Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma.

Introduction: Equine asthma (EA) is a common lower airway disease in horses, but whether its pathogenesis is allergic is ambiguous. Extrinsic stimuli like hay dust induce acute exacerbation of clinical signs and sustained local neutrophilic inflammation in susceptible horses. Aspergillus fumigatus is an EA stimulus, but it is unclear if it merely acts as an IgE-provoking allergen. We aimed to comprehensively analyze immunoglobulin (Ig) isotypes in EA, elucidating their binding to different A. fumigatus antigens, and their quantities systemically in serum and locally in bronchoalveolar lavage fluid (BALF). Methods: Serum and BALF from healthy horses (HE, n = 18) and horses with mild-moderate asthma (MEA, n = 20) or severe asthma (SEA, n = 24) were compared. Ig isotype (IgG1, IgG3/5, IgG4/7, IgG6, IgA, and IgE) binding to nine antigens (A. fumigatus lysate, and recombinant Asp f 1, Asp f 7, Asp f 8, dipeptidyl-peptidase 5, class II aldolase/adducin domain protein, glucoamylase, beta-hexosaminidase, and peptide hydrolase) was compared by enzyme-linked immunosorbent assays. Total Ig isotype contents were determined by bead-based assays. Results: MEA and SEA differed from HE but hardly from each other. Compared to HE, asthmatic horses showed increased anti-A. fumigatus binding of IgG (BALF and serum) and IgA (BALF). Serum and BALF IgE binding and total IgE contents were similar between HE and EA. Single antigens, as well as A. fumigatus lysate, yielded similar Ig binding patterns. Serum and BALF IgG1 binding to all antigens was increased in SEA and to several antigens in MEA. Serum IgG4/7 binding to two antigens was increased in SEA. BALF IgA binding to all antigens was increased in SEA and MEA. Total BALF IgG1 and IgG4/7 contents were increased in SEA, and serum IgG4/7 content was increased in MEA compared to HE. Yet, total isotype contents differentiated EA and HE less clearly than antigen-binding Ig. Discussion: A. fumigatus immunogenicity was confirmed without identification of single dominant antigens here. A. fumigatus provoked elevated BALF IgG1 and IgA binding, and these isotypes appear relevant for neutrophilic EA, which does not support allergy. BALF Ig isotype differentiation beyond IgE is crucial for a comprehensive analysis of immune responses to fungi in EA pathogenesis.

Successful intra-abdominal resection of a 24 kg ovarian granulosa cell tumor in a Warmblood mare.

Background: Granulosa cell tumors (GCTs) are the most common ovarian tumors in mares. The classical presentation of a GCT is a unilaterally enlarged ovary appearing as a multicystic honeycomb mass. In rare cases, GCTs cause hemoperitoneum as a result of the rapid growth of the tumor. The clinical diagnosis of GCT is usually based on history, rectal examination, ultrasonographic examination, and serum hormone analysis, and surgical removal of the affected ovary is the treatment of choice. The different surgical approaches are based on the dimension of the GCT. Case Description: A 7-year-old mare was referred to the department for horses due to suspicion of a large colon impaction. The mare presented with clinical signs of colic, fever, and signs of hypovolemic shock. Rectal and ultrasonographic examination showed hemoperitoneum and a honey-comb mass within the abdomen, and a GCT as the cause of an acute hemoperitoneum was diagnosed based on the serum level of anti-Müllerian hormone. After stabilization of the mare, the GCT was removed through a ventral midline incision. Because of the enormous dimensions of the GCT, intra-abdominal partial resection of the tumor using a tenotomy knife was necessary to exteriorize the ovarian pedicle. At 3 months follow-up, the mare was ridden for her intended use. Conclusion: This report provides an approach to an uncommon case of a very large and heavy GCT.

Horses as Sentinels for the Circulation of Flaviviruses in Eastern-Central Germany.

Since 2018, autochthonous West Nile virus (WNV) infections have been regularly reported in eastern-central Germany. While clinically apparent infections in humans and horses are not frequent, seroprevalence studies in horses may allow the tracing of WNV and related flaviviruses transmission, such as tick-borne encephalitis virus (TBEV) and Usutu virus (USUV), and consequently help to estimate the risk of human infections. Hence, the aim of our study was to follow the seropositive ratio against these three viruses in horses in Saxony, Saxony Anhalt, and Brandenburg and to describe their geographic distribution for the year 2021. In early 2022, i.e., before the virus transmission season, sera from 1232 unvaccinated horses were tested using a competitive pan-flavivirus ELISA (cELISA). In order to estimate the true seropositive ratio of infection with WNV, TBEV, and USUV for 2021, positive and equivocal results were confirmed by a virus neutralization test (VNT). In addition, possible risk factors for seropositivity using questionnaires were analyzed using logistic regression based on questionnaires similar to our previous study from 2020. In total, 125 horse sera reacted positive in the cELISA. Based on the VNT, 40 sera showed neutralizing antibodies against WNV, 69 against TBEV, and 5 against USUV. Three sera showed antibodies against more than one virus, and eight were negative based on the VNT. The overall seropositive ratio was 3.3% (95% CI: 2.38-4.40) for WNV, 5.6% (95% CI: 4.44-7.04) for TBEV, and 0.4% (95% CI: 0.14-0.98) for USUV infections. While age and number of horses on the holding were factors predicting TBEV seropositivity, no risk factors were discovered for WNV seropositivity. We conclude that horses are useful sentinels to determine the flavivirus circulation in eastern-central Germany, as long as they are not vaccinated against WNV.

[West-Nile-Virus infections in 12 horses in east-central Germany]. / West-Nil-Virus-Infektion bei 12 Pferden in Mitteldeutschland.

PURPOSE AND AIM: The presenting complaints, clinical signs, diagnostic evaluation, therapy, and outcome of 12 horses with clinically apparent West-Nile-Virus (WNV) infection are described. MATERIAL AND METHODS: Case series RESULTS: The adult horses (age 6-18 years, 7 mares, 5 geldings) from Saxony and Saxony-Anhalt were presented with various clinical histories between September 2018 and September 2020. All horses were presented in August or September and no horse was vaccinated against WNV. Fever as the most common general clinical sign was present in 8/12 horses. The most common neurological signs were muscle fasciculations (11/12 horses), ataxia (8/12 horses), hyperesthesia and head tilt (6/12 horses each). Diagnosis of WNV infection was confirmed by demonstrating IgM antibody and neutralizing antibody production in all horses; 2 euthanized horses also tested positive by PCR. Therapy was symptomatic and primarily included non-steroidal anti-inflammatories or dexamethasone as well as fluid therapy. Duration of hospitalization was 7.5 days on average. According to their owners, seven horses recovered completely, while information was missing for 2 horses. CONCLUSIONS AND CLINICAL RELEVANCE: In eastern-central Germany, WNV-encephalomyelitis must be considered a differential diagnosis for unvaccinated horses with acute neurologic disease occurring in summer and late summer. The reported clinical signs and the outcome of therapy are mostly congruent with reports from North America and other European countries.

Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences.

Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were analyzed. Using FC, BAL cells and PBMC were comprehensively characterized by cell surface markers ex vivo. An increased percentage of DH24A+ polymorphonuclear cells, and decreased percentages of CD14+ macrophages were detected in BAL from horses with sEA compared to healthy horses or horses with mEA, while lymphocyte proportions were similar between all groups. Independently of EA, macrophages in BAL were CD14+CD16+, which contrasts the majority of CD14+CD16- classical monocytes in PBMC. Percentages of CD16-expressing BAL macrophages were reduced in BAL from horses with sEA compared to healthy horses. While PBMC lymphocytes predominantly contain CD4+ T cells, B cells and few CD8+ T cells, BAL lymphocytes comprised mainly CD8+ T cells, fewer CD4+ T cells and hardly any B cells. These lymphocyte subsets' distributions were similar between all groups. After PMA/ionomycin stimulation in vitro, lymphocyte activation (CD154 and T helper cell cytokine expression) was analyzed in BAL cells of 26 of the horses and group differences were observed (p=0.01-0.11). Compared to healthy horses' BAL, CD154+ lymphocytes from horses with mEA, and CD4+IL-17A+ lymphocytes from horses with sEA were increased in frequency. Activated CD4+ T helper cells were more frequent in asthmatics' (mEA, sEA) compared to healthy horses' PBMC lymphocytes. In summary, FC analysis of BAL cells identified increased polymorphonuclear cells frequencies in sEA as established, while macrophage percentages were mildly reduced, and lymphocyte populations remained unaffected by EA. Cytokine production differences of BAL lymphocytes from horses with sEA compared to healthy horses' cells point towards a functional difference, namely increased local type 3 responses in sEA.

Seroprevalence and Risk Factors for Equine West Nile Virus Infections in Eastern Germany, 2020.

West Nile virus (WNV) infections were first detected in Germany in 2018, but information about WNV seroprevalence in horses is limited. The study's overall goal was to gather information that would help veterinarians, horse owners, and veterinary-, and public health- authorities understand the spread of WNV in Germany and direct protective measures. For this purpose, WNV seroprevalence was determined in counties with and without previously registered WNV infections in horses, and risk factors for seropositivity were estimated. The cohort consisted of privately owned horses from nine counties in Eastern Germany. A total of 940 serum samples was tested by competitive panflavivirus ELISA (cELISA), and reactive samples were further tested by WNV IgM capture ELISA and confirmed by virus neutralization test (VNT). Information about potential risk factors was recorded by questionnaire and analyzed by logistic regression. A total of 106 serum samples showed antibodies against flaviviruses by cELISA, of which six tested positive for WNV IgM. The VNT verified a WNV infection for 54 samples (50.9%), while 35 sera neutralized tick-borne encephalitis virus (33.0%), and eight sera neutralized Usutu virus (7.5%). Hence, seroprevalence for WNV infection was 5.8% on average and was significantly higher in counties with previously registered infections (p = 0.005). The risk factor analysis showed breed type (pony), housing in counties with previously registered infections, housing type (24 h turn-out), and presence of outdoor shelter as the main significant risk factors for seropositivity. In conclusion, we estimated the extent of WNV infection in the resident horse population in Eastern Germany and showed that seroprevalence was higher in counties with previously registered equine WNV infections.

Disease investigations for equine infectious anemia in Canada (2009-2012) - Retrospective evaluation and risk factor analysis.

This retrospective study describes the detection of equine infectious anemia (EIA) during Canadian Food Inspection Agency (CFIA) disease investigations in Canada, examines aspects of importance for disease control, and evaluates potential animal-level risk factors for EIA in high-risk horses. Based on review of all EIA-positive samples and all samples collected during disease investigations (N = 4553) over a 4-year period (2009 to 2012), 409 EIA cases were detected. Horse owners with EIA cases owned between 1 and 60 affected animals, and 49 horses seroconverted during a disease investigation period. Twenty-nine percent of cases (n = 68) for which this information was available had, or possibly had, clinical signs of EIA. Using a mixed effects logistic regression model, horses in older age groups were at greater odds of having a positive EIA status. The study emphasizes the importance of disease investigation activities when EIA is detected and identifies age as an animal-level risk factor in high-risk horses.

Enquêtes médicales pour l'anémie infectieuse équine au Canada (2009­2012) ­ Évaluation rétrospective et analyse des facteurs de risques. Cette étude rétrospective décrit la détection de l'anémie infectieuse équine (EIA) durant les enquêtes médicales de l'Agence canadienne d'inspection des aliments (CFIA) au Canada, examine les aspects importants pour la maitrise de la maladie, et évalue les facteurs de risque potentiels au niveau des animaux pour l'EIA chez les chevaux à risque élevé. Sur la base d'une revue de tous les échantillons positifs pour l'EIA et tous les échantillons prélevés durant les enquêtes (N = 4553) pendant une période de 4 ans (2009­2012), 409 cas d'EIA furent détectés. Les propriétaires de chevaux avec EIA possédaient entre 1 et 60 animaux affectés, et 49 chevaux ont séro-converti durant une période d'enquête. Vingt-neuf pourcents des cas (n = 68) pour lesquels l'information était disponible avaient, ou avaient possiblement eu, des signes cliniques d'EIA. Utilisant un modèle de régression logistique à effets mixtes, les chevaux des groupes d'animaux plus âgés étaient à plus grand risque d'avoir un statut positif pour l'EIA. Cette étude fait ressortir l'importance des activités d'enquêtes médicales lorsque l'EIA est détectée et identifie l'âge comme étant un facteur de risque au niveau de l'animal chez les chevaux à risque élevé.(Traduit par Dr Serge Messier).

A retrospective study of owner-requested testing as surveillance for equine infectious anemia in Canada (2009-2012).

This retrospective study was undertaken to estimate i) the surveillance coverage for equine infectious anemia (EIA) based on owner-requested testing, and ii) the incidence of case detection from this surveillance activity to inform a review of Canada's national disease control strategy. Based on sample submissions by accredited veterinarians to laboratories CFIA-approved for EIA testing between 2009 and 2012, the estimated national surveillance coverage was 14% for all years, and 72 cases of EIA were detected. The annual national incidence of EIA detection ranged from 0.03 to 0.08 cases/1000 horses. On average, a greater proportion of the horse population was tested in eastern Canada (32%) than in western Canada (6%, P < 0.0001). The cumulative incidence of EIA detection was higher in western Canada (0.25 cases/1000 horses) than in eastern Canada (0.02 cases/1000 horses, P < 0.0001). This study identified regional differences in owner-requested EIA testing and case detection resulting from this testing activity.

Étude rétrospective des tests demandés par les propriétaires comme surveillance pour l'anémie infectieuse équine au Canada (2009­2012). Cette étude rétrospective a été entreprise afin d'estimer i) la couverture de surveillance pour l'anémie infectieuse équine (AIE) basée sur les tests demandés par les propriétaires et ii) l'incidence de détection des cas à partir de cette activité de surveillance pour documenter un examen de la stratégie nationale de contrôle des maladies du Canada. L'estimation de la couverture nationale de surveillance, basée sur les soumissions d'échantillons par les vétérinaires autorisés aux laboratoires approuvés par l'ACIA pour l'AIE entre 2009 et 2012, était de 14 % pour toutes les années et 72 cas d'AIE ont été détectés. L'incidence nationale annuelle de la détection de l'AIE variait de 0,03 à 0,08 cas/1000 chevaux. En moyenne, une proportion supérieure de la population équine de l'Est du Canada (32 %) subissait des tests par rapport à l'Ouest canadien (6 %, P < 0,0001). L'incidence cumulative de la détection de l'AIE était supérieure dans l'Ouest canadien (0,25 cas/1000 chevaux) par rapport à l'Est du Canada (0,02 cas/1000 chevaux, P < 0,0001). Cette étude a identifié des différences régionales pour les tests de l'AIE demandés par les propriétaires et la détection des cas découlant de cette activité d'épreuve diagnostique.(Traduit par Isabelle Vallières).

Equine motor neuron disease in 2 horses from Saskatchewan.

Two horses from Saskatchewan were presented with signs of sweating, muscle fasciculations, weight loss, and generalized weakness. The horses were diagnosed with equine motor neuron disease (EMND), by histological assessment of a spinal accessory nerve or sacrocaudalis dorsalis medialis muscle biopsy. This is the first report of EMND in western Canada.

Maladie équine des motoneurones chez 2 chevaux de la Saskatchewan. Deux chevaux de la Saskatchewan ont été présentés avec des signes de sudation, de fibrillations musculaires, de perte de poids et de faiblesse généralisée. On a diagnostiqué la maladie équine des motoneurones (MEMN) chez les chevaux en effectuant une évaluation histologique d'un nerf accessoire de la colonne vertébrale ou une biopsie du muscle sacrocaudalis dorsalis medialis. Il s'agit du premier rapport de MEMN dans l'Ouest canadien.(Traduit par Isabelle Vallières).

Chronic active interstitial pancreatitis as a cause of transverse colonic obstruction and colic in a horse.

A mature Quarter horse was euthanized following colic of 3 days duration. Postmortem, the large intestine, except the descending colon, was diffusely distended and associated with adhesion of the transverse colon to the pancreas, which had changes consistent with chronic active interstitial pancreatitis. Other lesions included hepatic fibrosis, erosive gastritis, and bilateral adrenal cortical hyperplasia.

Pancréatite interstitielle chronique active comme cause d'une obstruction du côlon transversal et de coliques chez un cheval. Un cheval Quarter horse adulte a été euthanasié après des coliques d'une durée de 3 jours. À l'autopsie, il a été observé que le gros intestin, sauf le côlon descendant, présentait une distension diffuse et était associé à l'adhésion du côlon transversal au pancréas qui indiquait des changements conformes à une pancréatite interstitielle chronique active. Les autres lésions incluaient une fibrose hépatique, une gastrite érosive et une hyperplasie corticale bilatérale des surrénales.(Traduit par Isabelle Vallières).

Comparison between available serologic tests for detecting antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi in horses in Canada.

To investigate the agreement between available serologic tests for the detection of antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi, 50 serum samples from horses of unknown clinical status and at low risk for infection were tested. In addition to a point-of-care enzyme-linked immunosorbent assay (pocELISA), the evaluated tests included 2 indirect fluorescent antibody tests (IFATs) for antibodies against A. phagocytophilum and an IFAT, an ELISA confirmed with Western blot, and the Lyme multiplex assay for antibodies against B. burgdorferi. For each pair-wise comparison between serologic tests, the difference in the proportion of seropositive results as well as kappa and the prevalence-adjusted, bias-adjusted kappa were calculated. The proportion of seropositive results differed significantly in each pairwise comparison of tests for detection of antibodies against A. phagocytophilum, and between the pocELISA and IFAT as well as between the pocELISA and Lyme multiplex assay for detection of antibodies against B. burgdorferi. Agreement based on kappa varied from poor to fair while agreement was improved when evaluating prevalence-adjusted, bias-adjusted kappa. Lack of agreement may be explained by differences in methodology between the evaluated tests, cross-reactivity or false-positive and false-negative tests. In addition to the limitations of serologic test interpretation in the absence of clinical disease, this data suggest that screening of horses for exposure to tick-borne diseases in nonendemic areas may not be warranted.

Seroprevalence of equine granulocytic anaplasmosis and lyme borreliosis in Canada as determined by a point-of-care enzyme-linked immunosorbent assay (ELISA).

Equine granulocytic anaplasmosis (EGA) and Lyme borreliosis (LB) are an emerging concern in Canada. We estimated the seroprevalence of EGA and equine LB by testing 376 convenience serum samples from 3 provinces using a point-of-care SNAP(®) 4Dx(®) ELISA (IDEXX Laboratories, Westbrook, Maine, USA), and investigated the agreement between the point-of-care ELISA and laboratory-based serologic tests. The estimated seroprevalence for EGA was 0.53% overall (0.49% in Saskatchewan, 0.71% in Manitoba), while the estimated seroprevalence for LB was 1.6% overall (0.49% in Saskatchewan, 2.86% in Manitoba). There was limited agreement between the point-of-care ELISA and an indirect fluorescent antibody test for EGA (kappa 0.1, PABAK 0.47) and an ELISA/Western blot combination for LB (kappa 0.23, PABAK 0.71). While the SNAP(®) 4Dx(®) ELISA yielded expected seroprevalence estimates, further evaluation of serologic tests for the purposes of disease exposure recognition may be needed.

Séroprévalence de l'anaplasmose granulocytaire équine et de la borréliose de Lyme au Canada telle que déterminée par un test ELISA hors laboratoire. L'anaplasmose granulocytaire équine (AGE) et la borréliose de Lyme (BL) sont de nouvelles maladies émergentes au Canada. Nous avons estimé la séroprévalence de l'AGE et de la BL équine en testant 376 échantillons sériques de commodité provenant de trois provinces en utilisant un test ELISA SNAPMD 4DxMD hors laboratoire (IDEXX Laboratories, Westbrook, Maine, États-Unis) et nous avons analysé la concordance entre les tests ELISA hors laboratoire et des tests sérologiques faits en laboratoire. Le total des séroprévalences estimées pour l'AGE était de 0,53 % (0,49 % en Saskatchewan, 0,71 % au Manitoba), tandis que le total de la séroprévalence estimée de BL était de 1,6 % (0,49 % en Saskatchewan, 2,86 % au Manitoba). Il y avait une concordance limitée entre le test ELISA hors laboratoire et un test d'immunofluorescence indirecte pour l'AGE (kappa 0,1, PABAK 0,47) et une combinaison de tests ELISA/immunobuvardage pour BL (kappa 0,23, PABAK 0,71). Même si le test ELISA SNAPMD 4DxMD hors laboratoire a donné des estimations de séroprévalence attendues, une nouvelle évaluation des tests sérologiques à des fins de reconnaissance de l'exposition à une maladie peut être requise.(Traduit par Isabelle Vallières).

Passive surveillance for ticks on horses in Saskatchewan.

Passive surveillance of ticks on horses in Saskatchewan revealed that the horses were parasitized by 3 species, Dermacentor albipictus, D. andersoni, and D. variabilis. The nymphs and adults of D. albipictus occurred on horses earlier in the year than did adults of the 2 other species.

Surveillance passive des tiques sur des chevaux en Saskatchewan. Une surveillance passive des tiques chez des chevaux de la Saskatchewan a révélé que les chevaux étaient affectés par des parasites de trois espèces: Dermacentor albipictus, D. andersoni et D. variabilis. Les nymphes et les adultes de D. albipictus se présentaient chez les chevaux plus tôt dans l'année que les adultes des deux autres espèces.(Traduit par Isabelle Vallières).

Bilateral renal dysplasia, hydronephrosis, and hydroureter in a septic neonatal foal.

A Canadian warmblood foal treated for septic polyarthritis was diagnosed with bilateral congenital renal dysplasia, hydronephrosis, and hydroureter at 2 weeks of age based on abdominal ultrasound and postmortem examination. Intermittent abdominal pain throughout the course of treatment was attributed to hydronephrosis and hydroureter.

Dysplasie rénale bilatérale, hydronéphrose et urétérohydrose chez un poulain néonatal septique. Un poulain Warmblood canadien traité pour une polyarthrite septique a été diagnostiqué avec de la dysplasie rénale congénitale bilatérale, de l'hydronéphrose et de l'urétérohydrose à l'âge de 2 semaines en se fondant sur une échographie abdominale et une autopsie. Une douleur abdominale intermittente pendant l'administration du traitement a été attribuée à l'hydronéphrose et à l'urétérohydrose.(Traduit par Isabelle Vallières).

Mastitis in a neonatal filly.

Neonatal mastitis is a rare occurrence in the horse. This report documents a case of mastitis caused by an organism within the Streptococcus dysgalactiae group in a 1-week-old Paint filly.

Un cas de mastite chez une pouliche nouveau-née. La mastite est une maladie rare chez les pouliches nouveau-nées. Ce rapport documente un cas de la mastite causé par un organisme du groupe Streptococcus dysgalactiae qui se présente dans une pouliche Paint á l'âge de sept jours et discute la litérature pertinente au sujet de la mastite des nouveau-nés.(Traduit par les auteurs).

Management and long-term outcome of partial glossectomy in 2 horses.

Records were reviewed for 2 horses with partial glossectomy, 1 traumatic and 1 elective. According to long-term follow-up by telephone, both horses had recovered well, experiencing only temporary difficulty while eating, and went on to be ridden successfully using mouth bits. Partial glossectomy, therefore, had a favorable prognosis in 2 performance horses.

Gestion et résultat à long terme de la glossectomie partielle chez 2 chevaux. Les dossiers ont été examinés pour 2 chevaux ayant subi une glossectomie partielle, 1 étant une glossectomie traumatique et 1 étant une glossectomie non urgente. Selon le suivi à long terme par téléphone, les deux chevaux se sont bien rétablis, ont éprouvé seulement une difficulté temporaire à manger et ont ensuite pu être montés avec succès en utilisant des mors. Par conséquent, une glossectomie partielle a présenté un pronostic favorable chez 2 chevaux de performance.(Traduit par Isabelle Vallières).

Failure of a VapA/CpG oligodeoxynucleotide vaccine to protect foals against experimental Rhocococcus equi pneumonia despite induction of VapA-specific antibody and interferon-γ response.

We evaluated the immunogenic and protective potential of a recombinant VapA/CpG oligodeoxynucleotide (ODN) 2395 vaccine in neonatal foals undergoing experimental Rhodococcus equi challenge. Foals (n = 8) were vaccinated by intramuscular injection on days 1 and 15 of the study; control foals (n = 7) received a phosphate-buffered saline (PBS) solution. All foals were challenged by intrabronchial administration of 5 × 106 R. equi 103⁺ on day 29. Bronchoalveolar lavages were done on days 15, 29, and 36 and total cell count, differential cell count, rVapA-stimulated cell proliferation and interferon (IFN)-γ mRNA expression determined. Clinical examination, complete blood (cell) counts, serology for VapA-specific antibodies, and culture of nasal and fecal swabs were done on days 1, 15, 29, 36, 43, and 50. Foals were humanely euthanized on day 50 and severity of pneumonia scored on a 4-point scale. Vaccination resulted in a significant increase in VapA-specific immunoglobulin (Ig) production, with total IgG and IgG(T) being increased by day 15. Expression of VapA-specific IFN-γ mRNA by BAL cells was increased in the vaccinated foals following challenge. Postmortem lung severity scores did not differ between groups. Two foals shed virulent R. equi in feces; however, real-time polymerase chain reaction (PCR) revealed the isolates to be different from the challenge strain.

Nous avons évalué le potentiel immunogène et protecteur d'un vaccin recombinant VapA/oligodéoxynucléotide CpG (ODN) 2395 chez des poulains nouveau-nés soumis à une infection défi par Rhodococcus equi. Les poulains (n = 8) étaient vaccinés par voie intramusculaire aux jours 1 et 15 de l'étude; les poulains témoins (n = 7) ont reçu une injection d'une solution de saline tamponnée (PBS). Tous les poulains ont été challengés par administration intra-bronchique de 5 × 106R. equi 103+ au jour 29. Des lavages broncho-alvéolaires (LBA) ont été effectués aux jours 15, 29 et 36 et on détermina le nombre total de cellules, un dénombrement cellulaire différentiel, la prolifération des cellules rVapA stimulées et l'expression d'ARNm de l'interféron (IFN)-γ. Un examen clinique, des comptages cellulaires sanguins complets, une analyse sérologique pour détecter les anticorps spécifiques contre VapA, et une culture d'écouvillons nasal et fécal ont été effectués aux jours 1, 15, 29, 36, 43 et 50. Les poulains ont été euthanasiés au jour 50 et la sévérité de la pneumonie notée sur une échelle de 4 points. La vaccination a causé une augmentation significative de la production d'immunoglobulines (Ig) spécifiquement dirigées contre VapA, les quantités totales d'IgG et d'IgG(T) ayant augmentées au jour 15. L'expression d'ARNm de l'IFN-γ spécifique au VapA par les cellules des LBA était augmentée chez les poulains vaccinés suite au challenge. Aucune différence ne fut notée dans les pointages de sévérité des lésions pulmonaires lors des examens post-mortem. Deux poulains excrétaient du R. equi virulent dans leurs fèces; toutefois, l'analyse par réaction d'amplification en chaîne par la polymérase (PCR) a démontré que ces isolats étaient différents de la souche utilisée pour le challenge.(Traduit par Docteur Serge Messier).

Analyses of lipid rafts, Toll-like receptors 2 and 4, and cytokines in foals vaccinated with Virulence Associated Protein A/CpG oligonucleotide vaccine against Rhodococcus equi.

Rhodococcus equi establishes long-term pulmonary infection, survives in phagolysosomes of alveolar macrophages and causes pneumonia in foals. The failure of the foal to clear R. equi bacteria is believed to be due to its inability to produce IFN-γ and defects in Toll-like receptor(TLR) signaling. Lipid rafts sequester immune receptors such as TLRs and facilitate efficient cell signaling and therefore, a deficiency in accumulation of receptors in lipid rafts may result in failure to activate. We tested whether a Virulence Associated Protein A (VapA)/CpG vaccine against R. equi would impact the production of IL-10, IFN-γ and TNF-α in lung tissue and fluid samples, alter expression of TLR2 and TLR4 and alter their association with the lipid rafts in broncho-alveolar lavage (BAL) cells. Eight foals, 1­6 days of age, were vaccinated against R. equi followed by a booster at day 14 and challenged with R. equi (5 x 10(6) CFU/ml;10 ml) on day 28. This group was termed "vaccinated pre-challenge" before the infection and "vaccinated post-challenge" after the infection. A second group of foals (n = 7) was not vaccinated but challenged with R. equi on day 28 of the study. This group was termed "non-vaccinated pre-challenge" and after infection with R. equi was named "non-vaccinated post-challenged. We report adaptation of previous protocols to isolate plasma membrane fractions from BAL cells and identification of lipid raft fractions based on the presence of flotillin-1 and GM-1 and absence of transferrin receptor. TLR2 and TLR4 were restricted to plasma membrane fractions 7­9 of alveolar cells collected from vaccinated foals before and after the challenge. Western blots showed that vaccinated post-challenge foals had higher expression of TLR2 in their lung tissues compared to non-vaccinated pre-challenge foals. TNF- concentration was higher in BAL fluid collected from the vaccinated compared to the non-vaccinated foals on day 28. Lung tissue extracts collected on day 49 from the non-vaccinated R. equi challenged foals showed higher expression of IL-10 compared to the vaccinated-challenged foals. However, there were no differences among the groups with respect to the concentration of IFN-γ in BAL fluid or lung tissue extracts. Taken together, we modified previous protocols to isolate plasma membrane fractions from BAL cells of foals and report that the vaccination with a VapA/CPG vaccine increases association of TLR2 and TLR4 with lipid raft fractions and alters expression of TNF-α and IL-10. The data point to a subtle effect of vaccination on the association of TLR2 and TLR4 with lipid rafts in BAL cells.

Infusion of sodium bicarbonate in experimentally induced metabolic acidosis does not provoke cerebrospinal fluid (CSF) acidosis in calves.

In a crossover study, 5 calves were made acidotic by intermittent intravenous infusion of isotonic hydrochloric acid (HCl) over approximately 24 h. This was followed by rapid (4 h) or slow (24 h) correction of blood pH with isotonic sodium bicarbonate (NaHCO(3)) to determine if rapid correction of acidemia produced paradoxical cerebrospinal fluid (CSF) acidosis. Infusion of HCl produced a marked metabolic acidosis with respiratory compensation. Venous blood pH (mean ± S(x)) was 7.362 ± 0.021 and 7.116 ± 0.032, partial pressure of carbon dioxide (Pco(2), torr) 48.8 ± 1.3 and 34.8 ± 1.4, and bicarbonate (mmol/L), 27.2 ± 1.27 and 11 ± 0.96; CSF pH was 7.344 ± 0.031 and 7.240 ± 0.039, Pco(2) 42.8 ± 2.9 and 34.5 ± 1.4, and bicarbonate 23.5 ± 0.91 and 14.2 ± 1.09 for the period before the infusion of hydrochloric acid and immediately before the start of sodium bicarbonate correction, respectively. In calves treated with rapid infusion of sodium bicarbonate, correction of venous acidemia was significantly more rapid and increases in Pco(2) and bicarbonate in CSF were also more rapid. However, there was no significant difference in CSF pH. After 4 h of correction, CSF pH was 7.238 ± 0.040 and 7.256 ± 0.050, Pco(2) 44.4 ± 2.2 and 34.2 ± 2.1, and bicarbonate 17.8 ± 1.02 and 14.6 ± 1.4 for rapid and slow correction, respectively. Under the conditions of this experiment, rapid correction of acidemia did not provoke paradoxical CSF acidosis.

Pulmonary intravascular macrophages as proinflammatory cells in heaves, an asthma-like equine disease.

Heaves, an obstructive neutrophilic airway inflammation of horses, is triggered by dust components such as endotoxin and has similarities to human asthma. Pulmonary intravascular macrophages (PIMs) increase horses' sensitivity to endotoxin-induced lung inflammation; however, their role in an airborne pathology remains unknown. Therefore, we investigated the role of PIMs in the development of heaves in horses. Clinical and inflammatory responses were evaluated following induction of heaves by moldy hay exposure and PIM depletion with gadolinium chloride (GC). Mares (N = 9) were exposed to four treatments: alfalfa cubes (Cb), alfalfa cubes + GC (Cb-GC), moldy hay (MH), and moldy hay + GC (MH-GC). Clinical scores and neutrophil concentrations in bronchoalveolar lavage (BAL) fluid were higher when mares received MH compared with MH-GC. BAL cells from MH-GC-treated mares had significantly lower IL-8 and TLR4 mRNA expression compared with MH-treated mares. In vitro LPS challenge significantly increased IL-8 but not TLR4 mRNA expression in BAL cells recovered from horses fed with MH, but not from the MH-GC treatment. In summary, PIM depletion attenuated clinical scores, reduced the alveolar migration of neutrophils, and decreased the expression of proinflammatory molecules in BAL cells of heaves horses, suggesting a proinflammatory role of PIMs in the development of airborne pathology.