Integrated analysis of gut metabolome, microbiome, and exfoliome data in an equine model of intestinal injury.

BACKGROUND: The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-steroidal anti-inflammatory drug (NSAID)-induced intestinal inflammation and (2) apply computational data integration methods to examine host-microbiota interactions. METHODS: Twenty horses were randomly assigned to 1 of 2 groups (n = 10): control (placebo paste) or NSAID (phenylbutazone 4.4 mg/kg orally once daily for 9 days). Fecal samples were collected on days 0 and 10 and analyzed with respect to microbiota (16S rDNA gene sequencing), metabolomic (untargeted metabolites), and host exfoliated cell transcriptomic (exfoliome) changes. Data were analyzed and integrated using a variety of computational techniques, and underlying regulatory mechanisms were inferred from features that were commonly identified by all computational approaches. RESULTS: Phenylbutazone induced alterations in the microbiota, metabolome, and host transcriptome. Data integration identified correlation of specific bacterial genera with expression of several genes and metabolites that were linked to oxidative stress. Concomitant microbiota and metabolite changes resulted in the initiation of endoplasmic reticulum stress and unfolded protein response within the intestinal mucosa. CONCLUSIONS: Results of integrative analysis identified an important role for oxidative stress, and subsequent cell signaling responses, in a large animal model of GI inflammation. The computational approaches for combining non-invasive platforms for unbiased assessment of host GI responses (e.g., exfoliomics) with metabolomic and microbiota changes have broad application for the field of gastroenterology. Video Abstract.

Retrospective Analysis of Aetiological Agents Associated with Pulmonary Mycosis Secondary to Enteric Salmonellosis in Six Horses by Panfungal Polymerase Chain Reaction.

Pulmonary mycosis secondary to enterocolitis is an uncommon diagnosis in equine medicine, but is thought to result from mucosal compromise and translocation of enteric fungi. The aetiological agent associated with translocation is often identified based on fungal culture or hyphal features in histological sections. In order to understand better the aetiological agents involved, six horses diagnosed with Salmonella enteritis and concurrent pulmonary mycosis were identified retrospectively through a database search of veterinary teaching hospital records. Samples from these cases were subjected to polymerase chain reaction and sequencing of the internal transcribed spacer 2 (ITS-2) located between the 5.8S and 28S rRNA genes to identify the aetiological agent involved. Sequencing identified Aspergillus fumigatus, Aspergillus flavus, Fusarium spp., Cladosporium spp. and Curvularia spp. A single case had a dual infection with Fusarium spp. and A. fumigatus.

Culture-independent and dependent evaluation of the equine paranasal sinus microbiota in health and disease.

BACKGROUND: Horses with bacterial sinusitis frequently undergo empirical treatment with antimicrobials, however, in some cases bacterial culture of the affected sinus is used to direct therapy. Data regarding which organisms are part of the commensal microbiota of the equine sinus are lacking making it difficult to interpret culture results and guide empiric antimicrobial selection. OBJECTIVES: Our objectives were to describe the bacterial and fungal microbiota of the paranasal sinuses in clinically normal horses using culture-dependent and independent approaches and to compare the bacterial culture and susceptibility patterns of normal horses with those from horses affected with primary and secondary sinusitis. STUDY DESIGN: Experimental study and descriptive retrospective review of case records. METHODS: Sinus washes were collected from 23 healthy horses. Washes were submitted for routine culture and susceptibility testing and DNA was isolated for next generation sequencing of bacterial and fungal marker genes. For clinical cases of sinusitis, medical records from 2010 to 2017 were reviewed and horses diagnosed with primary and/or secondary sinusitis were included. RESULTS: The paranasal sinus cavity hosts multiple bacterial and fungal organisms. The bacterial microbiota of healthy horses consists largely of uncultivable, aerobic bacteria. While few anaerobes were isolated from normal horses, the majority of clinical cases resulted in growth of anaerobic organisms with no difference in the proportion of aerobic and anaerobic bacteria isolated from clinical cases. MAIN LIMITATIONS: Small sample size in both populations of horses and heterogeneity of the population prevent a more in-depth analysis. CONCLUSIONS: The microbiota of the paranasal sinuses of horses consists primarily of aerobic bacteria and fungal organisms, the majority of which are uncultivable via common clinical methods. Anaerobic bacteria are found in the majority of horses with clinical sinusitis. These findings suggest anaerobic bacteria are associated with sinusitis and their presence should be considered when treating horses with sinusitis.

Novel lipoprotein density profiling in laminitic, obese, and healthy horses.

Lipoproteins are water-miscible macromolecules enabling the transport of lipids in blood. In humans, altered proportions of lipoproteins are used to detect and classify metabolic diseases. Obesity and obesity-related comorbidities are common in horses. The pathophysiology of obesity is poorly understood and likely multifactorial. Development of new diagnostic tests to identify horses at risk of developing obesity to implement preventative measures is critical; however, a necessary first step to accomplish this goal is to improve our understanding of the pathophysiology of disease. Thus, the objective of this study was to characterize and compare lipoprotein profiles of horses with normal and excess body conditions, with and without laminitis using a novel method of continuous lipoprotein density profiling (CLPDP). Comparisons were made between 4 groups of horses: (1) laminitic, obese horses (n = 66); (2) laminitic, nonobese horses (n = 35); (3) nonlaminitic, obese horses (n = 41); and (4) nonlaminitic, nonobese horses (n = 95). Lipoprotein profiling, including evaluation of triglyceride-rich lipoprotein (TRL), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs) was performed using CLPDP, and all 4 groups were compared. A significant difference was observed among groups for the subfractions TRL, LDL1, LDL2, HDL2b, HDL2a, HDL3a, HDL3b, HDL3c, and total HDL. This is the first known description of CLPDP to characterize equine lipid profiles and holds promise as a useful method for lipid characterization of horses.

Ultrasonographic and CT accuracy in localising surgical- or necropsy- confirmed solitary hepatic masses in dogs.

OBJECTIVES: To estimate the sensitivity and specificity of ultrasound and CT for localising solitary hepatic masses to particular liver divisions. MATERIALS AND METHODS: Dogs diagnosed with a solitary liver mass by ultrasound and/or CT, with surgical or necropsy confirmation within 1 month of imaging. Ultrasound reports were reviewed for mass location. CT scans were reviewed by two radiologists and mass location was determined by consensus agreement. Sensitivity and specificity were calculated for ultrasound and CT for each liver division. RESULTS: Fourteen of 71 dogs had CT only, 27 of 71 had ultrasound only and 30 of 71 had both. Location was correctly predicted in 42 of 57 dogs (74%) by ultrasound and in 37 of 44 dogs (84%) by CT. Both CT and ultrasound had high specificity for localising masses in all divisions. Sensitivity varied among divisions and was highest for left division masses. CLINICAL SIGNIFICANCE: The results suggest that either imaging modality is appropriate for presurgical planning. Prospective studies are recommended to help identify additional factors that may aid in determination of hepatic mass location.

Use of Liposomal Gentamicin for Treatment of 5 Foals with Experimentally Induced Rhodococcus equi Pneumonia.

BACKGROUND: Adverse effects of, and bacterial resistance to, macrolides used to treat Rhodococcus equi infections have prompted search for clinically effective alternative antimicrobials. Liposomal gentamicin (LG) is effective against R. equi in vitro and decreases tissue concentrations of R. equi in experimentally infected mice. Effectiveness of LG treatment of foals with R. equi pneumonia, however, has not been described. HYPOTHESIS: Liposomal gentamicin is safe and effective for treating foals with R. equi pneumonia. ANIMALS: Ten foals with experimentally induced R. equi pneumonia. METHODS: Pilot treatment trial. Foals with pneumonia induced by intrabronchial instillation of R. equi were randomly allocated to receive either clarithromycin combined with rifampin (CLR + RIF) PO or LG IV, and followed by daily physical examinations and weekly thoracic ultrasonography and serum creatinine concentration determinations until the resolution of clinical signs. Treatment success was defined as the resolution of clinical signs and ultrasonographically identified pulmonary abscesses. RESULTS: All 10 foals were successfully treated. Two of 5 foals treated with LG developed azotemia within 1 week; LG was discontinued and treatment switched to CLR + RIF for these foals. None of the CLR + RIF treated foals developed azotemia. CONCLUSIONS AND CLINICAL IMPORTANCE: Liposomal gentamicin IV can be effective for treatment of R. equi pneumonia, but nephrotoxicity indicates that an alternative dosing interval or route (such as nebulization) will be needed before LG is adequately safe for clinical use. Larger comparative trials will be needed to evaluate the relative efficacy of a safer LG dosage regimen.

Concurrent ivermectin and Solanum spp. toxicosis in a herd of horses.

BACKGROUND: Representatives from a herd of horses with acute onset of neurologic signs after administration of ivermectin presented for evaluation and treatment. OBJECTIVES: Describe clinical signs of horses intoxicated by ingestion of Solanum sp. and administered ivermectin. ANIMALS: Six of 11 affected unrelated horses presented for evaluation and treatment. The remaining 5 affected horses were treated at the farm. Four additional horses, housed separately, were unaffected. METHODS: Case series is presented. Serum ivermectin concentrations were evaluated in the 6 hospitalized horses. The remnants of the tubes of ivermectin paste were analyzed for ivermectin concentration. The hay fed to the affected horses was analyzed for the presence of toxic plants. RESULTS: Serum ivermectin concentrations were higher than expected, given the dosage of ivermectin administered. The ivermectin concentration remaining in the administration tubes did not exceed specifications. The hay was heavily contaminated by 2 Solanum species. All horses returned to normal neurologic function with supportive care. CONCLUSIONS AND CLINICAL IMPORTANCE: Horses might exhibit signs of ivermectin toxicity after appropriate dosing of the drug if they concurrently consume toxic plants of the Solanum family.

Long-term prognosis of gastrojejunostomy in foals with gastric outflow obstruction: 16 cases (2001-2006).

REASONS FOR PERFORMING STUDY: It has been suggested that the success of gastric bypass surgery in foals for the treatment of gastric outflow obstruction is poor. However, few reports exist evaluating the long-term prognosis of these cases. OBJECTIVES: To determine the long-term success of foals, including racing records, surgically treated for gastric outflow obstruction secondary to gastroduodenal ulceration. METHODS: Medical records of foals undergoing surgical treatment of gastric outflow obstruction secondary to gastroduodenal ulceration were evaluated for clinical information. Owners, trainers and race records were evaluated regarding long-term survival and racing success. RESULTS AND CONCLUSIONS: Sixteen foals were included in the study, all treated with a gastrojejunostomy. All foals survived to immediate discharge from the hospital; 8 survived to racing age, with 7 of those entering training and 3 actually racing. Foals that did not survive to racing age had various post operative complications. The success rate for these foals appears somewhat better than that previously reported. POTENTIAL RELEVANCE: Gastrojejunostomy for the treatment of gastric outflow obstruction, secondary to gastric ulceration, is a valid treatment option for foals.

The effect of fertility control on the transmission of bovine tuberculosis in wild brushtail possums.

AIM: To determine the effect of fertility control on the rate of transmission of bovine tuberculosis (Tb), caused by Mycobacterium bovis, in brushtail possums (Trichosurus vulpecula). METHODS: At two study sites with a history of Tb infection in the resident possum population, a sample of adult male and female possums (n=50), determined by palpation to be Tb-free, was surgically sterilised by gonadectomy. A sample of untreated Tb-free male and female possums (n=54) served as controls. Each study site was trapped every 2 months over a 3-year period, and the Tb status of the individuals in the trial assessed. At the conclusion of the trial, all remaining experimental individuals were killed, necropsied and examined for characteristic lesions typical of Tb. The rate of transmission of Tb infection was estimated using the incidence of tuberculous cases in the experimental animals and comparing it between sites, sex and sterilisation treatment. RESULTS: Sterilisation by gonadectomy resulted in a reduction in the rate of transmission of Tb in male possums by 53%, but a corresponding increase in sterilised females for reasons that are still unclear. However, this interaction was statistically weak (p=0.10). When the sexes were combined, there was no overall effect of sterilisation on the rate of transmission of Tb (p=0.43). Sterility treatment notwithstanding, there was a highly significant difference in the rate of transmission between the sexes (p=0.01), being almost one order of magnitude higher in untreated males compared with untreated females. CONCLUSIONS: Although lacking strong statistical support, these results suggest that fertility control that targets endocrine control of fertility may provide some additional benefits for disease control over that achieved by reductions in population density, by reducing the rate of transmission of M. bovis in male possums. However, additional studies are needed to confirm the validity of these results. The large difference in the rate of transmission of M. bovis in untreated males compared with untreated females suggests that transmission of Tb among males may be an important driver of the dynamics of Tb infection in possums.

Trends in the incidence of tuberculosis in possums and livestock, associated with differing control intensities applied to possum populations.

AIM: To determine the trap-catch index (an estimate of abundance) of brushtail possum (Trichosurus vulpecula) populations infected with bovine tuberculosis (Tb; Mycobacterium bovis) that must be achieved, and the length of time such an index must be maintained, for Tb to be eliminated from possum populations and adjacent livestock. METHODS: Between 1997-1998 and 2000-2001, trap-catch surveys of possum populations naturally infected with Tb and subjected to population-control measures were undertaken at four forest sites and two farmland sites. At the same time, possum carcasses were collected at these sites and their Tb status determined, and all contiguous cattle and deer herds were Tb tested and abattoir slaughter data for these herds were interrogated. RESULTS: Trap-catch surveys indicated that numbers of possums on the farmland sites surveyed were usually very low and well below the control targets set (i.e. a 5% trap catch or approximately 0.5-1 possum/ha) for the study. In contrast, trap-catch surveys undertaken in forest sites indicated possum numbers were more variable, and often recovered rapidly from control operations to exceed control targets within 1-3 years. The annual rate of recovery of possum populations in half of the forest population surveys undertaken exceeded published intrinsic rates of increase for possums. The overall prevalence of Tb in possum populations was < or =1.9% at 5/6 sites, and was 6.5% at the sixth site. Juvenile possums infected with Tb were trapped within but near the edge of control zones and appeared to represent an immigrant source of infection. Mature infected possums survived control operations apparently by having home ranges in uncontrolled patches within control areas. Infection in possums appeared to be eliminated from one study site by the intensive control undertaken, but elimination at other sites appeared less likely. Levels of Tb in livestock on or adjacent to the study sites fell by at least 50% during the study, and cattle in one area tested clear for the first time in 20 years. CONCLUSIONS: Initial control of possums in forest appeared to achieve national control targets set by the Animal Health Board (AHB), despite trap-catch data often providing misleading population estimates. Such targets were often exceeded within 1-3 years. By comparison, possum control on farmland appeared to maintain populations at very low levels, while control on forest margins maintained populations at intermediate levels. Control was least effective in deep forest where human access was most difficult. Intensive population control measures appeared to have led to a reduced incidence of Tb in livestock at 3/4 sites, and elimination of Tb in livestock at one site. This result supports modelling studies that predict the eradication of Tb from possums through ongoing intensive control and may explain the lower success achieved with earlier less-intensive possum control.

Preparation and antimicrobial assessment of 2-thioether-linked quinolonyl-carbapenems.

This reports the synthesis and in vitro antimicrobial properties of a series of 2-thioether-linked quinolonyl-carbapenems. Although the title compounds exhibited broad spectrum activity, the MICs were generally higher than those observed for selected benchmark carbapenems, quinolonyl-penems, and quinolones. Enzyme assays suggested that the title compounds are potent inhibitors of penicillin binding proteins and inefficient inhibitors of bacterial DNA-gyrase. Uptake studies indicated that the new compounds are not substrates for the norA encoded quinolone efflux pump.

Effect of Alternaria solani culture filtrate on adventitious shoot regeneration in potato.

The effect of Alternaria solani culture filtrate on adventitious shoot regeneration from tuber discs was evaluated using five potato cultivars, which were selected based on their field reaction to Alternaria solani and which represented a range of disease reactions. The culture filtrate stimulated regeneration, a response that could prove to be very useful in the wider utilization of transformation and in vitro selection technology.

Synthesis and biological evaluation of 10-oxa-11-deoxyprostaglandin E1 and 10-nor-9,11-secoprostaglandin F1 and their derivatives.

Three 10-oxa-11-deoxyprostaglandin E1 and two 10-nor-9,11-secoprostaglandin F1 analogues were prepared. The compounds were evaluated for pregnancy interruptions, oxytocin-like activity (uterine strip), and antiprostaglandin activity. One of the 10-nor-9,11-secoprostaglandin F1 analogues displayed activity as a PGE2 antagonist in the gerbil colon smooth muscle preparation.

Etonitazene. An improved synthesis.

1-(BETA-Kiethylaminoethyl)-2-(p-ethoxybenzyl)-5-nitrobenzimidazole (u, etonitazene) is a potent analgesic that has value in drug addiction studies. We have developed a simple high-yield synthesis of 1 that is adaptable to alrge scale preparations. The synthesis involves the condensation of 2-(beta-diethylaminoethylamino)-5-mitroaniline and p-ethoxyphenylacetic acid in Thf in the presence of EEDQ.