PCR-AGE, automated and manual methods to identify Candida strains from veterinary sources: A comparative approach

The increasing incidence of candidiasis has drawn the attention of scientists and clinicians attempting to improve methods of studying Candida yeasts. PCR amplification followed by agarose gel electrophoresis (PCR-AGE) and the manual method (morphological characteristics, biochemical profiles and culturing on CHROMagar-Candida) and VITEK 2 automated method were used to test a total of 30 fungal strains from dog sources. The strains were obtained from cases of dermatitis, otitis externa and from the ears, oral mucosa, vaginal mucosa, prepuce and perianal region of clinically normal dogs. After identification as Candida yeasts by the manual method, the strains were analyzed using both VITEK and PCR-AGE methods. Isolates of C. parapsilosis ATCC 22019, C. krusei ATCC 6258 and C. albicans ATCC 10231 were included as controls. The universal primers ITS1, ITS3 and ITS4 were used in two independent PCR reactions. Of 30 yeast isolates, 3 isolates (Saccharomyces cerevisiae, C. rugosa and C. parapsilosis) that were incompletely identified by the manual method were identified with the PCR-AGE and VITEK methods. The results revealed a 96.7% and 86.7% concurrent identification between the PCR-AGE and VITEK methods versus the manual method, respectively. PCR-AGE showed a greater level of concordance with the manual method, besides being faster and more sensitive than the other methods examined, and is therefore indicated for routine diagnostic testing of Candida spp. strains from veterinary sources.     

Farnesol inhibits in vitro growth of the Cryptococcus neoformans species complex with no significant changes in virulence-related exoenzymes

Farnesol is a sesquiterpene alcohol that modulates cell-to-cell communication in Candida albicans. In recent years, several studies have shown that this molecule presents inhibitory effects against non-albicans Candida species, Paracoccidioides brasiliensis and bacteria. The present study aimed at determining the effect of farnesol on the growth of strains of the Cryptococcus neoformans species complex, through microdilution assays. In addition, the effect of farnesol on the synthesis of phospholipase and protease - important virulence-associated enzymes - by C. neoformans and Cryptococcus gattii was also investigated. A total of 36 strains were studied, out of which 20 were from veterinary sources, 8 were from human cases and 8 were from a reference collection. The minimum inhibitory concentrations (MICs) were determined in accordance with the M27-A3 protocol as described by the CLSI and farnesol was tested at a concentration range of 0.29-150μM. Phospholipase and protease activities were evaluated through growth on egg yolk agar and spectrophotometry, respectively, after pre-incubating the strains at different farnesol concentrations (MIC/4, MIC/2 and MIC). It was observed that farnesol presents an inhibitory activity against C. neoformans and C. gattii (MIC range: 0.29-75.0μM). Although farnesol did not significantly alter phospholipase activity, a tendency to decrease this activity was observed. Concerning protease, no statistically significant differences were observed when comparing the production before and after pre-incubation at different farnesol concentrations. Based on these findings, it can be concluded that farnesol has in vitro inhibitory activity against C. neoformans and C. gattii, but has little impact on the production of the analyzed virulence factors.     

3株非典型羊源布鲁菌的基因分型研究

[目的]探讨采用基因分型方法对非典型布鲁菌鉴定的实用性,为非典型菌株的鉴别提供参考。[方法]采用常规鉴定方法和VITEK 2.0全自动细菌鉴定分析系统对菌株进行初步鉴定,利用AMOS-PCR进行种型鉴定,应用多位点可变数目串联重复序列分析方法(MLVA)确定菌株的基因型。[结果]常规鉴定结果显示试验菌株为疑似布鲁菌;VITEK 2.0全自动细菌鉴定系统显示3株试验菌株均为布鲁菌;AMOS-PCR扩增表明试验菌株均为羊种菌,MLVA聚类分析表明试验菌株与羊种2型布鲁菌紧密地聚为一类,属东地中海基因型,并在Panel 2B中发现了新的基因型(4-4-3-7-5),命名为CN2B-45。[结论]MLVA基因分型方法对非典型布鲁菌具有极高的分辨力,是非典型布鲁菌分型鉴别的最佳策略。     

Evaluation of risk factors for Cryptococcus gattii infection in dogs and cats

OBJECTIVE: To determine risk factors associated with Cryptococcus gattii infection in dogs and cats residing on Vancouver Island in British Columbia, Canada. DESIGN: Matched case-control study. ANIMALS: 20 dogs and 29 cats with C gattii infection and matched controls. PROCEDURE: Dogs and cats with a confirmed or probable diagnosis of cryptococcosis resulting from infection with C gattii were enrolled by veterinarians, and owners completed a questionnaire designed to obtain information pertaining to potential risk factors for the disease. Owners of matched control animals were also interviewed. Odds ratios and 95% confidence intervals or paired t tests were calculated to determine significant associations. RESULTS: Animals were enrolled during 2 noncontiguous periods in August 2001 to February 2002 (8 dogs and 9 cats enrolled) and May to December 2003 (12 dogs and 20 cats enrolled). Risk factors significantly associated with development of cryptococcosis included residing within 10 km of a logging site or other area of commercial soil disturbance, above-average level of activity of the animal, travelling of the animal on Vancouver Island, hunting by the animal, and owners hiking or visiting a botanic garden. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that dogs and cats that were active or that lived near a site of commercial environmental disturbance had a significantly increased risk of developing C gattii infection. Veterinarians should communicate these risks to owners in context because cryptococcosis was an uncommon disease in this population.     

Bacteriology and mycology of otitis externa in dogs]

The bacterial and fungal flora of 1118 ears of dogs with otitis externa and 100 ears of healthy control dogs were studied in order to isolate the causative agents. The yeast Malassezia pachydermatis (56%) was by far the most common organism in otitic dogs followed by the bacteria Staphylococcus intermedius (23%), Pseudomonas aeruginosa (12%), Proteus spp. (6%) and Streptococcus canis (5%). A statistical analysis of observed results showed that the incidence of these organisms is significant in otitic dogs. Many strains of S.intermedius, P.aeruginosa and Proteus spp. are resistant to antimicrobial agents commonly used to treat otitis externa. Therefore an antimicrobial susceptibility testing was performed using "Cobas Bact" for these bacterias. Furthermore, 80 strains of M.pachydermatis were submitted to identification-kits (API 20 CAUX, API STAPH, Cobas Micro). The observed results showed that an identification with these tests was not possible.     

Rapid MALDI-TOF mass spectrometry identification of Leptospira organisms

Leptospirosis is a worldwide deadly zoonotic disease. Accurate identification of the causative Leptospira spp. spirochetes ascertains the pathogenic status of the isolates, identifies potential source of infection and recognises outbreaks. Species identification is currently based on technically demanding, time and resources consuming serological and molecular methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) recently emerged as a first-line method for the accurate identification of bacteria, yet no data issued for Leptospira spp. We investigated the potential of MALDI-TOF-MS for the rapid identification of Leptospira isolates. Starting from a 10(5)organisms/mL suspension, MALDI-TOF-MS yielded an unique protein profile for each one of 19 Leptospira species reference isolates with a 100% reproducibility over 12 repeats, allowing to create a Leptopsira database. MALDI-TOF-MS further accurately identified 20/21 additional reference isolates representative of various serogroups at the species level as Leptospira interrogans (n=12), Leptospira kirschneri (n=5), Leptospira borgpetersenii (n=3), Leptospira noguchii (n=1) with identification score value of 2-2.5. Furthermore, six clinical isolates previously identified by rpoB sequencing, were correctly identified by MALDI-TOF-MS as L. interrogans (n=5) and L. borgpetersenii (n=1) with identification score value of 2-2.6. Identification was achieved in 40 min starting from the Leptospira suspension. MALDI-TOF-MS could complement serological and sequencing-based methods for the first line, rapid identification of Leptospira isolates in the clinical microbiology laboratory.     

Evaluation of the API 20E system for the identification of gram-negative nonfermenters from animal origin.

The API 20E system was evaluated on isolates from animals of aerobic nonfermentative and cytochrome oxidase positive Gram-negative rods. An accuracy of identification of 80% (214/268 isolates) was achieved for those organisms included in the 1976-1977 API profile index. Members of the genera Pseudomonas and Acinetobacter were identified with 100% accuracy. Organisms not included in the API profile gave either an unacceptable profile number or were incorrectly identified as Moraxella spp. When the inoculum size was increased there was better identification.     

Clinical characteristics and predictors of mortality for Cryptococcus gattii infection in dogs and cats of southwestern British Columbia

Since 1999, Cryptococcus gattii has emerged as an important pathogen of humans and animals in southwestern British Columbia. Historically thought to be restricted to the tropics and subtropics, C. gattii has posed new diagnostic and treatment challenges to veterinary practitioners working within the recently identified endemic region. Clinical reports of canine and feline cryptococcosis caused by C. gattii diagnosed between January 1999 and December 2003 were included in this case series. The most common manifestations of disease were respiratory and central nervous system signs. Multivariate survival analysis revealed that the only significant predictor of mortality was the presence of central nervous system signs upon presentation or during therapy. Case fatality rates in both species were high. Further investigation into effective treatment regimes is warranted.     

Comparison of the API 20E and BBL Crystal E/NF Identification Systems for Differentiating Bacterial Isolates from Apparently Healthy Reared Sea Bass (Dicentrarchus labrax)

The ability of two commercial rapid identification systems, API 20E and BBL Crystal E/NF, to reliably identify bacterial isolates from the internal organs of reared sea bass were compared. The tests gave different results: API 20E identified bacteria as Pseudomonas spp. with 37% accuracy, while BBL Crystal E/NF identified them as Flavobacterium odoratum with 99% accuracy. Although F. odoratum is not a marine fish pathogen, conventional tests conducted with the same isolates were more indicative of them being Flavobacterium spp. than Pseudomonas spp., suggesting that BBL Crystal E/NF was more reliable in this identification. Both systems were found to be applicable for diagnostics of marine fish pathogens, but should be used with caution because of possible misinterpretation.     

Comparison of Two Biochemical Test Systems with Conventional Methods for the Identification of Bacteria Pathogenic to Warmwater Fish

Abstract

One hundred seven Aeromonas spp., 26 Edwardsiella ictaluri, 6 E. tarda, 12 Plesiomonas shigelloides, and 6 Pseudomonas spp. (131 piscine isolates and 26 reference isolates) were studied with 36 biochemical tests from the Minitek system, 20 tests from the API 20E system, and corresponding standard tube tests. Isolates were incubated at 25°C. Arginine dihydrolase, ornithine decarboxylase, mannose, and citrate showed less than 95% agreement between the Minitek system and the tube tests. Arginine dihydrolase, lysine decarboxylase, nitrite reductase, Voges-Proskauer, and citrate showed less than 95% agreement between the API 20E system and the tube tests. The 26 reference isolates were examined with the three systems and were incubated at both 25 and 37°C. There were no major differences between tests run at 25 and 37°C except with nine Aeromonas spp. that did not grow well at 37°C. Both the Minitek and API 20E systems will reproduce standard biochemical tube test results with at least 95% accuracy when used to test warmwater fish pathogens incubated at 25°C. However, the numerical identification databases for both the Minitek and API 20E systems were not usable for identifying fish pathogens.     

Successful treatment of cryptococcal pneumonia in a pony mare

A 20-year-old Welsh Mountain Pony (212 kg) mare was initially presented for a chronic cough, fever, weight loss and low grade abdominal pain. She later developed dyspnoea, tachypnoea and exercise intolerance. The presence of multiple masses (up to 17 cm diameter) in the pulmonary parenchyma was established using lateral thoracic radiography and transthoracic ultrasonography. Encapsulated, budding yeasts were observed in smears made from transtracheal washings and needle aspirates of the pulmonary lesions. Cryptococcus gattii (synonym: Cryptococcus neoformans variety gattii; Cryptococcus bacillisporus) was cultured from the transtracheal washings and aspirates of the lung masses. The pony was successfully treated using daily intravenous infusions of amphotericin B (typically 0.5 mg/kg in 1 L 5% dextrose in water over 1 h, following premedication with 50 mg flunixin intravenously) over a 1 month period, until a cumulative dose of 3 g had been administered. Treatment was considered to be successful on the basis of progressive improvement in clinical signs, reduction in the size of pulmonary cryptococcomas, 48 kg weight gain and a reduction in the cryptococcal antigen titre from 4096 to 256, 1 year after cessation of treatment.     

Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronic mastitis – A molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomérieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated.102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).     

First report of Yersinia ruckeri biotype 2 in the USA

A polyphasic characterization of atypical isolates of Yersinia ruckeri (causative agent of enteric redmouth disease in trout) obtained from hatchery-reared brown trout Salmo trutta in South Carolina was performed. The Y. ruckeri isolates were biochemically and genetically distinct from reference cultures, including the type strain, but were unequivocally ascribed to the species Y. ruckeri, based on API 20E, VITEK, fatty acid methyl ester profiles, and 16S rRNA gene sequencing analysis. These isolates were nonmotile and unable to hydrolyze Tween 20/80 and were therefore classified as Y. ruckeri biotype 2. Genetic fingerprint typing of the isolates via enterobacterial repetitive intergenic consensus (amplified by polymerase chain reaction) and fragment length polymorphism showed biotype 2 as a homogeneous group distinguishable from other Y. ruckeri isolates. This is the first report of Y. ruckeri biotype 2 in the USA.     

Streptococcus spp. and related bacteria: their identification and their pathogenic potential for chronic mastitis - a molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomérieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated.102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).     

Cryptococcosis in captive cheetah (Acinonyx jubatus): two cases

Cryptococcus neoformans is a yeast-like organism associated with pulmonary, meningoencephalitic, or systemic disease. This case report documents 2 cases of cryptococcosis with central nervous system involvement in captive cheetah (Acinonyx jubatus). In both cases the predominant post mortal lesions were pulmonary cryptococcomas and extensive meningoencephalomyelitis. Both cheetahs tested negative for feline immunodeficiency virus and feline leukaemia virus. The organism isolated in Case 2 was classified as Cryptococcus neoformans var. gattii, which is mainly associated with disease in immunocompetent hosts.     

Phenotypic characterization of mastitic Prototheca spp. isolates

Bovine mastitis associated with Prototheca is considered a rare pathology, but is increasing in prevalence all over the world and therefore becoming more relevant to the dairy industry. The biochemical characterization of 47 Prototheca isolates retrieved from mastitic milk was performed in this study using API 20C Aux and two BBL Crystal Kits, followed by an analysis with InforBio software. The usage of this methodology, allowed the identification of discriminative phenotypic characteristics for the strains tested. The differential-character-finding algorithm used by this software permitted the identification of new phenotypic characteristics to discriminate between Prototheca zopfii, P. blaschkeae and P. wickerhamii, such as, citrate, phosphorycholine and arabinoside. The main objective of this study was to determine new phenotypic characteristics that allowed a better characterization of Prototheca spp. Usage of recent bioinformatic tools improved the analyses of several features that are important for a better characterization of Prototheca spp.     

Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping

During the period of 2001-2003, a total of 591 Actinobacillus pleuropneumoniae field isolates from the Czech Republic were serotyped with a high occurrence of cross-reactions. The cross-reactions were observed in 416 isolates. Most frequently, in 401 isolates (67.9%), cross-reactions with antisera specific for serotypes 9, 11, and/or 1 were observed. Two additional molecular methods, ribotyping and restriction analysis of PCR amplified apxIVA gene (PCR-REA), were therefore used for detailed characterisation of A. pleuropneumoniae. In this subsequent analysis, reference strains representing serotypes 1-12 and 25 field isolates showing the most frequent serotype cross-reactions were examined. PCR-REA enabled all reference strains to be distinguished except for the strains of serotypes 9 and 11. Ribotyping distinguished all reference strains except two pairs of serotypes: 3 versus 6, and 9 versus 11, respectively. Field isolates with serotype cross-reactivity 9, 11, and/or 1 could not be differentiated by either of these methods.     

Phenotypic characterization and in vitro antifungal sensitivity of Candida spp. and Malassezia pachydermatis strains from dogs

Yeasts of the genera Candida and Malassezia can be found as commensal microorganisms in animals. The main species of importance in veterinary medicine are Malassezia pachydermatis and Candida albicans. The objectives of this study were to conduct a phenotypic characterization and to evaluate the in vitro antifungal sensitivity of strains of C. albicans (n=5), C. tropicalis (n=3) and M. pachydermatis (n=32) isolated from dogs. The phenotyping was based on macro and micromorphological features as well as biochemical analysis. The techniques of microdilution in broth and dilution in agar were used to evaluate the in vitro sensitivity of Candida spp. and M. pachydermatis, respectively. The tested drugs were ketoconazole (KTC), itraconazole (ITC), fluconazole (FLC) and amphotericin B (AMB). The morphological analysis of the strains of Candida spp. and M. pachydermatis did not show any noteworthy alterations when compared to standard strains. On the other hand, in the biochemical tests, 34.4% of the strains of M. pachydermatis were negative for the urease test. Four strains of C. albicans were resistant to FLC with a minimum inhibitory concentration (MIC) >64microg/mL and all were resistant to KTC and ITC (MIC>16microg/mL). The MIC for two strains of C. tropicalis were >16microg/mL for KTC and ITC, and >64microg/mL for FLC. It is worth highlighting that all of the strains tested were sensitive to AMB with the MIC varying from 0.25-1.0microg/mL. All strains of M. pachydermatis were sensitive to ITC with a minimum fungistatic concentration (MFC) 0.0075microg/mL. The MIC for 29 strains was the same (MFC0.0075microg/mL) for KTC. The MFCs for FLC varied from 1 to 16microg/mL, and for AMB, the MFC interval was 0.125-8microg/mL. There were no alterations in the classic phenotypic features of the strains of Candida spp. and M. pachydermatis isolated from dogs but, unlike M. pachydermatis, Candida spp. were much more resistant to azole antifungal agents.     

An evaluation of the API ZYM system as a means of identifying Haemophilus somnus and related taxa.

The commercially available API ZYM microbiological identification system was evaluated for the rapid identification of Haemophilus somnus. Eighty-seven isolates of the organism had API ZYM profiles which were characteristic. The API ZYM profiles demonstrate clear differences between H. somnus and other genera but suggest a close association to three related organisms. Enzyme activity of H. somnus isolates were similar to organisms identified as Histophilus ovis, Haemophilus agni and strains UQV of Actinobacillus actinoides and Actinobacillus seminis but was clearly different from isolates of Pasteurella haemolytica, Pasteurella multocida, Bordetella bronchiseptica and group EF4. The API ZYM system allowed more rapid identification of H. somnus than conventional biochemical tests and may be a useful adjunct to conventional methods used for identification of H. somnus isolates. The test did not reveal obvious differences between isolates from various anatomic locations.     

犬隐球菌病病原分离与ITS区序列分析鉴定

为确定引起格力犬皮肤病发生的病原菌种属并提供科学的诊治方案,从病犬发病部位真皮层刮取病料,采用沙氏培养基对病料进行分离、培养,根据菌落形态特征与镜检结果对分离到的病原菌进行初步鉴定。采用真菌鉴定通用引物ITS1及ITS4对分离菌的ITS区序列进行PCR扩增、测序,将测序结果与GenBank中的新生隐球菌进行比对分析并构建系统发育树。结果表明,分离到的病原菌经形态学鉴定初步判定为隐球菌属真菌,其ITS区序列与GenBank中的新生隐球菌（JN939462.1和JN939461.1）的ITS序列相似性为99%,且在系统发育树上属同一分支,提示引起该犬隐球菌病的病原菌为隐球菌属新生隐球菌。