THE MOLECULAR BASIS FOR RHODOCOCCUS EQUI SUSCEPTIBILITY OF FOALS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013226
• Project No.: KY014057
• Project Start Date: Aug 10, 2017
• Project End Date: Sep 30, 2021

Project Director
Horohov, DA, WA.

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Veterinary Science

Non Technical Summary
Respiratory diseases remain one of the major impediments to the equine industry leading to increased financial costs associated with prevention, treatment, and the loss of young animals. Rhodococcus equi, in particular, remains the most common cause of subacute or chronic abscessating bronchopneumonia in young foals. Epidemiological and experimental data indicate the majority of foals infected with the bacterium spontaneously resolve these infections. The mechanism involved in the recovery from infection is not known. A better understanding of this phenomenon could lead to improved approaches for preventing this costly disease. We will perform RNA sequence analysis to determine the molecular basis of this resistance using our previously described challenge model.

Animal Health Component

100%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3810	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1090 - Immunology;

Keywords

foal

rhodococcus

neonate

immunity

gene expression

Goals / Objectives
Rhodococcus equi remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals less than 5 months of age. Upwards of 80% of foals on endemic farms will show early signs of infection by thoracic ultrasonography. While most infected foals spontaneously resolve their lesions (regressors), other will develop a life-threatening bronchopneumonia (progressors). Our hypothesis is that lesion regression post-rhodococcal infection is the result of changes in the expression of specific genes that are associated with the protective mechanism.The overall goal of this project is to better understand the resistance mechanism of regressorsso that more effective prophylactic strategies may be dveloped. Our objective is be to assess total gene expression using transcriptional profiling on samples collected at critical time points pre- and post-infection of progressor and regressor foals. This approach will allow us to identify novel genes and pathways that will generate new insight into the molecular mechanisms that result in foals being at risk for bronchopneumonia.

Project Methods
A total of 30 healthy neonatal foals will be required for this study. Foals will be included if they have a normal complete physical exam, normal CBC and fibrinogen concentration, and normal thoracic ultrasound prior to challenge. In addition, only foals with adequate passive transfer (IgG >800g/dl evaluated using SNAP® foal IgG test) will be included. For the challenge, three (+ 1) day-old horse foals will receive 103 R equi (UKVDL206) in two mls of saline.Bacterial identification and confirmation of the presence of VapA will be performed by the University of Kentucky Veterinary Diagnostic Laboratory.Post challenge, the foals are returned to their dams and observed daily for any changes in attitude. Twice weekly, mares and foals will be placed together in individual stalls for a short period (2-3h) when complete physical exams including heart rate (HR), rectal temperature, respiration rate (RR), and auscultation of the lungs are performed. Particular attention will be paid on the identification of extrapulmonary lesions. To monitor for the development and progression of the lung lesions, thoracic ultrasound will be performed weekly after challenge. Peripheral blood samples and bronchoalveolar lavage samples will be collected from all of the foals at 4 weeks post challenge. This time point corresponds to that time when regressor foals begin to resolve their lesions.All foals will be euthanized at 8 weeks of age for definitive diagnosis of R equi pneumonia, unless clinical signs require earlier euthanasia. All necropsies will be performed by a board certified pathologist at Veterinary Diagnostic Laboratory, University of Kentucky who is blinded to the treatment. Pneumonia scores, from each lung lobe, will be added together to form a cumulative pneumonia score for each animal. The rest of the carcass will be evaluated for signs of extrapulmonary disease. In addition, representative tissue samples will be collected from each foal's lung and tracheobronchial lymph node routinely along with any extrapulmonary lesions that may be present for subsequent bacteriological culture and histopathologic confirmation of R. equi pneumonia.Peripheral blood samples and bronchoalveolar lavage cells will be collected for the evaluation of the immune response of the foals 4 weeks post challenge. This time point is associated with the appearance of lung lesions and is also when regression is likely to begin to occur. Duplicate BAL and peripheral blood tubes will be inoculated with VapA or media and cultured for 24 hours prior to transfer to Tempus tubes. Total RNA for gene expression analyses will be obtained from the Tempus tubes and both quantitative and qualitative parameters of the RNA preparations will be determined. All RNA samples will meet quality thresholds, 260/280 ratios of 1.7-2.0, 260/230 ratios of 1.8-2.1, and an Agilent RNA integrity number (RIN) of ≥ 7.0.Sequencing of the messenger RNA in each individual sample will be accomplished using Illumina's paired-end RNA-seq protocol with library preparation modified to generate strand-specific reads. This will enable resolution of transcripts from overlapping gene loci on opposite gDNA strands. Briefly, ~40 million 2x100bp stranded reads will be generated from each sample. Reads will be aligned to the equine reference genome (EquCab3) with MapSplice read alignment software. (http://www.netlab.uky.edu/p/bioinfo/MapSplice2). Relative expression at gene loci and at individual exon splice junctions will be determined directly by tabulating the number of aligned reads. For individual genes, the data will be normalized by the gene's cDNA length. Differential expression between tissues will be determined by comparing read depth values normalized to the total number of aligned reads in each individual sample using DESeq2, and open source Bioconductor software package (https://bioconductor.org/packages/release/bioc/html/DESeq2.html).Differential expression analysis. Differential expression between regressor/progressor phenotypes will also be analyzed using non-normalized gene count with EdgeR, which uses a negative binomial distribution to account for overdispersion within the RNA-seq data in a generalized linear model framework (GLM). In addition to regressor/progressor differences, the GLM allows for multiple regression modelling peripheral blood and BAL fluid differences in gene expression, and if these responses in these tissues are different between groups. Statistical significance for differential expression at a FDR of 10% after Benjamini-Hochberg multiple testing adjustment will be set at p < 0.05.In addition to the identification of differentially expressed genes between groups, biologic differences between gene expression both between phenotypes and across tissues will be further explored by (1) pathway overrepresentation analysis of differentially expressed gene lists; (2) gene set enrichment analysis; and (3) the construction of gene networks; and (4) comparative network analysis both within and across tissues.

Progress 08/10/17 to 09/30/21

Outputs
Target Audience:The work from this project would be of interest to equine veterinarians, farm managers and horse owners. There would also be scientific interest among other researchers involved in the investigation of bacterial pathogenesis, particularly as it relates to opportunistic infections caused by Rhodococcus and related bacteria. As this project also dealt with neonatal foals, there would be interested amongst those immunologist interested in the early development and maturation of the neonatal immune response. Changes/Problems:The combination of the ongoing coronavirus pandemic and the occurence of a Rotavirus-B outbreak on farms in the Lexington area precluded an additional sampling of foals during the past 2 years. Instead we have relied upon the analysis of archived samples. This resulted in a shift in emphasis of the project from transcriptional analyses to lipidomic analysis due to the nature of the samples (archived serum and plasma samples). What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?A paper outlining the results of this study has been published. The results will also be presented at the upcoming American Association of Equine Practitioners conference.. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We analyzed the plasma lipidome of infected and healthy foals in hopes of identifying a lipid biomarker that could differentiate infected from exposed but healthy foals.Both ageing and experimental infection altered the foal's plasma lipidome as demonstrated by multivariate statistical analysis. The intensities of 31 lipids were altered by ageing and 12 by infection (P < .05). Furthermore, nine lipids changed by more than twofold between clinical and subclinical groups.These experimental results provide the background for future work in the discovery of earlier biomarkers of R. equi pneumonia. Early identification of foals at risk of developing clinical pneumonia is key in order to decrease antimicrobial use and development of antimicrobial resistance.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: 1. Sanclemente, J.L., S.M. Rivera-Velez, N. Dasgupta, D.W. Horohov, P.L. Wood, and M.G. Sanz, Plasma lipidome of healthy and Rhodococcus equi-infected foals over time. Equine Veterinary Journal, 2021.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Equine veterinarians andhorses breeders. Changes/Problems:The disruption in breeding farm operations casued by CoVID-19 negatively impacted our ability to collect needed samples from regressor and progressor foals. It has also impacted our ability to continue laboratory analyses of those samples already collected as it also contibributed to the daparture of the technician who has worked on this project and was expected to complete the analysis. This was worsened by the fact that we are currently in the midst of a hiring pause. This delay, coupled with the difficulty in recruiting a person with the necessary experience may not allow us to complete all of the planned experiments before the end of the project period. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results from this and other studies were presented at a recent symposium on Life Stages of Foals held in Lexington, KY.. The audience consisted of equine veterinarians, horse owners and undergraduate students. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We continue to assess various clinical parameters to better define the regressor/progressor foal phenotype. Most recently we examined the effect of HIP on fecal bacterial counts both to further identify the efficacy of HIP and also to develop fecal bacterial culturing as an indicator of clinical status.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Sanz, M.G., D.S. Bradway, D.W. Horohov, and T.V. Baszler, Rhodococcus equi-specific hyperimmune plasma administration decreases faecal shedding of pathogenic R. equi in foals. Vet Rec, 2019. 185(1): p. 19

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Farm managers of large equine breeding operations. Veterinarians who oversee foal health plans. Horse owners. Changes/Problems:As noted above, the overall goal of this project is to identify the molecular basis for the progressor and regressor phenotypes. We thought it important to utilize foals naturally infected with field strains of R. equi in order to better define the resistance mechanism under non-experimental conditions. Based on our data, and those of other workers in this field, it would be expected that 80% of those foals initially identified as being infected with R. equi by thoracic ultrasonography would spontaneously regress their lung lesions. However, owing to the high value of the foals in this area, veterinary care is fairly aggressive and many foals with early signs of ultrasound lesions are treated, often without the aid of ancillary diagnostics. This phenomenon was noted during the current study as more than half of the foals were identified as progressors. As such, the RNA samples collected from these animals are likely contaminated with false positives (regressors identified as progressors). As a result, we will have to re-evaluate the use of naturally infected samples for this project. What opportunities for training and professional development has the project provided?A veterinary student from Lincoln Memorial University was involved in sample collection and analysis for this project. Through these activities the student gained a greater understanding of this important disease of newborn foals. A graduate student was involved in all aspects of this project. How have the results been disseminated to communities of interest?The results of our research are presented annually to local stakeholders during the Collage of Agriculture, Food and Environemtn's annual Equine Showcase. Those in attendence include local farm managers, veterinarians and horse owners. What do you plan to do during the next reporting period to accomplish the goals?Since it is imperative that we obtain accurate identification of both progressor and regressor foals, we will need to focus more on those foals involved in our experimental challenge studies. This will avoid the issue of false identification of progressor foals (i.e. those that are treated with the antibiotics even though they likely would have regressed their lesions). We are currently seeking funding to perform the challenge studies that will provide us with the necessary number of samples for analysis.

Impacts
What was accomplished under these goals? We have previously demonstrated, using both our low dose (103 cfu/foal) challenge model and in an initial study of naturally-infected foals, that the early antibody response of progressor foals is characterized by a significant increase in R. equi specificIgG(T) at 5-8 weeks of life. However, there was a small number of naturally infected foals in that study and a lack of confirmed diagnosis. In light of the challenge that identification of progressor foals currently presents, the objective of this study was to investigate the use of IgG(T) to identify progressor foals acquiring their infection on horse farms in the Lexington area. Foals were examined at 7 days of age and peripheral blood samples were collected. Thereafter, physical and lung ultrasonographic examinations were performed at 1, 2 and 3 months of age, or when specific clinical signs of pneumonia developed. The same physical examination and lung ultrasonographic parameters and guidelines were used to evaluate challenged and naturally infected foals. Four different veterinarians performed the examinations and assigned the respective scores to the naturally infected foals. When IgG(T) was further evaluated as a possible marker of progressor foals among the naturally infected population, the results indicated that it failed to predict the development of clinical disease. Overlap in individual titers between progressor and regressor groups appeared to limit thevalue of this assay for diagnostic purpose. Therefore, IgG(T) was not a reliable predictor of clinical pneumonia in the naturally infected foals on any of the sampling times. In addition, diagnostic accuracy was not increased by testing serial serum samples collected over the first 3 months of life in the studied populations. This study had several important limitations, including the pressure to treat foals with small ultrasonographic lung lesions as progressors. Studies including larger sample populations of progressor and regressor foals under field conditions where there is less pressure to treat subclinically affected foals could yield data more likely to be statistically significant. Another significant limitation of this study was that a definitive diagnosis could not be obtained from naturally infected foals. Thus, our negative results should be interpreted with caution based on the high positive predictive value of ultrasonographically detecting characteristic lung lesions in foals from endemic farms. More importantly, the presence of possible regressor foals being mislabeled as progressors calls into question the utility of these samples for future RNA sequence analysis.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: VACCINATION OF MARES WITH AN R. EQUI PILI-BASED VACCINE LEADS TO THE TRANSFER OF ANTI-PILI ANTIBODIES TO THEIR FOALS. International Equine Immunology Conference, Bern Switzerland. Oct 24-26, 2019.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Rhodococcus equi remains a serious health concern for foals. Current preventative measures are costly and ineffective. As such, our work in this area is of interest to equine practitioners, horse breeders and owners. Changes/Problems:The only challenge that we have thus far encountered is in obtaining accurate clinical definition of progressor and regressor cases in our field samples. As some foals are being treated with antibiotics fairly early in their infection, it may not be possible to definitively characterize them as progressor when they may well have resolved those lesions without intervention. This problem arose during our previous HATCH project where we attempted to correlate specific immunoglobulin isotype expression with progressor and regressor responses in the field. We will continue to work with our veterinarian collaborators in order to minimize this issue as the study continues. What opportunities for training and professional development has the project provided?The veterinary scientist working on this project will have the opportunity to gain experience with bioinformatic analysis techniques. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue to collect samples from well-characterized cases of Rhodococcus pneumonia as well as from those foals identified as regressors, i.e. those foals that apontaneously resolve their initial lesions as identified by ultronagraphy.

Impacts
What was accomplished under these goals? We are currently in the process of collecting BAL and peripheral blood samples from foals either experimentally infected or naturally infected with Rhodococcus equi. The latter samples are being provided by local practitioners from farms with endemic cases of Rhodococcus equi pneumonia. We also have samples collected from our own foals used in previous challenge studies.

Publications

Progress 08/10/17 to 09/30/17

Outputs
Target Audience:Rhodococcus equi remains a serious health concern for foals. Current preventative measures are costly and ineffective. As such, our work in this area is of interest to equine practitioners, horse breeders and owners. Changes/Problems:The only challenge that we have thus far encountered is in obtaining accurate clinical definition of progressor and regressor cases in our field samples. As some foals are being treated with antibiotics fairly early in their infection, it may not be possible to definitively characterize them as progressor when they may well have resolved those lesions without intervention. This problem arose during our previous HATCH project where we attempted to correlate specific immunoglobulin isotype expression with progressor and regressor responses in the field. We will continue to work with our veterinarian collaborators in order to minimize this issue as the study continues. What opportunities for training and professional development has the project provided?The veterinary scientist working on this project will have the opportunity to gain experience with bioinformatic analysis techniques. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue to collect samples from well-characterized cases of Rhodococcus pneumonia as well as from those foals identified as regressors, i.e. those foals that apontaneously resolve their initial lesions as identified by ultronagraphy.

Impacts
What was accomplished under these goals? We are currently in the process of collecting BAL and peripheral blood samples from foals either experimentally infected or naturally infected with Rhodococcus equi. The latter samples are being provided by local practitioners from farms with endemic cases of Rhodococcus equi pneumonia. We also have samples collected from our own foals used in previous challenge studies.

Publications