Association of anticardiolipin, antiphosphatidylserine, anti-ß2 glycoprotein I, and antiphosphatidylcholine autoantibodies with canine immune thrombocytopenia.

BACKGROUND: In humans, the presence of antiphospholipid antibodies (aPL) is frequently found in immune thrombocytopenia. The present study investigated whether aPL and any aPL subtypes are associated with canine thrombocytopenia, in particular, immune-mediated thrombocytopenia (immune thrombocytopenia) that usually manifests with severe thrombocytopenia. RESULTS: Sera were collected from 64 outpatient dogs with thrombocytopenia (Group I, platelet count 0 - 80 × 10(3)/uL), and 38 of which having severe thrombocytopenia (platelet count < 30 × 10(3)/uL) were further divided into subgroups based on the presence of positive antiplatelet antibodies (aPLT) (subgroup IA, immune thrombocytopenia, n =20) or the absence of aPLT (subgroup IB, severe thrombocytopenia negative for aPLT, n =18). In addition, sera of 30 outpatient dogs without thrombocytopenia (Group II), and 80 healthy dogs (Group III) were analyzed for comparison. Indirect ELISAs were performed to compare serum levels of aPL subtypes, including anticardiolipin antibodies (aCL), antiphosphatidylserine antibodies (aPS), antiphosphatidylcholine (aPC), and anti-ß2 glycoprotein I antibodies (aß2GPI), and antiphosphatidylinositol antibodies (aPI), among different groups or subgroups of dogs. Among outpatient dogs, aCL, being highly prevalent in outpatient dogs with thrombocytopenia (63/64, 98 %), is an important risk factor for thrombocytopenia (with a high relative risk of 8.3), immune thrombocytopenia (relative risk 5.3), or severe thrombocytopenia negative for aPLT (relative risk ∞, odds ratio 19). In addition, aPS is a risk factor for immune thrombocytopenia or severe thrombocytopenia negative for aPLT (moderate relative risks around 2), whereas aPC and aß2GPI are risk factors for immune thrombocytopenia (relative risks around 2). CONCLUSIONS: Of all the aPL subtypes tested here, aCL is highly associated with canine thrombocytopenia, including immune thrombocytopenia, severe thrombocytopenia negative for aPLT, and less severe thrombocytopenia. Furthermore, aPS is moderately associated with both canine immune thrombocytopenia and severe thrombocytopenia negative for aPLT, whereas aß2GPI, and aPC are moderately relevant to canine immune thrombocytopenia. In contrast, aPI is not significantly associated with canine immune thrombocytopenia.

Sequence and phylogenetic analysis of the gp200 protein of Ehrlichia canis from dogs in Taiwan.

Ehrlichia (E.) canis is a Gram-negative obligate intracellular bacterium responsible for canine monocytic ehrlichiosis. Currently, the genetic diversity of E. canis strains worldwide is poorly defined. In the present study, sequence analysis of the nearly full-length 16S rDNA (1,620 bp) and the complete coding region (4,269 bp) of the gp200 gene, which encodes the largest major immunoreactive protein in E. canis, from 17 Taiwanese samples was conducted. The resultant 16S rDNA sequences were found to be identical to each other and have very high homology (99.4~100%) with previously reported E. canis sequences. Additionally, phylogenetic analysis of gp200 demonstrated that the E. canis Taiwanese genotype was genetically distinct from other reported isolates obtained from the United States, Brazil, and Israel, and that it formed a separate clade. Remarkable variations unique to the Taiwanese genotype were found throughout the deduced amino acid sequence of gp200, including 15 substitutions occurring in two of five known species-specific epitopes. The gp200 amino acid sequences of the Taiwanese genotype bore 94.4~94.6 identities with those of the isolates from the United States and Brazil, and 93.7% homology with that of the Israeli isolate. Taken together, these results suggest that the Taiwanese genotype represents a novel strain of E. canis that has not yet been characterized.

Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein (TRAP) gene of Babesia gibsoni isolates from dogs in Taiwan.

The genetic diversity of Babesia gibsoni strains worldwide is currently poorly defined. The aim of the present study was to characterize B. gibsoni strains in naturally infected dogs in Taiwan using a combination of polymerase chain reaction (PCR) and sequence analysis of both 18S rDNA and the gene encoding thrombospondin-related adhesive protein (TRAP). Genomic DNA was extracted from 29 parasitemic dogs, and the target genes were separately amplified, sequenced and aligned with corresponding sequences available in GenBank. All 18S rDNA sequences (1,262 bp) amplified from the Taiwanese isolates were identical to each other and had very high similarity (99.9-100%) with previously reported B. gibsoni sequences. These results provide the first molecular evidence showing infection of dogs with B. gibsoni from Taiwan. On the other hand, a phylogenetic analysis based on the deduced amino acid sequence of the TRAP gene demonstrated that the Taiwanese isolates were closely related to strains previously identified from Okinawa Island, Japan, but genetically distinct from strains found on Honshu in Japan and Jeju Island in South Korea. The divergence of TRAP among the geographically dispersed strains examined in this study and others supports the conclusion that this gene is useful for molecular genotyping of B. gibsoni strains.

Detection and characterization of four novel genotypes of Ehrlichia canis from dogs.

The genetic diversity of Ehrlichia canis strains worldwide is currently poorly defined. The present study aimed to characterize E. canis strains in naturally infected dogs in Taiwan, using a combination of PCR and sequence analysis of the 16S rDNA and two antigen-encoding genes, gp19 and gp36. Genomic DNA was extracted from 34 parasitemic dogs and the genes of the pathogen were separately amplified, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rDNA sequences (1623 bp) amplified from the Taiwanese isolates were identical and had very high similarity (99.4-100%) with previously reported E. canis sequences. Nevertheless, most of the gp19 gene sequences (414 bp) from the Taiwanese isolates had three specific nucleotide substitutions at positions 9, 323 and 371 that resulted in three amino acid changes. The gp36 gene of the Taiwanese isolates consists of three regions: a 5' end pre-repeat region (426 bp), a tandem repeat region with variable numbers of the 27-bp repeat unit depending on the isolate, and a 3' end region (87 bp). The nucleotide sequences of the 5' end region of gp36 from Taiwanese isolates were identical to each other, but unexpectedly, quite distinct from the sequences of eleven other E. canis strains previously published, with 86.7-87.2% identities only. A phylogenetic tree of E. canis strains based on the gp36 amino acid sequences showed that the Taiwanese isolates fell into a separate clade, indicating the presence of a novel strain that had not yet been characterized.

Toxicity of jet fuel aliphatic and aromatic hydrocarbon mixtures on human epidermal keratinocytes: evaluation based on in vitro cytotoxicity and interleukin-8 release.

Jet fuels are complex mixtures of aliphatic (ALI) and aromatic (ARO) hydrocarbons that vary significantly in individual cytotoxicity and proinflammatory activity in human epidermal keratinocytes (HEK). In order to delineate the toxicological interactions among individual hydrocarbons in a mixture and their contributions to cutaneous toxicity, nine ALI and five ARO hydrocarbons were each divided into five (high/medium/low cytotoxic and strong/weak IL-8 induction) groups and intra/inter-mixed to assess for their mixture effects on HEK mortality and IL-8 release. Addition of single hydrocarbon to JP-8 fuel was also evaluated for their changes in fuel dermatotoxicity. The results indicated that when hydrocarbons were mixed, HEK mortality and IL-8 release were not all predictable by their individual ability affecting these two parameters. The lowest HEK mortality (7%) and the highest IL-8 production were induced with mixtures including high cytotoxic and weak IL-8 inductive ARO hydrocarbons. Antagonistic reactions not consistently correlated with ALI carbon chain length and ARO structure were evident and carried different weight in the overall mixture toxicities. Single addition of benzene, toluene, xylene or ethylbenzene for up to tenfold in JP-8 did not increase HEK mortality while single addition of ALI hydrocarbons exhibited dose-related differential response in IL-8. In an all ALI environment, no single hydrocarbon is the dominating factor in the determination of HEK cytotoxicity while deletion of hexadecane resulted in a 2.5-fold increase in IL-8 production. Overall, decane, undecane and dodecane were the major hydrocarbons associated with high cytotoxicity while tetradecane, pentadecane and hexadecane were those which had the greatest buffering effect attenuating dermatotoxicity. The mixture effects must be considered when evaluating jet fuel toxicity to HEK.