应用PCR检测犬埃立克体病的研究

用已确证为扁平埃立克体（Ehrlichia platys)和犬埃立克体（E.canis）混合感染的犬全血对6只格犬进行人工感染，通过对其临床症状，血流学变化，血涂片包涵体观察，并将其PCR结果与后来确诊为犬埃立克体病的18只病犬血的PCR结果比较，表明PCR方法能够对早期犬埃立克体病做出准确诊断。     

比格犬埃立克体病实验模型研究Ⅰ.血常规及血生化检测

为观察犬埃立克体病急性期血液学变化,对6只人工感染埃立克体的比格犬进行了血常规及血生化检测.采集比格犬感染前后的血液样本,测定血常规及各项血生化指标,并对测定结果作统计学处理.血常规检测发现感染后RBC、WBC、HGB、HCT、PLT、MPV、LYM和MONO值极显著降低(P＜0.01),RDW显著降低(P＜0.05);血生化检测发现感染后ALT和AST极显著升高(P＜0.01),ALB极显著降低(P＜0.01).结果表明,我国犬埃立克体病急性期血液学变化较国外报道的更为明显,这可能与埃立克体中国病原株的致病性不同有关.     

比格犬埃立克体病实验模型的建立

为犬埃立克体病的病理学、临床学、病原学的深入研究，我们用Beagle(比格犬）复制犬埃立克体病以建立犬埃立克体病实验模型。比格犬静脉接种E.platy和E.canis后，利用PCR和电镜技术确证感染成功。进一步的观察发现，接种后比格犬与最最感的德国牧羊犬自然感染埃立克体病的临床症关放病理学变化基本吻合。结果说明通过静脉接种病原成功建立了比格犬埃立克体病实验模型。     

我国犬埃立克体病病原分离与鉴定 Ⅱ.我国病原株与其他埃立克体间的遗传距离分析

应用clustalw 计算机分析软件,对我国南方某养犬基地的埃立克体病原株(Gzh981 和Gzh982)与基因库中其他相关埃立克体16SrRNA 基因进行了匹配分析。结果表明,我国的2 种分离株分别与E. platys和 E. canis 的遗传距离最小(0.001)。在系统发育中, Gzh981 和 E. platys、HGE、E. equi、E.phagocytophila 同属一群;Gzh982 和E. canis、E. ew ingii、E. chaffeensis、E. m uris同属一群。     

我国犬埃立克体病研究现状

犬埃立克体病是由严格细胞内寄生的埃立克体所致的疾病。目前,我国已经发现的犬埃立克体病原有犬埃立克体(Ehrlichia)和扁平埃立克体(E.platy)两种。我国已发现的犬埃立克体病均为E.canis和E.platy的混合感染。临床症状包括发热、体重严重减轻、流鼻血和腹泻带血等;白细胞和血小板计数同时下降是我国犬埃立克体病的特点;严重的病犬常因广泛性出血或继发感染而死亡。犬埃立克体病的病原学诊断主要依靠镜检、血清学试验和PCR等。犬血清埃立克体抗体的调查结果表明,我国犬埃立克体病的流行与蜱活动密切相关。四环素类抗生素是治疗犬埃立克体病的有效药物。     

我国犬埃立克体病病原分离与鉴定Ⅲ.病原的电镜观察

对埃立克体感染犬的单核细胞和血小板进行了透射电镜观察，结果表明，单核细胞的细胞质和血小板中均存在埃立克体包涵体，其中单核细胞的包涵体内病原多达８个，血小板的包涵体内至少有３个病原。这一结果从形态学角度进一步证实了引起了广州市郊栽养犬基地流行犬埃立克体病的病原为２处，即感染单核细胞的犬埃立克体（Ｅｈｒｌｉｃｈｉａ ｃａｎｉｓ）和感染血小板的扁平埃立克体（Ｅ．ｐｌａｔｙｓ）。     

我国犬埃立克体病病原分离与鉴定：Ⅱ.我国病原株与其他埃立克体间 …

应用Ｃｌｕｓｔａｌｗ计算机分析软件,对我国南方基地的埃立克体病原株（Ｇｚｈ９８１）和Ｇｚｈ９８２）与基因库中其他相关埃立克体１６ＳｒＲＮＡ基因进行了匹配分析。结果表明,我国的２种分离株分别与Ｅ．ｐｌａｔｙｓ和Ｅ．ｃａｎｉｓ的遗传距离最小（０．００１）。在系统发育中,Ｇｚｈ９８１和Ｅ．ｐｌａｔｙｓ、ＨＧＥ、Ｅ．ｅｑｕｉ、Ｅ．ｐｈａｇｏｃｙｔｏｐｈｉｌａ同属一群;Ｇｚｈ９８２和Ｅ．ｃａｎｉｓ、Ｅ．ｅｗ     

犬埃立克体实验模型初步研究

将犬埃立克体标准毒株（Florida株）体外细胞纯培养物和我国犬埃立克体病病犬血白细胞分别接种于C3H小鼠，接毒后d，经PCR检测证实均感染成功。本试验为犬埃立克体病小动物模型的建立打下了基础。     

我国犬埃立克体病病原的分离与鉴定——Ⅰ.病原16SrRNA基因序列测定与分析

用实验动物接种法,分离保存我国某养犬基地犬埃立克体病病原。以此病原对易感犬接毒,接毒犬发病后采集急性期全血提取基因组,对病原 １６ Ｓｒ Ｒ Ｎ Ａ 基因进行扩增、克隆测序。结果,病原被保存在病犬血细胞内,并能成功地使健康犬感染发病;检测到的 ２ 种致犬埃立克体病病原 １６ Ｓ ｒ Ｒ Ｎ Ａ 基因序列与国外已发现的相应病原基因序列有一定差异,显示其为不同菌株,分别命名为埃立克体 Ｇｚｈ ９８１ 株和 Ｇｚｈ９８２ 株。进一步分析表明,导致我国某养犬基地犬埃立克体病的病原为扁平埃立克体和犬埃立克体。     

vMDV感染雏鸡的Se-GSH-PX活性和LPO含量

以马立克氏病强毒（ｖＭＤＶ）人工感染１日龄ＡＡ肉用雏鸡,于感染后７,１４,２８,４２和５６日龄分别检测雏鸡中枢、外周淋巴器官及主要内脏器官的含硒谷胱甘肽过氧化物酶（Ｓｅ－ＧＳＨ－ＰＸ）活性和脂质过氧化物（ＬＰＯ）含量的变化。结果表明,ｖＭＤＶ感染雏鸡各器官Ｓｅ－ＧＳＨ－ＰＸ活性在检测各时期多显著低于健康对照雏鸡（Ｐ＜０．０５,Ｐ＜０．０１）,而各器官ＬＰＯ含量则多显著高于健康对照雏鸡（Ｐ＜０．０５,Ｐ＜０．０１）,提示马立克氏病（ＭＤ）及其肿瘤的发生和发展与Ｓｅ－ＧＳＨ－ＰＸ抗氧化活性降低和ＬＰＯ及其降解物对雏鸡的广泛病理损伤有关。     

Serological survey for Ehrlichia canis in urban dogs from the major population centres of northern Australia

OBJECTIVE: To detect evidence of Ehrlichia canis infection of dogs from the major population centres of northern Australia, if present. DESIGN: Serological investigation for E. canis. PROCEDURE: The sera of 316 domestic dogs, collected from the northern Australian population centres of Townsville, Cairns, Darwin, Kununurra and Broome from May 1997 to August 1999, were investigated for evidence of infection with E. canis. Samples were tested for antibodies to E. canis using an indirect fluorescent antibody (IFA) test. The buffy coats from blood of dogs whose serum reacted in the IFA test were subsequently tested with a nested PCR to detect E. canis DNA. When available, blood from these dogs was injected into suckling mice, which were then examined for clinical disease and tested for the presence of E. canis antibodies. RESULTS: Of the 316 samples tested seven reacted in the IFA test for E. canis. None of the dogs from which these samples were obtained exhibited clinical signs of acute or chronic ehrlichiosis. The six positive samples available for testing were negative when tested with the nested PCR. Suckling mice inoculated with blood from three of the dogs whose serum was positive by IFA test showed no signs of clinical disease nor did their give positive reactions in the IFA test. CONCLUSIONS: No evidence of E. canis infection was confirmed in any of the dogs examined. Northern Australia would appear to remain free of this obligate parasite.     

Doxycycline clearance of experimentally induced chronic Ehrlichia canis infection in dogs

BACKGROUND: Ineffective clearance of Ehrlichia canis after doxycycline administration has been reported despite the fact that the recommended treatment for canine ehrlichiosis is doxycycline. The effectiveness of doxycycline in clearing E canis infection from the blood and tissues of dogs requires additional evaluation. HYPOTHESIS: Doxycycline (5 mg/kg PO q12h), administered for 4 weeks, will eliminate E canis infection from the blood and tissues of experimentally infected dogs. ANIMALS: Fifteen Walker hound-mixed breed dogs were inoculated subcutaneously with E canis-infected canine histiocytic cells 4 months before doxycycline treatment. METHODS: Four dogs were treated with doxycycline (5 mg/kg PO q12h for 3 weeks), 5 dogs were treated with doxycycline at the same dosage for 4 weeks, and 5 control dogs were not treated. Dexamethasone (0.4 mg/kg i.v.) was given after treatment to precipitate recrudescence of any remaining E canis organisms. Platelet counts, anti-E canis immunofluorescent antibodies, and polymerase chain reaction (PCR) detection of E canis deoxyribonucleic acid (DNA) in blood and tissues were evaluated. RESULTS: E canis DNA was not detected in the blood and tissues of doxycycline-treated dogs after treatment. Platelet counts were within reference intervals, and E canis antibodies decreased. Spontaneous clearance of E canis infection occurred in 2 of 5 control dogs. Three control dogs had E canis DNA detected in blood and tissues, platelet counts remained low or within the reference interval, and E canis antibodies remained high. CONCLUSIONS AND CLINICAL IMPORTANCE: As administered in this study, doxycycline cleared E canis from the blood and tissues of experimentally infected dogs.     

Investigation of glomerular lesions in dogs with acute experimentally induced Ehrlichia canis infection.

Six male Beagles were inoculated with Ehrlichia canis. Transient proteinuria was confirmed during the acute phase of infection by serial determination of urinary protein-to-creatinine ratio. Peak urine protein loss, consisting principally of albumin, was observed 2.5 to 3.5 weeks after inoculation. Renal biopsy specimens were obtained before inoculation, during peak proteinuria, and 10 weeks after inoculation when proteinuria had resolved. Renal tissue was evaluated by use of light, immunofluorescent, and electron microscopy to correlate specific glomerular lesions with development of proteinuria. Histologic examination revealed perivenular and interstitial infiltrates of lymphocytes and plasma cells localized principally to the renal cortex. Glomerular lesions were minimal to absent. Immunofluorescent staining revealed moderate to marked deposition of anti-canine IgG and IgM in the glomerular tufts and mesangium. Depositions of anti-canine complement factor C3 were not observed. Immunofluorescent staining persisted 10 weeks after inoculation, despite resolution of proteinuria, and probably represented passive trapping of immunoglobulins. Ultrastructural examination revealed fusion of podocyte processes that coincided with development of proteinuria. Electron-dense deposits or changes in the basement membrane were not observed. Morphometric measurements of average podocyte process length and percentage of coverage of basement membrane by podocyte processes were used to quantify the degree of process fusion. Both measurements increased significantly (P < 0.05) during peak proteinuria, and returned to preinoculation values when proteinuria had resolved 10 weeks after E canis inoculation. These findings indicated possible minimal-change glomerulopathy, rather than immune-complex glomerulonephritis, during acute E canis infection and could explain transient proteinuria without histologic evidence of glomerular disease.     

Susceptibility of dogs to infection with Ehrlichia chaffeensis, causative agent of human ehrlichiosis.

Ehrlichia chaffeensis, the newly recognized agent of human ehrlichiosis, is closely related to E canis, the causative agent of canine ehrlichiosis. Eight pups were inoculated IV with E chaffeensis-, or with E canis-infected DH82 cells, or organisms released from these host cells. Two additional pups served as nonexposed controls. Marked thrombocytopenia was observed in the E canis-infected pups, but not in those infected with E chaffeensis. Homologous serologic response was observed in the E chaffeensis-exposed pups by postinoculation day (PID) 14 and in the E canis-exposed pups by PID 21. Ehrlichia chaffeensis and E canis were reisolated from the respective inoculated pups on each of 8 attempts from PID 7 to 26. One E chaffeensis-exposed pup that was challenge exposed with E canis via blood transfusion, developed fever, anorexia, and thrombocytopenia, suggesting lack of cross protection against E canis.     

Serosurvey of Ehrlichia canis and Hepatozoon canis infection in dogs in Yamaguchi Prefecture, Japan

Antibodies to Ehrlichia canis and Hepatozoon canis in dogs at the Animal Hospital in Yamaguchi University were surveyed and potential risk factors for both pathogens were evaluated. Among 430 dogs examined, 20 (4.7%) and 18 (4.2%) dogs showed positive findings for E. canis and H. canis, respectively. Neither, sex nor age was associated with the seropositivity of either pathogen, but the positive rate in dogs kept outside was slightly higher than that in dogs kept inside for both pathogens. A higher seropositive reaction to E. canis and H. canis was observed in dogs that lived in certain cities and towns. Beagles, golden retrievers and pointers had higher seropositivity than other breeds in E. canis, whereas shibas, akitas, beagles, pointers and mongrels had higher positive rates than other breeds in H. canis.     

Evaluation of neutrophil oxidative metabolism in canine monocytic ehrlichiosis

BACKGROUND: Canine monocytic ehrlichiosis (CME) is a tick-borne disease caused by Ehrlichia canis, a rickettsia that infects the monocytes of dogs. This infection can result in a chronic and life-threatening disease. Thrombocytopenia, mild anemia, and leukopenia are the most common hematologic findings in CME. OBJECTIVE: To investigate the role of peripheral blood neutrophils in CME, an evaluation was conducted of their functional state during the acute phase of the disease in dogs experimentally infected by E canis. METHODS: Seven dogs were inoculated with E canis, and 3 remained as uninfected controls. All dogs had physical exams and hematologic tests (CBC and nitroblue tetrazolium [NBT] reduction) during a 6-week period. RESULTS: There was no difference (P > .05) in spontaneous NBT reduction results between the 2 groups of dogs throughout the 6-week period of observation. Nevertheless, when stimulated, the neutrophils showed higher activity in the infected group (P = .01) on weeks 4 and 5 after infection. CONCLUSION: Infection by E canis has no influence on neutrophil oxidative metabolism even though during the remission period of the acute phase of the disease, the neutrophils seem to be more reactive under stimulation.     

Experimental inoculation of beagle dogs with Ehrlichia species detected from Ixodes ovatus

Three beagle dogs were inoculated with mice spleen/liver homogenate infected with Ehrlichia species detected from Ixodes ovatus (EIO) and one dog was used as a control. All three infected dogs did not show clinical signs of disease except for mild pyrexia throughout the 41-day study period. Splenomegaly was observed from Day 7 post-inoculation (p.i.) in two of the dogs. Hematological and biochemical abnormalities included mild thrombocytopenia, hypoproteinaemia, hypoalbuminaemia and increased C-reactive protein values. One of the dogs' splenic aspirate sample was PCR-positive for Ehrlichia Day 7 p.i. and another dogs' blood and bone marrow aspirate sample was PCR-positive Day 41 p.i. Sequence analysis of the PCR products showed 100% homology with the 16SrRNA partial gene sequence of Ehrlichia sp. HF565. Antibody titers to EIO were observed in all three experimentally infected dogs starting from the first week p.i. and cross-reactivity with Ehrlichia canis was detectable in one of the dogs starting Day 7 p.i. These data suggest that infection of dogs with EIO is possible, though is probably of low pathogenic importance. Cross-reactivity of EIO infected dog serum with E. canis raises the likelihood of false E. canis seropositive dogs.     

Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings

Clinical signs, humoral and cellular immune responses, and microscopic and gross tissue alterations resulting from acute experimental Ehrlichia canis infection in dogs were studied. Four dogs were inoculated with E. canis and four were used as uninfected controls. After a 10-14-day incubation period, infected dogs developed pyrexia up to 41 degrees C for 6-8 days. Antibody titers to E. canis antigen were demonstrable in all inoculated dogs at 30 days post-infection. Necropsy of infected animals revealed pale mucous membranes, generalized lymphadenopathy, splenomegaly, edema and ascites. Microcopically, the main lesions were: lymphoreticular hyperplasia in cortical areas of lymph nodes and spleenic white pulp, periportal accumulation of mononuclear cells and centrolobular fatty degeneration of the liver. Kidneys presented with glomerulonephritis characterized by interstitial mononuclear infiltration. Immunophenotyping of lymphocytes from lymph nodes and spleen sections displayed alterations in IgG, IgM, CD3+ and CD8+ cells population in infected dogs.