Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control.

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

Diagnosis of Porcine teschovirus encephalomyelitis in the Republic of Haiti.

In February and March 2009, approximately 1,500 backyard pigs of variable age became sick, and approximately 700 of them died or were euthanized in the Lower Artibonite Valley and the Lower Plateau of the Republic of Haiti. The main clinical sign was posterior ataxia followed by paresis and/or paralysis on the second or third day of illness. No gross lesions were observed at postmortem examinations. The morbidity and mortality were approximately 60% and 40%, respectively. Diagnostic samples (whole blood, brain, tonsil, lymph nodes, spleen, and lung) were negative for Classical swine fever virus and African swine fever virus. Porcine teschovirus type 1 was detected by reverse transcription polymerase chain reactions in brain samples. Results of virus isolation, electron microscopy of virus particles, histopathological analysis on brain tissues, nucleic acid sequencing, and phylogenetic analysis of the viral isolate supported the diagnosis of teschovirus encephalomyelitis. The outbreak of the disease in Haiti is the first appearance of the severe form of teschovirus encephalomyelitis in the Americas. This disease poses a potential threat to the swine industries in other Caribbean countries, as well as to Central and North American countries.

Identification of a major antibody binding epitope in the non-structural protein 3D of foot-and-mouth disease virus in cattle and the development of a monoclonal antibody with diagnostic applications.

Detection of FMDV non-structural protein 3D antibodies has been used as a complementary method for sero-epidemiological studies as an indirect indicator of FMDV infection. In order to develop a sensitive cELISA to detect FMDV antibodies, immune dominant epitopes in FMDV-3D protein were identified by peptide array analysis. Monoclonal antibodies were then raised to a selected epitope and used in cELISA. Ninety two peptides corresponding to the complete amino acid sequence of FMDV-3D were synthesized. The sera from 15 FMDV infected cows were tested for binding to the peptides in an indirect ELISA. One major peptide (3D-4) was recognized by antisera in 12 of the 15 infected cows (80%). The sequence was formed by amino acid residues 16-30 of FMDV-3D. The mAbs produced from the mice immunized with native 3D showed neither reactivity to this epitope nor competition with sera from FMDV infected cattle. However, the mAbs produced from the mice immunized with native 3D and boosted with the peptide 3D-4 showed reactivity with native 3D, recombinant 3D as well as competition with sera of FMDV infected cattle and sheep in ELISA assays. Immune response to FMDV-3D was determined using a cELISA. All cattle and sheep tested were positive at 9 dpi and remained positive until the end of the experiment on days 28-31 (>50% inhibition). This demonstrated that mAbs directed to the peptide 3D-4 were effective competitors to the polyclonal antibodies against 3D in infected sera. The approach described here provides a useful tool for specific mAb production in the development of new diagnostic tests.

Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR.

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription-PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding beta-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

Use of quantitative real-time and conventional PCR to assess the stability of the cp4 epsps transgene from Roundup Ready canola in the intestinal, ruminal, and fecal contents of sheep.

The stability of transgenic DNA encoding the synthetic cp4 epsps protein in a diet containing Roundup Ready (RR) canola meal was determined in duodenal fluid (DF) batch cultures from sheep. A real-time TaqMan PCR assay was designed to quantify the degradation of cp4 epsps DNA during incubation in DF at pH 5 or 7. The copy number of cp4 epsps DNA in the diet declined more rapidly (P < 0.05) in DF at pH 5 as compared to pH 7. The decrease was attributed mainly to microbial activity at pH 7 and perhaps to plant endogenous enzymes at pH 5. The 62-bp fragment of cp4 epsps DNA detected by real-time PCR reached a maximum of approximately 1600 copies in the aqueous phase of DF at pH 7, whereas less than 20 copies were detected during incubations in DF at pH 5. A 1363-bp sequence of cp4 epsps DNA was never detected in the aqueous fraction of DF. Additionally, genomic DNA isolated from RR canola seed was used to test the persistence of fragments of free DNA in DF at pH 3.2, 5, and 7, as well as in ruminal fluid and feces. Primers spanning the cp4 epsps DNA coding region amplified sequences ranging in size from 300 to 1363 bp. Free transgenic DNA was least stable in DF at pH 7 where fragments less than 527 bp were detected for up to 2 min and fragments as large as 1363 bp were detected for 0.5 min. This study shows that digestion of plant material and release of transgenic DNA can occur in the ovine small intestine. However, free DNA is rapidly degraded at neutral pH in DF, thus reducing the likelihood that intact transgenic DNA would be available for absorption through the Peyer's Patches in the distal ileum.

Construction and Characterization of Escherichia coli O157:H7 Strains Expressing Firefly Luciferase and Green Fluorescent Protein and Their Use in Survival Studies ‡.

The firefly ( Photinus pyralis ) luciferase (luc) gene on plasmid vector pBESTluc and the Aequorea victoria green fluorescent protein (gfp) gene on plasmid vector pGFP were introduced into strains of Escherichia coli O157:H7. The recombinant E. coli strains were indistinguishable from their parent strains in biochemical and immunological assays and in a multiplex PCR reaction. There was no significant difference in the growth kinetics of the luc-bearing recombinants and the parent strains. At 37°C all of the recombinant strains maintained the vectors and expressed luciferase and the green fluorescent protein when grown both with and without antibiotic selection. Individual colonies of luc-bearing E. coli strains were readily luminescent in the dark after being sprayed with a solution of 1 mM beetle luciferin. The recombinants containing pGFP emitted bright green fluorescence when excited with UV light and the addition of any other proteins, substrates, or cofactors was not required. The green fluorescent protein-expressing E. coli O157:H7 strains were used in studies examining the survival of the organism in apple cider and in orange juice. In apple cider the organism declined to undetectable levels in 24 days at refrigeration temperature while in orange juice the strains survived with only small decreases in number during the 24-day sampling period. These recombinant E. coli O157:H7 strains, containing readily identifiable and stable markers, could be useful as positive controls in microbial assays as well as in studies monitoring bacterial survival and the behavior of E. coli O157:H7 in foods and in a food processing environment.

A Multiplex PCR for Rapid Identification of Shiga-Like Toxin-Producing Escherichia coli O157:H7 Isolated from Foods ‡.

For rapid and specific identification of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 isolated from food samples, experimental conditions for a multiplex polymerase chain reaction (PCR) were optimized and a multiple digoxigenin (DIG)-labeled oligonucleotide probe hybridization (DLOPH) assay was developed. A suspect colony from MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indoxyl-ß-d-glucuronide (MSA-BCIG) was used for the multiplex PCR. Three different DNA sequences of E. coli O157:H7 were amplified simultaneously in the PCR: a specific fragment of an attaching and effacing gene (eae gene), conserved sequences of Shiga-like toxins (SLT) I and II, and a fragment of the 60-MDa plasmid. The identities of PCR products were confirmed by hybridization using DIG-labeled internal oligonucleotide probes and colorimetric detection with anti-DIG Fab fragments conjugated to alkaline phosphatase. This method yielded positive results with all reference strains of EHEC serogroup O157, including serotypes O157:H7, O157:NM, and O157:H-, and negative results were obtained with all strains of nontoxigenic E. coli serogroup O157, other serotypes of E. coli , and other bacterial species. The detection limit of the method was 65 colony-forming units (CFU) of E. coli O157:H7. All 29 cultures of EHEC O157:H7 isolated from meat samples and identified by biochemical and serological tests were positive in the multiplex PCR. EHEC O157:H7 was identified from all of 70 experimentally inoculated ground beef and milk samples which had initial inocula of 4 to 9 CFU/g (ml) and were subjected to a 6-h enrichment culturing. The multiplex PCR procedure could be very useful for routine examinations of food samples for the presence of EHEC O157.