Amino acid variations of the immuno-dominant domain of respiratory syncytial virus attachment glycoprotein (G) affect the antibody responses In BALB/c mice.

Respiratory syncytial virus (RSV) is the leading cause of respiratory illness in ruminants and infants. The G glycoprotein of RSV serves as the viral attachment ligand. Despite currently available vaccines, RSV immunity is insufficient, and re-infections occur. Vaccine studies employing the G-protein's 174-187 amino acids, representing the immunodominant domain, have protected mice and calves against infections. To investigate the causes of vaccination failure, we designed four synthetic peptides for the ruminant RSV isolates (391-2, Maryland-BRSV, European-BRSV, and ORSV) using the immune-dominant sequence and vaccinated mice groups with them. The produced antibodies targeting each peptide were evaluated using ELISA and flow cytometry to determine their reactivity against the linear antigen and the native form of the G protein, respectively. Antibodies responded to homologous and heterologous peptides as determined by ELISA. Using flow cytometry-analysis targeting the natively folded protein, most generated antibodies reacted only with their homologous strain. However, antibodies raised to 391-2 peptide reacted with homologous and heterologous Maryland-BRSV viral epitopes. Accordingly, inadequate immunity and recurring RSV infections might be attributed to variations of antibodies targeting the immunodominant region of the G-protein.

Molecular characterization of the L1 gene of papillomaviruses in epithelial lesions of cats and comparative analysis with corresponding gene sequences of human and feline papillomaviruses.

OBJECTIVE: To characterize the L1 gene of papillomaviruses detected in epithelial lesions of cats and to determine the relationship between those L1 gene nucleotide sequences and known L1 gene sequences of human and feline papillomaviruses. SAMPLE POPULATION: 10 tissue samples of epithelial lesions from 8 cats. PROCEDURES: DNA was extracted from tissue samples. Primers were designed to amplify the L1 gene of papillomaviruses. Amplicons of DNA were sequenced; nucleotide sequences were compared with known L1 gene nucleotide sequences of papillomaviruses and used for phylogenetic analysis. RESULTS: Tissue samples were obtained from lesions (diagnosed as dysplasia [n=1], squamous cell carcinoma in situ [3], or squamous cell carcinoma [6]) of the skin (9) and oral mucosa [1]. Two amplicons had 99% homology with the L1 gene nucleotide sequence of human papillomavirus type 38b subtype FA125. Another amplicon had 84% homology with the L1 gene nucleotide sequence of human papillomavirus type 80 and was considered to be a new type of papillomavirus. Phylogenetic tree analysis revealed that these 3 papillomaviruses were grouped into 2 clades that were not similar to the clades of Felis domesticus papillomavirus type 1 or F domesticus papillomavirus type 2 (FdPV2). The remaining 7 amplicons had 98% to 100% homology with the L1 gene nucleotide sequence of FdPV2. Phylogenetic tree analysis revealed that those 7 papillomaviruses were grouped nto a single clade with FdPV2. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the likelihood of transmission of papillomaviruses between humans and cats.

Influenza virus A (H1N1) in giant anteaters (Myrmecophaga tridactyla).

In February 2007, an outbreak of respiratory disease occurred in a group of giant anteaters (Myrmecophaga tridactyla) at the Nashville Zoo. Isolates from 2 affected animals were identified in March 2007 as a type A influenza virus related to human influenza subtype H1N1.

Development and validation of a TaqMan real-time reverse transcription-PCR for rapid detection of feline calicivirus.

Feline calicivirus (FCV) is a common cause of upper respiratory tract disease in cats and is associated with interstitial pneumonia, oral ulceration and polyarthritis. Recently, outbreaks have involved a highly virulent FCV that leads to multisystemic signs. Virus isolation and conventional RT-PCR are the most common methods used for FCV diagnosis. However, real-time RT-PCR offers a rapid, sensitive, specific and easy tool for nucleic acid detection. The objective of this study was to design a TaqMan probe-based, real-time RT-PCR assay for detection of FCV. It was determined in our previous study that the first 120 nucleotides of the 5' region of the genome are highly conserved among FCV isolates. Primers and a probe specific for this region were designed for a real-time RT-PCR assay to detect FCV. Initial validation was done using 15 genetically diverse isolates. Also, 122 samples were tested by the new assay and virus isolation. The real-time RT-PCR assay was as sensitive and specific as virus isolation and was far more rapid. This real-time RT-PCR assay targeting the conserved 5' region of the genome is a fast, economical and accurate method for detection of FCV.

Isolation of Propionibacterium acnes from a case of placentitis and abortion in a cow.

On the basis of the scarcity of reports in the veterinary literature, it appears that Propionibacterium spp. are rarely associated with disease or isolated from cattle tissues. Recently, Propionibacterium spp. has been associated with multifocal abscessation in cattle. This report describes a case of necrosuppurative placentitis and abortion in an adult Holstein cow. Numerous colonies of small, pleomorphic, Gram-positive, rod-shaped bacteria were observed within the fibrin lattice associated with placental lesions and within the fetal atelectatic lung. Propionibacterium acnes was isolated in high numbers from the placenta, fetal lung, and stomach contents. To the authors' knowledge, this is the first report of placentitis associated with propionibacteria in a cow.

Detection of feline panleukopenia virus using a commercial ELISA for canine parvovirus.

Feline panleukopenia virus (FPV) is a significant pathogen of cats. Rapid virus detection is critical for treatment and management, especially in populations in which spread may occur. This study investigated the ability of the SNAP Canine Parvovirus Antigen Test Kit (SNAP Parvo, IDEXX Laboratories) to detect FPV with confirmation of viral identity by polymerase chain reaction (PCR) assay and genetic sequencing on fecal samples (n = 97) from cats with suspected FPV infection. Fifty-five samples were positive by SNAP Parvo; 54 of 55 were also positive by conventional PCR assay and were identified as FPV by genetic sequencing. This study demonstrates that SNAP Parvo can detect FPV in clinical samples.

Abortion in a horse following Neorickettsia risticii infection.

A pregnant 18-year-old Quarterhorse mare presented with fever, anorexia, tachycardia, tachypnea, and gastrointestinal hypermotility at day 68 of gestation. Potomac horse fever was diagnosed based on polymerase chain reaction (PCR) analysis of whole blood and a high antibody titer to Neorickettsia risticii. The mare made a rapid clinical recovery following antibiotic therapy, but aborted 98 days later. Necropsy on the aborted fetus revealed lymphohistiocytic colitis, lymphadenitis, myocarditis, and hepatitis. The placenta was grossly and histologically normal. Formalin-fixed lymph node, thymus, liver, and colon taken from the aborted fetus were positive by PCR for N. risticii DNA. Potomac horse fever is a common disease in horses that may result in delayed abortion. The microscopic lesions in the fetus are characteristic, and the diagnosis can be confirmed by PCR on formalin-fixed tissues.

Evaluation of antibodies against feline coronavirus 7b protein for diagnosis of feline infectious peritonitis in cats.

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.

Development and validation of quantitative PCR for detection of Terrapene herpesvirus 1 utilizing free-ranging eastern box turtles (Terrapene carolina carolina).

Diseases that affect the upper respiratory tract (URT) in chelonians have been well described as a significant contributor of morbidity and mortality. Specifically, herpesviruses are common pathogens in captive chelonians worldwide, but their importance on free-ranging populations is less well known. Historical methods for the diagnosis of herpesvirus infections include virus isolation and conventional PCR. Real-time PCR has become an essential tool for detection and quantitation of many pathogens, but has not yet been developed for herpesviruses in box turtles. Two quantitative real-time TaqMan PCR assays, TerHV58 and TerHV64, were developed targeting the DNA polymerase gene of Terrapene herpesvirus 1 (TerHV1). The assay detected a viral DNA segment cloned within a plasmid with 10-fold serial dilutions from 1.04 × 10(7) to 1.04 × 10(1) viral copies per reaction. Even though both primers had acceptable levels of efficiency and variation, TerHV58 was utilized to test clinical samples based on less variation and increased efficiency. This assay detected as few as 10 viral copies per reaction and should be utilized in free-ranging and captive box turtles to aid in the characterization of the epidemiology of this disease.

Genetic analysis of feline caliciviruses associated with a hemorrhagic-like disease.

Feline calicivirus (FCV) is 1 of the most common causes of upper respiratory tract disease in cats. Other disease syndromes associated with FCV infection have been reported. Recently, calicivirus infection associated with a hemorrhagic-like disease leading to significant mortality in cats has been reported. The clinical signs are similar to those observed with the calicivirus of rabbit hemorrhagic disease. This study characterized 2 FCV isolates associated with hemorrhagic-like disease. Nucleotide sequencing of the complete genome has been done for these 2 isolates as well as for 4 additional isolates representing other disease syndromes. Previously reported sequence data for the entire genome of classical FCV (6 isolates) and a portion of the capsid gene for hemorrhagic-like FCV (3 isolates), isolated in different regions of United States were used in the genetic analysis. Sequence data were used to determine relationships among the isolates and any correlation with phenotype. Nucleotide sequence comparisons of the entire genome and individual open reading frames revealed high homology among all isolates. Data suggest that the virulence may have genetic determinants on the basis of phylogenetic clustering of the isolates associated with hemorrhagic-like disease.

Emergence of peste des petits ruminants virus lineage IV in Ismailia Province, Egypt.

Peste des petits ruminants (PPR) is an acute, highly contagious fatal disease of small ruminants characterized by high fever, ocular and nasal discharge, pneumonia, erosive stomatitis and severe enteritis that ultimately results in high mortalities. Peste des petits ruminants virus (PPRV) is widely distributed and endemic in several African, middle eastern and south Asian countries and it poses a threat to European countries. Egyptian veterinary medical authorities stated that Egypt is free from PPRV and the only measures for disease control are test and slaughter of infected population to maintain the free status. The aim of our investigation was to detect PPRV in Ismailia province as an indicator of the infection status in Egypt and perform molecular characterization of the emerging virus to gain insight into the origin of circulating virus. A total of 40 representative clinical samples, from a single goat case and goat flock in 2010 and sheep flock in 2012, were tested for PPRV by RT-PCR. About 21 (52.5%) samples were positive. The phylogenetic analysis of the detected viruses revealed circulation of PPRV lineage IV. The circulating viruses are closely related to Sudanese and Saudia Arabian strains with nucleotide identity ranged from 99.2% to 99.6%, respectively. Also, it is closely related to Moroccan 2008 viruses with identities ranged from 97.6% to 98%. Epidemiological investigation at the national level is recommended for monitoring PPRV spread and implementing an appropriate control program.

PCR prevalence of Ranavirus in free-ranging eastern box turtles (Terrapene carolina carolina) at rehabilitation centers in three southeastern US states.

Ranaviruses (genus Ranavirus) have been observed in disease epidemics and mass mortality events in free-ranging amphibian, turtle, and tortoise populations worldwide. Infection is highly fatal in turtles, and the potential impact on endangered populations could be devastating. Our objectives were to determine the prevalence of ranavirus DNA in blood and oral swabs, report associated clinical signs of infection, and determine spatial distribution of infected turtles. Blood and oral swabs were taken from 140 eastern box turtles (Terrapene carolina carolina) that were presented to the wildlife centers at the University of Tennessee (UT; n=39), Wildlife Center of Virginia (WCV; n=34), and North Carolina State University (NCSU; n=36), as well as a free-ranging nonrehabilitation population near Oak Ridge, Tennessee (OR; n=39) March-November 2007. Samples were evaluated for ranavirus infection using polymerase chain reaction (PCR) targeting a conserved portion of the major capsid protein. Two turtles, one from UT and one from NCSU, had evidence of ranavirus infection; sequences of PCR products were 100% homologous to Frog Virus 3. Prevalence of ranavirus DNA in blood was 3, 0, 3, and 0% for UT, WCV, NCSU, and OR, respectively. Prevalence in oral swab samples was 3, 0, and 0% for UT, WCV, and NCSU, respectively. Wildlife centers may be useful in detection of Ranavirus infection and may serve as a useful early monitoring point for regional disease outbreaks.