Since a vaccine is not available against Rhodococcus equi, R equi-specific hyperimmune plasma (HIP) is commonly used, although its efficacy remains controversial. The objective of this study was to evaluate the ability of a commercially available HIP to prevent clinical rhodococcal pneumonia in neonatal foals after experimental challenge.
- Respiratory disease
- Bacterial diseases
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Nine foals were randomly given intravenous HIP after birth, while nine foals remained as control. Foals were challenged the first week of life (103 cfu/foal R equi UKVDL206) and were monitored for eight weeks. One foal in the HIP group and four in the control group developed clinical pneumonia. HIP foals had significantly lower weekly (P<0.05) and cumulative ultrasonographic scores (P<0.001), lower white blood cell (WBC) counts (P=0.03), platelet counts (P=0.01) and fibrinogen concentration (P=0.01) than controls. Serum virulence-associated protein A (VapA)-specific IgG, IgGa and IgGb were significantly higher in HIP foals and IgGa and IgG(T) significantly increased (P<0.001) over time only in control foals. VapA-specific IgG (P=0.02) and IgGb (P=0.04) were significantly higher in bronchoalveolar lavage fluid (BALF) of HIP foals. While infection was not prevented by HIP administration after challenge, severity and development of clinical pneumonia were decreased by its use. Antibodies present in HIP transferred to BALF of foals.
Rhodococcus equi, a gram-positive facultative intracellular bacterium, causes life-threatening pyogranulomatous pneumonia in foals (Giguere and others 2011a) and has a major financial impact on the horse industry (Venner and others 2012). Because of the lack of an effective vaccine, farms with an endemic R equi problem attempt to prevent infections by other means. One method of choice is prophylactic intravenous administration of R equi-specific hyperimmune plasma (HIP) shortly after birth (Becu and others 1997). While the protective component of HIP remains uncertain (Perkins and others 2002), antibodies are thought to be important, especially those against virulence-associated protein A (VapA) (Hietala and Ardans 1987, Hooper-McGrevy and others 2001), a temperature-inducible surface-expressed lipoprotein, necessary for survival of R equi inside the macrophage (Takai and others 1991). Although the mechanism of antibody protection has not been fully elucidated, in vitro studies have shown that opsonisation by antibodies enhances uptake and killing of R equi by phagocytic cells (Martens and others 1987, 1989b). Thus, plasma from horses vaccinated with R equi augmented phagocytic activity of neutrophils (Grondahl and others 1997). Likewise, opsonisation of R equi with specific antibodies from commercially available HIP promoted phagocytic function of neutrophils and macrophages (Dawson and others 2011). Further, R equi-opsonising activity of serum increased after vaccination of mares with VapA and this opsonising activity was transferred from the vaccinated mares to their foals (Cauchard and others 2004). Thus, opsonising antibodies could be important in eliminating infection (Hietala and Ardans 1987).
In foals, endogenous production of IgG and IgA is not significant for the first four to five weeks of life (Sheoran and others 2000). This relative deficiency in antibodies expression may play a role in the development of R equi pneumonia (Lopez and others 2002). Since the half-life of Igs after intravenous administration ranges from 30 to 90 days, HIP may be an important source of antibodies until endogenous production occurs (Demmers and others 2001). The mechanism of protection associated with HIP is not fully understood, but the diffusion of antibodies into the airway after intravenous administration has been proposed (Dawson and others 2011).
While HIP appeared to be effective in some field studies (Madigan and others 1991, Becu and others 1997, Higuchi and others 1999), other showed no positive effect (Giguere and others 2002, Perkins and others 2002, Caston and others 2006). Field studies have the disadvantage of being affected by many variables that may influence outcome, such as year-to-year variation in disease prevalence and concurrent management changes. Further, in many of these studies, a definitive diagnosis of R equi by culture and cytology of tracheal fluid was not performed. Moreover, the efficacy of treatment is typically based on year-to-year comparisons (Becu and others 1997). Another confounder is the age of the foal at the time of administration of HIP (Madigan and others 1991, Higuchi and others 1999, Perkins and others 2002). Thus, early administration of HIP is critical (Chaffin and others 1991) as foals are most susceptible to infection within the first few weeks of life (Horowitz and others 2001, Chaffin and others 2008, Dawson and others 2010, Sanz and others 2013).
While the use of experimental challenge provides a more controlled environment for HIP evaluation, previous studies used large doses of R equi to infect foals (Martens and others 1989c, Perkins and others 2002, Caston and others 2006). This resulted in acute severe pneumonia characterised by large areas of pulmonary consolidation with the majority of foals being euthanased two to three weeks after challenge (Martens and others 1989a, Perkins and others 2002, Caston and others 2006). These particular clinicopathological findings do not reflect those typically observed after natural infection and call into question the use of such models to determine the effectiveness of HIP. While the onset of clinical signs of pneumonia, the number of bacteria isolated from the lungs and the lung weight to body weight ratio were lower in foals that received R equi HIP before challenge, pulmonary lesions were seen in all foals (Hooper-McGrevy and others 2001). In contrast, no significant effect of HIP treatment was seen in a different study (Caston and others 2006). This dichotomy could be explained by differences in the experimental challenge used or HIP composition, as antibodies in commercial HIP products differ (Sanz and others 2014b). Thus, despite its widespread use, convincing evidence to support HIP efficacy is lacking.
The objective of this study was to use the recently described (Sanz and others 2013) low-dose R equi experimental challenge model to evaluate the efficacy of HIP. This less aggressive challenge model may be more suitable to evaluate the protective effect of HIP.
Materials and methods
Foals were assigned immediately after birth to either treatment (HIP administration) or control (no HIP) groups using a balanced (2:2) block randomisation design. The study took place at the University of Kentucky Equine Research Farm, Lexington, Kentucky, USA, and all samples were processed at the Immunology Laboratory, Gluck Equine Research Center in Lexington, Kentucky, USA. This section is reported following Consolidated Standards of Reporting Trials (CONSORT) guidelines.
Eighteen newborn mix-bred foals were included; all had a normal complete physical exam, complete blood count, fibrinogen concentration and thoracic ultrasound before challenge. In addition, only foals with adequate passive transfer (IgG >800 g/dl), as determined using SNAP Foal IgG Test, were included. Foals were kept in the pasture with their mares for the duration of the study. The study concluded eight weeks after challenge or until resolution of pneumonia. All methods were approved by the University of Kentucky's Institutional Animal Care and Use Committee and the Institutional Biosafety Committee.
Four (±3)-day-old horse foals received 6×103 cfu/foal (range 4–9×103 cfu/foal) virulent R equi (UKVDL-206), as previously described (Sanz and others 2013). The exact number of bacteria in the inoculum was determined by serial dilution and culturing. The expression of VapA in each inoculum was confirmed by PCR.
Based on a previous study (Sanz and others 2014b), the HIP with the highest concentration of VapA-specific IgG and IgG subclasses and the least lot-to-lot variation (ReSolution, MgBiologics, Ames, Iowa, USA) in the market was used. All HIP bags belonging to the same lot were stored and thawed following manufacturer's instructions. Nine foals received HIP, while nine foals remained as control. For HIP administration, a short-term catheter (14G, 2.75″, Mila International) was aseptically placed. Each bag of HIP was administered slowly (0.5 ml/kg/h) using an in-line filter for the first 20 minutes during which time the foal was closely monitored for any signs of adverse reaction (muscle fasciculation, piloerection, increased heart and respiratory rates, increased temperature, respiratory distress) (Becu and others 1997). The speed of administration was increased (40 ml/kg/h) if no adverse reactions were observed.
Complete physical examination, including thoracic auscultation, rectal temperature, the presence and characterisation (serous, mucopurulent, purulent) of nasal discharge, respiratory effort or changes in attitude (anorexia, lethargy), was performed using minimal restraint twice a week, as previously described (Sanz and others 2013). In addition, foals were observed by experienced farm personnel twice a day for behavioural changes (failure to nurse, stand, increased respiratory effort, lethargy, etc).
Complete blood cell counts and fibrinogen concentrations were evaluated once a week using a QBC VetAutoread Hematological System (IDEXX Laboratories, Westbrook, Maine, USA).
To monitor the development and progression of lung lesions, thoracic ultrasound was performed twice a week. Both sides of the thorax were evaluated using a 7.5 MHz linear probe and a portable ultrasound machine (CTS-7700V-SIUI, Universal Medical System, Bedford Hills, New York, USA) using minimal restraint (Sanz and others 2013). Each abscess (focal hypoechoic areas of consolidation) was scored based on maximal diameter (Chaffin and others 2012); the sum of all abscesses was reported as the total score for a given time point (Venner and others 2012). A cumulative abscess score for each foal was developed by adding all the scores recorded in the study.
Foals were treated for R equi pneumonia if they had evidence of bronchopneumonia upon thoracic ultrasonography (lung abscessation as characterised by well-defined, hypoechoic nodules—relative to the surrounding parenchyma—or lung consolidation as characterised by ill-defined, hypoechoic regions with vessels and bronchi) (Ramirez and others 2004) and one of the following signs: fever for 36 hours (temp. >39°C) and/or signs of lower respiratory disease for 12 hours (either crackles or wheezes, tracheal rattles, tachypnoea, increased respiratory effort, productive cough or bilateral purulent nasal discharge) (Giguere and others 2011b).
Before treatment was instituted, a transtracheal wash (TTW) was performed, as previously described (Sellon and others 2001) and the fluid was submitted to the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL) for aerobic bacterial culture, VapA gene amplification by PCR and cytology evaluation of the fluid. A TTW was collected from all foals eight weeks after challenge or at the end of treatment and the fluid was analysed as described above.
Pneumonia was treated using oral azithromycin (10 mg/kg, every 24 hours for the first five days and every 48 hours thereafter) and oral rifampin (5 mg/kg every 12 hours) (Giguere and others 2011b) until complete resolution of the clinical signs and blood work abnormalities. Ultrasonographic findings were also used to evaluate treatment efficacy. During the treatment, foals were kept inside during the day and turned outside overnight to prevent hyperthermia (Traub-Dargatz and others 1996). Oral flunixin meglumine was used to reduce fever and to improve attitude and appetite (0.5 mg/kg, every 12–24 hours as needed) (Soma and others 1988). Rectal temperature and overall appearance were monitored twice a day for the duration of treatment.
Recombinant VapA protein was produced and purified using an Escherichia coli strain that contained a VapA plasmid fused to glutathione sodium dodecyl-transferase, as previously described (Hooper-McGrevy and others 2001, Sanz and others 2014b). The purity of the protein was assessed by SDS-PAGE (Bio-Rad, Philadelphia, Pennsylvania, USA) (Sanz and others 2014b).
Source of reference control sera
Positive (previously vaccinated mare) and negative controls (fetal equine serum and a four-month-old foal with negative lung tissue culture) as well as standard curves for each VapA-specific IgG, constructed as previously described, were included in each plate (Sanz and others 2014a). All reference sera were divided into 100 µl aliquots and stored at −20°C until used.
VapA-specific ELISA development
The ELISA for VapA-specific IgG was performed as previously described (Sanz and others 2014a). Murine anti-IgGa (CVS48), anti-IgGb (CVS39) or anti-IgG(T) (CVS40) hybridomas were kindly provided by P. Lunn, North Carolina State University. The absorbance was read using an ELISA Plate Reader (Bio-Rad, Philadelphia, Pennsylvania, USA) and the results were converted to ELISA units (EU) (Wright and others 1993). A coefficient of determination (r2) of ≥0.90 for the standard curve was required for the results to be considered valid (Page and others 2011).
Serum was collected from all foals by venepuncture before challenge (after HIP administration in treated groups) and weekly thereafter. Bronchoalvoelar lavage fluid (BALF) was collected before challenge from each foal. Serum was also collected from all the mares before foal challenge. A sample of plasma from each HIP bag was collected before administration. Bronchoalveolar lavage (BAL) samples were collected before challenge. All samples were stored at −20°C until analyses of VapA-specific IgG and IgG subclasses were performed using the ELISA as described above.
Data were analysed using a commercial software (SigmaPlot, SPSS, Chicago, Illinois, USA). Normality of the data and equality of variances were assessed using the Shapiro-Wilk and Levene's tests, respectively. The effect of treatment (HIP) on disease incidence was determined using Fisher's exact test. Differences in clinical parameters, blood work and antibodies over time were assessed using two-way repeated measures, analysis of variance (ANOVA) or repeated measures ANOVA on ranks. Differences in parameters between groups were assessed using Student's t test or Mann-Whitney rank-sum and multiple comparisons were performed using Dunn's or Holm-Sidak methods, respectively. The significance was set at P<0.05.
A total of 19 foals met the inclusion criteria. Nine newborn foals were included in each group; one foal was removed after inclusion but before experimental challenge because of naturally acquired Streptococcus zooepidemicus pneumonia that was confirmed by culture of fluid obtained from a TTW. Time from birth to HIP administration ranged from 8 to 48 hours. Foals were challenged on average at four days of age (ranged from one to eight days); age at challenge was not statistically significant between groups (P=0.63). The median challenge dose was 6.6×103 cfu/foal (range 4.2–9×103cfu/foal), which did not differ statistically between groups (P=0.41). The amount of VapA-specific IgG (all subclasses) in mare serum was not different between groups at foaling (P>0.05). No adverse reactions were observed post-HIP administration. The percentage coefficient of variation (CV per cent) between HIP bags was 43.3 per cent, 25.9 per cent, 14.3 per cent and 7.8 per cent for VapA-specific IgG, IgGa, IgGb and IgG(T), respectively. The CV (per cent) in foal's serum after HIP was administered was 63.3, 48.5, 48.8 and 17.7 for VapA-specific IgG, IgGa, IgGb and IgG(T), respectively.
One foal in the HIP group and four in the control group developed clinical signs of R equi pneumonia and required antimicrobial treatment. The cause of pneumonia was confirmed by culture, cytology and VapA gene PCR of fluid obtained from a TTW. A statistically significant association between HIP administration and development of disease was not found (P=0.29). As only one foal developed disease in the HIP group, statistical analysis of clinical parameters between pneumonic foals in each group could not be performed. Time from challenge to development of clinical signs was 30 days for the foal in the HIP group and ranged from 20 to 25 days (mean 23 days) in the control group. The duration of treatment with antimicrobials (azithromycin and rifampin) in the HIP foal was 16 days and averaged 22 days (ranged from 15 to 35 days) in control foals. Flunixin (1 ml orally, every 24 hours) was given six days to the HIP foal and for an average of seven days (ranged from six to eight days) to control foals.
Heart rate, respiratory rate and temperature significantly decreased (P<0.05) over time in all foals but were not significantly different between groups at any time point (see online supplementary table S1). White blood cell (WBC) counts (P<0.001) and fibrinogen concentration (P<0.001) significantly increased over time in both groups but were significantly higher in control foals (P=0.03 (WBC), P=0.01 (fibrinogen)) after challenge. Platelets also increased significantly over time (P=0.007) in control foals and were significantly higher than that of HIP foals (P=0.013) (Fig 1a–c, see online supplementary table S2).
While thoracic ultrasound scores significantly increased over time in both groups (P<0.05), they remained significantly higher and for a longer period of time in control foals (Fig 2a, see online supplementary table S3). In addition, the cumulative thoracic ultrasound score was significantly (P<0.001) higher in control foals (Fig 2b).
Serum VapA-specific IgG and IgGb significantly decreased over time (P<0.001) after HIP administration, but no significant changes in IgGa (P=0.89) and IgG(T) (P=0.23) over time were seen. In control foals, VapA-specific IgG and IgGb did not change significantly over time (P=0.141 and P=0.07, respectively); however, IgGa and IgG(T) significantly increased towards the end of the study period (P<0.001) (Fig 3a–d).
As expected, HIP foals had significantly higher VapA-specific IgG (P<0.001) and IgGb (P<0.001) than the control foals throughout the duration of the study. While VapA-specific IgGa was higher in foals after HIP administration (P<0.05), it was significantly higher in control foals by the end of the study. Similarly, VapA-specific IgG(T) was significantly higher in control foals late in the study (Fig 3a–d). There was no significant correlation between any of the evaluated VapA-specific IgGs and the total ultrasound scores (see online supplementary table S4).
Fluid from BAL of HIP foals had significantly higher VapA-specific IgG (P=0.02) and IgGb (P=0.04) than control foals before challenge. While VapA-specific IgGa was higher, the difference did not reach statistical significance (P=0.056). VapA-specific IgG(T) was not different between groups (P=0.18) (Fig 4).
To date, an effective vaccine that prevents R equi pneumonia is not available. Due to its worldwide distribution and severity, this condition has a significant financial impact on the horse industry (Venner and others 2012). Farms with an endemic R equi problem attempt to prevent infections by prophylactic intravenous administration of HIP shortly after birth (Becu and others 1997). The rationale behind using HIP is to provide foals with R equi-specific IgG, along with other factors, which may enhance humoral antibacterial effector (Dawson and others 2010). Despite its widespread use, convincing evidence to support HIP efficacy is lacking (Giguere and others 2002, Perkins and others 2002, Caston and others 2006).
As expected with the use of this particular challenge model, almost 50 per cent of the control foals developed clinical signs of pneumonia four to five weeks after challenge (4/9) (Sanz and others 2013). In contrast, only one of the nine HIP foals required treatment. This difference was not statistically significant, likely due to the low power of the study. Likewise, HIP did not prevent the development of pulmonary lesions after challenge. However, the severity of pneumonia was significantly reduced in the HIP group, which resulted in a lower number of foals requiring antimicrobial treatment. Furthermore, foals that received HIP had significantly lower weekly and cumulative ultrasonographic scores and abnormal ultrasonographic scores were present for a shorter period of time in HIP foals, indicating a less severe form of pneumonia. Similarly, the increase in WBC and fibrinogen, an expected non-specific response to bacterial infection (Leclere and others 2011, Venner and others 2012), was significantly less pronounced in the HIP group. Moreover, an increase in platelets, also a non-specific response to bacterial infection (Tafazzoli and others 2006), was only seen in control foals. It should be noted that regression of lesions that was seen here also normally occurs in the majority of naturally infected foals (Venner and others 2012, 2013).
Significant differences in physical exam parameters between groups were not seen. This was not entirely unexpected as these parameters are not good predictors of outcome (Leclere and others 2011). Nevertheless, fever (>39C°) only occurred in control foals. The decrease in heart and respiratory rates as well as rectal temperature over time in both groups was expected and resulted from normal ageing of the foals (Piccione and others 2006, Bernard and Barr 2011) as well as familiarisation with handling.
The variations seen in VapA-specific antibodies between bags of HIP and between foals after HIP administration were consistent with those previously reported for this product (Sanz and others 2014b). As expected, foals that received HIP had significantly higher serum VapA-specific IgG and IgGb for the entire duration of the study. This was predicted as IgGb is the predominant subclass in commercial HIP licensed for R equi in the USA (Sanz and others 2014b) and endogenous synthesis of IgGb does not begin until foals are at least two months of age (Sheoran and others 2000). Similarly, serum VapA-specific IgGa was initially higher after HIP administration; however, because of endogenous production that occurs as early as five weeks of age (Sheoran and others 2000, Sanz and others 2014a) as a response to infection (Sanz and others 2014a), control foals had significantly higher IgGa towards the end of the study. A significant increase in serum VapA-specific IgG(T) was only seen in control foals. This was also expected as this IgG subclass has been shown to increase when R equi pneumonia develops, and not as a result of environmental exposure to the pathogen (Hooper-McGrevy and others 2003, Sanz and others 2014a).
While the mechanism of protection associated with HIP administration is not fully understood, the diffusion of antibodies into the airways was proposed (Dawson and others 2011). VapA-specific IgG, IgGa and IgGb were higher in BALF of foals that received HIP, indicating that transfer of antibodies from serum to BALF does occur rapidly, as BALF was collected one to five days after HIP was given. The lack of VapA-specific IgG(T) in the BALF was consistent with its minimal presence in HIP (Sanz and others 2014b). The existence of these antibodies in BALF may explain the positive effect of HIP in this study; however, at this time, transferring of other molecules present in HIP such as complement factors, cytokines, collectins or fibronectin cannot be ruled out. To the author's knowledge, this is the first time that VapA-specific IgGs are measured in BALF after HIP administration.
Adverse effects have been reported after HIP administration (Becu and others 1997); however, none were seen in this study. This could be due to the use of a single HIP treatment, a common practice on most horse farms as it is largely accepted that foals are infected early in life (Horowitz and others 2001, Chaffin and others 2008, Sanz and others 2013). As the foals in this study were challenged within the first week of life, early administration of HIP was appropriate. It is not clear whether a second HIP treatment given at four weeks of age would have affected clinical outcomes.
Due to personnel availability, the study was not blinded as HIP administration was performed by the same person who performed the foal evaluations (MS). However, the authors are confident that bias was not introduced as a strict criterion for treatment of pneumonia with antimicrobials was used. As noted above, the power of the study to evaluate the effect of HIP treatment on infection rate was low because only a limited number of foals were available. The number of foals selected was, however, sufficient to reach adequate power (>0.8) in the majority of the parameters evaluated. While this study shows a protective effect of early HIP administration against the development of clinical rhodococcal pneumonia, these results cannot be extrapolated to all products available in the market, as high variability in the antibody content between and among HIP products exists (Sanz and others 2014b). In addition, unlike the foals in this study that were challenged one day, foals under natural conditions are constantly exposed to R equi. At this time, it is unknown if similar results will be seen under such circumstances. Though not statistically significant, the difference in the number of foals treated in each group does have clinical significance as macrolide resistance, a veterinary and human medicine concern, has been reported (Giguere and others 2010, Boyen and others 2011). While a larger R equi challenge dose would have resulted in an increased number of foals that developed clinical pneumonia, such a scenario is less representative of what occurs in natural conditions where the majority of foals recovers without antimicrobial aid (Venner and others 2012). Although much remains unknown about the protective components of HIP, its use may be of benefit to decrease antimicrobial usage in the equine industry until an effective vaccine is developed.
The authors would like to thank all UK farm personnel for their invaluable help. MGS was supported through a scholarship from Zoetis. Funding for this project was provided by the Jes E. and Clementine Schlaikjer endowment.
Provenance: Not commissioned; externally peer reviewed
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