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。     

实时荧光PCR检测牛边缘无浆体方法的建立及应用

根据牛边缘无浆体表面蛋白4的保守基因序列设计特异引物AMOC9/AMOC5、AMOC10/AMOC12和特异性探针MP,首次建立了牛边缘无浆体的实时荧光PCR检测方法,检测DNA的最低限度为200 fg。对中央无浆体、绵羊无浆体、牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫和伊氏锥虫进行检测,无荧光检测信号。本研究用所建立的方法检测采自江苏和哈尔滨的180份抗凝血（奶牛和肉牛）,其阳性率为8.9%。结果表明,建立的实时荧光PCR检测牛边缘无浆体的方法具有较高的特异性和敏感性,可用于牛边缘无浆体病的流行病学调查、检疫和监测。     

奶牛附红细胞体感染PCR诊断方法的建立

为建立特异、敏感、快速的奶牛附红细胞体感染诊断方法，该研究根据本实验室已测得的奶牛附红细胞体16S rRNA基因序列，设计1对种特异性引物，建立了奶牛附红细胞体的PCR诊断方法。特异性试验和敏感性试验结果表明，该诊断方法与猪肺炎支原体、鸡毒支原体、大肠杆菌、肠道沙门氏菌、葡萄球菌、鸡艾美耳球虫、牛双芽巴贝斯虫无交叉反应，能检测的奶牛附红细胞体最低DNA量为0．154fg，同时能检测出在4℃存放长达3个月的血样。通过临床血样检测，证明该方法可用于本病的早期诊断。     

驽巴贝斯虫病PCR检测方法的建立和应用

根据驽巴贝斯虫(Babesia caballi)18S rRNA基因序列设计1对特异性引物,扩增出452 bp核苷酸片段,建立了检测驽巴贝斯虫病的PCR方法。敏感性试验结果表明,该方法最低能检出0.01 fg/μL驽巴贝斯虫DNA模板。特异性试验结果显示,在被检测的6个巴贝斯虫株中,仅驽巴贝斯虫株能扩增出特异性片段,马泰勒虫、双芽巴贝斯虫、莫氏巴贝斯虫、卵形巴贝斯虫、大巴贝斯虫的扩增结果均为阴性。对45份马属动物血样进行检测,本研究建立的PCR方法测得驽巴贝斯虫病的阳性率为26.67%(12/45),与显微镜检测方法进行了比较,结果显示PCR检测方法可显著提高驽巴贝斯虫的检出率。     

牛双芽巴贝斯虫PCR检测方法的建立及初步应用

根据PCR技术原理,建立起了牛双芽巴贝斯虫病PCR检测方法,并测定出该方法的敏感性为108.66 fg。特异性试验表明,除牛双芽巴贝斯虫出现特异扩增带(295 bp)外,其他的虫株均未出现特异性扩增带。采用PCR检测方法,通过对35份牛血样的检测,测得牛双芽巴贝斯虫病的阳性率为28.57%(10/35),说明PCR方法敏感性强、特异性高,不失为牛双芽巴贝斯虫病临床诊断的好方法。     

新疆部分地区马驽巴贝斯虫病感染PCR检测调查

为了解新疆部分地区马匹感染马驽巴贝斯虫病的流行情况,通过PCR方法对不同地区所采集的样品进行检测,并对不同地区和不同年龄段马驽巴贝斯虫感染情况进行单因素方差分析。结果显示,所检测的504份马血样中,54份血样为阳性,阳性率为10.71%。其中北疆昭苏种马场、阿勒泰、吐鲁番托克逊马驽巴贝斯虫阳性率分别为20.61%、5.17%、0;巴音布鲁克牧场、和静地区马驽巴贝斯虫阳性率分别为0和8.59%。从阳性样品中都检测到了驽巴贝斯虫BC48基因片段,与已知序列的同源性为99%。X≤3,3X≤6,6X≤9,X9四个年龄段马驽巴贝斯虫感染率分别为9.76%、10.53%、11.21%和11.65%。单因素方差分析结果表明,昭苏种马场与阿勒泰、巴音布克牧场分别存在极显著性差异(P0.01);巴音布鲁克牧场与和静也存在极显著性差异(P0.01);昭苏种马场与吐鲁番托克逊存在显著性差异(P0.05),其余各地方差异不显著。各个年龄段马驽巴贝斯虫感染无显著性差异。本试验的研究数据可为研究新疆马驽巴贝斯虫分布特点提供数据支撑。     

犬巴贝斯虫实时荧光定量PCR检测方法的建立

为建立一种快速准确检测犬巴贝斯虫(Babesia cains,B.canis)的方法,试验根据GenBank中收录的巴贝斯虫18S rRNA序列设计一对特异性引物,对建立的基于SYBR GreenⅠ检测巴贝斯虫的实时荧光定量PCR方法进行了研究。结果表明:建立的实时荧光定量PCR方法可准确检测出10拷贝/μL的样本,灵敏度是常规PCR的1 000倍;该方法可以特异地检测巴贝斯虫,对6个对照组的检测结果均为阴性;批内和批间变异系数均低于5.00%;对豫西地区收集的21份临床样品进行检测,实时荧光定量PCR方法的阳性率为66.7%,普通PCR方法的阳性率为55.1%,血涂片法的阳性率为33.3%。说明该方法可用于临床疑似巴贝斯虫感染犬的早期检测和确诊。     

安格斯种牛暴发牛巴贝斯虫病的诊断及防治

2015年12月,云南曲靖市马龙县某规模化肉牛场新引进的澳大利亚安格斯种牛发生以高热、血尿、消瘦、血液稀薄及可视黏膜黄染为主要临床表现的疫情,发病初期经抗生素类药物治疗无好转,死亡数量不断增加。为了对此次暴发的疫病进行确诊,遂采集发病牛新鲜血液制备生理盐水悬液及血涂片染色镜检,同时制备EDTA-K2抗凝血进行血液细胞分析,并抽提血液基因组DNA进行巴贝斯焦虫(Babesia bovis)、泰勒焦虫(Theileria annulata)、无浆体(A.marginale/ovis)及埃利希氏体(E.ruminantium)PCR检测。结果发病牛血细胞数量稀少,大部分红细胞呈不规则样变形,红细胞膜边缘有数量不一的脊状凸起,凸起部位经染色后呈蓝紫色,血细胞分析结果也提示呈贫血血象。牛巴贝斯虫PCR检测扩增出约750bp大小的目的基因片段,PCR产物经切胶回收纯化后测序,所得序列在NCBI GenBank中blast,结果与2012年我国北方采集获得的巴贝斯焦虫ZJK15参考株(Accession No.:KP710223.1)18SrRNA基因序列同源性达99%,而无浆体、泰勒焦虫及埃利希氏体PCR检测均未扩增出目的基因片段。结合此次疫情的临床症状、病理变化、血涂片和生理盐水悬滴液镜检、血细胞分析、牛巴贝斯虫PCR检测及序列分析结果,最终确诊此次肉牛暴发的疫病为牛巴贝斯虫病,发病牛群经三氮脒(血虫净)、高糖、维生素B12、牲血素(右旋糖酐铁)静脉输液治疗后均全部康复,康复后2个月采集血液进行血细胞分析,结果显示各项血象指标均恢复正常。     

新疆部分地区牛梨形虫和无浆体感染情况调查分析

为了解新疆吐鲁番与阿勒泰地区牛梨形虫及牛无浆体的感染率和种类,采用聚合酶链反应(PCR)技术对两地区随机采集的120份牛血样品进行了泰勒虫、巴贝斯虫及无浆体的病原检测,对感染率进行统计学分析,并对其系统发育进行分析。结果显示,牛环形泰勒虫、牛双芽巴贝斯虫及牛无浆体感染率分别为20.8%(25/120)、14.2%(17/120)和11.7%(14/120),其中28头牛感染2种～3种病原,呈混合感染。调查结果可为上述两地区泰勒虫病、巴贝斯虫病及无浆体虫病防控提供参考。     

新疆和静县和吐鲁番地区牛感染巴贝斯虫病的血清学调查

为了解双芽巴贝斯虫和牛巴贝斯虫在新疆疫区牛感染的状况,从吐鲁番市周边散养户、和静县部分散养户采集了273份牛(牦牛)血清。采用间接ELISA方法对血清样本进行牛巴贝斯虫和双芽巴贝斯虫抗体检测。结果显示:和静县部分散养户被检牦牛血清抗牛巴贝斯虫(B.bovis)抗体阳性率为18.68%(17/91);被检牦牛血清抗双芽巴贝斯虫(B.bigemina)抗体阳性率为9.89%(9/91)。吐鲁番市周边散养户被检奶牛血清抗牛巴贝斯虫(B.bovis)抗体阳性率为15.38%(28/182);被检奶牛血清抗双芽巴贝斯虫(B.bigemina)抗体阳性率为9.34%(17/182)。通过调查发现牛巴贝斯虫和双芽巴贝斯虫均有混合感染的现象,其中和静牦牛混合感染率为6.59%(6/91);吐鲁番奶牛混合感染率为8.24%(15/182);不同品种牛均可感染牛巴贝斯虫和双芽巴贝斯虫,其感染程度及感染率具有一定的差异。本次试验结果可为有效防治新疆疫区牛梨形虫病提供重要依据。     

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%).     

Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA

An indirect enzyme linked immunosorbent assay (iELISA) for diagnosis of anaplasmosis using undiluted individual milk samples from dairy cows was developed. The recombinant 19 kDa major surface protein 5 (rMSP5) of Anaplasma marginale was used as antigen. A monoclonal antibody against bovine IgG1 conjugated with peroxidase and the chromogen 3,5,3',5'-tetramethylbenzidine were used in the test. Strong and weak, positive and negative milk samples were set up as reference controls. Results were expressed as percentage of positivity (PP) contrasting with the strongest positive control. The test was evaluated in two groups (G1 and G2) of lactating dairy cows from herds located in A. marginale non-endemic areas of Argentina. The infection status of both groups, G1 (n=128) sampled after anaplasmosis outbreak, and G2 (n=216) free of anaplasmosis was established by polymerase chain reaction (PCR). Serum samples of cows from G1 and G2 were analyzed by card agglutination test (CAT) and competitive ELISA (cELISA), while the novel iELISA was evaluated in their corresponding milk samples. At a cutoff of 42 PP, the ELISA has 98% sensitivity and 95% specificity. A significant difference (P<0.0001) was found between the mean PP value of negative samples from G1 (17.4+/-14.9), and G2 (8.6+/-7.1). The agreement and kappa (kappa) value between iELISA and PCR was 96%, kappa=0.919; between iELISA and CAT was 97%, kappa=0.880; and between iELISA and cELISA was 97%, kappa=0.899. These results strongly support the usefulness of iELISA to detect A. marginale antibodies in milk. Additional studies are necessary to define the ability of the milk iELISA to detect not only acutely infected, but also carrier cattle.     

利用套式PCR检测牛羊乏质体

为同时检测和鉴定牛边缘乏质体、中央乏质体及绵羊乏质体,根据这3种病原体的msp4基因核苷酸序列,自行设计、合成了针对3种乏质体的2对通用引物,及分别针对三者的特异引物,通过PCR条件优化,建立了检测乏质体及分别鉴定3种乏质体的套式PCR方法,并与OIE推荐的msp5半套式PCR比较.结果显示:该方法对牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫、伊氏锥虫均未扩增出特异性片段.套式PCR检测乏质体DNA量为0.2 pg(相当于6个感染红细胞).检测l 119份来自6个不同地区的奶牛、肉牛、水牛及羊的临床样品,阳性106份,经鉴定边缘乏质体46份,中央乏质体15份,绵羊乏质体35份,混合感染中央乏质体和绵羊乏质体4份,混合感染边缘乏质体和绵羊乏质体3份,混合感染边缘乏质体和中央乏质体3份.首次在分子生物学水平证明中央乏质体存在于中国.同时,证明牛可以混合感染边缘乏质体和中央乏质体或绵羊乏质体,以及混合感染中央乏质体和绵羊乏质体.上述848份样品用OIE推荐的msp5半套式PCR同时检测,两者符合率为98.5％(835/848).检测结果表明,msp4套式PCR特异、敏感,可用于边缘乏质体、中央乏质体、绵羊乏质体的检测和鉴定.     

Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.     

Comparison of blood smear and indirect fluorescent antibody techniques in detection of haemoparasite infections in trade cattle in Nigeria

The incidence of blood parasites in trade cattle was surveyed with emphasis on tick-borne parasites, using blood smears and immunofluorescent antibody (IFA) techniques. With the blood smear method, about 9 and 8.9% of cattle examined were found positive for Babesia bigemina and Anaplasma marginale, respectively. Percentage infections with other parasites were 3.33, 1.92, 0.75, 0.75 and 0.58, respectively, for Babesia bovis, Trypanosoma brucei, Anaplasma centrale, Eperythrozoon and Theileria species as well as Trypanosoma congolense. The incidence of A. marginale infection was at its peak during the rainy season while B. bigemina was most prevalent during the dry season. There were mixed infections of Anaplasma and Babesia (1.42%); Babesia and trypanosomes (1.00%); Babesia and Eperythrozoon (0.75%) and Babesia and Theileria (0.75%). Using the indirect fluorescent antibody test, 93, 55 and 68% of cattle sera examined were found to be positive for B. bigemina, B. bovis and A. marginale, respectively. Forty-nine percent of the positive sera of B. bigemina had highest titres. The importance of using serological means for determining the endemic levels of tick-borne diseases in cattle in Nigeria is discussed.     

Seroprevalences of vector-transmitted infections of small-holder dairy cattle in coastal Kenya

A cross-sectional study was carried out from July to September 1989 in Kaloleni Division, Coast Province, Kenya to estimate the prevalence of vector-transmitted diseases in small-holder dairy cattle and to identify the risk factors associated with different management systems. One hundred and thirty of the 157 herds with dairy cattle in Kaloleni Division were surveyed. These were from three agro-ecological zones (coconut-cassava, cashew nut-cassava and livestock-millet), comprised two management systems (stall-feeding and herded grazing) and were herds with either dairy cattle only or with Zebu and dairy cattle. A formal questionnaire sought answers to questions on cattle health and management practices. A total of 734 dairy and 205 Zebu cattle in 78 dairy and 52 mixed (dairy and Zebu) herds were sampled and screened for haemoparasites (Trypanosoma, Anaplasma, Babesia, and Theileria infections). Sera were tested for antibodies to Theileria parva, using the schizonts-antigen indirect fluorescent-antibody (IFA) test and to antibodies for Babesia bigemina and antigens to Anaplasma marginale by enzyme-linked immunosorbent assay (ELISA). Packed-cell volume (PCV) also was measured. Tick-control measures were practised by all except three of the farmers. Despite this, overall seroprevalence to T. parva was >70%--suggesting either that control practices were not strictly implemented or they were ineffective. The seroprevalence of T. parva in adult cattle kept in stall-feeding systems in the coconut-cassava zone was significantly lower (57+/-8% (S.E.)) than in herded-grazing systems (79+/-3%) and there was no association between antibody prevalence and age of cattle in this zone. Antibody prevalences in cattle in the cashew nut-cassava and the drier livestock-millet zone increased with age. Cattle in herded-grazing systems had an overall lower seroprevalence of T. parva infection in the livestock-millet zone (45+/-6%) than in the other two zones.Analysis was confined to the coconut-cassava zone for B. bigemina and to the coconut-cassava and cashew nut-cassava zones for A. marginale. Mean prevalences of B. bigemina were 40.9+/-9 and 73+/-6% for dairy cattle under stall-feeding and herded-grazing systems, respectively, and increased with age. Antigen prevalences of A. marginale were over 80% in all age groups of cattle in the coconut-cassava and cashew nut-cassava zones. Overall trypanosome prevalence in cattle was <1%. Trypanocidal treatment was uncommon. The variations in antibody prevalence associated with risk factors such as feeding system, agro-ecological zone and age of animal suggest that management system influenced exposure to tick-borne infection (particularly, T. parva infections) in small-holder dairy cattle in coastal Kenya.     

Serological evidence of exposure to tick fever organisms in young cattle on Queensland dairy farms

OBJECTIVES: To compare the features of farms on which the exposure of young cattle to tick fever organisms is sufficient to ensure that immunity is high and the risk of clinical disease is low (endemic stability) with those of farms on which exposure is insufficient to induce widespread immunity (hence without endemic stability); to examine the relationships between the management of ticks and tick fever, and endemic stability to Babesia bovis, B. bigemina and Anaplasma marginale. DESIGN: Cross-sectional study of 874 cattle between the ages of 6 and 15 months on 64 dairy farms, from three centres in south-eastern Queensland (Mutdapilly, Dayboro and Kenilworth) and one centre in far-north Queensland (Malanda). PROCEDURE: Blood samples collected from between 5 and 20 calves from each farm were submitted for serological assay to determine exposure to B. bovis, B. bigemina and A. marginale. A questionnaire about the farm characteristics and the management of ticks and tick fever was completed with each farmer. RESULTS: On 73% of farms, confirmed clinical cases of tick fever were recalled by the farmer, indicating that tick fever was a threat on most farms. The majority of herds in the study (54 of 64) did not have sufficient numbers of seropositive animals aged between 6 and 15 months to have a low risk of tick fever. Region had an effect on the likelihood of endemic stability for all tick fever organisms. Cattle near Malanda in Far-north Queensland were more likely to be seropositive to B. bovis and B. bigemina. The method, strategy and intensity of tick control were not related to the likelihood of endemic stability when the effect of region was considered. The decision to leave a few ticks on cattle in an effort to induce endemic stability did increase the likelihood of endemic stability to A. marginale. However, in practical terms, it was ineffective, because only 26% of these farms had endemic stability against all three organisms. CONCLUSIONS: Given the low proportion of farms that have endemic stability to the tick fever organisms and the high likelihood of clinical disease, vaccination is recommended to protect dairy cattle from tick fever throughout the tick infested area of Queensland. However, further work is required to determine the economic value of vaccination, taking into account the costs of vaccination, of outbreaks and the protective value of vaccination.     

Prevalence and genetic diversity of Babesia and Anaplasma species in cattle in Sudan

Disease prevalence studies are one of the most valuable tools to demonstrate the risk or impact of certain infections in local and global economies. The data obtained in these studies contribute to develop strategies for disease control. The present study aims to provide information about the prevalence of babesiosis and anaplasmosis in the northern regions of Sudan. Blood samples from four different states of Sudan were collected from apparently healthy cattle (n=692), DNA was extracted and the prevalence of Babesia and Anaplasma species was analyzed by PCR. The results confirmed the presence of Babesia bigemina, Babesia bovis and Anaplasma marginale in cattle in northern Sudan with overall prevalence rates of 4.0%, 1.9% and 6.1%, respectively. Statistical analysis revealed that the prevalence of B. bigemina, B. bovis and A. marginale varies significantly between Sudanese states as well as in different age groups, while gender seems not to have a significant effect on the prevalence of these pathogens among Sudanese cattle. The highest prevalence for B. bigemina was found in the Aljazirah State while the highest number of A. marginale positive samples was reported in River Nile.     

Diarrhea and loss of production on Dutch dairy farms caused by the Schmallenberg virus

At the end of August and the first two weeks of September 2011 dozens of veterinary practitioners reported to GD Veekijker (Animal Health Service) several dairy herds with cows with sudden decreased milk production, watery diarrhea and sometimes fever. In the beginning these reports came from the Eastern region of the Netherlands, after that also from the other three regions. The percentages of diseased herds per veterinary practice varied from a few till dozens per cent. Extensive bacteriological, virological and parasitological testing of the feces of sick cows did not reveal an infectious cause of the clinical problems. Recently, 50 stored blood samples of clinically diseased cattle were tested for the Schmallenbergvirus using a PCR, and 36% (18/50) tested positive. A large group of control cows (n=115) was also tested with the PCR and all cattle tested negative. Likely the Schmallenbergvirus was the primary cause of the clinical symptoms in the Dutch dairy herds. Further research will be done to confirm this.     

Babesia spp. infection in Boophilus microplus engorged females and eggs in Sao Paulo State, Brazil

Babesia spp. infections were investigated in Bos taurus x Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01).