The serological diagnosis of bovine brucellosis: an evaluation of the complement fixation, serum agglutination and rose bengal tests

In a collaborative investigation in which 4 laboratories took part, the Rose Bengal Test (RBT), Serum Agglutination Test (SAT) and 4 different Complement Fixation (CFT) techniques were evaluated in selected cattle for the diagnosis of bovine brucellosis, by comparing the results they gave with the bacteriological examination of a selection of lymph nodes taken from the same animals at slaughter. The RBT correctly classified all but 1 of 79 culture-positive cattle, but was more often positive in culture-negative animals than the other tests. The RBT may be most useful as a screen test. 11% of the culture-positive cattle had SAT titres below 100 iu and almost 4% of them had less than 30 iu. However, the SAT was more effective in cattle vaccinated with the 45/20 vaccine. The CFT, in 1 of 4 techniques used, identified all of the culture-positive cattle at a serum dilution of 1/4 or above and was considered to be far superior to the SAT as a diagnostic test, except perhaps in cattle vaccinated with the 45/20 vaccine.     

Mechanomyography and oxygen consumption during incremental cycle ergometry

Brucella abortus strain RB51 was recently approved as an official brucellosis calfhood vaccine for cattle by the Animal and Plant Health Inspection Service branch of the United States Department of Agriculture. Currently available serologic surveillance tests for B. abortus do not detect seroconversion following SRB51 vaccination. The purpose of this study was to evaluate a dot-blot assay using gamma-irradiated strain RB51 bacteria for its specificity and sensitivity to detect antibody responses of cattle vaccinated with strain RB51. Dot-blot titers of sera at a recommended dosage (10(10) colony-forming units) were similar to those of sera from cattle vaccinated with similar numbers of B. abortus strain 19 and greater (P < 0.05) than titers of nonvaccinated cattle. In the first 12 weeks after vaccination with 10(10) colony-forming units of strain RB51, the RB51 dot-blot assay had 100% specificity for titers of 80 or less and a 53% sensitivity for titers of 160 or greater. Sensitivity of the RB51 dot-blot assay peaked at 4 weeks after vaccination with 10(10) colony-forming units of strain RB51. Dot-blot responses of sera from cattle vaccinated with a reduced dosage of strain RB51 (10(9) colony-forming units) did not differ (P > 0.05) from titers of sera from nonvaccinated cattle. Following intraconjunctival challenge with B. abortus strain 2308, titers on the RB51 dot-blot assay did not differ (P > 0.05) between nonvaccinated cattle and cattle vaccinated at calfhood with strain 19 or strain RB51.     

Bacterial survival, lymph node pathology, and serological responses of bison (Bison bison) vaccinated with Brucella abortus strain RB51 or strain 19

From August 1993 to June 1994, 3 month-old bison (Bison bison) were vaccinated with Brucella abortus strain RB51 (SRB51, n = 6), strain 19 (S19, n = 3), or with saline (n = 1) and serologic responses and persistence of vaccine strains within lymph nodes were monitored. Bison vaccinated with S19 had granulomatous lymphadenitis and greater peak numbers of B. abortus than those vaccinated with SRB51. Bison vaccinated with RB51 had similar histological lesions and B. abortus were still present in lymph nodes at 16 weeks. Although antibodies against RB51 were produced, standard tube agglutination test responses of RB51-vaccinates remained negative. The histological lesions of B. abortus infections in bison were similar to those observed in cattle, but bison did not clear SRB51 as rapidly as cattle.     

Fee code creep among general practitioners and family physicians in Ontario: why does the ratio of intermediate to minor assessments keep climbing?

Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939-1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen.     

Effect of P39 gene deletion in live Brucella vaccine strains on residual virulence and protective activity in mice

The 39-kilodalton protein (P39) has previously been shown to be an immunodominant protein in Brucella infections. P39 gene deletion mutants of vaccine strains Brucella abortus S19 and Brucella melitensis Rev.1 were constructed by gene replacement. This deletion did not significantly modify the residual virulence of both vaccine strains in CD-1 mice. CD-1 mice vaccinated with the parent or mutant strains were protected against a virulent challenge. Mutant vaccine strains devoid of P39 could provide a means for differentiating vaccinated from infected animals.     

Studies on in vitro synthesis and secretion of human chorionic gonadtropin and its subunits

Data from cattle herds infected with brucellosis and from control (noninfected) herds were collected and analyzed using case control techniques. It appeared that herds located close to other infected herds and those herds whose owners made frequent purchases of cattle had an increased risk of acquiring brucellosis, particularly those who made purchases from other herds or from cattle dealers. Infected herds had a lower level of vaccination than noninfected herds. However, the percentage vaccinated was highly variable in each group. Vaccination per se did not appear to adversely influence the interpretation of serological test results nor did it appear to protect the individual animal. Once infected, the time required to become free of brucellosis was increased by large herd size and/or loose housing. Closed herds also took longer to become brucellosis free than more open herds. The percentage of animals removed from the herd was increased by active abortion. Those herds with multiple serological reactors (positives and questionables) at the first herd test after the imposition of quarantine had the highest percentage of cattle removed.     

The persistence of genetic homogeneity among Trypanosoma brucei rhodesiense isolates from patients in north-west Tanzania

The decrease in titer of PRV antibodies in serum was evaluated at 10, 37, 67, 109 and 173 days of age in 16 non-vaccinated pigs and 43 pigs vaccinated at 3, 67 and 80 days of age with a modified live TK/gIII gene deleted pseudorabies virus (PRV) vaccine. Serum samples were analyzed for antibodies to PRV by the serum-virus neutralization test (SN), a commercial competitive ELISA (CELISA), and the CELISA OMNIMARK PRV differential (OMD) diagnostic kit. At 10 days of age, all pigs had SN titers > or = 1:4 and were CELISA+/OMD+, indicating circulating antibodies to field strains of PRV. At 109 days, all non-vaccinated pigs had SN titers < 1:4. Forty-five percent of vaccinated pigs had SN titers > or = 1:4, 56% were CELISA positive and most were CELISA+/OMD-, indicating antibodies due to vaccination. At 24 weeks of age, all pigs had SN titers > or = 1:4 and were CELISA+/OMD+ due to exposure to field strains. Although circulating maternal antibodies interfere with the development of active immunity, vaccination at 3 days of age resulted in detectable antibodies by 67 days of age, and a limited immune response could be measured at 109 days of age.     

Determination of stability of Brucella abortus RB51 by use of genomic fingerprint, oxidative metabolism, and colonial morphology and differentiation of strain RB51 from B. abortus isolates from bison and elk

Brucella abortus RB51 and isolates from cattle, bison, and elk were characterized by pulsed-field gel electrophoresis and standard techniques for biotyping Brucella species, which included biochemical, morphological, and antigenic techniques, phage susceptibility, and antibiotic resistance. The objectives were to ascertain the stability of RB51 and to differentiate RB51 from other brucellae. Genomic restriction endonuclease patterns produced by pulsed-field gel electrophoresis demonstrated a unique fingerprint for RB51 relative to other brucellae. Comparisons of the oxidative metabolic profiles of RB51 after time in vivo (14 weeks) and in vitro (75 passages) showed no change in characteristic patterns of oxygen uptake on selected amino acid and carbohydrate substrates. Strain RB51 was biotyped as a typical rough B. abortus biovar 1 (not strain 19) after animal passage or a high number of passages in vitro and remained resistant to rifampin or penicillin and susceptible to tetracycline. No reactions with A or M antiserum or with a monoclonal antibody to the O antigen of Brucella lipopolysaccharides were detected; however, RB51 agglutinated with R antiserum. The results indicate that the genomic fingerprint and rough colonial morphology of RB51 are stable characteristics and can be used to differentiate this vaccine strain from Brucella isolates from cattle, bison, and elk.     

Sensitivity and specificity of serological and bacteriological tests for contagious bovine pleuropneumonia

In 1990 an outbreak of contagious bovine pleuropneumonia (CBPP) occurred in Italy. Subsequent surveillance for CBPP was based on random sampling in bovine herds, serological controls on all animals moved from the herd of origin and controls on slaughtered animals. Official tests employed were the complement fixation test (CFT) and bacteriological isolation and typing. A total of 33,856 serum samples collected from herds in CBPP-free regions were used to define CFT specificity, while samples from 595 animals from infected herds were employed to define the sensitivity. Ninety-nine animals from three infected herds were used to estimate the sensitivity of the isolation technique. Results showed the specificity of CFT (threshold +1:10) to be 98% and sensitivity to be 63.79%. The sensitivity of the test did not change significantly, regardless of whether the lesions were caused by acute or chronic infection. The sensitivity of the isolation technique was 54.1%.     

The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle

A diagnostic assay to differentiate antibodies induced by foot-and-mouth disease virus (FMDV) infection from those induced by vaccination was developed. The test is an indirect-trapping ELISA which uses a monoclonal antibody to trap the non-structural 3ABC-FMDV polypeptide expressed in E. coli. Experimental and field sera from naive, vaccinated and infected cattle were examined. Using the established threshold of 0.20 optical density units, the sensitivity of the assay was 100%, as all the experimental post-infection sera (n degree = 137) gave values greater than this threshold, irrespective of the FMDV serotype used for the infection. In contrast, more than 99% of sera from vaccinated animals were negative (225 out of 228 primo-vaccinates and 159 out of 159 multi-vaccinates). A high degree of specificity was also confirmed by the finding that 99.5% (442 out of 444) of sera from naive animals gave negative results. Serum conversion against 3ABC was first detected 8 days post-infection and demonstrable levels of 3ABC specific antibodies were detectable at least 1 year post-infection. The described 3ABC-ELISA is safe, cheap and also easy to perform in large scale serological surveys. The high specificity and sensitivity makes this test an ideal tool for FMD eradication campaigns and control programs.     

Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice

A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus

The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins. The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave a positive result in both the 3AB and the 3ABC ELISA's. Two cattle that had been both vaccinated and infected also gave positive results in both tests, suggesting that the 3AB and 3ABC ELISA's, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population.     

Adaptation and validation of antibody-ELISA using dried blood spots on filter paper for epidemiological surveys of tsetse-transmitted trypanosomosis in cattle

The indirect enzyme-linked immunosorbent assay (ELISA) for the detection of anti-trypanosomal antibodies in bovine serum was adapted for use with dried blood spots on filter paper. Absorbance (450 nm) results for samples were expressed as percent positivity, i.e. percentage of the median absorbance result of four replicates of the strong positive control serum. The antibody-ELISA was evaluated in Zambia for use in epidemiological surveys of the prevalence of tsetse-transmitted bovine trypanosomosis. Known negative samples (sera, n = 209; blood spots, n = 466) were obtained from cattle from closed herds in tsetse-free areas close to Lusaka. Known positive samples (sera, n = 367; blood spots, n = 278) were obtained from cattle in Zambia's Central, Lusaka and Eastern Provinces, diagnosed as being infected with Trypanosoma brucei, T. congolense, or T. vivax using the phase-contrast buffy-coat technique or Giemsa-stained thick and thin blood smears. For sera (at a cut-off value of 23.0% positivity) sensitivity and specificity were 86.1 and 95.2%, respectively. For bloodspots (at a cut-off value of 18.8% positivity) sensitivity and specificity were 96.8 and 95.7%, respectively. The implications of persistence of antibodies following treatment or self-cure are discussed.     

Sensitivity and specificity of antigen-capture ELISAs for diagnosis of Trypanosoma congolense and Trypanosoma vivax infections in cattle

Sensitivity and specificity of the FAO/IAEA antigen-ELISA kits for diagnosis of bovine trypanosomosis were investigated using sera from experimental cattle infected by tsetse challenge with cloned populations of Trypanosoma congolense (three populations) or T. vivax (one population). The kits are based on monoclonal antibodies that recognise internal antigens of tsetse-transmitted trypanosomes. Ten cattle were infected with each trypanosome population for at least 60 days, and in combination with uninfected cohorts (n = 16) were used in a double-blind study design. Sensitivity and specificity of the tests depended on the choice of positive-negative thresholds expressed as percent positivity with respect to the median OD of four replicates of the strong positive reference serum provided with the kit. In general, while overall specificities were high, sensitivities of the antigen-ELISAs were poor. For example, at a cut-off of 5% positivity, the sensitivities of the antigen-ELISAs were 11% for samples (n = 1162) from T. congolense infected cattle (n = 30), and 24% for samples (n = 283) from T. vivax infected cattle (n = 10). The corresponding specificity values were 95% and 79%, respectively. At a cut-off of 2.5% positivity sensitivity for T. congolense was 25%, and for T. vivax 35%; corresponding specificity values were 85% and 63% respectively. There were no values of the positive-negative threshold at which both sensitivity and specificity were satisfactory. Restricting the analyses to samples taken more than 2 weeks after tsetse challenge did little to improve sensitivity estimates. Trypanosome species specificities of the antigen-ELISAs were also poor. Sensitivity and species specificity of the antigen-ELISA for Trypanosoma brucei infections were not investigated. In contrast to the antigen-ELISA, the sensitivity of the buffy-coat technique when applied to the same experimental animals was fairly high at 67% for T. congolense infections and 60% for T. vivax infections. For samples taken more than 2 weeks after tsetse challenge, high sensitivity estimates of 96% for T. congolense and 76% for T. vivax infections were obtained.     

Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes

Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At approximately 1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61beta but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61beta- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER.     

Development of a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot for detection of CMV-specific IgM

We developed a new cytomegalovirus (CMV) immunoglobulin M (IgM) immunoblot to detect CMV-specific IgM in human sera. The new test contains four viral proteins (vp150, vp82, vp65, and vp28) purified from viral particles and four recombinant proteins (rp150, rp130, rp52, and rp38) purified from Escherichia coli. These antigens were individually loaded onto nitrocellulose strips, and the strips were then used to detect CMV-specific IgM by using a mu-specific conjugate. The new assay was evaluated in parallel with one or two IgM enzyme-linked immunosorbent assays (ELISAs) to test 592 serum samples from different groups of latently or acutely infected individuals. The sensitivity of the new assay with respect to the consensus of two ELISAs was 100%, the specificity was 98.6%, the positive predictive value was 96.9%, and the negative predictive value was 100%. We also evaluated the new test by testing sera from pregnant women and transplant recipients with a known clinical history. Our results suggest that the new test combines high sensitivity with high specificity, characteristics that are mutually exclusive with the other commercially available tests. Furthermore, a statistically significant correlation was observed between the number of IgM-reactive bands and the elevated risk of transmission from CMV-infected pregnant women to their offspring.     

Large-scale use of liquid-phase blocking sandwich ELISA for the evaluation of protective immunity against aphthovirus in cattle vaccinated with oil-adjuvanted vaccines in Argentina

Specific serum activity levels against four reference strains of foot-and-mouth disease virus (FMDV) were evaluated from 1634 animals vaccinated with commercial quadrivalent oil vaccines and from 746 unvaccinated, naive animals, using the liquid-phase blocking sandwich ELISA (lpELISA) test. Cows from the FMDV-free area of Argentina were tested for the absence of specific FMDV antibodies (sp FMDV Abs) and those showing lpELISA titres < 1.0 were grouped in lots of 16 animals. They were vaccinated and challenged at 90 days postvaccination (DPV) with one of four virus strains used for vaccine production and control (prototype strains). Serum samples from vaccinated and control cattle were collected 60 and 90 DPV and the level of sp FMDV Abs was determined by lpELISA. Animals were examined for clinical signs of disease. Results show that serum lpELISA titre levels directly correlate with the percentage of protected animals. It was seen that 100, 98, 93 and 87% of the vaccinated cattle with antibody titre levels > or = 2.1 were protected against challenge with serotypes C85, A87,01 Cas and A79, respectively. Evidence is also presented of seroconversion in a sample of 3-5-month-old calves vaccinated in the field, showing lpELISA titres compatible with protection against the four vaccine viruses as long as 150 DPV. Results reported in this paper strongly support the use of the lpELISA test for a rapid and reliable evaluation of the efficacy of FMDV commercial vaccines as well as for the assessment of the immunological status of cattle in FMDV-free and enzootic regions of South America.     

Characterization of recombinant adeno-associated virus-2 as a vehicle for gene delivery and expression into vascular cells

Investigations into the incidence of Salmonella abortus ovis (S. abortus ovis) infections should not be neglected in diagnosis of ovine abortion cases. For detection of this pathogen agent, direct cultivation, as well as pre-enrichment combined with enrichment procedures in tetrathionate or Rappaport-Vassiliadis medium, should be performed with respect to the features of S. abortus ovis. The detection limit of S. abortus ovis using pre-enrichment and enrichment media could be determined using 6.5 x 10(3)-6.5 x 10(4) bacteria. For subsequent cultivation of S. abortus ovis Gassner, XLD or Rambach agar are suitable. In infected sheep showing no excretion of S. abortus ovis, the pathogen can be detected by serological studies using microagglutination or the ELISA test. The ELISA test proved to be more sensitive than the microagglutination test, detecting antibodies against S. abortus ovis in 17% of the 814 sheep tested. The microagglutination test revealed positive results in only 2% of the sheep tested.     

Serological study of the occurrence of L. hardjo in sheep in The Netherlands

Sheep (3918) from 137 farms in the regions of North-, West- and Mid-Netherlands and Gelderland were serologically investigated for the presence of antibodies against Leptospira hardjo. Antibodies were detected in 3.3% of the sheep. There were large regional differences with respect to both the percentage of positive sheep and the percentage of positive flocks. All sera from sheep in Gelderland were negative. In West- en Mid-Netherlands there were 0.9 and 6.5% positive sera, respectively, and 19.4 and 32.7% positive flocks. The percentage of positive sheep per positive flocks varied from 1 tot 51.6. Serological positive sheep were from farms with and without cattle. On farms with cattle, there was no clear relationship between serologically sheep and the presence of hardjo antibodies in cattle. It thus seems plausible that sheep can be infected with hardjo independently of cattle.