Sensitivity of different morphological variants of Leptospira to the leptospirocidal activity of normal animal sera

The leptospirocidal activity of normal animal sera with respect to 23 Leptospira strains was experimentally studied in vitro. 91.3% of the strains under study proved to be sensitive to the lytic action of cattle serum and 86.9%, to sheep serum. The uncinate variants of the pathogenic strains showed resistance to the action of the above sera, and their nonuncinate analogs were subject to agglutination with subsequent lysis, similarly to saprophytes.     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.     

Bacteriostatic activity of serum against staphylococci

Cybulska, Janina (State Institute of Hygiene, Warsaw, Poland), and J. Jeljaszewicz. Bacteriostatic activity of serum against staphylococci. J. Bacteriol. 91:953-962. 1966.-Antistaphylococcal activity of normal serum against strains exhibiting various patterns of coagulase, clumping-factor, and staphylokinase production is not connected with the presence of these factors. Purified coagulase does not influence this property of serum. Coagulase-negative strains with clumping-factor activity grow in normal serum as typical pathogenic staphylococci. Serum bacteriostatic activity against staphylococci may be reversed by several nonspecific factors, such as sterile broth, supernatant fluids of coagulase-negative strains, and ammonium sulfate precipitates of culture supernatant fluids of various staphylococci. Immune sera with a high agglutinating titer for staphylococcal cells do not prevent growth of serum-resistant strains; serum-susceptible strains are inhibited as in normal serum control. Activation or blocking of the serum fibrinolytic system does not influence serum bacteriostatic activity. The growth rate of serum-resistant strains is identical in serum and in Todd-Hewitt broth; serum-susceptible strains are inhibited to the inoculum level, but decreases and increases in viable count are noted during a 24-hr observation period. Observations made with sera of 10 animal species clearly demonstrated differences in serum bacteriostatic activity, mouse serum being completely noninhibitory and cat serum only weakly inhibitory. The technique of quantitative determination of serum susceptibility of staphylococci is described, and the importance of serum antistaphylococcal activity in vitro is discussed. Experimental staphylococcal infection produced in rabbits by intravenous injection of different Staphylococcus aureus strains did not result in significant changes in serum antistaphylococcal activity. The technique of experimental infection used caused chronic infection, with a peak on the 14th day; this was proved by means of a newly developed 5'-nucleotidase test. At the same time, sera of infected animals exhibited slight inhibitory properties, which returned to initial values 1 week later. Infection was produced by strains recognized as nonpathogenic and was inhibited in vitro by sera from both normal and infected rabbits. It is concluded that antistaphylococcal activity of serum should be considered as an "in vitro" phenomenon, which seems to have no importance in defense mechanisms of rabbits infected intravenously with staphylococci.     

Adjuvant effect ofBacillus firmus in intranasal immunization of guinea pigs with inactivated type B influenza virus

Intranasal immunization of guinea pigs with inactivated type B influenza virus plus inactivated Bacillus firmus as an adjuvant compared to the virus alone yields higher titers of serum hemagglutination-inhibiting antibodies and virus-neutralizing antibodies. This phenomenon could be useful in standard serology, especially in the preparation of immune sera against highly pathogenic strains for in vitro diagnosis.     

Serologic distribution of antibodies against adenoviruses in sheep of large-scale farms.

The common soluble antigen of the first subgroup of bovine adenoviruses was used for assaying 793 sheep sera by the agar gel diffusion test. Of the 50 farms included in the study 43 were found infected. The ratio of reacting samples was 73.7% of the sera obtained from infected farms. Virus neutralization tests revealed that a considerable number of sera reacted specifically with all types of ovine adenoviruses, even with serotypes which had never been isolated in Hungary. The results yielded by the agar gel diffusion tests were compared with the results of virus neutralization tests. Of 850 cattle serum samples, agar gel diffusion tests gave positive results in 33.4%. Virus neutralization test was done only with the bovine and adenovirus type 2. No differences could be detected in antibody titres when the prototype strains (No. 19) and the strain isolated from sheep (ORT/111) were compared in parallel titrations. Both ruminant species were found to be infected with hovine adenovirus type 2. Neverthless, inapparent infection with these strains seemed to be less frequent among cattle than in sheep flocks.     

Recombinant ligA for leptospirosis diagnosis and ligA among the Leptospira spp. clinical isolates

Rapid diagnosis for differentiation of leptospirosis from other pyrogenic infections prevailing in the same locality is imperative for proper treatment. During infection, the pathogenic Leptospira spp. express virulence factors which induce antibody responses in the infected host. In this study, 50 referenced Leptospira spp. belonging to six genomospecies and 10 L. interrogans clinical isolates were studied for the presence of a gene encoding an in vivo expressed, surface exposed, immunoglobulin-like protein, LigA, by using PCR and southern hybridization specific to the 5' terminus sequence of the DNA. LigA was also detected in the Leptospira spp. whole cell homogenates by a direct ELISA using a mouse antiserum to the C-terminal portion of recombinant LigA (cLigA) as a detection reagent. All pathogenic Leptospira spp. except one of the two strains of L. santorasai were positive for the gene and its phenotype while all of the L. borgpetersenii and L. biflexa strains were negative. Recombinant cLigA was used as an antigen in ELISAs for detecting IgM and IgG in the sera of leptospirosis patients and in the sera of patients with other febrile illnesses and healthy subjects. When acute phase sera were tested by the cLigA IgM- and IgG-ELISAs, 92% and 100% of the MAT-positive sera were positive, respectively. The diagnostic sensitivity was 100% when both IgM- and IgG-ELISAs were performed on the same acute phase sera and the results were combined. Acute and convalescence sera of patients who were Leptospira culture positive but MAT/IgM-dipstick negative gave 88% and 100% positives by combined cLigA IgM/IgG ELISAs. The diagnostic specificities for the cLigA IgM- and IgG-ELISAs were 98% and 100%, respectively. Our cLigA based-serology has a high potential for early diagnosis of leptospirosis especially when the culture and MAT results are not yet available.     

Evaluation of a recombinant excretory secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay

The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24 kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep.     

Proteome response of an extraintestinal pathogenic Escherichia coli strain with zoonotic potential to human and chicken sera

A subset of extraintestinal pathogenic Escherichia coli is zoonotic and has developed strategies to adapt to different host-specific environments. However, the underlying mechanisms of these adaptive strategies have yet to be discerned. Here, the proteomic response of an avian pathogenic E. coli strain, which appears indistinguishable from neonatal meningitis E. coli, was compared following growth in human and avian sera to determine whether it uses the same mechanisms to overcome the antibacterial effects of sera from different host species. Proteins involved in biosynthesis of iron receptors were up-regulated under both sera, suggesting that serum, regardless of the host of origin, is an iron-limited environment. However, several proteins involved in synthesis of nucleic acids, sulfur-containing amino acids and fatty acids, were differentially expressed in response to the sera from different hosts. Mutational analysis showed that this APEC strain required nucleotide biosynthesis during incubation in human, but not avian serum, and deletion of genes involved in the biosynthesis of sulfur-containing amino acids increased its resistance to human serum. Continued investigation of the proteome of 'zoonotic' ExPEC strains, grown under other 'dual' host conditions, will contribute to our understanding of ExPEC pathogenesis and host specificity and development of effective therapies and control strategies.     

A survey of Clostridium perfringens enterotoxin antibody in human and animal sera in western Canada

Sera from human, cattle, sheep, swine, and horse populations in western Canada were tested for the presence of Clostridium perfringens enterotoxin antibody by the passive hemagglutination (PHA) test, supplemented by an immunodiffusion test and by counterimmunoelectrophoresis. A total of 224 human, 345 cattle, 165 sheep, 620 swine, and 768 horse serum samples were examined. Low-titer reactions in the PHA test were detected in human, cattle, horse, and swine sera, in that order, with no titers demonstrated in sheep. The titers in human sera ranged up to 1:128 and three of these samples were also positive in the other two serological tests. The significance of this antibody is not clear, but it is suggested that the low prevalence of the antibody may reflect a low prevalence of enterotoxigenic C. perfringens strains in western Canada. Such serological surveys may be applicable to epidemiological studies involving enterotoxigenic C perfringens.     

Detection of poliovirus antigens and antibodies: microindirect haemagglutination and haemagglutination inhibition tests for poliovirus types I, II, and III.

Microtitre indirect haemagglutination (IHA) and IHA-inhibition (IHAI) procedures were adapted to determine the reactivities of type I, II, and III poliovirus antibodies and antigens. Glutaraldehyde-fixed sheep erythrocytes were sensitized for these tests with concentrated, partially purified preparations of type I, II, and III poliovirus. Antibody titres by IHA were generally 10 to 100 times greater than serum microneutralization (SN) titres. The SN and IHA reactivities of three kinds of sera were compared. Of these sera, virus type specific antibodies, in monospecific guinea pig sera one week after immunization and in sera from hyperimmunized horses, could be readily differentiated and measured; antibodies in human diagnostic specimens, however, showed some intertypic cross reactivity. Monovalent one-week immune guinea pig sera reacted specifically in the IHAI test to differentiate viruses, and could be used for virus typing and differentiating strains of poliovirus type III.     

Laboratory diagnosis of leptospirosis

Percentage of serological positivity examined in 4205 blood sera by serological method microscopic agglutination test (MAT) on the hinterland territory of our laboratory (East Bohemia; 1999-2003) was 0.38-4.7 %. By the PCR method for detection of DNA of pathogenic leptospires (L. interrogans, L. borgpetersenii and L. kirschneri) from 57 samples of different biological materials from patients with fever of unknown etiology positive results were obtained in 4 specimens (7 %; 3 samples of urine and 1 sample of blood). This method was shown to distinguish between pathogenic and nonpathogenic strains and can detect 2.5-10 cells per mL of biological material. As an important presumption of successful detection of pathogenic leptospires a correct collecting of blood, urine samples or liquor is required before starting antibody therapy. The PCR method possesses a clear advantage over other methods, such as MAT, which relies on the detection of antibodies the presence of which cannot be detected until days after infection.     

The sheep antibody response to repeated infection with Lucilia cuprina.

, , and 1992. The sheep antibody response to repeated infection with Lucilia cuprina. International Journal for Parasitology 22: 1169–1174. The specific serum antibody responses of sheep exposed to 10 consecutive infections of L. cuprina have been analysed by enzyme-linked immuno-sorbent assay and immunoblotting using monoclonal antibodies specific for sheep immunoglobulin isotypes. Recognition of a number of larval excretory-secretory products by IgM antibodies appeared to be non-specific. IgG₁ was the major antibody class stimulated by the infection protocol and marked increases in antibody to specific excretory-secretory antigens were observed. Three molecules of 35, 30 and 25 kDa were particularly recognized although the extent of recognition of these molecules varied considerably between individual sheep serum. A pooled serum composed of sera collected after five to seven infections significantly inhibited larval growth in in vitro cultures when compared to a sera pool consisting of sera collected both prior to infection and after infections 1 and 2. The degree of inhibition was greater when serum with high specific antibody titre was used.     

Experimental Infection with Parent and L-Phase Variants of Neisseria meningitidis

Thirty-six strains of Neisseria meningitidis, including groups A, B, and C, produced L forms in vitro in the presence of an osmotic stabilizer and high concentrations of horse serum using penicillin as the transforming agent. Transformation to L growth occurred most readily among strains recently isolated from patients, and an unusually high rate of transformation was observed in 7 of the 36 strains. Revertant L strains developed diplococcal colonies on blood-agar and L colonies on sucrose-serum-penicillin-agar-always in a ratio of approximately 10 to 100 diplococcal colonies to 1 L colony. Using mucin as a host depressant, comparison was made between parent and revertant L strains of their initial pathogenicity and development of virulence by serial mouse passage. In general, revertant L strains showed the same pathogenic characteristics as the parent. Heart blood cultures from mice dying of infection with revertant L strains retained their ability to grow as L forms on penicillin media. Three stable L strains were completely avirulent for mice, although persistence of L forms could be demonstrated in peritoneal exudate for 6 days after inoculation.     

Toxoplasmosis II. Studies of Animal Sera Using Complement-Fixation Tests

Serological studies were performed in guinea pigs, a sheep, calf, goat and two pigs experimentally infected with toxoplasmosis. The direct complement-fixation method was effective in detecting antibodies in guinea-pig, goat and sheep sera. The modified complement-fixation technique supplementing complement with normal bovine serum fraction, was required when testing bovine serum. With swine sera best reactions occurred in the indirect complement-fixation test and definite but low grade reactions were produced in the direct test after pro-complementary activity was removed by pH treatment of the sera. Allergic skin reactions were produced in the experimental animals but improvement in the antigen is necessary before the test could be used generally in the field as a diagnostic method for animal toxoplasmosis.     

Echinococcus granulosus: Isolation and characterization of sheep hydatid fluid antigens

Five principal antigens are identifiable by immunoelectrophoresis using specific sera from rabbits immunized with sheep hydatid fluid and absorbed by polymerized sheep serum.     

Development of a western blotting assay to discriminate Brucella spp. and Yersinia enterocolitica O:9 infections in sheep

Outer membrane proteins (OMPs) of Rev-1 strain of Brucella melitensis were used in a Western blotting assay for the serological diagnosis of brucellosis in ovine sera. Fifty-four sheep sera were tested and divided into the following groups: Group A) n. 9 samples from one sheep that had been experimentally infected with Y. enterocolitica O:9; Group B) n. 10 samples collected from sheep infected with Brucella melitensis and 1 sample from a sheep vaccinated with the Rev 1 strain; Group C) n. 10 samples collected in "officially brucellosis-free" herds; Group D) n. 12 samples classified as "suspicious"; Group E) n. 12 samples classified as "positive". Antibodies were detected by routine tests performed for the diagnosis of brucellosis in serum samples of the sheep infected with Y. enterocolitica O:9 after the 2nd week post infection. In the WB assay, sera of group B recognised a 17 kDa protein, whereas sera of groups A, and D and 9 out of 12 of group E exhibited no reactivity to this protein. The results obtained encourage the use of the WB assay as a confirmatory test for the diagnosis of brucellosis.     

An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha and EQ1

AIMS: To examine Escherichia coli strains EQ1, DH5alpha, BLR and BL21 for known pathogenic mechanisms. METHODS AND RESULTS: Using specific DNA probes, the strains were shown not to carry the genes encoding invasion, various adhesion phenotypes or expression of a range of enterotoxins. The strains were unable to express long-chain lipopolysaccharide and were susceptible to the effects of serum complement. Using a BALB/c mouse model, the strains were shown to be unable to survive in selected tissues or to persist in the mouse gut. Using a chick model, strains EQ1, BLR and BL21 invaded livers but not spleens; only strain EQ1 persisted in the chick gut. In Merino sheep, only strain EQ1 was detected 6 d post-infection. CONCLUSIONS: Escherichia coli strains EQ1, DH5alpha, BLR and BL21 did not carry the well-recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli strains EQ1, DH5alpha, BLR and BL21 were considered to be non-pathogenic and unlikely to survive in host tissues and cause disease.