Hydration and endocrine responses to intravenous fluid and oral glycerol.

Athletes use intravenous (IV) saline in an attempt to maximize rehydration. The diuresis from IV rehydration may be circumvented through the concomitant use of oral glycerol. We examined the effects of rehydrating with differing regimes of oral and IV fluid, with or without oral glycerol, on hydration, urine, and endocrine indices. Nine endurance-trained men were dehydrated by 4% bodyweight, then rehydrated with 150% of the fluid lost via four protocols: (a) oral = oral fluid only; (b) oral glycerol = oral fluid with added glycerol (1.5 g/kg); (c) IV = 50% IV fluid, 50% oral fluid; and (d) IV with oral glycerol = 50% IV fluid, 50% oral fluid with added glycerol (1.5 g/kg), using a randomized, crossover design. They then completed a cycling performance test. Plasma volume restoration was highest in IV with oral glycerol > IV > oral glycerol > oral. Urine volume was reduced in both IV trials compared with oral. IV and IV with oral glycerol resulted in lower aldosterone levels during rehydration and performance, and lower cortisol levels during rehydration. IV with oral glycerol resulted in the greatest fluid retention. In summary, the IV conditions resulted in greater fluid retention compared with oral and lower levels of fluid regulatory and stress hormones compared with both oral conditions.

Immunological characterization of breakdown peptides of the 104 kilodalton hemolysin of Actinobacillus pleuropneumoniae serotype 1.

The 104 kilodalton (kDa) hemolysin of Actinobacillus pleuropneumoniae serotype 1, strain CM-5 was precipitated from RPMI-1640 culture supernatant using ammonium sulfate to 80% saturation. In immunoblots, a rabbit polyclonal antiserum against the 104 kDa hemolysin protein, recognized not only the original 104 kDa monomeric form of the hemolysin but other proteins in the crude antigen mixture ranging in molecular mass from 43 to greater than 125 kDa. The antiserum was able to crosslink these proteins to active hemolysin in RPMI-1640 culture supernatant resulting in bands of hemolysis in blood agar used in a contact assay. Corresponding to these bands of hemolysis, denatured peptides with molecular masses of 51, 85, 104 and greater than 125 kDa were excised and injected into rabbits. In immunoblots, the resultant antibodies recognized the injected peptide and the monomeric 104 kDa protein. However, only the rabbit antisera produced against the 104 and 125 kDa proteins contained antibodies which neutralized the active 104 kDa hemolysin in culture supernatant. These results indicate that (i) the 104 kDa protein hemolysin can exist in a higher molecular weight aggregate (greater than 125 kDa) but can also break down to peptides which have molecular masses smaller than the 104 kDa parent molecule and (ii) while several epitopes are present in the hemolysin molecule, there seems to be a restricted number of antigenic determinants responsible for inducing neutralizing antibodies and these seem to reside only in the 104 kDa parent molecule. This may have consequences, in terms of vaccine development, for the control of pleuropneumonia in swine herds.

Phenotypical and genotypical characteristics of paired isolates of Pasteurella multocida from the lungs and kidneys of slaughtered pigs.

Twenty-eight strains of Pasteurella multocida were isolated from 12 Danish and two Canadian abattoir pigs. Fourteen strains were isolated from pulmonary inflammatory lesions, and 14 strains were isolated from kidneys of the same animals. Phenotypical and genotypical characteristics of the strains were evaluated with a view to determine if P. multocida isolated from kidneys might have been disseminated from the lungs. All field strains were capsular type A. The biochemical reactivity in the API-20E and API-ZYM commercial test-kits was uniform with the exception of alpha-glucosidase activity which was present at low levels in only ten of the strains. One strain was markedly serum sensitive, six strains slightly sensitive and the remaining were serum resistant. The peptide patterns obtained by polyacrylamide gel electrophoresis of whole cell proteins of the strains were very uniform with the exception of differences in intensity of bands in the 38 and 34 kD regions. The pattern of oligonucleotides obtained after electrophoresis of total genomic DNA digested with BamHI showed that the paired isolates had identical patterns in eight of the 14 animals. It is therefore likely that isolates from kidney lesions represent blood borne dissemination from primary pulmonary lesions.

Protective efficacy of capsule extracts of Haemophilus pleuropneumoniae in pigs and mice.

Capsular extracts of Haemophilus pleuropneumoniae, Serotype 1, were mixed with AL(OH)3 gel (3 parts extract + 1 part Al(OH)3) and used as vaccines in pigs and mice. Four preparations were tested in Experiment I: NaCl and Cetavlon (hexadecyltrimethyl ammonium bromide) extracts of both low in vitro passage (LP) and high in vitro passage (HP) culture, respectively. Four pigs vaccinated with the NaCl extract of the LP strain survived, whereas one of four from each of the remaining vaccine groups and five of six from the control group died. All vaccines induced complement-fixing antibodies. No apparent boosting of titres occurred as a result of challenge with live bacteria. Mice were vaccinated in Experiment II with NaCl and Cetavlon extracts of the LP strain. Both were protective, although the Cetavlon vaccine appeared more efficacious than the NaCl extract. The use of Al(OH)3 adjuvant improved the efficacy of the NaCl vaccine in mice. In Experiment III six gnotobiotic pigs were vaccinated with a combined NaCl and Cetavlon vaccine and seven animals were given placebo. In Experiment IV seven specific pathogen-free (SPF) pigs were given the combined vaccine and eight pigs received placebo treatment. Both of these experiments indicated that the extract vaccines did not completely protect but reduced the mortality in pigs challenged with homologous virulent H. pleuropneumoniae bacteria. The results indicate that capsular antigens of H. pleuropneumoniae have some protective immunogenic efficacy in pigs and mice.

Biochemical and serological identification of strains of Haemophilus pleuropneumoniae.

Eighteen field isolates of Haemophilus pleuropneumoniae were studied biochemically and serotyped using the complement fixation test (CFT), agglutination test and the immunodiffusion test. Three biochemical tests (V-dependency, CAMP-reaction and urease activity) were found to be very useful for the biochemical characterization of the H. pleuropneumoniae. Haemolysis on blood agar plates, although present, was not sufficiently pronounced in all cases to warrant absolute dependence on this characteristic. Serological typing revealed the isolates belong to Serotypes 1 and 5. The immunodiffusion test proved to be the most serotype specific, while a marked cross-reaction was observed with the CFT.

Cytokines induced in vitro by Mycoplasma mycoides ssp. mycoides, large colony type.

Septicemic disease in goats and sheep caused by the large colony type of Mycoplasma mycoides ssp. mycoides has clinical and pathological features in common with septic endotoxemia. We studied the ability of the mycoplasma to induce mediators of biological responses to endotoxin, such as TNF alpha, IL-1 alpha, IL-6 and nitric oxide. Heat-killed suspensions of mycoplasmas in a concentration of 25 micrograms protein ml-1 induced all mediators in macrophages from peritoneal cavity and bone marrow of both C3H/HeN (LPS responsive) and C3H/HeJ (LPS low-responsive) mice. Lipopolysaccharide (LPS) in a concentration of 100 ng ml-1 induced the mediators in C3H/HeN derived macrophages, only. Simultaneous stimulation of macrophages with interferon-gamma enhanced the secretion of nitric oxide (measured as nitrite) but not the cytokines. We conclude that heat-killed Mycoplasma mycoides ssp. mycoides, large colony type, has cytokine inductive activity similar to bacterial endotoxin, but with an induction mechanism different from LPS.

Mycoplasma hyorhinis infection of pigs selectively bred for high and low immune response.

Pigs have been selected for high (H) or low (L) combined antibody and cell-mediated immune response to test the high immune response phenotype as a candidate for an indirect approach to improving health and productivity in livestock. Mycoplasma hyorhinis infection was induced in H and L pigs of the 4th generation of selection to test the hypothesis that immune response lines differ in response to infection. The major disease sign, arthritis, was more severe in the H pigs both clinically and at necropsy. M. hyorhinis was isolated at higher colony counts from synovial fluids of the H pigs. In contrast, pleuritis and peritonitis were less severe in pigs of the H than those of the L line. Pericarditis, although less in H than L pigs, did not differ significantly by line. Synovial fluid antibody to M. hyorhinis did not differ by line but H pigs produced serum antibody earlier and to a higher titre than did L pigs. Selection for H or L immune response therefore alters response to M. hyorhinis, however there is no indication of a consistent line-related health advantage.

Development and evaluation of a mixed-antigen ELISA for serodiagnosis of Actinobacillus pleuropneumoniae serotypes 1, 5, and 7 infections in commercial swine herds.

A mixed-antigen enzyme-linked immunosorbent assay (ELISA) containing serotype-specific polysaccharide antigens from serotypes 1, 5, and 7 of Actinobacillus pleuropneumoniae was developed. This ELISA was evaluated using sera from experimentally infected pigs. With a negative cutoff value of 0.250 (optical density at 405 nm), sensitivity and specificity were determined to be 96% and 99.5%, respectively. The ELISA was further evaluated using sera from commercial swine. Sows and suckling piglets were found to be the best age groups for detection of positive reactors to A. pleuropneumoniae. The mixed-antigen ELISA could be used in a herd health monitoring program for detection of A. pleuropneumoniae infections.

Infectious agents in acute respiratory disease in horses in Ontario.

A study of acute respiratory disease in horses in Ontario was undertaken to determine the identity of current causative infectious agents. A nasopharyngeal swab was designed and utilized to maximize isolation of viruses, mycoplasma, and pathogenic bacteria. Serum samples were collected for parallel determination of antibody titers to equine influenza virus type A subtype 1 (H7N7) and subtype 2 (H3N8), equine rhinovirus types 1 and 2, equine herpesvirus type 1, Mycoplasma equirhinius, and Mycoplasma felis. Equine rhinovirus type 2 was recovered from 28/92 horses tested, and equine influenza virus type A, subtype 2, was recovered from 5. The mycoplasma and bacteria isolated were consistent with those commonly associated with nonspecific respiratory diseases in horses, except that Streptococcus pneumoniae capsular type 3 was isolated from 10 horses.

Oral challenge of turkey poults with Mycoplasma iowae.

Day-old poults were inoculated orally each day for 7 days with 0.2 ml of Mycoplasma iowae, strain D112, 10(8) colony-forming units/ml. Cloacal swabs were taken from each poult during the inoculation period and at selected intervals until 21 days after the last inoculation. Most poults shed mycoplasmas persistently after inoculation. Cloacal swabs from eight out of ten poults were positive at 21 days after the last inoculation. Feces of poults in the infected group were normal, and there was no significant rise in cloacal temperature. At necropsy, mycoplasmas were recovered from tissues of the respiratory tract, gastrointestinal tract, spleen, and kidney. In the gastrointestinal tract, the most frequent recoveries were from the wall of the distal portion of the small intestine, cecum, and large intestine. Recovery of M. iowae from these organs and tissues indicated infection following oral challenge.

Colonization of the intestine of turkey embryos exposed to Mycoplasma iowae.

Eight-day-old turkey embryos were inoculated into the yolk sac with 3 X 10(5) colony-forming units of Mycoplasma iowae strain D112 in order to study the growth-depressing effect, the histopathological changes, and colonization of the intestinal tract. The embryo: egg weight ratio was significantly lower in the inoculated eggs than in controls. Histologically, there were infiltrations in parenchymatous organs and chorioallantoic membranes with heterophilic granulocytes. M. Iowae was demonstrated on the intestinal mucosa by antibody fluorescent microscopy and by transmission electron microscopy. Attaching mycoplasmas had a distinct morphology; the segment in contact with enterocytes was cone-shaped and had finely granulated cytoplasma which was abruptly separated from the distal coarsely granulated area. We conclude that M. iowae has a predilection for the intestinal tract of avian hosts.

Mycobacterium avium subspecies paratuberculosis triggers intestinal pathophysiologic changes in beige/scid mice.

We investigated whether infection of beige/scid mice with Mycobacterium avium subspecies paratuberculosis can induce intestinal pathophysiologic changes. Six-week-old beige/scid mice were inoculated intraperitoneally with M. paratuberculosis, then were killed 32 weeks after inoculation when the small intestine was evaluated for physiologic and morphologic abnormalities. All infected mice developed clinical disease. The lamina propria of the intestine from infected mice was mildly infiltrated with mononuclear cells containing acid-fast bacteria, and had significantly increased villus width. In vitro physiologic studies in Ussing chambers indicated that M. paratuberculosis infection caused significant abnormalities in intestinal transport parameters. Baseline short circuit current and potential difference were abnormally high in tissues from infected, compared with control mice, indicative of increased ion secretion. Baseline conductance was significantly decreased in infected mice, suggesting that intestinal tissue from infected mice was less permeable to ions. The change in short circuit current following transmural electrical and glucose stimulation was significantly reduced in intestines from infected mice, suggesting that inflamed intestine had neural and/or epithelial cell damage. We conclude that infection of beige/scid mice with M. paratuberculosis triggers significant intestinal pathophysiologic changes consistent with chronic inflammation. These functional abnormalities may contribute to the pathogenesis of the wasting syndrome seen in bovids with paratuberculosis. This animal model provides evidence that T cell-independent mechanisms are sufficient to cause mucosal pathophysiologic changes and inflammation in response to a specific pathogen, and may be of relevance to inflammatory bowel disease in humans.

Isolation of mycoplasmas from the canine genital tract: a survey of 108 healthy dogs.

Cultivation of preputial or vaginal swab samples from 108 apparently healthy dogs gave presumptive mycoplasma growth in 72 cases. Specific identification of 58 mycoplasma isolates based on biochemical studies, indirect immunofluorescence and growth inhibition tests showed association with eight different species or groups, only four of which had previously yielded genital isolates. Fourteen strains were related to M bovigenitalium.

Evaluation of growth of avian mycoplasmas on bile salt agar and in bile broth.

All nine Mycoplasma iowae strains and one strain of M gallinarum grew on 0.05 per cent 'bile' agar medium. The colony size of M iowae on this agar medium was similar to the size obtained on bile-free mycoplasma agar. One strain each of M maculosum, M arginini and M bovis also grew on 0.05 per cent bile agar. However, one strain each of M gallisepticum and M meleagridis were inhibited at this concentration. Six of the nine strains of M iowae were also resistant to 1 per cent 'bile' in broth medium but all were resistant to 0.5 per cent. The resistance of M iowae to 0.5 per cent 'bile' in broth may be a useful characteristic for differentiating it from some of the other avian mycoplasmas.

Endothelial cytotoxicity of Actinobacillus pleuropneumoniae.

The cytotoxicity of Actinobacillus pleuropneumoniae serotype 1 strain CM5 for porcine and bovine endothelial cells in vitro, was dose-dependent. This strain and its attenuated and avirulent substrain CM5A were equally cytotoxic. The cytotoxicity observed during five hours of exposure of endothelial cells to bacterial products was abolished if the bacteria were inactivated by heat or sonication. Exposure of the endothelial cells for five hours to 100 and 200 micrograms of purified lipopolysaccharide resulted in a partial cytotoxicity only, which was not enhanced in the presence of fresh guinea pig serum. The cytotoxicity of viable bacteria could be neutralised by a polyclonal rabbit antiserum to the purified 104kD haemolysin. A bacteria-free supernate of a culture of strain CM5 had both haemolytic and cytotoxic activity. The haemolytic activity could be neutralised completely by the anti-serum to the 104kD haemolysin, whereas the cytotoxic activity was only partially neutralisable. Hence A pleuropneumoniae is cytotoxic for endothelial cells and this cytotoxicity is possibly mediated by the 104kD haemolysin.

Identification of the heat-labile hemolysin of Actinobacillus pleuropneumoniae serotype 1.

The heat-labile hemolysin of Actinobacillus pleuropneumoniae serotype 1 strain CM-5 was partially purified using ammonium sulfate precipitation and gel permeation chromatography. This partially purified material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nylon filters. The filters were treated with convalescent pig serum and subsequently with CM-5 culture supernatant containing active hemolysin. A 104 kd peptide was identified as the hemolysin because it bound antibodies in convalescent pig serum which cross-linked active hemolysin. The same 104 kd protein when injected into a rabbit produced neutralizing antibodies to the CM-5 hemolysin in culture supernatant.

The association between serological evidence of mycoplasma infection and respiratory disease in feedlot calves.

Calves from five Ontario feedlots were bled on arrival and approximately 28 days later. Calves treated during this interval for undifferentiated respiratory disease were classified as cases and untreated calves were classified as controls. Serum was titrated blindly for antibodies to Mycoplasma bovis and Mycoplasma dispar. Indirect hemagglutination titers of 1:20 or more were assumed to reflect recent or current exposure, whereas 1:10 or less were not. The titers to M. bovis increased in all feedlots indicating active infection. The initial titers to M. dispar were higher than the titers against M. bovis, yet they increased in all feedlots except one, suggesting widespread infection with this organism. There was an increased risk (although not statistically significant) of being treated if the titer against M. bovis rose during the period. Calves with low M. dispar titers on arrival were at increased risk of being treated and titer increases were strongly associated with treatment (statistically significant). Thus, the serological results indicate high prevalence of M. bovis and M. dispar in the feedlot calves and that calves with increasing titers in particular to M. dispar are at increased risk of being treated for respiratory disease.

Cross protection among Haemophilus parasuis strains in immunized gnotobiotic pigs.

In an attempt to establish if cross protection can be induced by different strains of Haemophilus parasuis, three groups of 12 gnotobiotic pigs were immunized each with an aluminum hydroxide adsorbed whole cell bacterin of one of three H. parasuis strains. Two weeks later, four pigs within each vaccinated group were challenged with aerosols of live cultures of each of the three test strains and observed for response. Two virulent strains V1 and V2 protected all the vaccinated pigs, while all nonvaccinated controls succumbed to Glasser's disease when challenged with these strains. Vaccination with strain LV (of low virulence) protected the pigs against challenge with strain V2, but not against strain V1. Strain LV did not cause disease in the immunized animals and only in one of ten nonimmunized pigs upon second challenge. The results suggest that strains may differ in antigenicity and that virulence and immunoprotection are positively related. Strains to be used in commercial vaccines should therefore be selected carefully. Antibodies detected in the sera of vaccinated pigs were to outer membrane proteins of the bacteria, but not to lipopolysaccharides or capsular polysaccharides. This would suggest that for gnotobiotic pigs outer membrane proteins are more immunogenic than lipopolysaccharide or capsular antigens. Further work is needed to determine if outer membrane proteins also contribute protective immunogens.

Effect of time of exposure to rat coronavirus and Mycoplasma pulmonis on respiratory tract lesions in the Wistar rat.

The effects of time of exposure on the progression of pulmonary lesions in rats inoculated with Mycoplasma pulmonis and the rat coronavirus, sialodacryoadenitis virus (SDAV) were studied, using six groups of 18 SPF Wistar rats (n = 108). Rats were inoculated intranasally as follows: Group 1, sterile medium only; Group 2, sterile medium followed one week later by 150 TCID50 SDAV; Group 3, sterile medium followed by 10(5.7) colony forming units of M. pulmonis; Group 4, SDAV followed one week later by M. pulmonis; Group 5, M. pulmonis followed one week later by SDAV; Group 6, M. pulmonis followed two weeks later by SDAV. Six rats from each group were euthanized at one, two and three weeks after the final inoculation. In a separate experiment, six additional animals were inoculated in each of groups 3, 5 and 6 (n = 18) and were sampled at five weeks after they had received M. pulmonis. Bronchoalveolar lavage and quantitative lung mycoplasma cultures were conducted on two-thirds of the rats. Histopathological examination and scoring of lesion severity were performed on all animals. Based on the prevalence and extent of histopathological lesions, bronchoalveolar lavage cell numbers, neutrophil differential cell counts and the isolation of M. pulmonis, the most severe disease occurred in the groups that received both agents. There was no significant difference in lesion severity between the groups receiving both agents other than in those examined during the acute stages of SDAV infection. Based on these results, it is evident that SDAV enhances lower respiratory tract disease in Wistar rats whether exposure occurs at one week prior to or at various intervals following M. pulmonis infections.

Experimental pneumonia in rabbits inoculated with strains of Pasteurella multocida.

Domestic rabbits were inoculated with either a 3:A or 3:D serotype of Pasteurella multocida by aerosol, intravenous, or intratracheal inoculation. Different colony forming units of P. multocida were used. Animals which died or were killed after the 14 day observation period were examined macroscopically and microscopically for lesions in the lower respiratory tract. Pneumonic lesions were most consistently produced in rabbits inoculated intratracheally with serotype 3:A. Pulmonary and pleural lesions were observed in some animals inoculated intravenously with serotype 3:A. Lesions were minimal in rabbits inoculated with serotype 3:D. Of the three routes of inoculation evaluated, the intratracheal route appeared to be the best method to produce Pasteurella-associated lesions in the lower respiratory tract.