Hematologic and Clinical Aspects of Experimental Ovine Anaplasmosis Caused by Anaplasma ovis in Iran

Background

Anaplasma ovis infections can cause clinical symptoms in acute phase and lead to huge economic losses in flocks. The aim of the present study was to investigate the hematological and parasitological changes in experimental anaplasmosis in sheep with Iranian strain of A. ovis.

Method

Five male sheep without any blood parasite infection were selected. One hundred ml heparinized blood was collected from splenectomised sheep that showed 6% A. ovis parasitemia. Inoculums of 20 ml blood were administered intravenously to each test animal. Hematological, parasitological and clinical changes of experimental anaplasmosis were studied in 0-38 days post infection.

Result

Parasitemia was detected 3 days post infection and reached its maximum level on the day 12 of experiment in test animals. Then the parasitemia was declined, but the organism could be found persistently until the last day of study. The red cell counts, packed cell volume and hemoglobin concentration were decreased and mean corpuscular volume was increased significantly during the infection period. Reticulocytosis and basophilic stippling were also detected. No significant changes were observed in total and differential leukocyte count and animal body temperature.

Conclusion

Experimental A. ovis infection in sheep resulted in marked normocytic normochromic anemia at the beginning of the infection which became macrocytic normochromic by the development of the disease. There were negative correlations between parasitemia and RBC, PCV and Hb values, therefore hematological assessment can be considered as a practical diagnostic tool in ovine anaplasmosis.     

Serological investigation of vector-borne disease in dogs from rural areas of China

Objective

To evaluate the Anaplasma phagocytophilum (A. phagocytophilum), Ehrlichia canis (E. canis), Dirofilaria immitis (D. immitis) (canine heartworm), Borrelia burgdorferi (B. burgdorferi) infections in countryside dogs from Yunnan, Hainan and Anhui provinces.

Methods

Serum samples were collected from 26 dogs in Yunnan, Hainan and Anhui provinces. The samples were tested using a commercial ELISA rapid diagnostic assay kit (SNAP^® 4Dx^®; IDEXX Laboratories, Inc. U.S.A.). Meanwhile, indirect immunofluorescence assay (IFA) recommended by WHO was conducted to detect IgG to A. phagocytophilum. Two methods were analyzed and compared.

Results

The number of serologically positive dogs for IgG to A. phagocytophilum was only 2 which was from Hainan province and none of the 26 dogs responded positive for E. canis, D. immitis (canine heartworm), and B. burgdorferi by ELISA rapid diagnostic method. The number of serologically positive dogs for IgG to A. phagocytophilum was 13 (50%) by IFA method. Data of the two methods were analyzed by statistical software and the difference was statistically significant (P=0.002).

Conclusions

It can be concluded that IFA method was more sensitive than ELISA rapid diagnostic method. However, we need conduct further and intensive epidemiology survey on tick-born diseases pathogens including A. phagocytophilum, E. canis, D. immitis (canine heartworm), and B. burgdorferi which have public health significance.     

The Lyme Wars: time to listen

Lyme disease represents a public health threat of major proportions. The murky science and acrimonious politics of Lyme disease have created barriers to reliable diagnosis and effective treatment of this protean illness. Two major clinical problems with the disease are the absence of a therapeutic end point in treating Borrelia burgdorferi, the spirochetal agent of Lyme disease, and the presence of tick-borne co-infections, such as babesiosis, anaplasmosis and bartonellosis, that may complicate the course of the illness. From a pathophysiological standpoint, the affinity of B. burgdorferi for multiple cell types and the presence of non-replicating forms of the spirochete have contributed to persistent infection and failure of simple antibiotic regimens. Newer approaches to the treatment of Lyme disease should take into account its clinical complexity in co-infected patients and the possible need for prolonged combination therapy in patients with persistent symptoms of this potentially debilitating illness. The risk and prevention of human transmission of Lyme disease merit further study.     

New real-time PCR-based method for in vitro susceptibility testing of Anaplasma phagocytophilum against antimicrobial agents

Up to now, only a few isolates of Anaplasma phagocytophilum have been tested for their susceptibility against a small number of antimicrobial agents. In addition, as with other fastidious or intracellular bacteria, the test methods are laborious and neither minimal inhibitory concentration (MIC) definitions, nor the test conditions and the inocula are standardised to date. A new 16S-rDNA-based real-time PCR assay has been developed and used under standardised conditions to analyse the activity of seven antimicrobial agents against two A. phagocytophilum isolates. After 72 h incubation, MICs were determined by software-assisted calculation of bacterial growth in samples and controls from semi-quantitative PCR results. In our study, the rank order of potency on a mg/l basis for the antimicrobial agents with enhanced in vitro activity against A. phagocytophilum was moxifloxacin (MIC: ≤0.03 mg/l) > doxycycline (MIC: ≤0.125 mg/l) > ciprofloxacin (MIC: 0.125 mg/l). Gentamicin, ampicillin, azithromycin and cethromycin showed no activity against the isolates tested in this investigation. Our new 16S-rDNA-PCR-based microdilution test system was shown to be sensitive, reproducible and reliable. The assay is capable of testing larger numbers of isolates and antimicrobial agents under standardised and very precise test conditions and may therefore offer a competent technical solution of the difficulties known to be associated with in vitro testing of other bacterial pathogens that grow intracellularly, such as chlamydia or rickettsia.     

Tickborne pathogen detection, Western Siberia, Russia

Ixodes persulcatus (n = 125) and Dermacentor reticulatus (n = 84) ticks from Western Siberia, Russia, were tested for infection with Borrelia, Anaplasma/Ehrlichia, Bartonella, and Babesia spp. by using nested polymerase chain reaction assays with subsequent sequencing. I. persulcatus ticks were infected with Borrelia burgdorferi sensu lato (37.6% +/- 4.3% [standard deviation]), Anaplasma phagocytophilum (2.4% +/- 1.4%), Ehrlichia muris (8.8% +/- 2.5%), and Bartonella spp. (37.6% +/- 4.3%). D. reticulatus ticks contained DNA of B. burgdorferi sensu lato (3.6% +/- 2.0%), Bartonella spp. (21.4% +/- 4.5%), and Babesia canis canis (3.6% +/- 2.0%). Borrelia garinii, Borrelia afzelii, and their mixed infections were observed among I. persulcatus, whereas B. garinii NT29 DNA was seen in samples from D. reticulatus. Among the I. persulcatus ticks studied, no Babesia spp. were observed, whereas B. canis canis was the single subspecies found in D. reticulatus.     

Anaplasma phagocytophilum-infected ticks, Japan

We report Anaplasma phagocytophilum infection of Ixodes persulcatus and I. ovatus ticks in Japan. Unique p44/msp2 paralogs (and/or 16S rRNA genes) were detected in tick tissues, salivary glands, and spleens of experimentally infected mice. These findings indicate the public health threat of anaplasmosis in Japan.     

Antigenicity of ovine strains of Anaplasma phagocytophilum grown in tick cells and ovine granulocytes

Antigens prepared from ovine granulocytes and tick cells infected with ovine strains of Anaplasma phagocytophilum, the causative agent of tick-borne fever, were tested in respect of their suitability for the assay of antibodies in ovine sera by enzyme-linked immunosorbent assay (ELISA) and by indirect immunofluorescent antibody test (IFAT). Antigens prepared from tick cells were as sensitive and specific as those expressed in ovine granulocytes for the detection of specific antibodies by ELISA, but they failed to react in the IFAT with immune sera obtained from sheep previously infected with ovine strains of A. phagocytophilum.     

伯氏疏螺旋体与嗜吞噬细胞无浆体感染抑制Th1型细胞因子产生

目的 应用动物模型探讨蜱传病原体伯氏疏螺旋体和嗜吞噬细胞无浆体混合感染对Th1型细胞因子产生的影响.方法 建立三种伯氏疏螺旋体和嗜吞噬细胞无浆体混合感染小鼠模型和伯氏疏螺旋体单一感染小鼠模型.将小鼠分A组（同时感染组）、B组（伯氏疏螺旋体先于嗜吞噬细胞无浆体感染组）、C组（嗜吞噬细胞无浆体先于伯氏疏螺旋体感染组）、D组（伯氏疏螺旋体单感染组）和E组（无感染对照组）等共5组,每组16只,在不同时间点测定各组小鼠血清Th1型细胞因子浓度,并对数据进行统计学分析.结果 与单一感染组相比,在螺旋体感染后18d和30d,三种混合感染组小鼠血清中Th1型细胞因子显著偏低.结论 伯氏疏螺旋体和嗜吞噬细胞无浆体混合感染显著抑制Th1型细胞因子产生.     

荧光定量PCR检测嗜吞噬细胞无形体

目的采用新型TaqMan-MGB探针建立检测嗜吞噬细胞无形体的实时荧光定量PCR(quantitativereal-timePCR)方法。方法依据gltA基因序列设计嗜吞噬细胞无形体特异引物和探针,以克隆的嗜吞噬细胞无形体gltA基因片段作DNA模板,在荧光定量PCR检测仪(ABI7900HT)上建立实时荧光定量检测方法。结果建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.996);与套式PCR相比较,荧光定量PCR检测的灵敏度是其100倍。用荧光定量PCR检测其它相关立克次体和细菌DNA样本,检出结果几乎为0;对荧光定量PCR检测重复性进行分析,变异系数(CV)批内和批间误差在0~2.1%之间,证明该荧光定量PCR具有种特异性和良好的重复性。用荧光定量PCR检测体疑染嗜吞噬细胞无形体的10份蜱和30份小鼠脾脏标本,结果与套式PCR检测结果有密切相关性,但是定量PCR检测敏感性和准确率均高于套式PCR。结论本研究建立的检测嗜吞噬细胞无形体的实时荧光定量PCR具有很高的特异性和敏感性,特别适合检出样本中微量嗜吞噬细胞无形体。