Evaluation of surface antigen TF1.17 in feline Tritrichomonas foetus isolates.

Tritrichomonas foetus (T. foetus) is a flagellated protozoa that infects the distal ileum and proximal colon of domestic cats, as well as the urogenital tract of cattle. Feline trichomonosis is recognized as a prevalent cause of chronic diarrhea in cats worldwide. The suspected route of transmission is fecal-oral, with cats in densely crowded environments at highest risk for infection. Thus, the recommended strategy for minimizing spread of infection is to identify and isolate T. foetus-positive cats from the general population. Rapid identification of infected cats can be challenging due to the inability to accurately and quickly detect the organism in samples at point of care facilities. Thus, identification of targets for use in development of a novel diagnostic test, as well as a vaccine or therapy for T. foetus infection is a significant area of research. Despite a difference in organ tropism between T. foetus genotypes, evidence exists for conserved virulence factors between feline and bovine T. foetus. The bovine T. foetus surface antigen, TF1.17, is an adhesin that is conserved across isolates. Vaccination with the purified antigen results in amelioration of cytopathogenicity and more rapid clearance of infection in cattle. We previously showed that three feline isolates of T. foetus were positive for TF1.17 antigen so we further hypothesized that TF1.17 is conserved across feline T. foetus isolates and that this antigen would represent an attractive target for development of a novel diagnostic test or therapy for feline trichomonosis. In these studies, we used monoclonal antibodies previously generated against 1.15 and 1.17 epitopes of the bovine T. foetus TF1.17 antigen, to evaluate for the presence and role of TF1.17 in the cytopathogenicity of feline T. foetus. A previously validated in vitro co-culture approach was used to model feline T. foetus infection. Immunoblotting, immunofluorescence assays, and flow cytometric analysis confirmed the presence and surface localization of antigen TF1.17 across all feline T. foetus isolates tested. Antigen TF1.17 was notably absent in the presumably nonpathogenic intestinal trichomonad, Pentatrichomonas hominis, a parasite that can be confused microscopically with T. foetus. Similar to bovine trichomoniasis, TF1.17 was found to promote T. foetus adhesion to the intestinal epithelium. These results support further characterization and development of the TF1.17 antigen as a possible target for the diagnosis and prevention of feline T. foetus infection.

Relationship of adiponectin and its multimers to metabolic indices in cats during weight change.

Adiponectin is an important anti-inflammatory hormone secreted from adipose tissue. The high-molecular-weight form of adiponectin (HMW) closely correlates with insulin sensitivity in human beings. This study uses a novel method of size-exclusion gel chromatography combined with enzyme-linked immunosorbent assay to measure HMW feline adiponectin and determine its relationship to leptin, cholesterol, and insulin sensitivity as cats gain and lose weight. In addition, total adiponectin and its messenger RNA expression in subcutaneous adipose tissue were measured. No correlations were found between total serum adiponectin and subcutaneous adipose messenger RNA expression, fat mass, or measures of insulin sensitivity. This study demonstrates that cats have high percentages of HMW adiponectin. Although weak correlations between HMW adiponectin and fat mass were detected, additional cats are needed to determine if the correlations are significant.

Serial evaluation of neutrophil function in tumour-bearing dogs undergoing chemotherapy.

We hypothesized that neutrophil function in tumour-bearing dogs is negatively impacted by chemotherapy. Flow cytometric techniques were used to assess neutrophil oxidative burst and phagocytic activities at baseline, 7 and 21 days after induction chemotherapy in 20 dogs with lymphoma. Dogs had a lower percentage of neutrophils exhibiting oxidative burst activity after stimulation with Escherichia coli (day 7; P = 0.009) and phorbol 12-myristate 13-acetate (PMA) (days 7 and 21; P = 0.0003 and P = 0.01, respectively), compared with healthy controls. From day 0 to 7, the percentage of neutrophils exhibiting oxidative burst activity decreased after stimulation with E. coli (P = 0.016) and PMA (P = 0.0006). Induction chemotherapy suppresses the percentage of neutrophils capable of oxidative burst in dogs with lymphoma, with improvement in phagocytic activity over time (P = 0.03). The impact of neutrophil dysfunction on incidence and severity of sepsis in dogs receiving chemotherapy should be investigated.

Multilocus sequence typing for characterization of Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin.

The role of mecA and blaZ regulatory elements in mecA expression by regional clones of methicillin-resistant Staphylococcus pseudintermedius.

Two major regional clones of methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been identified in Europe and North America. They are designated multilocus sequence types (ST) 71 and 68 and contain staphylococcal chromosome cassette (SCCmec) types II-III and V(T), respectively. One notable difference between the two clones is a deletion in the mecI/mecR1 regulatory apparatus of ST 68 SCCmec V(T). This deletion in analogous methicillin-resistant Staphylococcus aureus (MRSA) results in more responsive and greater expression of the mecA encoded penicillin-binding protein 2a, and is associated with SCCmec types occurring in community-acquired MRSA lineages. The aim of this study was to characterize mec and bla regulatory apparatuses in MRSP and determine their effects on expression of mecA. Seventeen S. pseudintermedius isolates representing nine methicillin-resistant ST lineages were screened for the presence of the repressors blaI and mecI and sensors blaR1 and mecR1. The bla and mec operons for each isolate were sequenced and compared for homology between the repressor open-reading frames (ORF), sensor ORFs, and mecA promoter regions. A real-time reverse transcriptase PCR expression assay was developed, validated and applied to nine isolates determining the effect of oxacillin induction on mecA transcription. Significant differences were found in mecA expression between isolates with a full regulatory complement (mecI/mecR1 and blaI/blaR1) and those with truncated and/or absent regulatory elements. Isolates representative of European and North American MRSP ST regional clones have dissimilar mecA responses to oxacillin.

Identification of a predominant multilocus sequence type, pulsed-field gel electrophoresis cluster, and novel staphylococcal chromosomal cassette in clinical isolates of mecA-containing, methicillin-resistant Staphylococcus pseudintermedius.

Methicillin resistance encoded by the mecA gene is increasingly observed in Staphylococcus pseudintermedius. Little is known about the population genetics of veterinary staphylococci bearing methicillin resistance. The aim of this study was to determine the relatedness of resistant bacteria and to compare them with methicillin-susceptible isolates. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) fragment profiling were performed on methicillin-resistant S. pseudintermedius (MRSP) and methicillin-susceptible S. pseudintermedius (MSSP) isolates obtained from canine samples submitted to the veterinary teaching hospital bacteriology service between 2006 and 2008. Multilocus sequence typing detected 20 different sequence types, 16 of which were not previously described. Methicillin-resistant isolates were predominantly ST 68, possessed the Staphylococcus aureus-associated staphylococcal chromosomal cassette mec (SCCmec) type V(T) and fell within the largest PFGE cluster; whereas methicillin-susceptible strains were more genetically diverse. This suggests that most methicillin resistance within the population of isolates tested originated from a single source which has persisted and expanded for several years.

Role of chromodomain helicase DNA-binding protein 2 in DNA damage response signaling and tumorigenesis.

The chromodomain helicase DNA-binding proteins (CHDs) are known to affect transcription through their ability to remodel chromatin and modulate histone deacetylation. In an effort to understand the functional role of the CHD2 in mammals, we have generated a Chd2 mutant mouse model. Remarkably, the Chd2 protein appears to play a critical role in the development, hematopoiesis and tumor suppression. The Chd2 heterozygous mutant mice exhibit increased extramedullary hematopoiesis and susceptibility to lymphomas. At the cellular level, Chd2 mutants are defective in hematopoietic stem cell differentiation, accumulate higher levels of the chromatin-associated DNA damage response mediator, gamma H2AX, and exhibit an aberrant DNA damage response after X-ray irradiation. Our data suggest a direct role for the chromatin remodeling protein in DNA damage signaling and genome stability maintenance.

Changes in the testis seminiferous tubules and interstitium in prepubertal bull calves immunised against inhibin at the time of gonadotropin administration.

The aim of the present study was to evaluate the effects of gonadotropin administration at initiation of inhibin passive immunisation in Jersey bull calves (age 27 +/- 5 days) on testicular morphology and development. Primary treatments consisted of control (keyhole limpet haemocyanin, KLH; n = 9) or immunisation against inhibin (INH; n = 9). Subsets of calves were randomly assigned within primary treatments (TRT) to receive saline ( n = 3 per TRT), follicle-stimulating hormone (FSH; n = 3 per TRT) or gonadotrophin-releasing hormone (GnRH, n = 3 per TRT). The right testis was removed (age 118 +/- 5 days) to determine volumes of testicular components and cell numbers per testis using stereology. Data were analysed using the MIXED procedure of the SAS program. Antibody titres against inhibin were increased in INH bulls compared with KLH bulls (P < 0.05). In addition, a significant immunisation x hormone treatment interaction was noted for the number of germ cells. Administration of FSH at the time of initial immunisation against inhibin significantly increased the number of germ cells (92.2 +/- 9 x 10(6) cells) compared with INH+saline bulls (54.9 +/- 10 x 10(6) cells), with INH+GnRH bulls having an intermediate number of cells (64.5 +/- 9 x 10(6) cells; P < 0.05). These results suggest that gonadotropin administration at the time of inhibin immunisation increases the number of germ cells in the testis.

Influence of ambient atmosphere on charge transport in polycrystalline thin films of three simple aromatic hydrocarbons.

Drift mobility of charge carriers in p-terphenyl, p-quaterphenyl and tetracene polycrystalline thin films was measured using the classical time-of-flight method. The measurements were carried out both in the atmosphere and in vacuum of the order of 10( - 3) Torr. The mobility measured in the atmosphere is of the order of 10(-4)cm2 V(-1) s(-1) and turns out to be about 1 order of magnitude higher than that measured in vacuum. It is suggested that the hopping transport of charge dominates in the structures investigated. The increase in the mobility for the measurements in the atmosphere probably results from the additional localized states due to water and other atmosphere molecules.

Evaluation of an indirect enzyme-linked immunosorbent assay for the detection of feline lentivirus-reactive antibodies in wild felids, employing a puma lentivirus-derived synthetic peptide antigen.

An enzyme-linked immunosorbent assay (ELISA) using a puma lentivirus-derived synthetic peptide as coating antigen was evaluated as a diagnostic test for infection with feline immunodeficiency virus (FIV) or related lentiviruses in free-ranging lions. The sensitivity and specificity of the ELISA was determined using two approaches. In the first approach, the results were standardized according to certain statistical criteria, and in the second, the puma lentivirus western blot was used as the gold standard. The sensitivity of the test when compared with the standardized results was 85.4% and the specificity 100%. The sensitivity of the test when using the western blot as the gold standard was 78.6% and the specificity 100%. The test would therefore be well-suited to the screening of populations of wild felids in which FIV or related lentiviruses are endemic. The results also indicate that in spite of genetic divergence between lentiviruses isolated from Panthera and Felis spp., puma lentivirus-derived antigens can be used in immunoassays for the detection of antibodies in Panthera spp. reactive to FIV or related lentiviruses. The results also indicate that the lion population in the Hluhluwe-Umfolozi Game Reserve, South Africa is lentivirus negative.

The ovine respiratory syncytial virus F gene sequence and its diagnostic application.

Ruminant respiratory syncytial viruses (RSVs) are classified into 2 subgroups, ovine RSV and bovine RSV. Although ovine RSV infects cattle, its contribution to bovine respiratory tract disease has not been established, which is an important issue for vaccine development in cattle. Diagnosis by virus isolation or serology has low or variable sensitivity and/or specificity and polymerase chain reaction (PCR) has been recommended as a rapid and sensitive technique for RSV detection. A simple procedure has been developed to detect and identify bovine and ovine RSVs. First, the nucleotide sequence of the ovine RSV fusion (F) gene was determined and compared with representative strains of bovine RSV and human RSV subgroups A and B. The ovine RSV F gene has 85 and 72-73% nucleotide identity with those of bovine RSV and human RSV, respectively. The predicted amino acid sequence of the ovine RSV F gene has 94 and 83-84% amino acid identity with those of bovine RSV and human RSV, respectively. Then PCR primers targeting a specific F gene fragment of bovine and ovine RSV were designed. The primers represented bases 85-103 and the complementary sequence to bases 510-493 of the ovine RSV F gene. A similar PCR product (426 bp) was obtained on agarose gel electrophoresis from bovine RSV and from ovine RSV. The products, however, were unique to the parent virus and could be distinguished by EcoRI or MspI restriction endonuclease cleavage. EcoRI cleaved the ovine product into 2 bands (285 and 141 bp) but failed to affect the bovine RSV PCR product. However, MspI cleaved the bovine product into 2 bands (229 and 197 bp) but had no effect on the ovine product. Also, this assay did not amplify any PCR product with human RSV. The reverse transcription-polymerase chain reaction (RT-PCR) followed by restriction enzyme digestion is a useful and practical approach for detection and differentiation of ruminant respiratory syncytial viruses.

Identifying patients with gastroesophageal reflux disease in a managed care organization.

The ability of various strategies to identify patients with gastroesophageal reflux disease (GERD) and the relative economic impact on disease management programs for GERD were studied. A telephone interview was conducted of a random sample of patients enrolled in any of three health plans in a 100,000-member managed care organization who had either a pharmacy claim or an encounter claim during 1997. The telephone interview identified patients with GERD and served as the standard by which the sensitivity, specificity, and predictive values of the following patient-identification strategies were compared: (1) telephone interview, (2) chart review, (3) use of encounter claims, (4) use of pharmacy claims, (5) use of both encounter claims, and pharmacy claims, and (6) use of encounter claims or pharmacy claims. Conservative estimates of costs and projected savings were then used to model the potential return on investment of the strategies. A total of 1186 patients completed the telephone interview, of whom 390 (33%) met the case definition of GERD. The most sensitive method for identifying patients with GERD was using either pharmacy or encounter claims (26%). The most specific strategy with the highest positive predictive value (PPV) (87%) was using both pharmacy and encounter claims, but this approach had a case-detection rate of only 3%. Encounter claims were significantly more sensitive than pharmacy claims and yielded a higher estimate of prevalence. The telephone interview identified the most subjects who could have benefited from a disease management program and cost 84% less than chart review. While use of administrative data (pharmacy and encounter claims) was the least costly strategy, it identified 74% fewer patients expected to benefit from disease management. The efficiency of disease management programs for GERD may depend on the method of patient identification, which in turn may depend on whether PPV or negative predictive value (NPV) should be maximized. If there is a need to identify all cases (i.e., sensitivity and NPV are most important), then telephone interview may provide the greatest opportunity for disease management with the greatest return on investment, but at the expense of enrolling many patients who may not benefit.

Bordetella bronchiseptica fimbrial protein-enhanced immunogenicity of a Mannheimia haemolytica leukotoxin fragment.

Leukotoxin produced by Mannheimia (Pasteurella) haemolytica is an important virulence factor in shipping fever pneumonia in feedlot cattle and is a critical protective antigen. In this study, the immune response to a chimeric protein generated by combining a gene fragment encoding neutralizing epitopes of M. haemolytica leukotoxin and a fimbrial protein gene (fim N) from Bordetella bronchiseptica was evaluated. The recombinant gene was cloned in a bacterial expression vector under the control of the tac promoter and expressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli. Immunization of mice with the recombinant protein, GST-LTXFIM elicited a significantly stronger anti-leukotoxin antibody response than comparable immunizations with GST-LTX fusion proteins lacking FIM N. The GST-LTXFIM was also more stable than GST-LTX during storage at -80 degrees C, thus alleviating a stability problem inherent to leukotoxin. This chimeric protein may be a candidate for inclusion in new generation vaccines against shipping fever pneumonia.

Deletions in the 7a ORF of feline coronavirus associated with an epidemic of feline infectious peritonitis.

A population of Persian cats experienced an epidemic of feline infectious peritonitis (FIP) over 2 years. Twelve cases of FIP occurred in litters born during this period. Cats contracting FIP were all genetically related through the sire. Feline coronavirus (FCoV) genomic RNA was detected consistently in this study in biologic samples from adult cats, kittens suffering from FIP, and their siblings. Analysis of viral 7a/7b open reading frame (ORFs) were analyzed and revealed two distinct virus variants circulating in the population, one with an intact 7a ORF and one with two major deletions in the 7a ORF. The 7b ORFs were intact and similar among all virus isolates, although point mutations resulting in amino acid changes were present. The sire was determined to be infected with both variants, and was persistently virus-infected. We speculate the deletion variant arose from the non-deletion variant during viral replication in this population, possibly in the sire.

Validation of synthetic peptide enzyme immunoassays in differentiating two subgroups of ruminant respiratory syncytial virus.

Subgroup-specific peptide-based enzyme immunoassays from each respective G-glycoprotein of the ovine and the bovine respiratory syncytial virus (RSV) were developed to detect RSV-specific IgG responses in cattle. Antigenic peptides from the respective G-glycoprotein were identified from the extracellular central hydrophobic region (amino acids 158-189) located between 2 mucin-rich regions. These antigenic peptides identified by epitope mapping from each G-glycoprotein were synthesized and used to develop the subgroup-specific enzyme immunoassays. The negative cutoff for each enzyme immunoassay was established as the mean optical density of indirect immunofluorescent antibody-negative bovine sera plus 3 SDs. The sensitivity (82.9%) and specificity (100%) of the bovine enzyme immunoassay and the specificity (95.8%) of the ovine enzyme immunoassay were determined by comparison with indirect immunofluorescence (used as the "gold standard"). The negative and positive predictive values were calculated for each assay. The presence of serum antibody to ovine RSV in cattle implies that this virus infects cattle and may contribute to the pathogenesis of bovine respiratory disease.

Prevalence of ovine and bovine respiratory syncytial virus infections in cattle determined with a synthetic peptide-based immunoassay.

Subgroup-specific peptide-based enzyme-linked immunosorbent assays from the G-protein of the ovine and bovine respiratory syncytial virus (RSV), respectively, were used to determine the prevalence of the ovine and bovine subgroup strains of RSV infections in cattle. A total of 1,102 bovine serum samples were obtained from 6 diagnostic laboratories located in the northwestern and the southeastern USA and were tested for antibody to either the bovine or ovine subgroups of RSV. Antibody to viruses from each subgroup was present in samples from each region and all states tested. The Southeast had a higher prevalence of the bovine subgroup strains (69.5%). Then did the Northwest (40.9%). The prevalence of the ovine strain was similar for the two regions (16.7% in the southeast, 14.9% in the northwest). The overall prevalence was 56.6% for the bovine strain and 15.9% for the ovine strain. These results suggest members of the ovine subgroup of RSV circulate in the cattle population but with less frequency than those viruses of the bovine subgroup.

Enzyme replacement therapy in feline mucopolysaccharidosis I.

Enzyme replacement therapy (ERT) has long been considered an approach to treating lysosomal storage disorders caused by deficiency of lysosomal enzymes. ERT is currently used to treat Gaucher disease and is being developed for several lysosomal storage disorders now that recombinant sources of the enzymes have become available. We have continued development of ERT for mucopolysaccharidosis I (MPS I) using the feline model. Recombinant alpha-L-iduronidase was administered intravenously at low dose (approximately 0.1 mg/kg or 25,000 units/kg) to four cats and high dose (0.5 mg/kg or 125,000 units/kg) to two cats on a weekly basis for 3- or 6-month terms. Clinical examinations showed distinct clearing of corneal clouding in one cat although clinical effects in the others were not evident. Biochemical studies of the cats showed that the enzyme was distributed to a variety of tissues although the liver and spleen contained the highest enzyme activities. Glycosaminoglycan storage was decreased in liver and spleen, and the histologic appearance improved in liver, spleen, and renal cortex. Enzyme was not consistently detected in cerebral cortex, brainstem, or cerebellum and the histologic appearance and ganglioside profiles did not improve. A variety of other tissues showed low variable uptake of enzyme and no distinct improvement. IgG antibodies to alpha-L-iduronidase were observed in five cats with higher titers noted when higher doses were administered. Mild complement activation occurred in three cats. Enzyme replacement therapy was effective in reversing storage in some tissues at the biochemical and histologic level in MPS I cats but an improved tissue distribution and prevention of a significant immune response could make the therapy more effective.

Degradation of bovine complement C3 by trichomonad extracellular proteinase.

Bovine trichomoniasis is a local infection of the reproductive tract making interaction with mucosal host defenses crucial. Since the parasite is susceptible to killing by bovine complement, we investigated the role of the third component of complement (C3) in host parasite interactions. Bovine C3 was purified by anionic and cationic exchange chromatography. The purified protein was characterized by immunoreactivity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and peptide sequencing of the amino terminus of the beta chain. When purified bovine C3 was incubated for varying time periods with trichomonad extracellular proteinases, SDS-PAGE gels revealed digestion of the alpha chain to small fragments. Such degradation in vivo would prevent formation of C3b and completion of the complement cascade, resulting in evasion of killing. To evaluate the relevance of this data, we determined whether C3 was present in bovine genital secretions. With a quantitative ELISA assay, C3 could be demonstrated in both uterine and vaginal washes. To our knowledge, this is the first demonstration of bovine C3 in genital secretions. The C3 concentration increased significantly in vaginal secretions by 8 and 10 weeks in heifers infected with Tritrichomonas foetus. An increase was also seen in uterine secretions of infected heifers, but sample numbers were insufficient for statistical analysis. Transcription of the major extracellular cysteine proteinase (TFCP8) was demonstrated in T. foetus cells from uterine secretions of infected heifers by RT-PCR and Southern blotting. The results indicate that C3 may be important in genital defense and that trichomonad extracellular proteinases may play a role in evasion of complement-mediated killing.

Detection of feline coronavirus infection in captive cheetahs (Acinonyx jubatus) by polymerase chain reaction.

Feline coronavirus genetic elements were detected by polymerase chain reaction from blood, fecal samples, and effusive fluid collected from 33 cheetahs in the U.S.A. Feline coronavirus-specific serum antibodies were also measured by indirect immunofluorescence. Ten cheetahs were positive for viral shedding by polymerase chain reaction, whereas 13 were seropositive by immunofluorescence. Results of serology did not consistently correlate with shedding of virus, and the capture antigen used for detection of feline coronavirus-specific antibodies had a significant impact on results. Testing of samples from one population over a 1-yr period indicated chronic infection in some animals. These relatively healthy carrier animals were a source of virus for contact animals. Screening programs in cheetah populations for feline coronavirus infection may be most reliable if a combination of serologic analysis and viral detection by polymerase chain reaction is used.

Characterization of fimN, a new Bordetella bronchiseptica major fimbrial subunit gene.

Fimbrial proteins play an important role in the binding of Bordetella bronchiseptica to mammalian cells, an event that is key to the pathogenesis of this organism. The fimbrial phenotype of B. bronchiseptica isolates is usually defined serologically by Fim2 and Fim3 antigens. In this study, a previously unidentified fimbrial gene, fimN, was cloned and sequenced. The identity of fimN is based on several observations. The predicted FimN protein has 59.4 and 52. 2% homology with B. bronchiseptica Fim2 and Fim3, respectively, and is similar in size to these fimbriae. fimN, expressed as a recombinant protein, is recognized by mAb prepared against Fim2 from Bordetella pertussis. The fimN promoter region contains a stretch of cytosine residues similar in length to those of other fimbrial genes expressed by Bordetella species. It also has an activator binding region, upstream from the C-stretch, that closely resembles a corresponding bvg regulated region in fim2, fim3, and fimX. The fimN gene was isolated from a cosmid prepared with B. bronchiseptica genomic DNA that restored normal properties of cellular adhesion to an adhesion deficient strain of B. bronchiseptica. As such, FimN may be a previously overlooked fimbrial antigen and may play an important role in the pathogenicity of B. bronchiseptica.