Parasitology International 64 (2015) 319–323

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Co-immunization of cattle with a vaccine against babesiosis and Lactobacillus casei increases specific IgG1 levels to Babesia bovis and B. bigemina Carlos Ramón Bautista-Garfias a,⁎, Astrid Rodríguez Lozano a, Carmen Rojas Martínez a, Jesús Antonio Álvarez Martínez a, Julio Vicente Figueroa Millán a, Gustavo Román Reyes García a, Roberto Castañeda-Arriola b, Blanca Rosa Aguilar-Figueroa c a Unidad de Babesia, Centro Nacional de Investigación Disciplinaria en Parasitología Veterinaria (CENID-PAVET), Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Mexico b C.E.P. La Posta, km 22.4 Carretera Veracruz-Córdoba, Paso del Toro, Medellín de Bravo C.P. 91700, Veracruz, Mexico c Departamento de Parasitología, ENCB, IPN, Prolongación de Carpio y Plan de Ayala, Col. Sto. Tomás, Mexico, D.F., Mexico

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Article history: Received 5 December 2014 Received in revised form 4 March 2015 Accepted 22 April 2015 Available online 30 April 2015 Keywords: Bovine babesiosis vaccine Lactobacillus casei IgG1 IgG2

a b s t r a c t The effect of Lactobacillus casei administered along with a live attenuated vaccine vs. bovine babesiosis (VAC) on induction of IgG1 and IgG2 antibodies to Babesia bovis and Babesia bigemina was assessed by the indirect fluorescent antibody test (IFAT) in bovines of an endemic babesiosis area before (day 0) and after vaccination (days 15 and 30). We previously reported that L. casei increases the efficiency of VAC under controlled conditions and under extreme conditions in the field; however, the levels of IgG1 and IgG2 antibodies to B. bovis and B. bigemina are not known in vaccinated animals. Twenty-one dairy cows were allocated into three groups (seven animals per group): unvaccinated, vaccinated with VAC and vaccinated simultaneously with VAC and L. casei (VAC–LC). All animals were kept in a babesiosis endemic area at Tlalixcoyan, Veracruz. At days 15 and 30 after vaccination, the average levels of IgG1 to B. bovis and to B. bigemina were significantly higher in VAC– LC group than levels observed in VAC and control groups (P b 0.01). Levels of IgG2 were similar in VAC and VAC–LC groups but higher than in the control group (P b 0.01). When tested in in vitro cultures of B. bovis, sera from VAC–LC group significantly inhibited parasite growth as compared with the sera of the other two groups. It is suggested that the efficiency improvement of VAC, in part, is due to the L. casei boost of IgG1 over IgG2 antibodies to B. bovis and B. bigemina when the bacteria is co-inoculated with this vaccine. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction One of the most important parasitic diseases of bovines worldwide is babesiosis, a tick-transmitted disease provoked by protozoa of the Babesia genus, characterized by hemolytic anemia and fever with occasional hemoglobinuria and death [1–3]. The huge cost due to babesiosis has been estimated considering mortality, abortion, loss of cattle production (milk/meat), and control measures. In Mexico, 75% of the 23, 316,000 cattle [4] are at risk of acquiring babesiosis [5]. It is important to point out that, besides adults, cases of clinical babesiosis have been documented in calves younger than 9 months of age in endemic areas of this disease [6]. To curb the huge economic losses caused by bovine

⁎ Corresponding author at: CENID-PAVET, INIFAP. Carr. Fed. Cuernavaca-Cuautla No. 8534 Col. Progreso, Apdo. postal 206, CIVAC, C.P. 62550, Jiutepec, Morelos, Mexico. Tel.: +52 777 3192860x20; fax: +52 777 3192860x129. E-mail addresses: [email protected], [email protected] (C.R. Bautista-Garfias).

http://dx.doi.org/10.1016/j.parint.2015.04.005 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved.

babesiosis, live vaccines made of attenuated parasites were developed [7]. However, these vaccines have some drawbacks such as causing severe clinical signs after vaccination [8], even the death of some of the vaccinated animals [9]. At the National Institute for Forestry, Agricultural and Livestock Research (INIFAP) from the Mexican government, an attenuated live vaccine against Babesia bovis and Babesia bigemina (VAC) was developed. This vaccine protects 80% of the vaccinated cattle against virulent Babesia strains [10–12] and 70% of animals under extreme field conditions [13]. In this respect, both humoral and cellular immunity are important in protection against Babesia infection [14–16]; however, to date there is not a complete understanding of the protective immune response against Babesia in cattle after a natural infection or after vaccination. On the other hand, the use of the immunostimulant lactic-acid bacteria Lactobacillus casei as an alternative control measure of distinct parasitic diseases has been proposed [17]. In this context, it was demonstrated that L. casei, by itself, induces protective responses against Babesia microti in mice when inoculated before or the same day of the challenge infection [18] and

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against B. bovis and B. bigemina in cattle when the lactic-acid bacteria is administered with VAC under laboratory controlled conditions [19] and in extreme natural field conditions [20]. It is important to note that bovines immunized with VAC–LC do not show the clinical signs observed in cattle inoculated with VAC after vaccination [19,20]; however, the underlying mechanism responsible for this protection is not known. The only evidence we have is the recorded level of total IgG anti-Babesia [21], which is higher in animals treated with VAC and L. casei than in bovines immunized with VAC [18,19]. To date, there are no published studies on the levels of IgG1 and IgG2 against Babesia in bovines immunized with VAC or VAC and L. casei. Accordingly, the present investigation was carried out in order to document the levels of IgG1 and IgG2 anti-B. bovis and anti-B. bigemina antibodies in dairy cattle from an endemic area of babesiosis, vaccinated with VAC alone or together with L. casei (VAC–LC). 2. Materials and methods 2.1. Study animals Adult dairy cows (Swiss X Zebu), 3 to 4 years old, were distributed at random into three groups of seven bovines each: Control group, Vaccinated group (VAC), and VAC + L. casei (VAC–LC). Animals were handled in accordance with the NOM-062-ZOO-1999 “Technical specifications for production, care and use of laboratory animals” (http://www.senasica. gob.mx/?doc=743). 2.2. Bacteria The strain ATTCC7469 of L. casei [22] was grown in MRS broth at 37 °C for 18 h. The microorganisms were harvested and centrifuged at 5000 ×g for 10 min and then washed several times in PBS (pH 7.2). After a viable count, the L. casei was adjusted to a concentration of 109 bacteria/ml. 2.3. Bovine babesiosis bivalent attenuated vaccine The bivalent attenuated vaccine against bovine babesiosis was used. This consisted of a mixture of the attenuated B. bigemina BIS strain and of B. bovis BOR clones [13]. 2.4. Experimental design Animals were treated as follows: bovines in group CONTROL were not treated; those in group VAC were immunized intramuscularly (i.m.) with 1 × 108 infected erythrocytes of each Babesia species (B. bovis and B. bigemina) coming from in vitro cultures [13]; and bovines in group VAC–LC were inoculated i.m. in two different sites: one with VAC and the other with live L. casei (3 × 109 c.f.u.). They were kept in pens at a ranch in Tlalixcoyan (10 m above sea level, geographical coordinates 18° 48′ 0″ North, 96° 3′ 0″ West), Veracruz, an endemic area of bovine babesiosis with an estimated prevalence of bovine babesiosis higher than 90% [23] where they are occasionally exposed to Rhipicephalus (Boophilus) microplus ticks which are naturally infected with B. bovis and B. bigemina. These animals are subjected to a tick control program using anti-tick baths; however, sometimes cases of babesiosis do occur by the sporadic introduction to the herd of infected ticks coming from surrounding ranches. Rectal temperature was registered (°C) and blood samples were collected from the bovines for obtaining sera before (day 0) and after vaccination (days 15 and 30). After immunization the animals were isolated from tick exposure. 2.5. Indirect fluorescent antibody test (IFAT) Levels of IgG1 and IgG2 anti-B. bigemina and anti-B. bovis were assessed by the indirect fluorescent antibody test (IFAT) [24] using

commercial conjugates to bovine IgG1 (sheep anti-bovine IgG1: FITC) and IgG2 (sheep anti-bovine IgG2: FITC) (SEROTEC, Bio-Rad Laboratories Inc., Hercules, CA), following the manufacturer's instructions. Briefly, smears were prepared using a suspension of in vitro-cultured Babesia. These smears showed the following percentages of parasitized erythrocytes: 3% B. bovis (BOR strain), 3.5% B. bigemina (BIS strain). Microscopic detection of positive reactions was carried out using a LEICA DMLB microscope equipped for epifluorescence after mounting cover slides with 10% glycerol-PBS, pH 7.5. Positive and negative reference sera were included in each slide. According to the diagnostic cut-off value of IFA (1:80), all the animals were positive to B. bovis and B. bigemina antibodies at the beginning of the experiment. 2.6. In vitro culture of B. bovis B. bovis cultures were set up in accordance with Rojas et al. [25]. Briefly, the complete medium (CM) consisted of 60% medium 199 and 40% of bovine adult serum, supplemented with 25 mM TES (SigmaAldrich, Mexico) buffer with 100-g/ml streptomycin and 100 U/ml penicillin. The cultures were kept at 37 °C in a gas mixture comprising 5% O2, 5% CO2, 90% N2. The CM was replaced daily and subcultures were performed at 48 h. In subcultures, fresh normal bovine red blood cells in CM suspension were added to cultures to obtain a parasitemia of 1%. The treatments were set by triplicate as follows: 1) CM + normal bovine serum, 2) CM + pool of sera from 21 animals living in an enzootic area of babesiosis and obtained at the beginning of the experiment, 3) CM + pool of sera from seven animals vaccinated with VAC, and 4) CM + pool of sera from seven bovines vaccinated with VAC and L. casei (VAC–LC). Sera were obtained from animals in groups VAC and VAC–LC after 30 days of vaccination. 2.7. Percentage of parasitized erythrocytes (PPE) The PPE from each culture was estimated in the optical microscope by counting parasitized erythrocytes in 1000 red blood cells from a blood smear stained with Giemsa. 2.8. Statistical analysis The statistical significance of differences was determined from the means ± S.E.M. by analysis of variance (ANOVA) with software Paquete de diseños experimentales FAUNL. Version 2.5 [26]. When differences were found, the Tukey test was used and these were considered significant when P values were b0.01. 3. Results 3.1. Rectal temperature (RT) The mean RT was similar in Control and VAC–LC groups throughout the study; however, in VAC group an increase with respect to the Control and VAC–LC groups was observed at days 9 and 30 after vaccination (Fig. 1), but it was not statistically significant. 3.2. Levels of IgG1 and IgG2 to B. bovis A significant increase in IgG1 antibody levels was observed at days 15 and 30 after vaccination (av), which was higher in VAC–LC group (P b 0.01) with respect to the values of control and VAC groups (Fig. 2). The levels of IgG1 in the VAC–LC group were 113 and 84% higher at days 15 and 30 av, respectively, than the values of the VAC group while the levels of IgG2 were 11% higher at both dates with respect to the VAC group (data not shown). Similarly, IgG1 levels observed were higher than those of IgG2 in the three groups at days 15 and 30 av. However, levels of IgG2 were significantly different (P b 0.01) in VAC

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Fig. 1. Temperature (°C) registered before (day 0) and after vaccination (days 9, 15 and 30) to bovine babesiosis. White squares: control group; gray squares: VAC (vaccine vs. bovine babesiosis) group; black squares: VAC–LC (vaccine vs. bovine babesiosis and Lactobacillus casei) group. Each point represents the mean (±SEM) of seven dairy cows.

and VAC–LC groups with respect to the levels observed in the control group at days 15 and 30 after vaccination.

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Fig. 3. Levels of IgG1 and IgG2 to Babesia bigemina assessed by IFAT before (day 0) and after vaccination (days 15 and 30) to bovine babesiosis. White squares: control group; gray squares: VAC (vaccine vs. bovine babesiosis) group; black squares: VAC–LC (vaccine vs. bovine babesiosis and Lactobacillus casei) group. * (P b 0.01) with respect to control and VAC–LC groups. ** (P b 0.01) with respect to control group. Each point represents the mean (±SEM) of 21 dairy cows at day 0 and of seven animals at days 15 and 30.

3.3. Levels of IgG1 and IgG2 to B. bigemina A significant increase in IgG1 antibody levels was observed at days 15 and 30 after vaccination (av). It was higher in VAC–LC group (P b 0.01) with respect to the values of control and VAC groups (Fig. 3). The levels of IgG1 in the VAC–LC group were 185 and 84.6% higher at days 15 and 30 av, respectively, than the values of the VAC group while the levels of IgG2 were 82 and 11% higher, respectively, at days 15 and 30 av with respect to the VAC group (data not shown). Similarly, IgG1 levels were higher than those of IgG2 observed in the three groups at days 15 and 30 av. However, levels of IgG2 were significantly different (P b 0.01) in VAC–LC group with respect to the levels observed in VAC and control groups at day 15 av while this significant

Fig. 2. Levels of IgG1 and IgG2 to Babesia bovis assessed by IFAT before (day 0) and after vaccination (days 15 and 30) to bovine babesiosis. White squares: control group; gray squares: VAC (vaccine vs. bovine babesiosis) group; black squares: VAC–LC (vaccine vs. bovine babesiosis and Lactobacillus casei) group. * (P b 0.01) with respect to control and VAC–LC groups. ** (P b 0.01) with respect to control group. Each point represents the mean (±SEM) of 21 dairy cows at day 0 and of seven animals at days 15 and 30.

difference was noticed in VAC and VAC–LC groups with respect to the control group at day 30 after vaccination. 3.4. Effect of antibodies from vaccinated bovines on the growth of B. bovis in vitro After 48 h in vitro, the sera from VAC–LC group inhibited significantly (P b 0.01) the growth of B. bovis with respect to the other three groups (Fig. 4a) as evaluated by the percentage of parasitized erythrocytes

Fig. 4. a. Growth of Babesia bovis in vitro for 48 h in the presence of normal bovine serum (white circles), mixture of seven sera from VAC group (black circles); mixture of seven sera from VAC–LC group (white triangles); and mixture of 21 sera from bovines which live in an endemic area of babesiosis (white squares), obtained at the beginning of the study. b. Growth of B. bovis for 24 h after subculture carried out at 48 h: the percentage of parasitized erythrocytes was adjusted to 1 in each treatment. * (P b 0.01) with respect to the other three groups. Each point represents the mean (±SEM) of three repetitions. Sera from the VAC and VAC–LC groups were obtained at day 30 after vaccination.

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(PPE). Similarly, after subcultures were adjusted to 1 PPE, a significant difference between the control growth and the other three treatments which contained immune sera was observed (P b 0.01) after a further 24 h (Fig. 4b). The cultures that contained immune sera inhibited significantly parasite growth. The PPE averages (± SEM) were of 0.3333 (± 0.003) for VAC–LC group, 0.9400 (± 0.046) for VAC group and 1.1367 (+0.110) for the group of non-vaccinated animals from an enzootic area of bovine babesiosis. So, the highest inhibition was observed in VAC–LC group and the lowest in the last group. The difference among the three groups was significant (P b 0.01) (data not shown). Blood smears examined after 48 h of in vitro culture, in the presence of serum from a non-infected bovine, showed B. bovis with a normal structure (Fig. 5a); whereas, those blood smears from cultures which contained pooled sera from VAC–LC group showed free parasites outside the red blood cells and pyknosis in parasites inside the erythrocytes (Fig. 5b).

4. Discussion In spite of having anti-Babesia antibodies, the bovines living in endemic areas of babesiosis are susceptible to reinfections and calves born in these areas can show clinical signs of babesiosis also [6]. Animals that showed anti-Babesia antibodies were used; however, it has been pointed out that a persistent, detectable antibody titre is not a prerequisite for a protective immune response. However, it is a very effective indicator of recent infection, either naturally or by vaccination [2]. In the present study, animals responded to the live bivalent vaccine against Babesia and generated specific IgG1 and IgG2 antibodies to B. bovis and B. bigemina. IgG1 antibody levels were significantly higher in VAC–LC group at days 15 and 30 after vaccination as compared with VAC and control groups. It was supposed that L. casei participated in antibody production in the former group; however, we do not know what the responsible mechanism is. It might be that dendritic cells were activated by L. casei to process Babesia antigens and induce the production of IgG1 over IgG2 specific antibodies similarly to the study which demonstrated that bovine dendritic cells stimulate IgG1 production in vitro [27]. The difference on IgG1 and IgG2 anti-Babesia antibody levels over time in control animals (Figs. 2, 3) may be explained either on the basis of antibody fluctuation against Babesia over time [28], the stress due to handling provoked a recrudescence of a previous infection by both species of Babesia, or modulation of the immune response by the parasite [29,30]. Support of this fact is the finding that analyses of peripheral parasitemias in chronically infected calves with B. bovis showed cyclical patterns of extreme changes in parasite density over time [31]. It is also possible that the increase of IgG1 over IgG2 antibodies was due to the presence of more memory B cells producing IgG1 than those cells producing IgG2.

The levels of IgG1 anti-Babesia antibodies on days 15 and 30 after vaccination in the VAC–LC group were almost twice than in the VAC group (Figs. 2, 3). In other studies it has been shown that following B. bovis infection, IgG1, but not IgG2, complement-fixing antibodies are detected [32] and antibody-dependent cell-mediated cytotoxicity may be involved in the resolution of B. bovis infection in cattle [33]. On the other hand, it is important to note that IgG1 is the major IgG subclass present in ruminant colostrum and milk [34,35]. IgG1 represents 81% and 73% of total immunoglobulins in colostrum and milk, respectively [36]. The increased levels of IgG1 stimulated by L. casei along with the vaccine vs. babesiosis may be an advantage for the transference of passive immunity to B. bovis and B. bigemina from the cow to the calf. On the basis of the results and the observation that calves younger than 9 months of age and living in endemic areas of babesiosis are susceptible to the disease [6], the simultaneous vaccination of cows against babesiosis with the vaccine and L. casei would be desirable because it would promote high levels of IgG1 anti-Babesia antibodies which are the most important IgG sub-class of antibody transmitted passively through colostrum and milk to their offspring [37–41]. It has been pointed out that although the anti-Babesia antibodies by themselves do not eliminate Babesia, these may participate through several mechanisms to control parasitemia [42] such as antibodydependent cellular cytotoxicity (ADCC) mediated by IgG1 [33], opsonization mediated by IgG2, neutralization of adherence of the free merozoites to erythrocytes and of the cytoadherence of infected red blood cells to endothelial cells [16], and activation of complement mediated by IgG1 and IgG2 [30]. Results suggest that anti-Babesia antibodies blocked the reinfection of other erythrocytes by merozoites and that those parasites which got into normal erythrocytes were previously damaged by antibodies bound on their surfaces which in turn activated complement by the classic pathway as has been previously described [30]. It is worth noting that the IgG1/IgG2 proportion was almost 2/1 in VAC–LC group as compared with the other two groups, so it is probable that more IgG1 antibodies participated against Babesia than IgG2 antibodies. On the basis of what is known on the immunity to Babesia, such as the involvement of innate and acquired immunity and the involvement of cells and molecules such as macrophages, NK cells, T-regulatory cells, antibodies (IgG1, IgG2), cytokines (IL-12, IFN-gamma, TNF-alfa) and nitric oxide [15,16] and macrophage-antibody interactions [14], it is probable that the protective immune response elicited by L. casei and the vaccine against babesiosis in this study consisted of a combination of cellular and humoral elements of the immune response which should be further investigated. Our results, however, are in agreement with a recent study in which it has been indicated that for obtaining better vaccines the pathogen-associated molecular patterns recognized via Toll-like receptors (TLRs) may be used to induce an innate immune response which, in turn, promotes a better adaptive immunity [43]. In this context, it is known that L. casei stimulates the innate immune

Fig. 5. Growth of B. bovis in vitro after 48 h. a. Growth in the presence of serum from a non-infected bovine. b. Growth in the presence of a mixture of sera from VAC–LC group. Notice the free parasites (black arrows) and pyknosis in parasites inside the erythrocytes (white arrows).

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system through activation of TLRs and production of Th1 cytokines [44, 45]. TLRs not only induce innate immune responses but also modulate cellular and humoral adaptive immunity, giving rise to a better acquired immune response to a particular antigen such as those of vaccines [46, 47]. In this respect, it has been shown that a protective killed Leptospira borgpetersenii vaccine induces potent Th1 immunity comprising responses by CD4 and γδ T lymphocytes in cattle [48,49]. Under the conditions in which the present study was carried out, it is concluded that the simultaneous vaccination of dairy cows with a vaccine against bovine babesiosis and L. casei stimulates a significant increase of IgG1 over IgG2 antibodies to B. bovis and B. bigemina as compared with those bovines which only received the vaccine. We assume that these elicited antibodies were protective because they significantly inhibited B. bovis growth in vitro. Acknowledgments This study was carried out with funds from 1-1.6-9512932008-PP.11 INIFAP project. The authors thank José Carmen Lagunes Pacheco for allowing the use of his cattle at Tlalixcoyan, Veracruz, Mexico. References [1] Ristic M. In diseases of cattle in the tropics. In: Ristic M, McIntyre I, editors. The Hague. 1st ed., 1st ed.Martinus Nijhoff Publishers; 1981. p. 443–68. [2] Bock R, Jackson L, De Vos A, Jorgensen W. Babesiosis of cattle. Parasitology 2004;129: S247–69. [3] Hunfeld KP, Hildebrandt A, Gray JS. Babesiosis: recent insights into an ancient disease. Int J Parasitol 2008;38:1219–37. [4] INEGI. Estados Unidos Mexicanos. Censo Agropecuario 2007. VIII Censo Agrícola, Ganadero y Forestal. México: Aguascalientes, Ags; 2009. [5] Mosqueda JJ. Vacunas contra hemoparásitos en bovinos: Avances y perspectivas. In: Bautista Garfias CR, Figueroa Millán JV, editors. Perspectivas de control de parásitos de importancia veterinaria, 2. CENID-Parasitología Veterinaria, INIFAP, 2004; Publicación Técnica; 2004. p. 6–12. [6] Ojeda JJ, Orozco I, Flores R, Rojas C, Figueroa JV, Álvarez JA. Validation of an attenuated live vaccine against babesiosis in native cattle in an endemic area. Transbound Emerg Dis 2010;57:84–6. [7] Shkap V, de Vos AJ, Zweygarth E, Jongejan F. Attenuated vaccines for tropical theileriosis, babesiosis and heartwater: the continuing necessity. Trends Parasitol 2007;23:420–6. [8] Shkap V, Leivovitz B, Krigel Y, Hammerschlag J, Marcovics A, Fish L, et al. Vaccination of older Bos taurus bulls against bovine babesiosis. Vet Parasitol 2005;129:235–42. [9] Combrink MP, Carr G, Mans BJ, Marais F. Blocking Babesia bovis vaccine reactions of dairy cattle in milk. Onderstepoort J Vet Res 2012;79(1):E14. http://dx.doi.org/10. 4102/ojvr.v79il.491. [10] Cantó GJ, Figueroa JV, Álvarez JA, Vega CA. Capacidad inmunoprotectora de una clona irradiada de Babesia bovis derivada de cultivo in vitro. Tec Pecu Mex 1996; 34:127–35. [11] Figueroa JV, Cantó GJ, Álvarez JA, Lona GR, Ramos JA, Vega CA. Capacidad protectora en bovinos de una cepa de Babesia bigemina derivada de cultivo in vitro. Tec Pecu Mex 1998;36:95–107. [12] Álvarez JA, Ramos AJ, Rojas E, Mosqueda JJ, Vega MCA, Olvera A, et al. Field challenge of cattle vaccinated with a combined Babesia bovis and Babesia bigemina frozen immunogen. Ann NY Acad Sci 2004;1026:277–83. [13] Cantó GJ, Rojas EE, Álvarez JA, Ramos JA, Mosqueda JJ, Vega CA, et al. Protection against bovine babesiosis with a mixed in vitro culture derived B. bovis and B. bigemina vaccine under a field challenge. Immunization in an endemic area. Tec Pecu Mex 2003;41:307–15. [14] Bautista CR, Kreier JP. The action of macrophages and immune serum on growth of Babesia microti in short-term cultures. Tropenmed Parasitol 1980;31:313–24. [15] Aguilar-Delfin I, Wettstein PJ, Persing DH. Resistance to acute babesiosis is associated with interleukin-12- and gamma interferon-mediated responses and requires macrophages and natural killer cells. Infect Immun 2003;71:2002–8. [16] Brown WC, Norimine J, Knowles DP, Goff WL. Immune control of Babesia bovis infection. Vet Parasitol 2006;138:75–87. [17] Bautista Garfias CR. Inmunoestimulación con Lactobacillus casei como alternativa para el control de enfermedades parasitarias. In: Bautista Garfias CR, Figueroa Millán JV, editors. Perspectivas de control de parásitos de importancia veterinaria. CENID-Parasitología Veterinaria, INIFAP, 2. Publicación Técnica; 2004. p. 19–27. [18] Bautista-Garfias CR, Gómez MB, Aguilar BR, Ixta O, Martínez F, Mosqueda J. The treatment of mice with Lactobacillus casei induces protection against Babesia microti infection. Parasitol Res 2005;97:472–7. [19] Bautista CR, Alvarez JA, Mosqueda JJ, Falcon A, Ramos JA, Rojas C, et al. Enhancement of the Mexican bovine babesiosis vaccine efficacy by using Lactobacillus casei. NY Acad Sci 2008;1149:126–30.

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Co-immunization of cattle with a vaccine against babesiosis and Lactobacillus casei increases specific IgG1 levels to Babesia bovis and B. bigemina.

The effect of Lactobacillus casei administered along with a live attenuated vaccine vs. bovine babesiosis (VAC) on induction of IgG1 and IgG2 antibodi...
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