Ruminant Immunologic Disorders

George M. Barrington, Consulting Editor

Immunity at Birth and the Immune Development of the Calf

David J. Hurley

At birth, calves possess the appropriate immune components needed for a protective response; however, these components lack maturity and require time to become as functional as those in adults.

Prior to receiving colostrum, calves rely on macrophages located in tissues to manage tissue-level immune homeostasis. This includes mounting and managing the response to envi­ronmental microbes that share common antigenic determinants. The ability of macrophages to recruit monocytes, neutrophils, and other innate cells from neonatal circulation is a core component of the immediate innate immune response. Finally, in addition to macrophage activation and recruitment of circula­tory innate cells, the activities of antimicrobial peptides combine to form the functional limit of immunity in calves at birth.

The earliest production of antibody by neonatal calves is reported in circulation at approximately 4 days of age. IgM can regularly be measured at 8 to 10 days of age.² T cell­dependent IgG is not usually observed in the circulation of calves until at least two half-life decays of maternal antibody. A large volume of research supports the role of IgG1 in the health and immune development of calves and its importance in providing protection from invading microbes. In addition, IgA in colostrum and milk play an important role in managing the colonization of the GI tract by microbes and the manage­ment of GI disease. Maternal antibody, directed against local microbes, helps protect calves during development and growth.

In calves, some B cells can be found in the neonatal organized lymphoid tissue both before and immediately after birth. These cells are widely distributed and are not organized into germinal centers (organized B-cell areas following immune activation). Their exact function and origin are not clear. After ingestion of colostrum containing viable maternal cells and IgG, or noncellular maternal factors and IgG, an almost complete depletion of the IgG-bearing cells in organized lymphoid tissues of the neonatal calf is observed. A small number of IgM- and IgA-positive cells are also present in these organized lymphoid tissues, and these are not affected by feeding whole colostrum or maternal colostrum without live cells.³ Tracking of B cells in the peripheral blood of calves indicates that at birth about 5% of circulating mononuclear cells are B cells and that the number increases to about 20% by 20 weeks of age.⁴ Fetal B cells are rapidly deleted from organized lymphoid tissues of very young calves. B cells are found in low numbers in the blood of young calves but are not likely to represent a sufficient pool of naive B cells to produce all the antibody required to resist pathogens early in life. Thus antibody transferred from colostrum (or whey antibody in colostrum substitutes) is required to adequately protect the calf.

Colostrum has a significant effect on the development of the neonatal GI tract.⁵ Critical nutrients, vitamins, minerals and trace elements, and nonnutrient factors (Ig, growth factors, hormones, and cytokines) affect several processes, including the function of microbes; mucosal epithelial cell proliferation, migration, and differentiation; recruitment and function of innate immune cells; production of immune-active molecules; development of vascular beds and normal vascular tone; and mechanisms necessary for efficient transfer of nutrients and other components to the body. Serial microbial colonization of neonatal mucosal surfaces is critical for the development of both the innate and the adaptive immune networks.^6-8

Norrman and colleagues compared the effects on B- and T-cell production of feeding newborn calves either colostrum or milk replacer.9 They demonstrated that colostrum intake decreased neonatal adaptive immune responses compared with colostrum-deprived calves that lacked immune-priming assistance.

Once calves ingest colostrum, immunoglobulin (primar­ily IgG1) rapidly crosses the gut epithelial cells and enters the circulation. Once in circulation, IgG1is distributed to the tissues by mass action, where it acts as a key element in immediate immune protection against microbial agents. Because of its structural flexibility, IgG1 readily enters tissues and enhances the response by macrophages, monocytes, and neutrophils.

In addition to circulating innate immune cells, both T and B lymphocytes are present as part of the adaptive response, though both are considered naive, having never been activated. Core structures such as the spleen and lymph nodes will eventually serve as foci for lymphoid maturation.

There is evidence in calves that lymphocytes can be activated as early as 2 days after birth.^10,11 Both colostrum-deprived and colostrum-fed calves produced antibody- (primarily IgM) and antigen-responsive T cells after exposure to bacille Calmette- Guerin (BCG) and ovalbumin antigens, and calves receiving colostrum had less evidence of disease over the course of the study. Both groups showed the capacity to mount adaptive responses to test antigens. Thus it appears that a full comple­ment of experienced B and T cells and an adult-like organization of lymphoid tissues are not absolute requirements for successful production of at least some adaptive responses. However, this limited capacity is not sufficient to protect calves from disease.

It has long been established that maternal leukocytes present in colostrum successfully pass from the neonatal intestine into circulation. The exact function of these maternal cells is only beginning to be understood and is a topic of much debate. Nonetheless, while it is clear that calves can develop without the transfer of viable maternal cells, there are many reports of specific enhancements in immune function seen over the first 7 to 28 days after ingestion of colostrum with viable maternal cells.^12-24

The induction of the adaptive response strongly depends on expression of antigen in the context of MHC antigens.

Maternal cells originating from colostrum appear to home to lymph nodes and mucosal lymphoid structures of calves.²⁶ Circulating leukocytes in calves receiving maternal colostral cells showed enhanced capacity to respond to viral and bacterial antigens that the dam had exposure to for 12 to 48 hours after colostrum feeding.^16-18,21,27 Calves not receiving viable maternal cells from colostrum showed no response to viral antigens and suffered more severe GI disease.^16,22-24 The innate and adaptive immune responses are clearly enhanced by ingestion of viable maternal cells in colostrum.^{12-15,19,20,24}

In calves the number of circulating leukocytes declines rapidly beginning around day 2 after birth and reaching a minimum between days 7 and 11.^28,29 At birth, the predominant leukocyte in neonatal circulation is the segmented neutrophil, making up about two thirds of circulating WBCs. The other third includes lymphocytes, primarily T cells. As the total number of circulating leukocytes declines and then recovers over the first 2 weeks of life, the proportion of cell types also shifts. By day 14, neutrophils make up one third of total cells, and lymphocytes (including NK and gamma-delta cells) make up more than half of total cells. In the neonate, both NK and gamma-delta cells can leave the circulation and participate in immune protection within tissues.

Once DCs and neutrophils populate organized lymphoid tissues, the lymphoid environment changes to provide enhanced capacity for homing of lymphocytes and the structural develop­ment of germinal centers in these tissues. Although lymphoid germinal centers are rarely observed in mammals younger than 21 days of age, the seeds of the network required for their development begin in the first week of life. B cells, located in functional germinal centers, are under the influence of imbedded follicular DCs and surrounded by supporting Th cells. B cells and neutrophils (carrying whole antigen) provide important components for the development of an antibody response. Slowing development are low levels of MHC class II antigen expression by B cells, monocytes, DCs, and macrophages during the first 10 days to 2 weeks of life. Expression of MHC class II antigen is a limiting factor in the development of a rapid 25

adaptive response.²⁵

The process of calf immune development to adult levels takes time. Many potential pathways can lead to successful protection. The path a calf takes will be influenced primarily by the management strategies of the farm or ranch. For example, dairy calves can have a range of contact time with the dam (0 to 4 days) and may receive fresh, frozen, or processed colostrum, which is single-source or pooled. Dairy calves may be fed waste milk or milk replacer that may not possess populations of local microbes. In contrast, most beef calves are exposed to a number of herdmates and remain with their dam and herd during development. Beef calves are typically more aggressive than dairy calves, usually stand and nurse within 1 to 2 hours after birth, and continue to nurse ad libitum. In dairy calves the primary reason for FPT of maternal immunity involves poor-quality colostrum (low IgG1), whereas in beef calves FPT typically results from conditions that adversely affect the timely ingestion and absorption of colostral immune components.

Lethal Trait A46

Munashe Chigerwe

Lethal trait A46, also known as bovine hereditary zinc deficiency, Adema disease, hereditary thymus hypoplasia, and hereditary hyperkeratosis, is an autosomal-recessive immunodeficiency disorder with clinical presentation similar to acrodermatitis enteropathica (AE) in humans.

Zinc is an essential nutrient for several physiologic processes as a cofactor of many enzymes and transcription factors.⁴ The molecular basis in AE in humans has been linked to an identifica­tion of a defect in a solute carrier (SLC), referred to as SLC39A4. A defect in a bovine ortholog of SLC39A4 has been identified in cattle with lethal trait A46.⁵ The defect has been postulated to negatively affect a pore responsible for zinc transport, leading to impaired zinc absorption.⁵ Zinc is required for normal lymphocyte function. In a zinc-deficient state, lymphocyte numbers and function are reduced, and antibody responses are reduced.⁶ Clinical signs associated with lethal trait A46 include skin lesions, diarrhea, and bronchopneumonia. Calves appear normal at birth but develop skin lesions at 4 to 8 weeks of age. The skin lesions are characterized by alopecia, hyperkeratosis, and exanthema. The distribution of the skin lesions includes the perineum, lower legs, head, and neck. Skin lesions of the entire body of the animal may be observed in advanced cases. Death occurs at approximately 4 months, secondary to bronchopneumonia or complicated diarrhea. Necropsy findings in affected animals include acanthosis, hyperkeratosis, thymus atrophy, gut-associated lymphoid tissue, spleen, and lymph nodes. The disease is responsive to zinc supplementation. Doses of 0.5 to 1 g/day zinc oxide⁷ or 1 g/ day of zinc acetate in cattle younger than 2 years of age⁸ and of 45 g/day of zinc acetate in cattle older than 2 years have been recommended.⁵

Bovine Leukocyte Adhesion Deficiency

Bovine leukocyte adhesion deficiency (BLAD) is an autosomal- recessive congenital disease of Holstein calves characterized by chronic, recurrent bacterial infections and death. Leukocytes express cell surface glycoproteins called β₂ integrins, which mediate cell-extracellular matrix and cell-cell interactions.^9-11 The β₂ integrins include lymphocyte function-associated antigen-1 (LFA-1), complement receptor type 3 (CR3), and p150,95, and the family of these proteins is referred to as CD11/CD18 adhesion molecule.^9-11 The β₂ integrins LFA-1, CR3, and p150,95 consist of unique subunits—CD11a, CD11b, and CD11c, respectivelyand a common subunit CD18.^9,11 The CD11/CD18 adhesion molecules are essential for host defense against infections.^9-11 LFA-1 is expressed on all leukocytes and is essential for leukocyte adhesion and lymphocyte cytotoxicity.^9-11 CR3 and p150,95 are present on the surfaces of neutrophils, monocytes, macrophages, and NK cells.^9,12 Binding of CR3 and LFA-1 on the neutrophil surface with intercellular adhesion molecule 1 expressed on the vascular endothelium is required for neutrophil emigration into sites of inflammation.¹² Calves affected by BLAD lack expression of adhesion molecules of the CD11/CD18 family on the leukocyte surface.¹³ The molecular basis for BLAD is a single point mutation (adenine to guanine) of the CD18 gene, resulting in aspartic acid to glycine substitution at amino acid 128 (D128G) in the glyco­protein.¹⁴ Calves with BLAD are homozygous to the D128G allele, whereas β₂ integrin expression in heterozygotes occurs in 56% to 90% of cattle not affected by BLAD.¹⁵

Clinical signs in calves appear within a couple of weeks after birth and include anorexia, fever, chronic pneumonia, chronic diarrhea, severe ulcers on oral mucosal membranes, ulcerative stomatitis, gingivitis, periodontitis, loss of teeth, impaired wound healing, chronic dermatitis, decreased growth rate, and death.^16,17 Marked and persistent neutrophilia is a consistent hematologic finding in calves with BLAD due to lack of emigration of neutrophils out of the vessels.¹⁶ Consistent serum biochemical abnormalities include hypoalbuminemia, hyperglobulinemia, and hypoglycemia, consistent with chronic infections.¹⁷ In the United States, 14% of bulls and 6% of cows were reported to be carriers of the D128G allele.¹⁸ Diagnostic tests are available to detect heterozygotes and homozygotes. The information is available from the Holstein Association USA in Brattleboro, Vermont.

Selective IgG2 Deficiency

Rana Bozorgmanesh

IgG subclass deficiencies have been reported in ruminants. Primary (partial or complete) IgG2 deficiency in Red Danish milk cattle increases their susceptibility to gangrenous mastitis, bronchopneumonia, peritonitis, and abomasoenteritis.^1-3 There is also a report of IgG2 deficiency with transient hypogam­maglobulinemia and chronic respiratory disease in a 6-month- old Holstein heifer that was eventually euthanized due to lack of improvement with therapy and a poor prognosis.⁴ A transient IgG2 deficiency (with no adverse consequences) has been reported in neonatal lambs that ingested colostrum resulting in delayed synthesis of IgG2 to 5 to 6 weeks of age.²

Chediak-Higashi Syndrome

Chediak-Higashi syndrome is an inherited disorder resulting in partial oculocutaneous albinism, photophobia, increased susceptibility to infection, and coagulopathies. The condition affects cattle, mink, cats, mice, killer whales, and humans.^5,6 The condition is characterized by enlarged cytoplasmic granules (often lysosomes) in most types of granule-containing cells and defects in the function of leukocytes, renal tubular cells, and platelets. Neutrophils demonstrate abnormal intracellular killing due to a defect in the hexose monophosphate shunt as well as defective degranulation.⁶ The condition is well docu­mented in Japanese Black cattle, where inheritance has been identified as a simple autosomal recessive trait as it is in other animals and humans.^5,7

Combined Immunodeficiency

Both T- and B-cell deficiencies due to genetic failure result in combined immunodeficiency. This may go unnoticed until maternal antibodies wane at several weeks of age, leaving the animal susceptible to infections. There is one report of a 6-week- old Angus embryo-transfer calf that died as a result of general­ized systemic mycosis. This calf was hypogammaglobulinemic and Iymphopenic and had an absence of serum IgM. Necropsy confirmed the absence of lymphoid tissues and the diagnosis of combined immunodeficiency. Interestingly, the full sibling from the same embryo flush was healthy at 6 months.⁸

Viral and Bacterial-Induced Immunodeficiency

Bovine viral diarrhea (BVD) infection has been associated with altered immune function in infected calves and lambs.^9-11 Evidence suggests that the virus may have immunosuppressive effects on both the innate and the adaptive immune systems, making animals more susceptible to secondary infections and resulting in the establishment of persistent infection and immunotolerance.^9-10 Cattle infected with Johne's disease may show an inability to mount cell-mediated immune responses as well as the presence of humoral immunosuppressive factors contributing to chronic intracellular infection.¹² Furthermore, a state of tolerance may exist in the intestine of cows subclini- cally infected with Johne's disease organisms, in part due to proliferation of regulatory T cells that suppress mucosal immune responsiveness.¹³

Immunosuppression in the Periparturient Period

Barry J. Bradford • Jodi L. McGill

Introduction

In dairy cattle, the period 3 weeks prior to calving through 3 weeks after parturition, referred to as the transition period, is marked by dramatic changes in physiology, metabolism, and immunity.¹ The transition period is associated with a sharply increased incidence of metabolic and immunologic diseases, including endometriosis, mastitis, ketosis, and dystocia.^1-3 Further, the occurrence of health problems in the first few weeks postpartum is a major risk factor for continued production and reproductive performance problems.⁴ It is now widely accepted that the increased incidence of both metabolic and production-related diseases is directly related to a temporary state of immunosuppression, which is known to affect the transition cow for at least 3 to 5 weeks following parturition.²

IMMUNOLOGIC DISORDERS OF THE INNATE IMMUNE

SYSTEM. The innate immune system responds rapidly and nonspecifically to insults and thus plays a critical role in pro­tecting the cow during the early days following parturition. Neutrophil function is particularly affected during transition, demonstrating reductions in chemotaxis, phagocytosis, oxidative burst capacity,^5-9 and downregulated expression of pathogen recognition receptors.^10,11 Changes in glucocorticoid levels at or around the time of calving are thought to be a prime factor leading to impaired neutrophil functions,^12,13 and a number of studies have shown that increased circulating cortisol levels downregulate neutrophil adhesion molecules, leading to reduced neutrophil surveillance activity and immune response.^13-15 However, glucocorticoid levels are increased for only a short period post calving^16-18; thus additional factors likely contribute to the prolonged state of immunosuppression observed during transition. The final stages of fetal development and colostrogenesis require significant quantities of calcium, causing most cows to develop either subclinical or clinical hypocalcaemia in the transition period.¹⁹ Hypocalcemia is linked to elevated cortisol plasma concentrations,²⁰ and cows with decreased serum calcium concentrations have decreased neutrophil activity, delayed pathogen recognition by peripheral blood mononuclear cells, and increased susceptibility to several periparturient conditions.^21-24 Due to the high demands of early lactogenesis, transition cows also regularly enter a state of negative energy balance and mobilize adipose and other tissues for energy.^2,4,18 Increased mobilization leads to increased concentrations of non-esterified fatty acids (NEFAs) and ketone bodies, such as beta-hydroxybutyrate (BHB). BHB is clearly shown to negatively affect neutrophil functions, including formation of neutrophil extracellular traps (NETs), chemotaxis, and phagocytosis.^25-27 High circulating NEFA concentra­tions are associated with diseases such as hepatic lipidosis,²⁸ retained placenta,²⁹ and mastitis.^30,31 High circulating NEFA concentrations in vivo are associated with reduced neutrophil and mononuclear cell functions,^32,33 and a study by Scalia and colleagues showed in vitro that high levels of NEFAs were associated with increased neutrophil oxidative burst activity but severely reduced viability.³⁴

IMMUNOLOGIC DISORDERS OF THE ADAPTIVE IMMUNE SYSTEM. Like the innate immune system, the adaptive immune system of the transition cow is altered both qualitatively and quantitatively. Numbers of circulating lymphocytes and monocytes are reduced during the postpartum period.^35,36 Large quantities of serum IgG are transported into the mammary gland during colostrogenesis,³⁷ and physiologic reductions in serum IgM and IgG have been reported well into the postpartum period.³⁸ High levels of glucocorticoids are thought to be a primary factor in suppression of the adaptive immune system,¹² although lymphocyte blastogenesis and cytokine secretion are also adversely affected by metabolic factors such as hypocal- 22 3940 3241

caemia,²² ketosis,^39,40 and high circulating NEFA levels.^32,41

Epidemiology

Impaired immune function during the periparturient period likely contributes to increased risk of infectious disease at that time. It is well established that mastitis incidence is greater in the first 5 weeks of lactation than during the remainder of the lactation,⁴¹ along with high prevalence of metritis at this time.⁴² Although numerous possible mechanisms could explain increased infectious disease incidence in early lactation, there is now strong evidence that impaired innate immunity is at least a contributing factor.⁴³

Several functional attributes of neutrophils, including chemotaxis, phagocytosis, oxidative burst, and bacterial killing were found to be impaired prior to disease onset in cows that experienced retained placenta, mastitis, or metritis compared to healthy herdmates.^5,33,44 Mastitis and retained placenta were also associated with an increase in the proportion of immature neutrophils in circulation,⁴⁴ perhaps due to extravasion of the more mature, functional neutrophils. Shifts in monocyte populations were recently linked to postpartum disease; a high ratio of CD14+ to CD14- monocytes in the 6 weeks before parturition was associated with substantially greater risk of mastitis or metritis in early lactation.⁴⁵ Although there have been fewer studies linking adaptive immune function to transi­tion success, a pilot study showed that cows with a weak lymphocyte proliferation response to LPS 3 weeks prior to parturition subsequently suffered from clinical infections postpartum, and this functional classification was associated with a genetic polymorphism.⁴⁶

Interventions

A variety of approaches are being advanced to attempt to counteract periparturient immunosuppression and decrease disease incidence during this critical period.¹⁹ Among the most widely adopted methods is vaccination against the J5 component of E. coli endotoxin, which decreases the incidence of coliform mastitis.⁴⁷ Although not commercially available, early trials are also showing promise for a multitarget metritis vaccine strategy.⁴⁸ A longer-term approach already in use by dairies is genetic selection for superior immune response, with reported benefits for periparturient disease incidence.⁴⁹

An innate immune stimulant is now available to substantially increase circulating neutrophil populations and enhance their functionality.⁵⁰ This modified bovine G-CSF, when administered 7 days prior to and immediately after calving, decreases clinical incidence of mastitis in early lactation,^51,52 although the potential impacts on other infectious conditions are not yet known. Feed additives are increasingly investigated for impacts on immune function. Beyond the benefits of better understanding and meeting requirements for nutrients like vitamin E⁵³ and chromium,⁵⁴ some yeast-containing products may stimulate innate and adaptive immunity in ways that are not completely understood.^55-58 Another line of research is investigating the immune benefits of decreasing milk yield through decreased milking frequency or partial milking in the first week of lacta­tion.⁵⁹ Ultimately, the practices with the most impact for supporting immunity during the periparturient period are likely to be already established best management practices: appropriate stocking density,⁶⁰ prevention of heat stress⁶¹ and hypocalcemia,⁶² and body condition management.⁶³