Editorial

Summary

The possession of common antigens by three trematode parasites which commonly occur together in ruminants in the tropics, Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis was studied in relation to the reliability of serodiagnosis of infection with these helminths. The crude antigenic extracts of the three trematodes were subjected to Sephacryl S‐300 column chromatography and F. gigantica was fractionated into six peaks, S. bovis into nine peaks and D. hospes into seven peaks. Common antigens were found in these three trematodes in both the crude whole worm antigenic extracts and in the semi‐purified fractions obtained by Sephacryl S‐300 column chromatography.

The implications of this finding and the limitation it imposes on the usefulness of serodiagnostic tests in routine use as regards their specificity are discussed in relation to previous studies.     

The purification and characterization of a cysteine protease of Fasciola gigantica adult worms.

A 26-28 kDa protease was isolated from Fasciola gigantica adult worms by a two-stage purification process of column chromatography in a Sephacryl S-200 column and affinity chromatography in an L-phenylalanine-agarose column. This protease is a cysteine (thiol) proteinase with an optimum pH of 4.5 and is not inhibited by anti F. gigantica immunoglobulin G. The enzyme was inhibited by protease inhibitors known to inhibit cysteine proteases but not by metallo-, aspartate or serine protease inhibitors. The effect of several protease inhibitors and anti-F, gigantica IgG was also assessed on the total proteolytic activity of F. gigantica. There appears to be a preponderance of cysteine protease activity in F. gigantica and there was a significant inhibition of total proteolytic activity by anti-F. gigantica IgG.     

Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma.     

Separation of parasite antigens by molecular exclusion, anion exchange, and chromatofocusing utilizing FPLC protein fractionation systems

Excretory-secretory products (ESP) were harvested from balanced salt solutions in which adult Fasciola hepatica had been incubated for 4-6 h at 37 degrees C. The ESP was fractionated by standard low pressure molecular exclusion chromatography and FPLC (fast protein liquid chromatography) using the principles of molecular exclusion, anion exchange, and chromatofocusing. The dot-enzyme-linked immunosorbent assay (Dot-ELISA) was used to demonstrate the immunoreactivity of eluted fractions. Compared to Sephacryl S-200, separation by Superose-6 (FPLC) was faster and resolved more peaks (four with Sephacryl S-200 and nine with Superose-6). Peaks from Sephacryl S-200 were resolved by the first anion exchange (Mono Q) separation into seven peaks; when these peaks were subjected to a second anion exchange, 15 peaks were resolved. Thirty-eight peaks were resolved by chromatofocusing (Mono P) in the pH range 7-4. Immunoreactive fractions from narrow-range (single pH unit) chromatofocusing were identified by the Dot-ELISA. The FPLC system proved to be a means of rapid and high resolution separation of F. hepatica antigens.     

Purification of larval Taenia solium antigens by gel filtration.

Crude larval Taenia solium extracts were fractionated by Sephacryl S-200 gel filtration into four fractions (W1-W4). The sensitivities of the fractions to rabbit and pig antiserum against Taenia solium were tested by double immunodiffusion, immunoelectrophoresis, and ELISA. Fraction W2 which was highly sensitive to antisera was shown by immunoblotting to contain antigen B (95 and 105 kDa). The four fractions were shown to contain antigenic determinants common with pig serum proteins and crude extracts of other Platyhelminthes (especially Taenia hydatigena). Fraction W2 has the potential to be used as a serodiagnostic antigen.     

Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults

The study focused on characterizing and isolating Dicrocoelium dendriticum antigens or their fractions that could be used for the immunological diagnosis of dicrocoeliosis. Somatic (SoAg) and excretory-secretory antigens (ESAg) were analyzed by SDS-PAGE and their specificity was evaluated by Western blot with homologous and heterologous sera. The antigens were partially purified by chromatographic techniques of gel-filtration (Sephacryl S-300) and ion exchange (Hitrap-DEAE-Sepharose). Western blot analysis using sera of ovine infected with D. dendriticum revealed eight main antigenic polypeptides ranging from 24 to 205 kDa for SoAg and seven for ESAg with apparent molecular mass in the range of 26-205 kDa. We detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels, arranged as a 450 kDa tetramer in native conditions. It also showed strong immunoreactivity by Western blot against ovine sera experimentally infected with D. dendriticum. Gel filtration chromatography (Sephacryl S-300) also showed other specific proteins, one of about 24 kDa in SoAg and another of about 42 kDa in ESAg. The elution conditions of 450 kDa protein (130 kDa monomer) by DEAE chromatography were similar to those from the somatic antigen (pH 7.2, 0.1M NaCl, in 29-34 ml fractions) and from the excretion-secretion antigen (pH 8.0, 0.1M NaCl, in 29-35 ml fractions). The suitability of 130 kDa polypeptide for D. dendriticum infection diagnosis was confirmed by Western blot using a pool of sera as well as individual serum samples from experimentally infected sheep. The sequence of amino termini of 130 kDa polypeptide from both fractions was the same and identical to that reported for a peptide from D. dendriticum described as a globin. This sequence also revealed an appreciable similarity with the amino end of globins from some phylogenetically related worms.     

Serologically defined Rhipicephalus (Boophilus) microplus larval antigens in BmLF3, a partially pure Sephacryl S-300 fraction of crude larval proteins

This report is designed to provide additional information regarding larval soluble proteins toward the planned development of a comprehensive database of Rhipicephalus (Boophilus) microplus proteins that elicit a humoral immune response in cattle as a result of natural ectoparasite infestation. Larval proteins of R. microplus are complex and the protein profile is not dominated by any major proteins. This report focuses upon an S-300 Sephacryl (molecular sieve) column fraction, fraction 3 (BmLF3). With the use of SDS-PAGE (without-2ME) and Western blotting with a composite pool of pre- and post-R. microplus larval infestation antiserum BmLF3 was found to contain 7 apparent common ixodid major antigens (207.3, 171.9, 98.0, 86.5, 65.7, 58.9, and 38.0 kDa), those potentially shared with other ixodid species, and 2 apparent R. microplus specific antigens evidenced by low-level antibody binding in crude BmLF3 (149.4 kDa) and HPLC peak 8 of BmLF3 (116.0 kDa). In addition, BmLF3 contains potent inhibitors of trypsin activity. However, these inhibitors of trypsin did not appear to elicit host antibodies as a result of natural ectoparasite exposure, as defined by Western blotting of reduced and denatured trypsin binding proteins purified by affinity chromatography.     

Specificity of affinity-purified Trichinella spiralis antigens.

An affinity-purified fraction (APF) was obtained by passing crude somatic antigens of Trichinella spiralis muscle larvae through an Affi-Gel 10 column coupled with anti-Trichuris suis IgG. The fraction contained seven antigens with molecular weights ranging from 28 to 55 kDa. When tested with antiserum against other common nematodes of pigs from China, the APF was found to be markedly more specific than S3 antigens (prepared by a combination of cell fractionation and differential centrifugation according to Despommier and Lacetti, 1981) and fractions produced by Sephacryl S-200 gel filtration (F1 to F12). When the APF was used in an indirect IgG-enzyme-linked immunosorbent assay (IgG-ELISA) to screen serum samples from 2000 pigs imported from China, a positive rate of 7.5% was obtained. Similar screenings using the crude somatic antigens F1 and S3 gave a large number of cross-reactions and false positive reactions. Positive rates of 48%, 39% and 59.5% respectively were obtained for the three antigens.     

Main features of helminth parasitism in cattle in Ituri (Haut-Zaire). III. Geographic distribution and prevalence of the main helminths]

A necropsic survey, carried out in eleven slaughterhouses in Ituri (Haut-Za?re), has permitted to define the geographic distribution and prevalences of the main cattle helminths. Trematodes, except the paramphistomes, had a very heterogeneous distribution. Prevalence of Fasciola gigantica ranged from 9 to 72% according to the sites, but these variations were not linked to topographic or climatologic parameters. The infection with Schistosoma bovis was much variable as well (12.5 to 72%) and seemed absent from the central high altitude area. The occurrence of Dicrocoelium hospes was restricted to the northern part of Ituri with a moderate prevalence of about 35%. By contrast, nematodes had a fairly homogeneous distribution in Ituri. Prevalences were high for gastro-intestinal strongyles of the following genera, Haemonchus, Cooperia and Oesophagostomum (over 60%). Cysticercosis (Cysticercus bovis) occurred in 10 to 14% of cattle in the middle and south areas of Ituri whereas the north areas were nearly free.     

Purification of infectious bronchitis coronavirus by Sephacryl S-1000 gel chromatography

A procedure was developed to purify infectious bronchitis virus (IBV) by gel chromatography (GC) with a Sephacryl S-1000 column. Virus samples concentrated by centrifugation were applied to a Sephacryl S-1000 column and eluted by 0.02 M phosphate buffer (pH 7.2) containing 0.15 M NaCl. Virus particles were recovered mainly in the first peak. Purity of the samples was evaluated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy. Using electron microscopy, it was found that there were more spike-rich particles in the virus samples purified by GC than in those purified by sucrose density gradient centrifugation (SDGC). In addition, the hemagglutination unit [log10 (infectivity titer/hemagglutination titer)] of GC-purified virus samples was approximately 10 times lower than that of SDGC-purified virus samples. These results indicate that Sephacryl S-1000 gel chromatography is useful for purification of IBV.     

The development of a recombinant Babesia vaccine.

Crude extracts of Babesia bovis parasites were shown to induce levels of protection in susceptible cattle equivalent to that resulting from natural infection. The crude material was systematically fractionated and tested in numerous sequential vaccination/challenge experiments in adult cattle. Antigens in protective fractions were then purified by affinity chromatography with monoclonal antibodies. Three highly protective (more than 95% reduction in parasitaemias) antigens were thus identified. None of these antigens was immunodominant; a number of immunodominant antigens were identified and all were immunosuppressive and/or non-protective. The three protective antigens were cloned and expressed as either beta-galactosidase or glutathione-S-transferase (GST) fusion proteins. Two of these, GST-12D3 and GST-11C5, when used in combination were almost as protective as has been previously shown for the commercially available live attenuated vaccine. A short fragment of a third antigen (21B4) has also been shown to be protective. In two of the antigens, repetitive segments have been shown to be non-protective while the third antigen (12D3) does not contain repetitive domains. Homologues of these antigens exist in other Babesia species and it is anticipated that these may be candidate antigens for protective vaccines against those species.     

Bovine T cell responses to recombinant thioredoxin of Fasciola hepatica

Fasciolosis is an economically significant disease of ruminants, caused by infection with the digenetic trematodes, Fasciola hepatica and F. gigantica. Some vaccination trials using irradiated metacercariae or isolated proteins have been shown to afford significant protection. However, the mechanisms of specific immunity against this pathogen have not been elucidated. We have identified thioredoxin, a tegument antigen of F. hepatica, among several proteins that are common to both the juvenile and adult fluke within the mammalian host and have undertaken studies to characterize bovine T cell responses to recombinant thioredoxin protein (FH 2020). Peripheral blood mononuclear cells from immune cattle proliferated specifically to crude F. hepatica antigenic extract but not to FH 2020. However, after repeated stimulation of lymphocytes by alternating crude extract and FH 2020, FH 2020-specific proliferation by T cell lines was observed. T cell clones were subsequently generated and found to respond specifically but weakly to both crude antigen and FH 2020. Thioredoxin appears to be only weakly antigenic for bovine T cells and is, therefore, an unpromising candidate for inducing resistance to F. hepatica.     

Fractionation and characterisation of the cysticercus of Taenia solium

Fractionation by chromatography on Sephadex G-200 of a saline extract of Cysticercus cellulosae scolex antigen yielded three distinct fractions associated with distinct peaks. These fractions were analysed by double immunodiffusion (DID) and immunoelectrophoresis (IEP). The three peaks gave five, four and three antigenic determinants, respectively, by DID with homologous hyperimmune rabbit serum. However, the same serum gave nine antigenic determinants of scolex antigen by DID and 11 components by IEP. The IEP demonstrated seven and five antigenic components in the first two peaks. The first peak gave a stronger reaction in indirect haemagglutination than the others. There were common antigenic components in C cellulosae and C tenuicollis antigens.     

Clinical diagnosis of schistosomiasis in sudanese cattle

This study was done in the White Nile Province to characterise the history and signs of naturally occurring Schistosoma bovis infection in cattle (Gorag). Necropsy and laboratory examinations were performed on 10 animals six to 30 months of age which were in poor condition. They were selected because of a history suggestive of schistosomiasis. All the animals showed some degree of S. bovis infection; eight had a moderate or heavy degree of infection. Also all had liver damage due to either past or active Fasciola gigantica infection. Although concurrent infection with these two trematodes is common an owner who diagnoses Gorag is most likely referring to the syndrome caused by S. bovis as being the major cause of the poor performance observed. Fasciolicide treatment may eliminate active fascioliasis as the principal damaging agent. Also differentiating signs of haemorrhagic diarrhoea, severely sunken eyed appearance and only moderate inappetence are common in animals with acute schistosomiasis.     

Liver fluke infections of cattle in Mali. An abattoir survey on prevalence and geographic distribution

Six hundred and ninety two cattle, slaughtered in various abattoirs in Mali between August 1983 and November 1984 were examined for evidence of Fasciola gigantica and Dicrocoelium hospes infections. A great variation in geographic prevalence was observed. Sufficient knowledge was acquired to demonstrate both the importance of fascioliasis in some areas and also the high prevalence of D. hospes in other areas. We suggest that the difference in prevalence from one geographic area to another is influenced by local climatic conditions, presence or absence of rivers and lakes, and differences in livestock management practices by various ethnic groups.     

Identification and isolation of calcium-dependent and -independent bovine immunoglobulins reactive with sepharose 4B

Calcium-dependent and -independent Sepharose 4B-binding bovine serum proteins were isolated by affinity chromatography on unsubstituted Sepharose 4B using 2 mM ethylenediamine tetraacetic acid followed by 0.1 M galactose. They appeared in two different protein peaks by gel filtration on Sephacryl S-200. The earlier eluted protein was demonstrated to be immunologically IgM, whereas the retarded one IgG. From these findings, Sepharose 4B-binding bovine serum proteins are suggested to be calcium-dependent and -independent immunoglobulins IgM and IgG.     

Staphylococcosis of turkeys. 4. Characterization of a bacteriocin produced by an interfering Staphylococcus

A Staphylococcus epidermidis isolate designated strain 115, which is used as an interfering agent against staphylococcosis of turkeys, produces a bacteriocin that was partially purified and characterized in this study. This bacteriocin diffused through agar media, but it was not found in appreciable quantities in the supernatant fluid of broth cultures. Extraction of the bacterial cells with 7 M urea, 1% sodium dodecyl sulfate, or 1% Triton X-100 caused considerable amounts of the bacteriocin to go into solution. This substance was partially purified by selective chemical extraction and by gel filtration chromatography using a Sephacryl S-300 column. This bacteriocin had two active forms: an aggregate, and a small-molecular-weight form estimated by gel filtration chromatography to be less than 6500. Activity was not affected by heat, repeated freeze-thaw cycles, pH 2 and pH 10, or a variety of proteolytic enzymes, nucleases, a lipase, and lysozyme.     

Immunity of schistosomes using heterologous trematode antigens--a review

This review summarizes the field of cross-protection in schistosomiasis due to other parasitic infections and with subcellular fractions of the parasite trematodes. Regarding parasitic infections, the clearest evidence of cross-protection to Schistosoma mansoni was found with the trematodes, particularly with Fasciola hepatica. Evidence was also presented which demonstrated that the protective F. hepatica worm antigens were those which bound to antibodies to S. mansoni, and that as antigen purification proceeded, smaller amounts were required to obtain significantly high levels of protection. These 2 factors, cross-reactivity and improved protection with increasing antigen purity (and possibly improved immunogenicity), are both supportive of an immunological basis for protection against S. mansoni. A Fasciola/Schistosoma-defined immunity cross-reactive antigen from F. hepatica worms was isolated and designated as FhSmIII(M). An antiserum to this antigen was developed and used as a probe to detect the presence of this antigen (or its determinants) in different extracts of parasitic trematodes. In this manner, it was possible to demonstrate that FhSmIII(M) (or at least some of its determinants) were found on (or in) S. mansoni, S. bovis and Paragonimus westermani. Since mice immunized with P. westermani worm extracts acquire resistance to challenge with S. mansoni cercariae, a common link of cross-protection to the parasitic trematodes is suggested; i.e., FhSmIII(M). Although immunity to schistosomes is undoubtedly multifactorial, the demonstration of a common protective antigen (or determinant) will provide a handle for the evaluation of additional candidate protective antigens.     

Epidemiology and control of trematode infections in cattle in Zimbabwe: a review

In this paper the main epidemiological aspects of the major domestic ruminant trematode infections in Zimbabwe are reviewed and discussed with regard to the available options for control. Seasonal occurrence of amphistomes, Fasciola gigantica and Schistosoma mattheei are considered both in the definitive and intermediate hosts. The regional distribution of the trematodes is reviewed in relation to the distribution patterns of their snail intermediate hosts. Based on the epidemiological features of the trematodes, practical control measures are suggested.     

Modulation of host immune responses by protozoal DNA

The pathology caused by acute Babesia bovis infection is similar to that seen in severe human malaria caused by Plasmodium falciparum infection, which is related to dysregulated production of inflammatory cytokines and nitric oxide (NO). We have observed induction of NO, inducible nitric oxide synthase (iNOS) and inflammatory cytokines in macrophages by B. bovis. Furthermore, proliferation of lymphocytes from individuals never exposed to certain protozoal pathogens can be induced by crude protozoal parasite extracts. We have repeatedly observed stimulation of naive PBMC from cattle to antigenic extracts of Babesia bovis. Based on recent studies demonstrating the mitogenicity of bacterial and other non-vertebrate DNAs for murine B cells and macrophages, the mitogenic properties of B. bovis DNA were examined. B. bovis and E. coli DNAs induced proliferation of PBMC and purified B cells from non-exposed cattle. Stimulatory activity was reduced by DNase treatment and methylation with CpG methylase, indicating the presence of stimulatory non-methylated CpG motifs in the B. bovis genome. B. bovis and E. coli DNAs enhanced IgG secretion by cultured B cells, stimulating IgG1 and more strongly, IgG2. Several hexameric CpG immunostimulatory sequences (ISS) active for murine B cells were identified in an 11 kb fragment of B. bovis DNA. An oligodeoxyribonucleotide containing one of these (AACGTT), located in the rhoptry associated protein-1 (rap-1) open reading frame, stimulated B cell proliferation. These studies identify a potential mechanism by which protozoal parasites may modulate host immune responses, leading to consequences such as hypergammaglobulinemia and splenomegaly. These results also support the use of ISS as vaccine adjuvants to enhance Type 1 immune responses in cattle.