A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.
Animals ; Bacteria ; genetics ; isolation & purification ; Bacteriological Techniques ; methods ; Endometritis ; microbiology ; veterinary ; Female ; Multiplex Polymerase Chain Reaction ; standards ; Polymerase Chain Reaction ; veterinary ; Sensitivity and Specificity ; Sheep ; Sheep Diseases ; microbiology ; Tibet

OBJECTIVE: To investigate the prevalence and subtype distribution of Blastocystis sp. in pigs in Anhui Province. METHODS: A total of 500 stool samples were collected from large-scale pig farms in Bozhou, Anqing, Chuzhou, Hefei, Fuyang, and Lu'an cities in Anhui Province from October to December 2015. Blastocystis was detected in pig stool samples using a PCR assay based on the small subunit ribosomal RNA (SSU rRNA) gene, and positive samples were subjected to sequencing and sequence analysis. Blastocystis subtypes were characterized in the online PubMLST database, and verified using phylogenetic tree created with the neighbor-joining algorithm in the Meta software. RESULTS: The prevalence of Blastocystis infection was 43.2% (216/500) in pigs in 6 cities of Anhui Province, and all pig farms were tested positive for Blastocystis. There was a region-specific prevalence rate of Blastocystis (17.2% to 50.0%) (χ² = 26.084, P < 0.01), and there was a significant difference in the prevalence of Blastocystis sp. among nursery pigs (39.6%), preweaned pigs (19.1%), and growing pigs (62.3%) (χ² = 74.951, P < 0.01). Both online inquiry and phylogenetic analysis revealed ST1, ST3, and ST5 subtypes in pigs, with ST5 as the predominant subtype. CONCLUSIONS The prevalence of Blastocystis sp. is high in pigs in Anhui Province, with three zoonotic subtypes identified, including ST1, ST3, and ST5.
Animals ; Swine ; Blastocystis/genetics* ; Phylogeny ; Blastocystis Infections/veterinary* ; Polymerase Chain Reaction ; Prevalence ; Feces ; Genetic Variation

The aim of this study was to evaluate the presence of Helicobacter (H.) spp. in swine affected by gastric ulceration. Stomachs from 400 regularly slaughtered swine were subjected to gross pathological examination to evaluate the presence of gastric ulcers. Sixty-five samples collected from ulcerated pars esophagea and 15 samples from non-ulcerated pyloric portions were submitted to histopathological and molecular analyses, to detect Helicobacter spp., H. suis and H. pylori by PCR. Feces and saliva swabs were also collected from 25 animals in order to detect in vivo the presence of Helicobacter spp.. Gastric ulcers were detected in 373 cases (93%). The presence of ulcers in association with inflammatory processes was further confirmed by histological examination. Forty-nine percent (32/65) of the ulcerated esophageal portions as well as 53% (8/15) of the non-ulcerated pyloric portions were positive for Helicobacter spp. by PCR. The Helicobacter spp. positive samples were also positive for H. suis, while H. pylori was not detected. These results were confirmed by restriction enzyme analysis. With regard to feces and saliva samples, 15/25 (60%) and 16/25 (64%) were positive for Helicobacter spp. PCR, respectively but all were negative in H. suis and H. pylori specific PCR.
Animals ; Feces/*microbiology ; Helicobacter/*isolation & purification ; Polymerase Chain Reaction/veterinary ; Restriction Mapping/veterinary ; Saliva/*microbiology ; Stomach/*microbiology ; Stomach Ulcer/microbiology/pathology/*veterinary ; Swine ; Swine Diseases/*microbiology/pathology

Meningoencephalitis (ME) is a common inflammatory disorder of the central nervous system in dogs. Clinically, ME has both infectious and non-infectious causes. In the present study, a multiplex quantitative real-time polymerase chain reaction (mqPCR) panel was optimized for the detection of eight canine neurologic pathogens (Blastomyces dermatitidis, Cryptococcus spp., Neospora caninum, Borrelia burgdorferi, Bartonella spp., Toxoplasma gondii, Ehrlichia canis, and canine distemper virus [CDV]). The mqPCR panel was subsequently applied to 53 cerebrospinal fluid (CSF) samples collected from dogs with ME. The analytic sensitivity (i.e., limit of detection, expressed as molecules per 1 microL of recombinant vector) was 3.8 for CDV, 3.7 for Ehrlichia canis, 3.7 for Bartonella spp., 3.8 for Borrelia burgdorferi, 3.7 for Blastomyces dermatitidis, 3.7 for Cryptococcus spp., 38 for Neospora caninum, and 3.7 for Toxoplasma gondii. Among the tested CSF samples, seven (15%) were positive for the following pathogens in decreasing order of frequency: Cryptococcus spp. (3/7), Blastomyces dermatitidis (2/7), and Borrelia burgdorferi (2/7). In summary, use of an mqPCR panel with high analytic sensitivity as an initial screen for infectious agents in dogs with ME could facilitate the selection of early treatment strategies and improve outcomes.
Animals ; Bacteria/genetics/*isolation & purification ; Dog Diseases/*diagnosis/microbiology/parasitology ; Dogs ; Meningoencephalitis/diagnosis/microbiology/parasitology/*veterinary ; Multiplex Polymerase Chain Reaction/*veterinary ; Prevalence ; Real-Time Polymerase Chain Reaction/*veterinary ; Republic of Korea/epidemiology

OBJECTIVETo demonstrate a noninvasive large mammalian genetic sampling method using blood meal obtained from a tabanid fly.

METHODSBlood meal was recovered from the abdomen of an engorged tabanid fly (Haematopota sp.) which was captured immediately after biting a Sumatran rhino in captivity. The blood was applied on to a Whatman FTA(®) blood card. Subsequent laboratory work was conducted to extract, amplify and sequence the DNA from the sample. Validation was done by sampling the hair follicles and blood samples from the rhinoceros and subjecting it to the same laboratory process.

RESULTSBLAST search and constructed phylogenetic trees confirmed the blood meal samples were indeed from the rhino.

CONCLUSIONSThis method could be used in the field application to noninvasively collect genetic samples. Collection of tabanids and other haematophagous arthropods (e.g. mosquitoes and ticks) and other blood-sucking parasites (e.g. leeches and worms) could also provide information on vector-borne diseases.

Animals ; Diet ; veterinary ; Diptera ; genetics ; physiology ; Endangered Species ; Female ; Food Chain ; Indonesia ; Insect Bites and Stings ; blood ; veterinary ; Molecular Sequence Data ; Perissodactyla ; Phylogeny ; Polymerase Chain Reaction ; veterinary ; Sequence Analysis, DNA ; 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

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

Black rockfish (Sebastes schlegeli) is an important mariculture species in Korea. The production of this fish is drastically declined due to bacterial diseases, particularly streptococcosis caused by Lactococcus garvieae. The bacterial surface characteristics of SJ7 and TY6 were found to have capsule but not NB13 and YS18. The experiential evaluation of L. garvieae pathogenicity, the capsular isolates showed high cumulative mortality i.e. SJ7 (100%) and TY6 (60%) compared to non-capsular isolates. Based on this result the capsular isolates L. garvieae were highly suspected as the causative agent of streptococcosis in rockfish.
Agglutination Tests/veterinary ; Animals ; Bacterial Capsules ; DNA, Bacterial/genetics/isolation&purification ; Fish Diseases/*microbiology/mortality ; Fishes ; Fluorescent Antibody Technique, Indirect/veterinary ; Gram-Positive Bacterial Infections/microbiology/mortality/*veterinary ; Lactococcus/*pathogenicity ; Polymerase Chain Reaction/veterinary

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 x 10(3) cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.
Animals ; Cattle ; Colony Count, Microbial/veterinary ; DNA, Bacterial/chemistry/genetics ; Disease Outbreaks/prevention & control/veterinary ; Female ; Mastitis, Bovine/diagnosis/*microbiology ; Milk/cytology/*microbiology ; Mycoplasma/genetics/*isolation & purification ; Mycoplasma Infections/diagnosis/microbiology/*veterinary ; Polymerase Chain Reaction/veterinary

Streptococcus sp. is gram-positive coccus that causes streptococcal infections in fish due to intensification of aquaculture and caused significant economic losses in fish farm industry. A streptococcal infection occurred from cultured diseased olive flounder (Paralichthys olivaceus) in May, 2005 at a fish farm in Jeju Island, Korea. The diseased flounder exhibited bilateral exophthalmic eyes and rotten gills; water temperature was 16~18oC when samples were collected. Of the 22 fish samples collected, 3 samples were identified as Lactococcus garvieae and 18 samples were identified as Streptococcus parauberis by culture-based, biochemical test. Serological methods such as slide agglutination, hemolysis and antimicrobial susceptibility test were also used as well as multiplex PCRbased method to simultaneously detect and confirm the pathogens involved in the infection. S. parauberis and L. garvieae have a target region of 700 and 1100 bp., respectively. One fish sample was not identified because of the difference in the different biochemical and serological tests and was negative in PCR assay. In the present study, it showed that S. parauberis was the dominant species that caused streptococcosis in the cultured diseased flounder.
Agglutination Tests/veterinary ; Animals ; Aquaculture ; DNA, Bacterial/chemistry/genetics ; Fish Diseases/*microbiology ; *Flounder ; Hemolysis ; Korea ; Microbial Sensitivity Tests/veterinary ; Polymerase Chain Reaction/veterinary ; Streptococcal Infections/microbiology/*veterinary ; Streptococcus/genetics/*isolation&purification