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  • Author or Editor: Stephen A. Kania x
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Abstract

Objective—To evaluate the use of RNA interference targeted against feline herpesvirus 1 (FHV-1) glycoprotein D for inhibition of FHV-1 infection of feline kidney cells.

Sample Population—Crandell-Rees feline kidney cells.

Procedures—Crandell-Rees feline kidney cells were transfected with small interfering RNAs (siRNAs) that were designed to inhibit expression of FHV-1 glycoprotein D. The effectiveness of the treatment was determined via measurement of amounts of glycoprotein D mRNA, intracellular glycoprotein D, and glycoprotein D expressed on the surface of infected cells and comparison with appropriate control sample data.

Results—2 of 6 siRNAs tested were highly effective in reducing expression (ie, knockdown) of glycoprotein D mRNA; there were 77% and 85% reductions in mRNA in treated samples, compared with findings in the control samples. The knockdown of glycoprotein D mRNA resulted in reduced glycoprotein D protein production, as evidenced by 27% and 43% decreases in expression of glycoprotein D on the surface of siRNA-treated, FHV-1–infected cells and decreased expression of the protein within infected cells, compared with control samples. Treatment with these siRNAs also resulted in inhibition of FHV-1 replication, with reductions of 84% and 77% in amounts of virus released into cell culture supernatant, compared with findings in control samples.

Conclusions and Clinical Relevance—2 chemically produced siRNAs that targeted the glycoprotein D gene significantly reduced FHV-1 titers in treated cells, suggesting that glycoprotein D is necessary for production of infective virions. This gene is a potential target for RNA interference as a means of inhibition of FHV-1 infection of feline cells.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate the ability of 5 small interfering RNAs (siRNAs) targeting mRNA of the feline herpesvirus-1 (FHV-1) DNA polymerase gene to reduce in vitro viral replication and gene expression of FHV-1, to evaluate combinations of these siRNAs with siRNAs that target the glycoprotein D gene of FHV-1, and to determine the combination or combinations of siRNAs that yield the greatest inhibition of in vitro viral replication.

Sample Population—Cultured Crandell-Rees feline kidney (CRFK) cells.

Procedures—CRFK cells were transfected with siRNAs designed to target mRNA of the FHV-1 DNA polymerase gene. Effective treatment was determined by quantification of the inhibition of mRNA available for DNA polymerase translation, viral protein production, and viral replication. Combinations of 2 siRNAs that target mRNA of the FHV-1 DNA polymerase gene and 2 siRNAs that target the mRNA of the essential FHV-1 glycoprotein D gene were evaluated for the ability to inhibit viral replication.

Results—Verified by a reduction in viral gene expression, 2 of the 5 siRNAs designed to target mRNA of the FHV-1 DNA polymerase gene significantly suppressed viral replication. Two combinations of siRNAs that target mRNA of the FHV-1 DNA polymerase gene, the FHV-1 glycoprotein D gene, or both also significantly suppressed viral replication.

Conclusions and Clinical Relevance—Combinations of siRNAs that target mRNA of the FHV-1 DNA polymerase gene, FHV-1 glycoprotein D gene, or both could potentially be used as a treatment for the prevention of clinical disease associated with FHV-1 infection.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To analyze the 7a7b genes of the feline coronavirus (FCoV) of cheetahs, which are believed to play a role in virulence of this virus.

Sample Population—Biologic samples collected during a 4-year period from 5 cheetahs at the same institution and at 1 time point from 4 cheetahs at different institutions.

Procedures—Samples were first screened for FCoV via a reverse transcription-PCR procedure involving primers that encompassed the 3′-untranslated region. Samples that yielded positive assay results were analyzed by use of primers that targeted the 7a7b open reading frames. The nucleotide sequences of the 7a7b amplification products were determined and analyzed.

Results—In most isolates, substantial deletional mutations in the 7a gene were detected that would result in aberrant or no expression of the 7a product because of altered reading frames. Although the 7b gene was also found to contain mutations, these were primarily point mutations resulting in minor amino acid changes. The coronavirus associated with 1 cheetah with feline infectious peritonitis had intact 7a and 7b genes.

Conclusions and Clinical Relevance—The data suggest that mutations arise readily in the 7a region and may remain stable in FCoV of cheetahs. In contrast, an intact 7b gene may be necessary for in vivo virus infection and replication. Persistent infection with FCoV in a cheetah population results in continued virus circulation and may lead to a quasispecies of virus variants.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine frequency with which Staphylococcus schleiferi could be isolated from dogs with pyoderma and antimicrobial susceptibility patterns of isolates that were obtained.

Design—Prospective study.

Animals—54 dogs with a first (n = 14) or recurrent (40) episode of pyoderma.

Procedure—Specimens were obtained and submitted for bacterial culture. Isolates were identified as S schleiferi on the basis of growth and biochemical characteristics. Two isolates were submitted for DNA sequencing to confirm identification. Methicillin susceptibility was determined by means of disk diffusion with oxacillin-impregnated disks.

Results—3 of 14 dogs examined because of a first episode of pyoderma and 12 of 40 dogs examined because of a recurrent episode of pyoderma were receiving antimicrobials at the time of specimen collection. Staphylococcus schleiferi was not isolated from any dog with first-time pyoderma but was isolated from 5 dogs with recurrent pyoderma that were not receiving antimicrobials at the time of specimen collection and 10 dogs with recurrent pyoderma that were receiving antimicrobials. Nine isolates were identified as S schleiferi subsp schleiferi, and 6 were identified as S schleiferi subsp coagulans. All S schleiferi subsp schleiferi isolates were resistant to methicillin, but only 2 S schleiferi subsp coagulans isolates were. Two methicillin-resistant isolates were also resistant to fluoroquinolones, and 1 isolate had intermediate susceptibility to fluoroquinolones.

Conclusions and Clinical Relevance—Results suggest that S schleiferi subsp schleiferi and S schleiferi subsp coagulans may be isolated from dogs with recurrent pyoderma. Although isolates from dogs with pyoderma were frequently resistant to methicillin, multiple drug resistance was uncommon. (J Am Vet Med Assoc 2003;222:451–454)

Restricted access
in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine whether resistance to oxacillin and other antimicrobials in 3 Staphylococcus spp commonly isolated from dogs increased from 2001 to 2005.

Design—Retrospective case series.

Sample Population—1,772 clinical samples of various types obtained from dogs examined at the University of Tennessee Veterinary Teaching Hospital or at regional veterinary hospitals and submitted to the bacteriology and mycology laboratories associated with the teaching hospital.

Procedures—Samples were submitted by attending veterinarians to the bacteriology and mycology laboratories for routine aerobic microbial culture. Identification and antimicrobial susceptibility procedures were performed on all isolates. Susceptibility reports for each antimicrobial and Staphylococcus spp were determined from aggregate electronically archived test results. Oxacillin and multidrug resistance for Staphylococcus intermedius was analyzed by reviewing disk diffusion zone measurements.

Results—Oxacillin resistance increased among S intermedius isolates during the past 5 years, and the increase was associated with multidrug resistance. In 2005, 1 in 5 Staphylococcus spp isolates from canine clinical samples was resistant to oxacillin. The most common staphylococcal species isolated were S intermedius (n = 37), Staphylococcus schleiferi (21), and Staphylococcus aureus (4), and frequencies of oxacillin resistance in isolates of these species were 15.6%, 46.6%, and 23.5%, respectively.

Conclusions and Clinical Relevance—Veterinarians should be aware of the potential for empiric drug treatment failures in instances where Staphylococcus spp infections are common (eg, pyoderma). Judicious use of bacterial culture and susceptibility testing is recommended.

Restricted access
in Journal of the American Veterinary Medical Association

Abstract

Objective—To evaluate the ability of small interfering RNAs (siRNAs) to inhibit in vitro viral replication and gene expression of feline coronavirus (FCoV).

Sample—Cell cultures of Crandell-Rees feline kidney cells.

Procedures—5 synthetic siRNAs that each targeted a different region of the FCoV genome were tested individually and in various combinations for their antiviral effects against 2 strains of FCoV (feline infectious peritonitis virus WSU 79-1146 and feline enteric coronavirus WSU 79-1683) in cell cultures. Tested combinations targeted the FCoV leader and 3′ untranslated region, FCoV leader region and nucleocapsid gene, and FCoV leader region, 3′ untranslated region, and nucleocapsid gene. For each test condition, assessments included relative quantification of the inhibition of intracellular viral genomic RNA synthesis by means of real-time, reverse-transcription PCR analysis; flow cytometric evaluation of the reduction of viral protein expression in infected cells; and assessment of virus replication inhibition via titration of extracellular virus with a TCID50 infectivity assay.

Results—The 5 siRNAs had variable inhibitory effects on FCoV when used singly. Combinations of siRNAs that targeted different regions of the viral genome resulted in more effective viral inhibition than did individual siRNAs that targeted a single gene. The tested siRNA combinations resulted in approximately 95% reduction in viral replication (based on virus titration results), compared with findings in negative control, nontargeting siRNA–treated, FCoV-infected cells.

Conclusions and Clinical Relevance—In vitro replication of FCoV was specifically inhibited by siRNAs that targeted coding and noncoding regions of the viral genome, suggesting a potential therapeutic application of RNA interference in treatment of feline infectious peritonitis.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the methicillin-resistant profile of staphylococcal isolates from the skin of dogs with pyoderma.

Animals—90 dogs with pyoderma.

Procedure—Staphylococci isolated from dogs with pyoderma were tested for susceptibility to methicillin by use of a standard disk diffusion test with oxacillin disks. The DNA extracted from the isolates was tested for the mecA gene that encodes the penicillinbinding protein 2a (PBP2a) by use of a polymerase chain reaction (PCR) assay. The expression of PBP2a was determined with a commercial latex agglutination assay. Species of staphylococcal isolates were identified by use of morphologic, biochemical, and enzymatic tests.

Results—Most of the isolated staphylococci were methicillin-susceptible, coagulase-positive Staphylococcus intermedius isolates. Whereas only 2 of 57 S intermedius isolates were resistant to methicillin, approximately half of the isolates had the mecA gene and produced PBP2a. Staphylococcus schleiferi was the second most common isolate. Widespread resistance to methicillin was found among S schleiferi isolates. More coagulase-negative S schleiferi isolates were identified with mecA gene-mediated resistance to methicillin, compared with coagulase-positive S schleiferi isolates.

Conclusions and Clinical Relevance—The latex agglutination assay for the detection of PBP2a expression coupled with the PCR assay for the mecA gene may provide new information about emerging antimicrobial resistance among staphylococcal isolates. (Am J Vet Res2004;65:1265–1268)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate agents used for delivery of small interfering RNAs (siRNAs) into feline corneal cells, toxicity of the delivery agents, and functionality of anti-feline herpesvirus 1 (FHV-1)–specific siRNA combinations.

Sample—Feline primary corneal cells and 19 six-month-old colony-bred cats.

Procedures—siRNA delivery into corneal cells via various delivery agents was evaluated via flow cytometric detection of labeled siRNAs. Cellular toxicity was evaluated with a proliferation assay. Functionality was tested via quantitative reverse transcriptase PCR assay, plaque assay, and flow cytometry. In vivo safety was evaluated with an ocular scoring method following topical application of delivery agents containing siRNAs into eyes. Corneal biopsy specimens were used to assess safety and uptake of siRNAs into corneal cells.

Results—Use of 3 delivery agents resulted in > 95% transfection of primary corneal cells. Use of a peptide for ocular delivery yielded approximately 82% transfection of cells in vitro. In cultured corneal cells, use of the siRNA combinations resulted in approximately 76% to 89% reduction in FHV-1–specific mRNA, 63% to 67% reduction of FHV-1–specific proteins in treated cells, and 97% to 98% reduction in FHV-1 replication. The agents were nonirritating in eyes, caused no substantial clinical ocular signs, and were nontoxic. Histologically, corneal epithelium and stroma were normal in treated cats. However, none of the agents were effective in delivering siRNAs into the corneal cells in vivo.

Conclusions and Clinical Relevance—The tested anti–FHV-1–specific siRNAs could potentially be used as a treatment for FHV-1 if a successful means of in vivo delivery can be achieved.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine whether expression of feline coronavirus (FCoV) 7b protein, as indicated by the presence of specific serum antibodies, consistently correlated with occurrence of feline infectious peritonitis (FIP) in cats.

Sample Population—95 serum samples submitted for various diagnostic assays and 20 samples from specific-pathogen–free cats tested as negative control samples.

Procedures—The 7b gene from a virulent strain of FCoV was cloned into a protein expression vector. The resultant recombinant protein was produced and used in antibody detection assays via western blot analysis of serum samples. Results were compared with those of an immunofluorescence assay (IFA) for FCoV-specific antibody and correlated with health status.

Results—Healthy IFA-seronegative cats were seronegative for antibodies against the 7b protein. Some healthy cats with detectable FCoV-specific antibodies as determined via IFA were seronegative for antibodies against the 7b protein. Serum from cats with FIP had antibodies against the 7b protein, including cats with negative results via conventional IFA. However, some healthy cats, as well as cats with conditions other than FIP that were seropositive to FCoV via IFA, were also seropositive for the 7b protein.

Conclusions and Clinical Relevance—Expression of the 7b protein, as indicated by detection of antibodies against the protein, was found in most FCoV-infected cats. Seropositivity for this protein was not specific for the FCoV virulent biotype or a diagnosis of FIP.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the prevalence of stray dogs in eastern Tennessee seropositive to Ehrlichia canis and examine the correlation between results for an ELISA, indirect immunofluorescent antibody (IFA) test, and polymerase chain reaction (PCR) assay.

Sample Population—Blood samples obtained from 90 adult dogs admitted to an animal shelter in eastern Tennessee.

Procedure—Serum samples were analyzed for antibodies against E canis by use of a commercially available ELISA kit, 2 IFA tests, and a PCR assay; testing was performed at the University of Tennessee (TN) and North Carolina State University (NCSU). The PCR amplification was performed by use of DNA extracted from EDTA-anticoagulated blood and primers designed to amplify DNA of Ehrlichia spp.

Results—Antibodies against E canis were detected in only 1 dog by use of the ELISA. By IFA testing at TN, 10 of 90 (11%) dogs were seroreactive against E canis antigens, all of which had medium to high titers to E canis. Only 5 of the 10 TN seroreactors were also reactive against E canis antigens in IFA tests conducted at NCSU, and all 5 had low to medium titers. The DNA of Ehrlichia spp was not amplified in any blood samples by use of PCR assays conducted at the TN or NCSU.

Conclusions and Clinical Relevance—The discordant ELISA, IFA, and PCR results obtained in this study were unexpected and may have been related to exposure of dogs to an Ehrlichia species other than E canis, such as E ewingii. (Am J Vet Res 2004;65:1200–1203)

Full access
in American Journal of Veterinary Research