Effects of Administration of Live or Inactivated Virulent Rhodococccus equi and Age on the Fecal Microbiome of Neonatal Foals

BACKGROUND: Rhodococcus equi is an important pathogen of foals. Enteral administration of live, virulent R. equi during early life has been documented to protect against subsequent intrabronchial challenge with R. equi, indicating that enteral mucosal immunization may be protective. Evidence exists that mucosal immune responses develop against both live and inactivated micro-organisms. The extent to which live or inactivated R. equi might alter the intestinal microbiome of foals is unknown. This is an important question because the intestinal microbiome of neonates of other species is known to change over time and to influence host development. To our knowledge, changes in the intestinal microbiome of foals during early life have not been reported. Thus, the purpose of this study was to determine whether age (during the first month of life) or administration of either live virulent R. equi (at a dose reported to protect foals against subsequent intrabronchial challenge, viz., 1×10(10) colony forming units [CFU]) or inactivated virulent R. equi (at higher doses, viz., 2×10(10) and 1×10(11) [CFU]) altered the fecal microbiome of foals. METHODOLOGY/PRINCIPAL FINDINGS: Fecal swab samples from 42 healthy foals after vaccination with low-dose inactivated R. equi (n = 9), high-dose inactivated R. equi (n = 10), live R. equi (n = 6), control with cholera toxin B (CTB, n = 9), and control without CTB (n = 8) were evaluated by 454-pyrosequencing of the 16S rRNA gene and by qPCR. No impact of treatment was observed among vaccinated foals; however, marked and significant differences in microbial communities and diversity were observed between foals at 30 days of age relative to 2 days of age. CONCLUSIONS: The results suggest age-related changes in the fecal microbial population of healthy foals do occur, however, mucosal vaccination does not result in major changes of the fecal microbiome in foals

Oral Administration of Electron-Beam Inactivated Rhodococcus equi Failed to Protect Foals against Intrabronchial Infection with Live, Virulent R. equi

There is currently no licensed vaccine that protects foals against Rhodococcus equi-induced pneumonia. Oral administration of live, virulent R. equi to neonatal foals has been demonstrated to protect against subsequent intrabronchial challenge with virulent R. equi. Electron beam (eBeam)-inactivated R. equi are structurally intact and have been demonstrated to be immunogenic when administered orally to neonatal foals. Thus, we investigated whether eBeam inactivated R. equi could protect foals against developing pneumonia after experimental infection with live, virulent R. equi. Foals (n = 8) were vaccinated by gavaging with eBeam-inactivated R. equi at ages 2, 7, and 14 days, or gavaged with equal volume of saline solution (n = 4), and subsequently infected intrabronchially with live, virulent R. equi at age 21 days. The proportion of vaccinated foals that developed pneumonia following challenge was similar among the vaccinated (7/8; 88%) and unvaccinated foals (3/4; 75%). This vaccination regimen did not appear to be strongly immunogenic in foals. Alternative dosing regimens or routes of administration need further investigation and may prove to be immunogenic and protective

Intramuscular Administration of a Synthetic CpG-Oligodeoxynucleotide Modulates Functional Responses of Neutrophils of Neonatal Foals

Neutrophils play an important role in protecting against infection. Foals have age-dependent deficiencies in neutrophil function that may contribute to their predisposition to infection. Thus, we investigated the ability of a CpG-ODN formulated with Emulsigen to modulate functional responses of neutrophils in neonatal foals. Eighteen foals were randomly assigned to receive either a CpG-ODN with Emulsigen (N = 9) or saline intramuscularly at ages 1 and 7 days. At ages 1, 3, 9, 14, and 28, blood was collected and neutrophils were isolated from each foal. Neutrophils were assessed for basal and Rhodococcus equi-stimulated mRNA expression of the cytokines interferon-γ (IFN-γ), interleukin (IL)-4, IL-6, and IL-8 using real-time PCR, degranulation by quantifying the amount of β-D glucuronidase activity, and reactive oxygen species (ROS) generation using flow cytometry. In vivo administration of the CpG-ODN formulation on days 1 and 7 resulted in significantly (P<0.05) increased IFN-γ mRNA expression by foal neutrophils on days 3, 9, and 14. Degranulation was significantly (P<0.05) lower for foals in the CpG-ODN-treated group than the control group at days 3 and 14, but not at other days. No effect of treatment on ROS generation was detected. These results indicate that CpG-ODN administration to foals might improve innate and adaptive immune responses that could protect foals against infectious diseases and possibly improve responses to vaccination.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Differences in the genome, methylome, and transcriptome do not differentiate isolates of Streptococcus equi subsp. equi from horses with acute clinical signs from isolates of inapparent carriers

Streptococcus equi subsp. equi (SEE) is a host-restricted bacterium that causes the common infectious upper respiratory disease known as strangles in horses. Perpetuation of SEE infection appears attributable to inapparent carrier horses because it neither persists long-term in the environment nor infects other host mammals or vectors, and infection results in short-lived immunity. Whether pathogen factors enable SEE to remain in horses without causing clinical signs remains poorly understood. Thus, our objective was to use next-generation sequencing technologies to characterize the genome, methylome, and transcriptome of isolates of SEE from horses with acute clinical strangles and inapparent carrier horses-including isolates recovered from individual horses sampled repeatedly-to assess pathogen-associated changes that might reflect specific adaptions of SEE to the host that contribute to inapparent carriage. The accessory genome elements and methylome of SEE isolates from Sweden and Pennsylvania revealed no significant or consistent differences between acute clinical and inapparent carrier isolates of SEE. RNA sequencing of SEE isolates from Pennsylvania demonstrated no genes that were differentially expressed between acute clinical and inapparent carrier isolates of SEE. The absence of specific, consistent changes in the accessory genomes, methylomes, and transcriptomes of acute clinical and inapparent carrier isolates of SEE indicates that adaptations of SEE to the host are unlikely to explain the carrier state of SEE. Efforts to understand the carrier state of SEE should instead focus on host factors

Vaccination of yearling horses against poly-N-acetyl glucosamine fails to protect against infection with Streptococcus equi subspecies equi.

Strangles is a common disease of horses with worldwide distribution caused by the bacterium Streptococcus equi subspecies equi (SEE). Although vaccines against strangles are available commercially, these products have limitations in safety and efficacy. The microbial surface antigen β 1→6 poly-N-acetylglucosamine (PNAG) is expressed by SEE. Here we show that intramuscular (IM) injection alone or a combination of IM plus intranasal (IN) immunization generated antibodies to PNAG that functioned to deposit complement and mediate opsonophagocytic killing of SEE ex vivo. However, immunization strategies targeting PNAG either by either IM only injection or a combination of IM and IN immunizations failed to protect yearling horses against infection following contact with infected horses in an experimental setting. We speculate that a protective vaccine against strangles will require additional components, such as those targeting SEE enzymes that degrade or inactivate equine IgG

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

Abstract 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

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

Abstract 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

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

Abstract 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

Avaliação de técnicas de biópsia renal em ovinos

Devido à escassez de trabalhos sobre biópsias renais em ovinos foi desenvolvido um estudo comparativo entre três técnicas de biópsia renal nesta espécie. Neste estudo foram utilizadas nove ovelhas (26,64 kg ±4,86) mestiças (Santa Inês) em procedimentos seriados, com intervalos consecutivos de uma semana. Foram avaliados os aspectos clínicos, achados de patologia clínica, o peso das amostras renais, a qualidade histológica, o número de glomérulos e a presença de artefatos no corte histológico da técnica de biópsia percutânea cega, da biópsia guiada por ultrassonografia e do procedimento videolaparoscópico. Não foram observadas alterações hematológicas ou bioquímicas relevantes nos animais submetidos às biópsias renais e as manifestações clínicas detectadas foram leves e transitórias, exceto por um caso de obstrução uretral por coágulo sangüíneo. A técnica percutânea cega foi relacionada à maior ocorrência e gravidade de hematúria, com danos mais graves ao tecido renal e com o único caso de obstrução do fluxo urinário. Na técnica videolaparoscópica, o peso médio das amostras foi superior e a hematúria discreta e transitória. Verificou-se relação direta entre a ocorrência de hematúria grave e a presença de epitélio de transição nas amostras e o número de tentativas utilizado para a obtenção dos fragmentos