Patrick Joyce

Fasciola hepatica infection downregulates Th1 responses in mice

Immune responses induced with helminth parasites have been extensively studied, but there is limited information on those to Fasciola hepatica, especially on the subtype of T cell induced with this parasite. We investigated the local and systemic T cell responses of different strains of mice following oral infection with doses of metacercariae from F. hepatica. Spleen cells from BALB/c and 129Sv/Ev mice given a low‐dose (5 metacercariae) infection exhibited a Th2 response, producing high levels of the cytokines IL‐4 and IL‐5, and low levels of IFN‐γ and IL‐2. In contrast, C57BL/6 mice showed a mixed Th1/Th2 response. A more marked polarization to a Th2 response was observed in BALB/c, 129Sv/Ev exposed to a high‐dose (15 metacercariae) infection and the C57BL/6 mice also exhibited a clear Th2 response. IL‐4 defective (IL‐4−/−) C57BL/6 mice infected with 5 metacercariae produced less IFN‐γ and more IL‐5 compared to their wild‐type C57BL/6 counterparts, suggesting that IL‐4 is important in establishing the Th2 type response in murine fasciolosis. However, the secretion of IFN‐γ and IL‐2 was completely suppressed in the high‐dose infection and this was also observed in IL‐4−/− mice. Thus, liver flukes may secrete molecules that downregulate Th1 responses. T cell responses in the mesenteric (MLN) and hepatic lymph nodes (HLN) were also examined since newly excysted juveniles infect through the intestinal wall of their host before migrating to the hepatic tissue. Cells from both MLN and HLN secreted higher levels of IL‐4 and IL‐5 compared to spleen cells. We also observed a difference in cytokine profiles secreted by the MLN and HLN, which may reflect responses to antigens liberated by newly excysted juveniles and hepatic stage parasites, respectively.

Effect of decreased milking frequency of cows in late lactation on milk somatic cell count, polymorphonuclear leucocyte numbers, composition and proteolytic activity

The effects of reducing the frequency of milking of cows in late lactation on milk somatic cell count (SCC), polymorphonuclear leucocyte (PMN) content, chemical composition and proteolytic activity were investigated. Intermittent milking is frequently practised by Irish farmers in late lactation, and the objective of this study was to determine whether this procedure could be linked to altered quality of milk. Seventeen Holstein Friesian cows in late lactation (> 215 d in milk) were assigned to two treatment groups, and were either milked twice a day until drying-off (control group) or milked intermittently as the yield fell (test group). Milk composition and enzymic characteristics were measured on two occasions. At the first sampling, day 7, test cows were on once daily milking and at the second, day 15, the test cows were being milked every second day. Milk yields were significantly lower in test than control animals and decreased between days 7 and 15 in both groups. Milk SCC and PMN levels were increased on reducing milking frequency and, at day 15, the increase was not linked to decreased milk yield. Milk lactose levels were significantly decreased and pH, alpha-lactalbumin levels, plasmin activity and plasminogen activity significantly increased by reducing milking frequency. In conclusion, reduced frequency of milking in late lactation leads to the production of milk that is abnormal in character and this may be linked to reduced quality of dairy products manufactured from such milk.

Effects of stocking density and concentrate supplementation of grazing dairy cows on milk production, composition and processing characteristics

The effects on milk composition and processing characteristics of varying grass supply by changing stocking density and of offering a concentrate supplement were investigated. The experiment was conducted over 28 weeks of the lactation (April-October) using 48 spring-calved Friesian-Holstein cows. Three herds each of 16 cows were offered a restricted grass supply, a standard grass supply and a standard grass supply with a supplement of 3 kg concentrate/d. Treatment groups were grazed separately with a residence time of 3 d/paddock. Milk production, composition and processing characteristics such as renneting properties, ethanol stability and plasmin activity were measured weekly. Increasing stocking density above the standard system resulted in significant reductions in milk fat and protein yields, the concentrations of total protein, casein and whey proteins, and a deterioration in most processing characteristics. Imposing concentrate supplementation on the standard system increased total protein, casein and whey protein concentrations but generally did not improve processing characteristics except for ethanol stability. These results suggest that the standard grass supply in a rotational grazing paddock system can support efficient production of quality milk, and concentrate supplementation will not improve processing characteristics when an adequate supply of good quality herbage is available.

Type I hypersensitivity reactions in intestinal mucosae from rats infected with Fasciola hepatica

Type I hypersensitivity reactions in the intestinal tract of sensitized animals may contribute to resistance to reinfection with Fasciola hepatica. Colonic mucosae isolated from previously infected rats were voltage clamped in Ussing chambers. Antigen was prepared as a crude homogenate from adult liver fluke. Assay of serum antibodies against fluke antigen confirmed sensitization. Antigen challenge evoked a rapid onset, transient inward current in sensitized but not in control preparations. Chloride secretion accounted for at least part of the response since the loop diuretic bumetanide reduced the effect of antigen by 61%. Anti‐rat IgE mimicked the response to antigen and desensitized tissues to subsequent antigen challenge. Local synthesis of eicosanoids may mediate the response to antigen since the cyclo‐oxygenase inhibitor piroxicam reduced the response by 76%. In contrast, mepyramine which is a histamine receptor antagonist did not alter the ion transport response evoked by antigen. Tetrodotoxin reduced the response to antigen by 53% implicating intrinsic neurons within the lamina propria as effector cells in the responses of this tissue to antigen. We propose that antigen stimulation of electrogenic chloride movement and consequent fluid secretion in vivo may contribute to a local effector mechanism in prevention of reinfection of previously sensitized hosts.

Identification of phosphorylcholine containing antigens of Fasciola hepatica--successful tolerization against this epitope in experimental animals.

Summary Phosphorylcholine containing antigens have been identified in the parasite Fasciola hepatica by immunoblotting and ELISA. Immunoblots probed with polyclonal and monoclonal antibodies indicate that the majority of antigens identified in both the immature and mature parasite contain both phosphorylcholine and non‐phosphorylcholine epitopes. One antigen of 58 kDa appears to contain predominantly PC epitopes or at least this epitope is the major one responded to by host animals. Successful immunoiolerization against the epitope PC was achieved by injecting the PC conjugate, ovalbumin PC, into neonatal rats. Immurunolerization against PC resulted in a 25% reduction in worm burden upon subsequent infection with Fasciola hepatica.

Capture immunoassay for the diagnosis of bovine mastitis using a monoclonal antibody to polymorphonuclear granulocytes

A direct capture enzyme-linked immunosorbent assay (ELISA) was developed to measure elevated polymorphonuclear granulocyte (PMN) antigens using horseradish peroxidase (EC 1.11.1.7) conjugated rabbit polyclonal anti-PMN antisera and a monoclonal antibody specific for PMN cells. Optical densities obtained in the ELISA were used to predict the cell counts of milk samples. Predicted counts were not significantly different from actual somatic cell counts (SCC). In a total of 156 bovine milk samples the correlation coefficient between somatic cell counting, taking greater than 500,000 cells/ml as being indicative of mastitis, and the assay was 0.94, yielding an assay sensitivity of 95.2% and a specificity of 97.3%. In further trials the ELISA could detect elevated PMN antigens in milk with SCC as low as 100,000 cells/ml. The results indicate that the monoclonal antibody based direct ELISA has excellent potential in the detection and determination of bovine mastitis.

Characterization of surface glycoproteins and proteins of different developmental stages of Fasciola hepatica by surface radiolabeling.

velopmental changes take place in the tegumental cells that secrete the surface glycocalyx of Fasciola hepatica (Bennett and Threadgold, 1975, Experimental Parasitology 38: 38-55). Immunological assays using sera from infected animals (Hanna, 1980a, Experimental Parasitology 50: 155-170) and monoclonal antibodies (Hanna and Trudgett, 1983, Parasite Immunology 5: 409425; Aronstein et al., 1985, American Journal of Tropical Medicine and Hygiene 34: 889-897) indicate that corresponding developmental changes occur in the antigenic nature of the polypeptides secreted by these cells. These studies suggest that F. hepatica may express different molecules at the surface at different stages of development. To address this question directly, in this report we compare the polypeptides expressed on the surface of newly excysted juveniles (NEJ), 21-day-old immature flukes and 13wk-old mature flukes. The surface glycoproteins and proteins of each of these developmental stages were labeled using the galactose oxidase/sodium borohydride and radioiodination methods, respectively, and analyzed by SDS-polyacrylamide gel electrophoresis. Tissue autoradiography at the level of the light microscope was carried out to ensure that labeling was confined to the surface. Mature F. hepatica were removed from the bile ducts of rats 13 wk after oral infection with 20 metacercariae. Immature flukes were teased

Kinetic and immunological differences between the retinal specific C4- and B4-lactate dehydrogenase of the cichlid fish Oreochromis mossambicus

Abstract The eye-specific C4-lactate dehydrogenase (LDH) and the widely distributed B4-LDH isozymes from the fish Oreochromis mossambicus were purified to homogeneity using DEAE Sepharose ion-exchange chromatography and oxamate-linked sepharose affinity-chromatography. Kinetic analysis was performed on pure B4- and C4-LDH. The Michaelis-Menten constant (Km) for B4-LDH and pyruvate was 32.3μM, for B4-LDH and lactate 717 μM for C4-LDH and pyruvate 14.1 μM and for C4-LDH and lactate 1898 μM. The pure C4-isozyme was subjected to SDS-polyacrylamide gel electrophoresis and the Coomassie Brilliant Blue-stained band injected into a rat to produce antiserum. The antiserum proved to be C4-LDH monospecific, which will allow using it to localize the isozyme in the retina at a light and electron microscopical level.