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R J Boisvenue

Publications and source records attributed to R J Boisvenue.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and sequence comparisons of four distinct cysteine proteases expressed by Haemonchus contortus adult worms.

Three new members of a developmentally regulated cysteine protease gene family of the parasitic nematode Haemonchus contortus have been isolated and characterized. One of the new genes, AC-3, was found to be linked in tandem to the previously characterized AC-2 gene. Nucleotide sequence analyses revealed that the first 90 amino acids of AC-3 are organized into four exons, similar to the situation in AC-2. A cDNA that appears to be a near full-length copy of the AC-3 gene was isolated using the polymerase chain reaction (PCR) technique to amplify cDNAs from adult worm poly(A)+ mRNAs. In addition to AC-3, a distinct cysteine protease cDNA, AC-4, was amplified by the same oligonucleotide primers. cDNAs encoding a fifth cysteine protease, AC-5, were isolated from an adult worm cDNA expression library using specific rabbit antisera and by PCR. Comparison of the predicted amino acid sequences of AC-3, AC-4 and AC-5 reveal that they share 64-77% identity with one another and with the previously reported AC-1 and AC-2 sequences. The amino acids surrounding the active site cysteine are highly conserved, as are the positions of other cysteine residues in the mature protein sequences. The H. contortus proteases are more similar to one another than they are to human cathepsin B (38-44% amino acid identity) or to the Sm31 cysteine protease of Schistosoma mansoni (36-40% identity). Our studies indicate that H. contortus adult worms express mRNAs for several distinct cysteine proteases. The significant primary sequence differences between the proteases suggest that they differ in their substrate specificities and precise physiological functions.

Amino Acid Sequence↗
PubMed1992

Isolation of putative cysteine protease genes of Ostertagia ostertagi.

Recombinant phage containing putative Ostertagia ostertagi cysteine protease genes have been isolated from a lambda EMBL-3:genomic DNA library using a Haemonchus contortus cathepsin B-like cysteine protease cDNA as hybridization probe. Restriction enzyme maps of the phages suggest that they identify at least 3 genes, 2 of which appear to be linked in tandem. The complete nucleotide sequence of one gene, CP-1, was determined. The CP-1 gene appears to be organized into 12 exons than span 4.5 kb of DNA. The number and sizes of the exons are essentially identical to those in the H. contortus AC-2 cysteine protease gene. Partial nucleotide sequences obtained for a second O. ostertagi gene, CP-3, revealed a similar organization for exons 8-12 in this gene. Like other cathepsin B-like cysteine proteases, CP-1 appears to be synthesized initially as a preproprotein that is proteolytically processed to its mature form. The amino acid identity between the presumptive CP-1 and CP-3 proteins is 66%, which is similar to the level of homology between the presumed mature protein regions of CP-1 and AC-2. Amino acid identity between CP-1 and AC-2 is greatest in the mature protein region and lowest in the signal sequence and propeptide regions. The CP-3 protein appears to be most closely related to the H. contortus AC-5 protein. CP-1 and CP-3 display significantly greater homology to H. contortus cysteine proteases than they do to human cathepsin B or the Sm31 cysteine protease of Schistosoma mansoni (about 40% identity with each).

Amino Acid Sequence↗
PubMed1992

Inoculation of conventionally and specific-pathogen-free reared rabbits with Ostertagia ostertagi isolated from cattle.

Eleven trials were conducted to collect helminthologic and pathologic data from 27 conventionally reared (CR) and 53 specific-pathogen-free (SPF) 6- to 13-week-old male New Zealand White rabbits. These rabbits were given 50,000 to 100,000 ensheathed third-stage infective Ostertagia ostertagi larvae (L3) orally. The L3 had been isolated from the feces of cattle. Fecal egg counts were conducted and worm populations were determined after euthanasia 3 to 56 days after inoculation. At necropsy, nodules were observed in a confluent pattern in the mucosa of cardiac region of the stomach in all inoculated rabbits, except in CR rabbits inoculated at 6 weeks of age, which had no nodules or worm burdens 42 days after inoculation. Confluent areas were not observed in SPF rabbits euthanatized 5 days or less after inoculation; however, small, transparent nodules were evident in the mucosa of the cardiac region of the stomach. Fourth-stage larvae (L4) were obtained from the mucosal nodules after digestion of stomach of both types of rabbits. There were more L4 in SPF than CR rabbits. In addition, greater numbers of L4 were found in SPF rabbits euthanatized 14 days or less after inoculation. Petechial hemorrhages were in the fundic area of the stomach mucosa in SPF rabbits inoculated with 100,000 L3 and euthanatized 14 days later. Mature O ostertagi or worm eggs in feces were not found in inoculated CR or SPF rabbits. The pathologic changes had characteristics similar to those in cattle and goats infected with O ostertagi.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Fibrinogen-degrading proteins from Haemonchus contortus used to vaccinate sheep.

Sixteen nonsibling sheep, approximately 12 months old, that were raised in a helminth-free environment, were used for 2 protection studies 6 months apart. Sheep were vaccinated weekly for 5 weeks by IM injection of fibrinogen-degrading proteins derived from the intestinal tract of adult Haemonchus contortus. Ten days after the last vaccination, sheep were given 2,500 infective H contortus larvae by intraruminal injection. Vaccinated sheep produced specific antibodies, and were protected from the worm challenge. Significant differences in mean fecal worm egg counts for 56 days after worm challenge, in mean numbers of H contortus worms, and female fecundity ratios at necropsy were detected in vaccinated sheep, compared with those in control sheep. These data suggest that the fibrinogen-degrading proteins have a protective role in vaccination of sheep against H contortus.

Abomasum↗
PubMed1991

Protective studies in sheep immunized with cuticular collagen proteins and peptides of Haemonchus contortus.

Twenty-eight nonsibling sheep aged approximately 12 months and raised in a helminth-free environment were used in two protection studies. Immunizations were conducted by two intramuscular injections 30 days apart with a synthetic 18AA cuticle collagen peptide and native cuticle collagens derived from the third- and fourth-stage larvae of Haemonchus contortus. Ten days following the last immunization, the sheep were each given 500 infective H. contortus larvae per day for five consecutive days by intraruminal injection. Both collagen materials induced antibodies reactive with cuticle collagens; however, neither induced reproducible protection to H. contortus infections in vaccinated/infected sheep. In the most extensive test, there were no statistical differences in mean faecal worm egg count for 56 days post worm challenge, in mean numbers of H. contortus and female fecundity ratios at necropsy of immunized and unimmunized sheep. Failure to reproducibly immunize sheep with cuticle collagens may be due to the inability of antibodies or host immune cells to reach the collagen epitopes in the nematode cuticle without prior surface coat removal as postulated in human nematode studies.

Adjuvants, Immunologic↗
PubMed1990

Haemonchus contortus: evidence that the 3A3 collagen gene is a member of an evolutionarily conserved family of nematode cuticle collagens.

Rabbit antisera were raised against an 18 amino acid-long peptide that corresponds to the predicted sequence of the carboxy-terminal, nontriple helical region of the Haemonchus contortus 3A3 collagen gene. This sequence is highly conserved and diagnostic for members of the col-l collagen family, which includes the 3A3 gene. We find that these antisera react predominantly with multiple, high molecular weight (greater than 68 kDa) proteins on Western blots of whole worm extracts. The number and molecular weights of the reacting proteins vary depending upon the developmental stage of the worms analyzed. All of the reacting proteins are collagenase sensitive. The reacting collagens copurify with cuticles and are released from cuticles by reducing agents. In indirect immunofluorescence assays the antisera react only with the broken edges of isolated cuticles, suggesting that the antisera are reacting with an internal cuticle layer. This layer appears to be circular and to extend throughout the length of the worm. The antisera react on Western blots with multiple, high molecular weight collagens of eight other nematodes examined, representing two classes and several orders. These data provide additional support for the notion that the 3A3 collagen gene, and other members of the col-l collagen family, encode cuticle collagens. Collagens with this peptide sequence, presumably other members of the col-l collagen family, appear to be widely distributed in the phylum Nematoda.

Amino Acid Sequence↗
PubMed1990

Haemonchus contortus: a simple procedure for purifying surface proteins from third- and fourth-stage larvae.

Surface proteins were solubilized from exsheathed third (XL3)- and fourth (L4)-stage larvae of Haemonchus contortus by a one-step extraction procedure involving brief heat treatment of the worms in the presence of buffer and 100 mM sodium chloride. Surface proteins also could be preferentially extracted from XL3s, but not from L4s, by heating the worms briefly in 1% sodium dodecyl sulfate. The major proteins extracted by these procedures were similar in molecular weight to those detected by surface-labeling live worms with 125Iodine. Both extraction procedures solubilized a single, major protein with an apparent molecular weight of 68-97 kDa from XL3s. In contrast, extraction of L4s with 100 mM sodium chloride yielded four major proteins with relative molecular weights of 27, 29, 78, and 200 kDa. Antibodies raised in rabbits to surface proteins prepared by the sodium chloride procedure reacted with the surfaces of live worms in indirect immunofluorescence assays. The anti-XL3 surface protein serum was stage specific in immunofluorescence experiments using live worms and in immunoprecipitation experiments using 125Iodine-labeled XL3 and L4 surface proteins. The overall amino acid composition of the surface proteins is hydrophilic. Twenty-six percent of the amino acid residues of the XL3 surface proteins, which consist predominantly of the 68-97 kDa species, are glutamate or glutamine.

Amino Acids↗
PubMed1990

Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms.

We have cloned cDNAs encoding a 35-kilodalton cysteine protease that is a major component of protective extracts isolated from blood-feeding Haemonchus contortus adult worms. Near full-length cDNAs for the protease were isolated by immunoscreening an adult worm cDNA expression library with a rabbit antiserum prepared against the protein eluted from preparative SDS gels and by rescreening the library with oligonucleotide probes. The protein predicted from the nucleotide sequence of the cDNAs and of a genomic DNA clone comprises 342 amino acids and contains an N-terminal signal sequence, 16 cysteine residues and four potential N-linked glycosylation sites. The enzyme appears to be glycosylated in vivo. The H. contortus protease, called AC-1, displays an overall 42% sequence identity with the human lysosomal thiol protease cathepsin B. The similarities between cathepsin B and AC-1 are localized primarily to regions of cathepsin B that comprise the mature, active form of the enzyme. A stretch of six amino acids that includes the active site cysteine of cathepsin B is conserved, and is present in the same relative location in AC-1, suggesting that this region comprises the active site of the H. contortus enzyme.

Amino Acid Sequence↗
PubMed1990

A developmentally regulated cysteine protease gene family in Haemonchus contortus.

The nucleotide sequence of a gene encoding a 35-kDa thiol protease of the parasitic nematode Haemonchus contortus has been determined. The gene, designated AC-2, shares 97% nucleotide sequence identity and 98% amino acid identity with previously characterized AC-1 cDNAs encoding the thiol protease. The AC-2 gene spans 8 kb and appears to contain 11 introns, ranging in size from 57 bp to over 5.2 kb. One of the introns interrupts the proposed active site region that is conserved between the H. contortus protease and the related thiol proteases cathepsin B and papain. Southern blot hybridization experiments indicate that the protease is encoded by a small gene family in H. contortus. Rabbit antisera prepared against the recombinant protein react on Western blots with 35 and 37-kDa proteins of adult worms. These proteins were not detectable by Western blot analysis in three larval parasitic developmental stages of H. contortus. Northern blot hybridizations indicate that mRNA transcripts for the gene family are present at low levels in a mixed population of third- and fourth-stage larvae but highly abundant in adult worms. Expression of the protease correlates with blood-feeding and suggests a role for the protease in blood digestion.

Amino Acid Sequence↗
PubMed1989

Cuticle collagen genes of Haemonchus contortus and Caenorhabditis elegans are highly conserved.

Several genes and partial cDNAs encoding cuticle collagens have been isolated from the sheep parasitic nematode Haemonchus contortus. DNA sequencing and Southern blot hybridization studies reveal that H. contortus collagens comprise a large family of related, but non-identical genes. The genes appear to be dispersed throughout the genome. The predominant size of collagen mRNA in molting worms was found to be between 1.0 and 1.2 kb. The one complete gene that was sequenced contains two short introns and encodes a protein of about 300 amino acids. The predicted protein sequence contain several (Gly-X-Y)n triple helix-coding domains that are interrupted by short stretches of non-helix-coding amino acids. The size of the predicted protein and the organization of the triple-helix coding domains are similar to that of Caenorhabditis elegans collagens. All the H. contortus genes studied show a striking homology to the C. elegans collagen gene subfamily represented by col-1. In particular, the amino acid sequence of the carboxy-terminal non-(Gly-X-Y)n region and the positions of cysteine residues flanking the (Gly-X-Y)n domains were found to be highly conserved in the collagens of these two nematodes.

Amino Acid Sequence↗
PubMed1989

Identification and preliminary characterization of cuticular surface proteins of Haemonchus contortus.

Cuticular surface antigens of the XL3 and L4 stages of Haemonchus contortus have been studied by surface labeling and immunological techniques. Live worms were labeled with 125I and extracted with sodium dodecyl sulfate (SDS) followed by SDS + 2-mercaptoethanol. The SDS-soluble surface proteins of XL3s and L4s were found to consist of relatively few major species. The pattern of labeled polypeptides was distinctive for each developmental stage. These proteins are refractory to digestion by bacterial collagenase. Several of the proteins are glycosylated. Further extraction of labeled worms with SDS + 2-mercaptoethanol solubilized additional labeled proteins that appeared to be primarily collagens. Rabbit antisera prepared against native XL3 and L4-cuticles reacted strongly with the surfaces of live worms in immunofluorescence assays. In contrast, antisera prepared against SDS-extracted cuticles reacted weakly or not at all with live worms in similar experiments. Rabbit antisera prepared against adult cuticles failed to react with live XL3s or L4s. These studies suggest that the major surface antigens of XL3s and L4s are solubilized by SDS and that there are different antigens present on the cuticular surfaces of XL3s, L4s and adults. Stage-specificity in cuticular surface proteins may contribute to the successful parasitic lifestyle of this nematode.

Animals↗
PubMed1989

Polyfluoro 1,3-diketones as systemic insecticides.

A series of aryl polyfluoro 1,3-diketones were examined for systemic ectoparasiticidal activity in cattle. The compounds demonstrated efficacy against several economically important species of insects and acarina. At dosages of 5 mg/kg X1 or 0.35 mg/kg per day intraruminally, activity was observed against blowfly larvae (Phormia regina), adult stable fly (Stomoxys calcitrans), and lone star tick (Amblyomma americanum). In vivo activity was not directly related to in vitro activity, showing a stronger dependence on perfluoroalkyl-chain length and aryl-group substitution.

Animals↗
PubMed1989

Screening method for insecticidal activity using first instars of black blow fly (Diptera: Calliphoridae).

A bioassay method suitable for rapid mass screening of fermentation and synthetic organic compounds for insecticidal activity is described. The test, which uses first instars of susceptible black blow fly, Phormia regina (Meigen), in a bovine serum medium, detects insecticidal activity with reproducible results. It is capable of selecting the most active compound in structure-activity relationships by minimum effective dose concentration studies. The bioassay system is easy to operate and requires only a minute quantity of chemical compound.

Animals↗
PubMed1988

Anthelmintic activity of continuous low doses of fenbendazole into the rumen of sheep.

Fenbendazole (FBZ) was continuously infused for 30 days into the rumen of 103 lambs which had mature or developing benzimidazole-susceptible or thiabendazole-resistant Haemonchus contortus and susceptible Trichostrongylus colubriformis infections. Ovicidal, larvicidal and adulticidal activities were exhibited against benzimidazole-susceptible and benzimidazole-resistant H. contortus worms by FBZ at a dose level of greater than or equal to 0.2 mg kg-1 body weight day-1. Reasonably consistent high level efficacy against H. contortus was obtained with dose levels greater than 0.4 mg kg-1. Excellent control of susceptible T. colubriformis worms was achieved with the lowest dose tested of 0.4 mg kg-1 day-1. The intraruminal infusion critical study method is a tool to determine the feasibility of incorporating a candidate anthelmintic compound in a continuous sustained-release rumen device formulation. The anthelmintic profile of FBZ obtained by low-level intraruminal administration suggests that it would be a potential candidate.

Animals↗
PubMed1987

Protection of lambs with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae.

Helminth-free lambs (approx 4 months old) were inoculated SC with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae. The metabolite was obtained from in vitro cultivation and was identified as XL3FA1. After 3 periodic XL3FA1 inoculations, lambs were challenge exposed with H contortus. Multiple vaccinations with XL3FA1 did not inhibit the development of worm populations in the lambs; however, worm egg production seemed to be inhibited.

Animals↗
PubMed1985

Systemic activity of a benzimidazoline compound in cattle against ticks and biting flies.

A benzimidazoline compound [4-nitro-2-(1,1,2,2-tetrafluoroethyl)-6 -(trifluoromethyl)-1H-benzimidazol-2-, 01, sodium salt] referred to as EL-979 showed systemic acaricidal and insecticidal activity in cattle against 2 tick species, Amblyomma maculatum (Gulf Coast tick) and Dermacentor variabilis (American Dog tick) and adult Stomoxys calcitrans (stable flies). Larvae of black blow fly (Phormia regina) were fed serum collected from treated calves. A complete kill of larvae was obtained with a serum level of 3 parts per million (p.p.m.) of EL-979. Intravenous infusion at the rate of 0.25 mg kg-1 per day for 12 days provided a concentration of EL-979 in the blood of approximately 5 p.p.m. In A. maculatum this blood level prevented feeding in larvae and molting in nymphs, reduced numbers of females successfully feeding and prevented larvae production. D. variabilis adults were not as susceptible to a blood level of 5 p.p.m., but the number of feeding larvae and molting nymphs were reduced. Complete mortality of adult S. calcitrans occurred with repeated feedings of a 5 p.p.m. blood level. Fly control of 80% was found with 2.3 p.p.m. multiple feeds. Calf sera with a concentration range of 16--20 p.p.m. killed all flies which took a single feeding.

Animals↗
PubMed1983

In vitro activity of various anthelmintic compounds against Haemonchus contortus larvae.

Twenty-five known anthelmintic compounds were evaluated in vitro against the highly motile exsheathed non-feeding third-stage of Haemonchus contortus larvae. Activity was based on lack of motility or death of larvae after 24 h of chemical exposure. Six compounds (avermectins, closantel, levamisole, morantel, phenylhydrazone and ticarbodine) were active at a concentration of 100 micrograms cm-3 or less. The most active compounds were avermectins and levamisole. When higher in vitro concentrations were used, ten compounds (bephenium, coumaphos, dichlorovos, disophenol, hygromycin b, methyridine, parbendazole, phenothiazine, pyrantel and thiabendazole) exhibited activity. Nine compounds were found to be inactive; among these were the new benzimidazoles, i.e., albendazole, fenbendazole, mebendazole and oxibendazole. Because of the inactivity of the new benzimidazoles, this in vitro system is unsuitable as a routine screening tool. Also, the system appears to favor drugs that act quickly through percuticular entry. In an initial group of 5280 untested compounds, 254 (4.8%) exhibited in vitro activity at 100 micrograms cm-3 against the non-feeding larvae stage. The exogenous and in vitro cultivation techniques required for collecting, cleaning and exsheathing the larvae are described.

Animals↗