Some leptospira agglutinins detected in domestic animals in British Columbia.

During a period of six years 7,555 bovine sera, 421 canine sera, 251 porcine sera and 135 equine sera were tested for agglutinins to Leptospira interrogans serotypes canicola, grippotyphosa, hardjo, icterohemorrhagiae, pomona and sejroe. The bovine sera reacted predominantly with hardjo and/or sejroe at a rate of 15% compared to 3.5% with pomona. Breeding or abortion problems were associated with pomona but not with sejroe/hardjo agglutinins. The canine sera reacted to canicola (9.9%y and icterohemorrhagiae (5.4%), tcted predominantly with canicola (8.9%) and icterohemorrhagiae (8.1%).     

The demonstration and characterization of deoxyribonucleases of streptococci group A, B, C, G and L.

Streptococcus pyogenes, S. agalactiae, S. dysgalactiae, S. equi, S. equisimilis, S. zooepidemicus, Streptococcus group G and L were found to produce deoxyribonucleases (DNases) which were demonstrated using the Toluidine Blue DNA Agar (TDA) described for staphylococcal DNases. The activity of streptococcal DNases increased in the presence of Mg⁺⁺ and Ca⁺⁺ ions, the pH optimum was about 7.5 and native DNA was the best enzyme substrate. It is consequently recommended to modify the TDA according to these results for the demonstration of streptococcal DNases. All streptococcal DNases, except the DNase of S. zooepidemicus, were found to be heat-stable. Isoelectric focusing was a convenient technique for separation of streptococcal DNases and for estimation of the pI values of the DNases. S. agalactiae and S. dysgalactiae generally exhibited distinct species specific patterns in the isoelectric focusing experiments. The DNases produced by S. pyogenes were serologically related to the DNases of S. dysgalactiae and Streptococcus group G. A similar relationship was demonstrated between the DNases produced by S. equisimilis and Streptococcus group L.     

Serologic detection of adenovirus infections in specific-pathogen-free chickens

A specific-pathogen-free flock of White Leghorns, which were housed conventionally and were previously serologically negative for all common poultry pathogens including avian adenoviruses, incurred an outbreak of adenovirus that was detected at about 39 weeks of age. The infection was detected serologically through the agar-gel precipitin test (AGPT) and also by a microneutralization test (MNT) adapted for 11 serotypes of avian adenovirus. The MNT detected specific antibodies to serotype-3 avian adenovirus (IBHV-Tipton) but no other serotype. While AGPT-positive sera drawn from the flock gradually declined from 54% to 17%, neutralizing-antibody levels rose sharply at 46 weeks of age to a peak that was maintained over the remaining 18 weeks of the flock's production.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum

A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6 was used as antigen. The blocking ELISA was tested against sera from pigs experimentally infected with the 12 serotypes of Ap biotype 1. Cross-reaction with serotypes 3 and 8 but not with other serotypes was observed. The sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6, 8 or 12 and with sera from herds free of infection with any Ap serotype. The blocking ELISA showed a high herd sensitivity (1.00 (0.79-1.00)) and specificity (0.97 (0.93-0.99)).     

Development and evaluation of a mixed long-chain lipopolysaccharide based ELISA for serological surveillance of infection with Actinobacillus pleuropneumoniae serotypes 2, 6 and 12 in pig herds

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of antibodies against Actinobacillus pleuropneumoniae (Ap) serotypes 2, 6 and 12. The assay was designated MIX-ELISA. Lipopolysaccharide (LPS) from Ap serotypes 2, 6 and 12 was purified using hot phenol-water extraction followed by fractionation by size-exclusion chromatography. A mixture of fractions containing molecules with molecular weight above 50 kDa from all three serotypes was used as antigen. The MIX-ELISA was evaluated with sera from pigs experimentally infected with the serotypes 1, 2, 5b, 6, 7, 8, 10 and 12 of Ap biotype 1. In addition to reaction with sera from pigs inoculated with Ap serotypes 2, 6 and 12, reaction was observed with sera from pigs inoculated with serotype 8. Furthermore, the sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6 or 12 and with sera from herds free of infection with any Ap serotype of biotype 1. The ELISA showed a high herd sensitivity (0.98; 95% confidence interval: 0.89-1.00) and specificity (0.95; 0.88-0.99). The high diagnostic sensitivity and specificity of the assay indicate that screening of herds for Ap infection can be performed using this ELISA. Efficient serological surveillance can be achieved by using such mixed antigen ELISAs coated with size-selected LPS-antigens from the most prevalent serotypes.     

Serodiagnosis of swine pleuropneumonia due to Actinobacillus pleuropneumoniae serotypes 7 and 4 using long-chain lipopolysaccharides.

A saline boiled extract (SBE), capsular polysaccharides (CPS) and long-chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 7 have been evaluated in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. Mean optical densities (ODs) obtained with the 3 antigens using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotypes 7 or 4 were not statistically different. The positive ELISA reaction with anti-serotype 4 sera was unexpected with the CPS, which are supposed to be serotype-specific; LPS traces present in the CPS appeared to be responsible for this reaction. In addition, sera from animals exposed to A. pleuropneumoniae serotypes 5 or 10 presented cross-reactions with the SBE and the CPS, but not with the LC-LPS. Cross-reactions were mainly due to rough LPS, as shown by immunoblotting. The LC-LPS is easily obtainable and can be used for the detection of antibodies in animals infected with A. pleuropneumoniae serotypes 7 and 4.     

Serodiagnosis of pleuropneumonia using enzyme-linked immunosorbent assay with capsular polysaccharide antigens of Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7.

Capsular polysaccharide antigens of serotypes 1, 2, 5 and 7 of Actinobacillus pleuropneumoniae were used in enzyme-linked immunosorbent assays (ELISAs) to test sera from experimentally infected and field pigs. Specific reactions were found in sera of experimental pigs with antigens of serotypes 1, 5 and 7 whereas the serotype 2 antigen was cross-reactive. A 1:200 serum dilution was used for testing of 300 sera from 21 swine herds in southern Ontario. Cases of pleuropneumonia had occurred in 11 of these herds, but not in the others. The negative cut-off value was the mean optical density at 405 nm (OD405) + three standard deviations (SD) for 16 negative reference sera. Sera from four pigs naturally infected with Actinobacillus suis were tested and found to react to varying degrees with each of the antigens. Therefore a second cut-off value was determined as the mean OD405 + 2 SD for the A. suis sera. Sera which, in the ELISA produced OD readings above the latter cut-off were considered positive for antibodies to A. pleuropneumoniae; those which were lower than the former cut-off were considered negative. Readings between the two cut-off values may have been due to low positive titers or cross-reactivity, possibly with A. suis, and could not be used to predict pleuropneumonia. Of the pleuropneumonia-free herds, none had positive reactors to serotypes 5 or 7, whereas one and two herds had positive reactors to serotypes 1 and 2, respectively. Of the pleuropneumonia positive herds, six had positive reactors to serotype 1, one to serotype 2, four to serotype 5, and eight to serotype 7.     

Haemophilus parahaemolyticus serotypes. Serological response

Serotypes 1,2,4 and 5 of Haemophilus parahaemolyticus were inoculated into, respectively, 4,4,2 and 5 pigs. Serum samples were tested for circulating antibodies by the modified complement fixation test (CF test). When individual serotypes were used as antigen, titers were found only to the serotype which had been used for inoculation. Using antigen in which the serotypes were pooled, antibodies were demonstrated in sera from all the pigs. The CF titers obtained with the pooled antigen were equivalent to those found with each serotype separately. When the CF test was used for serological examination of field sera there was full agreement between the results obtained with the pooled antigen and those obtained with serotype 2 antigen alone. No cross reactions were found with the pooled antigen in herds that were sero-positive to Haemophilus parasuis, strain 4800. The experiment has shown that there is no serological cross reaction between serotypes 1, 2, 4 and 5 when they are used as antigen in the CF test. Also, the results imply that with a pool of the different serotypes of Haemophilus paralyticus as antigen similar results may be obtained as with the single serotype 2 antigen.     

Serological characterization of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 9

Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain.In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses.In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.     

Serotypic and biochemical characterization of Bacteroides nodosus isolates from Oregon.

Ninety-seven Bacteroides nodosus isolates were characterized by the tube agglutination test. Fourteen serotypes were identified including isolates that were serologically similar to Australian serotypes A, B and C. One additional isolate remains untyped and possibly represents another serotype. The isolates were cultured from 20 different flocks. Multiple isolates were obtained from 15 of the flocks and 13 of these had two to seven different B. nodosus serotypes. Eleven B. nodosus isolates representing one Australian and ten Oregon serotypes were nonfermentative in various carbohydrates and did not produce indole. These isolates all exhibited proteolytic activity. The prototype strains of 12 of the 14 serotypes demonstrated virulence as assessed by an elastase production assay.     

Serological Characterization of 8 Haemophilus Pleuropneumoniae Strains and Proposal of a New Serotype: Serotype 8

Eight strains of Haemophilus pleuropneumoniae isolated from 8 herd outbreaks of pleuropneumonia in pigs were studied by means of the slide agglutination test, the tube agglutination test, the IHA test and by gel diffusion.The 8 strains were antigenically homogeneous and serologically distinct from serotypes 1 through 7. It is therefore proposed to refer these strains to a new serotype: serotype 8, with strain 405 as the type strain.In addition to the serotype-specific capsular antigens, capsular antigen of serotype 3 (strain 1421) and serotype 6 (strain Femø) could be demonstrated in the 8 strains by means of the IHA test and by gel diffusion analyses.     

The differential diagnosis of Salmonella abortus ovis and Yersinia pseudotuberculosis in sheep abortions

55 strains of Salmonella abortus ovis and 3 strains of Yersinia pseudotuberculosis (two serotypes I, one serotype II), isolated out of aborted ovine fetuses from northern Bavaria, were investigated and compared culturally, biochemically and serologically. The identification of S. abortus ovis (group B) could not be achieved by use of Api 20 E. The code-number 4004500/47 identified S. typhi (group D) only. Y. pseudotuberculosis serotype II but not serotype I agglutinated with the polyvalent and the specific anti-0: 4-Salmonella serum. No agglutination was observed with the flagella anti-H-sera.     

Serological Properties of Neutralizing Antibodies Induced by Vaccination of Rainbow Trout with Distinct Strains of Infectious Pancreatic Necrosis Virus

Abstract

Adult rainbow trout Oncorhynchus mykiss were immunized with formalin-inactivated, concentrated infectious pancreatic necrosis virus (IPNV). Although the immune response was variable among fish inoculated with a given virus type, sera were obtained that contained high titers of antibodies against known representatives of each of the three major serotypes and several unclassified field isolates of IPNV. Preparations of semipurified macroglobulins from the rainbow trout were subsequently used for comparative cross-neutralization testing of viruses. Cross-reactions were generally low between serotypes; however, diversity and heterogeneity existed among viral isolates from North American hatcheries (e.g., within serotype 1). For example, the Jasper subtype was clearly serologically distinguishable from other western Canadian isolates and from typical eastern Canadian isolates, which were similar to U.S. isolate VR 299. Specific salmonid immunoglobulin is suggested as a possible supplemental reagent, together with mammalian polyclonal and monoclonal antibody, for determining the epidemiology of IPNV in North America.     

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.     

Evaluation of an enzyme-linked immunosorbent assay for serological surveillance of infection with Actinobacillus pleuropneumoniae serotype 5 in pig herds

An indirect enzyme-linked immunoassay for serological surveillance of infection of pigs with Actinobacillus pleuropneumoniae (Ap) serotype 5 was developed. The antigen used was prepared from Ap serotype 5b strain L20. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the antigen contained high molecular weight lipopolysaccharide (LPS) and presumably also capsular polysaccharide (CP). The Ap serotype 5 ELISA was tested using sera from pigs experimentally infected with the 12 different Ap serotypes of biotype 1 and with sera from herds naturally infected with Ap serotypes 5, 6, 7 and 12. Cross-reactions were shown in one pig from a herd naturally infected with Ap serotype 7 and in one pig from a herd naturally infected with Ap serotype 12. The herd sensitivities of the Ap5 ELISA and a complement fixation test (CFT) were both estimated to 1.0, on the basis of serum samples from six herds naturally infected with Ap serotype 5. The herd specificities of both tests were estimated to 0.98, based on serum samples from 123 pig herds (10 samples from each herd) from the Danish specific pathogen-free (SPF) programme for pig production.     

DNases in milk and blood sera from different species.

DNases were demonstrated in samples of colostrum and blood serum from man and various domestic animals. The measurable DNase activity recorded was highest in samples from cat and dog and lowest in samples from goat, horse, pig and sheep. In contrast to DNases produced by certain bacteria, these enzymes were thermo-labile and the activity was maximal in the area pH 5.0–5.5.A modification of an agar medium originally described for the demonstration of bacterial DNases was found to be suitable for assays of DNases from colostrum, milk and serum.     

Serological characterization of Actinobacillus pleuropneumoniae biotype 2 strains isolated from pigs in two Danish herds

Eight Actinobacillus pleuropneumoniae biotype 2 strains were isolated in pure culture from lungs of pigs originating from two Danish herds with growing and finishing pigs. The antigenic properties were studied by indirect haemagglutination (IHA) and immunodiffusion (ID) tests using soluble surface antigens and by enzyme-linked immunosorbent assays (ELISA) using capsular enriched fractions and LPS. In all tests the strains proved antigenically homogeneous and serologically distinct from the known biotype 1 and 2 serotypes. Thus, the strains represent a new serotype which is provisionally proposed as biotype 2 serotype 14.     

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum.

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum were analyzed by the rapid plate agglutination and cross-absorption tests. Encapsulated strain forming iridescent colony type of both serotypes 1 and 2 had at least three antigens: heat-labile and trypsin-sensitive (L), heat-labile and trypsin-resistant (HL), and heat-stable and trypsin-resistant (HS). The L was a major antigen located in a surface and divided serologically into three parts: L1, L2, and L3. The L1 was specifici to serotype 1, the L2 was specific to serotype 2, and the L3 was a common antigen shared by serotypes 1 and 2. The HL and HS were common antigens between serotypes. By trypsinization or heating at 121 C, L antigen lost its agglutinability and agglutinin-producing ability. Nonencapsulated organisms forming noniridescent colony type lacked the L antigen. These results suggested that antigenic structure of H paragallinarum serotypes 1 was L1, L3, HL and HS, while serotype 2 was L2, L3, HL, and HS.     

LEPTOSPIRAL AGGLUTININS IN DOGS IN SYDNEY

From June 1971 to June 1972, sera from 600 dogs in Sydney were tested for leptospiral agglutinins by a rapid slide agglutination method. The end-point titre was taken at 50 percent agglutination of the live organisms. Forty-one samples (6.8 percent) had a significant leptospiral titres (100 or greater) and 5 of these reacted to 2 serotypes. Thirty serums (5 percent) contained agglutinins against L. copenhageni, and 6 (1 percent) against L. pomona, while a few samples reacted against hardjo, tarassovi, australis, grippotyphosa or pyrogenes serotypes. No significant titres were found to L. canicola, L. hebdomadis, L. autumnalis or L. bataviae.     

Polypeptides associated with Pasteurella multocida infection in rabbits.

Polypeptides from whole cell preparations of Pasteurella multocida serotypes A:12 and A:3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Antigens were detected by immunoblot analysis, using sera from 3 groups of rabbits. Sera were obtained from rabbits inoculated intranasally with P multocida serotype A:12 or A:3, from rabbits maintained in a rabbitry with enzootic P multocida A:12 infection, and from rabbits maintained in a rabbitry with enzootic P multocida A:3 infection. Immunoblot analyses of pre- and postinoculation sera from experimentally infected rabbits, using serotype A:12 antigen, revealed 3 polypeptides with approximate molecular mass of 28, 30, and 37 kDa that consistently detected antibodies after P multocida-induced infection. Sera from rabbits naturally infected with either serotype, tested against serotype A:12 and A:3 antigens, detected the same polypeptides in both serotypes. Thus, immunologic reactivity to these polypeptides may be useful for serologic detection of P multocida infection.