Laser flaremetric evaluation of experimentally induced blood-aqueous barrier disruption in cats

OBJECTIVES: To determine whether aqueous humor flare, measured by use of laser flaremetry, was proportional to aqueous humor protein concentration and to use laser flaremetry to evaluate disruption of the blood-aqueous barrier (BAB) in cats. ANIMALS: 30 healthy adult cats. PROCEDURE: Laser flaremetry values for all eyes were compared with aqueous humor protein concentrations determined by use of a Coomassie blue microprotein assay. Laser flaremetry was then performed on both eyes before (0 hours) and 4, 8, and 26 hours after initiation of topical application of 2% pilocarpine (q 8 h) to 1 eye of 9 cats or paracentesis of the anterior chamber of 1 eye of 8 cats. Intraocular pressure and pupil size were also determined. Aqueous humor protein concentration was extrapolated from flare values by use of linear regression. RESULTS: There was a linear relationship between flare values and aqueous humor protein concentrations. Topical application of 2% pilocarpine and paracentesis of the anterior chamber caused a breakdown of the BAB that was detected by use of laser flaremetry. The highest mean flare readings after application of pilocarpine or paracentesis were 24.4 and 132.8 pc/ms, respectively, which corresponded to aqueous humor protein concentrations of 85.5 and 434.9 mg/dl, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Paracentesis of the anterior chamber resulted in a more severe breakdown of the BAB in cats than topical application of 2% pilocarpine. Laser flaremetry may be a useful clinical method to detect increases in aqueous flare and, hence, disruptions of the BAB in cats.     

Serological responses against the pathogenic dimorphic fungus Mucor amphibiorum in populations of platypus (Ornithorhynchus anatinus) with and without ulcerative mycotic dermatitis

Mucor amphibiorum, a dimorphic fungus, causes ulcerative dermatitis and systemic infections in the platypus Ornithorhynchus anatinus in some river systems in Tasmania but apparently not in other regions of Australia. As yet there are no suitable tests for population surveys, nor for detection of internal lesions in live animals. Consequently, immunoglobulins were purified from the serum of platypuses and anti-immunoglobulin antisera were prepared in rabbits in order to develop an enzyme-linked immunosorbent assay (ELISA) for anti-M. amphibiorum antibodies. Antigens from plate-grown cultures resulted in greater signal-to-noise ratios in indirect ELISA than those from broth-grown cultures. Platypuses with clinical ulcerative dermatitis had elevated anti-Mucor antibody levels compared to apparently unaffected individuals. Seroconversion was observed in one animal coincident with the development of cutaneous ulcers. The results suggested that platypuses in affected rivers were exposed to M. amphibiorum at a higher frequency than the occurrence of clinical disease. Some platypuses from New South Wales had elevated antibody levels but these increased significantly with age suggesting exposure to cross-reactive antigens, although exposure to M. amphibiorum cannot be excluded. Further studies are warranted to determine factors that result in progression from infection to disease, the occurrence of the fungus in areas where disease has not been observed and the specificity of antigen used in ELISA.     

Positive intraoperative cultures and canine total hip replacement: risk factors,periprosthetic infection,and surgical success

The results of closing intraoperative cultures from 27 canine total hip replacements (THR) were reviewed. The relationship between these culture results and presurgical and surgical factors, and the short- and long-term success of THR were assessed. Eleven out of 27 cases had a positive culture, but none of these 11 cases were diagnosed with periprosthetic infection at follow-up examination. The duration of the clinical signs of hip disease prior to THR was significantly greater for those cases with a positive culture (P=0.034). The incidence of positive cultures was not related to surgical success.     

Genetic Uniformity Among Isolates of Peronospora tabacina, the Tobacco Blue Mold Pathogen

ABSTRACT As a first step toward analysis of genetic variation and population structure in Peronospora tabacina, we used a collection of random genomic DNA fragments to survey for restriction fragment length polymorphisms (RFLPs) in DNA from a collection of isolates from Kentucky and other tobacco-growing regions of the United States. Also included in the study were isolates from the wild tobacco species, Nicotiana repanda, and from ornamental tobacco, N. alata. In a preliminary survey using DNA from 10 pathogen isolates, no polymorphisms were detected at six single-copy DNA loci using 22 probe-enzyme combinations. Moderately repetitive and highly repetitive regions of the genome were also remarkably similar between isolates, with only 6 of 15 different probes identifying genetic differences. Some of the polymorphic probes were then used to analyze a larger collection of isolates, most of which were from Kentucky. This resulted in the identification of very few additional polymorphisms, indicating that the population of P. tabacina that infects the Kentucky tobacco crop is genetically very homogeneous. The low level of polymorphism detected in this study overall, suggests that genetic variability may be lacking in P. tabacina populations throughout the United States. Two of the RFLP markers gave hybridization patterns that were consistent with P. tabacina being diploid. Frequencies of alleles at these loci and linkage disequilibrium between different marker loci indicated that genetic recombination does not occur frequently in the pathogen population. DNA polymorphisms that were identified in this study enabled us to differentiate the pathogen population into at least 10 haplotypes. One isolate was analyzed in detail and was shown to be genetically stable through several rounds of single-spore isolation and through several pathogenic cycles.     

Development of Contamination-Free Restriction Fragment Length Polymorphism Probes for the Obligate Biotroph Peronospora tabacina, an Oomycete Causing Blue Mold of Tobacco

ABSTRACT Peronospora tabacina is an obligately parasitic oomycete that causes blue mold, a devastating disease of tobacco. Genetic studies of this pathogen have been hampered by the lack of molecular markers. We generated a set of molecular markers for P. tabacina by collecting sporangiospores from infected tobacco leaves, extracting spore DNA, and cloning it in a plasmid vector. The resulting clones were then used to probe DNA from a collection of P. tabacina isolates to survey for polymorphisms. Most probes gave unexpected hybridization patterns with signal intensities that varied significantly from one DNA sample to another or between different DNA preparations of the same isolate. These results indicated that certain DNA preparations contained DNA from a source other than P. tabacina, which in turn suggested that some probes might have been derived from contaminating organisms present in the spore suspensions. Therefore, we characterized the inserts of several recombinant plasmids to determine their origins. Sequence analysis revealed that several of the inserts encoded peptides with similarity to bacterial proteins, suggesting that they were derived from bacterial contaminants. Of the remaining clones, five exhibited similarity to retroelements, one resembled eukaryotic helicase genes, and nine had no similarity to sequences in the databases. These were postulated to be true P. tabacina DNA clones. Verification of the origin of each probe was achieved by filtering a spore suspension, extracting DNA from the retentate and filtrate, and probing Southern blots of these DNA samples. These experiments confirmed the probe origins predicted by sequence analysis, resulting in the generation of 20 different restriction fragment length polymorphism probes that are specific for P. tabacina DNA. These probes should enable identification of reliable genetic markers for population studies of the blue mold organism.     

Development and evaluation of an enzyme-linked immunosorbent assay for the serodiagnosis of pythiosis in dogs

Pythiosis (caused by the aquatic oomycete Pythium insidiosum) is a devastating and often fatal cause of either severe transmural gastroenteritis or locally invasive subcutaneous disease in dogs living in the southeastern United States. Although early diagnosis is essential for successful treatment, tools available for this task are limited. Therefore, we developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-P insidiosum antibodies in canine serum. A soluble mycelial extract of P insidiosum was utilized as antigen in the ELISA, which was used to evaluate serum from 43 dogs with pythiosis, 8 dogs with lagenidiosis (another canine oomycosis), 16 dogs with nonoomycotic fungal or algal infections, 22 dogs with nonfungal gastrointestinal or skin disease, and 55 healthy dogs. Results were expressed as percent positivity (PP) relative to a strong positive control serum run on each plate. Medians and ranges for each of the 5 groups were as follows: pythiosis (81.7%, 50.6-98.5%), lagenidiosis (17.3%, 11.3-29.2%), other fungal or algal infections (8.2%, 4.7-15.4%), nonfungal gastrointestinal or skin disease (6.2%, 3.9-20.7%), and healthy dogs (6.7%, 3.0-15.2%). When using a cutoff value of 40% PP, the sensitivity and specificity of the ELISA both were 100%. In addition, ELISA values measured after successful surgical therapy in 2 dogs showed a decrease of anti-P insidiosum antibody concentrations into the normal range as early as 2 months after treatment. We conclude that the ELISA is a sensitive and specific test for the diagnosis of canine pythiosis, and may be a useful tool for monitoring response to medical or surgical therapy.     

Temporal patterns and quantification of excretion of Mycobacterium avium subsp paratuberculosis in sheep with Johne's disease

OBJECTIVES: To determine the frequency of excretion of Mycobacterium avium subsp paratuberculosis in Merino sheep with Johne's disease and to quantify excretion in a group of Merino sheep. DESIGN: A pen and laboratory experiment. PROCEDURE: Seven sheep selected from an affected flock on the basis of acid-fast bacilli in the sheep's faeces were housed and total daily faecal output was collected, weighed and subjected to culture for M avium subsp paratuberculosis. An end-point titration method was used to enumerate viable M avium subsp paratuberculosis in a 15 day pooled sample from five sheep that had acid-fast bacilli in their faeces while housed. RESULTS: Four sheep with subclinical multibacillary Johne's disease excreted M avium subsp paratuberculosis each day for 11 days of cultural observation. A further three sheep were intermittent excreters but lacked other evidence of infection with M avium subsp paratuberculosis. The average number of viable bacteria excreted was 1.09 x 10(8) per gram of faeces while total daily excretion was 8.36 x 10(10) viable M avium subsp paratuberculosis per sheep. Examination of faecal smears stained with Ziehl Neelsen was an unreliable means of assessing daily excretion in individual animals except in those with severe lesions. CONCLUSION: Excretion of M avium subsp paratuberculosis in Merino sheep with multibacillary Johne's disease occurred daily, proving that environmental contamination can be continuous on farms with endemic ovine Johne's disease. Faecal culture is a useful method for detecting infection as it does not appear to be affected by the timing of collection of a sample from sheep with multibacillary disease however, to maximise the sensitivity of disease surveillance using faecal culture, sampling rates should be adjusted to take account of the proportions of multibacillary and paucibacillary cases.