Application of PCR assays to determine the genotype of Babesia bovis parasites isolated from cattle with clinical babesiosis soon after vaccination against tick fever

OBJECTIVE: To demonstrate the value of PCR assays to determine the genotypes of Babesia bovis in cattle with clinical signs of babesiosis within 3 weeks after vaccination against tick fever. DESIGN: Samples from 5 cases of babesiosis in cattle soon after vaccination against tick fever were analysed in two PCR assays. PROCEDURE: Parasite DNA was purified from blood taken from cattle with signs of babesiosis within 3 weeks of vaccination against tick fever. DNA was also prepared from the tissues of animals that died of babesiosis. Two PCR assays that amplify repeat sequences of DNA within the B bovis genes, Bv80 and BvVA1, were used to differentiate the genotypes of field isolates and vaccine strains of B bovis. RESULTS: One of the five cases of babesiosis was found to be caused by a vaccine strain, but PCR analyses showed that the predominant isolate in the other four cases was not the vaccine strain. CONCLUSIONS: PCR assays on the DNA of B bovis obtained from the blood or tissues of cattle clinically affected with tick fever within 3 weeks after vaccination are useful to distinguish between vaccine strains and field isolates as the source of infection.     

Genotypic diversity in Babesia bovis field isolates and vaccine strains from South Africa

Genotypic diversity in Babesia bovis (cause of Asiatic redwater in cattle) vaccine strains and field isolates from South Africa were investigated using the Bv80 gene as well as microsatellites. The S11 vaccine strain possessed both A and B alleles of the Bv80 gene, as well as genotypic diversity within each allele type as defined by repeat variation resulting in different amplicon sizes. Rapid serial passage of vaccine strain from passage S10 to S24 resulted in loss of genotypic diversity that yielded a single allele A genotype with an amplicon size of 558 bp. This suggested that clonal selection occurred during rapid passaging. Extensive genotypic diversity exists in 44 field isolates characterized with both Bv80 A and B alleles, but can be readily distinguished from the S24 vaccine strain using either the Bv80 allele specific PCR assays or using multi-locus micro-satellite typing. This indicated that no recent documented clinical cases of Asiatic redwater were caused by the reversion to virulence of the current vaccine strain.     

用重组的牛巴贝斯虫棒状体蛋白1作为ELISA诊断抗原进行牛群中牛巴贝斯虫病的血清流行病学调查

用重组的牛巴贝斯虫棒状体蛋白1（Bc—RAP-1）作为ELISA诊断抗原,对采自青海省湟中县的120份奶牛血清样品,进行抗Bc—RAP-1特异性抗体的检测。结果检出7份阳性,阳性率为5．83％,说明该地区牛群存在牛巴贝斯虫的感染。     

牛巴贝斯虫PCR检测及体外培养试验

本试验通过PCR方法检测出牛巴贝斯虫单一感染的牛全血样本,并探讨了牛巴贝斯虫病患牛全血体外培养方法及培养条件。试验结果表明,PCR检测结果显示检测方法效果较好,单一感染样本的新鲜血液中虫体在含40%牛血清的完全培养液和37 ℃、5% CO₂的条件下能建立起牛巴贝斯虫的体外培养,虫体可以连续培养14 d,传代13次,最高染虫率可以达到12.5%,平均染虫率为4%。     

巴贝斯虫病水牛血清钙镁浓度变化与染虫率的关系

自水牛巴贝斯虫病非疫区购买的无任何血液原虫寄生的健康水牛犊２头，人工摘除脾脏后，用从疫区采集的饥饿镰形扇头蜱叮咬感染，观察巴贝斯虫的消长与血清钙、镁浓度的变化，结果发现二者关系密切，其中钙离子浓度与红细胞染虫率呈负相关，镁离子浓度与红细胞染虫率呈正相关，这一发现为进一步研究水牛巴贝斯虫病及在体内外研究巴贝斯虫的生长繁殖均具有重要意义。     

光生物素标记DNA探针检测牛巴贝斯虫研究初报

光生物素标记重组质粒DNA(c51A)探针检测牛巴贝斯虫研究的初步试验结果表明,能特异地检出感染牛血中的巴贝斯虫,灵敏度为10pg质粒DNA,相当于约150个感染红细胞的虫体DNA。     

Effect of breed of cattle on innate resistance to infection with <i>Babesia bovis, B bigemina and Anaplasma marginale

Objective To assess the innate resistance of naive Bos taurus, Bos taurus cross Bos indicus and Bos indicus cattle to virulent Babesia bovis, B bigemina and Anaplasma marginale parasites. Design Groups of 10, pure B indicus, fi B indicus cross,/B indicus cross and pure B taurus steers were infected with virulent B bovis, B bigemina and A marginale parasites. Procedure Sequential infections were carried out by intravenous inoculation of infected blood containing 1 times 10⁸ parasites of B bovis, followed by B bigemina and then A marginale. To assess resistance, measurements were made of parasitaemia, rectal temperature, packed cell volume and the number within a group requiring chemotherapy to control infection. There was a recovery period between each infection. Results Infection with B bovis showed that pure B indicus steers were significantly more resistant to B bovis infection than the other groups, with none of this group requiring treatment. There was no significant difference between fi B indicus cross and/B indicus cross with 30% and 20%, respectively, of steers in these groups requiring treatment. The pure B taurus steers were significantly more affected then those in the other three groups with 80% requiring treatment. Infections of B bigemina produced a mild response in comparison to that of B bovis and none of the steers required treatment. However, the pure B taurus group was significantly more affected than the other three groups for all other measurements. After the A marginale infection, B indicus steers were moderately affected with 50% requiring treatment, whereas 70% of the fi B indicus group, 80% of the /B indicus cross group and 100% of the pure B taurus group required treatment. Conclusions All breeds of cattle, ranging from pure B indicus to pure B taurus may be at risk of severe disease if exposed to virulent A marginale. The results confirm that pure B indicus cattle are relatively resistant to B bovis, but there could be a significant risk of severe mortalities if cross-bred herds are exposed to virulent infection.     

Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis and B bigemina transmitted by Boophilus microplus

OBJECTIVE: To assess the effect of breed of cattle on the transmission rates of and innate resistance to Babesia bovis and B bigemina parasites transmitted by Boophilus microplus ticks. DESIGN: Groups of 56 purebred B indicus and 52 B indicus cross B taurus (50%, F1 generation) steers were placed in a paddock seeded with and also naturally infested with B microplus which were the progeny of females ticks fed on B taurus cattle specifically infected with a virulent isolate of B bovis. The cattle were placed in the infested paddock 50 days after seeding had started. PROCEDURE: Cattle were inspected from horseback daily for 50 days. Clinically ill cattle were brought to yards and assessed by monitoring fever, depression of packed-cell volume, parasitaemia and severity of clinical signs. Any animals that met preset criteria were treated for babesiosis. Blood samples were collected from all cattle on day 28, 35 and 42 after exposure and antibodies to Babesia spp and packed cell volume measured. RESULTS: All steers, except for one crossbred, seroconverted to B bovis and B bigemina by day 35 and 75% of the crossbred steers showed a maximum depression in packed cell volume of more than 15% due to infection with Babesia spp compared with only 36% of the B indicus group. Ten of the 52 crossbreds and 1 of the 56 B indicus steers showed severe clinical signs. Two of the crossbreds required treatment of which one died 2 weeks after initial treatment. CONCLUSIONS: Pure-bred B indicus cattle have a high degree of resistance to babesiosis, but crossbred cattle are sufficiently susceptible to warrant the use of preventive measures such as vaccination. Transmission rates of B bovis and B bigemina to B indicus and crossbred cattle previously unexposed to B microplus were the same.     

牛巴贝斯虫巢式PCR诊断方法的建立

根据GenBank发表的XJ-MSA-2c核苷酸序列(登录号:EU328267)设计的2对特异性引物MS-1、MS-2、MS-3以及MS-4,建立牛巴贝斯虫病巢式PCR快速检测方法。在特异性检测试验中,仅从MSA-2c质粒样本中扩增出622、350bp2条目的片段,与预期片段大小相符,而作为对照样本的双芽巴贝斯虫、牛环形泰勒虫、东方巴贝斯虫基因组DNA均无此扩增目的条带出现。第1次和第2次扩增的敏感性分别为1.75、1.75×10-2μg/L。在对46份全血的DNA样本巢式PCR和显微镜检测中,阳性检出率分别为34.8%(16/46)和23.9%(11/46)。结果表明,所建立的巢式PCR方法准确、敏感、特异,作为牛巴贝斯虫病的快速检测和小范围的流行病学调查,具有重要的临床意义。     

利用聚合酶链反应技术检测牛结核杆菌病的研究

应用聚合酶链反应（ Ｐ Ｃ Ｒ） 技术检测牛结核分枝杆菌纯化 Ｄ Ｎ Ａ, 其敏感性为２５０ｆｇ 。所用引物序列对９种抗酸分枝杆菌 Ｄ Ｎ Ａ 进行扩增, 经琼脂糖凝胶电泳证实, 只有人型结核分枝杆菌、牛型结核分枝杆菌产生了３１７ｂｐ 的特异性扩增带。将 Ｐ Ｃ Ｒ 法与皮内变态反应试验（ Ｐ Ｐ Ｄ） 检测方法比较; ５４ 份血样标本中 Ｐ Ｃ Ｒ 的阳性率为１ ％ , Ｐ Ｐ Ｄ 试验的阳性率为０ 。同时对奶样标本的检测与血样结果一致。结果表明, Ｐ Ｃ Ｒ 在直接检测患牛血样、奶样标本中显示出快速、敏感、特异的优点。为今后牛结核病的检测工作提供了一条新的途径。     

牛巴贝斯虫新疆株MSA-2c基因的克隆表达及其重组蛋白免疫原性

本试验将新疆株牛巴贝斯虫的MSA-2c基因克隆,并构建重组质粒pGEX-4T-2/MSA-2c.利用大肠杆菌原核表达系统进行外源蛋白的表达,并成功诱导出GST-MSA-2c融合蛋白.通过优化诱导条件,得到了较高的可溶性表达;利用亲和层析技术纯化后的重组蛋白皮下免疫试验小鼠.间接ELISA检测发现,免疫接种56 d后,抗重组蛋白的抗体效价达到1∶220 000以上.用获得的抗血清进行Western-blot试验,可获得清晰的免疫反应条带.结果表明,本试验所表达的MSA-2c蛋白与文献报道的目的蛋白相符,并具有免疫原性.同时本试验也为建立以MSA-2c重组蛋白作为抗原的血清学诊断体系奠定了基础.     

DNA typing of Mycobacterium bovis strains from the Castlepoint area of the Wairarapa

Aim. To investigate isolates of Mycobacterium bovis from the Castlepoint area of the Wairarapa using three different methods of DNA typing.

Methods. Isolates of M. bovis, obtained from animals in the Castlepoint area between 1982–l998, were characterised by restriction endonuclease analysis. An isolate representing each restriction type was characterised by two newer DNA typing methods based on the polymorphic GC-rich repetitive sequence (PGRS) and spoligotyping.

Results. Over 300 isolates were distinguished into 26 restriction types.The 24 available restriction types were differentiated into 11 PGRS types and 7 spoligotypes. The three most common restriction types had the same PGRS type and the same spoligotype.

Conclusions. The relatively large number of restriction types found, indicated that restriction endonuclease analysis was well suited for detailed epidemiological studies at Castlepoint. Spoligotyping was less discriminatory than PGRS typing but both methods could be used to group isolates with different restriction types.     

Immune responses of cattle following vaccination with living and non-living Babesia bovis antigens

Twenty-four yearling Hereford (Bos taurus) cattle were vaccinated against Babesia bovis using either live parasites or non-living antigens obtained from the supernatant of in vitro cultures. A single dose of live parasites was given subcutaneously, while the non-living supernatant antigen (NLSA) was combined with saponin and 2 doses given, 2 weeks apart. Following vaccination with live parasites, serum antibodies remained at high levels for 6 months, but the lymphocyte transformation response was low and lasted only 10-18 days. In contrast, NLSA vaccination was followed, after 21-28 days, by a peak of serum antibodies which then slowly declined. The lymphocyte transformation response in these animals was much higher and persisted for 6 months. Following heterologous challenge all unvaccinated cattle had severe reactions and required treatment to prevent death. Cattle vaccinated with live parasites had mild reactions with only 1 of the 12 requiring treatment. Cattle vaccinated with NLSA were only partially protected and 6 of the 12 required treatment.     

Use of cerebellar brain smears in the diagnosis of babesiosis (Babesia bovis) in cattle

Summary Cerebral and cerebellar smears were made from 4 animals acutely reacting toBabesia bovis and 94 animals free from clinical babesiosis. The brain smears were stained by the Giemsa method and examined for the presence ofB. bovis parasites. In animals showing clinical babesiosis capillaries congested with parasitised erythrocytes were abundant in cerebral and cerebellar smears. Results obtained from both types of brain smears in animals free from clinical babesiosis agreed closely (83% conformity) as to the presence or absence of parasites. A third group of 39 animals from which cerebral and cerebellar smears were taken was also examined serologically by the indirect immunofluorescent antibody test (IFAT); about 69% of the IFAT positive and doubtful animals showed parasites in the cerebellar brain smears. The existence of false negatives in the IFAT test has been shown and discussed. It has been concluded that cerebellar samples obtained through the foramen occipitale can be used for the microscopic detection ofB. bovis parasites in latently infected bovines. This method can also be used in field cases suspected of cerebral babesiosis permitting brain sampling without resorting to the opening of the skull. Such an approach might prove particularly useful in areas where rabies occurs and the animal's head has to be sent to a diagnostic centre.

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El Empleo De Frotis De Cerebelo En El Diagnostico De Babesiosis (Babesia Bovis) En Bovinos

Resumen Se hicieron frotis de cerebro y cerebelo de 4 animales con síntomas agudos de babesiosis porBabesia bovis y de 94 animales clínicamente sanos. Los frotis se tiñeron con Giemsa y se examinaron por la presencia deB. bovis. En animales enfermos, los capilares del cerebro y cerebelo se encontraron congestionados y repletos de eritrocitos parasitados. Los resultados obtenidos en ambos tipos de frotis en animales libres de la enfermedad clínica, estuvieron de acuerdo (83% de conformidad) en cuanto a la presencia o ausencia de parásitos. Un tercer grupo de 39 animales de los cuales se tomaron frotis de cerebro y cerebelo, se examinaron también serológicamente mediante la técnica indirecta de anticuerpos fluorescentes (TIAF); cerca del 69% de animales positivos y dudosos por immunofluorescencia revelaron parásitos en los frotis de cerebelo. La existencia de falsos positivos se demostró mediante TIAF. Se concluye que los frotis de cerebelo tomados a través del agujero occipital pueden usarse para detectarB. bovis microscópicamente en animales con infecciones latentes. Este método se puede utilizar en el campo para no tener que abrir el craneo, sobre todo en áreas en donde existe rabia y las cabezas se envían al laboratorio.

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Examen De Frottis De Matière Cervelette Pour Le Diagnostic De La Babésiose (babesia bovis) Du Bétail

Résumé Des frottis de cerveau et de cervelet ont été effectués à partir de 4 animaux atteints de babésiose aiguë et de 94 autres n'ayant présenté aucun symptôme de la maladie. Les frottis ont été colorés au Giemsa et examinés pour mettre en évidence la présence deB. bovis. Chez les animaux atteints de babésiose clinique, les capillaires étaient congestionnés avec présence abondante d'érythrocytes parasités. Il existe une excellente concordance (83 p.100) entre les résultats des examens des deux types de frottis, qu'il s'agisse d'absence ou de présence des parasites. Un troisième groupe de 39 animaux dont les frottis de cerveau et de cervelet ont été faits ont également fait l'objet d'une étude sérologique pour déceler des anticorps par le test de l'immunofluorescence indirecte (I.F.A.T.); environ 69 p.100 des animaux reconnus positifs et douteux par l'I.F.A.T. ont montré des parasites dans les frottis du cervelet. L'existence de cas négatifs faux dans le test IFAT a été montrée et discutée. Il en a été conclu que les échantillons de cervelet obtenus à travers le foramen occipital peuvent être utilisés pour la détection microscopique deB. bovis chez des animaux en état d'infection latente. Cette méthode peut être également utilisée en brousse lors de suspicion de babésiose cérébrale car elle permet d'obtenir les prélèvements de tissus cérébraux nécessaires sans ouverture de la boite cranienne. Une telle approche peut être particulièrement utile dans les régions où sévit la rage et où il est nécessaire d'expédier la tête des animaux suspects à un centre de diagnostic.

     

High frequency of Mycobacterium bovis DNA in colostra from tuberculous cattle detected by nested PCR

We evaluated by nested PCR reaction, different cow secretions from a herd with 48% of prevalence of bovine tuberculosis (BTB), seeking to determine niches where Mycobacterium bovis could be found. Postmortem examination of 18 (75%) tuberculin reacting cows allowed demonstrates BTB-compatible lesions in six, all of them PCR positives in milk and four in colostra samples. Our results showed that up to 62% of the colostra analysed contained M. bovis DNA, whereas only 18% of milk gave a positive reaction. Moreover, in bronchoalveolar lavages from cattle with compatible lesions in lungs or lymph nodes, where macrophages account up to 90% of cells, we did not find evidences of M. bovis. Altogether, these results suggest that differences in the anti-bacterial capacity of bovine macrophages, dependent upon microenvironment and organ-specific factors, exist. Alternatively, we hypothesize that hypoxic conditions that are encountered in mammary glands macrophages could induce M. bovis entrance into a 'dormancy-like' state, and that the high number of colostra samples were M. bovis was detected, could be an indicator of reactivation during 'peripartum'.     

Evaluation of an enzyme-linked immunosorbent assay kit to detect Babesia bovis antibodies in cattle

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies; to Babesia bovis was evaluated in 1000 sera from Holstein heifers. Five hundred of them were from cattle naturally or experimentally infected with B. bovis and 500 from uninfected heifers born and raised in a region free of the vector of cattle babesiosis. Additionally, the ELISA was evaluated and compared with an indirect immunofluorescent antibody (IFA) test in 374 heifers inoculated with different kinds of B. bovis antigens in four trials. The cross-reaction was also evaluated in 50 heifers infected with Babesia bigemina and 50 heifers infected with Anaplasma marginale. The mean percentage positivity of negative sera in relation to the ELISA strong positive sera was 8%. The seropositive/seronegative cutoff point was set as twice the mean percentage positivity of negative cattle sera ( = 16%). The sensitivity of the ELISA was 98% with a 95% confidence interval (CI) of 96–99%. The specificity was 95% (CI 93–97%). The agreement was 97% and the kappa value was 0.93. The predictive values of positive and negative results were 95% and 98% respectively. ELISA showed a similar sensitivity to that of the IFA test to detect antibodies to different B. bovis antigens. Its sensitivity ranged from 97.1% to 100% (CI 89–100%), while the sensitivity of the IFA test ranged from 92.8% to 100% (CI 83–100%). ELISA cross-reacted in 8% and 6% of the sera carrying B. bigemina and A. marginale antibodies, respectively, while the IFA showed 4% cross-reaction in each situation. The ELISA evaluated has the advantages of a proper sensitivity, objectivity and capacity to be adapted to test large number of samples in a short period of time. The results indicate that the ELISA is a suitable replacement for the IFA test to detect B. bovis antibodies in cattle sera, especially in epidemiological studies.     

Serological Survey of Babesia bovis and Anaplasma marginale in cattle in Tete Province,Mozambique

A serological survey of bovine babesiosis and anaplasmosis in communal cattle was conducted in the northwestern province of Tete, Mozambique. Blood was collected from cattle ranging from 4 to 15 months old from randomly selected farms from six districts. Thirty-nine per cent of all 478 calves tested in Tete Province were seropositive to the ELISA for Babesia bovis antibodies and 63% of all calves were seropositive in the card agglutination test for Anaplasma marginale. Seroprevalence of B. bovis ranged from 22.8% in Tete City District to 48.1% in Angonia District. For A. marginale, it ranged from 34.4% in Angonia District to 87.3% in Moatize District. The dominant factor affecting seroprevalence for both haemoparasites was district and there was a trend for higher intensity of tick control to be associated with a higher seroprevalence of B. bovis and a lower seroprevalence of A. marginale. The obvious differences were the low prevalence of B. bovis in Tete City Council District and the low prevalence of A. marginale in Angonia District. The levels of exposure to B. bovis seen in our study are well below any that could be considered to be consistent with endemic stability, yet they are sufficiently high to ensure that clinical disease would be a risk. The seroprevalence of A. marginale, however, suggests that endemic stability with respect to this disease could exist in districts other than Angonia. There was no strong and consistent relationship between the intensity of control and the likelihood of seropositivity to either of the diseases.     

Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis

This paper reports a quantitative real-time polymerase chain reaction (q-PCR) based on the msa2c gene and standardized with Platinum SYBR Green/ROX for the detection of Babesia bovis in cattle. The msa2c q-PCR amplified a DNA fragment with average dissociation temperature of 77.41°C (± 0.25°C). No amplification was detected when DNA from B. bigemina, A. marginale or Bos taurus was used as the template. The detection limit of the msa2c q-PCR was 1000 copies per ml of blood sample, with a linear correlation between the number of msa2c copies and threshold cycle. The comparison between msa2c q-PCR and conventional PCR for cytochrome b revealed 88.8% agreement, with a Kappa index of 0.75. In the comparison between msa2c q-PCR and an enzyme-linked immunosorbent assay (ELISA) with semi-purified B. bovis antigen, agreement was 96.3% and the Kappa index was 0.91. The agreement between three tests was 85.8%. The msa2c q-PCR detected a higher number of positive cattle than conventional PCR in an enzootically stable area, but did not differ significantly from ELISA. No significant differences were detected between the three diagnostic tests with cattle from an enzootically unstable area. All animals raised on a tick-free facility were negative for B. bovis in the three tests. These results suggest that msa2c q-PCR is a useful test for the detection of B. bovis infection.