Protein A/G-based enzyme-linked immunosorbent assay for detection of anti-Pythium insidiosum antibodies in human and animal subjects.

OBJECTIVES: Pythiosis is a deadly infectious disease caused by Pythium insidiosum. Reports of both human and animal pythiosis are on the rise worldwide. Prognosis of the pythiosis patients relies on early diagnosis and prompt treatment. There are needs for an immunodiagnostic test that can detect the disease in both humans and animals. This study aims at reporting an optimized protocol for the development of a protein A/G-based enzyme-linked immunosorbent assay (ELISA) for the detection of anti-P. insidiosum antibody in multiple host species. RESULTS: A total of 25 pythiosis and 50 control sera, obtained from humans, horses, dogs, cats, and cows, were recruited for the assay development. With a proper ELISA cutoff point, all pythiosis sera can ultimately be distinguished from the control sera. The successfully-developed protein A/G-based ELISA can detect the anti-P. insidiosum antibodies in serum samples of both humans and animals. It is a versatile, feasible-to-develop, and functional immunodiagnostic assay for pythiosis.

First confirmed case of nasal pythiosis in a horse in Thailand.

INTRODUCTION: Pythiosis is caused by Pythium insidiosum, a fungus-like organism in the class Oomycetes. It can infect humans and a variety of animal species in tropical, subtropical and some temperate regions. Cases of animal pythiosis have occurred predominantly in horses in the skin and subcutaneous tissue at the limbs and in the ventral portion of thoracoabdominal wall - lesions in the nasal region are rarely reported. Moreover, although many human pythiosis cases have been reported in Thailand, no cases of animal pythiosis in Thailand have been reported. CASE PRESENTATION: We report a case of pythiosis in a horse infected at the nasal cavity. Diagnosis was performed by zoospore formation by bait technique, immunohistochemical stain, immunochromatography and sequence analysis. CONCLUSION: The sequences of rDNA were 99 % and 96 to 99 % identical to GenBank isolates of Pythium insidiosum from two Thai human patients and horses from various countries, respectively. This represents the first confirmed report of nasal equine pythiosis in Thailand.

Single nucleotide polymorphism-based multiplex PCR for identification and genotyping of the oomycete Pythium insidiosum from humans, animals and the environment.

Pythium insidiosum causes a life-threatening infectious disease, called pythiosis, in humans and animals worldwide. Diagnosis of pythiosis is difficult and often delayed. Surgical removal of infected tissue is the main treatment option. Disabilities and death are common outcomes for pythiosis patients. Reports of Py. insidiosum infections are rising. While it would be useful for clinical, epidemiological, and microbiological studies, information on genetic variation in Py. insidiosum strains is limited. This limitation is, at least in part, due to the cost and time-requirements of DNA sequencing procedures. rDNA-sequence-based phylogenetic analyses categorize Py. insidiosum into three groups, in relation to geographic distribution: Clade-I (American strains), Clade-II (American, Asian, and Australian strains), and Clade-III (Thai and American strains). In rDNA sequence analyses, we observed single nucleotide polymorphisms (SNP) that were associated with the phylogenetic clades of Py. insidiosum. In this study, we aim to develop a multiplex PCR assay, targeting the identified SNPs, for rapid genotyping of Py. insidiosum. We also aim to assess diagnostic efficiency of the assay for identification of Py. insidiosum. Fifty-three isolates of Py. insidiosum from humans (n=35), animals (n=14), and the environment (n=4), and 22 negative-control fungi were recruited for assay evaluation. Based on the pattern of amplicons, the multiplex PCR correctly assigned phylogenetic clades in 98% of the Py. insidiosum isolates tested. The assay exhibited 100% sensitivity and specificity for identification of Py. insidiosum. The assay successfully identified and genotyped the first proven isolate of Py. insidiosum from an animal with pythiosis in Thailand. In conclusion, the multiplex PCR provided accurate, sensitive and specific results for identifying and genotyping Py. insidiosum. Thus, this multiplex-PCR assay could be a simple, rapid, and cost-effective alternative to DNA sequencing for the identification and genotyping of Py. insidiosum.

Characterization of canine and feline methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) in small animal practice are very difficult to treat due to multi-resistance. In contrast to other countries, little is known about MRSP from Thailand. In particular, information on feline MRSP isolates in general is rare. In total, 39 MRSP isolates from dogs (n=28) and cats (n=11) from Thailand collected from independent clinical cases were used. Oxacillin resistance and presence of the mecA gene was confirmed. Susceptibility to additional 29 antimicrobial agents was tested according to CLSI recommendations. Antimicrobial resistance genes were detected by PCR assays. Molecular typing comprised spa typing, dru typing and macrorestriction analysis with subsequent pulsed-field gel electrophoresis (PFGE). For selected isolates, multi-locus sequence typing (MLST) was performed. All isolates were multi-resistant with resistance to at least six classes of antimicrobial agents. In all cases corresponding resistance genes were detected. In addition to mecA, the genes blaZ, catpC221, aacA/aphD, erm(B), dfrG, tet(M) and tet(K) were identified. Six spa types (t02, t05, t09, t10, t23, t72), eleven dru types (dt8ak, dt10ao, dt10cp, dt10cq, dt11a, dt11bo, dt11cb, dt11cj, dt11v, dt11y, dt11z) and 27 PFGE types (designated as A1-A10, B1-B8, C1-C2, D, E, F, G, H, I, J) were identified. MLST for one isolate of each main PFGE pattern A-J revealed seven types [ST45 (n=3), ST112, ST155, ST282 and the novel types ST432, ST433 (n=2) and ST434]. This study showed that MRSP isolates from clinical cases in individual dogs and cats in Thailand are multi-resistant with similar resistance genes and characteristics as isolates from Europe and North America.

Fatal melioidosis in goats in Bangkok, Thailand.

Bangkok, Thailand, is a city considered to be at low risk for melioidosis. We describe 10 goats that died of melioidosis in Bangkok. Half of them were born and reared in the city. Multilocus sequence typing ruled out an outbreak. This finding challenges the assumption that melioidosis is rarely acquired in central Thailand.

Prevalence of "Actinobacillus porcitonsillarum" in porcine tonsils and development of a diagnosis duplex PCR differentiating between "Actinobacillus porcitonsillarum" and Actinobacillus pleuropneumoniae.

"Actinobacillus porcitonsillarum" is a newly suggested commensal species colonizing porcine tonsils. In the diagnostic laboratory the sole difference to the porcine lung pathogen Actinobacillus pleuropneumoniae is a negative mannitol reaction. In order to substantiate and improve this important differentiation a PCR test was developed using the relevant reference strains including Actinobacillus minor. The practicability of the test was confirmed on 20 clinical isolates of Actinobacillus spp. cultured from 100 tonsil samples originating from 18 farms in Thailand. Applying the newly developed PCR test 10 isolates were identified as A. pleuropneumoniae, and 10 as "A. porcitonsillarum" with one of them being mannitol-positive in biochemical testing. Subsequent 16S rRNA sequencing confirmed classification of all 10 strains as "A. porcitonsillarum"/A. minor. These results emphasize that suspected A. pleuropneumoniae isolates, particularly from porcine tonsils, should be confirmed by PCR in order to prevent false positive diagnoses.

Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence.

A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.

Construction of an Actinobacillus pleuropneumoniae serotype 2 prototype live negative-marker vaccine.

Deletions were introduced into the ureC and apxIIA genes of an Actinobacillus pleuropneumoniae serotype 2 strain by homologous recombination and counterselection. The double-mutant contains no foreign DNA, is highly attenuated, protects pigs from homologous challenge upon a single aerosol application, and facilitates the serological discrimination of immunized and infected herds.