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To date, there is no clear standard to monitor drug treatment for canine Chagas disease. We used 2 real-time PCR (rtPCR) assays targeting Trypanosoma cruzi kinetoplast DNA (kDNA) and nuclear satellite DNA (nDNA) to detect T. cruzi in canine whole blood. Samples were collected randomly from 131 untreated dogs with unknown T. cruzi infection status in Texas. The kDNA-based rtPCR was slightly more sensitive (diagnostic sensitivity of kDNA = 49% vs. nDNA = 44%; p = 0.5732) but slightly less specific (diagnostic specificity of kDNA = 96% vs. nDNA = 97%; p > 0.9999) than the nDNA-based rtPCR. However, the differences in sensitivity and specificity between the nDNA- and kDNA-based rtPCR assays were not statistically significant. Using the nDNA- and kDNA-based qualitative rtPCR assays to monitor parasitemia from 137 itraconazole- and amiodarone-treated cases with nDNA- and kDNA-based PCR-positive baselines showed that the PCR positive rate decreased to 0% in 30 d. Using kDNA-based quantitative rtPCR to monitor normalized T. cruzi DNA copies in 4 representative dogs demonstrated that drug treatment could reduce parasite loads within 7-30 d. The kDNA-based qualitative rtPCR may be used for routine parasitemia screening of drug-treated Chagas-positive dogs, whereas nDNA-based qualitative rtPCR may be used for confirmation.

Objective: To evaluate clinical, serologic, parasitological, and histologic outcomes of dogs with naturally occurring Trypanosoma cruzi infection treated for 12 months with amiodarone and itraconazole., Animals: 121 dogs from southern Texas and southern Louisiana., Procedures: Treatment group dogs (n = 105) received a combination of amiodarone hydrochloride (approx 7.5 mg/kg [3.4 mg/lb], PO, q 24 h, with or without a loading dosage protocol) and itraconazole (approx 10 mg/kg [4.5 mg/lb], PO, q 24 h, adjusted to maintain a plasma concentration of 1 to 2 μg/mL) for 12 months. Control group dogs (n = 16) received no antitrypanosomal medications. Serologic assays for anti- T cruzi antibodies, PCR assays for T cruzi DNA in blood, and physical evaluations were performed 1, 6, 9, 12, and 24 months after study initiation. Adverse events were recorded. Outcomes of interest were recorded and compared between groups., Results: 86 of 105 treatment group dogs and 8 of 16 control group dogs survived and completed the study (5/19 and 6/7 deaths of treatment and control group dogs, respectively, were attributed to T cruzi infection). Mean survival time until death attributed to T cruzi was longer (23.19 vs 15.64 months) for the treatment group. Results of PCR assays were negative for all (n = 92) tested treatment group dogs (except for 1 dog at 1 time point) from 6 to 24 months after study initiation. Clinical improvement in ≥ 1 clinical sign was observed in 53 of 54 and 0 of 10 treatment and control group dogs, respectively; adverse drug events were minor and reversible., Conclusions and Clinical Relevance: Results suggested efficacy of this trypanocidal drug combination for the treatment of T cruzi infection in dogs.

A new simian retrovirus (SRV) subtype was discovered in China and the USA from Cambodian-origin cynomolgus monkeys. Histopathological examination from necropsied animals showed multifocal lymphoplasmacystic and histocytic inflammation. The complete genome sequences demonstrated that the US virus isolates were nearly identical (99.91-99.93 %) and differed only slightly (99.13-99.16 % identical) from the China isolate. Phylogenetic analysis showed that the new virus isolates formed a distinct branch of SRV-1 through -7, and therefore were named this subtype, SRV-8. This SRV-8 variant was also phylogenetically and serologically more closely related to SRV-4 than any other SRV subtype.

Fatal Yersinia pseudotuberculosis infection in cynomolgus macaques was diagnosed based upon pathology, microbiology and PCR for this study. Pathological findings included acute, erosive to ulcerative, necrohemorrhagic enterocolitis. Genotyping by PCR showed an O:3 pattern (gmd-fcl(+), ddhC-prt(+), manB(+), ddhA-B(+)), but an additional gene, wbyK, was detected. This is the second report to identify wbyK+ O:3 genotype as the cause of fatal yersiniosis. The first case was reported in 2008, and involved farm deer in the U.S. As the frequency of wbyK+ O:3 genotype is found more often in different carriers, O:3 genotype is proposed to be divided into two subtypes: O:3a without wbyK and O:3b with wbyK. Virulence gene analysis showed the presence of inv, ypmC, irp1, ybtP-ybtQ, yadA, yopB, yopH, lcrF, and suggested that this O:3b isolate could be a highly pathogenic strain to cynomolgus macaques., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi, the causative agent of Chagas disease, is endemic in south Texas due to the abundant vector and wild small mammalian reservoir populations. This situation predisposes nonhuman primate colonies exposed to outdoor housing to infection from ingestion or bite of triatomid insects. Using a T. cruzi-specific real-time PCR and Trypanosome spp.-specific ELISA, we revealed a prevalence rate of 8.5% in a colony of outdoor-housed cynomolgus macaques. By using a discriminating kinetoplastid minicircle PCR, we eliminated the possibility of mixed prevalence with nonpathogenic trypanosomes and showed the ELISA results were specific for T. cruzi. In this study, we found an inverse relationship between antibody titers and circulating parasite load. Also, 23% of T. cruzi IgG ELISA-positive macaques were negative by real-time PCR. Furthermore, in a subset of infected macaques, cardiac tissue was infiltrated by inflammatory mononuclear cells and contained T. cruzi genomic and kinetoplast DNA despite lacking microscopic evidence of discrete parasite stages. In addition, 19% of the infected macaques had titers for cardiac troponin I autoantibody, which could contribute to autoimmune myocarditis or interfere with circulating troponin I measurements. These findings indicate the possibility of T. cruzi to interfere with the assessment of cardiac safety signals in preclinical toxicology and safety pharmacology studies and the necessity for prestudy screening for T. cruzi in outdoor-housed nonhuman primates from endemic areas.

The nature of SRV-4 infection in cynomolgus macaques remains unclear to date. Here, we report the monitoring of 24 cynomolgus monkeys that were naturally infected with SRV-4 for virus isolation, proviral load and antibody. The results indicated that the SRV-4 antibody status was statistically correlated to environmental temperature.

At least 5 serotypes of exogenous simian retrovirus type D (SRV/D) have been found in nonhuman primates, but only SRV-1, 2 and 3 have been completely sequenced. SRV-4 was recovered once from cynomolgus macaques in California in 1984, but its genome sequences are unknown. Here we report the second identification of SRV-4 and its complete genome from infected cynomolgus macaques with Indochinese and Indonesian/Indochinese mixed ancestry. Phylogenetic analysis demonstrated that SRV-4 was distantly related to SRV-1, 2, 3, 5, 6 and 7. SRV/D-T, a new SRV/D recovered in 2005 from cynomolgus monkeys at Tsukuba Primate Center in Japan, clustered with the SRV-4 isolates from California and Texas and was shown to be another occurrence of SRV-4 infection. The repeated occurrence of SRV-4 in cynomolgus monkeys in different areas of the world and across 25years suggests that this species is the natural host of SRV-4., (Copyright 2010 Elsevier Inc. All rights reserved.)

G1P[6] rotaviruses were demonstrated previously to be associated with the neonatal nursery outbreak of gastroenteritis in Changhua Christian Hospital that is located in the central region of Taiwan, from September 1994 to May 1995. Meanwhile, rotaviruses were detected in children hospitalized for acute gastroenteritis. Our study characterizes the rotaviruses associated with the nursery outbreak by using genetic approaches. Nucleotide sequence analysis revealed that the VP7 genes of the nursery rotaviruses were distinct from those of the strains circulating in the community. The G1P[6] rotaviruses recovered from the nursery were closely related to another neonatal G1P[6] strain from the northern region of Taiwan in both the VP4 and VP7 genes. The VP4 genes of these nursery strains differed from those of the P[6] human reference strains 1076, M37, RV3, and ST3. Apparently, these nursery rotaviruses were distinct from the strains circulating in the community and seemed to be a variant when compared with P[6] strains reported previously.

The transmembrane glycoprotein NSP4 functions as a viral enterotoxin capable of inducing diarrhea in young mice. It has been suggested that NSP4 may be a key determinant of rotavirus pathogenicity and a target for vaccine development. Twenty two G1P[6] rotaviruses from babies with and without diarrhea were comparatively analyzed along with reference strains and another 22 Taiwanese human rotaviruses of G and P combination types different from the G1P[6] type. The sequence variations in the NSP4 genes were studied by direct sequencing analysis of the amplicons of reverse transcription-PCR. Two genetic groups could be identified in this analysis. While the majority of these strains were closely related to the Wa strain, the G2 viruses were closely related to the S2 strain. Furthermore, phylogenetic analysis of the NSP4 gene among the G2 rotaviruses revealed three distinct lineages associated with DS-1, S2, and E210, respectively, as has been reported previously for the VP7 gene. However, we found no apparent correlation in the deduced amino acid sequences corresponding to the proposed enterotoxic peptide region between the rotaviruses recovered from individuals with and without diarrhea. The NSP4 gene product being a pathogenic determinant may not be a generalized phenomenon.

G2 rotavirus was prevalent in a 1993 epidemic of acute gastroenteritis in Taiwan. In this study, the genetic relationship among G2 rotavirus strains was analysed. The VP7 genes were amplified and sequenced. Except for one strain isolated in 1981, the nucleotide sequences of the VP7 genes of most of the G2 rotaviruses were very similar (identity > 97%) and were closely related to that of a Japanese G2 reference strain, S2. The genetic relatedness of G2 rotaviruses was analysed further by RNA-RNA hybridization. The genomes of the major G2 strains of 1993 did not hybridize well with those of the G2 strains of previous seasons in RNA segments 1, 6 and 7. Partial nucleotide sequences of the VP1 gene were analysed and appeared to be similar among the major G2 strains from the same epidemic (identity > 98%), whereas the identity of the VP1 genes of the major G2 strains of the 1993 epidemic to those of previous seasons was only about 84%. Since the numbers of mutations accumulated in the VP1 and VP7 genes over a period of 10 years were comparable, the significant change in the VP1 genes of the major strains of the 1993 epidemic suggests that these G2 rotaviruses had evolved by genetic reassortment.

The deaD gene of Klebsiella pneumoniae was isolated and its nucleotide sequence determined. The K. pneumoniae gene is highly homologous with the Escherichia coli analog throughout most of the coding region. The deduced primary sequence of the K. pneumoniae deaD gene product is 659 amino acids in length, in contrast with the 571 amino acids of the E. coli deaD product published previously. Sequence comparison revealed several differences near the 3' end of the deaD genes which result in the frame-shift effect. The 3' end sequence of the E. coli deaD gene was therefore analyzed to verify the discrepancy. Our result indicates that the E. coli deaD gene encodes a product of comparable size to the K. pneumoniae DeaD protein, and the carboxyl terminal sequences of the two proteins are highly homologous. In vivo expression of the K. pneumoniae deaD gene in E. coli yielded a 65-kDa protein. Primer extension analysis of the mRNA from K. pneumoniae identified a major transcription start site at an A residue 44 nt upstream of the first in-frame ATG codon.