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Biomedical subjects

R W Fulton

Publications and source records attributed to R W Fulton.

At least 37 records · Page 2Linked to original sources

Co-migrating and shared antigens of selected Pasteurella haemolytica untypable strains.

Bacterial cell envelope preparations from eight untypable strains of Pasteurella haemolytica were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting with rabbit antisera prepared against the eight untypable strains (one untypable strain per rabbit) and with cattle antisera prepared against P. haemolytica serotypes 1, 2, 5, 6, 9 and against one heterologous untypable strain. Numerous comigrating and shared antigens were recognized by the eight rabbit antisera and the P. haemolytica serotype cattle antisera. Comigrating antigens at 43 and 30 kilodaltons (kDa) were recognized by all eight rabbit antisera. Shared antigens, detected by all eight rabbit antisera when reacting against P. haemolytica serotype 1, were recognized at 43, 32, 30, 20 and 15 kDa.

Animals↗

Porcine conceptus proteins: antiviral activity and effect on 2',5'-oligoadenylate synthetase.

Interferon-like proteins synthesized by conceptuses of domestic ruminants inhibit luteolysis during early pregnancy. Although pig conceptuses secrete trophoblast interferons during the period of CL maintenance, estrogen is involved with maintenance of the CL. The principal purposes of this work were to confirm production of trophoblast interferons by porcine conceptuses and to compare the effect of trophoblast interferons on endometrium of pigs and cattle. When measured using Madin-Darby bovine kidney (MDBK) cells challenged with vesicular stomatitis virus, antiviral activity in uterine flushings from cyclic gilts was not detectable throughout the estrous cycle; however, in pregnant gilts, antiviral activity increased from undetectable amounts to 4-11 x 10(3) U on Days 14, 16, and 18. Porcine embryos in culture produced 1,100 U/embryo/ml/24 h. Porcine conceptus secretory proteins induced 2',5'-oligo(A) synthetase in MDBK cells and in endometrial explants of cows but had no measurable effect on 2',5'-oligo(A) synthetase activity of endometrial explants of pigs. Similarly, endometrial 2',5'-oligo(A) synthetase of pregnant pigs was unaffected in vivo during the period of maximal synthesis of conceptus secretory proteins. Porcine conceptus secretory proteins produced no detectable increase in serum antiviral activity or 2',5'-oligo(A) synthetase activity of blood mononuclear leukocytes in utero-ovarian venous blood. These results suggest that conceptus interferons of pigs play different roles in the establishment of pregnancy compared to their roles in ruminants.

2',5'-Oligoadenylate Synthetase↗

Cause-specific mortality of white-tailed deer as influenced by military training activities in southwestern Oklahoma.

Radio-telemetry was used to monitor movements and mortality of 56 white-tailed deer (Odocoileus virginianus) in response to intensive military training activities on West Range (18,000 ha), Fort Sill Military Reservation, Oklahoma. Cause-specific mortality was determined for 22 radio-collared deer, including adults (greater than or equal to 2.0-yr-old), yearlings (0.6-1.9-yr-old), and fawns (less than or equal to 75-day-old age group) from 1987 to 1989. Winter home ranges were largely confined to a 14,411 ha impact area centrally located on West Range. The mean annual mortality rate was 0.50 for adults and yearlings combined. Fifty percent of all adult and yearling mortality was attributed to military training activities, 28% to hunting, 16% to collisions with automobiles, and 6% to unknown causes. The mean monthly mortality rate was 0.61 for neonatal fawns and predation accounted for three of four mortalities. All captured deer in the greater than or equal to 2.6-yr-old, 82% in the 1.6-yr-old, 10% in the 0.6-yr-old, and all deer in the less than 7-day-old age groups were seropositive for bluetongue virus (BTV). Our study strongly suggests that the consequences of military training activities should be considered in the management of white-tailed deer herds on military installations.

Animals↗

Comparison of antibody responses in cattle to outer membrane proteins from Pasteurella haemolytica serotype 1 and from eight untypeable strains.

Membrane associated proteins from 8 untypeable Pasteurella haemolytica strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with those of P haemolytica serotypes 1 and 2. Cattle antisera obtained from P haemolytica serotype 1 vaccine trials were used in immunoblotting assays to compare the membrane proteins from the 8 untypeable strains with those from P haemolytica serotypes 1 and 2. Densitometry was used to identify bands, and using linear regression analyses, the peak area optical densities (measuring antibody response) were correlated to lesion scores from the vaccinated calves. Significant antibody responses to proteins of 99, 69, 60, 55, 47, 45, 39, 33, 30, 16, and 14.5 kDa were detected for 4 or more of the 8 P haemolytica untypeable strains. Serotypes 1 and 2 of P haemolytica contained a comigrating 30-kDa protein. Antibody responses to proteins of 39, 33, and 32.5 kDa were significant for 3 of the untypeable strains and had significant correlation to lesion scores. Antibody responses to various other proteins were significant for 2 untypeable strains each.

Animals↗

Expression of antiviral activity and induction of 2',5'-oligoadenylate synthetase by conceptus secretory proteins enriched in bovine trophoblast protein-1.

Establishment of pregnancy in cattle has been proposed to depend on production of a conceptus protein, bovine trophoblast protein-1 (bTP-1), which has a high degree of sequence homology with bovine interferon-alpha (bIFN-alpha), especially the alpha II subfamily. A preparation of bovine conceptus secretory proteins enriched for bTP-1 has antiviral and physico-chemical properties similar to other bIFN-alpha. Antiviral activity is initially detectable in uterine flushings on Day 14 of pregnancy, when the conceptus measures 4-5 mm in length, and increases as the conceptus elongates through Day 18. Day 17 conceptuses produce more than 10(6) U antiviral activity during 24 h of culture. All IFNs induce the enzyme 2',5'-oligoadenylate synthetase, which catalyzes production of 2',5'-oligo(A), which in turn is involved in antiviral and growth inhibitory effects of IFNs. This enzyme activity is induced in Madin-Darby bovine kidney cells by the partially purified bTP-1 preparation similarly to IFN-alpha, -beta, and -gamma. Likewise, the partially purified bTP-1 and bIFN-alpha 1 induce 2',5'-oligo(A) synthetase activity in monolayers of endometrial epithelial and stromal cells. Compared to epithelial cells, stromal cells have higher baseline activity of 2'-5'-oligo(A) synthetase activity (p less than 0.01) and show a greater degree of induction in the presence of either the partially purified bTP-1 or bIFN-alpha 1 (p less than 0.01). Also, 2',5'-oligo(A) synthetase of endometrial stromal cells is induced to a greater degree by our enriched bTP-1 preparation than by bIFN-alpha 1 (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

2',5'-Oligoadenylate Synthetase↗

Endometrial surface and secretory alterations associated with embryonic mortality in gilts administered estradiol valerate on days 9 and 10 of gestation.

Administration of estrogen to gilts on Days 9 and 10 of pregnancy results in total embryonic loss by Day 18. The present study examined changes in the uterine endometrial surface and secretion during conceptus attachment in control and estrogen-treated (Days 9 and 10) pregnant gilts. Gilts were unilaterally hysterectomized on either Days 12 and 14 or Days 16 and 18 of gestation. Uterine horns were flushed with saline and conceptuses were evaluated. Intact conceptuses were recovered from all control gilts, whereas estrogen-treated gilts contained normal intact conceptuses only on Day 12 of gestation. Antiviral activity, which reflects conceptus viability, was reduced (p less than 0.01) in uterine flushings after Day 14 in estrogen-treated gilts. Culture of endometrial explants with [3H]glucosamine revealed several glycoproteins that are synthesized during the period of conceptus attachment; however, no difference in glycoprotein synthesis between treatment groups was detected by analysis with two-dimensional PAGE and fluorography. Analyses of the uterine epithelium by scanning and transmission electron microscopy demonstrated that estrogen administration caused an alteration in the uterine surface, a thinning of the uterine epithelial glycocalyx, and a reduction of cationic ferritin binding to the microvilli of the uterine epithelium. Results indicate that conceptus mortality after early administration of estrogen is associated with alterations in the uterine endometrial surface during the period of conceptus attachment in the pig.

Animals↗

In vitro and in vivo 2',5'-oligoadenylate synthetase activity induced by recombinant DNA-derived bovine interferon alpha I1 in bovine alveolar macrophages and blood mononuclear cells.

Biological responses to recombinant DNA-derived bovine interferon alpha (rBoIFN-alpha I1) by bovine alveolar macrophages were examined by measuring viral yield reduction and 2',5'-oligoadenylate synthetase (2',5'-OAS) production by IFN-treated cells. In vitro IFN pretreatment of alveolar macrophages reduced viral yield in cultures challenged exposed with parainfluenza-3 virus, compared with control cultures. In vitro treatment of alveolar macrophages with IFN also resulted in increased 2',5'-OAS activity. The 2',5'-OAS activity was measured in alveolar macrophages and blood mononuclear leukocytes of calves injected IM with 3.6 x 10(6) U of rBoIFN-alpha I1/kg of body weight. The IFN action was monitored by measuring 2',5'-OAS activity of blood mononuclear leukocytes beginning 6 days before and ending 24 hours after IFN treatment. The 2',5'-OAS activity in the blood mononuclear leukocytes sharply increased 24 hours after IFN treatment, indicating response to IFN. The alveolar macrophages collected from the same calves 24 hours after IFN administration also had increased 2',5'-OAS activity, compared with alveolar macrophages from the same calves collected 6 days before treatment. Increased 2',5'-OAS activity indicates: a possible mechanism of IFN action in cattle that may be responsible for viral yield reduction; potential use of high enzyme activity as a marker for IFN induction; and potential use of 2',5'-OAS activity as a marker for determining effects of IFN on bovine macrophages and other cells of the bovine immune system.

2',5'-Oligoadenylate Synthetase↗

Interferon and 2',5'-oligo(A) synthetase activities in serum and blood mononuclear leukocytes of cattle after injection of bovine interferon-alpha 1.

Cell extracts that were prepared from blood mononuclear leukocytes from 66 samples obtained from 6 clinically normal calves contained mean 2',5'-oligoadenylate (2',5'-oligo[A]) synthetase activity sufficient to synthesize 186 +/- 82 pmol of 2',5'-oligo(A)/h/10(6) cells. Calves had no measurable serum interferon (IFN) activity. Five calves were given IM injections of 10(4), 10(5), 5 x 10(5), 10(6), and 10(7) U of bovine IFN-alpha 1/kg of body weight at 2-week intervals. Five dosing sequences were used with a 5 x 5 Latin square design so that each calf received each dose once. Activity of 2',5'-oligo(A) synthetase increased at 24 hours in response to all dosages of IFN and then declined following first-order kinetics, with an apparent half-life (t1/2) of 2.1 +/- 0.5 days. The area under the concentration-time curve for 2',5'-oligo(A) synthetase increased with dose of IFN more rapidly than did peak response. Serum IFN that was measured at 1-day intervals following administration of IFN was consistently measurable only at dosages above 10(6) U of IFN/kg. The t1/2 for circulating IFN was 12.4 +/- 1.0 hours. Over all dosages, increases in 2',5'-oligo(A) synthetase activity were measurable for 3.5 days longer than were increases in IFN following IM injection of IFN. None of the calves developed detectable anti-IFN antibodies.

2',5'-Oligoadenylate Synthetase↗

Bovine leukosis virus transmission with mouthparts from Tabanus abactor after interrupted feeding.

A successful attempt was made to mechanically transmit bovine leukosis virus (BLV) from a BLV-infected cow with a normal lymphocyte count to sheep by inoculation with horse fly (Tabanus abactor) mouthparts. After interrupted natural feeding, horse flies were anesthetized with CO2. Mouthparts were severed and pooled into a tissue grinder containing medium. Five inocula containing the mouthparts of 10 flies each, and 5 inocula containing the mouthparts of 20 flies each, were prepared and inoculated SC in the right axilla of 10 BLV antibody-negative sheep. Five additional sheep served as controls. Serum samples were collected at 2-week intervals and tested by agar gel immunodiffusion for BLV antibodies. One sheep injected with 20 mouthparts developed antibodies to BLV at 10 weeks after inoculation. Six months after inoculation with fly mouthparts, 1 BLV antibody-negative sheep was randomly selected from each treatment group and injected, in the left axilla, with 3 ml of blood from the donor cow to confirm susceptibility of the sheep. All 3 sheep developed antibodies to BLV within 4 weeks.

Animals↗

Comparison of the Pasteurella haemolytica A1 envelope proteins obtained by two cell disruption methods.

The French pressure cell and sonication methods of bacterial cell disruption were compared for the evaluation of surface proteins from Pasteurella haemolytica A1. Several protein bands were quantitatively different when compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and densitometry. With densitometry, sonicated preparations had higher concentrations of the 102-, 83-, 50-, 38-, and 30-kilodalton (kDa) proteins; French pressure cell preparations had higher concentrations of the 96-, 65-, and 42-kDa proteins. Qualitative differences between these two disruption methods were evident at the 102-, 96-, 91-, 50-, 38-, and 30-kDa protein bands. However, significant differences (P less than 0.05) were detected between the two methods at only the 102-, 96-, 91-, and 50-kDa bands.

Bacterial Proteins↗

Neutralizing antibody responses to bluetongue and epizootic hemorrhagic disease virus serotypes in beef cattle.

Blood samples were obtained from sentinel beef cattle at monthly intervals, and the sera were tested for antibodies, using a bluetongue virus (BTV) immunodiffusion test (IDT) and virus-neutralization test (VNT), for 5 BTV serotypes (2, 10, 11, 13, and 17) and 2 epizootic hemorrhagic disease virus (EHDV) serotypes (1 and 2). The cattle tested were transported from Tennessee to Texas in 1984 and 1985. All cattle were seronegative by the BTV IDT at the initial bleeding in Texas in 1984 and 1985. In 1984, 16 of 40 (40%) cattle seroconverted as assessed by results of the BTV IDT. In the 16 seropositive cattle in 1984, neutralizing antibodies were detected to BTV serotypes 10 (n = 7), 11 (n = 3), and 17 (n = 11), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1984, no cattle seroconverted to BTV-2 or BTV-13. In 1985, 10 of 36 (27.8%) cattle seroconverted as assessed by results of the IDT. Of the 10 seropositive cattle in 1985, neutralizing antibodies were detected to BTV serotypes 10 (n = 10), 11 (n = 10), 13 (n = 7), and 17 (n = 5), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1985, no cattle seroconverted to BTV-2. Clinical diseases attributable to BTV or EHDV was not detected in these cattle in 1984 or 1985.

Animals↗

Effect of interferons on the replication of alcelaphine herpesvirus-1 of malignant catarrhal fever.

Four isolates of alcelaphine herpesvirus-1 of malignant catarrhal fever (MCF) were tested for their inducibility of and sensitivity to various interferons. Viral isolates from an Indian gaur (Bos gaurus), a greater kudu (Tragelaphus strepsiceros) and two wildebeest (Connochaetes gnou) calves did not induce measurable interferon (IFN) in bovine fetal kidney cells. However, these low passages of each virus were all highly cell-associated and viral replication was inhibited at these passages by IFN at 14 IFN units/0.05 ml recovered from NDV-infected MDBK cells and at 7.6 IFN units/0.05 ml of IFN from NDV-infected bovine macrophages. The herpesvirus from the Indian gaur and greater kudu and high passages (greater than 50) of the cell-free WC-11 strain of alcelaphine herpesvirus-1 also were inhibited in their replication by recombinant IFN of bovine and human origins as determined by a fluorescent focus unit (FFU) reduction assay. The concentrations of IFN required to produce a 50% reduction in herpesvirus-produced FFU ranged between 6.4 and 480 IFN units. These findings promote the use of IFN as part of the regimens of treatment of captive endangered ruminant species with clinical MCF.

Animals↗

Blocking dot-ELISA, using a monoclonal antibody for detection of antibodies to bluetongue virus in bovine and ovine sera.

A modified solid phase blocking enzyme immunosorbent assay (ELISA), using a monoclonal antibody (McAb) against the group specific bluetongue virus (BTV) antigen is described for detection of anti-BTV antibodies in cattle and sheep sera. Dots of an optimal dilution of BTV antigens were adsorbed to nitrocellulose (NC) paper (hence dot-ELISA) and then the remaining adsorptive sites were saturated with gelatin. After exposure to bovine or ovine test serum the NC strips were reacted with the McAb. The presence of McAb was detected with a peroxidase-conjugated anti-mouse IgG (H and L). In the absence of anti-BTV antibody in test sera, BTV antigen sites were reactive with McAb as indicated by a brown colored dot after enzyme degradation of hydrogen peroxide in the presence of diamino benzidine (DAB) or amino ethylcarbazole (AEC). In the presence of sufficient anti-BTV antibody no color reaction was observed. The blocking (B) dot-ELISA was superior to the agar gel immunodiffusion (AGID) in detecting anti-BTV antibodies in bovine and ovine sera early after experimental infection with BTV type 10. In 5 of 7 animals inoculated by combined intravenous and subcutaneous routes, anti-BTV antibodies in sera were detectable as early as 7 days post infection (DPI), all of which were AGID negative. Comparable B-dot-ELISA and AGID results were found in 23 paired sera (pre and 20 DPI) from cattle experimentally infected with different types of BTV and in 100 AGID negative sera from Ontario dairy and Alberta beef cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Induction and measurement of 2',5'-oligoadenylate synthetase in Madin-Darby bovine kidney cells and in cattle.

2',5'-Oligoadenylate [2',5'-oligo(A)] was separated from 14C-labeled nucleosides produced in the 2',5'-oligo(A) synthetase assay by using 100-microliters columns of Dowex 1. No detectable nucleoside remained on the column after elution with 20 column volumes of water, whereas less than 1% of oligonucleotides were eluted from the column. At least 99% of oligonucleotides were eluted from the column with 1 M NaCl, pH 2. The major product had properties consistent with 2',5'-oligo(A). Exposure to alpha-1 bovine interferon (IFN) caused an increase in cellular 2',5'-oligo(A) synthetase activity which was proportional to the concentration of IFN in the medium up to 10(4) U of IFN per ml and then leveled off at about 15 X control activity. Under the assay conditions used, 2',5'-oligo(A) synthetase activity was directly proportional to the amount of cell extract over a 10-fold range. Cattle inoculated with IBR/BVD/PI-3 modified live virus vaccine showed an increase in 2',5'-oligo(A) synthetase activity in peripheral blood mononuclear leukocytes which persisted for at least 3 days postvaccination. Intramuscular injection of cattle with IFN caused a similar increase in 2',5'-oligo(A) synthetase activity. Changes in 2',5'-oligo(A) synthetase activity should be of value in (i) assessing the response of cattle to experimental viral infections or inoculations with viral vaccines or IFN or (ii) indicating a possible viral etiology in disease.

2',5'-Oligoadenylate Synthetase↗

Immunologic response to Pasteurella haemolytica and resistance against experimental bovine pneumonic pasteurellosis, induced by bacterins in oil adjuvants.

Immunogenicity of and protection afforded by Pasteurella haemolytica bacterins were studied in calves. Bacterins contained an aluminum hydroxide in gel (ALH) adjuvant or one of the following oil-in-water adjuvants: Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and trehalose dimycolate (TDM). On days 0 and 7, calves were vaccinated with phosphate-buffered saline solution (PBSS), a bacterin, or live P haemolytica. Transthoracic intrapulmonic challenge exposure was done on day 21. In 3 experiments, there were no significant (P greater than 0.05) differences between lung lesions induced in PBSS-or ALH bacterin-vaccinated calves. Both FCA and FIA bacterins significantly (P less than 0.05) enhanced resistance against challenge exposure. Resistance induced by FCA and FIA bacterins was comparable with that induced by vaccination with live P haemolytica. Calves vaccinated with FIA bacterin and challenge-exposed to P haemolytica at a concentration of 4.5 X 10(9) colony-forming units (4.5 times greater than used in the first 3 experiments) resisted challenge exposure similar to calves given live organisms. The TDM bacterin failed to enhance resistance. All bacterins caused a significant increase (P less than 0.05) in serum antibody to P haemolytica somatic antigens, as measured by a quantitative fluorometric immunoassay. Pasteurella haemolytica leukotoxin neutralizing antibody titers did not increase significantly (P greater than 0.05) in sera after vaccination with any bacterin. Vaccination with FCA and FIA bacterins resulted in a significant increase (P less than 0.001) in serum antibody to a carbohydrate-protein subunit of P haemolytica, as measured by an enzyme-linked immunosorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗

A scanning and transmission electron microscopic study of rotavirus-induced intestinal lesions in neonatal gnotobiotic dogs.

Neonatal gnotobiotic dogs orally inoculated with canine rotavirus had ultrastructural changes limited to the jejunal and ileal regions of the small intestine. Early scanning electron microscopic findings consisted of swollen villus epithelial cells, denuded foci on intestinal villi, and slight to moderate villus atrophy. Later changes were slight villus atrophy with no denuded intestinal villi. Transmission electron microscopic changes in villus epithelial cells from 12 to 48 hours post-inoculation included: rotavirus particles associated with intracytoplasmic vacuoles near the terminal web and apical tubules; viral particles in dilated cisternae of rough endoplasmic reticulum; and moderate numbers of necrotic cells having no microvilli, swollen mitochondria, membrane-bound lipid-like material in the cytoplasm, clumped chromatin around the periphery of the nucleus, and disruption of the cytoplasmic membrane. In jejunum and ileum at 72 to 154 hours post-inoculation, there were fewer necrotic villus epithelial cells and fewer virus particles. In addition, the ultrastructural morphology of the majority of the villus epithelial cells was similar to crypt epithelium. These studies showed that rotavirus infected the villus epithelial cells with subsequent propagation of the rotavirus and destruction of villus epithelial cells.

Animals↗

Effect of recombinant DNA-derived bovine and human interferons on replication of bovine herpesvirus-1, parainfluenza-3, and respiratory syncytial viruses.

Antiviral effects of recombinant DNA-derived bovine (Bo) and human (Hu) interferons (IFN) on the replication of bovine herpesvirus-1, parainfluenza-3, and respiratory syncytial viruses were studied. Bovine monolayer cultures were treated with recombinant DNA-produced Bo IFN-alpha 1, Bo IFN-beta 2, Hu IFN-alpha A, or Hu IFN-alpha A/D and then challenge exposed with bovine herpesvirus-1, bovine parainfluenza-3 virus, bovine respiratory syncytial virus, or vesicular stomatitis virus. Treatment with each IFN reduced the viral yield for each of these viruses, compared with that of control cultures.

Animals↗

Immunologic response and resistance to experimentally induced pneumonic pasteurellosis in cattle vaccinated with various dosages of lyophilized Pasteurella haemolytica.

Pasteurella haemolytica was lyophilized in an enriched soybean polypeptone broth. Lyophilization in this medium resulted in a mean 10-fold loss in P haemolytica viability, as opposed to up to a 10(4)-fold loss in viability when other media were used. Lyophilized P haemolytica was reconstituted and used as a live vaccine in 3 experiments. Calves were challenge exposed by transthoracic injection with virulent P haemolytica. In experiment 1, 2 subcutaneous injections (7-day interval between injections) with 5 ml of recently harvested (1 X 10(9) colony-forming units [CFU]/ml) or lyophilized (1 X 10(8) CFU/ml) P haemolytica significantly (P less than 0.001) enhanced resistance against challenge exposure, compared with resistance in calves given saline solution or sterile medium (control calves) or calves vaccinated with lyophilized organisms at a concentration of 1 X 10(6) CFU/ml. In experiment two, 1, 2, or 5 ml of lyophilized P haemolytica (1 X 10(8) CFU/ml) significantly (P less than 0.05) enhanced resistance, compared with resistance in calves given saline solution (control calves). In experiment three, 1 or 2 injections of lyophilized P haemolytica significantly (P less than 0.01) enhanced resistance against challenge exposure, compared with that of calves given saline solution. The mean lesion score for calves given 1 injection was not significantly higher than the mean lesion score for the group given 2 injections. Vaccination with lyophilized P haemolytica vaccine caused significant (P less than 0.05) increases in serum antibody to P haemolytica somatic antigens, to a carbohydrate-protein subunit of the organism, and to leukotoxin.

Animals↗