Modified Mouse Model of Clostridioides difficile Infection as a Platform for Probiotic Efficacy Studies.

Probiotics may represent a promising approach for reducing Clostridioides (Clostridium) difficile infections (CDIs). A clinical trial conducted by our group demonstrated that CDI patients undergoing adjunctive treatment with Lactobacillus and Bifidobacterium probiotics had a reduction in diarrheal duration and compositional changes in their stool microbiomes. Here, we modified a CDI mouse model to represent clinical outcomes observed in patients and employed this model to identify evidence for the prevention of primary CDI and relapse with the same probiotic. Mice (n = 80) were administered 0.25 mg/ml cefoperazone over 5 days and subsequently challenged with 102C. difficile VPI 10463 spores. A subset of mice (n = 40) were administered 108 CFU of probiotics daily alongside cefoperazone pretreatment and until experimental endpoints were reached. Clinical scoring was performed daily on mice and used to evaluate CDI onset and severity. Moderate CDI in mice was defined by survival beyond day 3 postinfection, while mice with severe CDI were those who succumbed to infection prior to day 3 postinfection. Sequencing and analysis of 16S rRNA from stool content were performed to determine compositional alterations to the microbiota. Using total clinical scores, we identified an association between probiotic treatment and delayed onset of primary CDI and relapse by approximately 12 to 24 h (P < 0.001). The stool microbiome of mice with moderate CDI receiving probiotic treatment was significantly enriched with Lachnospiraceae during primary CDI (P < 0.05). The outcomes observed present an opportunity to use this modified CDI mouse model to examine the efficacy of nonantibiotic options for CDI management.

Enhanced bactericidal activity against Escherichia coli in calves fed Morinda citrifolia (Noni) puree.

BACKGROUND: Although adequate colostrum intake and properly used antibiotics can provide much protection for the bovine neonate, increased antibiotic scrutiny and consumer demand for organic products have prompted investigations of natural immunomodulators for enhancing calf health. One plant-based immunomodulator, Morinda citrifolia (noni) fruit, is a well-recognized natural product that has a broad range of immunomodulatory effects. HYPOTHESIS: Neonatal calves fed noni puree would demonstrate whole blood phagocytic capacity in Gram-negative and Gram-positive in vitro assays. ANIMALS: Blood samples from 18 neonatal Holstein bull calves. METHODS: Calves were divided into 2 groups: Group 1 comprised control calves, whereas Group 2 received 30 mL of noni puree twice a day in milk replacer. Day 0 blood samples were obtained between 36 and 48 hours of age before the first feeding of puree. Ethylenediaminetetraacetic acid anticoagulated blood was collected from each calf on days 0, 3, 7, and 14. Bactericidal assays were performed to estimate the percentage killing of Escherichia coli and Staphylococcus epidermidis. RESULTS: Blood samples from noni puree-fed calves displayed significantly more E. coli bacterial killing than did controls on day 14, and although differences were not significant on days 0, 3, and 7, bacterial killing progressively increased over time. There was no significant difference between the groups for S. epidermidis killing. CONCLUSIONS AND CLINICAL IMPORTANCE: The immunomodulatory effect of noni puree may prove valuable in the future as production animal antibiotic use becomes more restricted. Additional clinical trials are warranted to investigate the clinical application of noni puree in promoting calf health.

Quality Control of Compounded Crystalloid Fluids for Intravenous Delivery to Horses.

BACKGROUND: Periodic lack of availability and high cost of commercially produced isotonic fluids for intravenous (IV) use in horses have increasingly led to use of home-made or commercially compound fluids by veterinarians. Data regarding the quality control and safety of compounded fluids would be of benefit to equine veterinarians. OBJECTIVES: To compare electrolyte concentrations, sterility, and endotoxin contamination of commercially available fluids to 2 forms of compounded isotonic crystalloid fluids intended for IV use in horses. METHODS: Prospective study. Two methods of preparing compounded crystalloids formulated to replicate commercial Plasma-Lyte A (Abbott, Chicago, IL) were compared. One formulation was prepared by a hand-mixed method involving chlorinated drinking water commonly employed by equine practitioners, and the other was prepared by means of ingredients obtained from a commercial compounding pharmacy. The variables for comparison were electrolyte concentrations, sterility, and presence of endotoxin contamination. RESULTS: Electrolyte concentrations were consistent within each product but different between types of fluids (P < 0.0001). Hand-mixed fluids had significantly more bacterial contamination compared to commercial Plasma-Lyte A (P = 0.0014). One of the hand-mixed fluid samples had detectable endotoxin contamination. CONCLUSIONS AND CLINICAL IMPORTANCE: Chlorinated drinking water is not an acceptable source of water to compound isotonic fluids for IV administration. Equine practitioners should be aware of this risk and obtain the informed consent of their clients.

Leucocytoclastic vasculitis associated with Staphylococcus intermedius in the pastern of a horse.

A pregnant quarterhorse mare became acutely lame as a result of severe swelling of its right hind leg, thought to have been caused by a fracture or a muscle tear. Diagnostic procedures ruled out a traumatic musculoskeletal cause and a physical examination revealed chronic pastern dermatitis ('scratches'/'grease heel'). Histopathological evaluation of biopsy samples from the right hind leg was consistent with a leucocytoclastic vasculitis, and culture yielded Staphylococcus intermedius. The treatment and infectious causes of pastern dermatitis are discussed.

SK&F 86002, a dual cytokine and eicosanoid inhibitor, attenuates endotoxin-induced cardiopulmonary dysfunction in the pig.

Cytokines and eicosanoids are well documented important mediators of endotoxemia. Bicyclic imidazoles are a novel class of nonsteroidal anti-inflammatory compounds that display unique pharmacological profiles by reducing cytokine production and arachidonic acid metabolism. In this study, we evaluated the ability of the bicyclic imidazole, SK&F 86002, to attenuate endotoxin-induced cardiopulmonary dysfunction. Pigs were randomly assigned to one of four groups: LPS (n = 5), given .5 microgram/kg/h 055:B5 Escherichia coli lipopolysaccharide (LPS) intravenously (i.v.) for 6 h; saline (n = 5); SK&F 86002 (n = 3), given 50 mg/kg SK&F 86002 orally 30 min prior to anesthesia; and SK&F 86002 + LPS (n = 5). Administration of LPS resulted in cardiopulmonary dysfunction characterized by decreased stroke volume and arterial oxygen tension, and increased room air alveolar-arterial oxygen gradient, pulmonary arterial pressure, pulmonary vascular resistance, and peak intratracheal pressure. Additionally, LPS administration was associated with leukopenia and increased pulmonary myeloperoxidase activity. Pretreatment with SK&F 86002 attenuated LPS induced hypotension, hypoxemia and bronchoconstriction and blocked the pulmonary hypertension. SK&F 86002 blocked the LPS-induced increase in myeloperoxidase activity, indicating a reduction in pulmonary neutrophil infiltration, but had no effect on systemic leukopenia. Pretreatment with SK&F 86002 significantly attenuated LPS-induced increases in plasma thromboxane B2 and tumor necrosis factor-alpha. We hypothesize that ameliorating effects of SK&F 86002 in this endotoxin model of cardiopulmonary dysfunction are related to inhibition of cytokine and eicosanoid biosynthesis.

Morphometric analysis of oleic acid-induced permeability pulmonary edema: correlation with gravimetric lung water.

The technique used most commonly to quantitate pulmonary edema in in vivo animal models is postmortem gravimetric analysis (wet:dry) ratio. To determine whether lung water can be quantitated morphometrically, as accurately as by the commonly used gravimetric analysis, perivascular edema (cuff) area to vessel area ratio was correlated to wet:dry ratio. Anesthetized pigs were given either oleic acid (20 mg/kg/h, intravenously) or physiologic saline. At 4 h, lungs were excised and cuff:vessel and wet:dry ratio analysis was performed. The intermediate lobe was clamped across its main stem bronchus to maintain peak inspiratory inflation, excised, frozen in liquid nitrogen, and stored at -70 degrees C until cryostat sectioning and quantification of perivascular interstitial edema (cuff) area. Gravimetric analysis (wet:dry ratio) was performed on the remaining lung. Mean cuff:vessel and wet:dry analyzes showed that lung water increased significantly (p < .01) in the oleic-acid treated group (4.9 +/- .22 and 6.78 +/- .47, respectively), compared with the saline group (.03 +/- .02 and 2.55 +/- .27, respectively). The correlation coefficient between mean cuff:vessel and wet:dry ratios was .86 (p = .0016). This study demonstrates that cuff:vessel ratio analysis can be used to identify the distribution of edema fluid versus vessel diameter, and seems to be as effective a technique as gravimetric analysis to quantitate lung water changes in acute lung injury models. Moreover cuff:vessel ratio analysis can differentiate modest changes in pulmonary edema by direct quantitation, an important end-point not provided by wet:dry analysis. Therefore, it may be a more sensitive technique when investigating therapeutic interventions in in vivo models of acute lung injury.

Low molecular weight heparin prevents the pulmonary hemodynamic and pathomorphologic effects of endotoxin in a porcine acute lung injury model.

Tumor necrosis factor alpha (TNF-alpha) activity, platelet and neutrophil degranulation and margination, and increased vascular permeability are central to the pathophysiology of endotoxin-mediated acute lung injury. Nonanticoagulant activities of low molecular weight heparin (LMWH) include solubilization of the TNF-alpha receptor protein, inhibition of neutrophil adhesion, and regulation of thromboxane B2 (TXB2) biosynthesis. In this study, we evaluated the ability of LMWH to modulate TNF-alpha and TXB2 activity during endotoxemia and the subsequent effects on pulmonary hemodynamics. Domestic pigs 8-10 weeks old were anesthetized and catheterized for standard cardiopulmonary measurements and the lungs harvested for cuff:vessel ratio, myeloperoxidase activity, and permeability index. Pigs were randomly assigned to one of four groups: lipopolysaccharide (LPS) (n = 6), given .5 microg/kg/h Escherichia coli LPS intravenously for 6 h; saline control (n = 5); LMWH (n = 5), given .5 mg/kg LMWH for 30 min, followed by .5 mg/kg/h; and LMWH + LPS (same dosages, n = 6). Administration of LPS resulted in increased plasma TNF-alpha and TXB2 activity; increased pulmonary arterial pressure, pulmonary vascular resistance, and alveolar-arterial oxygen tension; decreased systemic arterial oxygen tension; and pulmonary edema. The cardiopulmonary parameters for the LMWH-treated pigs did not differ from those of the saline-treated control pigs. Pretreatment with LMWH attenuated the LPS-mediated TNF-alpha and TXB2 activity and attenuated LPS-mediated pulmonary hypertension, hypoxemia and neutrophil emigration, and edema formation. In conclusion, the data show that the protective effects of LMWH in this model of acute lung injury are associated with altered neutrophil adhesion and TNF-alpha and thromboxane activity.

Antithrombin III activity in horses with colic: an analysis of 46 cases.

Antithrombin-III (AT-III) activity was measured at the time of admission in 46 horses referred with colic. Differences in AT-III activities between animals treated medically or surgically and survivors or non-survivors were compared. The mean AT-III values for the horses treated medically (76.2 per cent), surgical survivors (69.5 per cent) and surgical non-survivors (55.9 per cent) were significantly different from the reference value for healthy adults (92 to 108 per cent). The mean AT-III activity of the survivors was significantly (P less than 0.01) greater than that of the nonsurvivors. The majority of the survivors (11 to 13 horses) had AT-III activities exceeding 60 per cent of normal, whereas that of 10 of the 14 non-survivors was less than 60 per cent of normal. There was an apparent distinction between the survivors and non-survivors at approximately 60 per cent of normal AT-III activity.

Comparison of transtracheal aspirate and bronchoalveolar lavage cytology in 50 horses with chronic lung disease.

Comparisons were made between transtracheal aspirate (TTA) and bronchoalveolar lavage (BAL) cytology obtained from 50 horses with chronic lung disease and from 10 control horses. There was no significant correlation between the TTA cytology and the BAL cytology, suggesting that the cell population in the trachea is not representative of the cell population in the lower airways. In control horses the range of differential cell counts obtained from TTA fluid was remarkably large, whereas the variability in cell populations observed in BAL fluid was smaller. In the principal horses the total and differential cell counts of the TTA and BAL fluids were within the 95 per cent confidence interval in 38 and 24 per cent of cases, respectively; and an increase in percentage neutrophils was most common. It was concluded that BAL may be a useful diagnostic aid when evaluating horses with chronic lung disease, but that the clinical usefulness of cytological evaluations of TTA fluid may be limited in these cases.

A tracheoscopic technique for obtaining uncontaminated lower airway secretions for bacterial culture in the horse.

Transtracheal aspirate (TTA) aerobic microbiology culture results from 30 ponies were compared to protected catheter brush (PCB) culture results obtained from the same ponies. The PCB procedure resulted in significantly fewer (P less than 0.05) isolates than those obtained by the TTA procedure and the PCB procedure resulted in significantly more samples with no bacteria growth than the TTA procedure. To address the possibility that the volume of tracheobronchial secretion obtained by PCB was insufficient as a microbiological sample, a sterile, double-sheathed, protected aspiration catheter (PAC) was developed. Comparisons were made between TTA and PAC aerobic microbiology culture results from 18 pneumonic foals and weanlings. There were no significant differences between the PAC and TTA procedures with respect to the number of bacteria isolates. It was concluded that the PAC technique may be used as an alternative to TTA for isolation of equine pulmonary bacteria and may be the preferred procedure because of the reduced risk of complications associated with the TTA.

Evaluation of transabdominal ultrasound-guided amniocentesis in the late gestational mare.

Serial transabdominal amniocentesis was performed on eight mares in late gestation. Ultrasonography was used to identify the two placental fluid sacs and the foetus. A biopsy guide attached to the transducer allowed visualisation of the needle path during insertion. Amniotic fluid was obtained in 85 per cent of the attempts, although as mares approached parturition substantial pools of amniotic fluid were more difficult to locate. Two mares aborted during the study probably because of complications of the procedures, whereas six mares produced normal foals at term. The abortions preclude clinical use of this technique currently. However, there was sufficient success in retrieval of amniotic fluid and outcome of pregnancies to continue the study of amniocentesis in larger groups of mares to determine the clinical and research value of this technique.

Cerebrospinal fluid changes in two horses with central nervous system nematodiasis (Micronema deletrix).

Two horses with cerebrospinal nematodiasis (Micronema deletrix) had signs similar to those of other neurologic diseases resulting from parasitic (fly larvae, protozoa, or other helminths) migration through the central nervous system (CNS). In one horse (horse 1), a 13-year-old Paso Fino stallion, the cerebrospinal fluid (CSF) was slightly xanthochromic (1+), with a pleocytosis (25 nucleated cells/microliter) and a normal protein level (69 mg/dl). A CSF differential cell count showed 15% neutrophils, 56% lymphocytes, 22% macrophages, 5% eosinophils, and 2% basophils. In the other horse (horse 2), a 19-year-old Tennessee Walking Horse stallion, the CSF was modestly xanthochromic (2+), with pleocytosis (81 nucleated cells/microliter) and a modestly elevated protein concentration (114 mg/dl). A CSF differential cell count showed 9% neutrophils, 41% lymphocytes, and 50% macrophages. The CSF changes were consistent with those described for equine protozoal myeloencephalitis and verminous encephalitis. The microscopic lesions in both brains contained multifocal areas of malacia and granulomatous inflammation. Meningeal vessels throughout the brain were greatly thickened and inflamed, and they contained parasites. The CSF changes were not specific and histopathologic examination was required for a definitive diagnosis.

Persistent urachal remnant causing intestinal strangulation in a cow.

A persistent urachal remnant causing small intestinal strangulation was found on exploratory laparotomy in a 2-year-old cow with colic. The persistent urachus consisted of a thin cord-like band that was attached ventrally to the umbilical region and caudally to the apex of the bladder. The band had formed an internal loop and had strangulated 6 to 8 cm of the distal portion of the ileum. The persistent urachus was detached manually at the umbilicus, ligated at the apex of the bladder, and removed; intestinal resection was not necessary. The urachal remnant was probably a congenital abnormality that may have become stretched during pregnancy, forming an internal hernial loop.

Use of a modified Roux-en-Y procedure for treatment of pyloroduodenal obstruction in a horse.

A modification of the Roux-en-Y anastomosis procedure was used to bypass a pyloroduodenal mass in a 12-year-old Arabian stallion. Clinical signs had consisted of a 4-week progression of ventral and hind limb edema, hypoproteinemia, fecal occult blood, intermittent abdominal pain, weight loss, and gastric reflux. On exploratory celiotomy, an obstructive mass was found in the pylorus and proximal portion of the duodenum. Gastrojejunostomy and duodenojejunostomy were performed by use of stapled side-to-side anastomosis techniques. Inaccessibility of the obstructed pyloric region prevented resection of the affected area.

Actinobacillus suis septicaemia in two foals.

A 24-hour-old Hackney ony filly developed signs of weakness, depression and a poor suck reflex, with harsh lung sounds over both fields, and a 48-hour-old Arabian colt from a normal birth which had sucked vigorously developed loose stools and became depressed, weak and anorectic. Both foals had serum IgG concentrations greater than 800 mg/dl, but each had a severe neutropenia with a left shift, and blood cultures from both of them yielded Actinobacillus suis. The A suis isolates had different antimicrobial susceptibility patterns and, in the case of the Arabian, the isolate was resistant to commonly used broad spectrum antimicrobial agents.

Congenital hepatoblastoma in a neonatal alpaca cria.

A neonatal alpaca cria found to have minimal clinical abnormalities was diagnosed postmortem with an epithelial-type hepatoblastoma with combined embryonal and fetal patterns, based on previously reported morphological features. Camelid neoplasia and domestic animal hepatoblastomas are very rare, with only a single case of congenital hepatoblastoma in a domestic animal previously reported.

Antithrombin III activity (residual thrombin activity) in plasma from non-medicated or heparinized horses.

Two synthetic substrate assays (fluorometric and chromogenic) were used to measure antithrombin-III (AT-III) activity (residual thrombin activity) in non-medicated and heparin (sodium) treated horses. In 18 non-medicated horses the fluorometric substrate assay (FSA) values were similar to previous reports but they reflected inconsistent trends and larger deviations in the heparin-treated groups (Group 2: 40 and 100 U/kg IV, n = 6; Group 3: 240 U/kg IV, n = 5; Group 4: 80 U/kg IV followed by 160 U/kg SC, n = 8) when compared to the chromogenic substrate assay (CSA) values. The CSA values for the 18 non-medicated horses indicated a higher AT-III activity (lower residual thrombin activity) than the FSA. AT-III activity was quantified in 18 non-medicated horses (29 mg/dl) and compared well with values for humans (30 mg/dl) and dogs (40 mg/dl). Plasma heparin concentrations, determined by the FSA, correlated well with the 'therapeutic range' (1.5 fold to 2.5 fold prolongation of the activated partial thromboplastin time (APTT) normal value) and values reported for humans. The effect of heparin therapy on AT-III activity in four treatment regimens was evaluated. AT-III activity was not significantly affected (with one exception) by a single dose of intravenous (IV) heparin (40 and 100 U/kg) nor by repeated subcutaneous (SC) injections of heparin (240 U/kg). A transient increase in residual thrombin activity was measured 12 h after an intravenous (80 U/kg) injection of heparin. Large doses of heparin (80 U/kg IV followed by 160 U/kg SC) given every 12 h produced a progressive prolongation of the APTT. In this group the APTT remained prolonged 48 h after the last treatment.

Ethnoveterinary application of Morinda citrifolia fruit puree on a commercial heifer rearing facility with endemic salmonellosis.

We have previously reported that Morinda citrifolia (noni) puree modulates neonatal calves developmental maturation of the innate and adaptive immune system. In this study, the effect of noni puree on respiratory and gastrointestinal (GI), health in preweaned dairy calves on a farm with endemic salmonellosis was examined. Two clinical trials were conducted whereby each trial evaluated one processing technique of noni puree. Trials 1 and 2 tested noni versions A and B, respectively. Puree analysis and trial methods were identical to each other, with the calf as the experimental unit. Calves were designated to 1 of 3 treatment groups in each trial and received either: 0, 15 or 30 mL every 12 hr of noni supplement for the first 3 weeks of life. Health scores, weaning age, weight gain from admission to weaning, and weaned by 6 weeks, were used as clinical endpoints for statistical analysis. In trial 1, calves supplemented with 15 mL noni puree of version A every 12 hr had a higher probability of being weaned by 6 weeks of age than control calves (P = 0.04). In trial 2, calves receiving 30 mL of version B every 12 hr had a 54.5% reduction in total medical treatments by 42 days of age when compared to controls (P = 0.02). There was a trend in reduced respiratory (61%), and GI (52%) medical treatments per calf when compared to controls (P = 0.06 and 0.08, respectively). There were no differences in weight gain or mortality for any treatment group in either trial.