First report of Giardia lamblia in different animals in the United Arab Emirates.

The aim of this study was to detect and characterize Giardia lamblia in animals in the UAE. Eighty-seven fecal samples were tested for G. lamblia using the conserved fragment of small subunit (SSU)-rRNA by nested PCR. Giardia-positive isolates were genotyped for assemblages A and B using assemblage specific primers of the triosephosphate isomerase (tpi) gene. Thirty samples (34.5%) were positive for G. lamblia. Conversely, neither genotype A nor B were detected using tpi genotyping on the studied samples. Further investigations are required using higher number of samples including both human and animals in the country taking into consideration the analysis of other genotypes to provide more detailed understanding about the zoonotic transmission of this parasite.

First report of Giardia lamblia in different animals in the United Arab Emirates

@#The aim of this study was to detect and characterize Giardia lamblia in animals in the UAE. Eighty-seven fecal samples were tested for G. lamblia using the conserved fragment of small subunit (SSU)-rRNA by nested PCR. Giardia-positive isolates were genotyped for assemblages A and B using assemblage specific primers of the triosephosphate isomerase (tpi) gene. Thirty samples (34.5%) were positive for G. lamblia. Conversely, neither genotype A nor B were detected using tpi genotyping on the studied samples. Further investigations are required using higher number of samples including both human and animals in the country taking into consideration the analysis of other genotypes to provide more detailed understanding about the zoonotic transmission of this parasite.

Comparative serological evaluation of 10 Blastomyces dermatitidis yeast phase lysate antigens from different sources.

Yeast phase lysate antigens from 10 isolates of Blastomyces dermatitidis (dog, ERC-2 and T-58; T-27, polar bear; woodpile, ER-3; bat lung, 48938; human, B5929, B5895, B5896, B5931, and CAPP) from different geographical regions, in addition to a Histoplasma capsulatum (G217B) lysate preparation were compared with respect to their reactivity against serum specimens from dogs, rabbits and humans positive for blastomycosis using an indirect enzyme-linked immunosorbent assay. In addition, the lysate antigens were also assayed against histoplasmosis-positive human serum samples to study their cross-reactivity. Variable results were obtained with T-58 and T-27 exhibiting the greatest reactivity. We also noticed that the lysate did not react consistently to serum samples across species with lesser reactivity evidenced when testing dog sera. Finally, T-58 gave the highest cross-reactivity with histoplasmosis-positive sera. The study may prove valuable in the development of antigen candidates for blastomycosis serodiagnosis.

Genetic diversity and transcriptional analysis of the bys1 gene from Blastomyces dermatitidis.

Blastomyces dermatitidis, a pathogenic fungal organism, is able to exist in two different morphologies, a multicellular mycelium or a unicellular yeast, according to temperature, 25 degrees C and 37 degrees C respectively. The switching between morphologies must be accompanied by a cascade of signaling events in which expression of genes responsible for the change of morphology is increased or decreased. bys1, a gene from B. dermatitidis isolate #58, is expressed at high levels in the unicellular yeast, but gradually diminishes as the temperature is lowered and the organism converts to the mycelial phase where there is no transcription of bys1. We explored if bys1 homologs are found in other B. dermatitidis isolates and if the transcription of the homologs were regulated by temperature. bys1 was identified in all B. dermatitidis isolates tested and could be grouped into two classes by Southern blot, PCR, and DNA sequence. Although the bys1 transcripts of both classes were regulated by temperature, transcription rates varied between the three isolates tested.

Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens.

Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis. A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A. tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia. This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia. Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy. A transformation frequency of 1 per 10(5) arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium: fungus ratio. This approach requires no special equipment that might complicate biocontainment. Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure. A. tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C. immitis and perhaps other fungi of medical importance.

Resistance to Coccidioides immitis in mice after immunization with recombinant protein or a DNA vaccine of a proline-rich antigen.

Two inbred strains of mice (BALB/c and C57BL/6) were vaccinated with either recombinant expression protein of a Coccidioides immitis spherule-derived proline-rich antigen (rPRA) in monophosphoryl lipid A-oil emulsion adjuvant or a DNA vaccine based on the same antigen. Four weeks after vaccination, mice were infected intraperitoneally with arthroconidia. By 2 weeks, groups of mice receiving saline or plasmids with no PRA insert exhibited significant weight loss, and quantitative CFUs in the lungs ranged from 5.9 to 6.4 log10. In contrast, groups of mice immunized with either rPRA or DNA vaccine had significantly smaller pulmonary fungal burdens, ranging from 3.0 to 4.5 log10 fewer CFUs. In vitro immunologic markers of lymphocyte proliferation and gamma interferon (IFN-gamma) release after splenocytes were stimulated with rPRA correlated with protection. Also, plasma concentrations of rPRA-specific total immunoglobulin G (IgG), IgG1, and IgG2a showed increases in vaccinated mice. These studies expand earlier work by demonstrating protection in mice which differ in H-2 background, by using an adjuvant that is potentially applicable to human use, and by achieving comparable protections with a DNA-based vaccine. Our in vitro results substantiate a Th1 response as evidenced by IFN-gamma release and increased IgG2a. However, IgG1 was also stimulated, suggesting some Th2 response as well. PRA is a promising vaccine candidate for prevention of coccidioidomycosis and warrants further investigation.

Comparative studies on the detection of antibodies and delayed hypersensitivity responses with 10 Blastomyces dermatitidis lysate antigens.

Yeast-phase lysate antigens were prepared from 10 different isolates of Blastomyces dermatitidis. Comparative studies were performed using the lysate antigens in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in sera from dogs with blastomycosis and histoplasmosis. In order to evaluate the ability of the lysate reagents to elicit delayed dermal hypersensitivity (DTH) responses, the lysates were compared as skin-testing antigens in hairless guinea pigs that were previously sensitized with B. dermatitidis or Histoplasma capsulatum killed whole yeast cells. All ten of the lysate reagents were able to detect antibody with the ELISA in the serum specimens from dogs with blastomycosis (absorbance values ranged from 0.184 to 0.272; mean value 0.235). In contrast, when the lysates were assayed against sera from dogs with histoplasmosis, the absorbance values ranged from 0.053 to 0.151, with a mean value of 0.092. All ten lysate antigens were able to elicit a DTH response in the B. dermatitidis-immunized animals (mean axes of induration values ranged from 7.0 to 14.4 mm; mean value 8.6 mm). On the other hand, only minimal reactivity was evidenced in the guinea pigs immunized with H. capsulatum (mean axes of induration values ranged from 0.8 to 2.9 mm; mean value 1.8 mm).

Induction and detection of cell-mediated reactions with different Blastomyces dermatitidis antigenic preparations.

Blastomyces dermatitidis yeast-phase antigens (killed cell, KWY; lysate, Lys; and filtrate, Fil) from canine isolate T-58 were compared with respect to the induction and detection of cellular immune responses in mice. The antigens exhibited good sensitivity and specificity when used to detect a delayed-type hypersensitivity (DTH) response in mice previously immunized with T-58 or Histoplasma capsulatum antigens. Greater reactivity was observed with the KWY and Lys antigens as DTH-inducing agents (immunogens) than with the Fil antigen. The antigens were also compared with regard to the induction and detection of a lymphoproliferative reaction using splenocytes from normal and sensitized mice, and optimal reactivity was demonstrated with the KWY and Fil antigen preparations both as immunogens (absorbance values 0.22-1.60 and 0.20-0.90, respectively) and as in vitro testing antigens (absorbance values 0.60-1.60 and 0.35-1.00 respectively) (P < 0.01). Peritoneal macrophages from mice sensitized with Fil and KWY antigens showed the greatest in vitro replication inhibition (RI) of B. dermatitidis yeast cells (RI values of 53% and 50% respectively) (P < 0.05). When mitogenic or antigenic lymphocytic supernatants were compared with respect to their ability to enhance the phagocytic activity of unelicited macrophages from normal mice to kill yeast cells, the T-cell mitogens (concanavalin A and phytohaemagglutinin) optimally activated the naive macrophages (45% and 44% RI respectively) compared with the B-cell mitogen (LPS) (23% RI) (P < 0.05). Similar results were obtained with the lymphocytic supernatants from mice immunized with KWY cells when activated using KWY or Fil antigens (46% and 40% RI respectively) (P < 0.05).

Sensitivity and specificity of an isoelectric focusing fraction of Blastomyces dermatitidis yeast lysate antigen for the detection of canine blastomycosis.

Blastomyces dermatitidis yeast lysate antigen (T-58, dog isolate) fractions prepared using the Rotofor preparative isoelectric focusing (IEF) cell (Bio-Rad) were compared with B. dermatitidis yeast lysate and filtrate reagents with respect to the detection of antibodies in sera from dogs with blastomycosis, histoplasmosis, coccidioidomycosis, cryptococcosis and aspergillosis. A horseradish peroxidase enzyme immunoassay with Turbo TMB substrate was used in the study. One particular IEF fraction (pH 4.3) was optimal in the assay, and it exhibited greater sensitivity (100%) and specificity (93%) than the lysate or filtrate preparations. The highest degree of cross-reactivity was encountered with the histoplasmosis and coccidioidomycosis specimens and considerably less with the cryptococcosis and aspergillosis sera. Studies are in progress to purify further the optimal IEF fraction.

Induction and detection of delayed dermal hypersensitivity in guinea-pig immunized with Blastomyces dermatitidis lysate and filtrate antigens.

Guinea-pigs were immunized using yeast phase antigens (lysate and filtrate preparations) from two strains of Blastomyces dermatitidis (T-58 and Le). Following a sensitization period, the animals were skin tested on days 40 and 216 using T-58 and Le yeast and mycelial phase lysate and filtrate antigen preparations for the detection of delayed dermal hypersensitivity (DTH). Using the Friedman's analysis of variance by rank test, significant differences were found in the efficacy of the immunogens to induce DTH in the animals when skin tested on both occasions (P < 0.05). Optimal reactivity was observed in guinea-pigs immunized with Le yeast lysate (mean axes of induration ranging from 12.0 to 18.8 mm and 7.0 to 18.5 mm on day 40 and 216, respectively) and T-58 yeast filtrate (mean axes of induration ranging from 7.5 to 18.0 mm and 8.0 to 17.0 mm on day 40 and 216, respectively) when the immunogens were administered with adjuvant. When the same data was analysed using the Friedman's test with regard to evaluating the efficacy of the skin test antigens to detect DTH, significant differences were found between them (P < 0.05) with optimal results using the Le mycelial filtrate and yeast lysate antigens (mean axes of induration ranging from 7.0 to 16.5 mm and 7.5 to 14.5 mm, respectively) when skin testing was done on day 40. When skin testing was done on day 216, the T-58 and Le mycelial lysate antigens gave optimal results (mean axes of induration ranging from 9.0 to 18.5 mm and 7.5 to 15.0 mm, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative studies on the detection of delayed dermal hypersensitivity in experimental animals with lysate and filtrate antigens of Blastomyces dermatitidis.

Yeast cell and mycelial lysate and filtrate antigens prepared in our laboratory from two strains of Blastomyces dermatitidis (canine isolate T-58 and human isolate Le) were evaluated with respect to the detection of delayed dermal hypersensitivity in hairless guinea pigs previously immunized with killed whole yeast cells from two strains of Blastomyces dermatitidis (T-58 and Le) and Histoplasma capsulatum (strain G-217A). The optimal potential reactivity (reactivity minus cross-reactivity) with regard to eliciting a dermal response in animals sensitized with B. dermatitidis was achieved with the yeast phase lysate and filtrate antigens prepared from both T-58 and Le isolates (mean axes of induration values ranging from 14.0 to 15.9 mm). In contrast, the mycelial phase reagents exhibited lower potential reactivity (mean axes of induration values ranging from 5.3 to 10.5 mm).

Comparative stability, sensitivity and specificity studies with different lots of Blastomyces dermatitidis yeast and mycelial lysate antigens.

Comparative studies were performed to assess the stability and lot-to-lot variation of Blastomyces dermatitidis yeast and mycelial phase lysate antigens. Four lots were prepared from each growth phase of B. dermatitidis strain T-58 (canine isolate) during a 14-month period. Serum specimens from dogs with blastomycosis, histoplasmosis, coccidioidomycosis, cryptococcosis and aspergillosis were assayed for antibody content using an alkaline phosphatase enzyme-linked immunosorbent assay (ELISA). The four lots of the yeast phase reagents were similar with respect to sensitivity and specificity, and the absorbance readings were approximately four times greater with sera from dogs with blastomycosis than with histoplasmosis or coccidioidomycosis. Even less cross-reactivity was evidenced when the sera from dogs with cryptococcosis and aspergillosis were assayed. In contrast, the four lots of the mycelial lysate reagents were considerably less reactive and more cross-reactive than the yeast phase antigens and, as above, the four reagents retained their activity after prolonged storage. Therefore the results indicated that the lysate antigens exhibited a great deal of stability and lot-to-lot variations in activity were not observed.