A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C.
Animals ; Foot-and-Mouth Disease/*diagnosis ; Foot-and-Mouth Disease Virus/genetics ; Nucleic Acid Amplification Techniques/*methods/veterinary ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Reverse Transcription/genetics ; Sensitivity and Specificity

In this study, peste des petits ruminants virus (PPRV) was detected in frozen pooled tissue samples from a dead Asiatic lion (Panthera leo persica). The samples were negative for canine distemper virus and positive for PPRV nucleic acids when tested with one-step RT-PCR using the appropriate virus-specific primers. Subsequent amplification, cloning, and sequencing of the partial nucleocapsid, matrix, and fusion genes confirmed the presence of PPRV nucleic acid. Comparative sequence and phylogenetic analyses of the structural genes of the isolated virus confirmed that the virus belonged to Asian lineage IV and was closely related to PPRV circulating in India.
Animals ; Cloning, Molecular ; *Lions ; Peste-des-petits-ruminants virus/*genetics/*isolation & purification ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction/veterinary

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.
Animals ; Distemper Virus, Canine/genetics/*isolation & purification ; Dogs ; Polymerase Chain Reaction/*methods/*veterinary ; RNA, Viral/genetics/isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Attenuated ; Viral Vaccines

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR.
Animals ; Bacteriological Techniques ; Biosensing Techniques/*veterinary ; Cattle ; Feces/*microbiology ; Mycobacterium avium subsp. paratuberculosis/*isolation & purification ; RNA, Bacterial/classification/isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity

H5N2 strains of low-pathogenicity avian influenza virus (LPAIV) have been circulating for at least 17 years in some Mexican chicken farms. We measured the rate and duration of viral excretion from Pekin ducks that were experimentally inoculated with an H5N2 LPAIV that causes death in embryonated chicken eggs (A/chicken/Mexico/2007). Leghorn chickens were used as susceptible host controls. The degree of viral excretion was evaluated with real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) using samples from oropharyngeal and cloacal swabs. We observed prolonged excretion from both species of birds lasting for at least 21 days. Prolonged excretion of LPAIV A/chicken/Mexico/2007 is atypical.
Animals ; Chickens ; Cloaca/virology ; *Ducks ; Influenza A Virus, H5N2 Subtype/*physiology ; Influenza in Birds/*physiopathology/virology ; Oropharynx/virology ; Poultry Diseases/physiopathology/virology ; Real-Time Polymerase Chain Reaction/veterinary ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Time Factors ; *Virus Shedding

It is essential to rapidly and precisely diagnose rabies. In this study, we evaluated four diagnostic methods, indirect fluorescent antibody test (FAT), virus isolation (VI), reverse transcriptase polymerase chain reaction (RT-PCR), and rapid immunodiagnostic assay (RIDA), to detect rabies in animal brain homogenates. Out of the 110 animal brain samples tested, 20 (18.2%) were positive for rabies according to the FAT. Compared to the FAT, the sensitivities of VI, RT-PCR, and RIDA were 100, 100, and 95%, respectively. The specificities of VI, RT-PCR and RIDA were found to be 100, 100, and 98.9%, respectively. Rabies viruses circulating in Korea were isolated and propagated in murine neuroblastoma (NG108-15) cells with titers ranging from 101.5 to 104.5 TCID50/mL. Although the RIDA findings did not completely coincide with results obtained from FAT, VI, and RT-PCR, RIDA appears to be a fast and reliable assay that can be used to analyze brain samples. In summary, the results from our study showed that VI, RT-PCR, and RIDA can be used as supplementary diagnostic tools for detecting rabies viruses in both laboratory and field settings.
Animals ; Antigens, Viral/blood ; Brain/virology ; Fluorescent Antibody Technique, Indirect/*veterinary ; Immunoassay/*veterinary ; RNA, Viral/genetics/isolation & purification ; Rabies/diagnosis/*veterinary/virology ; Rabies virus/genetics/*isolation & purification ; Republic of Korea ; Reverse Transcriptase Polymerase Chain Reaction/*veterinary ; Sensitivity and Specificity

Porcine endogenous retroviruses (PERVs) are members of family Retroviridae, genus Gamma retrovirus, and transmitted by both horizontally and vertically like other endogenous retroviruses (ERVs). PERV was initially described in the 1970s having inserted its gene in the host genome of different pig breeds, and three classes, PERV-A, PERV-B, and PERV-C are known. The therapeutic use of living cells, tissues, and organs from animals called xenotransplantation might relieve the limited supply of allografts in the treatment of organ dysfunction. Because of ethical considerations, compatible organ sizes, and physiology, the pig has been regarded as an alternative source for xenotransplantation. Sensitive duplex reverse transcription-polymerase chain reaction protocols for simultaneously detecting PERV gag mRNA and porcine glyceraldehydes 3-phosphate dehydrogenase mRNA in one tube was established. To compare the age-related PERV expression patterns of the lung, liver, spleen, kidney, heart, and pancreas in commercial pigs, 20 pigs from four age groups (5 heads each in 10 days-, 40 days-, 70 days-, and 110 days-old, respectively) were used in this study. The expression patterns of PERV were statistically different among age groups in lung, liver, and kidney (ANOVA, p<0.05). These data may support in the selection of appropriate donor pigs expressing low levels of PERV mRNA.
Animals ; Endogenous Retroviruses/*metabolism ; Gene Expression Regulation, Viral/*physiology ; RNA, Messenger/genetics/metabolism ; RNA, Viral/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/methods/*veterinary ; Sensitivity and Specificity ; Swine/*virology

OBJECTIVETo investigate the Hantavirus infection and their genotype in rodents in Huludao.

METHODSRodents were collected from the main epidemic areas to detect antigen of Hantavirus in rat lungs by indirect immunofluorescence assay. Antigen-positive samples were inoculated onto cultures of confluent Vero E6 cells for the isolation of virus. The genotypes of viruses in all antigen-positive samples were identified by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS200 rats were collected in the main epidemic areas, and 11 Hantavirus-positive samples were tested. The positive rate of Hantavirus in rats was 5.5%. Three strains of Hantavirus were isolated in Vero E6 cell culture. Data from the phylogenetic trees constructed by partial S segment (620-999 nt) or partial G1 segment (180-580 nt) showed that the three isolates carried by rats from Huludao were all genetic subtype SEOV 3. Furthermore, the phylogenetic tree constructed by partial G2 segment (2003-2302 nt) divided SEOV strains into 7 genetic subtypes, and the three isolates were having a closer evolutionary relationship with isolates CP211, ch302 and dc501 from Beijing, and the isolates SD10 and SD227 form Shandong.

CONCLUSIONData indicated that the rate of carrying virus was high and the main genetic subtype of Hantavirus was S3 of Seoul virus in Huludao area.

Animals ; Carrier State ; China ; Hantavirus ; classification ; genetics ; isolation & purification ; Hantavirus Infections ; veterinary ; Lung ; virology ; Phylogeny ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

The prevalence of feline herpesvirus-1 (FHV-1), feline calicivirus (FCV), and Chlamydophila (C.) felis was studied in cats of an animal shelter in Korea. Total 78 cats without ocular and upper respiratory tract disease were examined. Specimens were obtained from ocular conjunctiva and oropharynx. Using multiplex polymerase chain reaction (PCR) and reverse transcription PCR, three pathogens were simultaneously detected. In examined 78 cats, 49 (63%) cats were positive for FHV-1. However, all specimens were negative for C. felis and FCV. In conclusion, many cats recovered from FHV-1 infection remain subclinical carriers in shelter environment.
Animals ; Caliciviridae/genetics ; Caliciviridae Infections/epidemiology/*veterinary ; Cat Diseases/*epidemiology/*microbiology/*virology ; Cats ; Chlamydophila/genetics ; Chlamydophila Infections/epidemiology/*veterinary ; DNA Primers/genetics ; Herpesviridae/genetics ; Herpesviridae Infections/epidemiology/*veterinary ; Housing, Animal ; Korea/epidemiology ; Prevalence ; Reverse Transcriptase Polymerase Chain Reaction

Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein expressed in tissues in the intestine, uterus, placenta, kidney, pituitary gland and bone. Its exact function is unknown, but it is considered to regulate intracytoplasmic concentration and transport of free ions (Ca2+). CaBP-9k protein is involved in intestinal calcium absorption in the intestine and in the regulation of myometrial activity by intracellular calcium in the uterus. Renal CaBP-9k protein is expressed at the site of calcium re-absorption in the kidney and expressed in distal convoluted tubules, where it is thought to facilitate calcium re-absorption. Expression of the CaBP-9k gene has been explored in most mammalians except in a canine model. Presently, we elucidated the expression of CaBP-9k mRNA and protein in the duodenum, kidney and uterus in a canine model involving two adult (2.5-year-old) female beagles. To collect tissues, the dogs were euthanized and then the abdominal cavity was exposed by midline incision. The proximal duodenum, cortex of kidney and uterine horn were collected. Expression of CaBP-9k mRNA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. CaBP-9k protein expression and localization were ascertained by Western blot analysis and immunohistochemistry, respectively. CaBP-9k mRNA was detected in the duodenum, but not in the kidney and uterus. Its protein was expressed only in the enterocytes of the duodenum. Taken together, the results indicate that CaBP-9k mRNA and protein are highly expressed in the enterocytes of the duodenum of a canine model, consistent with findings in other mammalian species.
Animals ; Blotting, Western/veterinary ; Calcium-Binding Protein, Vitamin D-Dependent/*biosynthesis/genetics ; Dogs/*physiology ; Duodenum/*physiology ; Female ; Immunohistochemistry/veterinary ; Kidney/*physiology ; RNA, Messenger/biosynthesis/genetics ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Transcription, Genetic ; Uterus/*physiology