Babesia equi, also described as Theileria equi , is a tick-transmitted protozoan that infects horses. The parasite replicates within erythrocytes and causes anemia, which characterizes the acute stage of infection. Following resolution of acute parasitemia, horses remain life-long carriers but develop a long-term protective immunity against clinical disease (, ). Acquired immune responses are necessary to control parasitemia, although lysis of erythrocytes does not require adaptive immunity. Thus, following lysis of infected erythrocytes, parasites are accessible to antibody and associated mechanisms of antibody-mediated killing. Protective mechanisms associated with antibodies include neutralization of extracellular parasites by blocking of cell entry or opsonization of infected erythrocytes and complement lysis of either organisms or infected cells.

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Although high titers of antibody alone are not able to protect against Babesia infection, they are correlated with reduction of replication and clearance of the parasite (, ).Seven genes for immunoglobulin G (IgG) heavy chain constant in horses have recently been described ; however, only four IgG antibody isotypes have been characterized in equine serum, IgGa, IgGb, IgGc, and IgG(T). IgGa and IgGb antibodies are cytophilic and capable of both complement activation and opsonization, while IgG(T) and IgGc are noncytophilic and may block the protective effects of cytophilic antibodies by competitive binding to antigen (, ). In this study, the kinetics of the most prevalent IgG isotypes in horse serum, the IgGa, IgGb, and IgG(T) antibodies , were characterized during acute and chronic B. Equi infection; the potential use of these isotypes as immunological markers for infection control and vaccine development is discussed.Mixed-breed ponies, between 2.5 and 4 years of age and negative for B. Equi as tested by competitive enzyme-linked immunosorbent assay (ELISA) and nested PCR , were experimentally infected with B. Equi by two methods: parenteral inoculations and tick transmission. The use of horses in this study complied with all relevant national guidelines and Washington State University policies.

A stabilate of a B. Equi isolate obtained in Florida was used to infect horses and also to prepare the antigen for ELISA. For the parenteral inoculation group, four ponies were inoculated three times at 21-day intervals. The first injection was intravenous and contained 10 9 infected red blood cells; the second and third injections were intravenous and subcutaneous, respectively, and each contained 10 7 infected red blood cells. Two horses were infected with B. Equi by experimental Boophilus microplus tick transmission as described elsewhere.

During the experiments, the horses were regularly monitored for clinical signs and percentages of parasitized erythrocytes (PPE). Equi infection, all horses, infected by either method, had transitory parasitemia (Fig. ) and developed acute signs of infection, including fever and a decrease in packed cell volume (data not shown). ELISA titers of merozoite-specific IgGa, IgGb, and IgG(T) antibodies and PPE following experimental B. Equi infection in horses. Results are mean titers of antibody and mean PPE at each time point in a group of horses infected by parenteral injection ( n = 4) (A) or tick transmission ( n = 2) (B). Arrows represent B.

Equi injections., statistically significant; NS, nonsignificant difference in mean titer profiles of IgG isotypes or mean parasitemia between groups, as analyzed by MANOVA or ANOVA, respectively ( P. WPIMean ELISA titer (SD) of antibody after infection by: aParenteral injectionTick transmissionIgGaIgGbIgG(T)IgGaIgGbIgG(T)00 (0)0 (0)0 (0)0 (0)0 (0)0 (0)1336 (497).25 (33)0 (0)0 (0)0 (0)0 (0)3-4 b885 (236).507 (834).126 (142) b196 (21).66 (37).0 (0) b96,422 (1,993).22,226 (19,505).6,944 (5,388).506 (497).1,232 (122).762 (1,017)145,427 (1,707).23,805 (20,182).8,421 (2,338).540 (461).2,149 (653).4,230 (5,006).186,359 (3,349).17,284 (11,868).8,562 (3,489).914 (1,074).3,391 (1,681).3,617 (4,046).227,316 (2,111).24,332 (23,556).10,182 (3,460).1,077 (791).7,352 (897).4,182 (3,834). BData obtained at 4 weeks postinfection.Regardless of the method of infection, all three isotypes were present during chronic infection.

Fifty serum samples from carrier field horses, previously known to be positive for B. Equi by a complement fixation test and competitive ELISA (, ), were also tested to characterize the IgG isotype response during chronic B. Equi infection. The average titers (plus or minus standard deviations) of IgGa, IgGb, and IgG(T) antibodies in these naturally infected horses and in the horses experimentally infected in this study, tested at 22 WPI (parenteral inoculation) and 39 WPI (tick transmission), were 5,947 (±1,300), 12,028 (±4,959), and 6,954 (±3,474), respectively. The IgG isotype profiles were similar in all groups, as tested by MANOVA ( P 0.05), indicating that the isotypes produced during the chronic stage of B.

Equi infection are not affected by the mode of transmission of the parasite.The importance of cytophilic antibodies in controlling pathogen replication in hemoparasitic, viral, and bacterial infections has been reported previously (, -, ). Since the induction of cytophilic antibodies is dependent on the production of gamma interferon by T cells (, ), the IgG isotype profile may reflect the overall type of immune response (Th1 or Th2), as recently demonstrated for Rhodococcus equi infection. Herein, we showed that the B.

Equi-specific cytophilic antibodies, IgGa and IgGb, are correlated with parasitemia control, suggesting that the IgG isotype profile might also be useful as a potential immunological marker for B. Equi infection control and vaccine development. To test the hypothesis that the IgG antibody isotype profile induced by infection can be produced by a subunit antigen, recombinant equi merozoite antigen 1 (rEMA-1) in saponin was tested as an immunization model. EMA-1 is an immunodominant surface protein of B. Equi which induces the first measurable antibody response in horses during control of acute parasitemia (, ). REMA-1 was obtained and purified as previously reported (, ), and four horses (mixed-breed ponies, 3 to 6 years old) were immunized eight times with 80 μg of the protein (54 μg in the last immunization) in phosphate-buffered saline containing 6 mg of Quil A saponin during a period of 40 months. Equi IgGa and IgGb antibodies were induced by rEMA-1/saponin immunization, with no measurable IgG(T) (Fig.

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After the first three immunizations, the titers of IgGa and IgGb antibodies were significantly higher than the preimmunization titers ( P. ELISA titers of Babesia equi-specific IgGa, IgGb, and IgG(T) antibodies in sera from horses immunized with rEMA-1 in the adjuvant saponin ( n = 4). Results are mean titers at each time point, and error bars represent standard deviations. Arrows indicate rEMA-1/saponin immunizations.Since the horses did not produce IgG(T) antibodies following immunization, their ability to produce IgG(T) was tested by infecting them with 10 9 live B. Equi erythrocyte-stage parasites by intravenous injection at 10 days after the last immunization. All horses developed IgG(T) antibodies following B. Equi infection, with a mean titer of 3,058 at 2 WPI.

Although rEMA-1 immunization induced high titers of IgGa and IgGb (Fig. ), the horses developed clinical disease following B. Equi inoculation (data not shown). Several reasons related to the antigen used may explain this outcome. For instance, it is not known whether rEMA-1 is a replica of native EMA-1 for induction of immunity or whether native or recombinant EMA-1 is capable of inducing the necessary repertoire of protective immunity. These results reinforce the potential use of IgG isotypes as immunological markers for infection control and vaccine development.In conclusion, this study demonstrated the kinetics of IgG isotypes in B. Equi infection and the correlation of IgGa and IgGb antibodies with control of parasitemia.

It was also shown that the same IgG isotype profile developed during acute infection can be induced by a subunit immunization using saponin as an adjuvant.