Protective effects of clostridium sordellii LT and HT toxoids against challenge with spores in guinea pigs

The protective effects of Clostridium sordellii lethal toxin (LT) and hemorrhagic toxin (HT) toxoids against challenge with spores in guinea pigs were investigated. Purified LT and partially purified HT were obtained from the culture supernatant of C. sordellii strain 3703, and then were treated with formalin to make toxoids. LT. HT and combined LT and HT (LT/HT) toxoid vaccines were prepared by mixing each toxoid with an aluminum phosphate gel as adjuvant. Guinea pigs immunized twice with the respective toxoid vaccines were challenged with spores of strains 3703 or KZ1047. The latter strain does not produce HT. LT toxoid vaccine conferred protection against challenge with strain KZ1047, but not strain 3703, in guinea pigs. All guinea pigs immunized with HT toxoid vaccine died after challenge with spores of either strain. LT/HT toxoid vaccine gave complete protection against challenge with spores of strains 3703 and KZ1047 to guinea pigs. These results suggest that not only LT toxoid, but also HT toxoid, are essential protective antigens of C. sordellii.     

Characteristics of the Shiga-like toxin produced by Escherichia coli associated with porcine edema disease

Shiga-like toxin (SLT-IIv) from Escherichia coli strains associated with edema disease of pigs was characterized and compared with SLT-I, SLT-II, and the SLT of E. coli strain HI8 (SLT-HI8). SLT-IIv from an E. coli K12 in which the genes for SLT-IIv had been cloned was indistinguishable from SLT-IIv of wild strains of E. coli from edema disease. There was cross-neutralization among all SLTs except SLT-I. The different SLTs could be distinguished by heat lability, with the descending order of heat lability being SLT-IIv, SLT-II, SLT-I, and SLT-HI8. SLT-IIv and SLT-HI8 had lower cytotoxic titers on HeLa cells compared with Vero cells and were more active on MDBK cells than were the other SLTs. All SLTs were enterotoxic in rabbit but not in pig intestine and SLT-IIv was less enterotoxic than SLT-I. SLT-IIv had a lower LD50 in mice than did the other SLTs.     

Protective effect of Clostridium septicum alpha-toxoid vaccine against challenge with spores in guinea pigs

The protective effect of an alpha-toxoid vaccine of Clostridium septicum purified alpha-toxin was investigated in guinea pigs. Purified alpha-toxin was treated with formalin to make toxoid, and alpha-toxoid vaccine was prepared by mixing alpha-toxoid (4 to 64 microg/dose) with an aluminum phosphate gel as adjuvant. Guinea pigs were immunized twice with different doses of alpha-toxoid vaccine, and challenged with spores of C. septicum. The guinea pigs surviving after challenge had been immunized with 8 microg/dose or more of alpha-toxoid. All these animals produced titers of 20 units or higher of antitoxin at the challenge. The results suggest that C. septicum alpha-toxin plays an important role in protection against challenge with spores in guinea pigs.     

Reproduction of edema disease of swine with purified Shiga-like toxin-II variant.

Twenty-one weaned Yorkshire-Landrace pigs were injected intravenously with graded doses of purified Shiga-like toxin-II variant (edema disease toxin). In a preliminary study, three pigs (Nos. 1, 2, 3) were injected with 48, 24, and 12 ng, respectively, of SLT-IIv/kg of body weight. Subsequently, three groups (Nos. 4, 5, 6) of six pigs each were injected with 6, 3, and 1.5 ng, respectively, of SLT-IIv/kg of body weight. Severe clinical signs and histologic lesions characteristic of edema disease developed in pigs Nos. 1, 2, and 3, and all six pigs in group No. 4. Eight of these pigs were euthanatized in extremis (mean time to death was 34 hours) and one died of the disease (52 hours). Moderate signs and lesions of edema disease were observed in all pigs in group No. 5, and three pigs were euthanatized (mean time to euthanasia was 42 hours). Mild signs and lesions were observed in three pigs in group No. 6. The most common gross pathologic changes were edema of the eyelids, submucosa of the stomach, and mesentery of the spiral colon and hemorrhage of the colon and cerebellum. Microscopic lesions were associated with vascular injury and included vessel necrosis, perivascular edema and hemorrhage, and superficial colonic and cecal erosions. The vascular lesions were observed in the cerebellar folia, submucosa and mucosa of the stomach, cecum, colon, and sporadically in the retina. None of the clinical signs associated with endotoxin were observed.     

Protective immunity against Newcastle disease: the role of antibodies specific to Newcastle disease virus polypeptides

Studies were performed to determine if passive immunization with hyperimmune sera generated to specific Newcastle disease virus (NDV) proteins conferred protection against virus challenge. Six groups of 3-wk-old chickens were passively immunized with antiserum against either hemagglutinin-neuraminidase/fusion, (HN/F) protein, nucleoprotein/phosphoprotein (NP/P), Matrix (M) protein, a mixture of all NDV proteins (ALL), intact ultraviolet-inactivated NDV (UVNDV), or negative sera. Blood samples were collected 2 days postimmunization, and the birds were challenged with Texas GB strain of NDV. Antibody titers were detected from those recipient birds that had received the antisera against the HN/F, ALL, or UVNDV by a hemagglutination inhibition test, an enzyme-linked immunosorbent assay (ELISA), and a virus neutralization test. Antibodies were detected only by the ELISA from the birds that had received antisera against NP/P and M protein. Antibody titers in the recipient birds dropped by two dilutions (log2) after 2 days postinjection. Birds passively immunized with antisera against HN/F, ALL, and UVNDV were protected from challenge, whereas chickens passively immunized with antisera against NP/P and M protein and specific-pathogen-free sera developed clinical signs of Newcastle disease. The challenge virus was recovered from the tracheas of all passively immunized groups. The presence of neutralizing antibodies to NDV provided protection from clinical disease but was unable to prevent virus shedding from the trachea.     

Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response

Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1 × 10⁵ TCID₅₀ (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were E^rns antibody negative and had seroconverted against E^rns by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.     

表达猪瘟病毒E2蛋白的重组腺病毒对猪的免疫效力评价

为了进一步验证含有猪瘟病毒E2基因的重组腺病毒（rAdV—E2）在猪体上的免疫效力,将rAdV—E2按108TCID50／头接种猪2次,同时用野生型腺病毒wtAdV作为阴性对照,当抗体上升到一定程度后用致死剂量的猪瘟强毒石门株进行攻击。结果表明,rAdV—E2免疫组（n=5）所有免疫猪在加强免疫后均产生了猪瘟特异性中和抗体,并于加强免疫后3w达到峰值,攻毒后所有猪只抗体迅速升高,除了一头猪短期体温升高外,未出现任何其它临床症状;而野生型腺病毒wtAdV免疫组（n=5）猪只在攻毒前一直没有检出特异性抗体,攻毒后全部出现典型的猪瘟临床症状和严重的病毒血症,剖检时可见典型猪瘟病理变化。这表明构建的猪瘟病毒E2基因重组腺病毒rAdV—E2免疫猪后产生了很好的免疫效果,有望成为具有开发价值的活载体疫苗。     

Immunogenicity in Aujeszky's disease virus structural glycoprotein gVI (gp50) in swine.

Aujeszky's disease virus (ADV) envelope glycoprotein gVI (gp50) was purified from virus-infected Vero cells by ion-exchange and immunoaffinity chromatography and its usefulness as a subunit vaccine was evaluated in active and passive immunization studies. Four-week-old piglets were immunized intramuscularly (IM) with purified gVI twice two weeks apart and challenged intranasally (IN) 10 days after the second immunization with 30 LD50 (10(8)PFU) of a virulent strain of ADV. Pigs, vaccinated with 100 micrograms of purified gVI, produced virus neutralizing antibodies and did not develop clinical signs after challenge exposure. The challenge virus was not isolated from nasal swabs and tonsils of gVI-vaccinated pigs, whereas non-vaccinated control pigs developed illness after challenge exposure with the same virulent ADV strain which was later recovered from their nasal swabs and tonsils. Pregnant sows vaccinated twice with purified gVI (IM) at a three week interval produced virus neutralizing antibodies in colostrum. Four-day-old sucking piglets born of vaccinated sows were passively protected by colostral antibodies against intranasal challenge with a lethal dose of virulent ADV. Sera from gVI-vaccinated pigs were distinguished from experimentally infected swine sera by their differential reactivity in enzyme-linked immunosorbent assay (ELISA) using four major viral glycoproteins (excluding gVI) as antigen purified by the use of lentil-lectin.     

Protective effect of botulinum C/D mosaic toxoid against avian botulism

Avian botulism is a paralytic disease caused by a toxin produced by Clostridium botulinum type C. Since type C isolates from cases of avian botulism produced a neurotoxin consisting of a mosaic form of parts of type C and D neurotoxins, we examined the antitoxin titers in the convalescent sera of botulism-affected birds which belonged to family Anatidae. ELISA using the C/D mosaic neurotoxin as an antigen revealed that the antibody was detected in the sera at 2 weeks, but not at 5 weeks after the onset, suggesting that the antibody only appeared for a short period in the convalescent phase. However, we failed to detect the antibody titers with anti-chicken IgG instead of anti-duck IgG. We therefore examine the immunological properties of IgG among different families and species. The results revealed that different species of IgG in the same family exhibited strong cross-reactivity. Ducks immunized once with the toxoid together with a commercial oil-adjuvanted vaccine were found to develop sufficient antibody to protect against a challenge with a lethal toxin dose. The ELISA titers did not correspond to the neutralization titers in the sera of immunized ducks at the early stage during immunization. These findings suggest that the neutralizing titer was more useful than the ELISA titer for evaluating the protection against the toxin, but the ELISA technique may be applicable for detecting the occurrence of botulism.     

不同培养基制备精制破伤风类毒素免疫原性比较

用不同培养基制备的精制破伤风类毒素，加入福氏不完全佐剂免疫豚鼠，测血清中抗体效价。结果表明，加强免疫后，以酪蛋白培养基制备的精制破伤风类毒素，其免疫原性为最强，抗体的效价均值达３２６ＩＵ／ｍｌ，与其他各组相比差异显著，这为制备抗原用的培养基的筛选，提供了依据     

Protection by toxoid-induced antibody of gnotobiotic piglets challenged with the dermonecrotic toxin of Pasteurella multocida

A crude dermonecrotic toxin (DNT) of Pasteurella multocida (P.m.) type D was prepared by repeated sonication and freezing. It was sterilized by filtration. A toxoid was then made and pigs were hyperimmunized with it to get an antiserum. A control serum was obtained by hyperimmunization of pigs with a preparation derived from nontoxigenic P.m. type D in the same manner as the toxoid. Three gnotobiotic piglets were injected with the antiserum. This resulted in neutralization indices (NI) of 25 in their sera, as tested on mice. Three litter-mated controls were given the control serum. Their NI remained 1. All piglets were challenged intramuscularly 4 times, every third day, with 30 mouse LD50 of the DNT. When euthanized 15 days after the last DNT administration no snout lesions were found in passively immunized piglets, whereas control animals showed severe turbinate atrophy and other changes typical for atrophic rhinitis. The next experiment was identical to the previous one except for the challenge, which was given intranasally (4 times 300 mouse LD50). Also in this case circulating antitoxin protected the piglets from damage of the nasal turbinates caused by the DNT.     

Persistence of acquired immunity to Sarcocystis miescheriana infection in growing pigs

Sixteen 8-week-old pigs were each experimentally immunized by subclinical infections with 10(3) Sarcocystis miescheriana (syn. S. suicanis) sporocysts and 8 other pigs served as non-immunized controls. Four groups of pigs (each consisting of 4 immunized plus 2 control pigs) were then challenged by infection with 3 X 10(6) sporocysts at either 40, 80, 120 or 160 days post-immunization (dpi) to determine the persistence of the protective immunity against acute sarcocystosis. Pigs challenged 40 dpi demonstrated a solid immunity to lethal challenge and disease. They survived challenge following a mild fever phase whereas both controls died from acute disease. This immunity however, did not prevent the further establishment of parasitic cysts within the host musculature following challenge. The protective immunity against acute disease persisted to 80 dpi, but was not evident thereafter. At necropsy, the clinical and pathological findings in all pigs which had been subjected to challenge were consistent with anamnestic responses of sensitized hosts to re-infection.     

Adsorption of colostral antibodies against classical swine fever, persistence of maternal antibodies, and effect on response to vaccination in baby pigs.

OBJECTIVE: To determine kinetics of antibody absorption, persistence of antibody concentrations, and influence of titers on vaccination of baby pigs with a vaccine against classical swine fever (CSF). ANIMALS: 15 sows and their litters. PROCEDURE: Farrowings were supervised. Initial time of suckling was recorded. In the first experiment, blood samples were collected at farrowing, 2 and 4 hours after suckling, and hourly until 10 hours after initial suckling. Samples were assayed for CSF antibodies, using a serum neutralizing (SN) test. A second experiment included 33 baby pigs vaccinated as follows: 10 prior to ingestion of colostrum, 18 between 1 and 4 hours after ingestion of colostrum, and 5 at 12 hours after ingestion of colostrum. Fourteen pigs were vaccinated when 7 weeks old, and 15 pigs were not vaccinated. At 10 weeks of age, pigs were challenge-exposed with virulent CSF virus. Blood samples were collected and assayed for CSF antibodies and p125 antigen and p125 antibodies. RESULTS: CSF antibodies were detected in pigs beginning 2 hours after suckling. Colostral antibodies persisted for > 7 weeks (half-life, 79 days). Vaccination of pigs before suckling provided effective protection from severe disease after challenge-exposure. However, vaccination of neonates with antibody titers was not effective, because 19 of 23 (82%) pigs succumbed after challenge-exposure. All pigs vaccinated when 7 weeks old resisted challenge-exposure, whereas all unvaccinated control pigs succumbed. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination before ingestion of colostrum conferred good protection against CSF in baby pigs. Vaccination of 7-week-old pigs that had decreasing concentrations of passively acquired antibodies was efficacious.     

Vaccination with recombinant NetB toxin partially protects broiler chickens from necrotic enteritis

NetB toxin from Clostridium perfringens is a major virulence factor in necrotic enteritis in poultry. In this study the efficacy of NetB as a vaccine antigen to protect chickens from necrotic enteritis was examined. Broiler chickens were immunized subcutaneously with purified recombinant NetB (rNetB), formalin treated bacterin and cell free toxoid with or without rNetB supplementation. Intestinal lesion scores and NetB antibody levels were measured to determine protection after mild oral gavage, moderate in-feed and heavy in-feed challenges with virulent C. perfringens isolates. Birds immunized with rNetB were significantly protected against necrotic enteritis when challenged with a mild oral dose of virulent bacteria, but were not protected when a more robust challenge was used. Bacterin and cell free toxoid without rNetB supplementation did not protect birds from moderate and severe in-feed challenge. Only birds immunized with bacterin and cell free toxoid supplemented with rNetB showed significant protection against moderate and severe in-feed challenge, with the later giving the greatest protection. Higher NetB antibody titres were observed in birds immunized with rNetB compared to those vaccinated with bacterin or toxoid, suggesting that the in vitro levels of NetB produced by virulent C. perfringens isolates are too low to induce the development of a strong immune response. These results suggest that vaccination with NetB alone may not be sufficient to protect birds from necrotic enteritis in the field, but that in combination with other cellular or cell-free antigens it can significantly protect chickens from disease.     

Protection against bovine rotaviruses in newborn calves by continuous feeding of immune colostrum

Three pregnant cows were inoculated intramuscularly with inactivated vaccine to bovine rotavirus (BRV) serotype 1 (BRV-1) and serotype 2 (BRV-2). Serum neutralizing antibody (NA) titers against both serotypes increased significantly after immunization. NA titers of colostrum obtained from immunized cows against BRV-1 and BRV-2 were 29286 and 38109, respectively, which were significantly higher than those from non-immunized control cows. Nine and 6 colostrum deprived calves were orally challenged with BRV-1 and BRV-2, respectively, and monitored for clinical manifestation and viral shedding. Five calves of them, 3 with BRV-1 and 2 with BRV-2, received 2 l of milk replacer supplemented with 10% immune colostrum 2 hr before challenge and twice daily for the first 5 days after challenge. Other 10 calves, 6 with BRV-1 and 4 with BRV-2, were fed only milk replacer as controls. All control calves developed severe diarrhea and shed a large amount of BRV in feces, beginning from 24 to 48 hr after challenge inoculation. On the contrary, all calves but one fed colostrum supplement remained clinically healthy after challenge, and BRV was not detected in their feces during feeding immune colostrum. The possibility that continuous feeding of immune colostrum is capable of preventing newborn calves from diarrhea associated with BRV and viral shedding was suggested.     

A Clostridium botulinum type B vaccine for prevention of shaker foal syndrome

A toxoid was prepared from type B toxin of Clostridium botulinum by treatment with 0.6% formalin for 6 weeks. The toxoid was adsorbed to aluminium hydroxide and this vaccine was evaluated for safety in guinea pigs, mice and horses, and for immunogenicity in guinea pigs and horses. Neutralising antitoxin was demonstrated in adult horses receiving two 2 ml subcutaneous doses 6 weeks apart, and in a foal which suckled its vaccinated dam. Another vaccinated mare and the passively immunised foal were protected against subcutaneous injection of 1600 and 2000 mouse lethal doses of toxin per kg respectively.     

Genetic variation and cross-reactivity of Clostridium septicum alpha-toxin

Clostridium septicum alpha-toxin genes were sequenced with the polymerase chain reaction (PCR) products amplified from DNAs of 25 C. septicum strains, and were classified into 10 patterns. Alpha-toxins were purified from the culture supernatant of four C. septicum strains (strains No. 44, Kagoshima 8, Mie and Tokachi) which were specially chosen from patterns of the deduced amino acid sequences. The molecular weights of the alpha-toxins were not different according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. However, the isoelectric points between the alpha-toxins of No. 44 and Tokachi strains differed markedly. Cross-neutralization tests were performed with purified alpha-toxins and antitoxins in mice and in Vero cells. Each antitoxin showed roughly the same titers against the four alpha-toxins in mice and completely identical titers against these in Vero cells. Calves immunized with toxoid prepared from the culture supernatant of No.44 strain were challenged by exposure to spores of Mie strain. The toxoid conferred protection against the challenge in calves. From these results, although genetic variation has been observed within the C. septicum alpha-toxin gene, C. septicum strains toxoid of strain No.44 induces protective immunity against exposure to C. septicum that produce other subtypes of alpha-toxin containing several different amino acid residues.     

Verification of the protective effect of a toxoid vaccine against edema disease of weaned piglets in an infection model

105 piglets (56 vaccinated and 49 control animals) were utilized in 6 consecutive experiments. Each used litter was divided randomly into vaccine and control animals. One week prior to weaning each of the 56 piglets of the vaccine groups received 5 mg of nonpurified toxin treated with glutaraldehyd subcutaneously whereas to the remaining 49 control animals an extract of apathogenic E. coli was administered. During the first 12 hours post weaning each of the 105 piglets was challenged perorally with 10(10) cfu of edema principle toxin producing germs of E. coli serogroup O 139. 23 animals of the control groups (46.9%) and one animal of vaccine groups (1.8%) died due to the infection between days four and five post challenge. These control animals showed classical clinical symptoms as well as pathological findings typical for edema disease. In contrast, such findings as mentioned before could not be observed in the vaccinated piglets. The remaining part of the control animals and eight of those vaccinated ones exhibited edema disease symptoms. The vaccinated animals have shed the challenge strain one to three days, while the survivals of control groups shed those germs for two to six days. The vaccinated piglets showed a better growth rate than the remaining control animals. Presented data suggest that our toxoid immunizing procedure can be used successfully against edema disease of swine.     

Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2.

The protective efficacy of a live and killed non-encapsulated isogenic mutant of Streptococcus suis serotype 2 was determined in pigs, and compared with the efficacy of the capsulated wild-type strain. SPF pigs were vaccinated twice intramuscularly at 4 and 7 weeks of age with a dose of 1 x 10(9) formalin-killed CFU of the wild-type (WT-BAC), formalin-killed non-encapsulated mutant (CM-BAC) or live non-encapsulated mutant (CM-LIVE) strain. After 2 weeks, vaccinated pigs and non-vaccinated controls were challenged intravenously with 1 x 10(7) CFU of the homologous, wild-type S. suis serotype 2 strain. Protection was evaluated by clinical, bacteriological, serological and post-mortem examinations. All pigs vaccinated with WT-BAC were completely protected against challenge with the homologous serotype. Pigs vaccinated with CM-BAC were partially protected. Although all pigs vaccinated with CM-BAC survived the challenge, four out of five pigs developed clinical signs of disease for several days. Compared to the WT-BAC and CM-BAC, the CM-LIVE vaccine was less protective. Two out of five pigs vaccinated with CM-LIVE died in the course of the experiment and all of them developed specific clinical signs of disease for several days. The protective efficacy of the vaccines could be associated with serum antibody titers. Antibody titers against cells of wild-type and non-encapsulated mutant strains as well as against muramidase-released proteins (MRP) were high in pigs vaccinated with WT-BAC and CM-BAC. Pigs vaccinated with CM-LIVE showed lower antibody titers. Antibody titers against purified capsular polysaccharides (CPS) of S. suis serotype 2 were only found in pigs vaccinated with WT-BAC. These findings indicate that CPS and other bacterial components of WT-BAC are probably essential for full protection against homologous challenge.     

Respiratory tract protection upon challenge of pigs vaccinated with attenuated porcine reproductive and respiratory syndrome virus vaccines

In this study, the efficacy of two attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccines was assessed. The virological protection in the lungs of vaccinated pigs upon challenge was studied. Also, challenged pigs were exposed to lipopolysaccharide (LPS) to evaluate clinical protection. Six-week-old pigs were immunized intramuscularly with commercial vaccines based on either an attenuated American or an attenuated European virus strain. Non-immunized pigs and pigs intramuscularly inoculated with the virulent Lelystad strain were included as controls. Six weeks after immunization, pigs were challenged either intratracheally or intranasally with the Lelystad strain, and 3 and 6 days later intratracheally exposed to Escherichia coli LPS. After LPS administration, pigs were monitored for clinical signs. At 4 and 7 days after challenge, pigs were euthanized to determine virus quantities in broncho-alveolar lavage (BAL) fluids and in lungs. Challenge virus was recovered from three out of eight pigs that had been primo-inoculated with the Lelystad strain with titers ranging between 0.3 and 3.1 log(10). Fifteen out of sixteen pigs vaccinated with the attenuated American strain were positive for challenge virus and their mean virus titers were similar to those of non-immunized challenge controls. Eleven out of 16 pigs vaccinated with the attenuated European strain were positive for challenge virus and their mean virus titers were 2.0-2.5 log(10) lower than those of non-immunized challenge controls. Thus, the virological protection in the lungs of vaccinated pigs upon challenge was incomplete, but was more pronounced in the homologous situation. Clinical signs upon LPS exposure in both vaccinated groups were not reproducible in two experiments.