Modest Vengust

Diversity of Clostridium difficile in pigs and other animals in Slovenia

A study of Clostridium difficile diversity in pigs, calves and horses in Slovenia was conducted. A total of 547 samples were collected and C. difficile was isolated from 247/485 (50.9%) piglet samples, from 4/42 (9.5%) calf samples, and 1/20 (5%) horse samples. The isolates were characterized by toxinotyping, PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) using restriction endonuclease SmaI. Piglet isolates belonged to two toxinotypes (V and 0), four PCR-ribotypes (066, 029, SI 011, SI 010), and six pulsotypes. Bovine isolates were grouped into two toxinotypes (XIa and 0), three PCR-ribotypes (077, 002, 033), and three pulsotypes. The only equine isolate was indistinguishable from one calf isolate (XIa/033) in toxinotype, PCR-ribotype, and pulsotype. None of detected genotypes was present in all three animal hosts.

Equid Herpesvirus 2-Associated Oral and Esophageal Ulceration in a Foal

A case of a 1-month-old Thoroughbred foal with dysphagia, salivation, pyrexia, oral mucosal pustules, and esophageal ulceration is reported. Swabs from the ulcerated lesions yielded Equid herpesvirus 2 (EHV-2) in virus isolation assays, and histopathology of a biopsy from the esophageal lesion identified nuclear inclusions suggestive of herpesviruses. Immunohistochemical staining with antibodies specific for EHV-2 was positive for epithelial cells in the vicinity of the ulcer but not in more distant mucosa. Electron microscopic evaluation of the biopsy showed herpesviral particles in epithelial cells. The foal recovered over 5 days of supportive and gastroprotective therapy, and the esophageal ulcers healed. Serology and immunohistochemistry indicated that this foal likely had lesions associated with EHV-2 and not EHV-1, −4, or −5.

Transvascular fluid flux from the pulmonary vasculature at rest and during exercise in horses

Exercise causes changes in pulmonary haemodynamics through redistribution of blood flow, increase in the pulmonary surface area, and increase in pulmonary vascular pressures. These changes contribute to the increase in fluid exchange across the alveolar–capillary barrier. To determine the extent of the fluid exchange across the alveolar–capillary barrier at rest and during exercise, six horses were exercised on a high‐speed treadmill until fatigue. Arterial and mixed venous blood were sampled at rest and during exercise and recovery. Blood volume changes across the lung (ΔBV; measured in percentage) were calculated from changes in plasma protein and haemoglobin concentration, and haematocrit. Cardiac output (Q) was calculated using the Fick equation. Fluid flux (JV−A; measured in l min−1) across the alveolar–capillary barrier was then quantified based on Q and ΔBV. At rest, no fluid movement occurred across the pulmonary vasculature (0.6 ± 0.6 l min−1). During exercise, the amount of fluid moved from the pulmonary circulation was 8.3 ± 1.3 l min−1 at 1 min, 6.4 ± 2.9 l min−1 at 2 min, 10.1 ± 1.0 l min−1 at 3 min, 12.9 ± 2.5 l min−1 at 4 and 9.6 ± 1.5 l min−1 at fatigue (all P < 0.0001). Erythrocyte volume decreased by 6% (P < 0.01) across the lungs, which decreased the colloid osmotic gradient in the pulmonary vasculature. Decrease colloid osmotic gradient along with increased hydrostatic forces in the pulmonary vasculature would enhance displacement of fluid into the pulmonary interstitium. In conclusion, exercise caused large increases in transpulmonary fluid fluxes in horses. Here, we present a simple method to calculate transpulmonary fluid fluxes in different species, which can be used to elucidate mechanisms of lung fluid balance in vivo.

Zero prevalence of Clostridium difficile in wild passerine birds in Europe

Clostridium difficile is an important bacterial pathogen of humans and a variety of animal species, where it can cause significant medical problems. The major public health concern is the possibility of inapparent animal reservoirs of C. difficile and shedding of bacteria to noninfected individuals or populations, as well as being a source of food contamination. Migrating birds can be a key epizootiological factor for transmission and distribution of pathogens over a wide geographic range. Therefore, the purpose of this study was to investigate whether migrating passerine birds can be a source of spread of C. difficile along their migration routes. Cloacal samples were taken from 465 passerine birds during their migration south over the Alps. Selective enrichment was used for detection of C. difficile. Clostridium difficile was not isolated from any of the samples, which indicates that migrating passerine birds are unlikely to serve as a reservoir and a carrier of C. difficile.

Herpesvirus-Associated Neurological Disease in a Donkey

A 4-year-old donkey was evaluated for progressive neurological abnormalities consisting of depression, stupor, weakness, and recumbency. Diagnostic evaluation for viral involvement identified an asinine herpesvirus in DNA extracted from deep pharyngeal swabs. Specific primers were designed based on comparison with equine herpesviral DNA polymerase sequences and yielded an 875-base pair product from the donkey. This sequence had complete identity with short sequences of asinine herpesvirus previously identified in donkeys with interstitial pneumonia. Amino acid analysis of the entire sequence indicated high similarity with Equid herpesvirus 7 (91%), Zebra herpesvirus 1 (90%), and Equid herpesvirus 2 (89%). With supportive treatment and physical therapy, the donkey gradually recovered over 5 days of hospitalization and returned to normal function. The current case illustrates the potential of a novel asinine herpesvirus to induce neurological disease in donkeys and provides a large viral sequence allowing confident assignment of this virus to the subfamily Gammaherpesvirinae.

Prevalence and molecular characterization of Clostridium difficile isolated from European Barn Swallows (Hirundo rustica) during migration

BackgroundClostridium difficile is an important bacterial pathogen of humans and a variety of animal species. Birds, especially migratory passerine species, can play a role in the spread of many pathogens, including Clostridium difficile. Barn Swallows (Hirundo rustica) nest in close proximity to human habitats and their biology is closely associated with cattle farming. Therefore, we hypothesized that Barn Swallows can be the reservoir of Clostridium difficile.ResultsBarn Swallows (n = 175) were captured on their autumn migration across Europe to sub-Saharan Africa. Droppings were collected from juvenile (n = 152) and adult birds (n = 23). Overall prevalence of Clostridium difficile was 4% (7/175); 4.6% (7/152) in juvenile birds and 0/23 in adults. Clostridium difficile ribotypes 078, 002 and 014 were identified, which are commonly found in farm animals and humans. Three new Clostridium difficile ribotypes were also identified: SB3, SB159 and SB166, one of which was toxigenic, harbouring genes for toxins A and B.ConclusionsResults of this study indicate that Barn Swallows might play a role in national and international dissemination of Clostridium difficile and could serve as a source for human and animal infection. Clostridium difficile ribotype 078 was identified, which has been reported as an emerging cause of community-associated Clostridium difficile infection in humans. Based on this and other studies, however, it is more likely that Barn Swallows have a more indicative than perpetuating role in Clostridium difficile epidemiology.

An improved qPCR protocol for rapid detection and quantification of Clostridium difficile in cattle feces

Clostridium difficile (CD) can cause a significant and transmissible disease in animals and humans, with poorly understood epidemiology. Animals have been suggested as a possible source of infection and environment contamination. It is necessary that a precise and rapid diagnostic tool is available for the detection of CD from clinical and/or environmental samples. A quantitative real-time PCR (qPCR) protocol for CD detection defined by Penders et al. (FEMS Microbiol Lett, 243, 2005, 141-147) was modified. The modified protocol, supported by a novel extraction method, was tested on CD-spiked cattle feces and clinical fecal samples from calves. Quantification was performed targeting CD 16S rRNA gene. Three different commonly used TaqMan universal PCR master mixes were also compared. Results indicate that the modified protocol is very sensitive with an LOD of 7.72 CD cells per g CD-spiked feces. The protocol is capable of precise quantification with an LOQ of 77.2 CD cells per g CD-spiked feces, R(2) between 0.9957 and 0.9968, isolation efficiency from 87.89% to 90.96%, and an interassay CV ranging from 3.71% to 9.57%. The qPCR protocol for the detection and quantification of CD from animal feces investigated and described in this article using MIQE guidelines has the lowest detection and quantification limits published to date. Therefore, it can be implemented for precise epidemiological investigations of CD infections in animals and humans.

Acetazolamide attenuates transvascular fluid flux in equine lungs during intense exercise

•  During high intensity exercise approximately 4% of the cardiac output leaves the pulmonary circulation into the interstitium. This fluid flux has been attributed to an increase in pulmonary transmural hydrostatic (Starling) forces. •  Fluid efflux from erythrocytes may account for a considerable fraction of fluid exiting the pulmonary circulation. Transcapillary erythrocyte volume changes are largely determined by the Jacobs–Stewart cycle, a series of intracellular and extracellular diffusion and chemical reaction events of carbon dioxide, water, bicarbonate, hydrogen ions and chloride that are initiated when blood is exposed to a gradient such as when blood enters and traverses systemic and pulmonary capillaries. •  We tested the hypothesis that the Jacobs–Stewart cycle contributes to pulmonary transvascular fluid fluxes during exercise by inhibiting red cell carbonic anhydrase, the activity of which is critical to rapid completion of the Jacobs–Stewart cycle during capillary transit. •  Our results indicate that during exercise in horses, transvascular fluid fluxes in the lung appear to be dependent on the Jacobs–Stewart cycle and much less dependent upon transmural hydrostatic (Starling) forces. It also appears that pulmonary circulation transvascular fluid fluxes are mediated by chloride and water egress from erythrocytes directly into the interstitium without transit through plasma, which is likely the result of functional apposition of the erythrocyte and vascular endothelial membranes occurring during capillary transit.

Effect of frusemide on transvascular fluid fluxes across the lung in exercising horses

REASONS FOR PERFORMING STUDY Frusemide (Fru) is widely prescribed for management of racehorses experiencing EIPH. The effect of Fru in the lung appears to be a reduction in transcapillary pressures and inhibition of the erythrocyte anion exchange, which may lead to attenuation of transpulmonary fluid fluxes during exercise. HYPOTHESIS Treatment with Fru will attenuate transpulmonary fluid fluxes in horses during high intensity exercise. METHODS In a crossover study, 6 race-fit Standardbred horses were treated with 250 mg of Fru i.v. (FruTr) or placebo (Con) 4 h before exercise on a high speed treadmill until fatigue. Arterial and central mixed venous blood, as well as CO(2) elimination and O(2) uptake, were sampled. Volume changes across the lung and transvascular fluid fluxes were calculated from changes in haemoglobin, packed cell volume, plasma protein and cardiac output (Q). RESULTS During exercise, Q increased in both Con and FruTr, with Q being significantly lower in FruTr (mean ± s.e. 301.8 ± 8.5 l/min at fatigue) compared to Con (336.5 ± 15.6 l/min) (P<0.01). At rest frusemide had no effect on erythrocyte (J(ER)) and transvascular (J(V-A)) fluid fluxes across the lung. Exercise had a significant effect on J(ER) and J(V-A) (P ≤ 0.02). During exercise, J(ER) (at fatigue 14.6 ± 2.3 l/min and 11.6 ± 2.2 l/min in Con and FruTr, respectively) and J(V-A) (at fatigue 14.9 ± 2.3 l/min and 12.0 ± 2.2 l/min in Con and FruTr, respectively) were not significantly different between Con and FruTr (P = 0.6 and P = 0.8 for J(ER) and J(V-A), respectively). CONCLUSIONS AND CLINICAL IMPORTANCE Fru does not have a measurable effect on J(ER) and J(V-A). Cardiac output was reduced in FruTr, suggesting that there were also smaller changes in the capillary recruitment and transvascular transmural hydrostatic pressures; however, this did not effect J(V-A). Therefore, Fru at the dose of 250 mg does not appear to be an effective treatment for regulating pulmonary transvascular forces during exercise in horses.

Antimicrobial Susceptibility Patterns of Clostridium difficile Isolates from Family Dairy Farms

A significant risk factor for developing Clostridium difficile infection (CDI) in humans and animals is associated with the antimicrobial use. It has often been hypothesized that farm animals could be the source for human infection with Clostridium difficile (CD). In the European Union, family‐run dairy farms are the predominant farming model, which are more interlinked within the community compared to large‐scale intensive dairy or beef farms. Therefore, it is important to investigate antimicrobial susceptibility patterns of CD in such environment. A total of 159 CD isolates from 20 family dairy farms were tested with a customized broth microdilution plate for their antimicrobial resistance. Seventeen antimicrobials were selected (amoxicillin, ceftriaxone, clindamycin, daptomycin, erythromycin, fusidic acid, imipenem, levofloxacin, linezolid, metronidazole, moxifloxacin, oxacillin, rifampicin, tetracycline, tigecycline, trimethoprim/sulfamethoxazole and vancomycin), which are commonly used for treatment of CDI in veterinary and human medicine, or were previously applied in CD epidemiological studies. Antimicrobials, which are used for treatment of CDI in humans (metronidazole, vancomycin, fusidic acid, tigecycline, linezolid) inhibited CD growth in vitro. Most CD isolates were resistant to erythromycin (93.1%), daptomycin (69.2%) and clindamycin (46.5%). High multiple‐resistance was found in CD ribotype 012 (n = 5, 100%), some CD SLO 060 (n = 4, 25%) and one CD 033 (n = 1, 1.1%). High multiple‐resistance in this study was linked with CD ribotypes and not with the origin of CD. The low prevalence of these ribotypes (6.3%; 10/159) indicates that family‐run dairy farms are an unlikely source of CD with multiple‐resistance to antimicrobials.