Carol R. Wyatt

EVIDENCE FOR THE EMERGENCE OF A TYPE-1-LIKE IMMUNE RESPONSE IN INTESTINAL MUCOSA OF CALVES RECOVERING FROM CRYPTOSPORIDIOSIS

This study was undertaken to characterize the mucosal response to Cryptosporidium parvum in infected calves that had recovered from diarrhea. Flow cytometric surface phenotypes of lamina propria lymphocyte (LPL) suspensions from infected calves and age-matched controls revealed the presence of a significantly larger proportion of CD25+ LPL in infected calves than in controls. Freshly isolated LPL from infected calves expressed more iNOS and interferon (IFN)-γ than did controls. Infected calves excreted IgG1 and IgG2 isotype antibodies to C. parvum p23 by the end of the experiment. Moreover, immunohistochemistry of ileal sections revealed the presence of IgG1+ and IgG2+ B lymphocytes in the villi and IgG1+ but not IgG2+ B lymphocytes in continuous Peyers patch nodules. These data are consistent with the emergence of a type-1–like mucosal immune response in terminal ileal mucosa as calves recover from cryptosporidiosis.

Human xenografts are not rejected in a naturally occurring immunodeficient porcine line: a human tumor model in pigs.

Abstract Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments; however, therapies tested in such models often fail to translate into clinical settings. Therefore, a better preclinical model for cancer treatment testing is needed. Here we demonstrate that an immunodeficient line of pigs can host and support the growth of xenografted human tumors and has the potential to be an effective animal model for cancer therapy. Wild-type and immunodeficient pigs were injected subcutaneously in the left ear with human melanoma cells (A375SM cells) and in the right ear with human pancreatic carcinoma cells (PANC-1). All immunodeficient pigs developed tumors that were verified by histology and immunohistochemistry. Nonaffected littermates did not develop tumors. Immunodeficient pigs, which do not reject xenografted human tumors, have the potential to become an extremely useful animal model for cancer therapy because of their similarity in size, anatomy, and physiology to humans.

Cryptosporidiosis in Neonatal Calves

Cryptosporidiosis in calves is an ongoing problem, primarily because of the high prevalence and high morbidity associated with the infection. This article summarizes current knowledge of the host/parasite interactions associated with cryptosporidiosis. The infection process in intestinal mucosa, the pathophysiology of the disease process, and the immune responses initiated in the calf to control the infection are discussed. Methods for diagnosing C. parvum infection, treatments that have been tried, and management controls are also examined.

ASSOCIATION OF IL-10 EXPRESSION BY MUCOSAL LYMPHOCYTES WITH INCREASED EXPRESSION OF CRYPTOSPORIDIUM PARVUM EPITOPES IN INFECTED EPITHELIUM

The objective of this study was to determine whether changes in the ileal intraepithelial lymphocyte (IEL) phenotype and function occurred prior to development of diarrhea in Cryptosporidium parvum–infected calves. Calves were orally inoculated with 108 oocysts and maintained in enteric pathogen-free conditions until their use in experiments. Age-matched uninfected calves were used for comparisons. Ileal IELs were isolated and phenotyped to determine whether changes in lymphocyte population dynamics had occurred by 3 days postinoculation (PI). Ex vivo reverse transcriptase–polymerase chain reaction of messenger ribonucleic acid (mRNA) from IELs from infected calves was compared with controls to determine whether changes in cytokine expression had occurred by 3 days PI. No significant changes in lymphocyte population dynamics were documented; however, IELs isolated from 4 out of 8 infected calves, but not from 8 out of 8 control calves, expressed mRNA for interleukin-10 (IL-10). IL-10 expression by IELs was associated with the expression of a significantly larger (P < 0.001) proportion (0.75) of monoclonal antibody–defined C. parvum epitopes within infected ileal epithelium, as compared with a much smaller proportion (0.30) of epitopes with IL-10− lymphocytes. The results suggest that a temporal association exists between the expression of IL-10 by ileal IELs and the expression of C. parvum antigens in infected calf epithelium prior to development of cryptosporidiosis.

Preliminary Findings of a Previously Unrecognized Porcine Primary Immunodeficiency Disorder

Weaned pigs from a line bred for increased feed efficiency were enrolled in a study of the role of host genes in the response to infection with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Four of the pigs were euthanatized early in the study due to weight loss with illness and poor body condition; 2 pigs before PRRSV infection and the other 2 pigs approximately 2 weeks after virus inoculation. The 2 inoculated pigs failed to produce PRRSV-specific antibodies. Gross findings included pneumonia, absence of a detectable thymus, and small secondary lymphoid tissues. Histologically, lymph nodes, spleen, tonsils, and Peyer’s patches were sparsely cellular with decreased to absent T and B lymphocytes.

DETECTION OF ANTIBODIES TO A RECOMBINANT CRYPTOSPORIDIUM PARVUM P23 IN SERUM AND FECES FROM NEONATAL CALVES

Passive transfer of maternal antibodies via colostrum is important to protect newborn ruminants against microbial pathogens. In this study, 10 sets of calf serum, a sample of the colostrum fed to the calf, and serial fecal samples through the first 6 days after birth were collected from arbitrarily selected newborn Holstein heifers. A recombinant Cryptosporidium parvum p23, termed rC7, was used to determine whether anti–C. parvum antibodies can be detected in clinically normal neonates. The results demonstrated that serum, the associated colostrum, and fecal samples contained anti-rC7 antibodies. IgM and IgG1 anti-rC7 tended to be present in highest titers. The presence of specific antibodies to C. parvum was confirmed using Western blots of purified sporozoite membranes probed with serum and colostral whey. Collectively, the results indicated that neonatal calves had antibodies to C. parvum as early as 1 day after birth and suggested that the antibodies were passively transferred.

Not All SCID Pigs Are Created Equally: Two Independent Mutations in the Artemis Gene Cause SCID in Pigs

Mutations in >30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as SCID. SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T−B−NK+ SCID, representing, to our knowledge, the first example of naturally occurring SCID in pigs. In this study, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed that two mutations were present in the Artemis gene, which in the homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented in the present study reveals two mutations in the Artemis gene that cause T−B−NK+ SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues.

Analysis of blood leukocytes in a naturally occurring immunodeficiency of pigs shows the defect is localized to B and T cells.

Severe combined immunodeficiency (SCID) is the result of a set of inherited genetic defects which render components of the immune response nonfunctional. In Arabian horses, Jack Russell terriers, and mice, the disorder is a consequence of the absence of T and B lymphocytes, while natural killer (NK) cell and other leukocyte populations remain intact. Preliminary analysis of a naturally acquired form of inherited SCID in a line of pigs showed several defects in the architecture and composition of secondary lymphoid organs. In this study, a quantitative assessment of lymphocyte populations in affected and normal littermates showed depleted T or B lymphocyte populations in affected pigs; however, NK cells and neutrophils were present in numbers comparable to unaffected littermates. The results indicate that the immune defect in pigs shares the same features as other SCID-affected species.

Innate and T Cell-Mediated Immune Responses in Cryptosporidiosis

A variety of innate immune responses may help to control early parasite replication, initiate inflammation and generate signals for T cell activation. Ultimately, elimination of infection involves CD4 + T cells that are of the Th1 (cell-mediated immunity) phenotype, but there may be a protective role for lymphocytes of the Th2 type (antibody-dependent responses). Intraepithelial lymphocytes have increased activity as a result of infection and may be important in the anti-cryptosporidial immune response.

Characterization of Small Intestine Mucosal Lymphocytes During Cryptosporidiosis

Cryptosporidium parvum is a coccidian parasite that causes enteric disease in humans and animals[l]. Although CD4 T lymphocytes and interferon-gamma (IFN) are required to terminate infection(2,3 J, little is known about the gut mucosal immune response to the parasite. We are using a bovine model to examine mucosal immunity during cryptosporidiosis. and show that ileal intraepithelial T lymphocytes are activated coincident with enteric disease.