Cryptosporidium parvum spermidine/spermine N1-acetyltransferase exhibits different characteristics from the host enzyme.

Cryptosporidosis is a severe opportunistic infection of immuno-compromised individuals for which no reliable therapy exists. The parasite scavenges host-derived polyamines, particularly spermine, which is then converted to the lower polyamines by the combined action of spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine oxidase (PAO). We have isolated and expressed the Cryptosporidium parvum SSAT for kinetic and molecular comparison with the host enzyme. The CpSSAT is a homotetramer with a subunit molecular mass of 18 kDa and low sequence similarity to higher eukaryotes but maintains the critical arginine residues in the active site. The CpSSAT had an activity of 299 nmol(-1)min(-1)(mg of protein)(-1) and exhibits an ordered Bi-Bi kinetics with preferred substrate specificity for spermine. Polyamine analogues having unsaturated central carbons were found to exhibit mixed inhibition kinetics of the CpSSAT. The cis-analogues were more effective inhibitors of the CpSSAT with lower K(i) values than the trans-analogues. Experiments aimed at determining the ratio of the time of the analogue in the enzyme active site to that spent out (in-out time: delta ln E/deltat) confirmed the higher efficiency of the cis-analogues as inhibitors of the CpSSAT. The results of this study reveal that the C. parvum SSAT may provide a rational target for drug design.

Expression of lymphocyte homing and adhesion molecules during intramammary infection of cows with Serratiamarcescens or Streptococcusuberis: correlation with bacterial colonization and clinical signs.

We wished to determine the expression of trafficking/adhesion molecules on the surface of lymphocytes isolated from infected mammary glands of cows challenged with either Serratia marcescens or Staphylococcus uberis. Healthy Holstein cows in mid lactation were infected by intramammary infusion with S. marcescens or S. uberis. Following infection, milk samples were collected at various time points. Body temperatures of the cows were taken, and milk was analyzed for colony forming units (CFU) of bacteria and somatic cell counts (SCC). Leukocytes were isolated from the milk and analyzed by flow cytometry. Percentages and types of lymphocytes were determined as well as expression of CD62L, CD11a, LPAM-1 and CD44 on these cells. We found that the percentage of lymphocytes expressing either CD62L or CD11a showed a marked increase 12 h post infection (PI) with S. marcescens that was not seen in cows infected with S. uberis. Conversely, the percentage of lymphocytes expressing CD44 increased in cows infected with S. uberis at 12 h PI, but the increase was not seen in cows infected with S. marcescens. Expression of LPAM-1 was low at all time points in both groups of cows. Body temperatures became elevated in both groups of cows, peaking at 24 h PI in S. marcescens-infected cows and dropping thereafter. In contrast, temperatures of S. uberis-infected cows continued to rise and were still elevated 96 h PI. CFU of bacteria isolated from mammary glands of S. marcescens-infected cows dropped precipitously 24 h PI but continued at high levels in S. uberis-infected cows. SCC began falling in S. marcescens-infected cows 48 h PI but continued to increase in S. uberis-infected cows. Thus, a greater percentage of lymphocytes in milk had a phenotype consistent with recruitment from the peripheral pool following infection with S. marcescens than was seen following infection with S. uberis. Concurrent with the increases seen in percentages of this lymphocyte phenotype, clinical signs lessened in the S. marcescens-infected cows.

Characterization of a factor from bovine intestine that protects against Cryptosporidium parvum infection.

Cryptosporidium parvum is a protozoan parasite that causes intestinal infection in a variety of mammals. We have previously described a factor in adult rat or adult bovine intestinal mucosa that protects against C. parvum infection when fed to susceptible infant rats. This factor is absent in intestinal mucosa from bovine calves. In the present study we describe the further characterization of the active component of bovine intestinal mucosa. The ability to protect infant rats against C. parvum infection was found to be associated with the extrinsic membrane protein fraction of the intestinal mucosa. Extrinsic membrane preparations from adult cows, adult rats, and calves were separated by SDS-PAGE. A band with apparent molecular mass of 54 kDa was seen in preparations from adult rat and cow, but not calf. Protein was transferred to PVDF membrane and from this the band was excised and subjected to N-terminal sequence analysis using a gas-phase protein sequenator. A 15-amino acid consensus sequence was generated with homology to leucine aminopeptidase (LAP). Purified LAP was purchased from a commercial source and tested for ability to protect infant rats against C. parvum infection. Rats fed LAP from 7 to 11 days of age and challenged with C. parvum at 9 days were significantly less infected than controls upon necropsy at 15 days of age. These data suggest that a protein with N-terminal sequence homology to LAP may reduce susceptibility of infant rats to C. parvum infection.

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes during the periparturient period of dairy cattle.

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes (PMN) from periparturient dairy cattle was studied. Both percentages and the mean fluorescence intensity (MFI) of PMN expressing CD11a, CD44, CD62L, and LPAM-1 (alpha4 beta7) were evaluated at seven time points during the twenty-one day period post calving. CD11a and CD62L were expressed on 94-100% of PMN in both blood and milk and there were no significant differences in these percentages at any time point. LPAM-1 was expressed on 3-10% of the PMN in the blood and 13-45% in the milk and the percentage of cells expressing LPAM-1 in milk was significantly (P<0.05) greater than in blood at 0, 4, 10, 14, 18 and 21 days after calving. CD44 was expressed on 11-39% of the PMN in blood and 33-69% in the milk and the percentage of cells expressing CD44 in milk was significantly (P<0.05) greater than in blood at all time points. The MFI of CD11a on milk PMN was consistently higher than that of blood PMN throughout the study period and significantly (P<0.05) higher at days 4, 10 and 18 after calving.

Lymphocytes from one side of the bovine mammary gland migrate to the contra lateral gland and lymph node tissue.

The four quarters of bovine mammary glands are completely separated and two quarters on each side (right or left) are connected to ipsi lateral supra mammary lymph nodes. It is not clear whether cells infused into the cistern of the mammary gland are capable of migrating to lymph nodes or the general circulation. To examine cell migration, a prescapular lymph node was removed from each of two lactating and three non-lactating dairy cows, and isolated lymphocytes were stained with Hoechst 33342. Autologous stained cells were infused into the mammary gland and then activated by intramammary infusion of zymosan-stimulated serum (source of C5a). After 17 h, Escherichia coli J5 bacterin was infused into the contra lateral mammary gland to mimic infection. After 43 h cows were euthanized and tissue samples (mammary quarters, right and left supra mammary, mesenteric, ileocecal and prescapular lymph nodes, liver and spleen) were collected for microscopic examination as well as flow cytometric analysis. Hoechst stained cells were detected not only in infused quarters, but also in contra lateral quarters as well as in both supra mammary lymph nodes. This indicates that cells infused into the mammary gland migrate to contra lateral tissues and supra mammary lymph nodes.

Lymphocyte subsets and adhesion molecule expression in milk and blood of periparturient dairy cattle.

Fifteen Holstein dairy cattle were monitored for lymphocyte subsets and expression of adhesion molecules on cells in milk and blood at parturition and at intervals up to 21 days post-partum. Using flow cytometry, we determined percentages of T cells (CD4+, CD8+, gammadelta) and B cells from milk and blood of these cows. We also measured expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on lymphocytes in milk and blood. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points while significantly higher percentages of B cells were found in blood than in milk at all time points. There were minimal to no significant differences in percentages of CD4+ or gammadelta+ cells between milk and blood. Expression of adhesion molecules was consistently higher on all subsets of milk lymphocytes compared with blood lymphocytes. These differences were most pronounced and statistically significant at calving and in the first week following calving. CD62L, LPAM-1 and CD44 were expressed on a significantly higher percentage of lymphocytes in milk at calving than in milk at subsequent sampling times, while LFA-1 expression on lymphocytes in milk was significantly lower at calving than at subsequent times.

Expression of adhesion molecules on milk and blood lymphocytes from periparturient dairy cattle with Johne's disease.

Twelve dairy cows infected with Mycobacterium avium subsp. paratuberculosis were monitored for lymphocyte subsets and expression of adhesion molecules on cells in blood and milk at parturition and at intervals up to 21 days post-partum. Using fluorescent antibody labeling of cells and analysis by flow cytometry, we determined percentages of T cell subsets (CD4+, CD8+, gammadelta+) and expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on cells from blood and milk of these cows. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points; there were no significant differences in percentages of CD4+ or gammadelta+ cells. CD62L, LFA-1, and LPAM-1 were expressed on a significantly higher percentage of all T cell subsets in milk than in blood at various times after parturition. No differences were seen in expression of CD44. Increased percentages of T lymphocytes expressing adhesion molecules in milk compared to blood suggest that a migratory population of cells is being selectively recruited to the mammary gland from the circulation.

Cryptosporidium and host resistance: historical perspective and some novel approaches.

Cryptosporidium parvum is recognized as a major cause of diarrheal disease in neonatal bovine calves. In addition, this protozoan parasite has emerged as an important cause of disease in both immunocompromised and immunocompetent humans. Despite years of research, no consistently effective means of prevention or treatment are readily available for cryptosporidiosis in any species. Infection through ingestion of contaminated water has been widely documented; C. parvum was reported to be responsible for the largest waterborne outbreak of infectious disease in US history. In addition to its role as a primary disease agent, C. parvum has potential to initiate or exacerbate other gastrointestinal disorders, such as inflammatory bowel disease. Thus, control of C. parvum infection in both animals and humans remains an important objective. Research in our laboratory has focused on understanding mechanisms of resistance to C. parvum. We have demonstrated that acquisition of intestinal flora increases resistance to C. parvum. Substances present in the intestinal mucosa of adult animals can transfer resistance when fed to susceptible infants. Both expression of intestinal enzymes and rate of proliferation of epithelial cells may be altered following C. parvum infection. These and other changes may have profound effects on host resistance to C. parvum.

Cryptosporidium parvum infection in gene-targeted B cell-deficient mice.

The importance of B cells in host resistance to, and recovery from, Cryptosporidium parvum infection was examined in gene-targeted B cell-deficient (muMT-/-) mice. Neonatal muMT-/- mice infected with C. parvum at 5 days of age completely cleared the infection by day 20 PI. The kinetics of infection and clearance were similar to those seen with age-matched C57BL/6 control mice. Furthermore, B cells were not required to clear existing C. parvum infection in adult mice. Reconstitution of persistently infected Rag-1-/- adult mice with spleen cells from muMT-/- donor mice resulted in significant reduction of infection, as in the results seen with spleen cells from C57BL6 donors. These findings indicate clearly that B cells are not essential for host resistance to, and recovery from, C. parvum infection in mice.

Activities of DL-alpha-difluoromethylarginine and polyamine analogues against Cryptosporidium parvum infection in a T-cell receptor alpha-deficient mouse model.

The in vivo effectiveness of a series of conformationally restricted polyamine analogues alone and selected members in combination with DL-alpha-difluoromethylarginine against Cryptosporidium parvum infection in a T-cell receptor alpha-deficient mouse model was tested. Polyamine analogues were selected from the extended bis(ethyl)-sym-homospermidine or bis(ethyl)-spermine backbone having cis or trans double bonds at the center of the molecule. The cis isomers were found to have significantly greater efficacy in both preventing and curing infection in a mouse model than the trans polyamine analogues when tested in a T-cell receptor alpha-deficient mouse model. When tested in combination with DL-alpha-difluoromethylarginine, the cis-restricted analogues were found to be more effective in preventing oocyst shedding. This study demonstrates the potential of polyamine analogues as anticryptosporidial agents and highlights the presence of multiple points in polyamine synthesis by this parasite that are susceptible to inhibition resulting in growth inhibition.

Parasitic infections of the gastrointestinal tract.

Intestinal parasites continue to be a significant health problem in both developed and developing countries. In developed countries, protozoans are more commonly the cause of gastrointestinal infections than are helminths. Some protozoan parasites have stages in which, in addition to being resistant to chemicals used for water treatment, they are small enough to pass through commonly used filtration processes. The relatively large size of helminth eggs increases the likelihood of their removal during water filtration. The direct impact of protozoan parasites on both human and animal health is considerable, and there is some evidence that infection may contribute to the development of various forms of intestinal dysregulation as well as disseminated infection, especially in AIDS patients. Protozoans of special interest, due to either their frequency of isolation or their role as emerging pathogens, include Giardia duodenalis, Cryptosporidium parvum, Cyclospora cayetanensis, and the microsporidians, Enterocytozoon bieneusi and Encephalitozoon intestinalis.

Treatment with neurokinin-1 receptor antagonist reduces severity of inflammatory bowel disease induced by Cryptosporidium parvum.

Inflammatory bowel disease (IBD) is a chronic, debilitating disorder of uncertain and perhaps multiple etiologies. It is believed to be due in part to disregulation of the immune system. Neuroimmune interactions may be involved in induction or maintenance of IBD. In the present study, we examined the potential role of a neurotransmitter, substance P, in a mouse model of IBD. We found that binding sites for substance P, and more specifically, neurokinin-1 receptors, were upregulated in intestinal tissue of mice with IBD-like syndrome. Dosing of mice with LY303870, a neurokinin-1 receptor antagonist, reduced the severity of IBD, and treatment of mice with preexisting IBD allowed partial healing of lesions. We hypothesize that blocking the binding of substance P to the neurokinin-1 receptor interrupts the inflammatory cascade that triggers and maintains intestinal lesions of IBD.

Effects of mastectomy on composition of peripheral blood mononuclear cell populations in periparturient dairy cows.

There is an increased incidence of infectious disease in periparturient dairy cows. During the periparturient period there is a decline in T-lymphocyte cell subsets, which parallels a reduction in functional capacities of blood lymphocytes and neutrophils. Mechanisms responsible for these changes in immune function during the periparturient period are poorly characterized. Ten mastectomized and eight intact multiparous Jersey cows were used to determine whether the periparturient changes in peripheral blood mononuclear cell populations are the result of the physiological demands associated with the onset of lactation or whether they are a result of the act of parturition. Blood mononuclear cells were phenotyped with monoclonal antibodies against T-cell subsets, B-cells, and monocytes. Blood samples were taken frequently from before 4 to 4 wk after parturition. In intact cows, all T-cell subset populations (i.e., CD3-, CD4-, CD8-, and gamma-delta positive cells) decreased at the time of parturition, while the percentage of monocytes increased. Mastectomy eliminated the changes in leukocyte subset populations (CD3-, CD4-, and gamma-delta positive cells, and monocytes) observed in intact cows around parturition. These results indicate that the mammary gland and metabolic stresses associated with lactation influence the composition of peripheral blood mononuclear cell populations in dairy cows during the periparturient period.