Single domain antibodies against enteric pathogen virulence factors are active as curli fiber fusions on probiotic E. coli Nissle 1917.

Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic E. coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eukaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.

WHOLE BLOOD THROMBOELASTOGRAPHY IN HEALTHY ADULT CAMELIDS (VICUGNA PACOS AND CAMELUS DROMEDARIUS).

Thromboelastography (TEG) provides a global assessment of hemostasis and fibrinolysis and has broad applications to identify and monitor coagulation dysfunction in veterinary patients. Although alpacas are susceptible to a wide variety of coagulopathies, the assessment of TEG has not been reported in clinically healthy alpacas to date. The purpose of this study was to evaluate the analytical performance of recombinant human tissue factor (rhTF)- and kaolin-activated TEG and to establish reference intervals for TEG parameters (reaction [R] and clotting [K] times, angle [α], maximum amplitude [MA], and shear elastic modulus [G]) in healthy, adult alpacas. Kaolin and rhTF-activated TEG were performed using citrated whole blood samples from 20 clinically healthy, nonpregnant, adult Huacaya alpacas each after 30 min of sample storage at room temperature. Six individuals of a related species, dromedary camels, were also sampled for comparative purposes. All data were presented descriptively, assessed for normality, and compared using either independent-sample t tests or Mann-Whitney U tests, with P ≤ 0.05 considered significant. Reference intervals were calculated using a robust method and Box-Cox-transformed data. Mean TEG values (reference intervals) were determined for rhTF-activated TEG as follows: R 6.99 min (3.41-12.71), K 3.43 min (1.61-6.42), α 48.51° (27.21-67.38), MA 52.05 mm (21.53-65.92), and G 5.71 kdyn/cm2 (1.87-9.60), while mean values (reference intervals) for kaolin-activated TEG included R 7.72 min (4.48-11.43), K 4.24 min (2.03-9.20), α 45.06° (23.66-64.20), MA 52.18 mm (33.49-66.63), and G 5.78 kdyn/cm2 (NR-9.66). None of the measured TEG values differed significantly between activators, suggesting that activator choice may have a limited effect on TEG parameters in healthy alpacas. TEG results in alpacas were comparable to those of dromedary camels. These results will thus provide a useful starting point in the evaluation of hemostasis in adult camelids.

NEONATAL INTENSIVE CARE OF 10 HOSPITALIZED GIRAFFE CALVES (GIRAFFA CAMELOPARDALIS) REQUIRING HAND-REARING.

This retrospective case series describes the clinicopathologic findings, diagnoses, treatment, and outcomes of 10 hand-reared newborn giraffe (Giraffa camelopardalis) calves admitted to a university teaching hospital for intensive care. Ten calves (five males, five females; nine reticulated giraffes [Giraffa camelopardalis reticulata], one Masai giraffe [G. c. tippelskirchi]), were admitted under 2 days of age. Inadequate transfer of passive immunity was suspected in 5 of 10 calves based on assessment of serum total solids and globulin values. These calves were treated with oral frozen bovine colostrum and/or intravenous hyperimmune bovine plasma. Diarrhea occurred in 6 of 10 calves and was managed with supportive care, fecal microbiota transplantation, and limiting milk intake (offering 10% body weight [BW] in milk per day, while feeding <2 L per meal at 2- to 4-hr intervals). Less common diagnoses included pneumonia (n = 3) and mycoplasma-associated septic arthritis (n = 1). Eight calves received systemic antimicrobial therapy. Hyperlactatemia (lactate > 5 mmol/L; n = 8) and hypercreatininemia (creatinine > 2.0 mg/dl, n = 7) were the most common presenting laboratory abnormalities, which resolved with intravenous fluid therapy. All neonatal giraffes survived to discharge after a median hospitalization of 9.5 days (range, 5-37 days) and were successfully hand-reared at their place of birth. In conclusion, neonatal giraffe calves can be intensively managed in a hospital environment. Diarrhea was a common clinical problem and can be related to feeding regimens. Intravenous hyperimmune bovine plasma infusion was well tolerated to manage failure of transfer of passive immunity in calves with inadequate colostrum administration. The current study supports that compromised neonatal giraffe calves may carry an excellent prognosis after early, intensive intervention.

Neurologic Conditions Affecting the Equine Athlete.

EPM, CVSM, and EDM are currently recognized as the 3 most common neurologic diseases in US horses, with the latter 2 conditions being most prevalent in young animals. Moreover, horses competing at shows and performance events are at greater risk for exposure to highly contagious, neurologic EHV-1 outbreaks. A clinical diagnosis of any neurologic disease should be based on a careful history, complete neurologic examination, and appropriate diagnostic testing and interpretation. However, mild or early neurologic signs can often mimic or be mistaken for an orthopedic condition when horses present for performance-related concerns.

SURGICAL CORRECTION OF A CERVICAL SPINAL FRACTURE IN A BENNETT'S WALLABY (MACROPUS RUFOGRISEUS).

Macropods are susceptible to trauma, and fractures of the cervical vertebrae due to collisions are relatively common. A 4-yr-old, intact male Bennett's wallaby (Macropus rufogriseus) was presented acutely nonambulatory and tetraparetic but with motor function present in all limbs. Cervical radiographs revealed a C4 vertebral fracture. Surgical stabilization was obtained through a ventral midline approach whereby the vertebral bodies were distracted and then secured with screws and polymethylmethacrylate (PMMA). Postoperative radiographs revealed restoration of the spinal canal and appropriate implant placement. The patient was discharged 4 days postoperatively and gradually recovered full neurologic function per the owner over the ensuing 10 wk. Subsequent radiographs obtained 10.5 mo postoperatively revealed a healed fracture with stable implants. The ventral spinal distraction and stabilization technique using screws and PMMA, as are used in the domestic dog, was successful in this wallaby despite its smaller vertebral size and comparable lack of soft-tissue support.

An in vitro larval motility assay characterizes anthelmintic efficacy against Crenosoma vulpis, Angiostrongylus vasorum, and Aelurostrongylus abstrusus.

OBJECTIVE: This study determined the in vitro efficacy of 6 common anthelmintics (eprinomectin, ivermectin, milbemycin oxime, moxidectin, selamectin, and fenbendazole) on motility (viability) of infectious third-stage larvae (L3) of Crenosoma vulpis, Angiostrongylus vasorum, and Aelurostrongylus abstrusus, which are important causes of canine and feline cardiopulmonary disease. SAMPLES: First-stage larvae (L1) from C vulpis, An vasorum, and Ae abstrusus. PROCEDURES: Naïve Limax maximus slugs were fed 1,000 to 2,000 L1 and held at 16 °C for at least 4 weeks to produce live L3. Approximately 50 to 100 L3/well were subsequently incubated in culture media alone or media containing 6 separate test anthelmintics at 4 concentrations, to bracket expected in vivo drug plasma levels in anthelmintic-treated dogs and cats. Drug effects on L3 motility (viability) were analyzed by multilevel logistic models, generating dose-response relationships. Experiments were completed 1-9/2019. RESULTS: Drug concentration estimates corresponding to a 50% larval mortality rate identified that C vulpis was the most sensitive species to the anthelmintics tested. Ae abstrusus was most susceptible to moxidectin and selamectin, while An vasorum was insusceptible to all anthelmintics tested, except for selamectin at high drug concentrations. CLINICAL RELEVANCE: The in vitro anthelmintic response to antiparasitic agents may guide and improve disease therapy and prevention. Considering the observed lack of efficacy against L3, monthly anthelmintic treatment for protection against An vasorum infection in dogs would primarily rely on the anthelmintic's adulticidal activity. Maximal preventive control for An vasorum would, therefore, require at least 1 treatment administered a minimum of 1 week after the end of the transmission season.

Comparative pharmacokinetics of phenylbutazone in healthy young-adult and geriatric horses.

OBJECTIVE: To evaluate the effects of aging on phenylbutazone (PBZ) disposition in older horses (≥ 25 years old) compared to young adults (4 to 10 years old) by characterizing the pharmacokinetic profile of PBZ and its active metabolite, oxyphenbutazone (OPBZ), following a 2.2-mg/kg dose, IV. We hypothesized that the disposition of PBZ will be affected by age. ANIMALS: 16 healthy horses (8 young adults aged 4 to 10 years and 8 geriatric horses ≥ 25 years old). METHODS: Horses were administered a single 2.2-mg/kg PBZ dose, IV. Plasma samples were collected at designated time points and frozen at -80 °C until assayed using liquid chromatography-tandem mass spectrometry. Pharmacokinetic analyses were performed using Phoenix WinNonlin, version 8.0 (Certara). Both clinical and pharmacokinetic data were compared between age groups using independent samples t tests, with P < .05 considered significant. RESULTS: Baseline characteristics did not differ between groups, with the exception of age, weight, and plasma total solids. Plasma concentrations of PBZ were best described by a two-compartment model. The maximum plasma concentration of OPBZ was reached at 5 hours for both age groups, and the metabolite-to-parent-drug area-under-the-curve ratios were approximately 20% for both groups. None of the pharmacokinetic parameters of PBZ or its metabolite, OPBZ, differed significantly between age groups. CLINICAL RELEVANCE: The hypothesis was rejected as there was no significant difference in PBZ disposition in young-adult horses compared to geriatric horses. Our data do not support the need for dose adjustments of PBZ in clinically healthy geriatric horses.

Flow interruption compared to forced oscillatory maneuvers and esophageal balloon-pneumotachography for measurement of respiratory resistance in the horse.

Pulmonary function testing is critical to the diagnosis of equine asthma (EA), an important cause of respiratory disease in the horse, but its clinical use has remained elusive, unfortunately, due to the complexity of reference methods, esophageal balloon/pneumotachography (EBP) and forced oscillatory mechanics (FOM), so we sought a non-invasive, portable method for use in horses through rapid interruption of airflow for equilibration of alveolar pressure with proximal airway pressure, termed flow interruption (FI). Resistance (RINT) was computed as the relationship between the change in pressure at the nose before and immediately after interruption and flow immediately before interruption. A pilot study in 5 healthy university-owned animals using EBP and FI showed good correspondence between the two methods: RINT (0.33 +/- 0.05 cm H2O/l/s) and RL (0.31 +/- 0.06 cm H2O/l/s). In 2 separate populations of client-owned horses, with random assignment of methods to FI v EBP (n = 8), RINT showed good correlation with RL in horses, (rs =.995, p = .0002) and accords with RL, with no significant difference between RINT and RL. Using FOM (n = 12), RINT (0.67 +/- 0.31 cmH2O/l/s) has good correlation with RRS measured with FOM (r =.834, p = .0001), but is consistently smaller than RRS (0.74 +/- 0.33 cmH2O/l/s) . Histamine bronchoprovocation (HBP) was performed in a subset of these horses: FI classified one horse in 6 as less reactive than did EBP, and FI classified one horse in 7 as less reactive than did FOM.

A single VHH-based toxin-neutralizing agent and an effector antibody protect mice against challenge with Shiga toxins 1 and 2.

Shiga toxin-producing Escherichia coli (STEC) is a major cause of severe food-borne disease worldwide, and two Shiga toxins, Stx1 and Stx2, are primarily responsible for the serious disease consequence, hemolytic-uremic syndrome (HUS). Here we report identification of a panel of heavy-chain-only antibody (Ab) V(H) (VHH) domains that neutralize Stx1 and/or Stx2 in cell-based assays. VHH heterodimer toxin-neutralizing agents containing two linked Stx1-neutralizing VHHs or two Stx2-neutralizing VHHs were generally much more potent at Stx neutralization than a pool of the two-component monomers tested in cell-based assays and in vivo mouse models. We recently reported that clearance of toxins can be promoted by coadministering a VHH-based toxin-neutralizing agent with an antitag monoclonal antibody (MAb), called the "effector Ab," that indirectly decorates each toxin molecule with four Ab molecules. Decoration occurs because the Ab binds to a common epitopic tag present at two sites on each of the two VHH heterodimer molecules that bind to each toxin molecule. Here we show that coadministration of effector Ab substantially improved the efficacy of Stx toxin-neutralizing agents to prevent death or kidney damage in mice following challenge with Stx1 or Stx2. A single toxin-neutralizing agent consisting of a double-tagged VHH heterotrimer--one Stx1-specific VHH, one Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH--was effective in preventing all symptoms of intoxication from Stx1 and Stx2 when coadministered with effector Ab. Overall, the availability of simple, defined, recombinant proteins that provide cost-effective protection against HUS opens up new therapeutic approaches to managing disease.

A Longitudinal Analysis of Equine Asthma Presentation and Response to Treatment Using Lung Function Testing and BAL Cytology Analysis in Combination with Owner Perception.

(1) Background: Equine asthma (EA) is a pervasive and important cause of poor performance and respiratory morbidity in horses. Diagnosis of EA includes an owner complaint, clinical scoring, lung function testing, and cytological analysis of bronchoalveolar lavage (BAL) cytology. There is a paucity of information about the longitudinal course of the disease using these outcome assessments; thus, this study sought to describe and quantify, in horses with more than one visit to a specialty pulmonary clinic in New England, the type and range of clinical presentations with an eventual diagnosis of EA. It also aimed to develop and compare the outcomes of scoring systems for owner complaints and veterinary assessments, document and assess the diagnostic methods used, and evaluate the response of the horses to treatment and time. (2) Methods: This study was a retrospective, cross-sectional, STROBE-compliant observational analysis of equine patients who visited the Tufts Cummings Hospital for Large Animals (HLA) for evaluation of equine asthma (EA) from 1999-2023. The horses were categorized as having mild-moderate (mEA) or severe EA (sEA) using the ACVIM consensus statement guidelines. After excluding those with inadequate documentation or only one visit (n = 936), a total of 76 horses were included in the study. Of the 197 visits, 138 (70.0%) resulted in a diagnosis of mEA and 45 (22.8%) resulted in a diagnosis of sEA. Demographic information, owner complaints, clinical examination and scoring, lung function testing, BAL cytology, and recommendations for environmental remediation and pharmacologic treatment were recorded for all the visits. The data were analyzed for agreement between owner complaints (complaint score, CS) and clinical examination findings (examination score, ES), changes in CS and ES, lung function testing, and BAL cytology over time, with 197 visits recorded. (3) Results: A comparison between the CS and ES showed that the owners were more likely than veterinarians to detect cough, and a decrease in cough was the most common owner observation after treatment. The response to the histamine challenge, used to detect airway hyperreactivity, was significantly improved with treatment or time in the horses with mEA, whereas baseline lung function did not significantly change in mEA or sEA. (4) Conclusions: Owners can be astute observers of clinical signs, especially cough, in EA. Tests of airway hyperreactivity are more successful in detecting changes in mEA than are baseline lung function testing and assessment of BAL cytology.

Bronchoalveolar lavage hemosiderosis in lightly active or sedentary horses.

BACKGROUND: Hemosiderophages in bronchoalveolar lavage fluid (BALF) are commonly ascribed to exercise-induced pulmonary hemorrhage (EIPH). Little information exists regarding the presence of these cells in horses that perform light or no work and that are referred for respiratory problems. OBJECTIVES: Evaluate the presence of hemosiderophages in BALF of horses suspected of respiratory disease without history of or risk factors for EIPH and determine predictors of hemosiderophages in BALF in this population. METHODS: Observational retrospective cross-sectional study using STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines. Bronchoalveolar lavage fluid cytology reports of 353 horses evaluated for respiratory disease between 2012 and 2022 at the Cummings School for Veterinary Medicine were reviewed retrospectively. Horses with a history or likelihood of having performed past strenuous exercise were removed, and the remaining 91 horses were divided into hemosiderin-positive (HSD-POS) and hemosiderin-negative groups based on Perls' Prussian blue staining. Potential predictors for the presence of hemosiderophages in BALF (history, clinical evaluation, baseline lung function, airway reactivity, BALF cytology, and hemosiderin score) were compared between the 2 groups, using univariate and multivariate analyses. RESULTS: Horses with a diagnosis of severe equine asthma (sEA; odds ratio, 11.1; 95% confidence interval, 3.2-38.5; P < .001) were significantly more likely to be HSD-POS than horses with mild-to-moderate equine asthma. CONCLUSIONS AND CLINICAL IMPORTANCE: Hemosiderophages were found in the BALF cytology in a subset of horses that perform light or no work and presented for respiratory signs; these cells were found more frequently in horses with sEA. The link between hemosiderophages and sEA highlights previously unstudied pathology associated with this common disease.

A CONSORT-guided, randomized, double-blind, controlled pilot clinical trial of inhaled lidocaine for the treatment of equine asthma.

There are limited options for treatment of the common disease, equine asthma. The aim of this study was to estimate the feasibility and potential efficacy of using nebulized lidocaine for treating equine asthma, while at the same time treating a separate cohort of asthmatic horses with inhaled budesonide. Nineteen horses with a history consistent with equine asthma were recruited from our referral population for a double-blind, randomized, controlled pilot clinical trial using Consolidated Standards of Reporting Trials (CONSORT) guidelines. After screening, 16 horses met the inclusion criteria for equine asthma and 13 horses actually completed the study. Horses were treated by their owners at home for 14 d before returning to our hospital for follow-up assessment. Interventions consisted of nebulization q12h for 14 d with 1.0 mg/kg body weight (BW) of lidocaine or corticosteroid treatment (nebulized budesonide 1 µg/kg, q12h). Clinical and tracheal mucus score, pulmonary function testing, and respiratory secretion cytology were assessed after 2 weeks of treatment to determine the outcome. Both lidocaine and budesonide cohorts had significant decreases (P < 0.05) in clinical score; the lidocaine cohort showed a significant decrease in bronchoalveolar lavage (BAL) neutrophil percentage and tracheal mucus score. Neither treatment resulted in significant changes in lung function parameters. No adverse events occurred. Lidocaine may be an effective and safe treatment for equine asthma in horses that cannot tolerate treatment with corticosteroids.

Il existe des options limitées pour le traitement de la maladie répandue, l'asthme équin. Le but de cette étude était d'estimer la faisabilité et l'efficacité potentielle de l'utilisation de la lidocaïne nébulisée pour traiter l'asthme équin, tout en traitant en même temps une cohorte distincte de chevaux asthmatiques avec du budésonide inhalé. Dix-neuf chevaux ayant des antécédents compatibles avec l'asthme équin ont été recrutés dans notre population de référence pour un essai clinique pilote contrôlé, randomisé, en double aveugle, conformément aux directives CONSORT (Consolidated Standards of Reporting Trials). Après dépistage, 16 chevaux répondaient aux critères d'inclusion de l'asthme équin et 13 chevaux ont terminé l'étude. Les chevaux ont été traités par leurs propriétaires à domicile pendant 14 jours avant de retourner à notre hôpital pour une évaluation de suivi. Les interventions consistaient en une nébulisation deux fois par jour pendant 14 jours avec 1,0 mg/kg de poids corporel (PC) de lidocaïne ou un traitement aux corticostéroïdes (budésonide nébulisé 1 µg/kg, q12h). Le score clinique et de mucus trachéal, les tests de la fonction pulmonaire et la cytologie des sécrétions respiratoires ont été évalués après 2 semaines de traitement pour déterminer le résultat. Les cohortes de lidocaïne et de budésonide présentaient des diminutions significatives (P < 0,05) du score clinique; la cohorte de lidocaïne a montré une diminution significative du pourcentage de neutrophiles du lavage bronchoalvéolaire (BAL) et du score de mucus trachéal. Aucun des deux traitements n'a entraîné de changements significatifs dans les paramètres de la fonction pulmonaire. Aucun événement indésirable n'est survenu. La lidocaïne peut être un traitement efficace et sûr de l'asthme équin chez les chevaux qui ne tolèrent pas le traitement aux corticostéroïdes.(Traduit par Docteur Serge Messier).

The association between fecal microbiota, age and endoparasitism in adult alpacas.

Endoparasitism is a major cause of morbidity and mortality in alpacas (Lama pacos), with growing emergence of anthelmintic resistance. The purpose of the study was to correlate nematode worm burden and selected host phenotypic characteristics, such as age and weight, with the composition of the intestinal microbiota of adult alpacas. Fecal samples were collected per rectum from 102 healthy adult (2.1-11.2 years) alpacas at 3 separate timepoints (pre- and post-treatment with 8.8 mg/kg oral Levamisole HCL, and 4.6 months later) at a single farm. The profile of the fecal bacterial microbiota was characterized using 16S amplicon sequencing. Serial clinical exams and fecal egg counts were compared using related-samples analyses. The fecal microbiota of identically managed, healthy alpacas was characterized by a high level of temporal stability, as both α and ß-diversity significantly correlated between sampling timepoints. Pairwise ß-diversity between samples collected at each timepoint was low, ranging from 0.16-0.21 UniFrac distance units. The intensity of strongylid nematode infection (including Haemonchus, Ostertagia, Trichostrongylus) was only significantly correlated with microbiota composition in samples collected 14 days after treatment with levamisole. Analysis of similarity revealed no clustering of microbiota from anthelmintic responders or non-responders. Alpaca age explained the largest proportion of fecal microbiota variation and was the only consistently significant predictor of fecal microbiota taxonomic composition, by impacting the ratio of relative Bacteroidetes and Firmicutes abundance. Firmicutes, mostly Clostridiales, was the most abundant taxon across all collections.

Clinical effects and pharmacokinetics of nebulized lidocaine in healthy horses.

Background: Nebulized lidocaine appears promising as a novel corticosteroid-sparing therapeutic for equine asthma, but its safety and pharmacokinetic behavior have yet to be confirmed. Objective: To describe the effect of nebulized lidocaine on upper airway sensitivity, lung mechanics, and lower respiratory cellular response of healthy horses, as well as delivery of lidocaine to lower airways, and its subsequent absorption, clearance, and duration of detectability. Animals: Six healthy university- and client-owned horses with normal physical examination and serum amyloid A, and no history of respiratory disease within 6 months. Methods: Prospective, descriptive study evaluating the immediate effects of 1 mg/kg 4% preservative-free lidocaine following nebulization with the Flexineb®. Prior to and following nebulization, horses were assessed using upper airway endoscopy, bronchoalveolar lavage, and pulmonary function testing with esophageal balloon/pneumotachography and histamine bronchoprovocation. Additionally, blood and urine were collected at predetermined times following single-dose intravenous and nebulized lidocaine administration for pharmacokinetic analysis. Results: Upper airway sensitivity was unchanged following lidocaine nebulization, and no laryngospasm or excessive salivation was noted. Lidocaine nebulization (1 mg/kg) resulted in a mean epithelial lining fluid concentration of 9.63 ± 5.05 µg/mL, and a bioavailability of 29.7 ± 7.76%. Lidocaine concentrations were higher in epithelial lining fluid than in systemic circulation (Cmax 149.23 ± 78.74 µg/L, CELF:Cmaxplasma 64.4, range 26.5-136.8). Serum and urine lidocaine levels remained detectable for 24 and 48 h, respectively, following nebulization of a single dose. Baseline spirometry, lung resistance and dynamic compliance, remained normal following lidocaine nebulization, with resistance decreasing post-nebulization. Compared to the pre-nebulization group, two additional horses were hyperresponsive following lidocaine nebulization. There was a significant increase in mean airway responsiveness post-lidocaine nebulization, based on lung resistance, but not dynamic compliance. One horse had BAL cytology consistent with airway inflammation both before and after lidocaine treatment. Conclusions: Nebulized lidocaine was not associated with adverse effects on upper airway sensitivity or BAL cytology. While baseline lung resistance was unchanged, increased airway reactivity to histamine bronchoprovocation in the absence of clinical signs was seen in some horses following nebulization. Further research is necessary to evaluate drug delivery, adverse events, and efficacy in asthmatic horses.

Measurement of corneal sensitivity in 20 ophthalmologically normal alpacas.

OBJECTIVE: To determine corneal sensitivity in 20 healthy adult alpacas (40 eyes) in order to establish reference values. DESIGN: Prospective study. ANIMALS: Twenty healthy, adult alpacas. PROCEDURES: Corneal sensitivity was determined by the corneal touch threshold (CTT) using a Cochet-Bonnet esthesiometer. Five different regions of the cornea were evaluated (nasal, ventral, lateral, dorsal, and central). RESULTS: Corneal touch threshold values (in mm filament length) obtained from five corneal regions demonstrated varying corneal sensitivities. The central region (34.5 ± 7.1 mm) was the most sensitive, followed by the ventral (29.5 ± 7.2 mm), medial (29.3 ± 7.3 mm), dorsal (25.6 ± 6.3 mm), and lateral (21.8 ± 5.7 mm) regions. All pairwise comparisons of regional differences were statistically significant (P < 0.05), except for the comparison between the ventral and medial regions (P = 0.88). Evaluation of the CCT while controlling for age (2-5 years vs. 6-10 years) demonstrated an increased CTT with an increasing age for the central, ventral, medial and lateral regions. A decrease in the CTT of the dorsal region was noted with increasing age. CONCLUSION AND CLINICAL RELEVANCE: Corneal touch threshold values in 40 eyes of 20 healthy adult alpacas were determined using a Cochet-Bonnet esthesiometer. This study demonstrated the central corneal region to be most sensitive. Values obtained may serve as reference values in subsequent studies.

Comparative evaluation of clinical findings and prognostic outcome parameters in hospitalized, critically ill neonatal foals and crias.

OBJECTIVE: Species-related differences in the prevalence, manifestation, and outcome of neonatal illness may impact management practices of neonatal intensive care. The study aimed to elucidate similarities between disease manifestations and mortality risks of critically ill (CI) neonatal crias and foals admitted to the same referral center. DESIGN: A comparative, retrospective cohort evaluation of two species (camelid and equine). SETTING: The study was conducted in a University hospital. ANIMALS: Two hundred and forty-six CI neonatal crias (January 1999 to May 2016) and 356 neonatal foals (February 2001 to May 2016) under 4-week-old were admitted to a university hospital. INTERVENTION: All data are presented descriptively and compared between groups using univariate and multivariate analyses. MEASUREMENTS AND MAIN RESULTS: Female crias (142/246, 57.7%) were significantly overrepresented in comparison to fillies (132/352, 37.5%). Congenital defects and transfer failure of passive immunity were more often observed in neonatal crias, while colic, diarrhea, patent urachus, septic arthritis, and omphalitis were significantly more common in CI foals. Overall survival to discharge (excluding fatal congenital defects) was comparable between crias (174/224; 77.8%) and foals (287/347, 82.1%), while crias (26/48; 54.2%) were more likely than foals (21/60; 35%) to die naturally than undergo euthanasia. Lower respiratory disease and indications for oxygen or IV glucose support increased mortality in the multivariate outcome models of both species. Species-specific adaptations of pediatric diagnostic criteria for sepsis were significantly associated with mortality in the multivariate analysis of patients with complete hematological datasets. However, the diagnosis of systemic inflammatory response syndrome (SIRS) did not retain statistical significance as an independent outcome predictor. CONCLUSIONS: Lower respiratory disease and oxygen or glucose dysregulation increased mortality irrespective of species. However, despite species-specific differences in disease prevalence, the success of intensive care management was comparable.

Assessment of clinical and microbiota responses to fecal microbial transplantation in adult horses with diarrhea.

BACKGROUND AND AIMS: Fecal microbial transplantation (FMT) is empirically implemented in horses with colitis to facilitate resolution of diarrhea. The purpose of this study was to assess FMT as a clinical treatment and modulator of fecal microbiota in hospitalized horses with colitis. METHODS: A total of 22 horses with moderate to severe diarrhea, consistent with a diagnosis of colitis, were enrolled at two referral hospitals (L1: n = 12; L2: n = 10). FMT was performed in all 12 patients on 3 consecutive days at L1, while treatment at L2 consisted of standard care without FMT. Manure was collected once daily for 4 days from the rectum in all colitis horses, prior to FMT for horses at L1, and from each manure sample used for FMT. Fecal samples from 10 clinically healthy control horses housed at L2, and 30 healthy horses located at 5 barns in regional proximity to L1 were also obtained to characterize the regional healthy equine microbiome. All fecal microbiota were analyzed using 16S amplicon sequencing. RESULTS AND CONCLUSIONS: As expected, healthy horses at both locations showed a greater α-diversity and lower ß-diversity compared to horses with colitis. The fecal microbiome of healthy horses clustered by location, with L1 horses showing a higher prevalence of Kiritimatiellaeota. Improved manure consistency (lower diarrhea score) was associated with a greater α-diversity in horses with colitis at both locations (L1: r = -0.385, P = 0.006; L2: r = -0.479, P = 0.002). Fecal transplant recipients demonstrated a greater overall reduction in diarrhea score (median: 4±3 grades), compared to untreated horses (median: 1.5±3 grades, P = 0.021), with a higher incidence in day-over-day improvement in diarrhea (22/36 (61%) vs. 10/28 (36%) instances, P = 0.011). When comparing microbiota of diseased horses at study conclusion to that of healthy controls, FMT-treated horses showed a lower mean UniFrac distance (0.53±0.27) than untreated horses (0.62±0.26, P<0.001), indicating greater normalization of the microbiome in FMT-treated patients.

Assessment of the impact of age and of blood-derived inflammatory markers in horses with colitis.

OBJECTIVES: To determine the impact of age on survival in horses with colitis and to elucidate whether a lower type-1/type-2 cytokine ratio or an exaggerated inflammatory state contribute to reduced survival in aged horses. DESIGN: Part 1: Retrospective cohort analysis. Part 2: Analytic observational study. ANIMALS: Part 1: One hundred twenty-four adult horses with colitis. Part 2: Twenty-nine adult horses with new diarrhea onset while hospitalized. MEASUREMENTS AND MAIN RESULTS: Part 1: Patient signalment, select clinicopathological data, diagnoses, treatment, hospitalization length, and invoice were compared between survivors (n = 101) and nonsurvivors (n = 23). Only age and plasma transfusion retained statistical significance in the final multivariate outcome model, with 8.5 times lower odds of survival in transfused horses (95% confidence interval [CI], 2.6-27.2%). Additionally, the likelihood of nonsurvival increased by 11.8% (95% CI, 4-20.2%) for every year the horse aged (P = 0.002). Similarly, geriatric horses (≥20 years) were 15.2 times more likely to die than young-adults (2-12 years, P = 0.03), independent of financial investment, documented comorbidities, and duration of hospitalization. Part 2: Select cytokine analyses were performed on serum collected from hospitalized horses within 1 hour of diarrhea onset (T0) and 6 hours later. At T0, all recorded clinicopathological variables were comparable between geriatric and young-adult horses, suggesting a similar degree of systemic illness. The median concentration of type-2 cytokines interleukin-4 and interleukin-10, and type-1 cytokine interferon-γ did not differ between age groups. Inflammatory cytokines interleukin-6 and tumor necrosis factor-α were significantly higher in geriatric compared to young-adult horses at both sampling time points. CONCLUSIONS: Outcome of colitis was less favorable in aging horses and patients receiving a plasma transfusion. Although an exaggerated inflammatory state, based on increased interleukin-6 and tumor necrosis factor-α concentrations, in geriatric horses may contribute to reduced survival, a lower type-1/type-2 cytokines ratio was not identified in our geriatric population.

Comparison of Flowmetric Plethysmography and Forced Oscillatory Mechanics to Measure Airway Hyperresponsiveness in Horses.

Airway hyperresponsiveness (AHR) is linked to airway inflammation and is considered a key manifestation of mild/moderate equine asthma (EA). The study purpose was to determine whether two modalities of non-invasive lung function testing (FOM-forced oscillatory mechanics vs. FP-flowmetric plethysmography) establish the same clinical diagnosis of AHR in horses, using histamine bronchoprovocation. Nineteen horses (3-25 years, 335-650 kg) with clinical signs suggestive of mild/moderate equine asthma were enrolled. FOM and FP testing was performed in each horse on two consecutive days, using a randomized cross-over design. AHR was defined by the histamine dose needed to double FOM baseline resistance, or to achieve a 35% increase in FP delta flow. Bronchoalveolar lavage fluid (BALF) was subsequently collected and stained with modified Wright's and toluidine blue stains. Binary statistical tests (related samples T-test, Mann-Whitney U, Chi-square analyses) were performed to compare study groups, with P < 0.05 considered significant. Abnormal BALF cytology confirmed EA in 14/19 (73.7%) horses. Both FOM and FP revealed AHR in 7/14 (50%) of these EA horses. An additional 4/19 (21.1%) horses showed AHR based on FP but not FOM, including two horses with normal BALF cytology. A diagnosis of AHR was more often associated with FP than FOM (P = 0.013), although the prevalence of AHR was significantly higher in EA vs. non-EA horses, regardless of testing methodology. The phase angle between thoracic and abdominal components of breathing did not differ between test groups. In conclusion, FP diagnosed AHR more frequently than did FOM, including horses with no other diagnostic evidence of EA. Without further evaluation, these two testing modalities of AHR cannot be used interchangeably.

The fecal microbiota of healthy donor horses and geriatric recipients undergoing fecal microbial transplantation for the treatment of diarrhea.

BACKGROUND AND AIMS: Fecal microbial transplantation (FMT), a treatment for certain gastrointestinal conditions associated with dysbiosis in people, is also empirically employed in horses with colitis. This study used microbiota high-throughput sequencing to compare the fecal microbial profile of healthy horses to that of geriatric microbial transplant recipients experiencing diarrhea and tested whether FMT restores microbiota diversity. METHODS: To evaluate the effect of environment and donor characteristics on the intestinal microbiota, fecal samples were collected per rectum from 15 healthy young-adult (2-12 years) and 15 geriatric (≥20 years) horses. Additionally, FMT was performed for 3 consecutive days in 5 geriatric horses with diarrhea using feces from the same healthy donor. Fecal samples were collected from both donor and recipient prior to each FMT and from recipients 24 hours following the last FMT. The profile of the fecal bacterial microbiota was compared using 16S amplicon sequencing. RESULTS AND CONCLUSIONS: In contrast to diet and farm location, age did not significantly affect the healthy equine fecal microbiota, indicating that both healthy geriatric and young-adult horses may serve as FMT donors. The fecal microbiota of horses with diarrhea was significantly more variable in terms of ß-diversity than that of healthy horses. An inverse correlation between diarrhea score and relative abundance of Verrucomicrobia was identified in surviving FMT recipients. At study completion, the fecal microbiota of horses which responded to FMT had a higher α-diversity than prior to treatment and was phylogenetically more similar to that of the donor.