A comparative evaluation of parasitological tests and a PCR for Trypanosoma evansi diagnosis in experimentally infected water buffaloes

In this study five parasitological methods and a polymerase chain reaction (PCR) were compared for the diagnostic sensitivity for Trypanosoma evansi in experimentally infected water buffaloes over a period of 15 weeks. The combined estimates of sensitivity (CE(se)) of the PCR proved to be highest at 78.2%, closely followed by the mouse inoculation (MI), the micro-haematocrite centrifugation technique (MHCT) and the mini-anion-exchange centrifugation technique (MAECT) with CE(se) of, respectively, 74.0, 69.6 and 62.4%. The CE(se) of the buffy-coat technique (BCT) at 38.6% and the sodium dodecyl sulfate (SDS) clarification technique at 25.1% were considerably lower. PCR detected consistently all buffaloes infected from week 3 post-infection (PI) onwards. For MI this occurred after 5 weeks PI while for MHCT and MAECT these sustainable high levels were reached in the 7th week PI. BCT and SDS never detected all buffaloes infected. The influence of time and temperature on the viability of T. evansi in heparinized blood from water buffalo was also studied. In general we observed that the survival time tends to be longer when blood is kept at 4 degrees C. In samples kept in direct sunlight parasites became undetectable with the MHCT after 30min. After treatment of the water buffaloes with diminazene aceturate, the PCR signal disappeared within 24h.     

Evaluation of Diagnostic Tests for <Emphasis Type="Italic">Trypanosoma evansi</Emphasis> in Experimentally Infected Pigs and Subsequent Use in Field Surveys in North Vietnam and Thailand

This study is concerned with the evaluation of established diagnostic tests for diagnosis of Trypanosoma evansi in pigs. The immune trypanolysis test (TL), card agglutination test (CATT), latex agglutination test (LATEX), enzyme-linked immunosorbent assay (ELISA), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI) tests were initially evaluated in experimentally infected fattening pigs. All infected pigs were confirmed parasitologically positive with both MHCT and MI. Results of the serological assays indicated that the TL could be a reference test for the presence of RoTat 1.2 antibodies in pigs. The results of the CATT and LATEX were inconsistent with the TL while the ELISA results correlated with the TL results. The four serological assays were subsequently used in two field surveys in Vietnam and Thailand. Results of the two agglutination assays (CATT and LATEX) were not consistent and did not correlate with TL results. The ELISA at percentage positivity of 22 appeared to have good ability to discriminate between seropositive and seronegative animals. Of the 437 samples collected at smallholder pig premises in northern Vietnam, no positive pigs were detected with the TL test. In Thailand, 77 samples were collected from five farrowing farms with a history of surra. Two parasitologically positive sows were found and on each farm seropositive sows were detected.     

Detection of Trypanosoma evansi in camels using PCR and CATT/T. evansi tests in Kenya

Camel trypanosomosis (Surra) causes high morbidity and is an impediment to the camel husbandry in Kenya. The lack of a sensitive diagnostic test has hindered the collection of accurate epidemiological data and institution of control programmes. A cross-sectional study was conducted in three districts of Kenya to estimate the prevalence of Trypanosoma evansi (T. evansi) and to compare four diagnostic tests: polymerase chain reaction (PCR), card agglutination test (CATT/T. evansi), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI). A total of 549 camels were randomly sampled. The overall prevalence of Surra was 5.3% using MHCT, 26.6% using PCR and 45.9% using CATT/T.evansi. There was a significant difference (P < 0.001) between PCR and CATT/T.evansi test, MHCT and MI in detection of T. evansi. The prevalence of T. evansi was 39.8% in Samburu, 24.7% in Nanyuki and 14.4% in Isiolo districts using PCR. A male camel was 2.6 times more likely to be infected with T. evansi compared to a female camel (OR = 3.0% CI: 1.6, 4.1), while an adult camel was 2.2 times more likely to be infected compared to non-adults (OR = 2.2; 95% CI: 1.2, 5.0). There was a poor association between the presence of the published clinical signs and seropositivity (kappa = 0.12), PCR (kappa = 0.11) and MHCT (kappa = 0.05). However, there was a higher agreement between farmers' classification of disease with the PCR test (kappa = 0.5, n = 61). The mean PCV varied with age, presence of infection, locality and gender, with the lowest mean PCV being recorded in MHCT-positive animals (20.97 +/- 0.5) and from infected calves (19.5 +/- 1.2). This study shows that PCR was more sensitive in detecting T. evansi than other tests used. Further, the prevalence of T. evansi in the camel herds sampled is higher than that previously reported in Kenya, and that the judgment by camel keepers may be a reliable "pen-side" diagnostic test for Surra. Considering the low sensitivity of parasitological techniques in detection of chronic T. evansi infection and high cost of PCR, development of a sensitive pen side diagnostic test, with a low cost is still a priority.     

Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis

Loop-mediated isothermal amplification (LAMP) is a simple method that can amplify DNA with high specificity, sensitivity, and rapidity. In this study, we compared the performance of LAMP and real-time PCR assays for diagnosis of toxoplasmosis. We designed a real-time PCR assay targeting a 529 bp element repeated 200-300 times in the Toxoplasma gondii genome. The detection limits of the LAMP and real-time PCR assays were 10 fg/μL and 1 fg/μL of T. gondii DNA, respectively. Conventional PCR, LAMP, and real-time PCR methods were applied to detect T. gondii DNA in blood samples from 284 pigs and 292 sheep. Positive results were obtained with 0.4%, 3.2%, and 4.2% of the pig samples and 3.8%, 17.1%, and 17.8% of the sheep samples with conventional PCR, LAMP, and real-time PCR analyses, respectively. The real-time PCR assay provided the most sensitive diagnosis of toxoplasmosis, but the LAMP assay has potential as an alternative tool for detection of T. gondii in the field.     

Establishment and Preliminary Application of Nano-PCR and LAMP Methods for Bovine Parainfluenza Type-3 Virus

This study was aimed to establish Nano-PCR and LAMP which were new rapid type of molecular detection technologies of bovine parainfluenza type-3 virus (BPIV3).The comparative specificity and sensitivity of BPIV3 Nano-PCR and LAMP PCR were tested, and the assay was applied to detect 10 clinical samples. The specificity and sensitivity tests showed that Nano-PCR and LAMP were only sensitive to BPIV3,without cross reaction to other viruses, such as bovine respiratory syncytial virus,infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. In the sensitivity test, Nano-PCR and LAMP showed 10 times sensitivity than that of the traditional PCR technology,with the minimum detection of 4.16×10² copies/μL. Clinical test results showed that the coincidence rate of Nano-PCR and LAMP could reach to 100%, and the positive detection rate was much higher than that of normal PCR.Therefore, the Nano-PCR and LAMP methods established in this study provided a faster, sensitive and reliable tool for the clinical diagnosis of BPIV3.     

牛副流感3型病毒Nano-PCR、LAMP方法的建立及初步应用

试验旨在建立牛副流感3型病毒（bovine parainfluenza type-3 virus,BPIV3）纳米PCR（Nano-PCR）与环介导等温扩增技术（loop-mediated isothermal amplification,LAMP）新型快速分子检测技术,对其进行特异性和敏感性对比试验,并对10份临床样品进行了检测。特异性与敏感性试验结果显示,建立的Nano-PCR和LAMP方法只对BPIV3特异,而对牛呼吸道合胞体病毒、牛传染性鼻气管炎病毒、牛病毒性腹泻病毒无交叉反应;建立的Nano-PCR和LAMP方法具有相同的敏感性,均是普通PCR的10倍,最低核酸检出量均为4.16×10²拷贝/μL。临床检测结果显示,建立的两种方法阳性符合率为100%,且阳性检出率均高于普通PCR。因此,本试验建立的Nano-PCR和LAMP方法为BPIV3的临床诊断提供了更快速、敏感、可靠的工具。     

Detection of canine parvovirus in fecal samples using loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a novel, sensitive, and rapid technique for detection of genomic DNA. The end-product of the technique is a white precipitate of magnesium pyrophosphate that is visible without the use of gel electrophoresis. The LAMP method was applied to the detection of canine parvovirus (CPV) genomic DNA. A set of 4 primers, 2 outer and 2 inner, were designed from CPV genomic DNA targeting the VP2 gene. The optimal reaction time and temperature for LAMP were determined to be 60 minutes and 63 degrees C. On the basis of results for 50 canine fecal samples using polymerase chain reaction (PCR) analysis as the gold standard, the relative sensitivity of LAMP was 100% and the relative specificity was 76.9%. The detection limit of the LAMP method was 10(-1) median tissue culture infective doses (TCID50)/ml, compared with 10 TCID50/ml for PCR analysis. In addition to the advantage resulting from visual detection of the end product, the LAMP method is very rapid, requiring only 1 hour to complete. This assay would be a viable alterative to PCR analysis for diagnosis of CPV infection in dogs. The LAMP method holds promise for use as a diagnostic assay for CPV detection in a clinical setting.     

Preliminary application and evaluation of loop-mediated isothermal amplification (LAMP) for detection of bovine theileriosis and trypanosomosis in Tanzania

The sensitivity of LAMP, PCR and microscopy to detect Theileria spp. and Trypanosoma congolense in field-derived bovine blood samples from Tanzania was evaluated and compared. No parasites were detected by microscopy. Furthermore, no bovine Theileria spp. were detected by LAMP and PCR from all the 24 samples collected from Arusha. Four and one out of 24 samples were positive for Theileria congolense infection by LAMP and PCR respectively while, 18 and nine out of 40 samples from Dar es Salaam were positive by LAMP and PCR for Theileria spp. Infection, respectively. Although all samples from Dar es Salaam were negative for Trypanosoma congolense infections by PCR, 12 out of 40 samples were LAMP positive. Whilst PCR is an established gene amplification method for the detection of Theileria and trypanosome parasites, this study introduces LAMP as an alternative molecular diagnostic tool that could be used in large-scale epidemiological surveys.     

Preliminary evaluation of diagnostic tests using horses experimentally infected with trypanosoma evansi

Seven surra negative horses were intravenously inoculated with 3 x 10(6)Trypanosoma evansi parasites derived from a camel. One horse was maintained as an uninfected negative control. Three antigen and three antibody detection tests were evaluated for diagnosis of infection in horses. The microhaematocrit centrifugation test (MHCT) was the most sensitive, first detecting parasites between one and three days (x 2.4) post infection (p.i.). The antigen (ag)-ELISA detected antigen between three and ten days (x 6.6) p.i. The latex agglutination test (LAT) first gave positive results on day 3 (x 3.0) p.i. Following the treatment of horses with trypanocidal drugs, the MCHT and the mouse inoculation test (MIT) became negative. Antigen levels using LAT declined and reached pre-infection levels in five out of six horses during the period of observation (92-279 days). Antigen levels using the ag-ELISA declined as well but did not reach pre-infection levels in any of the six horses.Three antibody detection techniques, ab-ELISA, card agglutination test (CATT), and immunofluorescent antibody test (IFAT) detected antibodies in the blood of all seven infected horses but not in the uninfected control. However, the ab-ELISA did not discriminate clearly between sera from infected and uninfected horses because unacceptably high ELISA background readings were detected in 15% of the surra negative horses shipped to the UAE from the UK. The ELISA antibody increased above pre-infection levels in the six horses experimentally infected, but not in one horse. In this horse the ELISA antibody level exceeded the cut-off level only after the reoccurrence of the T. evansi infection. The IFAT detected antibodies 15.7 days p.i. in all infected horses.     

A comparative evaluation of parasitological, serological and DNA amplification methods for diagnosis of natural Trypanosoma evansi infection in camels

A representative number of 217 camels (Camelus dromedarius) from different areas of western Rajasthan State, India, were examined from July 2002 to May 2003 for Trypanosoma evansi infection. The tests used were parasitological (wet blood film, WBF; stained thin blood smear, TBS), immunodiagnostic (double antibody sandwich enzyme linked immunosorbent assay for antigen detection, Ag-ELISA), and DNA amplification by polymerase chain reaction (PCR). These techniques were compared and the best efficiency was found for the last named (PCR). A prevalence of T. evansi infection was detected in 17.05, 9.67, 4.60 and 4.14% by PCR, Ag-ELISA, TBS and WBF with a sensitivity of 100, 56.75, 27.02 and 24.32%, respectively. PCR revealed a specific 227bp band in positive samples. The intensity of PCR bands was variable in different test samples depending upon the level of infection in the test samples. The history of intermittent fever, emaciation, oedema, poor body condition significantly correlated with positive serological status in ELISA as well as trypanosome DNA detection by PCR.     

羊布鲁菌快速检测技术应用研究

通过用4种方法对羊布鲁菌的检测,确立适用于基层应用的羊布鲁菌快速检测技术。http://www.mdxycn.com/采用浊度仪LAMP、金属浴LAMP、荧光目视检测试剂盒LAMP和普通PCR共4种方法对羊血液中的布鲁菌进行检测,比较其检测结果。结果表明,共检测162份羊血液,布鲁菌检出率分别为浊度仪LAMP为8.6%、金属浴LAMP为8.6%、荧光目视检测试剂盒为8.6%、普通PCR为6.2%。LAMP较普通PCR方法特异性强且灵敏度高;金属浴LAMP方法操作简便,无需特殊设备,更适用于基层应用。     

Rapid diagnosis of duck plagues virus infection by loop-mediated isothermal amplification

Duck virus enteritis is a serious disease among farmed and free-living ducks (Anatidae) and a constant threat to the commercial duck industry in China. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to rapidly detect and diagnose duck plague virus (DPV) in both farmed and wild waterfowl, and compared with polymerase chain reaction (PCR) method and real-time PCR method in accuracy, sensitivity and specificity. A set of four specific primers was successfully designed to recognize six distinct genomic sequences of UL6 protein from DPV, including one forward inner primer, one back inner primer and two outer primers. The optimum reaction temperature and time were verified to be 61.5 °C and 60 min, respectively. Comparative experiments showed that LAMP assay was a simple, rapid, accurate, sensitive and specific method for detecting DPV, and was superior to PCR assay in sensitivity and specificity for DNA amplification. In addition, challenge tests indicated the newly developed LAMP method was more sensitive for the diagnosis of DPV infection than virus isolation and PCR. LAMP assay would be a good alternative method for on-farm disease diagnosis.     

3种新城疫病毒核酸扩增检测方法的比较研究

本研究利用3种方法对新城疫病毒(Newcastle disease virus,NDV)进行检测,并对这3种检测方法的灵敏度和特异性作出比较。根据新城疫病毒的融合蛋白基因(F基因)设计合成6条特异性引物,利用水浴LAMP法、PCR及LAMP实时浊度仪进行检测。通过对恒温水浴中的反应温度、反应时间及反应液中Mg^2＋浓度进行优化获得最佳反应条件,结果用凝胶电泳分析;用优化的温度进行LAMP实时浊度仪检测,同时都与PCR方法进行比较。结果显示,获得了最佳反应条件,水浴LAMP方法最低检测限是1.58 pg,比PCR高100倍,而LAMP实时浊度仪检测灵敏度比水浴LAMP方法高10倍,最低检测限是0.158 pg。3种方法对其他非新城疫病毒均无检出。结果表明,新城疫病毒水浴LAMP检测方法速度快、不需要高精密的仪器,而且具有灵敏度高、特异性强、操作简单等特点,有望在核酸扩增领域取代PCR技术,LAMP浊度仪法检测灵敏度更高,但是所需仪器和试剂比较昂贵。     

猪6种常见病毒多重PCR检测方法的建立及应用

旨在建立一种可同时检测猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)、猪细小病毒(PPV)和猪巨细胞病毒(PCMV)的多重PCR检测方法。参考相关文献及序列比对结果设计6对特异性引物,建立了可同时检测以上6种病毒的多重PCR方法并应用此方法对临床病例进行了检测。结果表明,所建立的多重PCR方法灵敏度高,对6种病毒的最低核酸检测量分别为12.8pg(PRRSV)、46pg(CSFV)、16pg(PRV)、23.5pg(PCV-2)、72pg(PPV)、8.6pg(PCMV),对猪流感病毒(SIV)、猪乙型脑炎病毒(JEV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)无特异性扩增。应用该方法对临床145份样品进行检测,总阳性率为83.45%,2种以上病毒混合感染阳性率为69.66%。说明所建立的多重PCR检测方法敏感,可用于猪群中上述6种猪病病原的单一感染或混合感染的鉴别诊断。     

Evaluation of a new in‐clinic test system to detect feline immunodeficiency virus and feline leukemia virus infection

Background: Many in‐house tests for the diagnosis of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infection are licensed for use in veterinary practice. A new test with unknown performance has recently appeared on the market. Objectives: The aims of this study were to define the efficacy of a new in‐clinic test system, the Anigen Rapid FIV Ab/FeLV Ag Test, and to compare it with the current leading in‐clinic test, the SNAP Kombi Plus FeLV Antigen/FIB Antibody Test. Methods: Three‐hundred serum samples from randomly selected healthy and diseased cats presented to the Clinic of Small Animal Medicine at Ludwig Maximilian University were tested using both the Anigen Rapid Test and the SNAP Kombi Plus Test. Diagnostic sensitivity, specificity, and positive and negative predictive values were calculated for both tests using Western blot as the gold standard for verification of FIV infection and PCR as the gold standard for FeLV infection. Results: The presence of antibodies against FIV was confirmed by Western blot in 9/300 samples (prevalence 3%). FeLV DNA was detected by PCR in 15/300 samples (prevalence 5%). For FIV infection the Anigen Rapid Test had a sensitivity of 88.9%, specificity of 99.7%, positive predictive value of 88.9%, and negative predictive value of 99.7%. For FeLV infection, the Anigen Rapid Test had a sensitivity of 40.0%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 96.9%. Diagnostic accuracy was similar to that of the SNAP Kombi Plus Test. Conclusion: The new Anigen Rapid FIV Ab/FeLV Ag Test performed very well and can be recommended for use in veterinary practice.     

牛轮状病毒三种核酸检测方法的比较

利用针对牛轮状病毒（BRV）的普通PCR、实时荧光定量PCR（Real-time PCR）和环介导等温扩增技术（LAMP）三种核酸检测方法对不同质粒浓度的样品和417份临床疑似样品进行检测,比较三种核酸检测方法的检出结果。三种核酸检测方法中LAMP最敏感敏感,能检测到1拷贝/μL,Real-time PCR能检测到100拷贝/μL,普通PCR只能检测到1×104拷贝/μL。但结合临床样品检测表明：常规PCR方法敏感度低会造成一部分漏检,LAMP灵敏度高又会造成错检。综合比较三种方法后,推荐用LAMP结合real-time PCR,不仅节约成本,且结果更为准确可靠,可提高牛轮状病毒的检出率。     

PCV2-DNA in formalin-fixed and paraffin embedded lymph nodes of wild boar (Sus scrofa ssp. scrofa): one sampling approach for two laboratory techniques

Superficial inguinal lymph nodes from 72 wild boars examined in a previous immunohistochemical (IHC) study on porcine circovirus type 2 (PCV2) were selected for a PCV2 polymerase chain reaction (PCR) analysis. Four of these lymph nodes were PCV2-IHC strongly positive with PMWS histological lesions (outcome 1), 6 weak to mild PCV2-IHC positive without PMWS histological lesions (outcome 2) and 62 PCV2-IHC negative. Considering IHC the gold standard for diagnosis, the aims of the study were to evaluate the suitability of the PCV2-DNA extraction from formalin-fixed and paraffin-embedded (FFPE) tissue and the sensitivity and specificity of PCR under two IHC interpretations criteria: (A) the sample was considered positive if the result was outcome 1; (B) the sample was considered positive if the result was outcome 1 or 2. Under (A) criteria, sensitivity and specificity of PCR were 100% and 89.7%, respectively; the Cohen''s Kappa coefficient was 0.49. Under (B) criteria, sensitivity and specificity of PCR were 80.0% and 95.2%, respectively; the Cohen''s Kappa coefficient was 0.72. The high Cohen''s Kappa coefficient under the (B) interpretative criteria indicates good agreement between the two methods. In conclusion, 1) DNA extracted from FFPE specimens of wild boar is suitable for PCR and further represents a screening test for PCV2/PCVD (PCV2 Diseases) investigations in wild boar as well; 2) routine histological sampling can also be useful for PCV2 virological studies in wild boar.     

Detection of rhodococcus equi by microbiological culture and by polymerase chain reaction in samples of tracheobronchial secretions of foals

The goal of the present study was to investigate whether new PCR-methods would improve diagnostic of R. equi. In a first step, sensitivity and specificity of the PCR-methods in respect to the"gold standard" microbiological culture were determined. Secondly, sensitivity and specificity of both microbiological methods were evaluated in respect to the clinical diagnosis. The tracheobronchial secretions of 48 foals with pulmonary abscesses and of 37 healthy foals were evaluated by bacteriological culture as well as by four PCR-methods: aceA-, ideR-, vapA- and VP-PCR. In respect to the"gold standard" microbiological culture, the sensitivity of most PCR methods lay between 63.9 and 69.4 % except the vapA-PCR (27.8 %).The specificity of all PCR methods in this comparison was between 98 to 100 %. In this analysis, clinical diagnosis had a low sensitivity (66.7 %) and a low specificity (51.0 %). In respect to the clinical diagnosis, microbiological culture sensitivity was 50.0 % and specificity 67.7 9%. In this analysis, sensitivity rates of aceA-, ideR and VP-PCR methods lay between 33.3 and 37.5 %, sensitivity of the vapA-PCR was lower (10.4 %).The specificity of all PCR methods ranged from 78.4 to 86.5 %. In conclusion, these results show that the diagnostic potential of the microbiological methods"Culture" and "PCR" is different and that for the diagnosis of R. equi-pneumonia in foals the combination of microbiological culture with PCR should be used for examination of samples of the airways of foals.