Robert D. Romanowski

Ascaris suum: Development of intestinal immunity to infective second-stage larvae in swine

The development of protective immunity to Ascaris suum was examined in pigs naturally exposed to eggs on a contaminated dirt lot. Pigs became almost totally immune to second-stage larvae migrating from the intestines because few larvae from a challenge inoculum could be found in the lungs, and liver white-spot lesions (an immunopathologic response to migrating larvae) were absent. Blood from these pigs contained lymphocytes that responded blastogenically to larval antigens in vitro, while the serum contained antibody to larval antigens. Immunity was related to parasite exposure and not to the age of the host, and was not affected by the removal of adult A. suum from the intestines. Naturally exposed pigs responded to a variety of A. suum antigens with an immediate-type skin reactivity, and their intestinal mucosa contained relatively large numbers of mast cells and eosinophils. Other pigs were maintained on a dirt lot not contaminated with A. suum eggs and the effects of common environmental conditions on development of resistance to A. suum were studied. Resistance also developed in these pigs because 72% fewer larvae were detected in their lungs following a challenge exposure than in control pigs confined indoors on concrete floors and challenged similarly. This response was not expressed at the intestinal level, however, because their livers had numerous, intense white-spot lesions. To verify that the intestinal immunity that developed in pigs after natural exposure to A. suum was a direct result of homologous infection and not related to other stimuli encountered on a dirt lot, pigs maintained indoors on concrete floors, free from inadvertent helminthic infection, were inoculated orally with A. suum eggs daily for 16 weeks. Intestinal immunity was induced because larvae from a challenge inoculum were not detected in the lungs, and few white-spot lesions appeared on the livers of these pigs. Apparently, continual exposure of the intestinal mucosa to larvae eventually elicits the appropriate effector components necessary to prevent larval migration from the intestines.

Ascaris suum: Protective immunity in pigs immunized with products from eggs and larvae

Parasite products were collected at three distinct phases of development of Ascaris suum, and their immunogenicity was determined after injection into rabbits and pigs. Products were derived from (1) the hatching fluid of infective eggs; (2) the conditioned medium of 2nd-stage larvae that developed to 3rd stage in vitro in defined medium; and (3) the conditioned medium of 3rd-stage larvae that developed to 4th stage in vitro in defined medium. Protein profiles from these three preparations, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were less complex than that of extracts from homogenized A. suum larvae. Hyperimmune rabbit antiserum raised against either egg products, 2nd- to 3rd-stage larval excretory-secretory products, or 3rd- to 4th-stage larval excretory-secretory products showed strong homologous reactions after immunoelectrophoresis, but relatively weak cross-reactions with the other preparations. A combined enteral immunization of pigs with egg products and parenteral immunization with the 2nd- to 3rd-stage larval excretory-secretory products, and 3rd- to 4th-stage larval excretory-secretory products induced antibody to each preparation and significant protective immunity to a challenge exposure with 10,000 A. suum eggs. However, a marked pathological response to larvae migrating in the liver after challenge exposure was also induced.

Multiresidue determination of β-lactam antibiotics in milk and tissues with the aid of high-performance liquid chromatographic fractionation for clean up

Screening of milk shipments for beta-lactam antibiotic residues is mandatory in the USA and is widely used in other countries. Interpretation of positive screening test results has been difficult. Only six beta-lactam antibiotics are approved for use in food-producing animals in the USA but many others are used in other countries. A multiresidue procedure was developed for identification and quantitation of unknown beta-lactam antibiotics. The residues were extracted with acetonitrile and tetraethylammonium chloride. The extract was concentrated by evaporation and filtered. The concentrated extract was then loaded onto an HPLC column in 100% 0.01 M KH2PO4 and eluted with an acetonitrile gradient. Fractions corresponding to analytes of interest were collected and tested for antibiotics using rapid milk screening tests. Fractions testing positive were analyzed by HPLC. The identity of beta-lactams was confirmed by treating a replicate with beta-lactamase.

Porcine interleukin 2: parameters of production and biochemical characterization.

Interleukin 2 (IL2) or T cell growth factor (TCGF) has been characterized in a number of species but not in porcines. Porcine IL2 was detected in supernates (SN) of cultures of pig lymphocytes by: 1) the stimulation of the IL2-sensitive murine T cell line, CT6; 2) a costimulator assay involving porcine thymocytes; and 3) by the in vitro maintenance of antigen or mitogen-induced porcine lymphoblastoid cells. Porcine IL2 production by pig lymphocytes was induced by the mitogens Concanavalin A (Con A) Phytohemagglutiniin (PHA), and Pokeweed mitogen (PWM), but not by lipopolysaccharide (LPS). IL2 activity was demonstrated in the SN of mitogen-stimulated lymphocyte cultures as early as 24 hr after initiation of culture, reached peak levels at 48 hr, and decreased by 72 hr. Mitogens induced IL2 secretion by pig peripheral blood mononuclear cells, lymph node cells, and spleen cells, but not thymus cells. The cells responsible for IL2 production are presumptive T cells because: 1) they are nylon wool non-adherent; and 2) are non-surface-Ig bearing. In contrast, SN from cultures of surface Ig-positive cells had minimal IL2 activity. Porcine IL2 resembles rat and human IL2 in that it has an apparent molecular weight of approximately 15,000, and does not bind to DEAE-cellulose (DE-52) ion exchange columns equilibrated in 0.05 M sodium phosphate buffer (pH 7.6).

The effect of immunization of pigs with Ascaris suum cuticle components on the development of resistance to parenteral migration during a challenge infection

The development of immunity to Ascaris suum was studied in pigs immunized with isolated cuticle fragments from A. suum second and third stage larvae (L2/L3) and adult worms, and compared with other methods that stimulate a strong protective response in pigs. A significant protective response was seen in animals immunized with isolated cuticle fragments from A. suum L2/L3 and adults, but it was less than that seen in animals inoculated with UV-irradiated eggs or naturally exposed to eggs on a dirt lot. Significant IgG responses to 2-mercaptoethanol (2ME)-soluble cuticle components were seen in all groups, but the level of the antibody response did not relate to protection. Group differences in antibody and lymphocyte blastogenic responses to cuticle proteins indicated quantitative and qualitative stage specific differences in 2ME-soluble and insoluble cuticular proteins. Intestinal immunity was notably absent from cuticle immunized pigs because a marked liver white spot response was observed following the challenge inoculation. Thus, cuticle fragments from larval and adult A. suum are capable of inducing a protective response to larval migration; however, the development of intestinal immunity is not a direct function of exposure to these antigens.

A Trichuris specific diagnostic antigen from culture fluids of Trichuris suis adult worms.

Trichuris suis is a nematode parasite of swine which can cause serious pathology in infected pigs, such as anorexia, diarrhea, anemia, and death in heavy infections, especially in young animals. Infection with Trichuris suis is difficult to diagnose because much of the pathology occurs when only larval stages are present, and because of sporadic egg production by adult female worms. We have isolated a 20 kDa excretory/secretory (E/S) glycoprotein from culture fluids of adult worms by column chromatography and preparative gel electrophoresis which is diagnostic for Trichuris suis infection in pigs. The antigen did not crossreact in Western blots or enzyme-linked immunosorbent assay with sera from pigs infected with Ascaris suum, Trichinella spiralis, Oesophagostomum dentatum, or Toxoplasma gondii. The antigen was also diagnostic for Trichuris vulpis infection in dogs, and did not crossreact with sera from dogs infected with Toxocara canis, Ancylostoma caninum, or Strongyloides stercoralis. The antigen may be useful in clinical serologic assays for diagnosis of Trichuris infection in pigs and dogs in cases of scouring diarrhea of unknown etiology, and in epidemiological surveys of Trichuris suis infection in swine feeder/finishing operations.

A DEVELOPMENTALLY REGULATED HYALURONIDASE OF HAEMONCHUS CONTORTUS

The trichostrongylid nematode Haemonchus contortus released a hyaluronic acid–degrading enzyme during in vitro development from the third (L3) to fourth (L4) larval stage. The enzyme did not degrade chondroitin sulfate A. Enzyme activity was optimal between pH 4.0 and 6.0, and the enzyme was inhibited by high concentrations of NaCl; the divalent cations Cu2+, Zn2+, Ca2+, and Mn2+ were not inhibitory. The hyaluronidase had a molecular mass estimated at 57 kDa by sucrose density gradient centrifugation and at 111 kDa by substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (reducing and nonreducing conditions), suggesting the formation of a dimer during the electrophoretic separation conditions. The level of hyaluronidase released during in vitro development peaked between 24 and 48 hr in culture and then gradually decreased, with little or no activity present in the 168-hr culture fluid. The enzyme was not detected in culture fluid from 24-hr incubations of either the mid-L4 stage (obtained from sheep 7 days postinfection) or the adult stage (obtained from sheep 30–35 days postinfection). The temporal expression of the hyaluronidase suggested a role for this enzyme in the early stages of the L3–L4 developmental process.

The secretory nature of the excretory gland cells of Stephanurus dentatus. II. Presence of hydrolytic enzymes

Abstract Enzymes catalyzing the hydrolysis of casein, N-benzoyl- l -tyrosine ethyl ester, p-nitrophenyl acetate, and l -leucyl-β-naphthylamine hydrochloride were found in extracts of the excretory gland cells of Stephanurus dentatus.

The secretory nature of the excretory gland cells of Stephanurus dentatus: III. Proteinase inhibitors

Abstract A proteinase inhibitor(s) was found in extracts of the excretory gland cells, intestines, esophagi, reproductive organs, and body walls from Stephanurus dentatus adults. The specific activity of the inhibitor(s) in the excretory gland cell extract was 45–175 times greater than in the other tissues. It is heat stable at pH 5.0 and inhibits the esterolytic activity of trypsin and chymotrypsin using p-toluenesulfonyl- l -arginine methyl ester hydrochloride (TAME) and benzoyl- l -tyrosine ethyl ester (BTEE) as the substrates, respectively, and also the proteolytic activity of both chymotrypsin and trypsin using casein as the substrate. S. dentatus adults maintained in NCTC 109 medium, secreted a trypsin inhibitor.