Non-specific seroreactions against Brucella abortus in ruminants in New Zealand and the presence of Yersinia enterocolitica 0:9

The level of non-specific reactions found in the brucellosis serology of ruminants in New Zealand was very low until July 1992. This changed when, in an export consignment of 1071 deer, 35% reacted in the Brucella abortus tube agglutination test with titres varying from 50 to 200 IU. The reactors were also positive in the Rose-Bengal agglutination test and most of them reacted in the complement fixation test with titres varying from 10 to 80 IU. Yersinia enterocolitica 0:9 was later isolated from one deer of this consignment. It was the first isolate of this serotype recovered in New Zealand from an animal. Shortly after, false reactors occurred more frequently than before in sera from Brucella abortus accredited free cattle herds. As the involvement of Yersinia enterocolitica 0:9 was suspected in these cases, faecal samples from reactors and in-contact animals were cultured for this organism. Yersinia enterocolitica 0:9 was isolated from nine of 19 herds showing one or more false Brucella abortus seroreactions. Prior to 1990, Yersinia enterocolitica serotype 0:9 had not been isolated in New Zealand, despite the recovery of a number of other bio- or serotypes of the organism from humans and animals. From 1990 onward, serotype 0:9 began to be isolated from human faecal samples with increasing frequency. Since the first isolations from deer and cattle in 1992, it has now also been recovered from a cat and an alpaca and from cattle without any association with false positive Brucella abortus reactions. All serotype 0:9 isolates were of biotype 2.     

鸡传染性法氏囊病新型疫苗的研究进展

鸡传染性法氏囊病(IBD)是由鸡传染性法氏囊病病毒(IBDV)引起的雏鸡的一种高度接触性传染病,自1962年报道以来,世界上主要养禽国家和地区均有流行,给养禽业带来严重经济损失。目前,IBD防控的主要方法是采用疫苗接种,使易感雏鸡获得主动或被动免疫保护,因此疫苗的质量对临床上IBD的防控起着至关重要的作用。虽然各国均有较好的商品化疫苗,但随着IBDV毒株的不断变异,商品化疫苗的抗原性与流行毒株不能完全匹配,临床上免疫失败时有发生,因此迫切需要研发与临床流行毒株相匹配的新型疫苗用于IBD的防控。对近期IBD的基因缺失苗、亚单位疫苗、DNA疫苗以及活载体疫苗等新型疫苗的研究进展进行概述,以此为IBD新型疫苗的研究提供参考。     

Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 III. Specificity of the in vitro antigen-specific gamma interferon test for bovine brucellosis diagnosis in experimentally Yersinia enterocolitica 0:9-infected cattle

The course of immunological reaction in 10 Yersinia enterocolitica 0:9 experimentally-infected heifers was followed using the conventional brucellosis tests complement fixation test (CFT), serum agglutination test (SAT) and brucella card test (BCT), and a recently developed Brucella antigen-specific gamma interferon (IFN-γ) test. Initially, the animals were exposed orally to 10¹⁰ colony-forming units (CFU) of Y. enterocolitica 0:9. Four weeks later, they were inoculated intravenously with 10⁸ CFU of Y. enterocolitica 0:9 cells. After oral inoculation, the response in the conventional brucellosis tests was minimal. Only after intravenous inoculation were CFT and SAT titres and BCT reactions comparable to natural, false positive brucellosis reactors. After oral exposure the Brucellergen-stimulated release of IFN-γ peaked at values above the cut-off stimulation index of 2.5 in 80% of the heifers. After intravenous inoculation, stimulation indices above 2.5 were present in only 10% of the animals. Two B. abortus infected control cattle showed stimulation indices of 3.1 and 3.4, and a negative control animal exhibited a stimulation index of 1.0. These findings show, in contrast to a previous study, that the Brucellergen-specific IFN-γ assay cannot be used as a specific and discriminatory test for B. abortus infections.     

抗鸡传染性法氏囊病病毒冻干卵黄抗体与同类产品的比较试验

本研究鸡传染性法氏囊病冻干卵黄抗体和市售精制液体卵黄抗体分别以10羽份和3 mL超剂量肌肉注射各10只18日龄SPF雏鸡,观察14 d均安全。两种抗体以1羽份剂量分别肌肉注射各20只18日龄SPF雏鸡24 h后,用IBDV强毒TL株攻击,连续观察144 h,2个试验组分别有20/20、17/20的雏鸡健活,剖检没有发现法氏囊病变;二者分别以1羽份剂量连续2 d肌肉注射各20只18日龄已经被IBDV强毒TL株攻击12 h的SPF雏鸡,连续观察96 h,2个试验组有19/20、17/20的雏鸡健活,而预防和治疗试验中的阳性对照组中20只鸡均有法氏囊特征性病变发生（20/20）,且死亡率在85%（17/20）以上,阴性对照组雏鸡均正常。     

Serological cross-reactivity between Brucella abortus and Yersinia enterocolitica 0:9:: IV. Evaluation of the M- and C-epitope antibody response for the specific detection of B. abortus infections

Smooth lipopolysaccharides (SLPS) from Brucella abortus contain A-epitopes against which the majority of serum antibodies are directed during infections. SLPS from Yersinia enterocolitica 0:9 possesses identical epitopes, which are the cause for serological cross-reactivity. All Brucella spp. possess M- and C-epitopes which are not present in Y. enterocolitica 0:9. In order to examine the usefulness of these M- and C-epitopes for discrimatory serological testing, a panel of sera were used in this study, comprising sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected cattle of comparable strength in the serological brucellosis tests to the sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected bovines with strong serological reactions and sera from animals free from B. abortus or Y. enterocolitica infections. These sera were tested in blocking ELISAs with seven M- and one C-epitope-specific monoclonal antibodies in combination with SLPS from B. melitensis M16 high in M-epitopes as antigen. Strong B. abortus sera inhibited most strongly, while negative sera showed no or little inhibition. Sera with weak or intermediate titres blocked to a lower extent. Unexpectedly, the sera from Y. enterocolitica 0:9-infected heifers showed inhibition behaviour virtually identical to the comparable sera from B. abortus infected animals. Absorbing out of the A-epitope specific serum antibodies with either Y. enterocolitica 0:9 SLPS or with Y. enterocolitica 0:9 bacteria, indicated the presence of M- or C-epitope-specific serum antibodies in some sera from B. abortus-infected cattle but not in the sera from Y. enterocolitica 0:9-infected animals. These results demonstrate that the M- or C-epitope-specific antibody response in sera from B. abortus infected cattle is only of limited value for the serological discrimination between B. abortus and Y. enterocolitica 0:9 infections.     

The serological response of cattle immunized with Yersinia enterocolitica O:9 or O:16 to Yersinia and Brucella abortus antigens in enzyme immunoassays

Sera from calves immunized with Yersinia enterocolitica serotypes O:9 or O:16 were tested by indirect enzyme linked immunosorbent assay (ELISA) using lipopolysaccharide (LPS) preparations from Brucella abortus or Y. enterocolitica O:9 or O:16 for their antibody content of the IgG1 or IgG2 subclasses. High IgG1 responses were present with the three antigens in both groups although some individual variations between animals were noted. The IgG2 responses were modest and in some cases not above background 'noise'. Thus IgG2 antibody was not measurable in sera from serotype O:9 injected calves when using serotype O:16 LPS or in serotype O:16 injected calves when using B. abortus or serotype O:9 LPSs. A competitive ELISA using B. abortus O-polysaccharide and a monoclonal antibody to B. abortus LPS (initially designed to differentiate the antibody responses of cattle naturally infected with B. abortus from those vaccinated with strain 19) was used on sera from both groups of calves. Using this test, no antibody was detected in the group immunized with serotype O:16 and except for one animal in the serotype O:9 immunized group, only low levels of antibody were transiently in evidence. One animal in this group responded with quite high levels of competing antibody which, however, declined towards the end of the test period. The competitive ELISA may prove a useful serological tool for differentiating vaccinal and field infection titers to B. abortus and also to eliminate cross-reactions observed with Y. enterocolitica serotypes.     

Reproduction of Brucella titers in swine using experimental infection with Yersinia enterocolitica, serotypes 0:9 and 0:6

Measurable Brucella titres were produced by slow agglutination, slow agglutination at 57 degrees C (heat test), and complement fixation reaction with Brucella antigen in infection experiments with gilts in which Yersinia enterocolitica Serotypes 0:9 and 0:6 were used. Slow agglutination gave brucellosis titres up to 1:1280 and titres against Yersinia enterocolitica, Serotype 0:9, up to 1:20480. The antibody titres stayed persistent throughout the 80 days of the experiment. Yersinia enterocolitica infection was found to be transmissible between the animals. Aspects relating to the development and course of the infection as well as to pathogen detection are discussed.     

传染性法氏囊病及其病原分子生物学研究进展

传染性法氏囊病(Infectious bursal disease,IBD)是由传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)引起的一种急性、高度接触性传染病.20世纪80年代中期以后,由于抗原变异和超强毒株在免疫鸡群的出现,使IBD的防控面临新的困难.文章简要概述近几年来,IBD在病原及分子生物学特性、抗原性、致病性、毒力与抗原的变异、疾病诊断和防制策略等方面的研究进展.     

Serologic response to Brucella abortus of cattle and guinea pigs experimentally inoculated with a Yersinia enterocolitica serotype from milk

Ten strains of Yersinia enterocolitica belonging to ten various serogroups isolated from raw milk were inoculated into groups of five guinea pigs and five calves. Y. enterocolitica serotype 0:16 was the only serotype tested that induced an antibody response to Brucella abortus in calves. No anti-Brucella response could be demonstrated serologically in guinea pigs. Activity of the anti-Y. enterocolitica 0:16 calf sera against B. abortus antigen was shown by the tube agglutination test, and by the complement fixation test. The early agglutinating antibody response was partly sensitive to reduction by 2-mercaptoethanol. This sensitivity decreased later in the response. This is the first report of anti-Brucella responses induced by a serotype of Y. enterocolitica other than 0:9; sera from a group of five calves inoculated with 0:9 were tested by the same serological techniques for comparison.     

外源病毒检验用抗鸡传染性法氏囊病病毒特异性血清的制备与检定

为制备外源病毒检验用抗鸡传染性法氏囊病病毒(Infectious Bursal Disease Virus,IBDV)的特异性血清,对300只3~4周龄的SPF鸡进行了基础免疫和加强免疫。最后一次免疫后21d采血并分离血清,血清经混合、分装、冷冻真空干燥后,对其进行了无菌检验、外源病毒检验、剩余水分测定、中和效价测定和特异性检验。结果表明,本研究制备的抗IBDV特异性血清无菌检验、外源病毒检验和剩余水分测定均符合《中华人民共和国兽药典》三部(二〇一〇年版)规定;血清中和效价为1:5747,与IBDV B87株毒种中和指数为104.3,与鸡新城疫病毒La Sota株、鸡传染性支气管炎病毒H120株、鸡传染性支气管炎病毒H52株、鸡传染性喉气管炎病毒、鸡痘病毒、禽呼肠孤病毒S1133株的中和指数均不大于10,通过AGP、ELISA、HI等试验确定血清中不含其他禽源外源病毒抗体。本研究为鸡传染性法氏囊病活疫苗或毒种的外源病毒检验提供了具有良好特异性和高效价的中和用血清。     

小肠结肠耶氏菌O:9血清型与布氏杆菌M_5株共同抗原的免疫印迹分析

应用SDS-聚丙烯酰胺凝胶电泳和免疫印迹技术,对经超声波打碎的小肠结肠耶氏菌O:9血清型和布氏杆菌M5株全菌体蛋白成分进行了分子量测定及抗原分析。结果表明,小肠结肠耶氏菌O:9血清型Y15株与布氏杆菌M5株存在一条发生交叉反应的蛋白质共同抗原带,其分子量为11400。Y15株与M5株有多个分子量相同的条带,但不发生交叉反应。Y15株与M5株皆有多个各自特异的条带。     

Differentiation of Brucella abortus and Yersinia enterocolitica serotype 09 infections in cattle: the use of specific lymphocyte transformation and brucellin skin tests

The lymphocyte transformation test (using an in vitro whole-blood lymphocyte stimulation procedure) and the Brucellin skin test were applied to five heifers infected with virulent Brucella abortus strain 544, five cows inoculated with Yersinia enterocolitica serotype 09, and four non-exposed cows. Lymphocytes from Brucella-inoculated animals persistently gave very high blastogenic reactions indicative of active Brucella infection. The test was persistently negative in Yersinia-infected and non-exposed cattle. Four of the five cows infected with Yersinia enterocolitica type 09 and all four control cattle were persistently negative to the delayed hypersensitivity skin reaction with brucellin. All cattle infected with Yersinia enterocolitica type 09 were strongly positive to the Rose Bengal, Serum agglutination, Complement fixation and Antibovine globulin tests using Brucella abortus antigens. One lactating cow infected with Yersinia enterocolitica type 09 was positive to Brucella milk ring test. These results indicate that standard Brucella serological tests are unreliable in differentiating the two infections in cattle and that both the Lymphocyte transformation and brucellin skin tests could be used to differentiate bovine brucellosis from yersiniosis.     

鸡传染性法氏囊病病毒XX08株VP2高变区基因的克隆与序列分析

应用反转录(RT-PCR)技术从河南新乡某鸡场分离的鸡传染性法氏囊病病毒XX08株总RNA中克隆出593bp的VP2高变区基因。序列分析表明,XX08毒株VP2高变区氨基酸序列与欧洲超强毒株UK661、日本超强毒株OKYM以及经典弱毒株D78的同源性均为96.8%。遗传进化分析表明,该毒株与超强毒株UK661和OKYM位于同一进化树。XX08毒株与超强毒株具有相似的氨基酸特征,但是222位的A被P替代,提示XX08毒株可能为中等强毒力毒株。     

Differentation of Brucella abortus and Yersinia enterocolitica serotype 09 infections in cattle: The use of specific lymphocyte transformation and brucellin skin tests

Summary

The lymphocyte transformation test (using an in vitro whole‐blood lymphocyte stimulation procedure) and the Brucellin skin test were applied to five heifers infected with virulent Brucella abortus strain 544, five cows inoculated with Yersinia enterocolitica serotype 09, and four non‐exposed cows. Lymphocytes from Brucella‐inoculated animals persistently gave very high blastogenic reactions indicative of active Brucella infection. The test was persistently negative in Yersinia‐infected and non‐exposed cattle. Four of thefive cowsinfected with Yersinia enterocolitica type 09 and allfour control cattle were persistently negative to the delayed hypersensitivity skin reaction with brucellin. All cattle infected with Yersinia enterocolitica type 09 were strongly positive to the Rose Bengal, Serum agglutination, Complement fixation and Antibovine globulin tests using Brucella abortus antigens. One lactating cow infected with Yersinia enterocolitica type 09 was positive to Brucella milk ring test. These results indicate that standard Brucella serological tests are unreliable in differentiating the two infections in cattle and that both the Lymphocyte transformation and brucellin skin tests could be used to differentiate bovine brucellosis from yersiniosis.     

Efficacy of a novel in ovo-attenuated live vaccine and recombinant vaccine against a very virulent infectious bursal disease virus in chickens

Infectious bursal disease (IBD) causes severe economic damage to the poultry industry worldwide. To prevent IBD virus (IBDV) infection, live virus vaccines have been widely used in chickens having wide-ranging levels of maternally derived antibodies. But, the risks of infection with other pathogens because of lesions related to atrophy of the bursa of Fabricius in vaccinated chickens are a concern. To resolve the problems, a recombinant turkey herpesvirus (HVT) vaccine expressing IBDV-VP2 protein (rHVT-IBD) has been developed. However, the induction of neutralizing antibodies by rHVT-IBD against a virulent IBDV might be delayed compared with that by the live IBD vaccine, leading to the high risks of IBDV infection for young chickens. To find the best selection of IBDV vaccine for the onset of immunity, we examine the protective efficacy of a novel in ovo-attenuated live IBDV (IBD-CA) vaccine and the rHVT-IBD vaccine in young chickens challenged with a very virulent IBDV (vvIBDV) strain. We show that the protective efficacy of IBD-CA vaccine was higher than that of the rHVT-IBD vaccine in 14-day-old chickens challenged with the vvIBDV strain, leading to the risk of IBDV infection for young chickens when vaccinated with rHVT-IBD. Our results suggest that farmers should select the best vaccines to maximize vaccine efficacy in consideration of the vaccine characteristics, prevalence levels of IBDV in the areas, and initial MDA levels of the chickens since the attenuated live and recombinant vaccines play a role in the different vaccine efficacies.     

Confirmation of the occurrence of the chewing louse Bovicola (Lepikentron) breviceps (Insecta: Phthiraptera: Trichodectidae) on alpacas (Lama pacos) in New Zealand

Abstract

AIM: To investigate the cause of classical swine fever (CSF) virus-seropositive animals in a nucleus pig-breeding herd in New Zealand, where porcine circovirus-associated disease had been diagnosed.

CASE HISTORY AND CLINICAL FINDINGS: An exotic disease investigation was undertaken to exclude CSF and porcine reproductive and respiratory syndrome (PRRS) on a nucleus pig-breeding herd comprising approximately 300 breeding sows, 1,000 weaners, and 650 grower pigs. The herd was experiencing poor reproductive performance in sows, and breeding records showed a declining farrowing rate attributable to a single manager. The growing pigs (10–15 weeks old) were experiencing respiratory disease and wasting, and the mortality rate by pen varied between 9 and 20%. Post-mortem changes in affected grower pigs were consistent with circovirus-associated diseases.

DIAGNOSTIC TESTING: Serological screening using an IDEXX-ELISA gave negative results for PRRS virus antibodies, but two grower pigs and one sow tested positive for CSF virus antibodies. These three seropositive animals remained positive to CSF virus, using three commercial ELISA test kits, over 27 weeks. A newly developed virus neutralisation test (VNT), using a New Zealand isolate of border disease (BD) virus, demonstrated that the seropositive pig sera had higher antibody titres to BD virus than to bovine viral diarrhoea (BVD) virus and CSF virus.

PCR performed on tonsil, kidney, ileum and spleen gave negative results for CSF virus, and histopathology on lymph nodes, intestine, lung, kidney, liver and brain showed no evidence of the disease. Virus isolation performed on a number of samples was negative.

CLINICAL RELEVANCE: The seropositive samples for CSF virus found in this investigation were likely to be a cross reaction to a pestivirus other than CSF virus. The finding of a possible endemic pestivirus capable of being transmitted between sheep and pigs on this farm may explain findings from previous serological survey work in New Zealand, and supports experience elsewhere, where BD virus was found to be the predominant ruminant pestivirus infecting pigs. The results show that pestivirus cross reactivity can result in unexpectedly high titres, and that testing with a full set of (local) pestiviruses is necessary to reach the correct conclusion. The investigation has direct relevance where pig herds with a low seroprevalence are encountered during surveillance for CSF.     

传染性法氏囊病病毒超强毒HZ株和XN株VP2基因的克隆和序列比较

根据已发表的５２／７０株ＩＢＤＶ基因组序列,设计并合成了一对特异扩增ＩＢＤＶ ＶＰ２基因的引物。以陕西地区分离的ＩＢＤＶ野毒ＸＮ株,ＨＺ株为材料,以其基因组为模板利用ＲＴ－ＰＣＲ技术扩增出了１．５ｋｂ的ｃＤＮＡ产物,将ＶＰ２基因克隆于ＰＵＣ１１９质粒上,得到重组ＰＵＣ１１９质粒。     

Comparison of the Q-fever complement fixation test and two commercial enzyme-linked immunosorbent assays for the detection of serum antibodies against Coxiella burnetii (Q-fever) in ruminants: Recommendations for use of serological tests on imported animals in New Zealand

Abstract

AIM: To make valid recommendations on the use of serological test methods for the detection of serum antibodies in ruminants against Coxiella burnetii (Q-fever), by comparing the performance of the complement fixation test (CFT) and two ELISA, and by identifying reasons for discrepancies between the test methods.

METHODS: A total of 73 serum samples from infected cattle, 69 from infected goats, and 100 samples from non-infected cattle and 57 samples from non-infected sheep, as well as 95 samples from infected cattle herds (mix of seropositive and seronegative samples), were tested using the CFT, the IDEXX ELISA (I-ELISA) and the Pourquier ELISA (P-ELISA). A mixed panel of 12 serum samples from sheep from inter-laboratory proficiency testing (proficiency panel) was also tested using the CFT and both ELISA, and further investigated using IgG- and IgM-specific ELISA.

RESULTS: Generally, the two commercial ELISA were more sensitive than the CFT for the detection of infected ruminants. Good agreement between ELISA for positive and negative results was found for samples from the infected herd, while results for the positive panels varied between the two ELISA. For the total of the positive serum panels, the I-ELISA detected 95% of samples as positive or suspicious, while the P-ELISA detected only 81%. In the P-ELISA, more samples were considered suspicious (18%) than in the I-ELISA (14%). All sera from noninfected sheep and cattle tested negative in the serological test methods employed, except for one positive sample from a sheep in the P-ELISA. Further investigation revealed that a CFT-positive but ELISA-negative result was due to high IgM and low IgG reactivity.

CONCLUSIONS: The two commercial ELISA were more sensitive than the CFT in all panels from infected ruminants. However, they could only detect IgG. The I-ELISA should be the serological test method of choice for cattle, sheep and goats for import testing of animals into New Zealand because it was more sensitive than the P-ELISA and was equally specific to the PELISA and the CFT. For other animal species, such as deer and camelids, the CFT should still be used since none of the ELISA has been evaluated for these species. This study has shown that the two commercial ELISA will detect the majority of infected ruminants but may miss animals that have not developed an IgG response.     

传染性法氏囊病病毒VP2基因高变区序列分析

根据传染性法氏囊病病毒（ＩＢＤＶ）ＶＰ２基因ＣＤＮＡ序列,在ＶＰ２基因高变区设计一对引物,用ＲＴ－ＰＣＲ方法扩增ＩＢＤＶ分离株ＪＳ３和ＪＳ４。将扩增片段克隆后以双脱氧链末端终止法测定核苷酸旬。ＪＳ３和ＪＳ４的同源性最高达９８％。与已发表的ｖｖＩＢＤＶ,ＩＢＤＶ变异要ＢＤＶ经典株为ＩＢＤＶ弱毒株核苷酸序列的同尖拨天９２￣９８％之间,根据ＩＢＤＶ的大ＯＲＦ推导出该片段蛋白的氨基酸序更,ＪＳ３和ＪＳ４的