N d R T M M a b c d a A R R A A K B B R R C 1 B l I i p K h 1 Ticks and Tick-borne Diseases 8 (2017) 335–340 Contents lists available at ScienceDirect Ticks and Tick-borne Diseases j ourna l h o me page: w ww.elsev ier .com/ locate / t tbd is either quantification by qPCR nor quantitative Elisa can be used to iscriminate Angus cattle for resistance/susceptibility to Babesia bovis odrigo Giglioti a,∗, Henrique Nunes Oliveiraa, Adriana Mércia Guaratini Ibelli b, alita Barban Bilhassi a, Thalita Athiê Néoc, Clarissa Helena Santanaa, árcio Dias Rabelod, Rosângela Zacarias Machadoa, Ana Carolina de Souza Chagasd, árcia Cristina de Sena Oliveirad Universidade Estadual Júlio de Mesquita Filho, Jaboticabal, SP, Brazil Embrapa Suínos e Aves, Concórdia, SC, Brazil Universidade Federal de, São Carlos, SP, Brazil Embrapa Pecuária Sudeste, São Carlos, SP, Brazil r t i c l e i n f o rticle history: eceived 27 July 2016 eceived in revised form 31 October 2016 ccepted 19 November 2016 vailable online 21 November 2016 eywords: abesia bovis eef cattle esistance epeatability orrelations a b s t r a c t With the aim of finding quantitative phenotypic traits that can be used to discriminate the levels of resistance/susceptibility to Babesia bovis, we estimated the repeatability and correlation between the level of infection, determined by the number of copies of a fragment of the gene that encodes cytochrome B (NC mt-cyB) of B. bovis, and the levels of the anti-B. bovis antibodies, in blood samples collected from 51 Angus cattle on two different occasions. Samples with the anticoagulant EDTA were used for DNA extraction and without anticoagulant for separation of the blood serum. The quantification of the NC mt-cyB of B. bovis was carried out by the quantitative PCR technique (qPCR), while the anti-B. bovis IgG antibody titers (S/P) were quantified by the ELISA method. The NC and S/P data were log10-transformed to improve the approximation to the normal distribution and were analyzed using mixed models. The correlations between NC mt-cyB and S/P were estimated, as well as the repeatability values for each trait. The results obtained showed the high sensitivity of the techniques, with 100% of the animals being positive for B. bovis, detected by both the serological and molecular tests. The correlations estimated between NC and S/P were low, 0.10 and 0.12, in the first and second collection, respectively. The repeatability estimated for NC was 0.06, whereas for the S/P it was 0.42. The low correlations between S/P and NC in the two collections demonstrated that the variation in the NC value is independent of the level of antibodies. This results indicated that animals with a higher levels of antibodies against B. bovis in the first collection continued to have a higher levels in the second one. However, the very low values for the repeatability value of NC, and for the correlations between S/P and NC, demonstrates that neither NC or S/P could be used to discriminate animals for resistance/susceptibility to B. bovis. © 2016 Elsevier GmbH. All rights reserved. . Introduction Bovine babesiosis is a disease caused by protozoa of the genus abesia, hemoparasite transmitted exclusively by ticks, causing osses to stock breeders throughout the world (McCOSKER, 1981). n Latin America, this disease affects both native cattle and animals mported from disease-free areas, causing significant losses to the roducers (Guglielmone, 1995). ∗ Corresponding author at: Embrapa Pecuária Sudeste, Rodovia Washington Luiz, m 234, CP 339, 13560−970 São Carlos, São Paulo, Brazil. E-mail address: gigliotirodrigo@gmail.com (R. Giglioti). ttp://dx.doi.org/10.1016/j.ttbdis.2016.11.008 877-959X/© 2016 Elsevier GmbH. All rights reserved. Babesia bovis is highly pathogenic and the infective forms, called sporozoites, are produced around two to three days after attach- ment of the tick larvae on the host (Riek, 1966). These evolutive forms invade the animals’ erythrocytes and multiply by merogony, provoking hemolysis and penetrating new cells until the host dies or develops immunity (Hunfeld et al., 2008). Erythrocytes infected by B. bovis accumulate in the capillaries of various organs, among them the brain, causing cerebral babesiosis (Gohil et al., 2013). Cat- tle born in areas that are endemic for babesiosis often show a certain degree of natural resistance to the disease (Bock et al., 2004). Greater resistance to babesiosis of zebu breeds (Bos taurus indi- cus) in comparison with taurine breeds (Bos taurus taurus) has been observed, mainly in the absence of clinical symptoms or the occur- dx.doi.org/10.1016/j.ttbdis.2016.11.008 http://www.sciencedirect.com/science/journal/1877959X http://www.elsevier.com/locate/ttbdis http://crossmark.crossref.org/dialog/?doi=10.1016/j.ttbdis.2016.11.008&domain=pdf mailto:gigliotirodrigo@gmail.com dx.doi.org/10.1016/j.ttbdis.2016.11.008 3 k-born r e g a t a a f b s g a i g a i c t c z ( 2 t f a s s a ( 2 R s l r c a m q c t e s r o B a a l 2 2 l 4 c i 2 t o 1 36 R. Giglioti et al. / Ticks and Tic ence of less severe symptoms (Bock et al., 1997, 1999; Jonsson t al., 2008; Piper et al., 2010). This pattern has prompted the reat majority of Brazilian producers to choose zebu animals, as lso occurs in Australia, in areas with high rates of tick infesta- ion (Jonsson, 2006). Nevertheless, higher resistance to babesiosis ppears not to be an exclusive trait of zebu animals, since Benavides nd Sacco, (2007), studying challenge of naïve animals with B. bovis, ound the occurrence of three distinct phenotypes in the taurine reeds Hereford and Aberdeen Angus: resistant, intermediate and usceptible animals. This variation between and within breeds sug- ests that resistance to babesiosis is a genetically determined trait nd also that it can respond to selection. However, the main issue s to identify a quantitative phenotypic trait that enables to distin- uish animals that are more resistant or sensitive to babesiosis. Serological tests, such as the enzyme-linked immunosorbent ssay (ELISA), can quantitatively determine the levels of antibod- es against babesiosis (Böse et al., 1990; Machado et al., 1997) and an be used to indicate resistance to the disease in genetic selec- ion studies. On the other hand, techniques based on polymerase hain reaction (PCR) allow amplification of the DNA of the proto- oa in cattle blood samples with high sensitivity and specificity Oliveira et al., 2005; Oliveira-Sequeira et al., 2005; Buling et al., 007; Guerrero et al., 2007). Bilhassi et al. (2014) used the qPCR echnique to study the level of infection by B. bovis in cattle of dif- erent genetic groups and found that blood samples from taurine nimals had a significantly higher number of copies of the para- ite’s DNA than samples from zebu and crossbred animals. Recently, everal research groups have conducted experiments involving the ssociation of immunological and molecular tests to detect Babesia Ferreri et al., 2008; Hüe et al., 2013; Ibrahim et al., 2013; Jaffer et al., 010; Machado et al., 2012; Mousa et al., 2013; Ramos et al., 2011; osales et al., 2013; Shebish et al., 2012). These associations have hown excellent efficiency in detecting infected animals, going a ong way toward resolving the problems of predicting the occur- ence of babesiosis outbreaks. No studies have been published reporting the repeatability and orrelation between the Babesia infection levels quantified by qPCR nd ELISA. The study by Giglioti et al. (2016) was the first to esti- ate the repeatability of B. bovis infection levels in Angus cattle uantified by qPCR. The authors observed low repeatability for both ollections analyzed. In light of the foregoing, we conducted the present experiment o evaluate the phenotypic traits that can discriminate the lev- ls of resistance/susceptibility to B. bovis, to be used in genetic election studies. Blood samples from Angus cattle (Bos taurus tau- us), reared in a stable endemic area for babesiosis, were collected n two occasions and were used to determine the NC mt-cyB of . bovis, using the qPCR technique, and the levels of anti-B. bovis ntibodies, employing the ELISA method. The resulting data were nalyzed to estimate the repeatability of each trait and the corre- ations between them, within and between collections. . Material and methods .1. Animals and sample collection The animals used in this experiment were reared on a farm ocated in the municipality of José Bonifácio (21◦ 03′ 10′′ S and 49◦ 1′ 18′′ W), in the state of São Paulo, where the cattle tick Rhipi- ephalus microplus infests herds in all months of the year. The area s considered to have endemic stability for babesiosis (Bilhassi et al., 014). The climate in the region is classified as AW, with average emperatures in May of 20 ◦C (minimum of 13 ◦C and maximum f 27 ◦C), with rainfall of 40 mm, and 21 ◦C in July (minimum of 1.7 ◦C and maximum of 26.0 ◦C), with rainfall of 23 mm. We used e Diseases 8 (2017) 335–340 51 clinically healthy Angus males, with ages near two years at the time of collection. Blood was drawn from each animal on two occa- sions in 2012, in Brazilian autumn (May) and winter (July). The samples were obtained by puncture of the jugular vein using the Vacutainer ® system in tubes containing the anticoagulant EDTA for extraction of DNA and without anticoagulant for separation of the serum. All the animals were infested by ticks, as confirmed by observation and palpation of the entire right side of the body. The animals were all considered healthy because they did not show signs of the disease or of hyperthermia. This experiment was in accordance with ethical principles for animal experimentation of the Embrapa Pecuária Sudeste ethics committee for animal experiments (CEUA-EMBRAPA/CPPSE). 2.2. Extraction of DNA and preparation of serum The DNA was extracted from the blood samples using the illustra blood genomic Prep Mini Spin kit (GE Healthcare, Little Chalfont, UK), according to the manufacturer’s recommendations, using a blood volume of 300 �L. The blood samples without anticoagu- lant were centrifuged to separate the serum, which was placed in microtubes and frozen at −20 ◦C until the analysis. 2.3. ELISA for Babesia bovis The ELISA protocols were conducted in the Immunoparasitol- ogy Laboratory of the Department of Veterinary Pathology of FCAV/UNESP Jaboticabal, according to the method described by Machado et al. (1997). The concentration of the B. bovis anti- gen was determined by the bicinchoninic acid assay (BCA), using the PierceTM Micro BCATM Protein Assay Kit (Thermo Scientific, Waltham, Massachusetts, US), with the samples diluted to a con- centration of 5 �g/mL. As controls for the qPCR and ELISA testing, serum from experimentally infected cattle with high titers of anti- bodies against B. bovis was used for positive control, while as negative control the serum samples were collected prior to infec- tion and from calves before they suckled colostrum (Machado et al., 1997). In each ELISA run, two negative and two posi- tive control samples were used. In summary, 100 �L of antigen diluted in carbonate-bicarbonate buffer 0.05 M, pH 9.6, with anti- gen concentrations of 5 �g/mL, was placed in wells of polystyrene ELISA microplates (96 wells, Thermo Fisher Scientific, Rochester, NY, US), which were sealed and incubated at 4 ◦C overnight. The plates were blocked with a 6% rabbit serum solution in PBS- Tween buffer (phosphate-buffered saline, pH 7.2 and PBS-Tween with 0.05% Tween 20) for one hour in a humid chamber at 37 ◦C. The plates were then washed three times with PBS-Tween buffer, after which 100 �L of bovine serum was added, diluted at 1:400 in PBS-Tween. The positive and negative control sera were also diluted at 1:400 in the same buffer. The plates were incubated at 37 ◦C in a humid chamber for 90 min and then washed three times with the same PBS-Tween solution. An aliquots of 100 �L of solution diluted to 1:10,000 of anti-IgG bovine conjugated with alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, US) in PBS- Tween buffer was added to each well and the plates were incubated at 37 ◦C in the same conditions described above, for 90 min. The plates were again washed three times with PBS-Tween solution and 100 �L of the substrate for phosphatase and liquid p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO, US) was added in each well. The plates were sealed and incubated at room tempera- ture for 30 min. At the end of this period, the optical densities of the samples were read by a spectrophotometer (Dynex Tech- nologies, MRX TC plus), with a 405 nm filter. The enzyme activity from each serum sample was calculated by determining the sam- ple to positive serum ratio (S/P), considering positive and negative sera as reference, using the following equation: S/P = (mean sample k-born a a a ( ( d t m i B t T w 2 C v r 3 fl a D m w l ( fi i u E fi e g g s d w t U a f t D a ( o b ( N l u ( m a p 5 t r d S considered positive. The mean absorbance observed for the posi- tive controls was within EL 9. All the serum samples from the two collections were positive for B. bovis (EL ≥ 4). The means ± standard errors for S/P values obtained in the ELISA were significantly differ- Table 1 ELISA level (EL) scale according to the antibody titers (S/P) value ranges. (The serum samples were considered positive when the S/P values were greater than or equal to EL 4). EL S/P Ranges 1 0.000–0.098 2 0.099–0.133 3 0.134–0.181 4 0.182–0.245 5 0.246–0.332 R. Giglioti et al. / Ticks and Tic bsorbance−mean absorbance of negative serum reference)/(mean bsorbance of positive reference serum−mean absorbance of neg- tive serum reference). S/P values were grouped into ELISA levels EL), which ranged from 0 (lowest level) to 9 (highest level) Machado et al., 1997). The maximum amplitude of the EL = 0 was etermined by the mean of the absorbance values of the nega- ive samples, plus two standard deviations from the corresponding ean. From this limit, the intervals between the other EL were ncreased by 35% each, as described by Machado et al. (1997), for the abesia spp. system. The cutoff point was obtained by multiplying he maximum amplitude value of the EL zero by the coefficient 2.5. he serum samples were considered positive when the S/P values ere greater than or equal to the cutoff point. .4. qPCR reactions The CFXTM Real-Time PCR Detection System (BioRad, Hercules, A, USA) was used in the qPCR assays to obtain the NC mt-cyB alues, according to Buling et al. (2007). The primers used in the eactions were cbosg 1 (forward) 5′- TGTTCCTGGAAGCGTTGATTC- ′ and cbosg 2 (reverse) 5′-AGCGTGAAAATAACGCATTGC-3′, which ank a fragment of the mt-cyB gene of B. bovis and produce an mplicon with 88 base pairs (Salem et al., 1999; Buling et al., 2007). NA samples extracted from B. bovis isolates (donated by the Ani- al Pathology Department, UNESP Jaboticabal, São Paulo, Brazil) ere used to plot the calibration curve and as a positive control. To construct the calibration curve, a DNA sample from the iso- ate of B. bovis was amplified using the specific primers for B. bovis cbosg1 forward and cbosg2 reverse). The PCR products were puri- ed using the PureLinkTM PCR purification kit (Invitrogen), cloned nto pGEMT Easy Vector Systems (Promega) according to the man- facturer’s protocols. The recombinant clones were transformed in . coli DH5� cells and the white colonies were selected and ampli- ed to confirm the vector insert (Silva et al., 2014). The DNA was xtracted using the PureLink ® Quick Plasmid Miniprep Kit (Invitro- en), sequenced with an Applied Biosystems ABI Prism 3130 Avant ® enetic analyzer and submitted to BLAST analysis to confirm the equences obtained (Altschul et al., 1990). To estimate the NC mt-cyB values, calibration curves were stan- ardized based on the products cloned from the B. bovis isolate, hich were quantified in a NanoDrop ND-1000 spectrophotome- er (Thermo Scientific NanoDrop Products, Wilmington, Delaware, S) and based on the known value of each product they were seri- lly diluted tenfold. The dilutions of the products of the amplicons rom part of the cloned isolate were submitted to the qPCR tests ogether with the samples and controls to estimate the NC of the NA from B. bovis. The reaction efficiencies (E) were calculated ccording to the formula of Pfaffl (2001) and Vandesompele et al. 2002): E = 10 (−1/Slope), where Slope is the derivative (tangent line) f the calibration curve. The number of copies (NC) was computed y the formula described by Ke et al. (2006): NC (�l) = (6.022 × 1023 copies/mol) × concentration (g/mol))/molecular mass (g/�l). The C values for the dilutions from the calibration curves were uti- ized to calculate the NC values of the B. bovis DNA in each sample sing the software that comes with the CFX96 system (BioRad). The PCR mixture contained 0.3 �L of 10 �M of each primer cbosg forward and reverse), 5 �L of SsoFastTM EvaGreen ® Super- ix (BioRad), 4.4 �L of ultrapure water (Invitrogen, Carlsbad, CA), nd 2.0 �L of template DNA in a total volume of 12 �L. The qPCR rotocol started at 95 ◦C for 2 min, followed by 45 cycles of 95 ◦C for s and 57 ◦C for 30 s (annealing/extension). Following the last cycle, he melting curve was generated by heating from 65 ◦C to 95 ◦C at a ate of 0.5 ◦C/s. These conditions were according to the recommen- ations of the manufacturer of the reagent SsoFastTM EvaGreen ® upermix (BioRad), with some modifications. To prevent contami- e Diseases 8 (2017) 335–340 337 nation, barrier pipettes were used. All the samples were analyzed in duplicate, as were the positive and negative controls (with an addition of water instead of DNA in the negative control). 2.5. Statistical analysis The data from the S/P and NC were transformed into log10 (n + 1) for the purpose of approximating the normal distribution. After transformation, the data were analyzed using the mixed models method. A mixed model with repeated measures from the same animal, including the effect of the collection, quantification method (S/P and NC) and their interaction as fixed effects was used to ana- lyze S/P and NC values. The structure of the (co)variance matrix (symmetric) was specified according to the following model: ⎡ ⎢⎢⎢⎢⎣ �2 N �NN12 �NA11 �NA12 �NN12 �2 N �NA12 �NA11 �NA11 �NA12 �2 A �AA12 �NA12 �NA11 �AA12 �2 A ⎤ ⎥⎥⎥⎥⎦ where �2 K =variance component of NC (K = N) or S/P (K = A) common to both collections; and �KLij = covariance component between K and L (K,L = N,A) in collection i and j (i, = 1j = 1,2) (Littell et al., 2006). For each trait, the repeatability was estimated from the (co)variance matrix, as the covariance of the two collections of the same trait divided by the variance of the trait. The repeata- bility for each trait indicates how much of variation in a trait can be attributed to the inner effect of the animal, and can be interpreted as the correlation between the measures for the same individual in the two collections. Likewise, correlations were estimated between the values from two variables taken from the same animal, in the same or different collections. The MIXED routine from the SAS statistical package was employed for the analyses and the option LIN(q) from the command line REPEATED was used to specify the (co)variance matrix (SAS, 2002; Littell et al., 2006). 3. Results 3.1. ELISA test and qPCR assay The ELISA results obtained demonstrated high sensitivity in detecting IgG antibodies against B. bovis in the serum samples. The mean absorbance values of the positive and negative sera were 0.857 ± 0.152 and 0.090 ± 0.004, respectively. The ELISA scale was defined according to Table 1. Starting at EL 4, the animals were 6 0.333–0.449 7 0.450–0.607 8 0.608–0.820 9 <0.821 338 R. Giglioti et al. / Ticks and Tick-borne Diseases 8 (2017) 335–340 F level a e fi g c a t r a t 1 N i c t fi t a t m F e t p a i e t t 0 w F q ig. 1. Histogram of frequencies of the number of copies in the qPCR (NC) and ELISA nd July 2012. nt between the two collections (p < 0.05), with 0.24 ± 0.01 in the rst and 0.22 ± 0.01 in the second. The qPCR tests allowed detecting the fragment of the mt-cyB ene of B. bovis in the blood samples from all the cattle. The negative ontrols did not show amplification and the mean ± standard devi- tion of the quantitative cycle (Cq) of the controls was 20.4 ± 0.15, hus indicating little variation among the tests. The means of the eaction efficiency (E) were within the 95–105%, range accepted for n efficient PCR reaction. The means and standard errors of the transformed NC values for he mt-cyB gene of B. bovis for the first and second collection were .69 ± 0.08 and 1.33 ± 0.08, respectively. Fig. 1 presents the EL and C frequencies for the two collections (seasons of the year). In Fig. 1 t’s possible to note that the qPCR of the samples from the second ollection, the frequencies of the NC values greater than 100 (non- ransformed data) declined. Likewise, with respect to the EL, in the rst collection (May 2012) levels 7 and 8 predominated, while in he second (July 2012), EL 6 increased, at the expense of levels 7 nd 8 in relation to the first collection. EL 9 also declined in relation o the first collection. For both collections (autumn and winter), the correlations esti- ated between NC and S/P were low, 0.10 and 0.12 respectively. ig. 2 presents the distributions of the EL and NC values consid- ring all the samples evaluated in both collections. It can be seen hat except for EL 4, which showed low NC values, the values were ractically independent. Only one animal in the autumn collection nd three in the winter presented EL 4. Starting at EL 5, the same ndividals with low NC values presented high EL value. The high- st NC value found for a sample fell in the EL 6 range. Besides this, he animals with S/P values corresponding to EL 9 presented varia- ions of NC for the autumn and winter collections of 1.12–2.75 and .82–2.13, respectively. The repeatability values for S/P and NC in the two collections ere 0.42 and 0.06 respectively. The very low repeatability value ig. 2. Distribution of the log10-transformed number of copies (NC) determined by PCR according to ELISA levels (EL) in blood samples from Angus cattle. number (EL) for blood samples collected from Angus cattle on two occasions: May for NC and moderate value for S/P demonstrate that: (i) the ani- mals with a higher number of antibodies against B. bovis in the first collection continued to have a higher number in the second; and (ii) the number of copies of the mt-cyB gene detected by the qPCR in an animal in the first collection had little relationship with the number found in the second one. 4. Discussion The present experiment involved measuring the levels of anti- bodies and number of copies of the mt-cyB gene of B. bovis in blood samples of Angus cattle bred in an area of the state of São Paulo that according to a recent study (Bilhassi et al., 2014) is stably endemic for babesiosis. The results of this experiment con- firm those findings, and the fact that the animals reared in this region have developed resistance to the clinical form of the disease through repeated infections during the year. The ELISA results indi- cated that 100% of animals were positive for B. bovis, with antibody titers both in the first and second collections. Various studies car- ried out in Brazil have found similar results to ours here. Juliano et al. (2007) found prevalence rates higher than 92% on two occa- sions, in studying the levels of antibodies in Curraleira cattle bred in a cerrado (savanna) area of northeastern Brazil. Guedes Junior et al. (2008) found 98.8% prevalence in zebu and crossbred cattle in the northern region, and Trindade et al. (2010) observed prevalence of 91.7% in zebu calves in the state of Tocantins in the midwestern region of Brazil. Our results differ from those reported in southern Brazil by Osaki et al. (2002), who observed the prevalence of 64.2% among Nelore cattle, and Barros et al. (2005), who found 63.7% in cattle reared in the semi-arid region of northeastern Brazil. The South American continent is very heterogeneous regarding types of cattle production systems, and factors such as endemicity, susceptibility of host to tick, tick density and climate conditions can determine unique qualitative and quantitative characteristics for transmis- sion of babesiosis (Nari, 1995). Since Brazil is the largest country in South America, the occurrence of these variations is understand- able. Besides the high frequency of infection by B. bovis detected in this study, there were significant differences in the EL and S/P val- ues between the two collections. The month of May marks the end of autumn in southeastern Brazil, and this season has been identi- fied as having peak infestation by cattle ticks (Oliveira and Alencar, 1989; Andrade et al., 1998; Silva et al., 2007). This fact can explain, at least partly, the higher levels of antibodies observed in the first collection. The qPCR technique proved to be highly sensitive and specific in detecting DNA of B. bovis, comparable to the results reported by Buling et al. (2007). All the animals were positive for B. bovis in both collections. The high sensitivity of qPCR to assess the level of k-born i a b v m 2 i u m f l d i d n a R n ( B o n e t w s s c p l r w f t i b t t h D t t f c a t b c t r t s b a i a s i m i o t i R. Giglioti et al. / Ticks and Tic nfection by B. bovis has also been reported by Bilhassi et al. (2014) nd Giglioti et al. (2016). The association of the molecular and serological techniques has een used to improve babesiosis monitoring and diagnostic ser- ices. Various research groups have observed that ELISA can be a ore sensitive technique than molecular tests (Terkawi et al., 2011, 012; Machado et al., 2012; Ibrahim et al., 2013). Ferreri et al. (2008) nvestigated the diagnosis of B. bovis in water buffaloes in Argentina sing the nested-PCR and ELISA techniques. They found that the olecular technique was more sensitive. Different results were ound by Rosales et al. (2013) in diagnosing B. caballi in Venezue- an horses by means of qPCR and ELISA. They did not manage to etect the hemoparasites by the molecular technique while ELISA dentified infection prevalence of 23.2%. The authors attributed this iscrepancy to problems inherent to the principles of each tech- ique since qPCR detects DNA from the parasite while ELISA detects ntibodies against the parasite. Our results corroborate those of amos et al. (2011), who compared molecular and serological tech- iques in Brazil to monitor two regions, one with endemic stability Juíz de Fora, MG, Brazil) and another with instability (Bagé, RS, razil) for babesiosis. In the stable endemic area, the prevalences f infection by B. bovis measured by the qPCR, PCR, and ELISA tech- iques were 95.91, 82.65 and 92.85%, respectively. In the unstable ndemic area, they only detected B. bovis by qPCR, and the infec- ion rate was only 10%. One of the aspects that should be observed hen using PCR is the nature of the gene used to amplify the para- ite’s DNA. Salem et al. (1999) developed a PCR test and assessed its ensitivity and specificity in detecting B. bigemina and B. bovis by omparing episomal and ribosomal sequences of DNA from these arasites. With the episomal technique they detected 85% preva- ence of B. bovis, while with the ribosomal, technique the detection ate was only 65%. This information reinforces our findings, since e used primers that flank the region of the mt-cytB gene, specific or B. bovis, and that according to Buling et al. (2007) increase the est’s sensitivity more than 100 times. The main goal of this experiment was to check the repeatabil- ty of the qPCR and ELISA methods, in animals chronically infected y B. bovis, as well as the occurrence of associations between these wo tests. We did not find in the literature any studies of this nature o be used for comparison. Our results indicate that both tests are ighly sensitive in detection, be it of antibodies against B. bovis or NA from this parasite. With respect to the fluctuation in the quan- ity of DNA from the hemoparasite in the animals’ blood, it is hard o determine the cause. Considering the repeatability values found or the NC metric, we can infer that a large part of this variation an be attributed to environmental factors that act on the animal t the collection time, and only a small part can be attributed to he animals’ ability to maintain low infection levels, as also verified y Giglioti et al. (2016). Although tick infestation can vary between ollections, previous studies have shown no association between ick count and NC (Giglioti et al., 2016) and indicate this cannot be eported as the cause of the variation. The greater repeatability of he serological tests in relation to quantification of DNA of para- ites in the bloodstream shows the existence, at least in peripheral lood, of a greater individual variation in the NC than in the level of ntibodies. The immune response of animals frequently exposed to noculations of the antigen is sufficient to keep the hemoparasites t low levels, but without eliminating them completely. As demon- trated by the low correlations between S/P and NC, the variation n the NC value is independent of the level of antibodies. In conclusion, we can state that although the test used to esti- ate the NC in this experiment was able to detect 100% of B. bovis nfection, the repeatability estimates showed that, in the conditions f this study, this test does not discriminate animals for resis- ance to babesiosis. On the other hand, higher repeatability of S/P ndicates that animals with higher titers in the first sampling also e Diseases 8 (2017) 335–340 339 exhibit higher titers in the second collection. However, the cor- relations between the S/P and NC were low in both collections, indicating that higher level of antibodies does not imply better ability to maintain low levels of infection. Therefore, it is possi- ble that although detectable by the technique, the variations in the NC of this group of animals are not significant enough either to produce clinical signs in the animals or to trigger a momentaneous immune response. New studies needed to be carried out to try to identify quantitative traits that can be used in studies of selection for resistance to babesiosis. Acknowledgments Grants for this project were provided by Fundaç ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grants #2012/00067- 5 and #2011/23833-2), UNESP/FCAV, Jaboticabal and Embrapa Pecuária Sudeste. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Andrade, A.B.F., Silva, R.G., Costa, A.J., Rocha, U.F., Landim, V.J.C., 1998. Genetic and environmental aspects of the resistance of zebu cattle to the tick Boophilus microplus. In: World Congress on Genetics Applied to Livestock Production, 6. Proceedings. . . Armidale, Australia, pp. 339–342. Böse, R., Jacobson, R.H., Gale, K.R., Waltisbuhl, D.J., Wright, I.G., 1990. An improved ELISA for the detection of antibodies against Babesia bovis using either a native or a recombinant B. bovis antigen. Parasitol. Res. 76, 648–652. Barros, S.L., Madruga, C.R., Araújo, F.R., Menk, C.F., De Almeida, M.A.O., Melo, E.P.S., Kessler, R.H., 2005. Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays. Mem. Inst. Oswaldo Cruz. Benavides, M.V., Sacco, A.M.S., 2007. Differential Bos taurus cattle response to Babesia bovis infection. Vet. Parasitol. 150, 54–64. Bilhassi, T.B., Oliveira, H.N., Ibelli, A.M.G., Giglioti, R., Regitano, L.C.A., Oliveira-Sequeira, T.C.G., Bressani, F.A., Malagó, W., Resende, F.D., Oliveira, M.C.S., 2014. Quantitative study of Babesia bovis infection in beef cattle from São Paulo state, Brazil. Ticks Tick. Borne. Dis. 5, 234–238. Bock, R.E., de Vos, A.J., Kingston, T.G., McLellan, D.J., 1997. Effect of breed of cattle on innate resistance to infection with Babesia bovis, B. bigemina and Anaplasma marginale. Aust. Vet. J. 75, 337–340. Bock, R.E., Kingston, T.G., de Vos, A.J., 1999. Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis, Babesia bigemina transmitted by Boophilus microplus. Aust. Vet. 77, 461–464. Bock, R., Jackson, L., de Vos, A., Jorgensen, W., 2004. Babesiosis of cattle. Parasitology 129, S247–S269. Buling, A., Criado-Fornelio, A., Asenzo, G., Benitez, D., Barba-Carretero, J.C., Florin-Christensen, M., 2007. A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina. Vet. Parasitol. 147, 16–25. Ferreri, L., Benitez, D., Dominguez, M., Rodriguez, A., Asenzo, G., Mesplet, M., Florin-Christensen, M., Schnittger, L., 2008. Water buffalos as carriers of Babesia bovis in Argentina. Ann. N. Y. Acad. Sci., 149–151. Giglioti, R., Oliveira, H.N., Santana, C.H., Ibelli, A.M.G., Néo, T.A., Bilhassi, T.B., Rabelo, M.D., Machado, R.Z., Brito, L.G., Oliveira, M.C.S., 2016. Babesia bovis and Babesia bigemina infection levels estimated by qPCR in Angus cattle from an endemic area of São Paulo state, Brazil. Ticks Tick. Borne Dis. 7 (5), 657–662. Gohil, S., Herrmann, S., Günther, S., Cooke, B.M., 2013. Bovine babesiosis in the 21 st century: advances in biology and functional genomics. Int. J. Parasitol. 43, 125–312. Guedes Junior, D.S., Araújo, F.R., Silva, F.J.M., Rangel, C.P., Barbosa Neto, J.D., Fonseca, A.H., 2008. Frequency of antibodies to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burdgorferi in cattle from the northeastern region of the state of Pará. Braz. Rev. Bras. Parasitol. Vet. 17, 105–109. Guerrero, F.D., Bendele, K.G., Davey, R.B., George, J.E., 2007. Detection of Babesia bigemina infection in strains of Rhipicephalus (Boophilus) microplus collected from outbreaks in South Texas. Vet. Parasitol. 145, 156–163. Guglielmone, A.A., 1995. Epidemiology of babesiosis and anaplasmosis in South and Central America. Vet. Parasitol. 57, 109–119. Hüe, T., Graille, M., Mortelecque, A., Desoutter, D., Delathière, J., Marchal, C., Teurlai, M., Barré, N., 2013. Diagnostic methods used to monitor an outbreak of babesiosis (Babesia bovis) in a herd of feral cattle in New Caledonia. Aust. Vet. J. 91, 254–258. Hunfeld, K.P., Hildebrandt, A., Gray, J.S., 2008. Babesiosis: recent insights into an ancient disease. Int. J. Parasitol. 38, 1219–1237. Ibrahim, H.M., Adjou Moumouni, P.F., Mohammed-Geba, K., Sheir, S.K., Hashem, I.S.Y., Cao, S., Terkawi, M.A., Kamyingkird, K., Nishikawa, Y., Suzuki, H., Xuan, X., 2013. Molecular and serological prevalence of Babesia bigemina and Babesia http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0005 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0010 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0015 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0020 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0025 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0030 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0035 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0040 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0045 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0045 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0045 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0045 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0045 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0045 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0045 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0045 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0050 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0055 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0060 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0065 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0070 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0075 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0080 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0085 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0090 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 3 k-born J J J J K L M M M M N O O O O P Brazil. Rev. Bras. Parasitol. Vet. 19, 169–173. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., 40 R. Giglioti et al. / Ticks and Tic bovis in cattle and water buffalos under small-scale dairy farming in Beheira and Faiyum Provinces. Egypt. Vet. Parasitol. 198, 187–192. affer, O., Abdishakur, F., Hakimuddin, F., Riya, A., Wernery, U., Schuster, R.K., 2010. A comparative study of serological tests and PCR for the diagnosis of equine piroplasmosis. Parasitol. Res. 106, 709–713. onsson, N.N., Bock, R.E., Jorgensen, W.K., 2008. Productivity and health effects of anaplasmosis and babesiosis on Bos indicus cattle and their crosses, and the effects of differing intensity of tick control in Australia. Vet. Parasitol. onsson, N.N., 2006. The productivity effects of cattle tick (Boophilus microplus) infestation on cattle, with particular reference to Bos indicus cattle and their crosses. Vet. Parasitol. 137 (1), 1–10. uliano, R.S., Machado, R.Z., Fioravanti, M.C.S., Andrade, G.M., de Sá, Jyme V., 2007. Soroepidemiologia da babesiosis em rebanho de bovinos da raç a Curraleiro. Cien. Rural 37, 1387–1392. e, G.M., Cheng, H.L., Ke, L.Y., Ji, W.T., Chulu, J.L.C., Liao, M.H., Chang, T.J., Liu, H.J., 2006. Development of a quantitative Light Cycler real-time RT-PCR for detection of avian reovirus. J. Virol. Methods 133, 6–13. ittell, R.C., Milliken, G.A., Stroup, W.W., Wolfinger, R.D., Schabenberger, O., 2006. SAS for Mixed Models, 2nd ed. SAS Institute Inc, Cary, NC. achado, R.Z., Montassier, H.J., Pinto, A.A., Lemos, E.G., Machado, M.R., Valadão, I.F., Barci, L.G., Malheiros, E.B., 1997. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Babesia bovis in cattle. Vet. Parasitol. 71, 17–26. achado, R.Z., Toledo, C.Z.P., Teixeira, M.C.A., André, M.R., Freschi, C.R., Sampaio, P.H., 2012. Molecular and serological detection of Theileria equi and Babesia caballi in donkeys (Equus asinus) in Brazil. Vet. Parasitol. 186, 461–465. cCOSKER, P.J., 1981. In: Ristic, M., Kreier, J.P. (Eds.), The Global Importance of Babesiosis, in Babesiosis. Academic Press, New York. ousa, A.A., Cao, S., Aboge, G.O., Terkawi, M.A., Kirdasy, A., El Salama, A., Attia, M., Aboulaila, M., Zhou, M., Kamyingkird, K., Moumouni, P.F.A., Masatani, T., Aziz, S.A.A., El Moussa, W.M., Chahan, B., Fukumoto, S., Nishikawa, Y., Ballal, S.S., El Xuan, X., 2013. Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP). Exp. Parasitol. 135, 414–420. ari, A., 1995. Strategies for the control of one-host ticks and relationship with tick-borne diseases in South America. Vet. Parasitol., 153–165. liveira, G.P., Alencar, M.M., Freitas, A.R., 1989. Resistance of cattle to the tick Boophilus microplus, II Natural infestation. Pesq. Agropec. Bras. 24, 1267–1271. liveira, M.C.S., Oliveira-Sequeira, T.C.G., Araujo Jr, J.P., Amarante, A.F.T., Oliveira, H.N., 2005. Babesia spp. infection in Boophilus microplus engorged female and eggs in São Paulo State, Brazil. Vet. Parasitol. 130, 61–67. liveira-Sequeira, T.C.G., Oliveira, M.C.S., Araujo, J.P., Amarante, A.F.T., 2005. PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle. Int. J. Parasitol. 35, 105–111. saki, S.C., Vidotto, O., Marana, E.R.M., Vidotto, M.C., Yoshihara, E., Pacheco, R.C., Igarashi, M., Minho, A.P., 2002. Occurrence of antibodies against Babesia bovis and studies on natural infection in Nelore cattle, in Umuarama municipality, Paraná State, Brazil. J. Vet. Parasitol. 11, 77–83. faffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45. e Diseases 8 (2017) 335–340 Piper, E.K., Jackson, L.A., Bielefeldt-Ohmann, H., Gondro, C., Lew-Tabor, A.E., Jonsson, N.N., 2010. Tick-susceptible Bos taurus cattle display an increased cellular response at the site of larval Rhipicephalus (Boophilus) microplus attachment, compared with tick-resistant Bos indicus cattle. Int. J. Parasitol. 40, 431–441. Ramos, C.A.N., Araújo, F.R., Souza, I.I.F., Bacanelli, G., Luiz, H.L., Russi, L.S., Oliveira, R.H.M., Soares, C.O., Rosinha, G.M.S., Alves, L.C., 2011. Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis. Vet. Parasitol. 176, 79–83. Riek, R.F., 1966. The cycle of Babesia argentina (Lignières, 1903) (Sporozoa: piroplasmidea) in the tick vector Boophilus microplus (Canestrini). Aust. J. Agric. Res. 17, 247–254. Rosales, R., Rangel-Rivas, A., Escalona, A., Jordan, L.S., Gonzatti, M.I., Aso, P.M., Perrone, T., Silva-Iturriza, A., Mijares, A., 2013. Detection of Theileria equi and Babesia caballi infections in Venezuelan horses using Competitive-Inhibition ELISA and PCR. Vet. Parasitol. 196, 37–43. Statistical Analyses System −SAS, 2002. SAS/INSIGHT User’s Guide, Version 9 1.3, Version to Windows. SAS Institute, CD-ROM, Cary. Salem, G.H., Liu, X., Johnsrude, J.D., Dame, J.B., Roman Reddy, G., 1999. Development and evaluation of an extra chromosomal DNA-based PCR test for diagnosing bovine babesiosis. Mol. Cell. Probes 13, 107–113. Shebish, E., Vemulapalli, R., Oseto, C., 2012. Prevalence and molecular detection of Anaplasma marginale, Babesia bovis and Babesia bigemina in cattle from Puntarenas Province, Costa Rica. Vet. Parasitol. 188, 164–167. Silva, A.M., Alencar, M.M., Regitano, L.C.A., Oliveira, Barioni Jr., M.C.S., W, 2007. Artificial infestation of Boophilus microplus in beef cattle heifers of four genetic groups. Genet. Mol. Biol. 30, 1150–1155. Silva, M.L.B., Mezzari, M.P., Ibelli, A.M.G., Gregory, K.B., 2014. Sulfide removal from livestock biogas by Azospirillum-like anaerobic phototrophic bacteria consortium. Int. Biodeter. Biodegr. 86, 248–251. Terkawi, M.A., Huyen, N.X., Shinuo, C., Inpankaew, T., Maklon, K., Aboulaila, M., Ueno, A., Goo, Y.K., Yokoyama, N., Jittapalapong, S., Xuan, X., Igarashi, I., 2011. Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the northeast region of Thailand. Vet. Parasitol. 178, 201–207. Terkawi, M.A., Alhasan, H., Huyen, N.X., Sabagh, A., Awier, K., Cao, S., Goo, Y.K., Aboge, G., Yokoyama, N., Nishikawa, Y., Kalb-Allouz, A.K., Tabbaa, D., Igarashi, I., Xuan, X., 2012. Molecular and serological prevalence of Babesia bovis and Babesia bigemina in cattle from central region of Syria. Vet. Parasitol. 187, 307–311. Trindade, H.I., Silva, G.R.A., Teixeira, M.C.A., Sousa, M.G., Machado, R.Z., Freitas, F.L.C., Almeida, K.S., 2010. Detection of antibodies against Babesia bovis and Babesia bigemina in calves from the region of Araguaína State of Tocantins. Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3 (RESEARCH0034). http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0095 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0100 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0105 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0110 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0115 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0120 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0125 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0130 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0135 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0140 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0145 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0150 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0155 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0160 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0165 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0170 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0175 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0180 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0185 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0190 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0195 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0200 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0205 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0210 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0215 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0220 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0220 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0220 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0220 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0220 http://refhub.elsevier.com/S1877-959X(16)30227-8/sbref0220 http://refhub.elsevier.c