Use of dried blood on filter paper in the ELISA for Brucella abortus.

An ELISA for Brucella abortus antibody detection using blood collected on filter paper is described. The method gave similar results to the complement fixation test. The signal-noise ratio was good. The system offers considerable advantages when transport of serum samples to the laboratory causes problems.

Q fever is absent from New Zealand.

To investigate the presence of Coxiella burnetii in sheep and cattle, the two major ruminant populations of New Zealand, its seroprevalence was determined in aborting cattle and sheepdogs. These groups of animals were chosen because of their accessibility and the fact that they would be good indicators for the presence of the organism. A total of 2181 bovine and 12,556 canine samples were all seronegative. On the basis of these results and previous reports it is argued that New Zealand is free of coxiellosis or Q fever.

Restriction endonuclease analysis and plasmid profiling of Actinobacillus pleuropneumoniae serotype 7 strains.

Seventeen serotype 7 Actinobacillus pleuropneumoniae strains isolated in New Zealand and A. pleuropneumoniae serotypes 1-12 reference strains were typed by restriction endonuclease analysis of chromosomal DNA and plasmid profiling. All serotype 7 strains produced similar DNA cleavage patterns and were significantly different to other reference serotype strains. Minor differences in the cleavage patterns enabled the 17 serotype 7 strains to be grouped into seven profiles. Plasmids were identified in all but three strains but the banding patterns did not account for the differences in the chromosomal profiles. The study showed that restriction endonuclease analysis and plasmid profiling are useful in epidemiological studies of porcine pleuropneumonia.

Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 II the use of Yersinia outer proteins for the specific detection of Yersinia enterocolitica infections in ruminants.

Yersinia outer protein (YOP) preparations from Y. enterocolitica and Y. pseudotuberculosis were used as antigens in immunoblots for the detection of Yersinia infections in experimentally and naturally infected ruminants. Sera from 9 groups of animals were used: (1) 51 sera from cattle which were false-positive in the standard brucellosis serological tests, (2) 52 sera from brucellosis-negative cattle, (3) 51 sera from a deer herd in which 16 animals were positive in the brucellosis tests and Yersina species were isolated from 5 animals, (4) 50 sera from a deer herd in which sera from all animals were negative in the brucellosis tests, (5) 107 sera from brucellosis-negative cattle which were received from throughout New Zealand, (6) 30 sera from cattle naturally infected with B. abortus and from which B. abortus was isolated, (7) 55 sera from cattle naturally infected with B. abortus, (8) 26 sera from cattle experimentally infected with B. abortus, with mostly high titres in the conventional brucellosis tests, and (9) sera taken weekly from 3 cattle experimentally infected with Y. enterocolitica 0:9. In all 3 Y. enterocolitica 0:9 experimentally infected animals the antibody reactivity against major YOPs in the Y. enterocolitica and in the Y. pseudotuberculosis YOP preparation correlated well with the strength in the classical brucellosis tests and with the staining of smooth lipopolysaccharides (SLPS) in blots, thus confirming the usefulness of YOPs for the detection of Yersinia infections. Sera from naturally infected cattle and deer herds, regardless of whether they were false positive or negative in the brucellosis tests, showed high frequencies of staining in YOP blots (53-58% in cattle and 80-100% in deer), indicating a high prevalence of field infections with Yersinia species in New Zealand. In two of the three sera groups from B. abortus infected animals, antibodies against YOPs were detected with high frequency, showing that dual infections may be common and may interfere with differential serological testing.

Comparison of a complement fixation test, a gel diffusion test and two absorbed and unabsorbed ELISAs for the diagnosis of paratuberculosis in sheep.

A complement fixation test for paratuberculosis, a gel diffusion test and two enzyme-linked immunosorbent assays (ELISA) were evaluated using sera from Mycobacterium paratuberculosis infected and non-infected sheep. Gross pathology and histopathology were used as parameters of infection. The two ELISAs, one of which is commercially available for testing cattle, were used before and after sera had been absorbed with a soluble sonicate of Mycobacterium phlei. Differences between the various tests and between ELISAs before and after absorption were non-significant (P > 0.05) in non-infected sheep or in animals with gross or histopathological lesions. The specificity of all the tests was at least 97%. Sensitivity in histopathologically positive sheep was at least 98%. Sheep from infected flocks but without histopathological lesions showed serological results which were poorly correlated between the various tests.

Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 I immunoblot analysis of the antibody response to Brucella protein antigens in bovine brucellosis.

Sera from three groups of Brucella abortus infected cattle were examined in immunoblots with the following antigens: sodium dodecyl sulfate/mercapto ethanol (SDS/ME) extracts of two rought B. abortus strains (45/20 and RB51) and rough B. ovis, smooth lipopolysaccharides (SLPS) from B. abortus strain 99 and Y. enterocolitica 0:9, and a cytoplasmic extract from smooth B. abortus strain 19-S. The sera groups were: (1) 26 sera from animals, experimentally infected with B. abortus strain 544, which were all positive in the conventional brucellosis serological tests; (2) 152 sera from naturally infected cattle herds with varying titres in the conventional brucellosis tests, and (3) 30 sera from naturally infected cattle with varying titres in the conventional brucellosis tests and from which B. abortus was cultured. B. abortus strain 99 and Y. enterocolitica serotype 0:9 SLPS staining showed up frequently in all sera groups and correlated well with the strength in the conventional brucellosis tests, confirming the immunodominance of SLPS in B. abortus infections. Another immunodominant component of 50-80 kDa was found in the rough B. abortus 45/20 antigen preparation but not in the B. abortus RB51 and in the B. ovis cell extracts. This component was also recognised by sera from Y. enterocolitica 0:9 infected cattle and is probably a protein-lipopolysaccharide complex. Although many of the sera from B. abortus infected cattle with high titres in the conventional brucellosis tests showed complex protein staining patterns in blots, no protein bands other than the 50-80 kDa bands were found to be immunodominant.

Sensitive silver-staining detection of bacterial lipopolysaccharides in polyacrylamide gels.

For sensitive detection of bacterial lipopolysaccharides in the nanogram range, three almost identical silver-staining methods are often used, which are based on ammoniacal silver solutions and an acidic developer. We modified a method used for proteins, based on neutral silver nitrate solution and an alkaline developer, for the visualization of lipopolysaccharides in polyacrylamide gels, which yields better sensitivity and is much less prone to formation of non-specific background staining than the acidic developer-based silver stains.

A sensitive immunoblotting technique for the serodiagnosis of Brucella ovis infections.

A simplified electrophoretic immunoblotting technique based on antigen extracted from Brucella ovis cells with sodium dodecyl sulfate/mercaptoethanol was compared with the complement fixation test (CFT), the enzyme-linked immunosorbent assay, and the gel diffusion test. Sera from 89 chronically infected, semen culture-positive rams, 378 sera from B. ovis-infected flocks, 300 sera from accredited disease-free flocks, and 29 sera from specific-pathogen-free sheep were used. The immunoblotting technique had sensitivity and specificity comparable to those of the standard tests and was able to identify several CFT-negative or -borderline sera as positive. The major immunoreactive antigens of B. ovis had molecular masses of 63, 29, 19 kD (proteins) and 8-12 kD (rough lipopolysaccharide). Antibodies against these antigens were present in 96% of CFT-positive sera from infected flocks and in 100% of sera from semen culture-positive rams. However, immunoblotting also identified antibodies to components other than the major antigens in 1% of CFT-negative sera from infected flocks and in 7.7% of the sera from flocks with a history of freedom from the disease. These reactions probably represent cross-reactivities with other microorganisms and were distinguishable from truly positive reactions.

Identification and characterization of immunodominant antigens during the course of infection with Brucella ovis.

The seroresponse against Brucella ovis of 8 intrapreputially and 6 intravenously infected rams and 9 ewes infected through mating was analyzed by electrophoretic immunoblotting. Additionally, 87 sera from chronically infected rams that were shedding B. ovis in their semen, 226 sera from rams belonging to infected flocks, and 324 sera from false-positive complement fixation test (CFT) reactors were examined. In all infected animals, antibody reactivity was predominantly found against 5 B. ovis components of 8-12, 17, 19, 29, and 63 kD, of which the 29-kD antigen was most dominant in the seroresponse. Antibodies to the 29-kD component were present in 93-100% of the infected sheep in each infected group, whereas the frequency of antibodies to the 4 other components varied considerably among and within the different groups. No reactivity against the 29-kD antigen was found in the false-positive CFT reactors. By using monoclonal antibodies against known bacterial macromolecules, the immunodominant antigens were identified as rough lipopolysaccharide (8-12 kD), outer membrane proteins (17, 19, 29 kD), and a heat-shock protein (63 kD).

Virulence of five live vaccines against avian infectious laryngotracheitis and their immunogenicity and spread after eyedrop or spray application.

Five live virus vaccines against avian infectious laryngotracheitis were studied with regard to safety, immunogenicity and route of administration. Significant differences in virulence between the vaccine strains were found. Reduced virulence was accompanied by a reduction of immunogenicity and capacity to spread. After eyedrop application, a low virulent vaccine induced 90-100% flock immunity for the first 10 weeks after vaccination (PV), followed by a slow decline to 50% at 31 weeks PV, whereas flock immunity induced with the more virulent types remained at about 90% till the end of the experiments (24 and 48 weeks PV). Aerosol vaccination induced 70-100% flock immunity but vaccine reactions were severe. Application of vaccine in a coarse spray did not result in adverse vaccine reactions but induced a maximal protection rate of only 50%. Microneutralisation titres provided a useful indicator of immunity from the onset of immunity until immunity started to decline. A vaccine virus carrier state was demonstrated by means of sentinel birds.

Eradication of Brucella ovis from the Falkland Islands 1977-1993.

A campaign to eradicate Brucella ovis from sheep in the Falkland Islands, using a combination of serological tests and culling, was initiated in 1977 and continued until 1993 when, after a total of 65,266 tests had been carried out, the organism had been eradicated from the national flock. The paper discusses the relative values of the serological tests and suggests guidelines for maintaining the flock free of the infection.

[Indications for the circulation of lentogenic Newcastle disease field virus in broiler chicks]. / Aanwijzingen voor het circuleren van lentogeen NCD-veldvirus in slachtkuikens.

Haemagglutinin of nine out of eighteen lentogenic isolates from broilers showing respiratory problems was more stable than that of the La Sota strain (licensed for vaccination) and the Hitchner B1 strain (licensed for production but not for vaccination). This is indicative of the circulation of a lentogenic field virus during the period of isolation (1979-1981), the origin of which is not directly attributable to vaccination. The possible origin of this field virus is discussed.

[Susceptibility of birds other than chickens to infectious laryngotracheitis]. / Vatbaarheid voor infectieuze laryngotracheïtis van andere vogels dan kippen.

Susceptibility to infectious laryngotracheitis virus was studied in peafowl (Pavo cristatus), various species of pheasant (Phasianus colchicus, Lophura swinhoeii, Lophophorus impejanus), guinea-fowl (Numida meleagris), canaries (Serinus canaria), budgerigars (Melopsittacus undulatus) and Japanese quail (Coturnix coturnic japonica). Apart from clinical observations, experiments were evaluated in terms of histopathology, immunofluorescence, serology and recovery of virus. Only peafowl and pheasants were found to be susceptible, pheasants responding more strongly than chickens to ocular vaccination and intratracheal inoculation. The other species were found to be refractory.

[A microtitration and neutralization test for infectious laryngotracheitis virus. Their reproducibility and the effect of various tests conditions]. / Een microtitratie en -neutralisatietest voor infectieuze Laryngotracheïtisvirus. Hun reproduceerbaarheid en de invloed van verschillende testvoorwaarden.

The reproducibility of micro-assays for ILTV was assessed. The standard deviation of the virus assay in three successive determinations was 0.4, 0.4 and 0.3 log TCID50. The micro-neutralisation test produced standard deviations of 0.6, 0.8, 0.5, 1.2 and 1.1 log2 in various experiments. Preincubation for 24 hours at 39 degrees C, rather than for sixty minutes at 22 degrees C, which was suggested by Bitsch to enhance sensitivity, resulted in antibody titres which were approximately 1 log2 higher, though the specificity of the test declined markedly. The dose-response relationship between virus and antibody titre was curvilinear, the steepest part of the slope being in the lower range. Thermal inactivation and freezing and thawing resulted in a significant reduction of serum titres (P less than 0.01). The following method is suggested as a practical standardised procedure: freezing of sera prior to processing: omission of thermal inactivation and preincubation for sixty minutes at 22 degrees C.

Use of the double immuno gel diffusion test and the enzyme-linked immunosorbent assay to distinguish false from true reactors in the complement fixation test for Brucella ovis.

A gel diffusion test with sonicated Brucella ovis antigen and an enzyme-linked immunosorbent assay based on heat-extracted antigen were used to distinguish false from true reactions in a complement fixation test based on heat-extracted antigen. Of 142 complement fixing reactors (occurring in supposedly Brucella ovis-free, accredited flocks), the gel diffusion test correctly identified the status of 139 animals as compared to 128 with the enzyme-linked immunosorbent assay. A combination of the two methods resulted in a correct identification of 141 animals. The procedures provide an easy, cheap and quick way to determine the true status of reactors that show up during routine use of the complement fixation test in Brucella ovis re-accreditation procedures.

Antibodies in dogs against Leptospira interrogans serovars copenhageni, ballum and canicola.

In a nationwide survey carried out during 1990-91 of more than 5800 dogs to detect antibodies against Leptospira interrogans serovars copenhageni, ballum and canicola, only one weak reactor against serovar canicola was found. Reactors of varying titre were found against serovar ballum in 0.7% of dogs tested, indicating sporadic infection with this serovar. Reactors (0.9%) to serovar copenhageni came mainly from the Waikato, Northland and the Auckland region. This was in agreement with the reported occurrence of the clinical syndrome and with the results of a smaller survey in urban Auckland, in which more than 5% of dogs tested were seropositive to serovar copenhageni.

Drinking water vaccination against infectious laryngotracheitis.

Chickens varying in age from ten days to five years were vaccinated with 10(1.3), 10(2.3) and 10(3.3) EID50 per bird of a commercial infectious laryngotracheitis drinking water vaccine. The vaccine gave no adverse reaction in the dose range tested. Five weeks after administration of 10(3.3) EID50 per bird 70% were protected against the intratracheal challenge with virulent infectious laryngotracheitis virus. Doses of 10(1.3) and 10(2.3) EID50 per bird did not give protection. No serological response could be detected by the neutralization test even in the group that had received 10(3.3) EID50 per bird. No contact spread of virus was detected from 14 days post-vaccination. Carriers of vaccine virus could not be demonstrated.

Enzootic bovine leukosis in New Zealand--a case report and update.

Enzootic bovine leukosis was diagnosed in an 8-year-old Friesian cow which had lost condition and was milking poorly. The cow had a severe nonregenerative anaemia, panhypoproteinaemia and lymphoid leukaemia. At necropsy there was widespread lymphoid infiltration of many organs, including the abomasal mucosa, myocardium, uterus, kidney, lymph nodes and bone marrow. Antibodies against bovine leukaemia virus were detected in the serum. Although clinical enzootic bovine leukosis is rare in New Zealand, having been confirmed on only five properties, infection with the causative agent, bovine leukaemia virus, is more widespread. The results of a recently completed survey of bulk milk samples using an enzyme-linked immunosorbent assay for bovine leukaemia virus antibodies suggest that there are about 300 dairy herds in the country with a bovine leukaemia virus infection level of at least 5-10%. The economic impact of enzootic bovine leukosis on the productivity of New Zealand's dairy cattle population is probably still negligible but the introduction of control or eradication schemes in Europe and North America could sooner or later lead to restrictions on the export of live cattle and genetic material from New Zealand.