Polymerase chain reaction analysis of eae gene subtypes present in attaching and effacing Escherichia coli isolated from pigs with diarrhea.

In this study the subtype of eae gene was determined by polymerase chain reaction for a total of 59 attaching and effacing Escherichia coli isolated from preweaned (38 isolates) and postweaned (21 isolates) pigs. The eae(beta) gene detected in 19 E. coli from preweaned pigs and 10 E. coli from postweaned pigs was found to be the most common subtype, followed by eae(gamma), eae(epsilon), and eae(zeta) genes. Subtypes were not determined for 7 E. coli isolates. No other subtype of the eae gene was detected in eae+ E. coli evaluated in this study.     

Genotypic Prevalence of the Adhesin Involved in Diffuse Adherence in Escherichia coli Isolates in Pre‐weaned Pigs with Diarrhoea in Korea

A total of 1002 Escherichia coli strains isolated from pre‐weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat‐stable (STa, STb) and heat‐labile (LT) enterotoxin, enteroaggregative E. coli heat‐stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes. Twenty‐three (2.3%) of the 1002 E. coli isolates carried the gene for AIDA. Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor. Other isolates carried other virulence factor genes in addition to AIDA. Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins. Sixteen isolates carried genes for enterotoxins only. The AIDA may represent an additional virulence determinant in pre‐weaned pigs with diarrhoea.     

Herd-level seroprevalence of swine-influenza virus in Korea

A total of 911 serum samples from 130 herds (an average of nine serum samples per herd) in Korea were examined for antibody to swine H1N1-influenza virus using enzyme-linked immunosorbent assay (ELISA). The list of farms was obtained from the Korean Swine Association, and herds were included from all five of the country’s states. Farms were selected using a random-numbers table for swine within farms and for farms. All serum samples were collected from 22- to 24-week-old finishing pigs between September 2000 and March 2001. By ELISA, 93 out of 130 sampled herds (71.5%) were positive against swine H1N1-influenza virus. Our data suggested that seropositive herds for swine H1N1-influenza virus are distributed diffusely throughout the Republic of Korea.     

In situ hybridization for the detection of the apxIV gene in the lungs of pigs experimentally infected with twelve Actinobacillus pleuropneumoniae serotypes

The detection of the apxlV gene in lung tissues from pigs experimentally infected with the 12 major A. pleuropneumoniae serotype (1 to 12) reference strains was studied by in situ hybridization using a non-radioactive digoxigenin-labeled DNA probe. In situ hybridization produced a distinct positive signal in all pigs inoculated with the 12 A. pleuropneumoniae serotypes. Positive hybridization typically exhibited a dark-brown to black reaction product in intracellular and extracellular locations, without background staining. A strong hybridization signal was seen in degenerated alveolar leukocytes ("oat cells") adjacent to the foci of coagulative necrosis and in the alveolar spaces. The in situ hybridization methodology developed for the detection of the apxIV gene is a valuable tool for the diagnosis of porcine pleuropneumonia caused by A. pleuropneumoniae when only formalin-fixed tissues are submitted for diagnosis.     

Transmission of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in horses using experimentally infected ticks (Ixodes scapularis)

Most human granulocytic ehrlichiosis (HGE) studies carried out in horses use needle inoculation of infected leucocytes or cell cultures. This route of inoculation does not accurately reflect natural infection of the tick-borne agent. To investigate whether tick transmission influences the course of granulocytic ehrlichiosis in the horse model, experimental transmission through infected laboratory-reared Ixodes scapularis ticks was attempted into two healthy horses. One additional horse served as negative control and was exposed to uninfected ticks. Eleven days after exposure to nymphal or adult ticks infected with Anaplasma phagocytophila (HGE agent) the two horses developed severe clinical and laboratory signs consistent with granulocytic ehrlichiosis. Bacteraemia was determined at various time points in the two horses by observation of morulae within neutrophils and by detection of A. phagocytophila genomic DNA by PCR of peripheral blood leucocytes. Further, both horses seroconverted. In contrast the control horse stayed uninfected. The results demonstrate that A. phagocytophila can be experimentally transmitted by infected nymphal and adult ticks and that the agent is able to produce a severe disease, similar to naturally occurring cases. Therefore, tick transmission is highly reproducible and can be successfully used in the equine animal model in order to study HGE.     

Isolation of Scalarane-Type Sesterterpenoids from the Marine Sponge Dysidea sp. and Stereochemical Reassignment of 12-epi-Phyllactone D/E

The chemical investigation of the marine sponge Dysidea sp., which was collected from Bohol province in the Philippines, resulted in the identification of 15 new scalarane-type sesterterpenoids (1–14, 16), together with 15 known compounds. The chemical structures of the new compounds were elucidated based on NMR spectroscopy and HRMS. The structure of 12-epi-phyllactone D/E (15) isolated during this study was originally identified in 2007. However, careful inspection of our experimental ¹³C NMR spectrum revealed considerable discrepancies with the reported data at C-9, C-12, C-14, and C-23, leading to the correction of the reported compound to the C-12 epimer of 15, phyllactone D/E. The biological properties of compounds 1–16 were evaluated using the MDA-MB-231 cancer cell line. Compound 7, which bears a pentenone E-ring, exhibits significant cytotoxicity with a GI₅₀ value of 4.21 μM.     

Monoclonal antibody-based immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fixed, paraffin-embedded intestinal tissues.

An immunohistochemistry technique was developed for the diagnosis of porcine epidemic diarrhea virus (PEDV). The technique was tested on formalin-fixed, paraffin-embedded intestinal tissues from piglets naturally infected with PEDV. Five different monoclonal antibodies (MAbs) were tested in this study. PEDV antigen was consistently detected in the PLP (4% paraformaldehyde, 100 mM L-lysine dihydrochloride, 10 mM sodium m-periodate in phosphate-buffered saline)-fixed PEDV-infected Vero cells or formalin-fixed, paraffin-embedded intestinal tissues from piglets naturally infected with PEDV. The C9-2-2 MAb gave the strongest reactivity and least background staining, detecting 10 of 10 infected pigs. The positive reaction was cytoplasmic. Positive enterocytes were distributed over the tip and along the sides of atrophied or fused villi in the jejunum and ileum. Positive-staining cells were not detected in the crypts. No staining was observed in cecum and colon. No positive cells were observed when the C9-2-2 MAb was reacted with the tissue sections from noninfected piglets or from transmissible gastroenteritus virus (TGEV)- and rotavirus-infected piglets. The selected anti-PEDV MAbs tested on formalin-fixed, paraffin-embedded tissue sections are useful for diagnosis when virus isolation is not available. This method would be of particular value in countries where both PEDV and TGEV are epizootic and would aid in differentiating between PEDV and TGEV infection.     

Hydrochemical and stable isotopic assessment of nitrate contamination in an alluvial aquifer underneath a riverside agricultural field

Major ions and stable isotopic (δD_water, δ¹⁸O_water, δ¹⁵N_nitrate, δ¹⁸O_nitrate) measurements in concert with hydrochemical modeling were used in order to elucidate the sources and geochemical processes controlling nitrate contamination of shallow alluvial groundwater underneath a riverside agricultural field in the Buyeo area, Korea. Beneath vegetable fields in the sandy soil, the mean nitrate concentration of groundwater was 148.6 mg/L, which is significantly higher than in groundwater (mean 28.8 mg/L) beneath silty soils underneath rice paddy fields. Nitrogen isotope data indicate that synthetic fertilizers are the predominant source of groundwater nitrate in the study area. Denitrification during recharge through rice paddy soils appears to be responsible for the lower nitrate concentrations in groundwater beneath the silty soil zone. The relationship between nitrogen and oxygen isotope data of nitrate also suggests mixing of two different groundwater bodies with nitrates from the silt zone and the sand zone. Geochemical mass balance modeling on hydrochemical data indicates that various agricultural chemicals such as urea, lime, magnesium sulfate and potassium chloride dissolve in vegetable fields of the sandy zone, resulting in significant enrichment of various solutes such as K⁺, Ca²⁺, Mg²⁺, NO₃⁻, SO₄²⁻ and Cl⁻. As a consequence of over-utilization of synthetic nitrogen fertilizers, the sand zone is characterized by very high nitrate concentrations in the groundwater. This study suggests that a reduction of over-fertilization especially on vegetable fields in the riverside sand zone is required to minimize the nitrate contamination of groundwater. This study also shows that combination of geochemical and isotopic techniques with simple mass balance modeling provides information about the causes and processes of nitrate contamination of groundwater underneath a riverside agricultural field. The study also provides sustainable measures to optimize fertilization rate as an important basis of eco-friendly agriculture.