Serum activities of complement 1q and antibodies to the virulence-associated protein A are lower in foals that develop rhodococcal pneumonia.

OBJECTIVE: To determine the effects of transfusion of Rhodococcus equi hyperimmune plasma (REHIP) on serum concentrations of complement component 1q (C1q) and to examine the association of serum C1q and anti-rhodococcal antibodies of newborn foals with subsequent development of rhodococcal pneumonia. ANIMALS/SAMPLES: Foals (n = 205) from 2 Thoroughbred breeding farms in New York transfused with REHIP between January 1, 2022, and December 1, 2022. PROCEDURES: Blood was collected immediately before transfusion with REHIP and again from the contralateral vein immediately after transfusion. Foals were followed through weaning for clinical and ultrasonographic evidence of rhodococcal pneumonia. Serum samples were tested by ELISA for concentrations of C1q and for activity of IgG1 and IgG4/7 recognizing the virulence-associated protein A (VapA) of R equi. Logistic regression analysis was used to determine the association between rhodococcal pneumonia and levels of C1q and anti-VapA IgG1 and IgG4/7. RESULTS: REHIP significantly decreased C1q concentrations immediately after transfusion. Accounting for effects of farm and birth month, estimated odds of pneumonia were 2.1-fold (P = .0330) higher for foals with pretransfusion C1q concentrations less than or equal to the population median and 3.3-fold (P = .0051) higher for foals with posttransfusion IgG1 activity in the lower quartile. CLINICAL RELEVANCE: Both C1q and IgG appear to contribute to protection against R equi, and IgG1 appears to be especially important. Increasing IgG1 concentrations targeting rhodococcal proteins in REHIP or serum of foals might improve protection against R equi foal pneumonia.

Intramuscular but not nebulized administration of a mRNA vaccine against Rhodococcus equi stimulated humoral immune responses in neonatal foals.

OBJECTIVE: Design and evaluate immune responses of neonatal foals to a mRNA vaccine expressing the virulence-associated protein A (VapA) of Rhodococcus equi. ANIMALS: Cultured primary equine respiratory tract cells; Serum, bronchoalveolar lavage fluid (BALF), and peripheral blood mononuclear cells (PBMCs) from 30 healthy Quarter Horse foals. METHODS: VapA expression was evaluated by western immunoblot in cultured equine bronchial cells transfected with 4 mRNA constructs encoding VapA. The mRNA construct with greatest expression was used to immunize foals at ages 2 and 21 days in 5 groups: (1) 300 µg nebulized mRNA (n = 6); (2) 600 µg nebulized mRNA (n = 4); (3) 300 µg mRNA administered intramuscularly (IM) (n = 5); (4) 300 µg VapA IM (positive controls; n = 6); or (5) nebulized water (negative controls; n = 6). Serum, BALF, and PBMCs were collected at ages 3, 22, and 35 days and tested for relative anti-VapA IgG1, IgG4/7, and IgA activities using ELISA and cell-mediated immunity by ELISpot. RESULTS: As formulated, nebulized mRNA was not immunogenic. However, a significant increase in anti-VapA IgG4/7 activity (P < .05) was noted exclusively in foals immunized IM with VapA mRNA by age 35 days. The proportion of foals with anti-VapA IgG1 activity > 30% of positive control differed significantly (P = .0441) between negative controls (50%; 3/6), IM mRNA foals (100%; 5/5), and IM VapA (100%; 6/6) groups. Natural exposure to virulent R equi was immunogenic in some negative control foals. CLINICAL RELEVANCE: Further evaluation of the immunogenicity and efficacy of IM mRNA encoding VapA in foals is warranted.

Transfusion of hyperimmune plasma for protecting foals against Rhodococcus equi pneumonia.

The bacterium Rhodococcus equi causes pneumonia in foals that is prevalent at breeding farms worldwide. In the absence of an effective vaccine, transfusion of commercial plasma from donor horses hyperimmunised against R. equi is used by many farms to reduce the incidence of pneumonia among foals at farms where the disease is endemic. The effectiveness of hyperimmune plasma for controlling R. equi pneumonia in foals has varied considerably among reports. The purposes of this narrative review are: (1) to review early studies that provided a foundational basis for the practice of transfusion of hyperimmune plasma that is widespread in the United States and in many other countries; (2) to summarise current knowledge of hyperimmune plasma for preventing R. equi pneumonia; (3) to provide an interpretive summary of probable explanations for the variable results among studies evaluating the effectiveness of transfusion of hyperimmune plasma for reducing the incidence of R. equi pneumonia; (4) to review mechanisms by which hyperimmune plasma might mediate protection; and (5) to consider risks of transfusing foals with hyperimmune plasma. Although the weight of evidence supports the practice of transfusing foals with hyperimmune plasma to prevent R. equi pneumonia, many important gaps in our knowledge of this topic remain including the volume/dose of hyperimmune plasma to be transfused, the timing(s) of transfusion, and the mechanism(s) by which hyperimmune plasma mediates protection. Transfusing foals with hyperimmune plasma is expensive, labour-intensive, and carries risks for foals; therefore, alternative approaches for passive and active immunisation to prevent R. equi pneumonia are greatly needed.

Fecal concentration of Rhodococcus equi determined by quantitative polymerase chain reaction of rectal swab samples to differentiate foals with pneumonia from healthy foals.

BACKGROUND: Diagnostic accuracy of real-time, quantitative PCR (qPCR) assays to quantify virulent Rhodococcus equi using rectal swab samples has not been systematically evaluated. OBJECTIVE: To evaluate the accuracy of qPCR of rectal swab samples to differentiate foals with pneumonia from healthy foals of similar age from the same environment. ANIMALS: One hundred privately owned foals born in 2021 from 2 farms in New York. METHODS: An incident case-control study design was used. Rectal swabs were collected from all foals diagnosed with R. equi pneumonia at 2 horse-breeding farms (n = 47). Eligible pneumonia cases (n = 39) were matched by age to up to 2 healthy (n = 53) control foals; rectal swabs were collected from control foals on the day of diagnosis of the index case. DNA was extracted from fecal swabs and the concentration of virulent R. equi (ie, copy numbers of the virulence-associated protein A gene [vapA] per 100 ng fecal DNA) was estimated by qPCR. RESULTS: The area under the ROC curve for qPCR of fecal swabs was 83.7% (95% CI, 74.9-92.6). At a threshold of 14 883 copies of vapA per 100 ng fecal DNA, specificity of the assay was 83.0% (95% CI, 71.7-92.4) and sensitivity was 79.5% (95% CI, 66.7-92.3). CONCLUSIONS AND CLINICAL IMPORTANCE: Although fecal concentrations of virulent R. equi are significantly higher in pneumonic foals than healthy foals of similar age in the same environment, qPCR of rectal swabs as reported here lacks adequate diagnostic accuracy for clinical use.

Association of pneumonia with concentrations of virulent Rhodococcus equi in fecal swabs of foals before and after intrabronchial infection with virulent R. equi.

BACKGROUND: Intragastric administration of virulent Rhodococcus equi protects foals against subsequent experimental intrabronchial (IB) infection, but it is unknown whether R. equi naturally ingested by foals contributes to their susceptibility to pneumonia. HYPOTHESIS: Fecal concentration of virulent R. equi before IB infection with R. equi is positively associated with protection from pneumonia in foals. ANIMALS: Twenty-one university-owned foals. METHODS: Samples were collected from experimental studies. Five foals were gavaged with live, virulent R. equi (LVRE) at age 2 and 4 days; the remaining 16 foals were not gavaged with LVRE (controls). Fecal swabs were collected from foals at ages 28 days, immediately before IB infection. Foals were monitored for clinical signs of pneumonia, and fecal swabs were collected approximately 2 weeks after IB infection. Swabs were tested by quantitative PCR for concentration of virulent R. equi (ie, copy numbers of the virulence-associated protein A gene [vapA] per 100 ng fecal DNA). RESULTS: Fecal concentrations of virulent R. equi (vapA) before IB infection were significantly (P < .05) lower in control foals (25 copies/100 ng DNA [95% CI, 5 to 118 copies/100 ng DNA) that developed pneumonia (n = 8) than in healthy control foals (n = 8; 280 copies/100 ng DNA; 95% CI, 30 to 2552 copies/100 ng DNA) or those gavaged with LVRE (707 copies/100 ng DNA, 95% CI, 54 to 9207 copies/100 ng DNA). CONCLUSIONS AND CLINICAL IMPORTANCE: Greater natural ingestion of LVRE might contribute to protection against pneumonia among foals.

Randomized, controlled trial comparing Rhodococcus equi and poly-N-acetyl glucosamine hyperimmune plasma to prevent R equi pneumonia in foals.

BACKGROUND: Hyperimmune plasma raised against ß-1→6-poly-N-acetyl glucosamine (PNAG HIP) mediates more opsonophagocytic killing of Rhodococcus equi (R equi) than does R equi hyperimmune plasma (RE HIP) in vitro. The relative efficacy of PNAG HIP and RE HIP to protect foals against R equi pneumonia, however, has not been evaluated. HYPOTHESIS: Transfusion with PNAG HIP will be superior to RE HIP in foals for protection against R equi pneumonia in a randomized, controlled, blinded clinical trial. ANIMALS: Four hundred sixty Quarter Horse and Thoroughbred foals at 5 large breeding farms in the United States. METHODS: A randomized, controlled, blinded clinical trial was conducted in which foals were transfused within 24 hours after birth with 2 L of either RE HIP or PNAG HIP. Study foals were monitored through weaning for clinical signs of pneumonia by farm veterinarians. The primary outcome was the proportion of foals that developed pneumonia after receiving each type of plasma. RESULTS: The proportion of foals that developed pneumonia was the same between foals transfused with RE HIP (14%; 32/228) and PNAG HIP (14%; 30/215). CONCLUSIONS AND CLINICAL IMPORTANCE: Results indicate that PNAG HIP was not superior to a commercially available, United States Department of Agriculture-licensed RE HIP product for protecting foals against R equi pneumonia under field conditions.

Antibody activities in hyperimmune plasma against the Rhodococcus equi virulence -associated protein A or poly-N-acetyl glucosamine are associated with protection of foals against rhodococcal pneumonia.

The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide ß-1→6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C՛1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n = 119) or subclinical pneumonia at Farm B (n = 114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity ≤ the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities ≤ the population median for PNAG or C՛1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C՛1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals.

Serum Antibody Activity against Poly-N-Acetyl Glucosamine (PNAG), but Not PNAG Vaccination Status, Is Associated with Protecting Newborn Foals against Intrabronchial Infection with Rhodococcus equi.

Rhodococcus equi is a prevalent cause of pneumonia in foals worldwide. Our laboratory has demonstrated that vaccination against the surface polysaccharide ß-1→6-poly-N-acetylglucosamine (PNAG) protects foals against intrabronchial infection with R. equi when challenged at age 28 days. However, it is important that the efficacy of this vaccine be evaluated in foals when they are infected at an earlier age, because foals are naturally exposed to virulent R. equi in their environment from birth and because susceptibility is inversely related to age in foals. Using a randomized, blind experimental design, we evaluated whether maternal vaccination against PNAG protected foals against intrabronchial infection with R. equi 6 days after birth. Vaccination of mares per se did not significantly reduce the incidence of pneumonia in foals; however, activities of antibody against PNAG or for deposition of complement component 1q onto PNAG was significantly (P < 0.05) higher among foals that did not develop pneumonia than among foals that developed pneumonia. Results differed between years, with evidence of protection during 2018 but not 2020. In the absence of a licensed vaccine, further evaluation of the PNAG vaccine is warranted, including efforts to optimize the formulation and dose of this vaccine. IMPORTANCE Pneumonia caused by R. equi is an important cause of disease and death in foals worldwide for which a licensed vaccine is lacking. Foals are exposed to R. equi in their environment from birth, and they appear to be infected soon after parturition at an age when innate and adaptive immune responses are diminished. Results of this study indicate that higher activity of antibodies recognizing PNAG was associated with protection against R. equi pneumonia, indicating the need for further optimization of maternal vaccination against PNAG to protect foals against R. equi pneumonia.

Transfusion With 2 L of Hyperimmune Plasma is Superior to Transfusion of 1 L or Less for Protecting Foals Against Subclinical Pneumonia Attributed to Rhodococcus equi.

Transfusing foals with Rhodococcus equi hyperimmune plasma (REHIP) is a standard practice at many horse-breeding farms to help prevent R. equi pneumonia. At many large breeding farms, pneumonia is most commonly recognized as subclinical based on thoracic ultrasonography findings. The efficacy of REHIP transfusion and the impact of the volume of plasma transfused for reducing the cumulative incidence of subclinical R. equi pneumonia are unknown. A retrospective cohort study was conducted among foals born and residing through weaning at a large breeding farm. Foals were transfused with either 0 L (n = 2 foals), 1 L (n = 85 foals), or 2 L (n = 62 foals) of REHIP within 36 hours of birth. Volume transfused was principally based on intended use of the foals. All foals at the ranch were routinely screened using thoracic ultrasonography at 5, 7, and 9 weeks of age to detect subclinical pneumonia attributed to R. equi based on farm history. The proportion of the foals receiving < 1 L REHIP that developed subclinical pneumonia (32%; 26/82) was significantly (P = .0068; chi-squared test) greater than that among foals transfused with 2 L of REHIP (12%; 8/68). Despite the important limitations of this observational study, it provides evidence supporting the need for well-designed clinical trials to evaluate the impact of the use and dose of REHIP for preventing subclinical pneumonia. Reducing the incidence of subclinical pneumonia is important because reducing antibiotic treatment of subclinical cases will decrease selection pressure for antimicrobial resistance in R. equi.

In vitro evaluation of complement deposition and opsonophagocytic killing of Rhodococcus equi mediated by poly-N-acetyl glucosamine hyperimmune plasma compared to commercial plasma products.

BACKGROUND: The bacterium Rhodococcus equi can cause severe pneumonia in foals. The absence of a licensed vaccine and limited effectiveness of commercial R. equi hyperimmune plasma (RE-HIP) create a great need for improved prevention of this disease. HYPOTHESIS: Plasma hyperimmune to the capsular polysaccharide poly-N-acetyl glucosamine (PNAG) would be significantly more effective than RE-HIP at mediating complement deposition and opsonophagocytic killing (OPK) of R. equi. ANIMALS: Venipuncture was performed on 9 Quarter Horses. METHODS: The ability of the following plasma sources to mediate complement component 1 (C1) deposition onto either PNAG or R. equi was determined by ELISA: (1) PNAG hyperimmune plasma (PNAG-HIP), (2) RE-HIP, and (3) standard non-hyperimmune commercial plasma (SP). For OPK, each plasma type was combined with R. equi, equine complement, and neutrophils isolated from horses (n = 9); after 4 hours, the number of R. equi in each well was determined by quantitative culture. Data were analyzed using linear mixed-effects regression with significance set at P < .05. RESULTS: The PNAG-HIP and RE-HIP were able to deposit significantly (P < .05) more complement onto their respective targets than the other plasmas. The mean proportional survival of R. equi opsonized with PNAG-HIP was significantly (P < .05) less (14.7%) than that for SP (51.1%) or RE-HIP (42.2%). CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma hyperimmune to PNAG is superior to RE-HIP for opsonizing and killing R. equi in vitro. Comparison of these 2 plasmas in field trials is warranted because of the reported incomplete effectiveness of RE-HIP.