Seasonal Variations in the Presence of Antibodies against Coxiella burnetii in Dairy Cattle in Hokkaido,Japan

The prevalence and seasonal variations of infection by Coxiella burnetii in cattle were investigated seroepidemiologically on a farm in Hokkaido, Japan, by an immunofluorescent antibody test. A total of 364 serum samples from 28 cows were collected from August 1993 to October 1995 in two barns on the farm. It was found that the number of antibody-positive cows and their antibody titers were significantly elevated in winter and decreased in summer. In addition, antibodies were detectable in seroconverted cows for about five months.     

Isolation of Coxiella burnetii from Dairy Cattle and Ticks,and Some Characteristics of the Isolates in Japan

Coxiella burnetii was isolated from raw milk (36/214, 16.8%) and uterus swab samples (13/61, 21.3%) originating from dairy cattle with reproductive disorders, aborted bovine fetus samples (2/4, 50%), mammary gland samples (4/50, 8%) originating from healthy dairy cattle, and tick samples (4/15, 26.7%) originating from 2 pastures. Fifty-nine strains had various degrees of pathogenicity, high (8; 13.6%), moderate (28; 47.5%) and low (23; 39%), for guinea pigs. The results of isolation suggested a high prevalence of Coxiella infection in dairy cattle with reproductive problems in Japan. Twelve strains (7, 2 and 3 strains from cattle, ticks and humans, respectively) and the reference Nine Mile strain of phases I and II were propagated in both yolk sacs of embryonated hen eggs and Buffalo green monkey (BGM) cell cultures. Protein profiles of these strains were similar to those of the reference strain of phase I. Lipopolysaccharide (LPS) profiles of 12 strains were similar to those of the reference strain of phase I and different from those of the reference strain of phase II. The LPS profiles of 12 strains suggested that these strains are associated with an acute form of Q fever.     

Isolation of Coxiella burnetii from Children with Influenza-Like Symptoms in Japan

The prevalence of Coxiella burnetii antibodies was investigated by indirect immunofluorescence (IF) test in 55 paired sera (acute and convalescent phases) of school children who had influenza-like symptoms. Of the convalescent serum samples examined, 18 (32.7%) sera reacted positively to phase II antigen of C. burnetii. Coxiella-like organism was isolated from the sera of 13 children after injection of the 18 acute phase sera into mice. The organism was identified as C. burnetii by Giemsa staining and the IF antigen test of mouse spleen smears, the polymerase chain reaction (PCR) method, electron microscopic observations of the mouse spleen cells, and the IF antibody test of mouse sera. This is the first report of isolation of C. burnetii from serum specimens of children having influenza-like symptoms. The evidence that C. burnetii was isolated from people indigenous to Japan at a considerably high incidence suggested that C. burnetii may be widespread as a cause of influenza-like symptoms in Japan.     

Seroepidemiological Survey of Coxiella burnetii in Domestic Cats in Japan

Cats are assumed to be one of the most important reservoirs of causative agent of human Q fever especially in urban areas. There is no evidence of Coxiella burnetii infection in cats in Japan prior to this. Sera from 100 cats, collected in various parts of Japan, were examined for antibody against C. burnetii. Sixteen out of the 100 samples contained antibodies against C. burnetii. The prevalence of the antibody decreased from the northeastern to the southwestern part of Japan. A high prevalence of the antibodies was observed in sera from cats of more than four years of age. It is difficult to deny that cats would be one of the important sources of human Q fever in Japan.     

Antigenic Characteristics of Polypeptides of Coxiella burnetii Isolates

Eighteen Coxiella burnetii strains from a variety of clinical and geographical sources were screened for antigenic variation of polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with Coomassie brilliant blue (CBB) staining or immunoblotting. These polypeptide profiles showed the greatest variability in the region from 33 to 8.1 kDa. Such differences in the antigenicity of the polypeptides were also recognized by immunoblotting with 15 various mouse anti-C. burnetii antisera. In addition, we detected a polypeptide at about 28 kDa which was immunodominant in strains from human cases of acute Q fever, milk and ticks but not immunogenic in strains from human cases of chronic Q fever. These findings suggest that this polypeptide is a marker to distinguish between acute and chronic strains.     

In vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of Coxiella burnetii

Coxiella burnetii is the agent of the worldwide zoonosis, Q fever. The in vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of C. burnetii was evaluated for the first time. The MICs against Japanese isolates were almost the same as the MICs against the foreign reference isolates. The results suggest that the common antibiotics therapy for Q fever used in other countries is also effective for Japanese Q fever patients.     

Comparison of Japanese isolates of Coxiella burnetii by PCR-RFLP and sequence analysis

The genetic variation of Japanese isolates of Coxiella burnetii, the agent of Q fever, was found for the first time. Forty-nine out of 72 isolates had the chronic pattern of the isocitrate hydrogenase gene. Sequence analysis revealed that the isolates have a specific nucleotide sequence. The putative amino acid sequence was the same as that of chronic reference strains. These results suggest the variation of C. burnetii isolates in Japan.     

Evaluation of a Recombinant 27-kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Antibodies Are Generated during Infection to Coxiella burnetii Macrophage Infectivity Potentiator Protein (Cb-Mip)

Antisera from rabbits immunized with formalin inactivated Coxiella burnetii isolates associated with either acute (Nine Mile, phase I or phase II) or chronic (Priscilla) Q fever showed reactivity to a C. burnetii macrophage infectivity potentiator protein (Cb-Mip) cloned in Escherichia coli. Further, antisera generated in BALB/c mice after infection with live Nine Mile phase I or Priscilla isolates also showed reactivity to Cb-Mip by immunoblot analysis. In addition, human serum from an individual with previous serological and clinical evidence of Q fever showed reactivity to Cb-Mip. This study indicates that Cb-Mip is immunogenic in both experimental and natural infections, and is the first report on the presence of antibodies to Mip/Mip-like proteins of intracellular bacteria in human sera. Cb-Mip may serve as a potential target antigen for developing recombinant vaccines or diagnostic assays for Q fever.     

Differentiation of Coxiella burnetii by Sequence Analysis of the Gene (com1) Encoding a 27-kDa Outer Membrane Protein

The gene (com1) encoding a 27-kDa outer membrane protein in 21 strains of Coxiella burnetii from a variety of clinical and geographical sources was sequenced for strain differentiation. The com1 gene was highly conserved among all the strains tested but there were several differences in nucleotide and deduced amino acid sequences. Based on the com1 gene-specific nucleotides and deduced amino acids, the 21 strains were divided into four groups. Group 1 contained 14 strains originating from ticks, cattle and human cases of acute Q fever. Groups 2 and 3 included 2 and 3 strains, respectively, originating from human cases of chronic Q fever. Group 4 contained 2 strains originating from a human case of acute Q fever and a goat with abortion. The results indicated that the strains originating from ticks, cattle and human cases of acute Q fever differed at the molecular level from those of human chronic Q fever. This study suggests that a sequence analysis of the com1 gene can be used for strain differentiation of C. burnetii.     

快速准确监测奶牛体细胞数的方法

实验通过荧光染色的方法,运用荧光显微术对3个取样点总计39头奶牛的体细胞在不同时间计数,表明吖啶橙染色是一种快速、准确的监测奶牛体细胞数的方法,同时还表明不同的饲养条件及管理会造成牛群健康状况的显著差异,并依据实验结果对改善牛群的健康状况提出了建议。     

Genetics of Coxiella burnetii

Abstract Those organisms considered to be obligate intracellular bacteria are interesting objects for genetic studies. Little is known about their mechanisms for natural genetic exchange. Many genes from the bacterium Coxiella burnetii , an obligate intraphagolysosomal pathogen, have therefore been cloned and characterized using the heterologous host Escherichia coli . Recently, use of electroporation methodology followed by long-term selection periods have provided initial data on genetic transformation in C. burnetii .     

Evaluation of Two PCR Tests for Coxiella burnetii Detection in Dairy Cattle Farms Using Latent Class Analysis

Different tests performed on bulk tank milk samples (BTM) are available to determine the C. burnetii status of herds. However, these tests, which are based on the detection of either antibodies directed against C. burnetii (ELISA) or bacterial DNA (PCR), have limitations. A currently disease-free herd infected in the past may continue to test positive with ELISA due to the persistence of antibodies in animals that were infected and that subsequently cleared the infection. Infectious herds can also be misclassified using PCR because of the absence of bacteria in the BTM when the test is performed. Recently, PCR has been used for bacterial DNA detection in the farm environment, which constitutes the main reservoir of C. burnetii. The objectives of this study were to assess and compare the sensitivities and specificities of one commonly used PCR test in BTM (PCR BTM) and of a PCR applied to environmental samples (PCR DUST) in dairy cattle farms. BTM and dust samples were collected (using environmental swabs) in 95 herds. The evaluation of the performance of the 2 tests was conducted using latent class models accounting for within herd disease dynamics. Parameter estimation was carried out using MCMC, within a Bayesian framework. Two types of priors were used for the specificity of PCR DUST. A model with a uniform prior on 0–1 fitted the data better than a model with a uniform prior on 0.95–1. With the best model PCR DUST had a lower sensitivity than PCR BTM (0.75 versus 0.83) and a specificity of 0.72. The moderately low value for the specificity of PCR DUST suggests that the presence of bacteria on farm is not always associated with persistent infections and shedding of bacteria in milk.     

Expression of beta-lactamase in Coxiella burnetii transformants

Coxiella burnetii can be transformed to ampicillin resistance by electroporation with plasmids encoding beta-lactamase. However, non-plasmid emergence of resistance to ampicillin also develops. To validate the usefulness of the bla gene marker for selection and detection, transformed C. burnetii were examined for beta-lactamase expression by use of immunoblotting after SDS-PAGE. The 29-kDa mature form of the beta-lactamase protein was detected in C. burnetii lysates. Quantitation of these immunoblot signals showed that C. burnetii surprisingly expressed low levels of beta-lactamase. The results validate the use of plasmid-encoded ampicillin resistance as a means for selecting C. burnetii transformants; they also suggest that levels of ampicillin used for selection pressure should be empirically determined and that detection of beta-lactamase by antibody blotting done to confirm transformants.     

日粮能量水平对奶牛生殖激素分泌的研究

选择发情周期为21天、产后9天~63天的第一胎中国荷斯坦奶牛24头,分为三组,设置三个能量水平,研究对奶牛生殖激素分泌的影响。结果表明:母牛的能量状况与促乳素的相关系数r=-0.9618(P<0.01),与生长激素的相关系数r=-0.7435,在能量负平衡阶段,能量状况与胰岛素值为负相关,在能量正平衡阶段,能量状况与胰岛素值为正相关。发情持续期前第7周的能量值与雌二醇浓度相关系数r=0.9675(P<0.01),与孕酮的相关系数r=-0.7129,黄体期前第7周和第8周的能量值与雌二醇的相关系数分别为0.8099和0.5775,与孕酮的相关系数分别为0.8263和0.6502,因此,在分娩前2周提高日粮能量水平,不仅会提高发情鉴定率,更能提高受胎率,对于高产奶牛,同时控制产后2周泌乳量,可有效缓解能量负平衡,达到提高发情鉴定率和受胎率的目的。     

Establishment of a genotyping scheme for Coxiella burnetii

Coxiella burnetii is the causative agent of Q fever. The bacterium is highly infectious and is classified as a category B biological weapon. The tools of molecular biology are of utmost importance in a rapid and unambiguous identification of C. burnetii in naturally occurring Q fever outbreaks, or in cases of a deliberate release of the infectious agent. In this work, development of a multiple locus variable number tandem repeats (VNTR) analysis (MLVA) for the characterization of C. burnetii is described. Sixteen C. burnetii isolates and five passage history/laboratory variants were characterized. The VNTR markers revealed many polymorphisms resulting in nine unique MLVA types that cluster into five different clusters. This proves that the MLVA system is highly discriminatory. The selected VNTR markers were stable. The MLVA method developed in this report is a promising tool for the characterization of C. burnetii isolates and their epidemiological study.     

Characterization of the Coxiella burnetti sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase.

The Coxiella burnetii sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme was cloned by immunological screening of a lambda EMBL3 genomic library prepared from strain Nine Mile DNA and sequenced. The homology of the cloned gene product to the counterpart in Escherichia coli was 54.3%, but the homology of the N-terminal region was only 42%. The gene was expressed in E. coli as an independent unit from its own promoter, producing an immunoreactive protein of about 50 kDa on SDS-PAGE which reacted with antisera from laboratory animals and sera from human patients with acute Q fever. The study results suggest that the C. burnetii E2o enzyme may serve as a potential target antigen for diagnostic assays for Q fever.     

灭活贝氏柯克斯体免疫小鼠抗登革病毒感染的实验研究

目的 研究灭活贝氏柯克斯体增强小鼠非特异性抗登革病毒感染的免疫效能。方法 用灭活贝氏柯克斯体全细胞疫苗(WCV)免疫BALB/c小鼠,每只小鼠皮下注射50μg WCV,初次免疫后第5周及第7周分别腹腔注射20μg WCV加强免疫。1周后,用10~5 TCID_(50)剂量的登革病毒2型经尾静脉注入感染小鼠,于感染后48、72h分别解剖小鼠.自血和脑组织提取RNA样本,用实时荧光定量聚合酶链反应(PCR)检测样本。结果 用登革病毒特异的定量PCR检测感染小鼠血RNA样本,结果显示感染72h血样本中病毒含量显著低于48h样本,但免疫小鼠与未免疫小鼠之间无显著差异。检测脑组织RNA样本,未免疫小鼠和免疫小鼠的感染48h样本检出病毒均为少量;但未免疫小鼠感染96h样本检出病毒量明显增加,显著高于免疫小鼠。结论 灭活贝氏柯克斯体可诱导机体产生非特异的免疫应答,具有一定增强小鼠抗登革病毒感染的能力,但具体机制有待进一步研究。     

PCR法对鸡卵黄中Coxiella burnetii菌的测定

[目的]通过用定量PCR加巢式PCR方法,提高了对Coxiella burnetii (C.b)CoMl基因的检出率;通过对鸡卵中病原微生物Coxiella burnetii的基因检测,明确鸡卵的食品安全性;并对明确Coxiella burnetii的流行病学有重要意义.[方法]提取鸡卵DNA,用定量PCR加巢式PCR方法检测上述基因,并对PCR产物进行测序分析,通过间接免疫荧光法观察鸡血白细胞中的微生物.[结果]用定量PCR加巢式PCR方法可检出4个以上的Coxiella burnetii Coml基因,用此方法可测出鸡卵中Coxiella burnetii Coml基因达104-106个,阳性率为5％-22％;对阳性鸡卵Coml基因PCR产物的测序结果显示有变异菌株的存在;免疫荧光法可见鸡卵中含有该微生物.[结论]由此认为鸡卵中存在病原微生物Coxiella burnetii,可能是Q热传染源.     

感染小鼠组织中Q热立克次体的分子病理学检测

腹腔注射Q热立克次体悬液感染BALB／c小鼠,发病后解剖,取主要组织脏器,应用免疫组化和原位杂交检测感染小鼠体内Q热立克次体的抗原分布和DNA在靶细胞中的表达,探讨Q热病变规律及致病机理。结果显示阳性信号多位于单核-巨噬细胞系统细胞胞浆内。结果提示免疫组化和原位杂交技术可以作为Q热特异性的诊断方法,并为致病机理的研究提供线索。