Immunity in the Developing Fetus and the Newborn, Maternal Immunity in Ruminants, the Role of the Gut Microbiota

Ian Tizzard (From Proceedings Ruvasa Congress 2016)

When a mammal is born, it emerges from the sterile uterus into an environment where it is immediately exposed to a host of microorganisms. Its surfaces acquire a complex microbial flora within hours. If it is to survive, the newborn animal must be able to control this microbial invasion. In practice, the adaptive immune system takes some time to become fully functional, and innate mechanisms are responsible for the initial resistance to infection. In the domestic mammals, the adaptive immune system is fully developed at birth but cannot function at adult levels for several weeks. The complete development of adaptive immunity depends on antigenic stimulation. Thus, newborn mammals are vulnerable to infection for the first few weeks of life. They need assistance in defending themselves at this time. This temporary help is provided by the mother in the form of antibodies from colostrum and milk. The passive transfer of immunity from mother to newborn is essential for survival. Although the gestation period of the cow is 280 days, the fetal thymus is recognizable by 40 days postconception. The bone marrow and spleen appear at 55 days. Lymph nodes are found at 60 days, but Peyer’s patches do not appear until 175 days. Blood lymphocytes are seen in fetal calves by day 45, IgM+ B cells by day 59, and IgG+ B cells by day 135. The time of appearance of serum antibodies depends on the sensitivity of the techniques used. It is therefore no accident that the earliest detectable immune responses are those directed against viruses, using highly sensitive virus neutralization tests. Fetal calves have been reported to respond to rotavirus at 73 days, to parvovirus at 93 days, and to parainfluenza 3 virus at 120 days. Fetal blood lymphocytes can respond to mitogens between 75 and 80 days, but this ability is temporarily lost near the time of birth as a result of high serum steroid levels. T cells are present in newborn calves at levels comparable to adults, but B cell numbers increase significantly during the first 6 months after birth.

Lamb

The gestation period of the ewe is about 145 days. Major histocompatibility complex (MHC) class I positive cells can be detected by day 19, and MHC class II positive cells can be found by day 25. The thymus and lymph nodes are recognizable by 35 and 50 days postconception, respectively. Gut-associated follicles appear in the colon at 60 days, jejunal Peyer’s patches at about days 75 to 80, and ileal Peyer’s Patches at days 110 to 115. Blood lymphocytes are seen in fetal lambs by day 32, and CD4+ and CD8+ cells appear in the thymus by 35 to 38 days. B cells are detectable at 48 days in the spleen and by that time have already begun to rearrange their IGLV genes.

C3 receptors appear by day 120, but Fc receptors do not appear until the animal is born. Fetal liver lymphocytes can respond to stimulants by 38 days. Lambs can produce antibodies to phage fX174 at day 41 and reject skin allografts by day 77. Some fetal lambs can produce antibodies to Akabane virus by as early as 50 days postconception. Antibodies to Cache Valley virus can be provoked by day 76, to SV40 virus by day 90, to T4 phage by day 105, to bluetongue virus by day 122, and to lymphocytic choriomeningitis virus by day 140. The proportions of α/β and γ/δ T cells change as lambs mature. Thus, 1 month before birth, 18% of blood T cells are γ/δ positive. By 1 month after birth, they constitute 60% of blood T cells.

Piglet

The gestation period of the sow is about 115 days. B cells appear in the yolk sac at day 20, progress to the fetal liver by day 30, and to the bone marrow by day 45. The first leukocytes can be found in the yolk sac and liver on day 17. The thymus develops by 40 days postconception and is colonized by T cell progenitors beginning on day 38. γ/δ T cells appear first in the thymus and in peripheral blood about 10 days later. α/β T cells develop by day 55, but their numbers grow rapidly so that they predominate late in gestation.The intestinal lymphoid tissues are devoid of T cells at birth. CD4+ T cells appear in the intestine at 2 weeks of age, and CD8+ T cells appear at 4 weeks.

Their proliferation is driven by the intestinal microflora. IgM+ B cells can be found in liver at 40 days, spleen by day 50, and bone marrow by day 60. Fetal piglets can produce antibodies to parvoviruses at 58 days and can reject allografts at about the same time. Blood lymphocytes can respond to mitogens between 48 and 54 days. Natural killer (NK) cell activity does not develop until several weeks after birth, although cells with an NK phenotype can be identified at 45 days’ gestation in spleen and umbilical blood.

B cells are the first lymphocytes to appear in peripheral blood. The number of circulating B cells rises significantly between 70 and 80 days’ gestation. The molecular development of the antibody repertoire has been followed in the developing pig. Thus, VDJ rearrangement is first seen in the fetal liver at day 30. However, the fetal piglet does not initially use all available genes. IgM, IgA, and IgG transcripts are present from 50 days in all major lymphoid organs. Piglets are thus born with relatively limited B cell diversity. B cell numbers increase for the first 4 weeks after birth, but their antigen-binding repertoire does not begin to expand until 4 to 6 weeks of age.

Maternal Immunity in Ruminants

The placenta of ruminants is syndesmochorial; that is, the chorionic epithelium is in direct contact with uterine tissues, whereas the placenta of horses and pigs is epitheliochorial and the fetal chorionic epithelium is in contact with intact uterine epithelium. In mammals with both these types of placenta, the transplacental passage of immunoglobulin molecules is totally prevented. Thus their newborns are entirely dependent on antibodies received through the colostrum.

Secretion and Composition of Colostrum and Milk

Colostrum contains the accumulated secretions of the mammary gland over the last few weeks of pregnancy together with proteins actively transferred from the bloodstream under the influence of estrogens and progesterone. Therefore, it is rich in IgG and IgA and contains some IgM. The predominant immunoglobulin in the colostrum of most of the major domestic mammals is IgG, which may account for 65% to 90% of its total antibody content; IgA and the other immunoglobulins are usually minor but significant components. As lactation progresses and colostrum changes to milk, differences among species emerge. In pigs and horses, IgG predominates in colostrum, but its concentration drops rapidly as lactation proceeds so that IgA predominates in milk. In ruminants, IgG1 is the predominant immunoglobulin in both milk and colostrum.

All of the IgG, most of the IgM, and about half of the IgA in bovine colostrum are derived by transfer from the bloodstream. In milk, in contrast, only 30% of the IgG and 10% of the IgA are so derived; the rest is produced locally by lymphoid tissue within the udder. Colostrum also contains secretory component both in the free form and bound to IgA. Colostrum is rich in cytokines. For example, bovine colostrum contains significant amounts of IL-1β, IL-6, TNF-α and IFN-γ. These cytokines may promote the development of the immune system in the young animal.

Absorption of Colostrum

Young mammals that suckle soon after birth ingest colostrum. Thus naturally suckled calves ingest an average of 2 L of colostrum, although individual calves may ingest as much as 6 L. In these young mammals, protease activity in the digestive tract is low and is further reduced by trypsin inhibitors in colostrum. Therefore, colostral proteins are not degraded but can reach the small intestine intact. Colostral immunoglobulins bind to receptors on intestinal epithelial cells. Once bound to these receptors, immunoglobulin molecules are taken up by intestinal epithelial cells and transferred to the lacteals and possibly the intestinal capillaries. Eventually the absorbed immunoglobulin reaches the bloodstream, and newborn mammals obtain a massive transfusion of maternal immunoglobulins.

Newborn mammals differ in the selectivity and duration of intestinal permeability. In the horse and pig, protein absorption is selective. IgG and IgM are preferentially absorbed, whereas IgA mainly remains in the intestine. In ruminants, immunoglobulin absorption is unselective, and all classes are absorbed, although IgA is gradually excreted. Young pigs and probably other young mammals have large amounts of free secretory component in their intestine. Colostral IgA and, to a lesser extent, IgM can bind this secretory component, which may then inhibit their absorption. The duration of intestinal permeability varies among species and immunoglobulin classes. In general, permeability is highest immediately after birth and declines after about 6 hours, perhaps because of the replacement of receptor-bearing intestinal epithelial cells by cells that do not express this receptor. As a rule, absorption of all immunoglobulin classes drops to a very low level after about 24 hours. Feeding colostrum tends to hasten this closure, whereas a delay in feeding results in a slight delay in closure (up to 33 hours). In piglets, the ability to absorb immunoglobulins may be retained for up to 4 days if milk products are withheld. The presence of the mother may be associated with increased immunoglobulin absorption. Thus calves fed measured amounts of colostrum in the presence of the mother will absorb more immunoglobulins than calves fed the same amount in her absence. In laboratory studies in which measured amounts of colostrum are fed, there is a great variation (25% to 35%) in the quantity of immunoglobulins absorbed. Management should ensure that calves ingest at least 1 L of colostrum within 6 hours of birth.

Unsuckled mammals normally have very low levels of immunoglobulins in their serum. The successful absorption of colostral immunoglobulins immediately supplies them with serum IgG at a level approaching that found in adults. Peak serum immunoglobulin levels are normally reached between 12 and 24 hours after birth. After absorption ceases, these passively acquired antibodies decline through normal metabolic processes. The rate of decline differs among immunoglobulin classes, and the time taken to decline to nonprotective levels depends on their initial concentration.

The secretions of the mammary gland gradually change from colostrum to milk. Ruminant milk is rich in IgG1 and IgA. Nonruminant milk is rich in IgA. For the first few weeks in life, while protease activity is low, these immunoglobulins can be found throughout the intestine and in the feces of young mammals. As the digestive ability of the intestine increases, eventually only secretory IgA molecules remain intact. The amount of IgA provided by milk can be large; for instance, a 3-week-old piglet may receive 1.6 g daily from sow’s milk.

The IgG transferred through a mother’s colostrum represents the results of her history of antigen exposure, B cell responses, and somatic mutation. This maternal IgG in effect represents the immunological experiences of the mother. Maternal antibodies act on the immune system of the newborn during a critical imprinting period and appear to exert a lifelong influence on the newborn’s immune development. This influence may be stronger than some genetic predispositions! Thus maternal antibodies can enhance the newborn immune responses to some antigens and suppress their responses to others. They may also influence Th1/Th2 polarization and the subsequent development of allergies.

Failure of Passive Transfer

The absorption of IgG from colostrum is required for the protection of a newborn against septicaemic disease. The continuous intake of IgA or IgG1 from milk is required for protection against enteric disease. Failure of these processes predisposes a young animal to infection. There are three major reasons for failure of passive transfer through colostrum. First, the mother may produce insufficient or poor-quality colostrum (production failure). Second, there may be sufficient colostrum produced but inadequate intake by the newborn animal (ingestion failure). Third, there may be a failure of absorption from the intestine despite an adequate intake of colostrum (absorption failure).

Production Failure

Since colostrum represents the accumulated secretions of the udder in late pregnancy, premature births may mean that insufficient colostrum has accumulated. Valuable colostrum may also be lost from mammals as a result of premature lactation or excessive dripping before birth. Colostral IgG levels also vary among individuals, with up to 28% of mares producing low-quality colostrum. It is not possible to assess colostral quality simply by looking at it. Its IgG content should be assessed using a colostrometer (a modified hydrometer) to measure its specific gravity. This is normally in the range of 1.060 to 1.085, equivalent to an IgG concentration of 3000 to 8500 mg/dL.

Ingestion Failure

In sheep or pigs, an inadequate intake may result from multiple births simply because the amount of colostrum produced does not rise in proportion to the number of newborn. It may be due to poor mothering, an important problem among young, inexperienced mothers. It also may be due to weakness in the newborn, to a poor suckling drive, or to physical problems such as damaged teats or jaw defects.

Absorption Failure

Failure of intestinal absorption is a major cause for concern in any species. It is especially important in horses not only because of the value of many foals but also because even with good husbandry, about 25% of newborn foals fail to absorb sufficient quantities of immunoglobulins.

Diagnosis of Failure of Passive Transfer

The success of passive transfer cannot be evaluated until 18 to 24 hours after birth when antibody absorption is essentially complete. Several assays for serum immunoglobulins are available. The most rapid and economic procedure is the zinc sulfate turbidity test, which involves mixing a zinc sulfate solution with serum. Zinc sulfate makes globulins insoluble. In total failure of transfer, the reaction mixture remains clear. In sera with an IgG level of more than 400 mg/dL, the mixture becomes cloudy. As an alternative to visual inspection, the optical density of the mixtures can be read in a spectrophotometer and the IgG concentration read off a standard curve. Other similar techniques include precipitation by glutaraldehyde or by sodium sulfite.

Single radial immunodiffusion is a more accurate method in that it is both quantitative and specific for IgG. Known standards are compared with the test serum by measuring the diameter of precipitation produced in agar gel containing an antiserum to bovine IgG. Unfortunately radial immunodiffusion is slow. It takes 18 to 24 hours to give a result and is thus impractical when a rapid diagnosis is required. It is also possible to use a semiquantitative membrane-filter enzyme-linked immunosorbent assay (ELISA) test to measure IgG in serum. The color intensity of the reaction on the test filter is compared with color calibration spots. A variant technique uses a dipstick ELISA. Less satisfactory techniques include serum protein electrophoresis and refractometry. (Refractometry is an effective and practical test in calves but is less reliable in foals, in which the wide range of values leads to inaccuracy).

Management of Failure of Passive Transfer

Calves with serum IgG of less than 1000 mg/dL at 24 to 48 hours of age have mortality rates more than twice those of calves with higher IgG levels. A minimum of 150 to 200 g of colostral IgG is required for optimal passive transfer. Three liters of colostrum should be administered by oropharyngeal tube to calves within 2 hours of birth. Substantially larger quantities of IgG must be administered after 2 hours to achieve optimal protection. Commercially available colostrum may be enriched in specific antibodies to protect the calf against potential pathogens such as K99 Escherichia coli, rotaviruses, and coronaviruses, the major causes of calf diarrhea.

Cell-Mediated Immunity and Colostrum

Colostrum is full of lymphocytes, but milk is not. For example, sow colostrum contains between 1 x 105 and 1 x 106 lymphocytes/mL. Of these lymphocytes, 70% to 80% are T cells. Bovine colostrum also contains up to 106 lymphocytes/mL, about half of which are T cells. Colostral lymphocytes may survive up to 36 hours in the intestine of newborn calves, and some may penetrate the epithelium of Peyer’s patches and reach the lacteal ducts or the mesenteric lymph nodes. Within 2 hours after receiving colostrum that contained labeled cells, maternal lymphocytes appeared in the bloodstream of piglets. It is possible that cell-mediated immunity is transferred to newborn mammals in this way. Piglets that had received these colostral cells showed enhanced responses to mitogens compared with control mammals. Cell-containing and cell-free colostrum have been compared for their ability to protect calves against enteropathic E. coli. The calves receiving colostral cells excreted significantly fewer bacteria than the mammals receiving cell-free colostrum. The concentration of IgA- and IgM-specific antibodies against E. coli in the serum of neonatal calves was higher in those that received colostral cells than in those that did not. The calves that received colostral cells had better responses to the mitogen concanavalin A and to foreign antigens such as sheep erythrocytes. The mechanisms of this protective effect are unclear.

The CD8+ T cells in bovine colostrum can produce large quantities of IFN-γ which may influence the early development of Th1 responses in neonatal calves. Thus ingestion of maternal colostral cells appears to accelerate the development of activated calf lymphocytes. Transfer of cell-mediated immunity by bovine milk lymphocytes has been demonstrated. Pregnant cows were vaccinated against BVDV. Blood lymphocytes from calves that received cell-free colostrum from these cows were unresponsive to BVDV antigen. In contrast, lymphocytes from calves that received colostrum containing live cells showed enhanced responses to BVDV antigen at 1 and 2 days after colostral ingestion. The lymphocytes of calves that received whole colostrum showed enhanced responses to maternal and unrelated leukocytes after 24 hours. They also responded to the nonspecific stimulant staphylococcal enterotoxin B. In contrast, the lymphocytes of calves that received acellular colostrum did not. Clearly, ingestion of maternal colostral leukocytes immediately after birth stimulates the development of the neonatal immune system.

Development of Adaptive Immunity

Although a fetus is not totally defenseless, it is less capable than an adult of combating infection. Its adaptive immune system is not fully functional; as a result, some infections may be mild or unapparent in the mother but severe or lethal in the fetus. Examples include bluetongue, infectious bovine rhinotracheitis [bovine herpesvirus 1 (BHV-1)], bovine viral diarrhea, rubella in humans, and toxoplasmosis. Fetal infections commonly trigger an immune response as shown by lymphoid hyperplasia and elevated immunoglobulin levels. For this reason the presence of any immunoglobulins in the serum of a newborn, unsuckled animal suggests infection in utero.

In general, the response to these viruses is determined by the state of immunological development of the fetus. For example, if live bluetongue virus vaccine, which is nonpathogenic for normal adult sheep, is given to pregnant ewes at 50 days postconception, it causes severe lesions in the nervous system of fetal lambs, including hydranencephaly and retinal dysplasia, whereas if it is given at 100 days postconception or to newborn lambs, only a mild inflammatory response is seen. Bluetongue vaccine virus given to fetal lambs between 50 and 70 days postconception may be isolated from lamb tissues for several weeks, but if given after 100 days, reisolation is not usually possible. Akabane virus acts in a similar fashion in lambs. If given before 30 to 36 days postconception, it causes congenital deformities. If given to older fetuses, it provokes antibody formation and is much less likely to cause malformations. Piglets that receive parvovirus before 55 days postconception will usually be aborted or stillborn. After 72 days, however, piglets will normally develop high levels of antibodies to the parvovirus and survive. Prenatal infection of calves with BHV-1 results in a fatal disease, in contrast to postnatal infections, which are relatively mild. The transition between these two types of infection occurs during the last month of pregnancy.

The effects of the timing of viral infection are well seen with bovine viral diarrhea virus (BVDV). If a cow is infected early in pregnancy (up to 50 days), she may abort. On the other hand, infections occurring between 50 and 120 days, before the fetus develops immune competence, lead to asymptomatic persistent infection because the calves develop tolerance to the virus. These calves are viremic yet, because of their tolerance, fail to make antibodies or T cells against the virus. Some of these calves may show minor neurologic problems and failure to thrive, but many are clinically normal. If the cow is infected with BVDV between 100 and 180 days postconception, calves may be born with severe malformations involving the central nervous system and eye, as well as jaw defects, atrophy, and growth retardation. Vaccines containing modified live BVDV may have a similar effect if administered at the same time. Calves infected after 150 to 180 days’ gestation are usually clinically normal. Since they are specifically tolerant to BVDV, persistently infected calves shed large quantities of virus in body secretions and excretions and so act as a major source of infectious virus. The persistently infected calves may also produce neutralizing antibodies if immunized with a live BVDV vaccine of a serotype different from that of the persistent virus. Despite this, the original virus will persist in these animals. These persistently infected calves grow slowly and often die of opportunistic infections such as pneumonia before reaching adulthood. (BVDV has a tropism for lymphocytes and is immunosuppressive.) Their neutrophil functions are also depressed.

BVD viruses occur in two distinct biotypes: cytopathic and noncytopathic. (The name derives from their behavior in cell culture, not their pathogenicity in animals.) Noncytopathic strains do not trigger type I interferon (IFN) production and therefore can survive in calves and cause persistent infections. Cytopathic strains induce IFN production and cannot cause persistent infection. These cytopathic strains, however, do cause mucosal disease (MD), a severe enteric disease leading to profuse diarrhea and death. Mucosal disease develops as a result of a mutation in a nonstructural viral gene that changes the BVDV biotype from noncytopathic to cytopathic while the animal fails to produce neutralizing antibodies or T cells. The cytopathic strain can spread between tolerant animals and lead to a severe mucosal disease outbreak. Both cytopathic and noncytopathic viruses can be isolated from these animals. Recombination may also occur between persistent noncytopathic strains and cytopathic strains in vaccines and lead to MD outbreaks. Although some of the lesions in MD are attributable to the direct pathogenic effects of BVDV, glomerulonephritis and other immune complex-mediated lesions also develop. The reasons for this are unclear but may reflect superinfection or the production of non-neutralizing antibodies. Because persistently infected calves can reach adulthood and breed, it is possible for BVD infection to persist indefinitely within carrier animals and their progeny. Epidemiological studies suggest that between 0.4% and 1.7% of cattle in the United States are persistently infected in this way.

The Role of the Gut Microbiota

The development of the newborn immune system is largely driven by the intestinal microbiota. In its absence, “germ-free” mammals fail to fully develop their mucosal lymphoid tissues. The commensal flora generates a complex mixture of pathogen-associated molecular patterns that act through epithelial cell toll-like receptors. Likewise microbial antigens are taken up by dendritic cells and presented to CD4+ T cells. These signals collectively promote the functional development of the immune system. The intestinal microbiota also plays a key role in determining any Th1 or Th2 bias in immune function. This is the basis of the “hygiene hypothesis,” the idea that the development of allergies is influenced by microbial exposure early in life.

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