Immune evaluation of dogs with recurrent infections (Proceedings)

The patient that presents with recurrent infections may have an underlying immune deficiency or dysfunction. Depending upon the age of the patient and the age of onset, the problem may be genetic (primary immunodeficiency) or acquired. In either case it is possible to carefully dissect the function of the immune system to determine which if any of the immune functions are abnormal. A typical history might be that the patient developed a bacterial infection, which responded well to antibiotics, and then when the antibiotics were discontinued, the patient relapsed. If the recurrent infections have been occurring since early in life, the possibility of a primary immunodeficiency is high. Many primary immunodeficiencies have been described in dogs, including those affecting innate immunity, humoral, cellular, and complement. Older dogs may have predisposing conditions that have compromised one or more of their immune constituents.

A focused approach to immune system evaluation

There are a variety of assays available to test immune function in the dog. The choice of assay should be based on: 1) the type of pathogen causing the infection and the body system of the patient that is most frequently infected, 2) breed, age, and history of the patient, 3) evaluation of the complete blood count (CBC). For example, a dog that has a high neutrophil count and evidence of a bacterial infection that recurs after antibiotic withdrawal may have a neutrophil defect (such as inability to phagocytose and kill). In contrast, a dog with chronic fungal infection would be more likely to have a T cell defect. A young German Shepherd with recurrent lung or gastrointestinal infection may have a selective IgA deficiency. For each of these there are diagnostic assays that can assist with pin pointing the defect.

Evaluation of phagocytic cell function

The first parameter to assess is cell number and morphology. Dogs afflicted with Cyclic Neutropenia will show normal neutrophil numbers when they are non-symptomatic, but will have drastically lowered numbers when they are sick. This syndrome occurs in grey coat color collies, which is a big hint that neutrophils need to be examined. For other dogs neutrophil numbers may be normal, increased, or decreased in the presence of bacterial infection. Once it is established that the level of cells is not abnormally low, the various processes required for them to find and kill infecting pathogens must be examined.

Determining the ability of the neutrophils to carry out their function involves independent assessment of: adhesion molecule expression, response to chemotactic stimuli, ability to engulf, and finally intracellular killing (both oxidative and non-oxidative systems are involved). The genetic defect resulting in defective expression of the integrin CD11b/CD18 causes an inability of the neutrophil to respond to chemotactic stimuli and emigrate from the blood to the site of infection in tissues. This uncommon condition is called Canine Leukocyte Adhesion Deficiency (CLAD) has been described in the Irish Setter breed. Affected dogs die early due to recurrent bacterial infections. An extreme leukocytosis coupled with an absence of pus in the peripheral tissues in the presence of bacterial infection is typical of a CLAD afflicted dog. Defects in chemotaxis can be evaluated using a Boyden chamber, in which the patient's neutrophils are separated from a chemotactic stimulus by a semi permeable membrane. When patient is compared with control cells depression of chemotactic activity can be documented. Another assay is used to evaluate the ability of the patient's cells to engulf bacteria. Incubation with bacteria or with opsonized particles followed by fixation of the cells and observation of the stained slide under the microscope is used to calculate a phagocytic index, to evaluate intake by phagocytosis. There are several tests available to evaluate killing. Oxidative killing is examined using the nitroblue tetrazolium test. Finally, overall ability to engulf and kill can be determined using a bactericidal assay. This assay is laborious and thus expensive, but it is perhaps the best way to evaluate the effectiveness of neutrophil killing. These assays are performed in clinical veterinary immunology laboratories, but they do require prior notice and cells that are fresh and viable. Most labs will also ask that you submit a sample from a normal control for comparison when the bactericidal assay is performed.

Evaluation of humoral immunity

The absence of antibody-mediated protective immunity or more commonly the depression of antibody levels is reflected in infections by bacteria normally handled well by humoral responses, as well as some organisms not normally associated with disease. In dogs selective deficiencies of a single antibody class have been reported. The most common antibody deficiency is IgA. Affected dogs have normal IgM and IgG levels, but depressed production of IgA. The German Shepherd breed is most commonly affected, although the problem is also seen in the Shar pei breed. Affected German Shepherds most often have clinical signs associated with infection of mucosal surfaces: anal furunculosis and bacterial overgrowth in the intestinal tract. The Shar pei dogs often have clinical signs associated with the respiratory tract: pneumonia, recurrent cough, and conjunctivitis. To identify an IgA deficiency the single radial immunodiffusion test (SRID) can be performed using serum form affected dogs. The amount of IgA present is compared to standards and is used to determine if the sample is within or below a normal range. These deficiencies are genetic defects and as such are present from an early age (after maternal immunity wanes).

The adult dog that has recurrent infections may have an acquired defect in humoral immunity. Poor nutrition, stress, age, and administration of immunosuppressive drugs for therapy of autoimmune, allergic, or neoplastic disease can depress immune responses. For example, depressed antibody production can result from chronic zinc deficiency. In dogs fed high quality dog food, this is an unlikely cause, but is possible in animals on home cooked fare without vitamin mineral supplementation. Canine distemper virus grows in T and B lymphocytes and ultimately invades and destroys secondary lymphoid organs. Immunosuppression is thus a component of distemper infection and frequently causes secondary infection with a variety of normally non- or moderately pathogenic organisms.

Numbers of B lymphocytes can be evaluated in peripheral blood using flow cytometry. IgG and IgM levels can be measured as described for IgA using SRID. Low levels of B cells could indicate a problem with humoral immunity that is either associated with a primary defect (genetic) or a secondary deficiency. Most veterinary immunology laboratories are capable of performing this evaluation, although it will require that fresh cells in anticoagulant are submitted to the laboratory preferably on the day of sampling.

Evaluation of cell mediated immunity

When the recurrent infections are with a fungal or viral pathogen it is likely that the immune defect is in cellular immunity, specifically the T cell arm of the immune response. There are some primary immune deficiencies in which T cells are reduced or defective. Most notable there is a combined immunodeficiency disorder that has been recognized in Jack Russell terriers; a similar defect occurs in basset hounds and Cardigan Welsh Corgis. In the Basset hounds and Corgis the increased susceptibility to infection, particularly viral, but also deep pyoderma and other bacterial infection, is notable. These animals have defective T cell and humoral immune responses. There is a thymic aplasia, absence of lymph nodes, and stunted growth in affected dogs. The status of humoral immunity in pups may be difficult to evaluate when maternal antibodies are present, but after maternal immunity wanes at 6 to 8 weeks of age the SRID, as described above, will show an inability to produce IgG and IgA. Interestingly the B cell numbers are normal. The defect has been identified as a mutation that affects the interleukin 2 (IL-2) receptor. This cytokine is critical for proliferation of T lymphocytes. When flow cytometry is used to evaluate CD4 and CD8 positive lymphocytes in these dogs, there is a skew of the ratio towards CD4 cells.

Other genetic defects in T cell immunity have been described in several breeds. For example cases of thymic aplasia associated with dwarfism have been described in the Weimaraner breed. In the German Shepherds with severe nonresponsive staphylococcal pyoderma T cell function has been shown to be impaired.

There are not as many in vitro methods available for evaluation of cell mediated as there are for humoral immunity and phagocyte function. However, stimulation of lymphocytes with mitogens (plant lectins that activate all T cells) can be used to measure responsiveness of a patient's peripheral blood lymphocytes. To perform this test a sample of heparinized blood is submitted to the laboratory, where the cells are incubated with these mitogens in culture for several days. An indicator of cell division is added and used to determine whether or not the activation (blastogenesis) is comparable to a normal control. This assay is called lymphocyte stimulation. A related assay involves a similar incubation in culture with mitogens but the read-out is a measurement of cytokine production, specifically interferon gamma, an important T cell cytokine. Stress, chronic disease, neoplasia and old age are factors that have been shown to be associated with impaired T cell stimulation.

Evaluation of complement activity

The complement system is an important part of the immune defense system. This series of proteins can be activated by one of several innate mechanisms as well as by antibody binding to antigen (immune complex formation). The effects of complement include chemotaxis of inflammatory leukocytes, mast cell degranulation with subsequent local increases in vascular permeability, opsonization of cells and pathogens, and finally lysis of target cells. When these defenses are directed towards pathogens they comprise a strong defense mechanism. Problems with complement are rarely noted among our veterinary patients. There have been some isolated incidences of genetic deficiencies of complement components. The only one that is usually manifested as increased incidence of recurrent infection is deficiency of C3. That is because C3 is the component common to all pathways and without it none of the important functions of complement can be implemented. Brittany spaniels affected with C3 deficiency have low levels of IgG and recurrent infections. A genetic mutation has been identified that is expressed as no C3 (homozygous) or low C3 (heterozygous). To evaluate complement function an assay called CH50 can be performed. In this assay the patient's plasma is used as a complement source in an erythrocyte lysis assay. While it doesn't identify which component is decreased, it gives an overall evaluation of how well the complement system is working in the patient.

A patient with recurrent infection may have a primary or secondary immune defect that is causing the immune system to function at a lower than normal level. There are assays available to help the clinician dissect the immune system and determine where the problem lies. Table 1 summarizes this approach.