快速检测双芽巴贝斯虫LAMP方法的建立

为提高双芽巴贝斯虫(Babesia bigemina)检出率,本研究采用环介导等温扩增技术(LAMP)建立一种快速、灵敏、特异的B.bigemina检测方法。根据GenBank上公布的Babesia bigemina细胞色素b(Cytochrome b,cyt b)基因序列,设计4条特异地识别B.bigemina的cyt b基因6个特殊区域的LAMP引物,优化反应体系和条件,在Bst DNA聚合酶的作用下,65 ℃反应60 min,加入SYBR Green Ⅰ后观察。结果表明,该LAMP检测方法特异性强,与牛巴贝斯虫(Babesia bovis)等DNA不发生交叉反应;敏感性高,对B.bigemina的cyt b基因最小检测值为0.085 fg/μL,是一般PCR方法的1000倍。该方法具有简单、快速、低成本的特点,可用于B.bigemina的基层现场快速检测。     

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.     

Erythrocytic oxidative damage in crossbred cattle naturally infected with Babesia bigemina

This study aimed to determine the erythrocytic lipid peroxidation and haemoglobin oxidation as contributory factors causing anaemia in cattle (Friesian × Egyptian native breed) infected with Babesia bigemina. Blood was collected from 32 cows infected with B. bigemina along with 18 healthy cows as controls for determination of erythrocytic malondialdehyde (MDA), blood methaemoglobin (MetHb), plasma free haemoglobin (PHb), corpuscular osmotic fragility (COF), red blood cell count (RBC), total haemoglobin (Hb) and packed cell volume (PCV). Percentage of parasitaemia varied from 14% to 36%. MDA, MetHb, COF and PHb were significantly increased (P < 0.001) in infected cows versus controls. Parasitaemia was positively correlated (P < 0.001) with MDA, MetHb, COF and PHb. MDA was positively correlated (P < 0.001) with COF and PHb and negatively correlated (P < 0.001) with RBC, Hb and PCV. MetHb was negatively correlated (P < 0.001) with RBC, Hb and PCV and positively correlated (P < 0.001) with COF. In conclusion, B. bigemina infection in cattle is associated with a parasitic burden-dependent corpuscular oxidative damage as indicated by membrane lipid peroxidation and methaemoglobin formation, which are contributed to COF and intravascular haemolysis.     

Genotyping of Babesia bigemina from cattle from a non-endemic area (Switzerland)

In August 2002, bovine anaplasmosis and concurrent infections with Mycoplasma sp. and piroplasms were reported in a cattle herd in an alpine region of Switzerland. The piroplasms were identified by PCR/sequencing of part of the 18S rRNA gene as Babesia bigemina and Theileria of the buffeli/sergenti/orientalis-complex, which have never been diagnosed in Switzerland before. The B. bigemina isolate was genetically characterised at two loci and compared with isolates from Italy, Spain, Turkey, Kenya and Mexico. Analysis of the internal transcribed spacer 2 (ITS2) of the rRNA genes revealed high polymorphism not only among the isolates but even within the isolates, and the presence of two types of the ITS2 in every isolate was confirmed. A dendrogram based on ITS2 sequences showed that the Swiss isolate was most closely related to a Spanish isolate but no sequences of the isolate from Switzerland were identical to any of the other isolates. The isolate from Italy was not positioned in the same cluster as the Swiss and the Spanish isolate. This had been anticipated as the nearest known endemic area of B. bigemina in Central Italy. Sequence analysis of the rhoptry-associated protein-1c gene (rap1c) confirmed the similarity of the Swiss and Spanish isolate. Hence, our molecular analyses of the Swiss B. bigemina isolate did not unequivocally track its geographical origin and the way of introduction remains obscure.     

Detection of Babesia bigemina in cattle of different genetic groups and in Rhipicephalus (Boophilus) microplus tick

Babesia bigemina infections were investigated in four genetic groups of beef cattle and in Rhipicephalus (Boophilus) microplus engorged female ticks. Blood samples and engorged female ticks were collected from 15 cows and 15 calves from each of the following genetic groups: Nelore, Angus x Nelore, Canchim x Nelore, and Simmental x Nelore. Microscopic examination of blood smears and tick hemolymph revealed that merozoites of B. bigemina (6/60) as well as kinetes of Babesia spp. (9/549) were only detected in samples (blood and ticks, respectively) originated from calves. PCR-based methods using primers for specific detection of B. bigemina revealed 100% infection in both calves and cows, regardless the genetic group. Tick infection was detected by nested-PCR amplifications showing that the frequency of B. bigemina was higher (P<0.01) in female ticks collected from calves (134/549) than in those collected from cows (52/553). The frequency of B. bigemina was similar in ticks collected from animals, either cows or calves, of the four genetic groups (P>0.05).     

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in cattle from central region of Syria

A total of 207 bovine blood samples were collected from clinically healthy cattle bred in central region of Syria and examined by Giemsa-stained blood smears, nested PCR, ELISA, and IFAT to determine the molecular and serological prevalence of Babesia bovis and B. bigemina. All samples were negative to Babesia spp. by microscopic examination of blood smears. On the other hand, the overall prevalence of B. bovis and B. bigemina was 9.18% and 15.46% by nPCR, 15.46% and 18.84% by ELISA, and 18.36% and 21.74% by IFAT, respectively. Mixed infections were detected in a total of 5 samples (2.4%) by nPCR, 16 (7.73%) by ELISA and 27 (13.04%) by IFAT. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location. These data provide valuable information regarding the occurrence and epidemiology of B. bovis and B. bigemina infections in Syrian cattle, which can be employed in developing rational strategies for disease control and management.     

Evaluation of some acute phase proteins in cattle naturally infected with Babesia bigemina

Bovine babesiosis due to Babesia bigemina infection induces systemic inflammation, evidenced by increased sialic acid (SA) levels and declined cholinesterase activity. The current study was undertaken to assess further indicators of the systemic inflammation in the naturally infected cattle. To this end, serum levels of some selected acute phase-proteins (APPs) including serum amyloid A (SAA), haptoglobin (Hp), ceruloplasmin (Cp), and fibrinogen were measured. Additionally, sensitivity and specificity of the APPs were calculated by receiver operating characteristic curve. The correlation among APPs, SA and cholinesterase activity were also assessed. Our previous blood specimens were used to measure APPs. Briefly, the diseased animals were divided into two groups according to the parasitemia: 12 mildly (20 % <) and 8 severely (20 %>) infected animals. Moreover, 10 healthy animals as the control were included. The levels of all APPs were measured to be significantly elevated in a parasitemia burden-dependent fashion as compared to the control. Furthermore, all the APPs showed 100 % specificity, but only SAA and Cp had 100 % sensitivity. A strong and positive correlation was calculated between the APPs and SA; however, cholinesterase activity was inversely correlated with AAPs and SA. In conclusion, inflammatory reactions play a pivotal role in the pathogenesis of bovine babesiosis and APPs can be considered as the potential indicators of inflammation.     

Cholinesterases as markers of the inflammatory process associated oxidative stress in cattle infected by Babesia bigemina

The objective of this study was to assess the influence of an asymptomatic experimental infection by Babesia bigemina on cholinesterase’s as markers of the inflammatory process and biomarkers of oxidative imbalance. For this purpose, eight naive animals were used, as follows: four as controls or uninfected; and four infected with an attenuated strain of B. bigemina. Blood samples were collected on days 0, 7 and 11 post-inoculation (PI). Parasitemia was determined by blood smear evaluation, showing that the infection by B. bigemina resulted in mean 0.725 and 0.025% on day 7 and 11 PI, respectively, as well as mild anemia. The activities of acetylcholinesterase, butyrylcholinesterase and catalase were lower, while levels of thiobarbituric acid reactive substances and superoxide dismutase activity were higher in infected animals, when compared with the control group. This attenuated strain of B. bigemina induced an oxidative stress condition, as well as it reduces the cholinesterasés activity in infected and asymptomatic cattle. Therefore, this decrease of cholinesterase in infection by B. bigemina purpose is to inhibit inflammation, for thereby increasing acetylcholine levels, potent anti-inflammatory molecules.     

Detection of Babesia bigemina DNA in ticks by DNA hybridization using a nonradioactive probe generated by arbitrary PCR

The Dig-labeled probe specific to Babesia bigemina generated from monomorphic RAPD fragment of approximately 873 bp size amplified by a 10 mer CGGTGGCGAA, detected up to 100 ng of template DNA. This nonradioactive probe also detected B. bigemina in preparations of larval tick DNA from two of the five samples on dot-blot hybridization.     

Seroprevalence of Babesia bigemina in smallholder dairy cattle in Tanzania and associated risk factors

Variations in the seroprevalence of antibody to Babesia bigemina infection by farm and animal level risk factors were investigated for 2 contrasting regions of Tanga and Iringa in Tanzania. Tanga is situated in the eastern part of the country and has typical tropical coast climate while Iringa is situated in the Southern Highlands and has a tropical highland climate. Two hundred farms from each region were selected using simple random sampling procedure and visited once between January 1999 and April 1999. Blood samples were collected from 1329 smallholder dairy animals on selected farms for harvesting serum which was subsequently used for serodiagnosis of B. bigemina using an indirect enzyme linked immuno-sorbent assay (ELISA). Of the 1329 sera samples screened, 34.9 % were positive for B. bigemina. The prevalence was higher in Iringa Region [43%, 95% confidence intervals (CI) = 39.5-47.3] than in Tanga Region (27%, CI = 23.6-30.5). Using a logistic binomial regression model as an analytical method for predicting the likelihood of animal seropositivity, we found (in both regions) that the risk of positive reaction varied with the animal's age, history of grazing and geographical location. Seroprevalence increased with age (beta = 0.01 and 0.01 per year of age, P < 0.005 in Tanga and Iringa, respectively). Animals located in Lushoto and Iringa urban district were associated with increased risk of seropositivity [Odds ratio (OR) = 4.24, P = 0.001, for Lushoto, and OR = 1.81, P = 0.040, for Iringa Urban, respectively). Animals grazed 3 months prior to sampling had higher odds for seropositivity than zero/semi-grazed, despite farmer-reported high frequency of tick control (OR = 2.71, P = 0.0087, for Tanga, and OR = 4.53, P = 0.001, for Iringa). Our study suggests that even though herd sizes are small, B. bigemina infection is widespread in many smallholder dairy farms and endemic stability with respect to this disease has not yet been attained, but the observed levels are sufficiently high to ensure that clinical disease would be a risk.     

Use of long-acting oxytetracycline in the immunisation of cattle against Babesia bovis and B bigemina

Forty Friesian one-year-old calves were vaccinated simultaneously with live Babesia bovis and B bigemina vaccines. Three groups of 10 calves each were treated with two, three or four doses of 20 mg kg-1 long-acting oxytetracycline (OTC/LA) at six- to seven-day intervals starting from day 6 after vaccination. Ten animals remained untreated. The treated calves showed considerably fewer days of patency and higher packed cell volumes than the vaccinated untreated calves. All calves developed serum antibodies to both parasites following vaccination. Five months later the 40 vaccinated and 30 new calves were challenged with syringe-transferred virulent parasites of both species. The vaccinated calves showed no parasites or clinical manifestations while calves of the new group exhibited severe clinical babesiosis. These results show that when OTC/LA is administered following anti-babesial vaccination, parasitaemia and red blood cell destruction are significantly reduced without, however, inhibiting the development of immunity.     

In vitro cultivation of Babesia bigemina

A strain of Babesia bigemina was isolated from an infected calf and propagated in vitro. Culture conditions included washing of infected and normal bovine erythrocytes in a special solution, and the use of a 5% to 10% (v/v) erythrocyte suspension in medium 199 (with 20% to 50% fresh normal bovine serum) at a depth of 4 mm in a 5% CO2, 2% O2, 93% N2 atmosphere. After 36 days in vitro and 9 subcultures, the cultured organism was inoculated into a susceptible calf. This calf developed clinical signs of disease and recovered when treated with 1% trypan blue solution. The strain was also reisolated from the second calf. The original isolate had been maintained in continuous in vitro cultivation for more than 99 days.     

Cloning of in vitro propagated Babesia bigemina

The original Babesia bigemina culture conditions were modified with regard to infected bovine erythrocyte concentration and atmospheric environment. A procedure was designed which would yield a homogeneous parasite population, beginning with a single infected erythrocyte. Calculated dilutions were made in 96-well tissue culture plates to approach one infected erythrocyte per four wells. Growth of parasites in wells was detected between 16 and 28 days after cultures were initiated. Clones were transferred to 24-well tissue culture plates for regular maintenance. Three primary clones were selected for additional recloning. The probability that the parasites detected in one well are the progeny of a single infected erythrocyte approaches 0.99 for tertiary clones.     

Characterization of a repetitive DNA probe for Babesia bigemina

A plasmid (p16) containing a Babesia bigemina DNA insert was selected and labeled with 32P. This probe was evaluated for specificity and sensitivity by dot blot hybridization. The probe was specific and hybridized with only Babesia bigemina DNA, and not DNA from Babesia bovis, bovine leukocyte, Trypanosoma brucei or Anaplasma marginale. The DNA probe detected as little as 10 pg of Babesia bigemina DNA. The probe hybridized with Babesia bigemina isolates from Mexico, the Caribbean region and Kenya. Genomic Babesia bigemina DNA of a Kenyan isolate was digested with restriction endonucleases, and the fragments were separated by gel electrophoresis and Southern blotted. The filter was hybridized with labeled p16 and each endonuclease digestion produced at least 16 resolvable DNA fragments. The inserted Babesia bigemina DNA was approximately 6.3 kb in size. A partial restriction map was constructed. A simple whole blood dot blot procedure was utilized to evaluate the sensitivity of the DNA probe. This probe would detect as few as 150 Babesia bigemina infected erythrocytes contained in a 1-microliter sample. The DNA probe has the potential to be a very sensitive and specific diagnostic tool.     

Babesia bovis and B. bigemina DNA detected in cattle and ticks from Zimbabwe by polymerase chain reaction

From blood collected from 94 cattle at 12 locations in the eastern and northeastern areas of Zimbabwe, DNA was extracted and analysed by polymerase chain reaction with primers previously reported to be specific for Babesia bigemina and Babesia borvis. Overall, DNA of Babesia bigemina was detected in the blood of 33/94 (35%) cattle and DNA from B. bovis was detected in 27/58 (47%) of cattle. The prevalence of DNA of B. bigemina was significantly higher in young animals (<2 years) (23/46) than in animals over 2 years of age (10/48; chi2= 8.77; P <0.01%). Although tick sampling was not thorough, Boophilus decoloratus could be collected at 7/9 sites sampled and Boophilus microplus at 4/9 sites. Of the 20 B. decoloratus allowed to oviposit before PCR analysis, 1 (5%) contained DNA that could be amplified with primers for B. bigemina while 12 (60%) were positive with primers for B. bovis. Of the B. microplus allowed to oviposit, 11/16 (69%) were positive for B. bovis DNA by PCR and 2/16 (12%) were positive for B. bigemina.     

Prevalence and molecular detection of Anaplasma marginale, Babesia bovis and Babesia bigemina in cattle from Puntarenas Province, Costa Rica

A study was conducted in 2008 to determine the prevalence of Anaplasma and Babesia infections in cattle in the Puntarenas Province of Costa Rica. Blood samples were taken from a total of 449 cattle during the month of March at 30 farms in the region of Espiritu Santu, Costa Rica. Commercially available enzyme-linked immunosorbent assays (ELISA) were used to determine presence of antibodies to Babesia bigemina and Anaplasma marginale, and real-time PCR was used to determine the presence of DNA from the disease-causing organisms. The ELISA results indicated that 87.5% of the cattle sampled were positive for antibodies to A. marginale, while 59.1% were positive for antibodies to B. bigemina. The real-time PCR results showed that 235 cattle were carrying A. marginale DNA (56.9%), 6 with B. bigemina DNA (1.34%), and 2 with B. bovis DNA (0.45%).