Immitis Isolates Research Articles

Establishment of macrocyclic lactone resistant Dirofilaria immitis isolates in experimentally infected laboratory dogs.

BackgroundStrains of Dirofilaria immitis suspected of lack of efficacy (LOE) to macrocyclic lactone (ML) preventive drugs have been increasingly reported in dogs by practicing veterinarians since 2005 in the Lower Mississippi Delta region. If proven, and not controlled in the early stages, the emergence of ML drug resistance threatens to become a widespread problem in the US that may limit the effectiveness of current preventive drug treatment methods.MethodsTo validate practice reports, a statewide survey of Louisiana veterinarians was done to define the extent of the problem and identify focal ‘hotspots’ of reported ML LOEs using Geographic Information Systems (GIS) methods. The present study then utilized microfilariae (Mf) from two canine field cases from different state locations that fit criteria for a high index of suspicion of LOE against heartworms by ML drugs. Blood containing Mf from the canine field cases was used to infect and produce L3 in Aedes aegypti for experimental infection of two groups of dogs, each of which contained two laboratory dogs, one treated with prophylactic ivermectin (12 μg/kg) monthly for 6 months at twice the label dose (6 μg/kg), and one untreated control.ResultsBoth treated and untreated dogs from Group I and Group II developed patent D. immitis infections by 218 DPI and 189 DPI, respectively, as evidenced by a positive occult heartworm antigen test and microfilaremia by the Knott’s test. Mf counts gradually increased post-patency in test and control dogs. Infective larvae raised from microfilariae from the treated Group I dog were used to successfully establish a second generation isolate, confirming heritability of resistance in the face of a monthly ivermectin challenge dose of 24 μg/kg, given monthly for 3 months.ConclusionsThese experimental infection studies provide in vivo evidence of the existence of ML drug resistance in dogs infected by D. immitis L3 from suspect field LOE cases in the Lower Mississippi Delta. Results encourage further work on mechanisms underlying the emergence of ML resistance in D. immitis and development of evidence-based resistance management strategies for heartworm preventives in order to extend the useful life of current drugs.

Establishment of macrocyclic lactone resistant Dirofilaria immitis isolates in experimentally infected laboratory dogs

Establishment of macrocyclic lactone resistant Dirofilaria immitis isolates in experimentally infected laboratory dogs

Next-Generation Sequencing ofCoccidioides immitisIsolated during Cluster Investigation

Next-generation sequencing enables use of whole-genome sequence typing (WGST) as a viable and discriminatory tool for genotyping and molecular epidemiologic analysis. We used WGST to confirm the linkage of a cluster of Coccidioides immitis isolates from 3 patients who received organ transplants from a single donor who later had positive test results for coccidioidomycosis. Isolates from the 3 patients were nearly genetically identical (a total of 3 single-nucleotide polymorphisms identified among them), thereby demonstrating direct descent of the 3 isolates from an original isolate. We used WGST to demonstrate the genotypic relatedness of C. immitis isolates that were also epidemiologically linked. Thus, WGST offers unique benefits to public health for investigation of clusters considered to be linked to a single source.

Population genomic sequencing of Coccidioides fungi reveals recent hybridization and transposon control.

We have sequenced the genomes of 18 isolates of the closely related human pathogenic fungi Coccidioides immitis and Coccidioides posadasii to more clearly elucidate population genomic structure, bringing the total number of sequenced genomes for each species to 10. Our data confirm earlier microsatellite-based findings that these species are genetically differentiated, but our population genomics approach reveals that hybridization and genetic introgression have recently occurred between the two species. The directionality of introgression is primarily from C. posadasii to C. immitis, and we find more than 800 genes exhibiting strong evidence of introgression in one or more sequenced isolates. We performed PCR-based sequencing of one region exhibiting introgression in 40 C. immitis isolates to confirm and better define the extent of gene flow between the species. We find more coding sequence than expected by chance in the introgressed regions, suggesting that natural selection may play a role in the observed genetic exchange. We find notable heterogeneity in repetitive sequence composition among the sequenced genomes and present the first detailed genome-wide profile of a repeat-induced point mutation (RIP) process distinctly different from what has been observed in Neurospora. We identify promiscuous HLA-I and HLA-II epitopes in both proteomes and discuss the possible implications of introgression and population genomic data for public health and vaccine candidate prioritization. This study highlights the importance of population genomic data for detecting subtle but potentially important phenomena such as introgression.

Antifungal susceptibility profiles of Coccidioides immitis and Coccidioides posadasii from endemic and non-endemic areas

Coccidioidomycosis is a systemic fungal infection endemic in Southwestern United States, Mexico, Central and South America. The causal agents are Coccidioides immitis and C. posadasii. A large number of cases of coccidioidomycosis in New York State residents were identified. We compared susceptibility profiles of these isolates and of C. immitis isolates from California using mycelial phase inoculum and CLSI (NCCLS) M38-A broth microdilution protocol. Minimum fungicidal concentrations (MFC) were also determined. Results indicated that geometric mean MICs of amphotericin B (AMB, 0.06 microg/ml), fluconazole (FLC, 8.0 microg/ml), itraconazole (ITC, 0.07 microg/ml), ketoconazole (KTC, 0.04 microg/ml), voriconazole (VRC, 0.04 microg/ml), posaconazole (PSC, 0.17 microg/ml) and caspofungin (CSP, 0.15 microg/ml) were in susceptible range as per breakpoints published for pathogenic Candida species. However, geometric MFC for FLC was relatively higher (52.4 microg/ml). Also, no significant difference in MIC and MFC values was evident for C. immitis and C. posadasii isolates. In conclusion, current methods for antifungal susceptibility testing yield reproducible profiles for Coccidioides species, which appear to be highly susceptible to most antifungal agents.

Molecular and biochemical characterization of a Coccidioides immitis-specific antigen.

Results of earlier investigations have indicated that the saprobic phase of Coccidioides immitis produces a heat-stable, 19-kDa antigen with serine proteinase activity which has been suggested to be specific for this pathogenic fungus. In the present study we have determined the N-terminal and partial internal amino acid sequences of the purified, 19-kDa antigen, cloned the gene which encodes this polypeptide, and confirmed that the secreted proteinase is a Coccidioides-specific antigen (CS-Ag). Both the genomic and cDNA sequences are reported and reveal that the csa gene which encodes this antigen has no introns. A 543-bp open reading frame encodes a 181-amino-acid-containing protein with a predicted molecular mass of 19.8 kDa and an isoelectric point of 8.3. The csa gene was localized on chromosome I of three representative C. immitis clinical isolates on the basis of Southern hybridizations. Expression of the csa gene in Escherichia coli using the pET21a plasmid vector yielded a recombinant protein that was recognized in immunoblot assays by antibody raised to the purified 19-kDa CS-Ag. Secretion of the native antigen is suggested to occur by cleavage of a putative 23-residue signal peptide. The native CS-Ag showed a low degree of glycosylation. Analysis of the carbohydrate composition of the CS-Ag revealed xylose, mannose, galactose, and glucose. However, the purified antigen showed no affinity for concanavalin A. A PCR method with specificity and high sensitivity for detection of C. immitis genomic DNA, using a pair of synthetic oligonucleotide primers whose sequences were based on that of the csa gene, was developed. A 520-bp product was amplified only when C. immitis genomic DNA was used as the template. The lower limits of DNA detection using this PCR method were 1 pg of C. immitis genomic DNA by ethidium bromide staining and 100 fg after Southern hybridization. The csa gene-based PCR method for detection of C. immitis DNA is useful for culture identification and may have clinical applications for the diagnosis of coccidioidal infections.

Characterization of Coccidioides immitis isolates by restriction fragment length polymorphisms

The marked increase in the number of cases of coccidioidomycosis in California in 1992 led to a study of isolates from various patients and environmental sources by restriction fragment length polymorphism (RFLP) analysis. Of 15 different isolates, most of the isolates (13 of 15) from California and 1 from Venezuela yielded one main RFLP pattern with evidence of two subgroups. The other two isolates (both from patients in the San Joaquin Valley of California) yielded a different RFLP pattern.

Comparative evaluation of chemiluminescent DNA probe assays and exoantigen tests for rapid identification of Blastomyces dermatitidis and Coccidioides immitis

Chemiluminescent DNA probe (Accuprobe) assays developed by Gen-Probe, Inc. (San Diego, Calif.), for the rapid identification of Blastomyces dermatitidis and Coccidioides immitis were evaluated and compared with the exoantigen test by using 74 mycelial cultures of B. dermatitidis and 72 mycelial cultures of C. immitis. Seventeen isolates of the dimorphic pathogen Paracoccidioides brasiliensis were included because of their gross morphologic and antigenic relatedness to B. dermatitidis. The heterologous fungi, namely, species of Chrysosporium, which are often confused with B. dermatitidis, and species of Malbranchea, which morphologically resemble C. immitis, were tested. All 74 of the B. dermatitidis mycelial isolates were correctly identified by the Accuprobe assay for B. dermatitidis within 2 h. However, the B. dermatitidis probe cross-hybridized with rRNA extracts of 10 of the 17 P. brasiliensis isolates, misidentifying them as B. dermatitidis. All 72 of the C. immitis isolates were identified correctly with the C. immitis probe. None of the other heterologous fungi belonging to Chrysosporium spp., Malbranchea spp., Onychocola canadensis, and Geotrichum sp. were cross-reactive with the B. dermatitidis and C. immitis probes. The exoantigen tests specifically identified 74 B. dermatitidis, 72 C. immitis, and 17 P. brasiliensis isolates within 48 to 72 h and differentiated the related heterologous fungi from the three dimorphic fungal pathogens.

Review of human and animal cases of coccidioidomycosis diagnosed in Canada

The first Canadian case of coccidioidomycosis in a human was reported in 1952 and 11 more cases since then. This study provides details of other cases of coccidioidomycosis that have been diagnosed in Canada. Based on clinical details, isolation of Coccidioides immitis, detection of a specific antibody (F band) for coccidioidomycosis by macro- or microimmunodiffusion tests, concurrently used with the complement fixation procedure, and histopathological findings, 116 more cases of this disease were verified. The great majority (94%) of these cases were diagnosed in the western Canadian provinces of British Columbia, Alberta, Saskatchewan and Manitoba, and the others in Quebec, Ontario and Nova Scotia (5, 1, and 1 cases, respectively). Available information indicates that the C. immitis infections were contracted during visits to endemic areas in the United States (Arizona, California and New Mexico), Mexico, and Bolivia. Pulmonary infections were the most common type of coccidioidomycosis (93%) followed by the disseminated or meningeal types C. immitis infections occurred in individuals with or without predisposing factor(s) and were more common in males than in females. The exoantigen procedure was very useful and reliable in the accurate and rapid identification of suspected C. immitis isolates. Two cases of coccidioidomycosis were reported in animals in Ontario, Canada.

Evaluation of commercial reagents to identify the exoantigens of Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma species cultures.

Commercially available individual serologic reagents (Nolan/Scott Biological Laboratories, Inc.) and an "exoantigen identification system" (a kit supplied by Immuno-Mycologics, Inc.) were evaluated at two laboratories for their possible application in the microimmunodiffusion test to identify Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma species. Both laboratories accurately identified all the cultures, and comparison of the results obtained with the commercial products and those established with the conventional methods performed by a third control laboratory demonstrated full agreement. The use of these two commercial products is recommended for the immunologic identification of cultures suspected of being B. dermatitidis, C. immitis, and Histoplasma species. It was observed, however, that the exoantigen reactions for four B. dermatitidis and two C. immitis isolates were much sharper and more readable with the individual reagents than the reactions observed with the kit.

Immuno-identification of cultures of fungi pathogenic to man

Because isolates of the fungal pathogensBlastomyces dermatitidis. Coccidioides immitis, Histoplasma capsulatum, andParacoccidioides brasiliensis frequently vary widely in gross and microscopic features and are often difficult or impossible to convert to their tissue forms, a simple diagnostic procedure not dependent upon sporulation is needed to identify them specifically and rapidly. The exoantigen technique has been found to meet this need effectively. On the basis of studies with 166Histoplasma spp. isolates, 128C. immitis isolates, 59B. dermatitidis isolates, 30P. brasiliensis isolates, and 181 saprophytes, we determined that the exoantigen test is valuable for the presumptive identification of the four fungal pathogens studied. All of the positive reactions have correlated with the cultural, histologic, or other available laboratory data, and we are unaware of any false positive or flase negative reactions.

Immunological procedure for the rapid and specific identification of Coccidioides immitis cultures.

An immunological procedure for the rapid and specific identification of Coccidioides immitis isolates has been developed. The specificity of the procedure is based on the fact that C. immitis produces antigens that are not produced by morphologically similar fungi. The procedure involved the transfer of heavy mold-form inocula to flasks that contained small volumes of brain heart infusion broth. Shake cultures were grown at 25 degrees C on a gyratory shaker at 150 rpm. The concentrated supernatant antigens so obtained from broth cultures were tested in parallel with coccidioidin by a microimmunodiffusion technique against C. immitis antiserum. The ability of the immunological procedure to identify C. immitis cultures was evaluated by testing the supernatant antigens of 166 unknown isolates, many of which, by gross or microscopic examination or both, resembled C. immitis or belonged to the family Gymnoascaceae. Each culture was also identified by conventional laboratory procedures. Comparative evaluation showed that the immunological test for C. immitis was 100% sensitive and specific. The diagnostic precipitin bands that were produced by the interaction of the C. immitis supernatants and antisera were shown to consist of (i) a heat-stable antigen comparable to that active in the tube precipitin test and (ii) two heat-labile antigens, one of which was associated with complement fixation reactivity. With pure cultures, this immunological procedure permitted the identification of C. immitis isolates within 5 days.