Laboratory diagnosis of psittacine beak and feather disease by haemagglutination and haemagglutination inhibition

SUMMARY Simple and sensitive haemagglutination and haemagglutination Inhibition assays were developed for psittacine beak and feather disease (PBFD) virus and serum antibody, respectively. The assays were used in the examination of samples from 73 birds clinically affected with PBFD. High antigen titres (log₂ 9 to log₂ 12) were detected In feathers, faeces and cloacal contents of PBFD-affected birds. Antigen was not detected In either faecal or feather samples from 20 normal galahs (Eolophus roselcapillus) and 9 normal sulphur crested cockatoos (Cacatua galerita). After kaolin treatment and haemadsorption of serum, haemagglutination inhibition (HI) antibody titres could not be detected in serum from 42 PBFD-affected birds, whereas serum HI titres from 64 normal psittacine birds ranged from less than log₂ 1 to log₂ 8. Serum and yolk HI antibody responses of 6 PBFD virus-inoculated layer hens were measured. Pre-inoculation chicken sera contained high concentrations of non-specific haemagglutination inhibitors (not detected in chloroform-extracted yolk), which were removed by kaolin treatment and haemadsorption.     

A COMPLEMENT-FIXATION TEST FOR ENZOOTIC PNEUMONIA OF PIGS USING A COMPLEMENT DILUTION METHOD

Complement-fixing antibody to Mycoplasma hyopneumoniae in the serums of pigs experimentally infected with enzootic pneumonia was demonstrated by comparing the haemolytic titre of guinea-pig complement titrated in the presence of heated test serum, M. hyopneumoniae antigen and unheated normal pig serum with the titre obtained when the antigen was omitted. The haemolytic titres against sensitised sheep erythrocytes were determined after a fixation period of 16 to 18 hours at 5°C. When serums, collected at intervals of 3 to 7 days, from 43 pigs exposed to pigs experimentally infected with enzootic pneumonia were tested, 4.6 or more complement units were first fixed 14 to 44 (mean 23.4) days after contact began. Serums collected subsequently fixed from 4.6 to more than 31 complement units. This positive reaction usually persisted until the pigs were killed 4 to 35 weeks after contact began. Thirty-three had gross enzootic pneumonia lesions and 9 had lung lesions detected microscopically. Serum antibody was not detected in 73 weaned pigs aged 7 weeks in a pneumonia-free herd but serums from 9 of 15 unweaned piglets aged 9 to 14 days in the same herd, fixed between 3 and 7 complement units.     

间接血凝抑制试验检测血清和卵黄中鸡毒支原体抗体的研究

将13只正处于产蛋期的试验鸡在接种疫苗前后分别采血、分离血清；收集鸡蛋，分离蛋黄，并分别用16％NaCl、8％柠檬酸纳和氯仿处理后，检测鸡毒支原体(MG)间接血凝抑制(HI)抗体。结果表明：接种疫苗前的血清和16％NaCl、8％柠檬酸钠和氯仿处理卵黄抗体平均效价分别为4.7，4．4，4．1和4.2 log2；第一次接种疫苗后的血清和16％NaCl、8％柠檬酸纳和氯仿处理卵黄抗体平均效价分别为7．4、6．7、7．1、6．9log2；第二次接种疫苗后第1次采集的血清和16％NaCl、8％柠檬酸钠、氯仿处理卵黄抗体平均效价分别为10．4、9．6、10．1、9．710g2，第2次采集的血清和16％NaCl、8％柠檬酸钠、氯仿处理卵黄抗体平均效价分别为7．9、7.1、7．6和7．4log2。8％柠檬酸钠处理后的卵黄抗体效价最高，与血清抗体效价最接近。血清HI抗体效价与卵黄HI抗体效价具有良好的一致性和相关性，用卵黄可以代替血清进行HI试验。     

In vitro studies on feline panleucopaenia virus. Standardisation of haemagglutination-inhibition test for feline panleucopaenia virus antibody

Standardised procedure for obtaining reproducible haemagglutination-inhibition results for FPV antibody which correlate with serum-neutralization titres was described. Optimal conditions were found to be Alsevers anticoagulant, PBS/0.05% BSA (pH 6.8) as buffer, especially washed round bottom microplates, determination of maximally sensitive porcine erythrocytes, use of reproducible erythrocyte concentrations, inactivation of serum samples at 56 degrees C for 30 min and serum treatment with koalin pH 9.0. The concentration of erythrocyte used for estimation of haemagglutination units in H1 test should not differ from that used as indicator in the test. Predilution of serum beyond 1:4 associated with false results. Reproducible method for removing natural agglutinins in serum by adsorption with erythrocytes was described.     

Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus

An enzyme linked immunosorbent assay (ELISA) was developed to detect antibody to bovine viral diarrhoea virus (BVDV) in bovine serum. The ELISA results were compared with those of the serum neutralisation test (SNT) using serums from 6 experimentally infected calves bled at intervals from 0 to 154 days postinfection and 886 field samples. The optical density (OD) produced by a single dilution of test serum was compared with a standard curve and the result expressed in ELISA units. Despite wide variation between absolute ELISA and SNT results, an agreement of 97% was obtained when reciprocal SNT titres greater than or equal to 8 and ELISA units greater than or equal to 10 were taken as indicative of a specific reaction. The ELISA was shown to be an efficient method of measuring antibody in bovine serum samples and would assist in any large scale screening of cattle herds for BVDV antibody.     

Immunodiffusion test for ovine progressive pneumonia.

An agar gel immunodiffusion test was developed to detect precipitating antibody against ovine progressive pneumonia (OPP) virus. The test was conducted in plastic petri dishes containing 6 ml of 1% purified agar in tris buffer and 8% sodium chloride. Wells for serum and antigen were 8 mm in diameter and were cut in a hexagonal pattern 3 cm from a central well. Tests were read at 24 and 48 hours. Soluble antigen for the test consisted of concentrated nutrient medium removed every 2 weeks from a cell culture persistently infected with isolate WLC 1 of OPP virus. Specificity of results was verified by testing serums from experimentally exposed sheep and appropriate controls. Two lines of precipitate formed with some serums from experimentally inoculated sheep. Serums taken soon after exposure of sheep to the virus and those taken 3 to 4 years after exposure frequently formed only 1 line of precipitate. Of 37 lambs inoculated with OPP virus, 25% of those tested were positive by postinoculation (PI) month 1, 79% of those tested were positive by PI month 3, and all of those tested were positive by PI month 6. The test appears adequate to detect exposure of sheep to OPP virus.     

Evaluation of the results of ELISA in the quantitative determination of antibodies against infectious bursitis in poultry]

Specific antibodies were investigated in serums of chicks vaccinated with live vaccine and revaccinated with inactivated vaccine against the infectious bursal disease virus, using three methods. An ELISA technique was used to determine antibody titres at a fourfold serum dilution; the reaction was evaluated visually. The results were compared with the titres of neutralizing antibodies and percentage of samples with precipitin activity (Fig. 1). The average values of neutralizing antibody titres and ELISA titres were found to have an analogical pattern; a decrease in maternal antibodies on the first days of chick life and an increase in the antibodies after vaccination, accompanied by an increase in precipitin activity, were typical in these tests. Using the different ELISA technique, 138 samples of fowl serum were examined (Fig. 2). The high correlation, r = 0.85, was found between the ELISA titres determined by visual reading of the reaction within the fourfold serum dilutions and the absorbance values determined at single serum dilution. Applying a spectrophotometer programme, a scale of antibody quantification was made up pursuant to the intensity of immunoenzymatic reaction. For the purposes of method reproducibility, the evaluation was made within the average values of absorbance of positive serum on the one hand and of negative serum on the other. The span of these values was divided into ten equal parts, designated by degrees 0 to 9. Corresponding degrees of positivity were assigned to the examined samples in dependence on the determined value of absorbance (Fig. 3). The correlation r = 0.61 was found between the ELISA values and the titres of neutralizing antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemagglutination and antigenic comparison of rabbit hemorrhagic disease virus

The hemagglutinating activity and serological properties of three strains of rabbit hemorrhagic disease virus, Chinese, Korean and Shizuoka, which was first isolated in Japan, were examined by hemagglutination (HA) and cross hemagglutination inhibition (HI) test with human erythrocytes. Similar results were observed between the Chinese and Korean strains, both of which gave positive HA at 4 degrees C with O, A, B and AB, and at 22 degrees C with B and AB blood groups. In the Shizuoka strain, positive HA was observed at 4 degrees C with O, A, B and AB, at 22 degrees C with A, B And AB, and at 37 degrees C with B blood group. In experimentally infected rabbits, HI antibody in these animals showed a titer of 16,384 or 32,768 at 4 weeks after inoculation. No serological difference was observed in three strains by cross HI test.     

几种鸡新城疫-减蛋综合征二联灭活苗免疫效果比较试验

用自制的鸡新城疫-减蛋综合征二联油乳剂灭活苗接种140日龄的商品蛋鸡,并以其他公司及进口的同种二联油苗、自制的ND单苗和EDS76单苗作对照,抽测不同免疫时期鸡血清,测定各种疫苗接种后ND和EDS76HI抗体消长规律。结果表明,自制二联灭活苗与国外某公司生产的ND-EDS76二联灭活疫苗相当,EDS76抗体和ND抗体的增长曲线无明显差异。     

A microplate enzyme-linked immunorsorbent assay for measuring antibody to Anaplasma marginale in cattle serum

An enzyme-linked immunosorbent assay (ELISA) method is described for measuring antibody against Anaplasma marginale in cattle serum. This method was more sensitive and objective than a previously described ELISA method for A. marginale and possible reasons for this are discussed. All 83 cattle experimentally infected with A. marginale (81) or A. centrale (2) developed demonstrable specific antibody but the serums of 98.8% of 839 cattle from cattle tick-free areas did not react by ELISA; 378 serums containing antibody to Babesia bovis were tested for cross reactions in the A. marginale ELISA. There were no significant cross-reactions except when cattle had been inoculated at least twice with B. bovis-infected erythrocytes, presumably due to antibodies reacting with erythrocyte material in the ELISA antigen. The ELISA detected antibodies for more than 3 years after infection, at least 2 years longer than did a complement fixation test. When A. marginale infections in cattle were eliminated by long acting oxytetracycline, their serums ceased to react by ELISA. An ELISA score for serum antibody level was shown to have a statistically significant correlation with ELISA titre.     

不同鸡新城疫疫苗免疫鸡血清HI抗体的测定

将试验鸡分成3个试验组和1个对照组。A组鸡接种鸡新城疫系疫苗,B组鸡接种油乳剂灭活苗,C组鸡接种鸡新城疫系疫苗,并在接种疫苗后第3、4、5、6、7、9、11、13、15、20、25d采取各组鸡血并分离血清,检测HI抗体。结果表明,接种系疫苗的组,HI抗体效价均值从4.67log2上升到10log2,接种后第5d开始上升,接种后第11d达到峰值,持续6d保持高滴度抗体水平。接种系疫苗的组,HI抗体效价均值从4.67log2上升到7log2,接种后第4d开始上升,接种后第9d达到峰值。接种油乳剂灭活苗的组,HI抗体效价均值从4.67log2上升到9.33log2,接种后第5d开始上升,接种后第11d达到峰值,持续16d保持高滴度抗体水平。系疫苗HI抗体效价上升快,效价高,较适合于紧急接种,油乳剂灭活苗HI抗体效价可在高水平维持较长时间,较适合于预防接种。     

Evaluation of a gel diffusion precipitin test for porcine parvovirus

The use of a gel diffusion precipitin (GDP) test for the detection of porcine parvovirus (PPV) infection in pigs is described. The close correlation between gel diffusion precipitin and haemagglutination inhibiting (HI) antibody titres indicates that, with careful standardisation, a high level of sensitivity can be achieved with the GDP test and that it is a simple and relatively inexpensive alternative to the more commonly used HI test. Experimental infection of 2 groups of pigs showed that GDP and HI antibody responses were closely correlated and that GDP antibodies to PPV persisted for at least 41 weeks after infection. In a commercial herd study, serological evidence of declining passive immunity and subsequent acquisition of active immunity was demonstrated by measuring the GDP and HI antibody titres in sequential serum samples of pigs from a known PPV endemic farm. The GDP test described was shown to be less sensitive than haemagglutination (HA) in the detection of viral antigen but was, nevertheless, considered useful as a simple screening test for the amounts of antigen usually present in PPV infected mummified foetuses.     

The effect of including anti-Ig sera in the haemagglutination inhibition test forMycoplasma gallisepticum

Addition of anti-immunoglobulin M (anti-IgM), G (anti-IgG) and A (anti-IgA) sera to the haemagglutination-inhibition (HI) test (anti-Ig HI test) forMycoplasma gallisepticum resulted in 2- to 8-fold increases in the HI titres. On investigating the anti-Ig HI reaction using IgM and IgG antibodies separated by affinity chromatography, it was confirmed that, in the enhanced HI titres, specificity existed between the chicken Ig classes having antibody activity and the antisera used in the test. Four days after inoculation ofM. gallisepticum, the anti-Ig HI reaction was markedly enhanced by anti-IgM antiserum in the intravenously inoculated chickens and by anti-IgA serum in the nasally inoculated chickens. Ten days after inoculation ofM. gallisepticum marked enhancement of the reaction was produced by anti-IgG serum in both intravenously and nasally inoculated chickens, but the enhancement of the anti-Ig HI reaction diminished from the second week after inoculation.     

Alternative complement pathway in bovine serum: lysis of human erythrocytes.

The hemolysis of unsensitized human erythrocytes by fresh bovine serums was investigated. Lysis occurred in ethylene glycol bis-amino tetraacetate buffers and with serums depleted of Clq. Serums extensively absorbed with packed human erythrocytes at 0 C effectively lysed human erythrocytes, but optimal lytic capacity required target cells "sensitized" with a heat-stable serum factor. Lysis did not occur with serums absorbed with zymosan at 17 C or heat inactivated at 50 C. These results indicate that human erythrocytes can activate the alternative pathway of complement in bovine serums. Lysis can proceed in the apparent absence of antibodies, although their presence may enhance the reaction.     

IMMUNOGLOBULINS IN BLOOD SERUM OF FOETAL PIGS

A total of 1,147 samples of blood serum, collected from porcine foetuses, were examined for the presence of immunoglobulin. The foetuses, from 182 sows, were sampled at abattoirs in Queensland during 1975. For detection and measurement of immunoglobulins, rabbit anti-pig serum and monospecific anti-pig IgG, anti-pig IgM and anti-pig IgA were employed in immunoelectrophoresis, double diffusion and single radial immuno-diffusion assays. Twenty-four foetuses (from 7 litters) had detectable IgG or IgM. None of the samples were positive for IgA. Two of the serums (from siblings) had high antibody titres to porcine parvovirus but in the remainder of the immunoglobulin-positive serums no antibody activity was detected.     

A SEROLOGICAL SURVEY FOR BLUETONGUE VIRUS ANTIBODY IN WESTERN AUSTRALIA

A serological survey was carried out to detect specific (serotype 20) and a group bluetongue virus antibody in cattle and sheep serums collected in Western Australia during the period January 1 1978 to June 30 1979. Of 18,849 cattle serums examined by the gel diffusion precipitin test (GDPT), 9.7% were positive and 6.1% gave doubtful results. All 1949 sheep serums tested were negative. Precipitin antibody was demonstrated in 22.5% of serums from Kimberley cattle and 3.6% of cattle serums from the Northwest. Serums collected from cattle in the South were consistently negative in GDPT. When 915 serums that reacted in the GDPT were further tested by the complement fixation test (CFT), 164 were positive. The percentage of CFT positive serums increased as the GDPT reaction became stronger. 2467 serums collected from cattle in Kimberley and Northwest areas and tested by the CFT, 175 (7.1%) were positive. These 175 positive serums were also examined by GDPT and 164 doubtful or positive reactions were obtained. The virus neutralisation (VNT) using serotype 20 virus was carried out on 3804 serums, including all serums that reacted in the GDPT, and 57 were positive. When the VNT positive serums were examined in the other 2 tests, 47 serums were either positive or doubtful in the GDPT and 8 were positive in the CFT. The presence of bluetongue virus group antibody in cattle serums closely followed the suggested distribution pattern of Culicoides brevitarsis but specific serotype 20 neutralising antibody was limited to cattle serums from stations situated north of latitude 17 degrees S in an area of mean annual rainfall higher than 700 mm.     

Comparison of serological tests for the measurement of the primary immune response to avian infectious bronchitis virus vaccines

The immune response of individual chickens exposed in an aerosol apparatus to live commercial avian infectious bronchitis virus (IBV) vaccines was measured by serum neutralization (SN), haemagglutination inhibition (HI), complement fixation (CF) and agar gel precipitin (AGP) tests over a period of 14 weeks.The SN titres showed considerable variation for individual chickens and in a large number of birds were negative until 14 weeks after infection.Positive HI titres were recorded for most birds at one week after infection and persisted throughout the observation period. Some relationship was seen between HI and SN titres particularly in birds showing a high immune response.The results obtained with the AGP were transient, variable and did not compare well with results obtained by the other tests. The highest number of positive AGP reactors were seen two to three weeks after infection.Most birds showed positive titres by the CF test at some time after infection but titres were always low and did not correlate with results obtained by the other tests.Twenty-two weeks after vaccination 23 chickens were challenged by aerosol exposure to the Massachusetts 41 strain of IBV and four days later the trachea, kidneys and oviducts were removed from each bird for attempted virus isolation. Virus was recovered from only one kidney and 11 trachea samples. The mean pre-challenge HI and SN titres of birds from which no virus was recovered were significantly higher than the mash titres of vaccinated birds from which virus was isolated after challenge.     

肉仔鸡新城疫疫苗免疫效果观察

为了解宝鸡某鸡场对内仔鸡新城疫的免疫情况,对免疫接种后鸡群随机采血分离血清,通过血凝抑制试验测定血清抗体效价,评价疫苗免疫对鸡群的保护作用.结果显示,用Clone-30株、La Sota株做4单位毒测得血清抗体效价均在25以上,而用当地分离株测得同样血清的抗体效价在21～22之间,并且试验后期鸡群发病.结果表明此次免疫...     

Humoral immune response of sheep to infection with Eperythrozoon ovis

Circulating antibody was detected by an indirect fluorescent antibody test (IFAT) in the serum of sheep infected experimentally with Eperythrozoon ovis. Antibodies were first detected 15 to 32 days after infection with E ovis and titres peaked at 41 days. This antibody may be associated, at least in part, with protection against infection with E ovis since the initial increase in antibody titre coincided with a fall in the primary parasitaemia. A role for antibody is suggested further by the fact that the prepatent period of infection was prolonged by one day and the parasitaemia initially remained at low levels in infected sheep protected by passively transferred hyperimmune serum. Moreover, following primary infection, acquired immunity was manifest by a lack of parasitaemia following challenge infections while increased IFA titres were observed. No evidence of opsonic activity was observed in an in vitro erythrophagocytosis test in that neither mouse macrophages nor sheep monocytes phagocytosed E ovis infected or uninfected erythrocytes sensitised with hyperimmune serum.     

Immunogenicity of a Locally Produced Newcastle Disease I-2 Thermostable Vaccine in Chickens in Uganda

A locally-produced Newcastle disease (ND) I-2 thermostable vaccine of embryo-infective dose (EID50) 10(8.5) per ml was administered to 100 laboratory chickens in four test groups, each of 25 birds. It was given by the eye-drop method, in drinking water, in drinking water freshly medicated with levamisole, or using millet grains as a vaccine carrier. A fifth control group consisting of 25 birds received the heat-sensitive La Sota vaccine (EID50 10(9) per ml) by the eye-drop method. The immunological responses were monitored by the enzyme-linked immunosorbent assay (ELISA) ND antibody technique using serum samples collected from 18 birds in each group at 3-week intervals for 3 months. The overall mean ND antibody log(10) titres and percentage positivities were 3.1, 88%; 2.9, 70%; 3.0, 83%; 3.2, 87% and 3.3, 87%, respectively. The use of water alone or medicated with levamisole for vaccine administration produced significantly lower ND antibody titres only in the first 3 weeks. The immunogenicity shown by the I-2 vaccine as a potential vaccine is discussed in relation to free-range poultry management conditions in Uganda.