Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiolog y of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep.

METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp.

RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected.

CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks.     

Induction of mRNAs in response to acclimation of trout cells to different osmolalities

The direct effect of osmolality on growth and mRNA population were investigated in the rainbow trout cell line (RTG-2). These cells can grow in the media of osmolalities ranging from 200 to 600 mosmol kg-1. With two-dimensional electrophoresis, the in vitro translation of poly(A⁺) RNA isolated from these cells showed osmoresponsive changes in the population of translatable mRNAs. Using differential mRNA display polymerase chain reaction, however, we identified inducible cDNA products in hyper-osmotic and hypo-osmotic media as third component of complement, and as homologues of known genes: an atypical protein kinase regulated by the thyrotropin-dependent mitogenic pathway, nucleolin and CHD3. The remaining cDNAs have no significant homology in GenBank. Northern blots demonstrate that their mRNA levels were induced in hyper-osmotic and hypo-osmotic media, but not by other stresses. The expressed proteins of these mRNAs may be involved directly or indirectly in the adaptation of RTG-2 cells to different osmolalities probably through the osmotic signal transduction and adjustment in cellular metabolism to osmotic stress.     

补体结合酶联免疫吸附试验方法的建立

为改进免疫学诊断技术的准确性,研究了一种基于补体结合的免疫学检测新技术———补体结合酶联免疫吸附试验（CF-ELISA）。CF-ELISA技术采用酶标记抗菊糖纯化豚鼠补体C3抗体及其酶显色系统作为补体参与反应的指示系统,用ELISA方法进行补体结合试验。经对布氏菌病抗体检测的初步试验结果显示,CF-ELISA技术可检测到0.01 IU的布氏菌病抗体,灵敏度与间接酶联免疫吸附试验（iELISA）相当,是虎红平板凝集试验（RBPT）试管凝集试验（SAT）的5 000倍、补体结合试验（CFT）的10 000倍。对349份确诊布氏菌病感染群牛、羊血清的检测结果显示,CF-ELISAi、ELISA、CFT、SAT、RBPT的阳性率分别为35.82%3、6.39%、31.81%、30.09%、36.1%,CF-ELISA与iELISA、CFT、SAT、RBPT的阳性符合率分别为：98.4%、88.8%、80.0%、90.6%。CF-ELISAi、ELISA、CFT、SAT、RBPT对490份布氏菌病阴性群牛、羊血清的阴性率分别为100%、99.6%、100%、99.4%、99.8%,CF-ELISA与iELISA、CFT、SAT、RBPT的阴性符合率分别为：99.6%1、00%、99.4%、99.8%。研究表明,CF-ELISA是具有高特异性和高敏感性的布氏菌病免疫学检测技术。     

仿刺参补体类似物活性的测定

用3种方法对仿刺参Apostichopus japonicus体腔液、体腔液上清液及体腔细胞中的总补体溶血活性和补体类似物AjC3、AjC4活性进行测定.结果表明:在仿刺参体腔液、体腔液上清液和体腔细胞中能够检测到总补体溶血活性和补体类似物AjC3、AjC4,表明仿刺参体内存在有脊椎动物的补体类似物,且活性与正常人补体活性相似.仿刺参体内总补体溶血活性和补体类似物AjC3、AjC4的活性依次顺序均为体腔液＞体腔细胞＞体腔液上清液.     

Comparison of ELISA and CFT assays for Chlamydophila abortus antibodies in ovine sera

OBJECTIVE: To compare the sensitivity and specificity of Chlamydophila abortus antibody assays, to find a suitable serological assay for testing sheep for export. DESIGN: Comparison of results from known positive and negative sheep populations. PROCEDURE: Fifty-five positive and fifty negative sera were analysed by four enzyme linked immunosorbent assays (ELISA), three using recombinant antigens based on the chlamydial polymorphic outer membrane proteins (POMP90-3, POMP90-4, POMP80-90) and one using a synthetic peptide based on chlamydial major outer membrane proteins (MOMP-P). They were also analysed by complement fixation tests (CFT) using crude antigens from chlamydia isolated from an Australian sheep, a Californian parakeet and a Texan turkey. Assay sensitivity and specificity were expressed as point estimates and 95% confidence intervals. Results were compared using McNemar's test for paired samples. RESULTS: ELISA sensitivity ranged from 70 to 98% and complement fixation test sensitivity from 60 to 96%; with POMP90-3 > POMP90-4 > CFT (parakeet) > CFT (turkey) > POMP80-90 > MOMP-P > CFT (sheep). There was no significant difference from POMP90-3 to POMP80-90 (P > 0.05). ELISA specificity ranged from 88 to 100% and CFT specificity was 100% for all three antigens; with CFT and POMP90-4 > MOMP-P > POMP80-90 > POMP90-3. There was no significant difference from CFT to POMP80-90 (P > 0.05). Changing the CFT cut-off from 1:32 to 1:4 substantially reduced the specificity with little improvement in sensitivity. CONCLUSION: Assays using POMP90-4, POMP80-90, CFT (parakeet) and CFT (turkey) had equivalent sensitivity and specificity; none of the ELISAs were more specific than any CFT. The POMP80-90 ELISA is recommended as an alternative to CFT (parakeet) but as its specificity is not ideal the search for a more specific assay should continue.     

Complement, polymorphonuclear leukocytes and platelets in acute experimental respiratory reactions to Aspergillus

An experimental complement-dependent model of an acute allergic response to inhalation of Aspergillus spores has been used to examine the roles of polymorphonuclear leukocytes (PMN) and platelets in mediating this reaction as measured by decreases in arterial oxygen tensions (Pa_O2). Normal rabbits selectively depleted of PMN by nitrogen mustard (HN₂) showed a decrease in Pa_O2 later than that observed in untreated animals. The HN₂-treated group also showed a marked decrease in platelets after challenge, not seen in the similarly challenged untreated group or in unchallenged HN₂-treated animals. These data demonstrate the importance of PMN in complement-mediated host responses to aerosols of fungal spores. In the absence of the PMN, platelets appear capable of initiating a similar, albeit delayed, reaction, thus further emphasizing the flexibility of pulmonary defense mechanisms against inhaled particles.     

Experimental maedi infection in sheep. 1. Detection of virus, clinical course, histopathology

Eight sheep were inoculated with Icelandic maedi strain M 88; 2 sheep served as control sheep and were in close contact with the inoculated ones. Four of the sheep were inoculated via the respiratory tract with 7×10⁶ TGID50 of strain M88 and the other 4 intracerebrally with 5×10⁵ TGID50 of the same strain.Maedi M88 strain was isolated from peripheral blood leukocytes of all inoculated sheep. There was a striking difference between the 2 groups in the appearance of demonstrable viremia after inoculation. Viremia could be demonstrated in the intrapulmonarily inoculated sheep within 2–6 months but not until 8–11 months after inoculation in the intracerebrally inoculated ones. This finding is thought most probably to reflect a weak neurotropism of the strain used. After the first demonstration of viremia, maedi virus has been recovered quite reqularly in peripheral leukocytes of all intrapulmonarily inoculated sheep, but less regularly in the intracerebrally inoculated ones. Maedi virus was isolated from 1 of the uninoculated control sheep 15 months after inoculation.The first clinical case with a clinical appearance suggesting combined involvement of maedi and visna was found among the intrapulmonarily inoculated sheep, 8% months after inoculation. Histopathological examination and virus isolation confirmed maedi. The cause of paraplegia could not be confirmed. No histopathological changes were found and no virus isolation was made from the central nervous system of this animal.One of the intracerebrally inoculated sheep died suddenly without any observed clinical signs 11 months after inoculation. Histopathological examination revealed pulmonary lesions of maedi, but no visna lesions in the central nervous system, although maedi virus was isolated from various parts of brain.None of the other experimental sheep displayed clinical signs of maedi or visna during the observation period of 18 months.     

Serological Diagnosis of Classical Swine Fever: A Comparison of a Modified Direct Complement Fixation Test with an Immunofluorescence Plaque Neutralization Test in the Diagnosis of Experimental Subclinical Infection

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera.In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies.During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.