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.     

Effects of Escherichia coli Shiga-like toxins (verotoxins) in pigs.

Escherichia coli K12 strains TB1(pCG5), with the genes for Shiga-like toxin IIv from an edema disease isolate of E. coli and TB1(pCG5-1), with the toxin genes inactivated by transposon mutagenesis, were used to test the hypothesis that Shiga-like toxin IIv was the same as edema disease principle. Ammonium sulfate precipitated culture supernatants from the pair of E. coli K12 strains and from a wild edema disease isolate of E. coli (E145) were tested for their ability to induce signs and lesions of edema disease in intravenously inoculated weaned pigs. Similar preparations from E. coli which produce Shiga-like toxins I and II were also tested. Preparations from E. coli TB1 (pCG5) and E145 contained high levels of Shiga-like toxin IIv and induced signs and lesions similar to those seen in edema disease, whereas preparations from E. coli TB1 (pCG5-1) failed to induce signs or lesions of edema disease. All Shiga-like toxin preparations produced delayed neurological signs, fibrinoid necrosis of arterioles and hemorrhages in the cerebellum of pigs. High doses of Shiga-like toxin IIv were associated with superficial necrosis of the colonic epithelium and vasculitis. Shiga-like toxins I and II resulted in kidney lesions but no enteric pathology. Shiga-like toxin II preparations had the lowest median lethal dose for pigs, Shiga-like toxin IIv was intermediate and Shiga-like toxin I was the least toxic.     

Prevention of edema disease in pigs by vaccination with verotoxin 2e toxoid.

Pigs in 2 herds with persistent problems with post weaning edema disease caused by infection with verotoxin-2e (VT2e)-producing Escherichia coli O139 were treated with a VT2e-toxoid vaccine. Treatment was performed as a randomized blind field trial with parallel treatment and non-vaccinated control groups. In 1 herd, a group of pigs was injected with adjuvant alone. Pigs were vaccinated at 1 and 3 wk of age and weaned at 4 wk of age. The effect of vaccination was measured by average daily weight gain (ADG), mortality due to edema disease within the 1st 4 wk after weaning, and weight at 3-6 mo of age. Pathological and microbiological examinations were performed on all pigs that died during the 1st 4 wk post weaning. Only pigs from which VT2e+, F18+ E. coli O139 was isolated were categorized as "death due to edema disease." The serological response to vaccination was evaluated by an indirect ELISA. Vaccination had a statistically significant effect on the level of antibodies specific for VT2e in both herds. Vaccination resulted in a statistically significant increase in ADG in the nursery period but not in the grower-finishing period. Vaccination had a statistically significant effect on mortality due to edema disease with an odds ratio of 0.039, indicating that there was almost total elimination of mortality due to the disease in the vaccine groups.     

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.     

Immunization of pigs with culture antigens of Taenia solium

An evaluation has been made of the protective effect of immunizing pigs with excretory-secretory homologous antigens on Taenia solium infections. This procedure reduced the number of cysticerci established from a challenge infection.     

Shiga-like toxin production and attaching effacing activity of Escherichia coli associated with calf diarrhea

Four hundred twenty-nine isolates of Escherichia coli from calves were tested for the production of HeLa cell cytotoxin(s). Isolates that produced enough cytotoxin to be detected in culture supernatants of iron-depleted broth were considered to produce increased amounts of cytotoxins. Isolates also were tested for homology with a DNA probe for a gene that encodes localized adherence of human enteropathogenic E coli. Four isolates produced increased amounts of cytotoxin that was neutralized by Shiga antitoxin (toxin designated as Shiga-like toxin-I [SLT-I]). A 5th isolate produced increased amounts of cytotoxin (SLT+) that was not neutralized by the Shiga antitoxin, but was neutralized by antitoxin against a variant of SLT (toxin designated as SLT-II). None of the isolates hybridized with the probe for the localized adherence gene. Three of the SLT+ isolates belonged to human enteropathogenic E coli serogroups O26 and O111. All 5 of the SLT+ isolates were from calves with diarrhea, but none of the 5 SLT+ isolates contained genes for classic heat-labile or heat-stable enterotoxins, for K99 fimbriae, or for invasiveness; neither did any of them adhere to HeLa cells in culture. Three of the 5 SLT+ isolates had attaching and effacing activities when inoculated into ligated intestinal loops of rabbits. One of the isolates with attaching and effacing activity in rabbits was originally isolated from a calf with lesions characteristic of those produced by attaching effacing E coli (AEEC). Calves inoculated with this SLT+ AEEC isolate developed focal colonic lesions characteristic of those produced by AEEC, but did not develop diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turbinate osteoporosis in pigs following intranasal inoculation of purified Pasteurella toxin: histomorphometric and ultrastructural studies

Turbinate osteoporosis, induced by intranasal inoculation of purified toxin isolated from serotype D Pasteurella multocida, was investigated in 3- to 5-week-old, caesarean-derived, colostrum-deprived, isolation-reared pigs. Marked bilateral reduction in relative volume of trabecular bone occurred in osseous cores of turbinates of toxin-treated pigs relative to control pigs on post-inoculation day (p.i.d.) 3, 6, 9, 12, and 15. The fractional resorptive surface along turbinate bone was greater in toxin-treated pigs when compared to controls on p.i.d. 3 and 6. A significant decrease in resorptive surface occurred over time in toxin-treated pigs, whereas the fractional resorptive surface was constant over time in control pigs. Osteoclasts in medullary spaces separating bony trabeculae of turbinates were abundant in toxin-treated pigs and scant in controls on p.i.d. 3, 6, and 9. Degeneration and necrosis of bone forming cells, principally osteoblasts, were progressively more extensive with time and were associated with decreased mineralization and reduced thickness of osteoid and woven bone matrix. Osteoclasts along resorptive surfaces of turbinate bone in toxin-treated pigs had more abundant, more highly vacuolated cytoplasm, a more prominent microvillous border, and a greater number of nuclei per cell than osteoclasts from control pigs on p.i.d. 3 and 6. We conclude that this Pasteurella toxin stimulates osteoclastic osteolysis and inhibits osteogenesis in turbinates by causing degeneration and death of osteoblasts.     

Immunization of pigs against Streptococcus suis serotype 2 infection using a live avirulent strain.

Streptococcus suis capsular type 2 is still an important cause of economic losses in the swine industry. At the present time, vaccination of pigs against this infection is generally carried out with autogenous bacterins and results are equivocal. In this study, the protective effect of a live avirulent S. suis type 2 strain (#1330) which had induced a good protection in mice, was evaluated in swine. The experiment was performed in triplicate using 4 week-old piglets. A total of 15 piglets were vaccinated 3 times, 15 others were vaccinated 2 times, and 15 piglets were injected 3 times with sterile Todd-Hewitt broth. Using an indirect ELISA, an increase in the IgG response to S. suis antigens was noted in 27 of the 30 vaccinated piglets. On day 21 post-vaccination, all animals were challenged intravenously with a virulent S. suis type 2 strain (#999). In the 2 vaccinated groups, 26 animals were fully protected. Only 1 out of the 15 piglets vaccinated 3 times developed mild clinical signs. In the group vaccinated twice, 3 piglets showed clinical signs and 1 of them died after the challenge. In the control group, 7 animals died out of the 11 with clinical signs of infection. In conclusion, a protective immunity was observed in swine when using strain 1330. However, more studies are needed to assess the use of a live S. suis strain in a vaccine for pigs.     

Induction of pneumonia in rabbits by use of a purified protein toxin from Pasteurella multocida.

Heat-labile toxin from a cell sonicate of a virulent type-D strain of Pasteurella multocida was purified by ammonium sulfate precipitation followed by ion exchange chromatography, gel filtration chromatography, and polyacrylamide gel electrophoresis. Toxic activity was assayed during toxin purification by cytopathic effect in Vero or bovine embryonic lung cell cultures. Toxicity for cells correlated with dermonecrosis in guinea pig skin. Toxicity was accounted for by a single protein with a molecular weight of 149,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbits were inoculated intranasally with purified toxin to determine whether toxin had a role in the induction of pneumonia in rabbits infected with P multocida. Pneumonia, pleuritis, acute hepatic necrosis, and splenic lymphoid atrophy were found in 4 of 5 rabbits. One of 5 rabbits had bilateral turbinate atrophy. Western blotting with monoclonal antibodies to toxin from a P multocida isolate causing atrophic rhinitis in pigs revealed the toxin that induces pleuritis and pneumonia in rabbits to be the same or a closely related toxin.     

Antibodies to Aujeszky's disease virus in pigs immunized with purified virus glycoproteins

Antibodies to Aujeszky's disease virus (ADV) glycoproteins gII, gIII, and gp50 were compared using four in vitro tests. Antibodies generated by vaccination with a modified-live vaccine (MLV) were also compared. The serological assays employed were: serum neutralization test (SNT), complement facilitated serum neutralization test (C'SNT), complement-mediated cytolysis and antibody dependent cellular cytotoxicity (ADCC). Pigs were immunized with single glycoproteins twice 14 days apart, or once with the modified-live vaccine. Fourteen days after the second immunization, sera were collected. Virus neutralizing activity (SNT) was demonstrated in the sera from all pigs immunized with gp50 and in one out of three immunized with gIII. Sera from the MLV group all had neutralization titers higher than animals immunized with single glycoproteins. Addition of guinea pig complement to the serum neutralization test (i.e., C'SNT) produced an enhancement of antibody titers in all groups except the pigs immunized with gIII. The complement-mediated cytolysis test rendered antibody titers similar in magnitude for all pigs immunized with single glycoproteins, but slightly lower than values for MLV vaccinated pigs. ADCC activity was clearly displayed in sera from pigs immunized with gIII or vaccinated with MLV, whereas sera from pigs immunized with gII or gp50 had a minimal response. The results indicate that the relative efficiency of antibodies against ADV glycoproteins in protection should be considered for selecting or producing gene-deleted strains for use in vaccine production.     

Immunization with recombinant alpha toxin partially protects broiler chicks against experimental challenge with Clostridium perfringens

This study described the first report of BTV-16 in Croatia. Serological evidence occurred in cattle at the end of September and continued during October and November 2004. All positive animals were in the Dubrovnik-Neretva County, a region located in the southernmost part of Croatia. BTV-16 infection was also detected in goats and sheep. Apart from few cases reported in Greece between 1999 and 2000, BTV-16 has never been reported in the Balkanic peninsula before. The BTV strain was isolated from cattle blood samples and typed as BTV-16. When the S5 was sequenced, it showed 100% homology with the BTV-16 vaccine isolate produced by Ondersterpoort Biological Product (SA) and used in Italy during the 2004 BT vaccination campaign. On the other hand no complete homology was found when the same RNA segment sequence was compared with that of the homologous Italian field isolate. As no evidence of livestock movements from Italy was demonstrated, an eolic transmission of the infection through infected Culicoides was hypothesised. According to the local meteostations, in several occasions, during the 2004 summer months, the west–east breeze blew with a speed above 50 km/h from Italy towards the Dubrovnik County. It is concluded that the BTV-16 which infected Croatian livestock was similar to the homologous OBP vaccine isolate and it is likely that it was introduced from Italy into the Southern regions of Croatia through infected Culicoides carried by the wind.