Comparison between six parasitological methods for diagnosis of Trypanosoma evansi in the subtropical area of Argentina

In a total of 165 blood samples from horses in the Province of Formosa (Argentina), the diagnosis for equine trypanosomiasis (T. evansi) was made using Giemsa-stained smears (GSS), wet blood films (WBF), Strout's concentration method (SCM), haematocrit centrifuge technique (HCT), buffy coat method (BCM) and mouse inoculation of blood (MBI). Trypanosoma evansi was demonstrated in 52 samples. Mouse inoculation gave a sensitivity of 88.2%; HCT 71.1%; BCM 63.4%; WBF 53.8%; SCM 46.1% and GSS 45.6%. No single method alone was totally effective. The haematocrit centrifuge technique, mouse inoculation of blood and Giemsa-stained smears were proposed as the most effective diagnostic combination.     

Influence of Trypanosoma evansi infection on milk yield of dairy cattle in northeast Thailand.

Effect of subclinical Trypanosoma evansi infection on the milk yield of newly introduced Holstein Friesian dairy cattle were investigated. Five hundred pregnant heifers were introduced in Loei Province, northeast Thailand and a total of 168 blood samples were collected at 20 farms during 6 visits over 2 years. Trypanosomes were found in cattle in June and November 1996, after which the parasite was rarely seen. On the other hand, the infection prevalences by antigen-detection ELISA (Ag-ELISA) were around 40% from the first sampling through October 1997; then, antigenemic cattle decreased to 20% by June 1998. Milk yields of the cattle with detectable parasitaemia in June and November 1996 were significantly lower than those of the non-infected cattle by Student's t-test. Similarly, the milk yields of Ag-ELISA positive cattle were lower than those of negative cattle at every sampling and significant differences were observed during the first year and in February, 1998 (tested by 2-way ANOVA; T. evansi status and herd as factors). This study suggested that subclinical trypanosomosis caused decrease in milk yield of newly introduced dairy.     

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.     

Direct and sensitive detection of Trypanosoma evansi by polymerase chain reaction.

The mechanically transmitted haemoflagellate, Trypanosoma evansi causes 'surra', a wasting disease of domestic animals and is highly endemic in distribution in Southeast Asia. The detection of T. evansi is important for improving the epizootiological and animal health status of the region. The specificity and sensitivity of polymerase chain reaction (PCR) using oligonucleotide primers constructed from T. evansi repetitive DNA sequences were studied in the present investigation. Using the assay, it was possible to amplify template DNA of T. evansi derived from buffaloes, camels and horses to a threshold sensitivity level of 0.5 pg and to detect DNA from as few as five organisms in 10 microliters crude blood samples. Following experimental infection of calves with 5 x 10(5) T. evansi, positive signals could be observed as early as 12 h post-infection. DNAs from two common haemoflagellates of cattle, Babesia bigemina and Theileria annulata were not amplified with the primers.     

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.     

奶牛附红细胞体和伊氏锥虫二重PCR诊断方法的建立

为建立奶牛附红细胞体和伊氏锥虫两种病原诊断方法并探索二者之间在奶牛感染中的关系,本研究针对两病原分别设计两对特异性引物,建立了奶牛附红细胞体和伊氏锥虫感染的二重PCR诊断方法,其扩增片段大小分别为415bp和237bp。敏感性试验和特异性试验表明,附红细胞体和伊氏锥虫的DNA的最低检测量为0.154pg和0.105pg;与猪肺炎支原体、大肠杆菌、葡萄球菌、鸡艾美耳球虫、牛双芽巴贝斯虫无交叉反应。35份临床血样检测结果为:奶牛附红细胞体阳性率22.89%,伊氏锥虫阳性率8.89%,其中共感染率为2.89%。临床试验表明,该方法可用于奶牛附红细胞体和伊氏锥虫的诊断,特别适用于早期诊断。     

PCR amplification of RoTat 1.2 VSG gene in Trypanosoma evansi isolates in Kenya

A direct card agglutination test for Trypanosoma evansi, CATT/T. evansi based on the predominant variable antigen-type (pVAT) RoTat 1.2 was evaluated previously in the field in Isiolo District, Kenya. Sixteen out of 51 (31.4%) parasitologically positive camels were negative by the antibody detection test. In the present study, trypanosomes isolated from the camels were analysed in an attempt to determine the cause of the false negative results of CATT/T. evansi. A total of 20 field isolates comprised 16 stocks from camels that were negative by CATT/T. evansi, and 4 from CATT/T. evansi-positive camels. In addition, 15 known T. evansi and four T. brucei were used as reference. Purified DNA samples were tested using an established RoTat 1.2-based polymerase chain reaction (PCR) that yields a 488 bp product for the specific detection of T. evansi. Antibodies to RoTat 1.2 variant surface glycoprotein (VSG) were used in Western blotting to detect RoTat 1.2 VSG linear epitopes. Results of PCR and Western blot showed that the 16 stocks isolated from CATT/T. evansi-negative camels fell into three groups. In Group 1, both the RoTat 1.2 VSG gene and the VSG were absent in three stocks. In five trypanosome stocks in Group 2, the RoTat 1.2 VSG gene was detected, but Western blot was negative indicating absence of the expressed VSG. Five other stocks containing the RoTat 1.2 VSG gene were also in this group. The RoTat 1.2 VSG gene was detected and Western blot was positive in all four trypanosome stocks in Group 3. All four stocks from CATT/T. evansi-positive camels contained the RoTat 1.2 VSG gene and the expressed VSG. The reference T. evansi KETRI 2479 lacked the RoTat 1.2 VSG gene and there was no immune reactivity detected by Western blot. The rest of the reference T. evansi stocks examined contained the RoTat 1.2 VSG gene. All the four T. brucei samples examined were negative by PCR and Western blot. In conclusion, this study showed that the RoTat 1.2 VSG gene was absent from some T. evansi trypanosomes in Kenya.     

Longitudinal study of an outbreak of Trypanosoma evansi infection in equids and dromedary camels in Israel

An outbreak of trypanosomoasis caused by Trypanosoma evansi involving horses, camels and donkeys occurred in a farm in Israel. A longitudinal study of two outbreak phases was conducted which included clinical monitoring, blood smears, packed cell volume (PCV), serology and polymerase chain reaction (PCR) followed by reverse dot blot (RDB) for the molecular detection of infection. This was the first reported T. evansi outbreak in domestic animals in Israel. Most of the camels on the farm (8/10; 80%) were diagnosed with T. evansi infection whereas infection was less prevalent in the horses (3/7; 43%) and donkeys (6/13; 46%). Clinical disease was evident in 4 camels and 1 horse exhibiting characteristic clinical signs, anemia and parasitemia detected on blood smears and by positive RDB. Six other animals were diagnosed as asymptomatic latent carriers by positive RDB and 6 additional animals were only seropositive and were considered suspected carriers. A significant difference was found in the mean PCV between symptomatic and latent carriers with severe anemia observed only in the symptomatic animals. An anaphylactic-like reaction, fatal in one case, was observed in 2 camels diagnosed with severe trypanosome parasitemia immediately following treatment with melarsenoxide cysteamine. Furthermore, recurrence of infection was documented in one camel 4 months post treatment.     

犬附红细胞体PCR检测方法的建立

目的 步建立犬附红细胞体PCR检测方法。方法 据已发表的附红细胞体基因序列，设计了一对特异性引物，以犬附红细胞体基因组DNA和附红细胞体可疑病犬样品DNA为模板，聚合酶链式反应（PCR）扩增，扩增产物经克隆测序分析。结果 CR扩增产物大小为541bp，序列分析表明与GenBank数据中发表的序列一致，表明这套引物成功扩增出目的基因序列，但正常犬血样品DNA和弓形虫、伊氏锥虫、吉氏巴贝斯虫、犬细小病毒、犬瘟热的DNA样品都不能扩增出目的基因片段。结论 研究建立的PCR方法可以用于犬附红细胞体的检测，为犬附红细胞体病的诊断及分子流行病学的调查提供了新的手段。     

Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north Vietnam

In the present study, a collection of 415 water buffalo serum samples originating from the north of Vietnam was used for evaluation of different diagnostic antibody detection methods available to detect infections with Trypanosoma evansi. The diagnostic sensitivity and specificity of a direct card agglutination test (CATT/T. evansi), an indirect card agglutination test (LATEX/T. evansi) and a newly developed antibody detection ELISA (ELISA/T. evansi) was calculated on the basis of parasitological results, obtained by mouse inoculation, and compared for all assays. The immume trypanolysis assay with the predominant T. evansi RoTat 1.2 variable antigen type was used as reference test for antibody presence. All parasitologically confirmed animals (n=8) were positive in all tests. Diagnostic specificity was highest in CATT/T. evansi (98%) followed by the ELISA/T. evansi (95%) and the LATEX/T. evansi (82%). Concordance of the variant specific immune trypanolysis test with the other tests was calculated and revealed that few (1-8%) false positive results were actually due to a specific reactions, and that LATEX/T. evansi and ELISA/T. evansi detected more immune trypanolysis positives than the CATT/T. evansi. It was concluded that, apart from the immune trypanolysis test, which is not generally applicable, ELISA/T. evansi with a 30% positivity cut-off and LATEX/T. evansi, thanks to their superior capacity of detecting T. evansi specific antibodies, would be suitable as epidemiological tools detecting both active infections and persisting T. evansi specific antibodies. The ELISA/T. evansi with a 50% positivity cut-off and the CATT/T. evansi on the other hand, seem more appropriate to detect true infected water buffaloes.     

PCR detection of Porcine circovirus type 2 DNA in whole blood,serum, oropharyngeal swab,nasal swab,and feces from experimentally infected pigs and field cases

Porcine circovirus type 2 (PCV2) shedding patterns were investigated by polymerase chain reaction (PCR) for the detection of PCV2 DNA, and the diagnostic suitability of a sample for the PCR was examined by using different types of samples. In the experimental infection, sixteen pigs were inoculated intranasally with PCV2. The samples, including oropharyngeal and nasal swabs, feces, whole blood and serum became positive for PCV2 DNA by PCR immediately after the inoculation, and almost all samples remained positive during the observation period, post-inoculation-day 70. Field samples were collected from 313 pigs in five different age groups. The overall percentages of positive samples in the whole blood, nasal swabs, and feces detected by PCR were 30.4%, 19.2%, and 20.4%, respectively. The frequency of positive samples increased after the nursery stages and reached a peak in the 3 to 4-month-old pigs. These results indicate that PCV2 infection may occur after weaning, that PCV2 DNA may be present in whole blood for a long period after infection, and that whole blood and serum are the most suitable sample types for the PCR analysis of PCV2.     

Impairment in the pituitary-thyroid axis of the Camelus dromedarius infected with Trypanosoma evansi

Camels infected with Trypanosoma evansi were diagnosed by the Passive Haemagglutination Test and Ag-ELISA and subjected to hormonal blood analyses to estimate the function of the thyroid gland. The results indicated that T. evansi caused a significant case of hypothyroidism evidenced as a decrease in the thyroid triiodothyronine (T3) and thyroxine (T4) blood levels associated with a parallel decrease in the level of blood pituitary thyrotropic hormone (TSH) as measured by the RIA technique.     

Development of a TaqMan PCR assay for the detection of Trypanosoma evansi, the agent of surra

A TaqMan PCR assay was developed for the detection of Trypanosoma evansi. The assay targets the internal transcribed spacer 1 (ITS-1) region of rRNA. The ITS-1 region of eleven strains of T. evansi from widely separated geographical regions were sequenced and alignments compared. Primers and probe for the test were designed from these sequence data. The assay was tested using blood from infected rats and was found to be sensitive, detecting less than one genomic equivalent of T. evansi. The assay has been tested against 10 different species of trypanosomes found in native animals in Australia and did not detect any of these trypanosome species. Time course experiments using rats infected with T. evansi were performed to compare the TaqMan assay with the Haematocrit centrifugation test (HCT) and the mouse inoculation (MI) assay. The assay was more sensitive than the HCT but not as sensitive as the MI. The TaqMan assay has the ability to rapidly detect T. evansi and determine the number of organisms present in a blood sample from an infected animal. This is the first time a TaqMan assay has been developed for the detection of T. evansi.     

弓形虫Real-time PCR检测方法的建立和初步应用

根据已发表的弓形虫529 bp高拷贝序列,设计特异性引物和探针,建立TaqMan探针法的实时荧光定量PCR检测方法。将该方法用于360份临床样本的检测,并与常规PCR和环介导等温扩增(LAMP)技术进行对比分析。结果表明:Real-time PCR检测的阳性率(11.11%)高于LAMP(7.5%)和常规PCR(3.61%)。采用本实验室建立的Real-time PCR,对收集于不同地区4种动物的750份临床样本检测发现,不同种类动物血液样本中均可检测到弓形虫感染,其中羊的感染率最高(17.8%),猪次之(4.22%),牛较低(1.98%),犬最低(1.37%)。     

Enzootiology of Trypanosoma evansi in Pantanal, Brazil

In order to better understand the enzootiology of trypanosomiasis caused by Trypanosoma evansi in the Brazilian Pantanal we examined domestic and wild mammals by microhematocrit centrifuge technique (MHCT), immunofluorescence antibody test (IFAT) and polymerase chain reaction (PCR). T. evansi infection was detected in all species sampled with exception of the sheep and the feral pig. High parasitemias were observed in capybaras (5/24), coatis (18/115), horses (31/321) and dogs (3/112). Among these species, only the capybaras did not develop anemia. Low parasitemias, only detected by PCR, were found in buffaloes (18/43), bovines (29/331), marsupials (1/4), small rodents (14/67), bats (7/18), and one armadillo (1/8). The highest prevalence of T. evansi infection was recorded in horses (73%), although no neurological signs in infected horses were observed. Diagnosis through standard parasitological tests and IFAT should be used with caution since they may overlook comprovedly infected horses. The relationship between ranch management and T. evansi infection in horse was investigated. The importance of other transmission mechanisms apart from the tabanids and reservoir hosts are discussed.     

猪附红细胞体小鼠感染模型建立条件的优化

为探究猪附红细胞体感染特性,进而优化猪附红细胞体小鼠感染模型建立的条件,本试验设计了不同小鼠处理方式感染试验（A试验）、不同病原形式感染试验（B试验）、冻存病原感染试验（C试验）和小鼠模型血液再感染小鼠试验（D试验）。通过对每个试验中各组小鼠血液感染情况、感染首现时间、临床症状和平均感染时间的综合评估,分析不同小鼠处理方式、不同病原形式、冻存病原及小鼠模型血液再感染是否对小鼠模型的建立有影响。通过PCR检测、电镜观察和特异基因片段序列测定,进一步鉴定小鼠模型感染的病原与猪附红细胞体是否一致。结果显示,A试验中,昆明小鼠切除脾脏组感染效果最优;B试验中,猪附红细胞体阳性血液样本组感染效果最优;C试验中,阳性血液未冻存组感染效果优于冻存组;D试验中,猪阳性血液样本组、小鼠模型阳性血液样本组无明显差异。PCR检测、电镜观察和特异基因片段序列测定结果显示,小鼠模型感染的病原与猪附红细胞体一致。结果表明,不同小鼠处理方式、不同病原形式和病原的冻存等条件对猪附红细胞体小鼠感染模型的感染效果均有影响,通过对比,以猪阳性血液感染切除脾脏的昆明小鼠的方式建立小鼠感染模型效果最优。     

Evaluation of the card agglutination test (CATT/T. evansi) for detection of Trypanosoma evansi infection in water buffaloes (Bubalus bubalis) in Egypt

A card agglutination test (CATT/T. evansi) was evaluated for detection of antibodies against Trypanosoma evansi (T. evansi) in experimentally and naturally infected buffaloes. Four calves were inoculated with a strain of T. evansi isolated from a dromedary camel. Parasitological examination of the calves revealed trypanosomes in the blood from days 4 to 9 post-inoculation (PI). General emaciation appeared from day 26 PI and aggravated until the end of the experiment (day 88 PI). Antibodies against T. evansi were detectable from day 8 PI till the end of the experiment. Parasitological examination of 200 water buffalo blood samples obtained from slaughterhouses revealed negative results. Serological examination of these animals showed that 48 (24%) water buffaloes had anti-T. evansi antibodies.     

Optimization of Establishment Conditions of Mouse Model of Mycoplasma suis

In order to explore the infection characteristics of Mycoplasma suis(M.suis), and optimize the establishment of infection model of M.suis,this research designed the infection test with different mice (A test), different pathogen forms (B test), frozen pathogens (C test), and studied on mice model of blood reinfection (D test). On the basis of a comprehensive assessment of each test in the blood of mice infection, the first infection time, the clinical symptoms and the average duration of infection, to analyze whether the different treatments and different pathogenic mice forms, cryopreservation of pathogen and the mouse model of blood infection had influence on the establishment of mouse model. PCR detection, electron microscope observation and specific gene fragment sequencing were used to determine whether the pathogen of mice infected with the model was consistent with that of M.suis. The results showed that the infection effect of the spleen group was the best in the A test. In the B test, the infection effect of the M.suis positive blood samples group was the best. In the C test, the infection effect of the non-frozen positive blood group was better than that of the cryopreserved group. In the D test, the positive model of pig blood samples and mice model positive blood group had no significant difference. The results of PCR detection, electron microscope observation and specific gene fragment sequencing showed that the pathogen of mouse model was identical with that of M.suis. The results showed that the infection condition of the mice infected with the mouse model was influenced by the different treatment of the mice, the different forms of pathogens and the cryopreservation of the pathogen,and the model of Kunming mice without spleen infected with M.suis positive blood samples was the best.     

Surveys in Papua New Guinea to detect the presence of Trypanosoma evansi infection

OBJECTIVE: To confirm serological evidence that Trypanosoma evansi is present in Papua New Guinea. DESIGN: Three surveys were undertaken in PNG during 1997/1998. Animals were selected for sampling on the basis of convenience. Samples of blood were examined for the presence of T evansi by the haematocrit centrifugation technique (HCT) and mouse inoculation test (MI). Sera were tested in the field using the card agglutination test for trypanosomiasis/T evansi (CATT). Bovine sera were tested at James Cook University using an antibody-detection ELISA (Ab-ELISA). Results from testing bovine sera with the Ab-ELISA and sera from wallabies with the CATT were analysed using FreeCalc to determine the probability that animals in these populations were infected with T evansi. RESULTS: A total of 545 serum samples were collected, during the three surveys of which 39 cattle, two pig and three agile wallaby samples were positive with the CATT. All bovine sera collected were negative when tested with an Ab-ELISA. T evansi was not isolated using the HCT or the MI from any of these animals. CONCLUSION: Based on the Ab-ELISA results it was concluded that T evansi infection was not present in cattle in villages around Balimo at a minimum expected prevalence of 10% (P < 0.05) and, based on the CATT results, that infection was not present in wallabies on the Bula plain at a minimum expected prevalence of 10% (P < 0.1). These results indicate that it is unlikely that T evansi is endemic in PNG.     

Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques

The use of non invasive sampling, such as collection of conjunctival swabs, as a diagnostic tool for the detection of Leishmania DNA is of interest. The purpose of this study was to evaluate the diagnostic utility of detecting Leishmania infection with the use of conjunctival swab samples in dogs living in a highly endemic area for leishmaniosis and to investigate, for the first time, the presence of Leishmania DNA in oral swabs in the same population. One hundred sixty-three dogs living outdoor and recruited in various provinces of Sicily were studied. Leishmania infantum indirect fluorescent antibody test (IFAT), delayed-type hypersensitivity reaction to leishmanin (DTH) and real-time PCR of blood (BL), lymph node (LN), conjunctival (CS) and oral swab (OS) samples were performed. The positive PCR percentages in LN, CS, OS and BL samples were: 24.5%, 22.1%, 8.7% and 5.5%, respectively. Serological and DTH positive percentages were 27.0% and 73.8%, respectively. Seropositive and LN-PCR positive dogs had a high likelihood to be positive by CS-PCR. The similar positive PCR percentages found in CS and LN samples suggest the use of CS-PCR as non-invasive alternative technique to LN-PCR for the detection of Leishmania infection in dogs. In addition, this study demonstrated, for the first time, the presence of Leishmania DNA in oral swabs in dogs.