgA is also present in greater concentration than in canine serum (Heddle and Rowley,1975). Newborn puppies have a serum IgG concentration of 1.2 mg/ml which increases to 23 mg/ml 12 h after ingestion of colostrum. At the same time the concentration of serum IgA is 0.45 mg/ml and IgM 0.2 mg/ml (Kolb, 2003). Feline colostrum is also rich in both IgG and IgA, and both immunoglobulins are present in concentration greater than in the serum of the queen. One intriguing species di¡erence is the observation that feline milk continues to be dominated by IgG.There is debate as to the relative concentration of IgG in feline colostrum and milk; early studies suggested that the cat did not have a speci¢c colostral phase, with similar IgG concentrations in colostrum and milk (Casal et al., 1996) but more recently, reduced concentrations of IgG and IgA have been demonstrated in milk relative to colostrum (Claus et al., 2006). Newborn kittens may have undetectable serum IgG but small quantities of IgM (Casal et al.,1996; Levy et al., 2001). After ingestion of colostrum, total serum IgG and IgA levels peak and then gradually decline. Kittens may then have undetectable serum IgA between weeks 1and 6 of life. Endogenous IgG production starts by 5^6 weeks of age and IgA production shortly after. By contrast, total serum IgM concentration in newborn kittens steadily increases to plateau at about day 60 of life (Casal et al., 1996).
Table 1 Immunoglobulin concentrations in canine and feline serum, colostrum and milk Parameter Serum IgG Serum IgM Serum IgA Colostral IgG Colostral IgM Colostral IgA Milk IgG Milk IgM Milk IgA
Dog concentration in mg/ml
Cat concentration in mg/ml
5.2^17.3 (mean 9.8) 0.7^2.7 (mean 1.7) 0.2^1.2 (mean 0.5) 15.68 (160% of mean serum concentration) 0.23 (14% of mean serum concentration) 2.5 (500% of mean serum concentration) 0.098 (1% of mean serum concentration; days 25^50) 0.15 (9% of mean serum concentration; days 25^50) 1.35 (270% of mean serum concentration; days 25^50)
15.075.4 3.7 to 6.4 1.971.4 62.0723.8 0.4 to 2.0 14.3711.6 5.377.3 (day 7) 2.071.3 (day 42) 2.972.3 (day 7 and 42)
Canine data from Heddle and Rowley (1975), Feline data from Claus et al. (2006), and Casal et al. (1996) for IgM.
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M.J. Day
The uptake of maternally derived immunity (MDA) is a two-edged sword to these species. On the one hand, this is an essential process, the failure of which rapidly leads to neonatal infection and death, but on the other, the presence of high concentrations of maternal immunoglobulin inhibits development of the endogenous neonatal immune response until such time as su⁄cient of this maternal protein has degraded to permit endogenous responsiveness.The reported half-life for maternal IgG is approximately 8 days in pups, but only 4.4 days in kittens (Casal et al., 1996). In addition, it has been suggested that the growth rate of the newborn contributes to the speed of degradation of maternal immunity, with rapidly growing breeds more quickly eliminating this immunoglobulin (Chappuis, 1998). It is known that in the absence of passive transfer of maternal immunity, newborn pups are able to respond to antigen (e.g. parvovirus vaccine) as early as 2 weeks of age (Toman et al., 2002). Day-old pups lacking MDA when vaccinated with modi¢ed live parvovirus make a serological response 21^91 days post-vaccination similar in magnitude to that in older vaccinated puppies (Chappuis,1998). The point at which a newborn pup or kitten becomes immune competent (generally considered to be somewhere between 6 and 12 weeks of age) is thus determined by the concentration of colostral immunoglobulin ingested. This means that it is not possible to predict accurately the onset of immunocompetence for any one individual newborn. Therein lies the conundrum which has underpinned neonatal vaccination of pups and kittens for the past four decades: the requirement (using conventional vaccines) for administration of at least two doses of vaccine to ensure that at least one is given during the period of immunocompetence. It is clear that one advantage of new vaccine technology (e.g. molecular or vectored vaccines) is the ability of such products to stimulate endogenous immunity in the face of MDA.
Neonatal Maturation There has been minimal study of the development of the immune system within the ¢rst 6 months of the life of the pup or kitten. The majority of studies of immunity are conducted in animals over 12 months of age when these species are regarded as fully immunocompetent. Although it is likely that substantial changes occur within the immune system from birth to 1 year, they are poorly documented. It is accepted that the concentration of serum immunoglobulins (IgG, IgM and IgA) do not reach full adult levels until 12 months of age but few data support this supposition and there are no breed-speci¢c reference ranges for serum immunoglobulin concentration for
dogs or cats up to this time. One study reports serum IgG, IgM and IgA concentrations in 10 month old beagles; these concentrations are less than those in older dogs (Schreiber et al., 1992). The concentration of other serum proteins also increases with age from 6 weeks to adulthood in the dog, although that of the alpha-1 globulins shows the reverse trend (Kraft and Dusch, 2004). Little is known about maturation of lymphoid populations in the developing puppy or kitten. The blood lymphocyte count of dogs increases over the ¢rst week of life whilst neutrophil numbers decline. During the ¢rst three months of life, pups have higher blood lymphocyte counts than adult dogs but proportionally more of these cells are CD21+ B cells. These B cells decline in number to 16 weeks of age. Although the percentage of blood CD4+ Tcells remains relatively stable from birth to adulthood in dogs, the percentage of CD8+ T lymphocytes is low at birth (thus causing a high CD4:CD8 ratio) and increases with age (Faldyna et al., 2001). These blood lymphocytes are able to respond to stimulation with mitogen (e.g. Concanavalin A), but more weakly than cells from adult dogs (Toman et al., 2002). Canine blood T lymphocytes may express MHC class II molecules and this expression develops over the ¢rst months of life (Holmes and Lunn, 1994). The thymus of newborn dogs comprises approximately 12 per cent CD4+ and 3 per cent CD8+ Tcells, with 69 per cent double-positive and 13 per cent doublenegative cells (Somberg et al.,1994). Similar studies of blood lymphocyte subsets have been performed for speci¢c pathogen-free cats between birth and 90 days of age (Sellon et al., 1996; Bortnick et al., 1999). Total blood lymphocyte count increases over this period with the most marked elevation being in B cells and CD8+ T cells, leading to a reduction in CD4:CD8 ratio as described for the dog. However, in contrast to the dog (and humans), the percentage of CD4+ Tcells also increases over the same time period. A similar change in T-cell subsets occurs within the thymus and lymph nodes, suggesting active thymic output of (particularly CD8+) T cells in early life. At day 23 of life, the cat achieves the greatest thymus to body weight ratio. The thymus involutes during the ¢rst year in both species. In the cat this commences at 6^8 months of age (Woo et al.,1997) and in the dog there is progressive decline between 6 and 23 months of age (Ploeman et al., 2003). The early life maturation of the complement pathways and the function of phagocytic cells (neutrophils and macrophages) or antigen-presenting cells (dendritic cells), or other aspects of innate immunity, has not been investigated to any degree in the dog and cat.
ARTICLE IN PRESS Immune Development in Dogs and Cats
Some studies have investigated developmental aspects of mucosal immunity in the dog. Physiological and immunological changes occur within the gastrointestinal tract during maturation. During the ingestion of colostrum, the canine small intestinal villi increase in size due to hypertrophy of enterocytes with cytoplasmic vacuolation and dilation of the lacteals (Paulsen et al., 2003). These changes are less prominent in the feline intestine 24 h after birth (Buddington and Paulsen,1998). Our own investigations have compared the composition and function of the immune population in the respiratory mucosa of pups (mean age 2.3 months) and adult dogs (mean age 7.4 years). In general, adult dogs have more Tcells, plasma cells and dendritic cells at all levels of the respiratory tract, but the puppies had signi¢cantly more mast cells and macrophages within the mucosa (Peeters et al., 2005a). Despite this, there were no signi¢cant di¡erences in transcription of genes encoding a range of cytokines and chemokines between puppies and adult dogs (Peeters et al., 2005b). Similarly, in the canine nasopharyngeal mucosa, adult dogs (mean age 8.8 years) had more plasma cells than puppies (mean age 0.3 years), but pups had more dendritic cells, macrophages and T cells than the adult animals (Billen et al., 2006). Another study has reported concentrations of IgG, IgM and IgA in nasal secretions from puppies from birth to 6 weeks of age. IgG is the dominant class of immunoglobulin with the exception of time points between weeks 1 and 3, where IgA was predominant. Signi¢cant variation was noted between individual animals and between dogs of di¡erent litters (Schafer-Somi et al., 2005). A recent study has investigated gestational and early life canine immunity in the context of Toxocara canis infection (Torina et al., 2005). In this project, blood samples were taken from infected and control bitches throughout gestation and from their pups between 4 and 10 weeks of age. Blood lymphocytes were stimulated with mitogen or parasite antigen and the production of IL-10 and IFN-g cytokines within these cultures determined by enzyme linked immunosorbent assay (ELISA). Cells from both infected and uninfected bitches produced increasing concentrations of IL-10 throughout the 8 weeks of monitoring, with higher levels generated in cultures from infected animals. By contrast, IFN-g production decreased throughout pregnancy. These data suggest an immunomodulatory milieu in the pregnant bitch which is enhanced by concurrent parasitism. After birth, both infected and uninfected puppies produce IL-10 in culture, but the concentration of this cytokine declines to 10 weeks of age, whereas IFN-g production increases. This is the ¢rst evidence for a switch from a Th2/Treg milieu in
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the neonatal dog to one dominated by Th1 immunity with increasing antigenic exposure.
Genetic Aspects of Immune Competence In these species there is an extraordinary heterogeneity in phenotype through the establishment of pure breeds; a change which has largely occurred over the past 200 years. With such selective inbreeding comes recognition that there is likely to be great diversity in the functioning of the immune system between (particularly canine) breeds. This has been clear for many years, based on the unique susceptibility of particular dog breeds to immune-mediated, infectious and neoplastic disease. The key genetic elements of immune responsiveness lie within the genes of the major histocompatibility complex (MHC); present as the dog leukocyte antigen (DLA) and feline leukocyte antigen (FLA) systems in the species under discussion. Recent studies of DLA haplotypes and genome-wide screening in the dog have shown distinct breed variation but limited intra-breed heterogeneity in genetic background (Kennedy et al., 2002). This would suggest that speci¢c dog breeds have genetically determined immune function, and recent studies con¢rm breed-speci¢c serological response patterns to vaccination (Kennedy et al., 2005). Moreover, there is breed variation in the proportions of blood lymphocyte subsets (Faldyna et al., 2001). Although it was originally suspected that the cat had more limited diversity in immune response genes of the MHC, recent work has shown allotypic variation at FLA class II loci as found in the dog. Such genetic background is also likely to impinge on maturation of the immune system in these species.
Immunodeficiency Disease One area in which we have been informed about immune system development in the dog has been in the study of the rare primary immunode¢ciency disorders which a¡ect this species (but are almost never documented in cats). Although many of these are poorly characterized at the immunological and genetic level, some (such as the canine leukocyte adhesion de¢ciency [CLAD], X-linked severe combined immunode¢ciency [X-SCID] and cyclic haematopoiesis of grey collies) have been fully de¢ned, at least in part due to their importance as clinical models for the human homologues. As an example, investigations of immune function in X-SCID dogs has con¢rmed limited immune system development in pups with illness, and death of these pups generally occurs following loss of maternal immune protection (Somberg et al., 1994; Felsburg et al.,1999).
ARTICLE IN PRESS S14
M.J. Day
Experimental Manipulation of Immune Maturation The dog, in particular, is a useful large animal model for human disease. Consequently, in an experimental setting various manipulations of the developing immune system have been described. It has proven possible to sensitize newborn dogs to aeroallergens or food allergens by repeated injection of adjuvanted antigen over the ¢rst weeks of life (Day, 2005). Such animals mount the expected hypersensitivity response following antigenic challenge later in life but this can be prevented by inducing oral tolerance to the same antigen (Zemann et al., 2003). Selective breeding for immunological traits has also been documented; for example the o¡spring of bitches and sires sensitized to aeroallergen in this manner have greater propensity to mount allergen-speci¢c IgE responses and display clinical signs following challenge (Barrett et al., 2003).
Vaccination During the Period of Immune Maturation It is clear that the immune system of newborn pups and kittens, whilst likely fully capable of response to antigenic challenge, will not respond in the same fashion as that of an adult dog or cat. Although poorly de¢ned, maturational events do take place throughout the ¢rst year of life in these species. The immunological challenge that we deliver to neonates of these species through vaccination must clearly impact on this developing immune response. Our anticipation is that this will be in a positive fashion; by generating solid protective immunity and antigen-speci¢c memory cells. However, we should not lose sight of the fact that at the same time we are challenging a na|« ve and evolving immune system and the full impact of such challenge might not be appreciated.Vaccination of adult animals does lead to alterations in immune parameters in the post-vaccinal period (Strasser et al., 2003) and at least in a small proportion of neonatal animals, vaccination may potentially lead to one of a wide array of possible adverse consequences. Some studies have investigated the e¡ects of vaccination on the immune system of puppies and shown immunological alterations. For example, one group reported lymphopenia and increased response of blood lymphocytes to mitogens 7 days post-vaccination (Miyamoto et al., 1992) but this ¢nding was not replicated in a study of greyhound puppy vaccination in which there was also no e¡ect on circulating proportions of CD4+ and IgG+ lymphocytes (McMillen et al., 1995). Despite these observations, there is no doubt that in terms of risk bene¢t, neonatal vaccination is crucial for the protection of
the individual and the population at large from lifethreatening infectious disease.
References Barrett, E. G., Rudolph, K., Bowen, L. E. and Bice, D. E. (2003). Parental allergic status in£uences the risk of developing allergic sensitization and an asthmatic-like phenotype in canine o¡spring. Immunology, 110, 493^500. Billen, F., Peeters, D., Dehard, S., Day, M. J. and Clercx, C. (2006). Distribution of leucocyte subsets in the canine pharyngeal tonsil. Journal of Comparative Pathology, 135, 63^73. Bortnick, S. J., Orandle, M. S., Papadi, G. P. andJohnson, C. M. (1999). Lymphocyte subsets in neonatal and juvenile cats: comparison of blood and lymphoid tissues. Laboratory Animal Science, 49, 395^400. Bryant, B. J. and Shifrine, M. (1972). Histogenesis of lymph nodes during development of the dog. Journal of the Reticuloendothelial Society, 12, 96^107. Bryant, B. J., Shifrine, M. and McNeil, C. (1973). Cellmediated immune response in the developing dog. International Archives of Allergy, 45, 937^942. Casal, M. L., Jezyk, P. F. and Giger, U. (1996).Transfer of Colostral antibodies from queens to their kittens. American Journal of Veterinary Research, 57,1653^1658. Buddington, R. K. and Paulsen, D. B. (1998). Development of the canine and feline gastrointestinal tract. In: Recent Advances in Canine and Feline Nutrition, Vol. 2, G.A. Reinhart and D.P. Carey, Eds, Orange Frazer Press, Wilmington, Ohio, pp. 195^215. Chappuis, G. (1998). Neonatal immunity and immunisation in early age: lessons from veterinary medicine.Vaccine, 16, 1468^1472. Claus, M. A., Levy, J. K., MacDonald, K., Tucker, S. J. and Crawford, P. C. (2006). Immunoglobulin concentrations in feline colostrum and milk, and the requirement of colostrum for passive transfer of immunity to neonatal kittens. Journal of Feline Medicine and Surgery, 8,184^191. Day, M. J. (2005). The canine model of dietary hypersensitivity. Proceedings of the Nutrition Society, 64, 458^464. Faldyna, M., Leva, L., Knotigova, P. and Toman, M. (2001). Lymphocyte subsets in peripheral blood of dogsça £ow cytometric study.Veterinary Immunology and Immunopathology, 82, 2^37. Felsburg, P. J., Hartnett, B. J., Henthorn, P. S., Moore, P. F., Krakowka, S. and Ochs, H. D. (1999). Canine X-linked severe combined immunode¢ciency. Veterinary Immunology and Immunopathology, 69,127^135. Felsburg, P. J. (2002). Overview of immune system development in the dog: comparison with humans. Human and ExperimentalToxicology, 21, 487^492. Heddle, R. J. and Rowley, D. (1975). Dog immunoglobulins. I. Immunochemical characterization of dog serum, parotid saliva, colostrum, milk and small bowel £uid. Immunology, 29,185^195. Holmes, M. A. and Lunn, D. P. (1994). Variation of MHC II expression on canine lymphocytes with age. Tissue Antigens, 43,179^183.
ARTICLE IN PRESS Immune Development in Dogs and Cats
Kennedy, L. J., Barnes, A., Happ, G. M., Quinnell, R. J., Bennett, D., Angles, J. M., Day, M. J., Carmichael, N., Innes, J. F., Isherwood, D., Carter, S. D., Thomson, W. and Ollier,W. E. R. (2002). Extensive interbreed, but minimal intrabreed, variation of DLA class II alleles and haplotypes in dogs.Tissue Antigens, 59,194^204. Kennedy, L.J., Lunt, M., Barnes, A., McElhinney, L.M., Fooks, A.R. and Ollier,W.E.R. (2005). Do dogs vary in their response to rabies vaccination? Proceedings of the 48th Annual Congress of the British Small AnimalVeterinaryAssociation, p. 550. Kolb, E. (2003). The signi¢cance and composition of the colostrum and milk of the bitch: a review. Tierarztliche Umschau, 58,125. Kraft, W. and Dusch, R. (2004). Serum total protein, albumin, and globulins in relation to age in the dog.Tierarztlcihe Praxis, 32, 45^49. Krakowka, S. (1998). Immunology of the dog: ontogeny of the immune system. In: Handbook of Vertebrate Immunology, P.-P. Pastoret, H. Bazin, P. Briebel and A. Govaerts, Eds, Academic Press, San Diego, p. 271. Levy, J. K., Crawford, P. C., Collante, W. R. and Papich, M. G. (2001). Use of adult cat serum to correct failure of passive transfer in kittens. Journal of the American Veterinary Medical Association, 219,1401^1405. McMillen, G. L., Briggs, D. J., McVey, D. S., Phillips, R. M. and Jordan, F. R. (1995). Vaccination of racing greyhounds: e¡ects on humoral and cellular immunity.Veterinary Immunology and Immunopathology, 49,101^113. Miyamoto, T., Taura, Y., Une, S., Yoshitake, M., Nakama, S. andWatanabe, S. (1992). Changes in blastogenic responses of lymphocytes and delayed type hypersensitivity responses after vaccination in dogs. Journal of Veterinary Medicine and Science, 54, 945^950. Paulsen, D. B., Buddington, K. K. and Buddington, R. K. (2003). Dimensions and histologic characteristics of the small intestine of dogs during postnatal development. AmericanJournal of Veterinary Research, 64, 618^626. Peeters, D., Day, M. J., Farnir, F., Moore, P. and Clercx, C. (2005a). Distribution of leucocyte subsets in the canine respiratory tract. Journal of Comparative Pathology, 132, 261^272. Peeters, D., Peters, I. R., Farnir, F., Clercx, C. and Day, M. J. (2005b). Real-time RT-PCR quanti¢cation of mRNA encoding cytokines and chemokines in histologically normal canine nasal, bronchial and pulmonary tissue. Veterinary Immunology and Immunopathology, 104,195^204. Ploemen, J. -P. H.T. M., Ravesloot,W.T. M. andVan Esch, E. (2003). The incidence of thymic B lymphoid follicles in healthy beagle dogs.Toxicologic Pathology, 31, 214^219.
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Schafer-Somi, S., Bar-schadler, S. and Aurich, J. E. (2005). Immunoglobulins in nasal secretions of dog puppies from birth to six weeks of age. Research in Veterinary Science, 78, 143^150. Schreiber, M., Kantimm, D., Kirchho¡, D., Heimann, G. and Bhargava, A. S. (1992). Concentrations in serum of IgG, IgM and IgA and their age-dependence in beagle dogs as determined by a newly developed enzyme-linked immunosorbent assay (ELISA). European Journal of Clinical Chemistry and Clinical Biochemistry, 30, 775^778. Sellon, R. K., Levy, J. K., Jordan, H. L., Gebhard, D. H., Tompkins, M. B. and Tompkins, W. A. (1996). Changes in lymphocyte subsets with age in perinatal cats : late gestation through eight weeks.Veterinary Immunology and Immunopathology, 53,105^113. Somberg, R. L., Robinson, J. P. and Felsburg, P. J. (1994). T lymphocyte development and function in dogs with X-linked severe combined immunode¢ciency. Journal of Immunology, 153, 4006^4015. Strasser, A., May, B., Teltscher, A., Wistrela, E. and Niedermuller, H. (2003). Immune modulation following immunization with polyvalent vaccines in dogs. Veterinary Immunology and Immunopathology, 94,113^121. Tiedemann, K. and van Ooyen, B. (1978). Prenatal hematopoiesis and blood characteristics of the cat. Anatomy and Embryology, 153, 243^267. Toman, M., Faldyna, M., Knotigova, P., Pokorova, D. and Sinkora, J. (2002). Postnatal development of leukocyte subset composition and activity in dogs.Veterinary Immunology and Immunopathology, 87, 321^326. Torina, A., Caracappa, S., Barera, A., Dieli, F., Sireci, G., Genchi, C., Deplazes, P. and Salerno, A. (2005). Toxocara canis infection induces antigen-speci¢c IL-10 and IFN-g production in pregnant dogs and their puppies. Veterinary Immunology and Immunopathology, 108, 247^251. Woo, J. C., Dean, G. A., Pedersen, N. C. and Moore, P. F. (1997). Immunopathologic changes in the thymus during the acute stage of experimentally induced feline immunode¢ciency virus infection in juvenile cats. Journal of Virology, 71, 8632. Zemann, B., Schwaerzier, C., Griot-Wenk, M., Nefzger, M., Mayer, P., Schneider, H., de Weck, A., Carballido, J. M. and Liehl, E. (2003). Oral administration of speci¢c antigens to allergy-prone infant dogs induces IL-10 and TGF-b expression and prevents allergy in adult life. Journal of Allergy and Clinical Immunology, 111, 1069^1075.
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Immune System Development in the Dog and Cat M. J. Day Division of Veterinary Pathology, Infection and Immunity, School of ClinicalVeterinary Science, University of Bristol, Langford BS40 5DU, UK
Summary Routine vaccination of young puppies and kittens takes place within the ¢rst 16 weeks of life, during which time there is considerable change in the immune system of these animals. Newborn pups and kittens must obtain passive immune protection through the ingestion of colostrum within the ¢rst hours of life. The timing of early life vaccination is determined by the period of time required for passively acquired immunoglobulin to degrade, thereby permitting an endogenous immune response to be generated by the neonate. In the absence of inhibitory maternally derived antibody (MDA), pups and kittens are capable of mounting a protective immune response at an early age. New generation molecular vaccines appear able to circumvent the inhibitory e¡ects of MDA. In addition to changes in serum immunoglobulin concentrations, there are alterations in the numbers and proportions of blood and tissue leucocytes (particularly CD4+ and CD8+ Tcells, and B cells) during the ¢rst year of life. The qualitative nature of the newborn immune system may also alter fromTh2 regulation in utero to Th1 regulation in the neonatal period. Immune function is likely to be genetically determined, and in dogs there is evidence for breed e¡ects on immune function which likely relate to the inheritance of particular haplotypes of major histocompatibility complex (MHC) genes. The design of vaccines for young animals of these species must take into account these immunological changes and the potential modulatory e¡ect of vaccines on immune development. r 2007 Elsevier Ltd. All rights reserved. Keywords: dog; cat; immune development; colostrum; vaccine
Introduction The focus of this overview is the in utero development and neonatal maturation (to the age of approximately 3^6 months) of the immune system of the dog and cat. Despite the obvious importance of clear understanding of this area for optimizing neonatal vaccination of these species, there is in reality a sparse evidence-base concerning the development of the immune system in puppies and kittens.
In utero Development As for other domestic animal species, there is an assumption that the newborn pup or kitten has in place the constitutive components of a functional, but yet na|« ve, immune system which has to date been under the regulatory in£uence of the maternal immune milieu Correspondence to: M.J. Day (e-mail: [email protected]). 0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2007.04.005
(Felsburg, 2002). The in utero ontogeny of lymphoid tissue in the dog and cat was de¢ned many years ago, but there is little knowledge of the development of immune cell subsets and soluble proteins in these species. In the dog, development of the thymus commences on day 27 of gestation and is complete by day 45. Lymphocytes appear in the thymus on day 35, in lymph nodes on day 46 (Bryant and Shifrine, 1972) and in the spleen around day 50^55. Canine fetal splenic and thymic lymphocytes can be stimulated by mitogen from day 45 and 50, respectively (Bryant et al., 1973). Lymphocytes are known to appear in the circulation of fetal cats at approximately day 25 of gestation (Tiedemann and van Ooyen, 1978). In the ¢nal 2 weeks of gestation, the proportions of blood lymphocytes in the fetal kitten alters; with a marked elevation inTcells and reduction in ‘null cells’ (Sellon et al.,1996). Few studies have investigated whether antigenic challenge to the developing canine or feline fetus induces premature immune r 2007 Elsevier Ltd. All rights reserved.
ARTICLE IN PRESS Immune Development in Dogs and Cats
responsiveness in utero, but in the dog such responses to a range of antigens are possible in the ¢nal trimester of gestation (Krakowka,1998).
Maternal Immunity The best documented aspect of immune system development in the dog and cat relates to the essential requirement for passive transfer of immunity via the ingestion of colostral immunoglobulin (Ig). These species have endotheliochorial placentation in which there is a relatively impenetrable barrier (comprising maternal endothelium and the chorionic epithelium) to the in utero transfer of maternal immunoglobulin. It is generally accepted that small quantities of IgG may pass through this barrier, such that the newborn pup or kitten has a serum concentration of IgG that approximates to 5 per cent of the adult level. In the ¢rst 24 h after birth, the pup or kitten must ingest immunoglobulin-rich colostrum which provides passively acquired immune protection throughout the neonatal period. In newborn kittens, such absorption does not occur after the ¢rst 16 h of life (Casal et al., 1996). Although not speci¢cally documented in the dog and cat, it is assumed that this transfer is permitted to occur via mechanisms such as low concentration of intestinal proteolytic enzymes and the transient expression of the intestinal FcgRn immunoglobulin receptor allowing absorption of IgG into the neonatal vascular and lymphatic circulations. IgM and IgA are also absorbed, but it is not speci¢cally known whether colostral IgA is signi¢cantly absorbed and re-secretedçor largely remains within the intestinal lumen in these species. Similarly, there is little published information as to whether there might be absorption of other proteins (e.g. cytokines) or maternal leucocytes from this colostral source. There may be considerable variation between littermates in the e⁄ciency of uptake of colostral immunoglobulin; relating
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to the size and strength of the individual newborn and the maternal abilities of the bitch or queen. There may also be variation between the individual bitch/queen in the concentration of speci¢c antibodies within the colostral immunoglobulin. Several studies have examined the immunoglobulin composition of canine and feline colostrum and milk (Table 1). Canine colostrum is rich in both IgG and IgA and both immunoglobulins are present in higher concentration than in the serum of the bitch. By contrast, milk contains signi¢cantly more IgA than IgG and this IgA is also present in greater concentration than in canine serum (Heddle and Rowley,1975). Newborn puppies have a serum IgG concentration of 1.2 mg/ml which increases to 23 mg/ml 12 h after ingestion of colostrum. At the same time the concentration of serum IgA is 0.45 mg/ml and IgM 0.2 mg/ml (Kolb, 2003). Feline colostrum is also rich in both IgG and IgA, and both immunoglobulins are present in concentration greater than in the serum of the queen. One intriguing species di¡erence is the observation that feline milk continues to be dominated by IgG.There is debate as to the relative concentration of IgG in feline colostrum and milk; early studies suggested that the cat did not have a speci¢c colostral phase, with similar IgG concentrations in colostrum and milk (Casal et al., 1996) but more recently, reduced concentrations of IgG and IgA have been demonstrated in milk relative to colostrum (Claus et al., 2006). Newborn kittens may have undetectable serum IgG but small quantities of IgM (Casal et al.,1996; Levy et al., 2001). After ingestion of colostrum, total serum IgG and IgA levels peak and then gradually decline. Kittens may then have undetectable serum IgA between weeks 1and 6 of life. Endogenous IgG production starts by 5^6 weeks of age and IgA production shortly after. By contrast, total serum IgM concentration in newborn kittens steadily increases to plateau at about day 60 of life (Casal et al., 1996).
Table 1 Immunoglobulin concentrations in canine and feline serum, colostrum and milk Parameter Serum IgG Serum IgM Serum IgA Colostral IgG Colostral IgM Colostral IgA Milk IgG Milk IgM Milk IgA
Dog concentration in mg/ml
Cat concentration in mg/ml
5.2^17.3 (mean 9.8) 0.7^2.7 (mean 1.7) 0.2^1.2 (mean 0.5) 15.68 (160% of mean serum concentration) 0.23 (14% of mean serum concentration) 2.5 (500% of mean serum concentration) 0.098 (1% of mean serum concentration; days 25^50) 0.15 (9% of mean serum concentration; days 25^50) 1.35 (270% of mean serum concentration; days 25^50)
15.075.4 3.7 to 6.4 1.971.4 62.0723.8 0.4 to 2.0 14.3711.6 5.377.3 (day 7) 2.071.3 (day 42) 2.972.3 (day 7 and 42)
Canine data from Heddle and Rowley (1975), Feline data from Claus et al. (2006), and Casal et al. (1996) for IgM.
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The uptake of maternally derived immunity (MDA) is a two-edged sword to these species. On the one hand, this is an essential process, the failure of which rapidly leads to neonatal infection and death, but on the other, the presence of high concentrations of maternal immunoglobulin inhibits development of the endogenous neonatal immune response until such time as su⁄cient of this maternal protein has degraded to permit endogenous responsiveness.The reported half-life for maternal IgG is approximately 8 days in pups, but only 4.4 days in kittens (Casal et al., 1996). In addition, it has been suggested that the growth rate of the newborn contributes to the speed of degradation of maternal immunity, with rapidly growing breeds more quickly eliminating this immunoglobulin (Chappuis, 1998). It is known that in the absence of passive transfer of maternal immunity, newborn pups are able to respond to antigen (e.g. parvovirus vaccine) as early as 2 weeks of age (Toman et al., 2002). Day-old pups lacking MDA when vaccinated with modi¢ed live parvovirus make a serological response 21^91 days post-vaccination similar in magnitude to that in older vaccinated puppies (Chappuis,1998). The point at which a newborn pup or kitten becomes immune competent (generally considered to be somewhere between 6 and 12 weeks of age) is thus determined by the concentration of colostral immunoglobulin ingested. This means that it is not possible to predict accurately the onset of immunocompetence for any one individual newborn. Therein lies the conundrum which has underpinned neonatal vaccination of pups and kittens for the past four decades: the requirement (using conventional vaccines) for administration of at least two doses of vaccine to ensure that at least one is given during the period of immunocompetence. It is clear that one advantage of new vaccine technology (e.g. molecular or vectored vaccines) is the ability of such products to stimulate endogenous immunity in the face of MDA.
Neonatal Maturation There has been minimal study of the development of the immune system within the ¢rst 6 months of the life of the pup or kitten. The majority of studies of immunity are conducted in animals over 12 months of age when these species are regarded as fully immunocompetent. Although it is likely that substantial changes occur within the immune system from birth to 1 year, they are poorly documented. It is accepted that the concentration of serum immunoglobulins (IgG, IgM and IgA) do not reach full adult levels until 12 months of age but few data support this supposition and there are no breed-speci¢c reference ranges for serum immunoglobulin concentration for
dogs or cats up to this time. One study reports serum IgG, IgM and IgA concentrations in 10 month old beagles; these concentrations are less than those in older dogs (Schreiber et al., 1992). The concentration of other serum proteins also increases with age from 6 weeks to adulthood in the dog, although that of the alpha-1 globulins shows the reverse trend (Kraft and Dusch, 2004). Little is known about maturation of lymphoid populations in the developing puppy or kitten. The blood lymphocyte count of dogs increases over the ¢rst week of life whilst neutrophil numbers decline. During the ¢rst three months of life, pups have higher blood lymphocyte counts than adult dogs but proportionally more of these cells are CD21+ B cells. These B cells decline in number to 16 weeks of age. Although the percentage of blood CD4+ Tcells remains relatively stable from birth to adulthood in dogs, the percentage of CD8+ T lymphocytes is low at birth (thus causing a high CD4:CD8 ratio) and increases with age (Faldyna et al., 2001). These blood lymphocytes are able to respond to stimulation with mitogen (e.g. Concanavalin A), but more weakly than cells from adult dogs (Toman et al., 2002). Canine blood T lymphocytes may express MHC class II molecules and this expression develops over the ¢rst months of life (Holmes and Lunn, 1994). The thymus of newborn dogs comprises approximately 12 per cent CD4+ and 3 per cent CD8+ Tcells, with 69 per cent double-positive and 13 per cent doublenegative cells (Somberg et al.,1994). Similar studies of blood lymphocyte subsets have been performed for speci¢c pathogen-free cats between birth and 90 days of age (Sellon et al., 1996; Bortnick et al., 1999). Total blood lymphocyte count increases over this period with the most marked elevation being in B cells and CD8+ T cells, leading to a reduction in CD4:CD8 ratio as described for the dog. However, in contrast to the dog (and humans), the percentage of CD4+ Tcells also increases over the same time period. A similar change in T-cell subsets occurs within the thymus and lymph nodes, suggesting active thymic output of (particularly CD8+) T cells in early life. At day 23 of life, the cat achieves the greatest thymus to body weight ratio. The thymus involutes during the ¢rst year in both species. In the cat this commences at 6^8 months of age (Woo et al.,1997) and in the dog there is progressive decline between 6 and 23 months of age (Ploeman et al., 2003). The early life maturation of the complement pathways and the function of phagocytic cells (neutrophils and macrophages) or antigen-presenting cells (dendritic cells), or other aspects of innate immunity, has not been investigated to any degree in the dog and cat.
ARTICLE IN PRESS Immune Development in Dogs and Cats
Some studies have investigated developmental aspects of mucosal immunity in the dog. Physiological and immunological changes occur within the gastrointestinal tract during maturation. During the ingestion of colostrum, the canine small intestinal villi increase in size due to hypertrophy of enterocytes with cytoplasmic vacuolation and dilation of the lacteals (Paulsen et al., 2003). These changes are less prominent in the feline intestine 24 h after birth (Buddington and Paulsen,1998). Our own investigations have compared the composition and function of the immune population in the respiratory mucosa of pups (mean age 2.3 months) and adult dogs (mean age 7.4 years). In general, adult dogs have more Tcells, plasma cells and dendritic cells at all levels of the respiratory tract, but the puppies had signi¢cantly more mast cells and macrophages within the mucosa (Peeters et al., 2005a). Despite this, there were no signi¢cant di¡erences in transcription of genes encoding a range of cytokines and chemokines between puppies and adult dogs (Peeters et al., 2005b). Similarly, in the canine nasopharyngeal mucosa, adult dogs (mean age 8.8 years) had more plasma cells than puppies (mean age 0.3 years), but pups had more dendritic cells, macrophages and T cells than the adult animals (Billen et al., 2006). Another study has reported concentrations of IgG, IgM and IgA in nasal secretions from puppies from birth to 6 weeks of age. IgG is the dominant class of immunoglobulin with the exception of time points between weeks 1 and 3, where IgA was predominant. Signi¢cant variation was noted between individual animals and between dogs of di¡erent litters (Schafer-Somi et al., 2005). A recent study has investigated gestational and early life canine immunity in the context of Toxocara canis infection (Torina et al., 2005). In this project, blood samples were taken from infected and control bitches throughout gestation and from their pups between 4 and 10 weeks of age. Blood lymphocytes were stimulated with mitogen or parasite antigen and the production of IL-10 and IFN-g cytokines within these cultures determined by enzyme linked immunosorbent assay (ELISA). Cells from both infected and uninfected bitches produced increasing concentrations of IL-10 throughout the 8 weeks of monitoring, with higher levels generated in cultures from infected animals. By contrast, IFN-g production decreased throughout pregnancy. These data suggest an immunomodulatory milieu in the pregnant bitch which is enhanced by concurrent parasitism. After birth, both infected and uninfected puppies produce IL-10 in culture, but the concentration of this cytokine declines to 10 weeks of age, whereas IFN-g production increases. This is the ¢rst evidence for a switch from a Th2/Treg milieu in
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the neonatal dog to one dominated by Th1 immunity with increasing antigenic exposure.
Genetic Aspects of Immune Competence In these species there is an extraordinary heterogeneity in phenotype through the establishment of pure breeds; a change which has largely occurred over the past 200 years. With such selective inbreeding comes recognition that there is likely to be great diversity in the functioning of the immune system between (particularly canine) breeds. This has been clear for many years, based on the unique susceptibility of particular dog breeds to immune-mediated, infectious and neoplastic disease. The key genetic elements of immune responsiveness lie within the genes of the major histocompatibility complex (MHC); present as the dog leukocyte antigen (DLA) and feline leukocyte antigen (FLA) systems in the species under discussion. Recent studies of DLA haplotypes and genome-wide screening in the dog have shown distinct breed variation but limited intra-breed heterogeneity in genetic background (Kennedy et al., 2002). This would suggest that speci¢c dog breeds have genetically determined immune function, and recent studies con¢rm breed-speci¢c serological response patterns to vaccination (Kennedy et al., 2005). Moreover, there is breed variation in the proportions of blood lymphocyte subsets (Faldyna et al., 2001). Although it was originally suspected that the cat had more limited diversity in immune response genes of the MHC, recent work has shown allotypic variation at FLA class II loci as found in the dog. Such genetic background is also likely to impinge on maturation of the immune system in these species.
Immunodeficiency Disease One area in which we have been informed about immune system development in the dog has been in the study of the rare primary immunode¢ciency disorders which a¡ect this species (but are almost never documented in cats). Although many of these are poorly characterized at the immunological and genetic level, some (such as the canine leukocyte adhesion de¢ciency [CLAD], X-linked severe combined immunode¢ciency [X-SCID] and cyclic haematopoiesis of grey collies) have been fully de¢ned, at least in part due to their importance as clinical models for the human homologues. As an example, investigations of immune function in X-SCID dogs has con¢rmed limited immune system development in pups with illness, and death of these pups generally occurs following loss of maternal immune protection (Somberg et al., 1994; Felsburg et al.,1999).
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Experimental Manipulation of Immune Maturation The dog, in particular, is a useful large animal model for human disease. Consequently, in an experimental setting various manipulations of the developing immune system have been described. It has proven possible to sensitize newborn dogs to aeroallergens or food allergens by repeated injection of adjuvanted antigen over the ¢rst weeks of life (Day, 2005). Such animals mount the expected hypersensitivity response following antigenic challenge later in life but this can be prevented by inducing oral tolerance to the same antigen (Zemann et al., 2003). Selective breeding for immunological traits has also been documented; for example the o¡spring of bitches and sires sensitized to aeroallergen in this manner have greater propensity to mount allergen-speci¢c IgE responses and display clinical signs following challenge (Barrett et al., 2003).
Vaccination During the Period of Immune Maturation It is clear that the immune system of newborn pups and kittens, whilst likely fully capable of response to antigenic challenge, will not respond in the same fashion as that of an adult dog or cat. Although poorly de¢ned, maturational events do take place throughout the ¢rst year of life in these species. The immunological challenge that we deliver to neonates of these species through vaccination must clearly impact on this developing immune response. Our anticipation is that this will be in a positive fashion; by generating solid protective immunity and antigen-speci¢c memory cells. However, we should not lose sight of the fact that at the same time we are challenging a na|« ve and evolving immune system and the full impact of such challenge might not be appreciated.Vaccination of adult animals does lead to alterations in immune parameters in the post-vaccinal period (Strasser et al., 2003) and at least in a small proportion of neonatal animals, vaccination may potentially lead to one of a wide array of possible adverse consequences. Some studies have investigated the e¡ects of vaccination on the immune system of puppies and shown immunological alterations. For example, one group reported lymphopenia and increased response of blood lymphocytes to mitogens 7 days post-vaccination (Miyamoto et al., 1992) but this ¢nding was not replicated in a study of greyhound puppy vaccination in which there was also no e¡ect on circulating proportions of CD4+ and IgG+ lymphocytes (McMillen et al., 1995). Despite these observations, there is no doubt that in terms of risk bene¢t, neonatal vaccination is crucial for the protection of
the individual and the population at large from lifethreatening infectious disease.
References Barrett, E. G., Rudolph, K., Bowen, L. E. and Bice, D. E. (2003). Parental allergic status in£uences the risk of developing allergic sensitization and an asthmatic-like phenotype in canine o¡spring. Immunology, 110, 493^500. Billen, F., Peeters, D., Dehard, S., Day, M. J. and Clercx, C. (2006). Distribution of leucocyte subsets in the canine pharyngeal tonsil. Journal of Comparative Pathology, 135, 63^73. Bortnick, S. J., Orandle, M. S., Papadi, G. P. andJohnson, C. M. (1999). Lymphocyte subsets in neonatal and juvenile cats: comparison of blood and lymphoid tissues. Laboratory Animal Science, 49, 395^400. Bryant, B. J. and Shifrine, M. (1972). Histogenesis of lymph nodes during development of the dog. Journal of the Reticuloendothelial Society, 12, 96^107. Bryant, B. J., Shifrine, M. and McNeil, C. (1973). Cellmediated immune response in the developing dog. International Archives of Allergy, 45, 937^942. Casal, M. L., Jezyk, P. F. and Giger, U. (1996).Transfer of Colostral antibodies from queens to their kittens. American Journal of Veterinary Research, 57,1653^1658. Buddington, R. K. and Paulsen, D. B. (1998). Development of the canine and feline gastrointestinal tract. In: Recent Advances in Canine and Feline Nutrition, Vol. 2, G.A. Reinhart and D.P. Carey, Eds, Orange Frazer Press, Wilmington, Ohio, pp. 195^215. Chappuis, G. (1998). Neonatal immunity and immunisation in early age: lessons from veterinary medicine.Vaccine, 16, 1468^1472. Claus, M. A., Levy, J. K., MacDonald, K., Tucker, S. J. and Crawford, P. C. (2006). Immunoglobulin concentrations in feline colostrum and milk, and the requirement of colostrum for passive transfer of immunity to neonatal kittens. Journal of Feline Medicine and Surgery, 8,184^191. Day, M. J. (2005). The canine model of dietary hypersensitivity. Proceedings of the Nutrition Society, 64, 458^464. Faldyna, M., Leva, L., Knotigova, P. and Toman, M. (2001). Lymphocyte subsets in peripheral blood of dogsça £ow cytometric study.Veterinary Immunology and Immunopathology, 82, 2^37. Felsburg, P. J., Hartnett, B. J., Henthorn, P. S., Moore, P. F., Krakowka, S. and Ochs, H. D. (1999). Canine X-linked severe combined immunode¢ciency. Veterinary Immunology and Immunopathology, 69,127^135. Felsburg, P. J. (2002). Overview of immune system development in the dog: comparison with humans. Human and ExperimentalToxicology, 21, 487^492. Heddle, R. J. and Rowley, D. (1975). Dog immunoglobulins. I. Immunochemical characterization of dog serum, parotid saliva, colostrum, milk and small bowel £uid. Immunology, 29,185^195. Holmes, M. A. and Lunn, D. P. (1994). Variation of MHC II expression on canine lymphocytes with age. Tissue Antigens, 43,179^183.
ARTICLE IN PRESS Immune Development in Dogs and Cats
Kennedy, L. J., Barnes, A., Happ, G. M., Quinnell, R. J., Bennett, D., Angles, J. M., Day, M. J., Carmichael, N., Innes, J. F., Isherwood, D., Carter, S. D., Thomson, W. and Ollier,W. E. R. (2002). Extensive interbreed, but minimal intrabreed, variation of DLA class II alleles and haplotypes in dogs.Tissue Antigens, 59,194^204. Kennedy, L.J., Lunt, M., Barnes, A., McElhinney, L.M., Fooks, A.R. and Ollier,W.E.R. (2005). Do dogs vary in their response to rabies vaccination? Proceedings of the 48th Annual Congress of the British Small AnimalVeterinaryAssociation, p. 550. Kolb, E. (2003). The signi¢cance and composition of the colostrum and milk of the bitch: a review. Tierarztliche Umschau, 58,125. Kraft, W. and Dusch, R. (2004). Serum total protein, albumin, and globulins in relation to age in the dog.Tierarztlcihe Praxis, 32, 45^49. Krakowka, S. (1998). Immunology of the dog: ontogeny of the immune system. In: Handbook of Vertebrate Immunology, P.-P. Pastoret, H. Bazin, P. Briebel and A. Govaerts, Eds, Academic Press, San Diego, p. 271. Levy, J. K., Crawford, P. C., Collante, W. R. and Papich, M. G. (2001). Use of adult cat serum to correct failure of passive transfer in kittens. Journal of the American Veterinary Medical Association, 219,1401^1405. McMillen, G. L., Briggs, D. J., McVey, D. S., Phillips, R. M. and Jordan, F. R. (1995). Vaccination of racing greyhounds: e¡ects on humoral and cellular immunity.Veterinary Immunology and Immunopathology, 49,101^113. Miyamoto, T., Taura, Y., Une, S., Yoshitake, M., Nakama, S. andWatanabe, S. (1992). Changes in blastogenic responses of lymphocytes and delayed type hypersensitivity responses after vaccination in dogs. Journal of Veterinary Medicine and Science, 54, 945^950. Paulsen, D. B., Buddington, K. K. and Buddington, R. K. (2003). Dimensions and histologic characteristics of the small intestine of dogs during postnatal development. AmericanJournal of Veterinary Research, 64, 618^626. Peeters, D., Day, M. J., Farnir, F., Moore, P. and Clercx, C. (2005a). Distribution of leucocyte subsets in the canine respiratory tract. Journal of Comparative Pathology, 132, 261^272. Peeters, D., Peters, I. R., Farnir, F., Clercx, C. and Day, M. J. (2005b). Real-time RT-PCR quanti¢cation of mRNA encoding cytokines and chemokines in histologically normal canine nasal, bronchial and pulmonary tissue. Veterinary Immunology and Immunopathology, 104,195^204. Ploemen, J. -P. H.T. M., Ravesloot,W.T. M. andVan Esch, E. (2003). The incidence of thymic B lymphoid follicles in healthy beagle dogs.Toxicologic Pathology, 31, 214^219.
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Schafer-Somi, S., Bar-schadler, S. and Aurich, J. E. (2005). Immunoglobulins in nasal secretions of dog puppies from birth to six weeks of age. Research in Veterinary Science, 78, 143^150. Schreiber, M., Kantimm, D., Kirchho¡, D., Heimann, G. and Bhargava, A. S. (1992). Concentrations in serum of IgG, IgM and IgA and their age-dependence in beagle dogs as determined by a newly developed enzyme-linked immunosorbent assay (ELISA). European Journal of Clinical Chemistry and Clinical Biochemistry, 30, 775^778. Sellon, R. K., Levy, J. K., Jordan, H. L., Gebhard, D. H., Tompkins, M. B. and Tompkins, W. A. (1996). Changes in lymphocyte subsets with age in perinatal cats : late gestation through eight weeks.Veterinary Immunology and Immunopathology, 53,105^113. Somberg, R. L., Robinson, J. P. and Felsburg, P. J. (1994). T lymphocyte development and function in dogs with X-linked severe combined immunode¢ciency. Journal of Immunology, 153, 4006^4015. Strasser, A., May, B., Teltscher, A., Wistrela, E. and Niedermuller, H. (2003). Immune modulation following immunization with polyvalent vaccines in dogs. Veterinary Immunology and Immunopathology, 94,113^121. Tiedemann, K. and van Ooyen, B. (1978). Prenatal hematopoiesis and blood characteristics of the cat. Anatomy and Embryology, 153, 243^267. Toman, M., Faldyna, M., Knotigova, P., Pokorova, D. and Sinkora, J. (2002). Postnatal development of leukocyte subset composition and activity in dogs.Veterinary Immunology and Immunopathology, 87, 321^326. Torina, A., Caracappa, S., Barera, A., Dieli, F., Sireci, G., Genchi, C., Deplazes, P. and Salerno, A. (2005). Toxocara canis infection induces antigen-speci¢c IL-10 and IFN-g production in pregnant dogs and their puppies. Veterinary Immunology and Immunopathology, 108, 247^251. Woo, J. C., Dean, G. A., Pedersen, N. C. and Moore, P. F. (1997). Immunopathologic changes in the thymus during the acute stage of experimentally induced feline immunode¢ciency virus infection in juvenile cats. Journal of Virology, 71, 8632. Zemann, B., Schwaerzier, C., Griot-Wenk, M., Nefzger, M., Mayer, P., Schneider, H., de Weck, A., Carballido, J. M. and Liehl, E. (2003). Oral administration of speci¢c antigens to allergy-prone infant dogs induces IL-10 and TGF-b expression and prevents allergy in adult life. Journal of Allergy and Clinical Immunology, 111, 1069^1075.