The Effectiveness of Anti-R. equi Hyperimmune Plasma against R. equi Challenge in Thoroughbred Arabian Foals of Mares Vaccinated with R. equi Vaccine

This study aimed to determine the effectiveness of a pregnant mare immunization of a Rhodococcus equi (R. equi) vaccine candidate containing a water-based nanoparticle mineral oil adjuvanted (Montanide IMS 3012) inactive bacterin and virulence-associated protein A (VapA), as well as the administration of anti-R. equi hyperimmune (HI) plasma against R. equi challenge in the mares' foals. The efficacy of passive immunizations (colostral passive immunity by mare vaccination and artificial passive immunity by HI plasma administration) was evaluated based on clinical signs, complete blood count, blood gas analysis, serological response (ELISA), interleukin-4 (IL-4) and interferon gamma (IFN-γ), total cell count of the bronchoalveolar lavage fluids (BALF) samples, reisolation rate of R. equi from BALF samples (CFU/mL), lung samples (CFU/gr), and lesion scores of the organs and tissue according to pathological findings after necropsy in the foals. The vaccination of pregnant mares and HI plasma administration in the foals reduced the severity of R. equi pneumonia and lesion scores of the organs and tissue by 3.54-fold compared to the control foals. This study thus indicates that immunization of pregnant mares with R. equi vaccine candidate and administration of HI plasma in mares' foals effectively protect foals against R. equi challenge.


Introduction
Rhodococcus equi (R. equi) is a Gram-positive, nonmotile, obligate aerobe, intracellular microorganism. Virulent R. equi causes pyogranulomatous bronchopneumonia in young foals aged from 1 to 6 months [1]. Young foals may also develop extrapulmonary disease, such as septic arthritis, osteomyelitis, ulcerative enterocolitis, mesenteric lymphadenopathy, neonatal diarrhea, and sudden death. R. equi is additionally considered as an opportunistic pathogen of immunosuppressed people, especially AIDS patients [2]. R. equi was initially isolated from pulmonary lesions of foals by Magnusson in 1923 [3]. R. equi bacterium is present in soil and horse feces. Foals are thought to become infected when, within the first few days of life, they ingest or breathe in soil, dust, or fecal particles harboring the bacteria [2,4]. Inhalation of aerosolized virulent R. equi from the environment and intracellular replication within alveolar macrophages is essential components of pathogenesis of R. equi pneumonia in foals [5]. Virulence in foals is associated with the presence of 80-90 kb plasmids that encode the 15-17 kDa lipoprotein "virulence-associated protein A" (VapA) [6]. The disease is endemic on some farms and sporadic on other farms, but nonexistent on most farms. Recent epidemiologic studies indicate that the difference in the disease's prevalence on farms directly relates to differences in foal population density, 2 The Scientific World Journal farm management, and environmental factors, such as temperature, dust, soil pH, and the number of virulent R. equi organisms in the soil [7].
Several antimicrobial agents are active against R. equi in vitro. However, since R. equi is a facultative intracellular pathogen that survives, replicates in macrophages, and causes pyogranulomatous lesions, many of these agents are ineffective in vivo [8]. R. equi pneumonia significantly impacts the equine industry by posing financial losses since foals that recover from the disease are less likely to race as adults. The cost of therapy and occasional death of foals also pose financial risks. Furthermore, treatment with long-term antibiotics does not guarantee full recovery [9].
Due to the epitheliochorial placentation of equines, foals must obtain all maternally derived antibodies from the ingestion of colostrum [10]. Ingestion of colostrum from hyperimmunized mares was found to be associated with protection against R. equi in foals normally hypogammaglobulinemic at birth [11,12]. Foals become infected approximately when maternal antibody concentrations wane [13]. Immunization of mares has been suggested by several researchers to prevent R. equi infection in foals [11,12,[14][15][16][17]. Traditional hyperimmune plasma therapy is currently the only proven method for prevention of R. equi in foals, especially those exhibiting passive antibody transfer failure [11,12,15].
Due to the presence of the maternal antibody and the immaturity of foals' immune system, vaccination of neonate presents different results [18][19][20], yet none of the control strategies to protect horses from R. equi infection have proven successful. Several vaccines have been investigated for the prevention of R. equi, though none have been developed for widespread vaccination [21].
This study thus aimed to determine the effectiveness of a pregnant mare immunization with a R. equi vaccine candidate and the administration of anti-R. equi hyperimmune plasma against R. equi challenge in these mares' foals.

Immunization of Mares. Four pregnant thoroughbred
Arabian mares were vaccinated three times at months 8, 9, and 10 during pregnancy. Vaccination was performed intramuscularly with the R. equi vaccine candidate containing a water-based nanoparticle mineral oil adjuvanted (IMS 3012, SEPPIC, Paris, France) inactive antigen and VapA. Four mares not vaccinated formed the control group. Serum samples were collected from each mare at birth to test the presence of an anti-R. equi-specific antibody by ELISA. ELISA was carried out according to Takai et al. [22].
Nine healthy Arabian mares were selected for the production of anti-R. equi hyperimmune plasma. After proving to be free of equine infectious anemia (EIA), dourine, glanders, African horse sickness, and S. abortus equi, the mares were hyperimmunized 5 days apart with four doses of inactive R. equi. After 10 to 15 days, mares were vaccinated 21 days apart with 3 doses of R. equi vaccine candidate [23,24]. After 15 to 20 days following the most previous immunization, serum samples were obtained from the mares and tested by ELISA for anti-R. equi antibody titers [22]. Horses having anti-R. equi antibody titers ≥1/12800 by ELISA were selected as plasma donors. Donor horses were bled, and the hyperimmune plasma was separated from the blood cells by plasmapheresis (PCS2, Haemonetics, Braintree, MA, USA). The plasma samples were packed in 200 mL sterile bottles in a BSL 2 cabinet and stored at 4 ∘ C. Sterility tests for aerobic, anaerobic bacteria, mycoplasma, and mycotic agents as well as mouse safety tests were performed, after which the hyperimmune plasma samples were used. Donor horses were subsequently vaccinated at intervals of 50 to 60 days and tested 10 to 15 days later, and if the titers were again satisfactory, they were again bled.

Challenge.
To determine the effectiveness of a pregnant mare immunization using a R. equi vaccine candidate and HI plasma activity against R. equi infection in foals, 4 weeks old mares which born four vaccinated and four unvaccinated mares challenged the 2 mL of 1.0 × 10 5 CFU pathogen R. equi by intercostal injection in the lobe of the left lung [25,26]. Before receiving the challenge, foals were kept together with their dams approximately 3 weeks after birth to ingest a sufficient amount of colostrum. Two days before the challenge, 150 mL of HI plasma was administered to each foal of the vaccinated mares by intravenous infusion and 50 mL by subcutaneous infusion at days 1, 5, 9, 13, and 17 after the challenge. HI plasma was not given to the foals of unvaccinated mares.

Laboratory
Tests. Blood samples were obtained from the challenged foals to determine the presence of anti-R. equi specific antibodies using a ELISA on the challenge day (day 0) and on days 10 and 20, to measure the interleukin-4 (IL-4) and interferon gamma (IFN-) concentrations on days 0, 1, 10, 20, and 30 and for complete blood count and blood gas analysis on days 0, 1, 5, 10, 14, 20, and 30 [25]. BALF samples were taken by passing a nasotracheal tube and in fusing 20 mL of sterile saline solution to bacterial culture and measure total cell count (TCC) on days 0, 1, 5, 10, 14, 20, and 30 [25,26]. BALF samples were collected according to the method described by Mansmann and Knight [27] and Higuchi et al. [28]. The IL-4 and IFN-levels were measured using the Horse IL-4 ELISA kit (product code CSB-E14223Hs [96 T], Cusabio, Wuhan, Hubei, China) and Equine IFN-ELISA kit (ALP) (product code 3117-1A-6, MabTech, Thomastown, VIC, Australia) according to the manufacturer's instructions. The optimal dilutions of the serum samples, conjugate, substrate, and concentration of the coating antigen were standardized in our laboratory.
The Scientific World Journal 3 Samples from these organs were fixed in 10% formalin and embedded in paraffin wax blocks. Block sections of 5 m thickness were stained with Luna [29]. All sections were evaluated under a light microscope.

Statistical Analysis.
All statistical analyses were performed using the Student's -test.

Results
Two foals (numbers 5 and 7) of unvaccinated mares died before the challenge, and R. equi, Streptococcus sp., and Corynebacterium sp. were isolated from the lungs of these dead foals. In the place of the dead foals, two unvaccinated mares' foals were included in the study. One of these foals died on day 7 and another on day 13 after the challenge.
Anti-R. equi antibody titer measured in vaccinated mares was higher than in the unvaccinated mares at birth by ELISA. In the foals of vaccinated mares, anti-R. equi antibody titer was determined to be 1/1600 of the maximum after the administration of hyperimmune plasma, and 1/200 titer was also determined in control foals ( Table 2).
The IL-4 concentration was measured to have a mean of <16 pg/mL at days 0, 10, and 20 and a mean of 80 pg/mL at day 30 in foals of the vaccinated group, as well as a mean of 32 pg/mL in control foals. IFN-concentration increased on day 10 compared to the challenge day in both vaccine and control groups. By day 20, the control group remained stable, while the vaccine group exhibited doubling. By day 30, it increased in both groups but was measured to be 4.1fold more in the vaccine group than in the control group ( Table 3).
The reisolation rate of R. equi from BALF samples was determined to have increased by day 10, decreased by day 20, and increased again by day 30 in the vaccinated and HI plasma-administered group. An increase was observed in the control group compared to day 1 (Table 4, Figure 1).
A decrease in the concentration of pO2 and SO2 in the vaccine group was observed on day 5. The concentration of pCO2 and tCO2 increased, while the concentration of SO2 decreased on day 10. A decrease in the concentration of pO2 and SO2 in the control group was observed on day 30 ( < 0.05) ( Table 6).
An insignificant ( > 0.05) increase was observed in WBC concentration in both the control and vaccine group during the study period. Significant ( < 0.05) differences were determined in percentages of LYM, MON, 4 The Scientific World Journal  and GRAN concentration. The RBC concentration decreased significantly ( < 0.05) on days 14 and 30 ( Table 6). TCC of BALF and lung scores had increased in both groups by day 14 (Table 5). These increases were statistically significant ( < 0.05) in the control group, yet insignificant ( > 0.05) in the vaccine group.
The clinical and lung auscultation findings indicate that clinical signs commenced on day 5 and increased until day 10 in both groups. In the vaccine group, however, it began to decrease after day 10 and because of the sepsis two foals died on days 7 and 15 in the control group.
The mean of total lesion scores of the organs and tissue was determined to be 78 in control group and 22 in the vaccine group. According to pathological findings, the severity of R. equi pneumonia and lesion scores of the organs and tissue was observed 3.54-fold less in the vaccinated and HI plasmaadministered foals compared to the control foals (Table 7, Figure 2).

Discussion
Cell-mediated immunity is thought to play an important role in eliminating the facultative intracellular pathogen from foals, yet humoral immunity seems to be critically involved in the early protection in young foals. Foals are the most susceptible to the effects of virulent organisms when maternal antibody levels wane [30,31]. The passive transfer of immunity plays a critical role in the foals' resistance to a variety of infectious agents. Due to the epitheliochorial placentation of equines, foals must obtain all of their maternally derived antibodies by ingesting colostrum [10]. The lowest circulating antibody titers in foals appear from 1 to 6 months via the combined effects of waning maternally derived antibodies and low endogenous antibody production [32]. As a result, foals are susceptible to R. equi pneumonia during this period. Due to age-dependent susceptibility to R. equi, foals need to develop anti-R. equi immunity shortly after birth [25].
Since R. equi lives within macrophages, it resists many common antibiotics, and antibiotics-based therapy is prolonged, expensive, possibly associated with adverse effects, and inconsistently successful [33].
Studies investigating the active immunization of mares as a means of enhancing the passive transfer of virulent R. equi antibodies in colostrum and protecting foals from R. equi pneumonia have yielded mixed results. Solo vaccination of mares has not proven protective against R. equi pneumonia in foals, despite a significant increase in a colostral-specific antibody [14,34]. Martens et al. [34], Madigan et al. [35], and Varga et al. [36] did not observe protection in foals against The Scientific World Journal 5  1  0  65000  135  1400  140  50  2  0  1000  55  1220  0  21000  3  0  1300  120   R. equi pneumonia after mare vaccination. Moreover, Hines et al. [31] reported that immunoglobulin in mares may not be efficiently transferred via colostrum. However, according to other researchers, passive antibody transfer from ingested colostrum was found to be associated with protection against R. equi in foals normally hypogammaglobulinemic at birth [11,12,14,15]. Immunization of pregnant mares with virulent R. equi and VapA protein antigen associated with a water-based nanoparticle adjuvant as a candidate vaccine developed a higher serum IgG and opsonic activity, which resulted in passive antibody-mediated protection of foals [14]. Muscatello [17] reported that the protective effect was associated with an increase in the opsonic capacity of polymorphonuclear leucocytes against virulent R. equi in foals from immunized mares.
Martens et al. [37] were the first to show the immunoprophylactic capacity of specific hyperimmune plasma in an experimental model of R. equi pneumonia in foals. Other researchers have reported a reduction in foal morbidity and mortality due to R. equi as a result of HI plasma administration [14,35,[37][38][39]. However, several other studies report no protective effects of HI plasma [11,12,40].
According to our results, the immunization of pregnant mares with a R. equi vaccine candidate and the administration of the anti-R. equi HI plasma in vaccinated mares' foals proved to have protective effects during experimental R. equi infection. The clinical signs of pneumonia were significantly delayed, and the reisolation rate of R. equi from BALF samples decreased. The natural mortality rate due to R. equi infection was less than 50% in these foals compared to the controls. The severity of R. equi pneumonia and lesion scores of the organs and tissue determined 3.54-fold less than control foals.
The protective components of HI plasma are not completely known. Antibodies to Vap proteins, specifically VapA, appear to be crucially important [17]. It has been reported that there is no correlation between the total serum IgG levels and concentration of the specific anti-R. equi antibody [34,35]. The phagocytic ability of foal neutrophils has been found comparable to adults, but the lymphocyte stimulation response alone did not influence the course of R. equi infection, while the opsonic ability of foal serum was found to be a limited factor for phagocytosis from the ages of 1 to 6 weeks [41,42]. Phagocytic activity of foal neutrophils was found to improve when mixed with adult serum or plasma [43], which may be related to unknown, nonspecific immune factors provided by HI plasma and normal adult equine plasma that are absent from colostrum, such as fibronectin, complement, and cytokines [12,40,44]. The effectiveness of HI plasma is likely to be affected by the dosage, timing of administration, innate immune system competence, management conditions, and number of virulent bacterin in the environment [45].
Cytokines IFN-and IL-4 are major macrophage and neutrophil-activating factors, as well as upregulated microbial killing pathways [46]. It is reported that newborn foals had a deficiency of IFN-/IL-4 and levels not reaching adult status until approximately 4 months of age [47]. Reduced IFN-and IL-4 expression have a limited killing capacity of phagocytes in young foals [47,48]. In our study, 6 The Scientific World Journal The Scientific World Journal 7   the IFN-and IL-4 concentration measured 4.1-fold and 2.5-fold more in the foals of vaccinated mares and HI plasma-administered, respectively, compared to the control foals. According to changes in blood gases of foals, it was observed that lung ventilation in the challenge group had been affected by day 5, intensified after day 10, and continued to increase during the study period.
The important decrease ( < 0.05) of the RBC concentration on days 14 and 30 and significant differences ( < 0.05) of the MCV and RDW values in the vaccine and control group were evaluated as microcytic/normocyticnormochromic-regenerative anemia which was interpreted as a response to emerging infectious.
Given the decrease in the number in the controls, BALF-TCC parameters were detected as statistically insignificant ( > 0.05).
According to clinical scores and laboratory findings, the effect of infection in the vaccine group began on day 5, increased from day 9 to 14, and was constant from days 20 to 30. The effect of infection was similar in the control group, though the clinical findings increased from days 7 to 15, and two foals died of sepsis during this latter period. These findings show that resistance to infection was low in the control group.
As yet there is no licensed vaccine for the prevention of R. equi. However, on January 27, 2011, Intervet/Schering-Plough Animal Health announced that a vaccine against Rhodococcus equi infection in foals had entered the final stages of development [49].

Conclusions
Our results indicate that the immunization of pregnant mares with a water-based nanoparticle mineral oil adjuvanted (IMS 3012) inactive bacterin and VapA and the administration of HI plasma in foals of these mares effectively protect foals against R. equi challenge. Foals are born into the R. equi contaminated environment due to mares carrying the R. equi in their intestines. R. equi infection can be controlled by both the mares' vaccination and anti-R. equi-HI plasma administration in foals of such dams.