Detection of mixed infections with "Candidatus Mycoplasma haemominutum" and Mycoplasma haemofelis using real-time TaqMan polymerase chain reaction.

The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with "Candidatus Mycoplasma haemominutum" (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.     

Endotracheal intubation in horses: a study of two cuff inflation pressures, correlation with liquid aspiration, and tracheal wall damage

Nine adult horses were anesthetized for a nonsurvival abdominal adhesion study. Horses were randomly assigned into two groups to receive endotracheal tube cuff pressures of either 80 cm H₂O (Group P80) or 120 cm H₂O (Group P120). After intubation (Bivona 30 mm ID), anesthesia was maintained with isoflurane. Horses were ventilated 10 times per minute with a suitable inspiratory pressure to maintain Pe ′CO₂ in the 35–40 mm Hg (4.7–6.0 kPa) range. Cuff pressure was continuously monitored with a pressure transducer (TruWave, Baxter) calibrated to the atmospheric pressure and maintained at a constant pressure. Twenty‐five millilitres of methylene blue dye in saline were instilled proximal to the cuff over 5 minutes. The horses were euthanized 123 ± 23 minutes later (mean ± SD). Immediately, the trachea was opened distal to the tip of the endotracheal tube, and the mucosa was observed for evidence of dye leaking past the cuff. The cervical trachea was resected and the lumen exposed by a ventral longitudinal incision. Biopsies (1–2 rings) were obtained at mid‐cuff level and distal to the tip of the endotracheal tube, and placed in formalin for later histologic examinations (H&E stain). Methylene blue stain was not observed distal to the endotracheal tube cuff in any horse. Visual examination of the tracheal mucosa revealed hyperemic or hemorrhagic lesions at the level of cuff contact both ventrally and dorsally. Histologic changes included epithelium damage, submucosal neutrophil infiltrates, and acute submucosal hemorrhages. P80 horses had none or focal to multifocal lesions on the ventral and dorsal aspects of the rings. P120 horses had multifocal to diffuse lesions on all aspects (dorsal, ventral, and lateral). We concluded that the endotracheal tube cuff produced a seal sufficient to prevent leakage at both pressures. Tracheal damages on gross and microscopic examinations were more severe and occurred more frequently at the higher cuff pressure.     

Malassezia species isolated from the intermammary and preputial fossa areas of horses

BACKGROUND: Malassezia-type yeasts previously have been observed on cytologic examination of the intermammary region of mares that presented with tail-head pruritus; topical antiyeast treatment resolved the pruritus. Further, Malassezia dermatitis has been observed in horses in intertriginous areas such as the udder and prepuce; the species of yeast was not confirmed. It is not known whether healthy mares or male horses can be carriers of this yeast in these body areas. HYPOTHESIS: Malassezia spp. are present in the intermammary region in healthy mares and the preputial fossa in healthy geldings. ANIMALS: Eleven healthy horses (5 mares and 6 geldings). METHODS: Samples of surface material were taken digitally from the intermammary area of 5 mares and the preputial fossa region of 6 geldings. The samples were examined cytologically and were cultured on modified Sabouraud's dextrose agar. The DNA from yeast colonies grown on the agar was extracted, and samples were assayed using fungal generic polymerase chain reaction (PCR) analysis. Amplicons with positive PCR results were sequenced and compared with sequences in the BLAST database search program. RESULTS: Of 44 attempts at culture, 5 yielded a species identified as Malassezia equi, and 2 yielded M slooffiae. In contrast, of 44 cytologic examinations, yeasts with the morphology of Malassezia spp. were seen in 40 samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Due to its presence in healthy horses, finding of Malassezia-type yeast on cytologic examination may not incriminate it as a pathogen. Despite difficulty in culturing, cytologic examination was an effective tool to rapidly demonstrate the organism.     

Monitoring and surveillance in abattoirs of residues of antibacterial substances – a description of programs in Australia and the USA

SUMMARY Australia and the USA are major international meat exporting countries. Both countries conduct programs to monitor and survey for antibacterial residues. Australian programs use a urine screening test, whereas the US programs use tissue fluids as the test medium. The development of surveillance programs to provide rapid feedback to producers is a feature of the Australian programs. The programs in each country compare favourably with regard to numbers of animals tested, results and action taken to prevent residues. The results of the Australian programs justify the promotion of a ‘clean food’ image for Australian meat products with respect to antibacterial drugs.     

Electrochemical principles for active control of liquids on submillimeter scales

Electrochemical methods were combined with redox-active surfactants to actively control the motions and positions of aqueous and organic liquids on millimeter and smaller scales. Surfactant species generated at one electrode and consumed at another were used to manipulate the magnitude and direction of spatial gradients in surface tension and guide droplets of organic liquids through simple fluidic networks. Solid microparticles could be transported across unconfined surfaces. Electrochemical control of the position of surface-active species within aqueous films of liquid supported on homogeneous surfaces was used to direct these films into periodic arrays of droplets with deterministic shapes and sizes.     

Molecular epidemiologic features of Corynebacterium pseudotuberculosis isolated from horses

OBJECTIVE: To characterize isolates of Corynebacterium pseudotuberculosis from horses, cattle, and sheep in Colorado, Kentucky, Utah, and California in samples collected during perceived epidemics of infection (increased numbers of cases identified) in 2002 and 2003, and determine how closely isolates were related and their possible source. SAMPLE POPULATION: 54 isolates of C pseudotuberculosis from 49 horses, 4 cattle, and 1 sheep. PROCEDURES: Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) assay, PCR assay for the gene encoding the phospholipase D (PLD) toxin, biochemical analyses, and tests for susceptibility to 17 antimicrobial drugs were performed. RESULTS: All isolates reduced nitrate to nitrite, most yielded positive results for the PLD toxin gene, and all were susceptible to antimicrobial drugs. Ten genetic types were detected by use of RAPD PCR assay; types III to X were isolated from horses, cattle, or both in 1 or more states. Types III and IX were isolated from both horses and cattle. Types VII and VIII were isolated in only 1 state, but the number of isolates in these groups was small. In contrast, all other types were isolated in 2 or more states. All isolates from Utah were type III, but the other 3 states had isolates from more than 1 type. CONCLUSIONS AND CLINICAL RELEVANCE: These data are consistent with a clonally expanding epidemic of infection in Utah and an increase in number of infections caused by multiple strains of C pseudotuberculosis not derived from a single source in the other states. The increase in number of infections could be the result of reporting bias, environmental factors facilitating infection, or host factors such as greater herd susceptibility.