National Veterinary Services Laboratories Research Articles

Validation of a Real-Time Reverse Transcriptase–PCR Assay for the Detection of H7 Avian Influenza Virus

This report describes the validation of an avian influenza virus (AIV) H7 subtype-specific real-time reverse transcriptase-PCR (rRT-PCR) assay developed at the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 AI in North and South American wild aquatic birds and poultry. The validation was a collaborative effort by the SEPRL and the National Veterinary Services Laboratories. The 2008 H7 rRT-PCR assay detects 10(1) 50% embryo infectious doses per reaction, or 10(3)-10(4) copies of transcribed H7 RNA. Diagnostic sensitivity and specificity were estimated to be 97.5% and 82.4%, respectively; the assay was shown to be specific for H7 AI when tested with > 270 wild birds and poultry viruses. Following validation, the 2008 H7 rRT-PCR procedure was adopted as an official U.S. Department of Agriculture procedure for the detection of H7 AIV. The 2008 H7 assay replaced the previously used (2002) assay, which does not detect H7 viruses currently circulating in wild birds in North and South America.

Evaluation of field and laboratory protocols used to detect avian influenza viruses in wild aquatic birds

Careful selection and observance of standard field and laboratory protocols are critical for successful detection and characterization of avian influenza viruses (AIV) from wild birds. Cloacal swabs were collected from hunter-killed or nesting waterfowl and shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Swab samples were inoculated into embryonated eggs followed by hemagglutination (HA) test to determine the presence of hemagglutinating agents. Antigen capture-ELISA (AC-ELISA) and real-time reverse transcription-PCR (RRT-PCR) were used to detect AIV from both allantoic fluids (AF) and swab specimens of HA-positive samples. Hemagglutination inhibition test was used to detect Newcastle disease virus, another hemagglutinating virus common in wild birds. The HA-positive AF were sent to the National Veterinary Services Laboratory for subtyping of the isolates. Out of 825 samples tested, 19 AIV and 3 avian paramyxovirus subtypes were identified by the National Veterinary Services Laboratory. Without egg passage, AC-ELISA did not detect virus, whereas matrix gene of 13 AIV were detected using RRT-PCR. When testing was done on AF, 14 were positive for influenza A by AC-ELISA and 20 by RRT-PCR. Antigen capture-ELISA did not detect influenza A when the HA titer was lower than 125, whereas RRT-PCR detected AIV from AF with HA titer as low as 4. The highest isolation rate was from Florida, where out of 109 samples analyzed, 14 AIV were detected by RRT-PCR from AF. Real-time reverse transcription-PCR was more sensitive, specific, and cost-effective than AC-ELISA. However, to avoid false-negative results, testing should be performed on AF and not directly from cloacal swabs. Our procedures to detect AIV directly from cloacal swabs need further optimization for improved sensitivity.

Primary diagnosis and surveillance of white spot syndrome virus in wild and farmed crawfish (Procambarus clarkii, P. zonangulus) in Louisiana, USA

This is the first report of natural white spot syndrome virus (WSSV) infection in wild and large-scale farmed crawfish. In the spring of 2007, 3 crawfish farms experienced heavy mortality in ponds populated by Procambarus clarkii and P. zonangulus. Histological examination revealed findings consistent with severe viral infection characterized by numerous intranuclear inclusions in ectodermal and mesodermal tissues. Samples tested by in situ hybridization, injection bioassay in Litopenaeus vannamei, and PCR (nested and real time) were all positive for WSSV. Samples were sent to the National Veterinary Services Laboratory in Ames, Iowa, USA, where WSSV was verified. Subsequently, a multi-parish survey of 184 sites in Louisiana (including farm and wild basin samples) using real-time PCR determined that >60% of sites sampled were positive for WSSV, including wild basin samples.

A real-time polymerase chain reaction assay to detect single nucleotide polymorphisms at codon 171 in the prion gene for the genotyping of scrapie susceptibility in sheep

The objective of this study was to report a reliable real-time polymerase chain reaction assay compatible with the Roche LightCycler 2.0 capable of genotyping sheep for scrapie susceptibility at codon 171. The single nucleotide polymorphisms (SNPs) in the prion protein gene in sheep that may govern resistance to scrapie at codon 171 encode for lysine (K), histidine (H), glutamine (Q), and arginine (R). A modified proteinase K method for leukocytes or whole blood was used to isolate genomic DNA from sheep blood. Fluoresentric developed and optimized primers and probes for the codon 171 SNP assay. The assay was initially validated using 218 determinations from whole blood of known genotypes with 100% correct identity. The assay was further validated through a whole-blood check test provided annually by the National Veterinary Services Laboratory with a correct identification rate of 100%. From January 2005 to December 2006, 3,672 samples from blood were genotyped at codon 171. The genotypes were QR(171) (n = 1,838, 50.05%), RR(171) (n = 1,423, 38.75%), QQ(171) (n = 407, 11.08%), HR(171) (n = 2, 0.05%), and HQ(171) (n = 2, 0.05%). The combination of this simple extraction method and the novel Fluoresentric assay is very accurate, is capable of identifying all 4 SNPs at codon 171 in one reaction, and has proven to be a useful tool for producers in their selective breeding programs.

Brain Heart Infusion Broth May Not Be a Required Component for the Decontamination Process for the Isolation of Mycobacterium Avium Subspecies Paratuberculosis from Fecal Samples using ESP® Broth Cultures

Based on the authors' laboratory experience indicating that increased bacterial contamination in Mycobacterium avium ssp. paratuberculosis (MAP) cultures may be because of the addition of brain heart infusion broth (BHI) during the decontamination process, this study was designed to examine whether BHI is a required component for the isolation of MAP from ESP(R) broth cultures. Twenty-six National Veterinary Services Laboratory (NVSL) proficiency test samples supplied for the year 2005 were used for the comparison. Two paired sets of samples were processed in the experiment. In one set, the hexadecylpyridinium chloride monohydrate (HPC) and antibiotic brew were prepared in half strength BHI and for the other set, HPC and antibiotic brew were prepared in sterile distilled water. Culture of the 26 samples using the BHI/HPC decontamination method identified 13 (50%) positives, whereas culture using the water/HPC decontamination method identified 14 (54%) positives. The proportions of samples with a positive result did not differ significantly between the 2 decontamination methods. Although in most cases it took longer to identify a positive result by the BHI method, the difference between methods with respect to the number of days to a positive culture result was not statistically significant. Retrospective data collected from the Animal Disease Diagnostic Laboratory, Ohio also suggest that inclusion of BHI in the decontamination protocol may not have any effect on MAP recovery or contamination rate. Elimination of BHI from broth cultures may increase the sensitivity of MAP isolation, and reduce the cost of testing.

Monoclonal antibodies to equine IgM improve the sensitivity of West Nile virus-specific IgM detection in horses

West Nile virus (WNV) is a zoonotic pathogen of global importance. In horses with neurological signs, detection of WNV-specific immunoglobulin M (IgM) in serum is widely used to identify clinical cases of WNV encephalitis. Here, we describe the development of two monoclonal antibodies (mAbs) to equine IgM which were used in a WNV IgM-specific enzyme-linked immunosorbent assay (ELISA). Their performance was compared to an established assay based on polyclonal anti-IgM. Check test serum samples from the National Veterinary Service Laboratory (NVSL) were used to evaluate the performance of the three anti-IgM antibodies. The anti-IgM 1-22 mAb correctly identified all NVSL samples. Both the polyclonal antibody and monoclonal anti-IgM 2B-63 identified eight out of ten samples correctly. The three assays were then compared using serum samples from clinically healthy animals ( n = 33) and horses with neurological signs ( n = 21). High Spearman rank correlations (0.76–0.86) were found among the ELISA results. Inter-test agreements (weighted kappa) for assay interpretation resulted in strong agreement (0.95) of the results obtained by the mAbs and moderate agreements when monoclonal and polyclonal anti-IgM-based assays were compared. To determine the analytical sensitivities of anti-WNV IgM detection, serial dilutions of WNV IgM-positive serum samples were analyzed. The highest sensitivity was obtained by using the anti-IgM 1-22 mAb to capture IgM from equine serum. In conclusion, the use of monoclonal anti-IgM antibodies can improve the sensitivity of IgM detection in the acute phase of WN disease.

An Automated High-Throughput Screening Enzyme Linked Immunosorbent Assay for Johne's Disease Antibodies in Bovine Serum

Amanual Mycobacterium avium subspecies paratuberculosis antibody enzyme linked immunosorbent assay (ELISA) (IDEXX Inc.) was fully automated using an automation workstation system (AWS). The IDEXX commercial kit is used to screen for exposure to the Johne's disease (JD) bacterium in cattle on a herd basis. Due to the large number of sera involved in this type of surveillance project, there is a need to increase the cost effectiveness and decrease the turn around time. The purpose of the work was to develop an automated ELISA (a-ELISA) for JD antibodies screening and examine the productivity advantages, quality assured performance characteristics, the laboratory information management (LIM) work, and the multitasking provisions under this format. The AWS was assembled using a modular approach design. The high-throughput screening configuration consisted of a Beckman Coulter hybrid Biomek FX liquid-handling system integrated with a Molecular Devices Spectramax microplate reader, a Biotek ELx405 microplate washer, a Kendro Cytomat 6000 hotel, and an Axis PTZ Network camera. The integrated system function was programmed with an IBM computer using the SAMI software. This setup was housed in a Class 2 Biosafety Cabinet. This fully automated system provided flexibility to accommodate the multistep requirements of the immunoassay procedure. Complexity of the working steps was kept to a minimum by using one unique operation program. The complete robotic workstation with its peripheral equipment was simple to operate, robust, and easily maintained. These characteristics enabled the elimination of forced downtime due to human and/or machine failure. A 5 to 10 fold improvement in cost, test capacity, and productivity was achieved. With this system, one diagnostician is able to carry out over 1000 antibody screening tests a day. Based on the automated nature of the test, there is ample walk away time between the ELISA tests, enabling the technologist to multitask–performing other important duties such as sample receiving and preparation, test validation, result interpretation, information dissemination, and other related LIM work. The performance characteristics–such as repeatability, reproducibility, optimal cutoff values, identity score, test sensitivity, and specificity were evaluated. These test validation results were based on data accumulated in an 8-year proficiency testing with the US National Veterinary Services Laboratory. Our laboratory test has received certification from the United States Department of Agriculture to perform serologic testing for JD since 1998. In 2000–2001, this test scope was accredited by the Standard Council of Canada. In 2004, this a-ELISA was validated according to the requirements of the ISO/IEC 17025 standard.

Surveillance for Avian Influenza in the United States

In the United States, some 1.7 million agar gel immuno diffusion (AGID) tests for avian influenza (AI) are conducted yearly by various poultry groups, governmental sectors, and private industry. In addition to the AGID test, additional testing includes virus isolations, enzyme-linked immunosorbent assays, real-time reverse transcriptase polymerase chain reactions, and hemagglutination inhibition (HI) tests. HI and neuraminidase inhibition tests are conducted on positive AGID samples to determine the subtype. Directigen, a type of antigen capture test, is used in the field in some cases. If monitoring and surveillance activities give rise to a suspicious test result, the accredited veterinarian and official State laboratory are required to report these to the governmental authorities. A thorough investigation in collaboration with the National Veterinary Services Laboratories (a World Organization for Animal Health--AI reference laboratory), State and Federal veterinarians, and others is conducted. Testing conducted as part of the National Poultry Improvement Plan (NPIP) effectively monitors the status of breeder and multiplier flocks. A new commercial poultry program is being added and will expand NPIP AI testing to all commercial flocks. Private poultry companies conduct additional tests; and in the poultry-producing States, there are active state-wide programs to monitor poultry health. All components of the live-bird market system (source flocks, haulers, dealers, and markets) are tested under the Low Pathogenicity AI Live-Bird Market Program.

An Unusual Koi Herpesvirus Associated with a Mortality Event of Common Carp Cyprinus carpio in New York State, USA

Koi herpesvirus (KHV), a highly contagious and lethal virus that affects both koi (Cyprinus carpio koi) and common carp (Cyprinus carpio), was isolated in 1998 from two outbreaks of koi suffering mass mortality in New York State, USA, and in Israel. The disease had been described as early as 1996 in Europe. In July 2004, this virus was found associated with a mass mortality event in wild common carp in the Chadakoin River, New York, USA (42 degrees 07' N, 79 degrees W). Affected fish typically showed marked hyperplasia of gill tissues, abdominal adhesions, and severe multifocal to diffuse external hemorrhages. The virus isolated in this outbreak was somewhat unusual in that it initially replicated well in fathead minnow cell cultures, which is typical of spring viremia of carp virus. Testing at the National Veterinary Services Laboratories, Ames, Iowa, USA, confirmed the virus's identity to be KHV. Koi herpesvirus is not currently on the OIE (World Organisation for Animal Health) list of notifiable diseases; however, it is capable of causing mass mortality in susceptible fish at permissive temperatures.

Evaluation of the Efficacy of a Live Fowlpox-Vectored Infectious Laryngotracheitis/Avian Encephalomyelitis Vaccine Against ILT Viral Challenge

Infectious laryngotracheitis (ILT) is caused by an alphaherpesvirus, and latency can be produced by previous exposure to vaccine virus. The main sites of latency for the ILT virus have been shown to be the trigeminal ganglion and the trachea. Reactivation of latent virus is one factor related to the production of clinical signs. The development of a genetically engineered ILT vaccine has been suggested for many years as a tool to eliminate viral latency. Several approaches have been suggested. Included among them is the development of a thymidine kinase-deficient mutant or the insertion of ILT viral glycoproteins into a viral vector such as a poxvirus. A commercially available, live, fowlpox-vectored infectious laryngotracheitis + avian encephalomyelitis (FP-LT+AE) vaccine was used in field trials in leghorn pullet flocks and evaluated by tracheal challenge in a laboratory setting with the use of the National Veterinary Services Laboratory (Ames, IA) ILT challenge virus. Interference of the pigeon pox vaccine, which is often administered concurrently with fowlpox vaccine, was also evaluated when given in conjunction with the FP-LT+AE vaccine. Overall, the results indicate that the FP-LT+AE vaccine provides adequate protection against ILT viral challenge. Proper administration is essential. In one flock, inadequate protection was most likely a result of either poor vaccine administration or previous exposure to pox virus. In addition, the simultaneous administration of pigeon pox vaccine did not appear to interfere with protection against ILT viral challenge.

Influenza Virus Infection in Racing Greyhounds

To the Editor: Influenza is globally the most economically important respiratory disease in humans, pigs, horses, and fowl (1). Influenza virus is known for its continuous genetic and antigenic changes, which impedes effective influenza control (1,2). More importantly, emergence of a new subtype by genetic reassortment or interspecies transmission is of great concern for preventing influenza epidemics and pandemics (1). Recently, influenza outbreaks have occurred in species (feline and canine) that historically do not carry influenza virus (3,4), which alerted both regulatory and scientific communities to expansion of the host range of influenza virus. We report an outbreak of respiratory disease by influenza virus infection in Iowa racing greyhounds after influenza outbreaks in Florida in 2004. In mid-April, an influx of racing greyhounds into Iowa greyhound tracks resulted in outbreaks of respiratory disease within the track compounds. The disease was characterized by rapid onset of fever and cough, rapid respiration, and hemorrhagic nasal discharge. The illness rate was almost 100% in both racetrack compounds, although the death rate was <5%. Most affected dogs recovered, yet many died of hemorrhagic pneumonia. Therapeutic administration of broad-spectrum antimicrobial drugs reduced the severity of the disease but could not control it. Tissue samples from 4 animals that died of severe pneumonia were submitted to the Iowa State University Veterinary Diagnostic Laboratory. The animals represented 2 different racing tracks located in eastern and western Iowa. On gross examination, lungs exhibited extensive red to red-black discoloration with moderate to marked palpable firmness. Mild fibrinous pleuritis was also noted. Microscopically, lung sections were characterized by severe hemorrhagic interstitial to bronchointerstitial pneumonia. Patchy interstitial change with alveolar septal thickening, coagula of debris in alveoli, and associated atelectasis were evident. Focally extensive pyogranulomatous bronchointerstitial pneumonia with dilatation of airways by degenerate cells and debris was observed. Scattered vasculitis and vascular thrombi were apparent. Microbiologic testing for conventional viral and bacterial agents did not show any important pathogens except Streptococcus equi subsp. zooepidemicus from lung tissues of all animals examined. Two of the 4 lung samples were positive for influenza A virus by a real-time reverse transcription–polymerase chain reaction (RT-PCR) (5). Viral pneumonic lesions of both lungs were positive for immunohistochemistry (IHC) with monoclonal antibody specific for the nucleoprotein of influenza A virus (6) and with antigen-capturing enzyme-linked immunosorbent assay (Directigen Flu A, Becton-Dickinson, Sparks, MD, USA). Bronchioalveolar lavage samples from the 2 positive lungs were also positive by RT-PCR for influenza A virus. Virus isolation was attempted; the influenza virus in canine lungs was unexpected since no influenza virus infection in dogs had been reported, except a recent communication at a meeting of veterinary diagnosticians (4). A virus that was able to agglutinate rooster erythrocytes was isolated in Madin-Darby canine kidney cells from lung and bronchioalveolar lavage fluid of 1 of the 2 animals in which influenza virus was detected by IHC and RT-PCR. Isolates were determined by RT-PCR to be influenza A virus of H3 subtype. The US Department of Agriculture National Veterinary Services Laboratory (Ames, IA) subtyped the virus isolates (A/Canine/Iowa/13628/2005) as H3N8 by using hemagglutination-inhibition assay and neuraminidase-inhibition assay. Sequencing hemagglutinin (HA) and neuraminidase (NA) genes of both isolates showed 100% and 99.8% identity, respectively, between the 2 isolates. Phylogenetically, the HA gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:DQ146419,term_id:76362990}}DQ146419) of the isolates was genetically close (96%–98% nucleotide homology) to the HA gene of recent H3N8 equine influenza viruses (7). The NA gene ({type:entrez-nucleotide,attrs:{text:DQ146420,term_id:76574407}}DQ146420) of the isolates also showed 96%–98% homology with the NA gene of recent H3N8 equine influenza viruses. Internal genes remain to be sequenced. In conclusion, recent outbreaks of hemorrhagic pneumonia and associated deaths in Iowa racing greyhounds were primarily due to infection by an H3N8 influenza virus genetically and antigenically similar to equine influenza viruses. This conclusion can be supported by a previous report of fatal hemorrhagic pneumonia by H3N8 virus infection in racing greyhounds in Florida (4). The fact that greyhounds in 2 different racetracks, which are in geographically remote sites in Iowa, simultaneously died of the disease without the involvement of sick horses suggests that the influenza virus isolate is likely a canine-adapted strain and able to perpetuate and spread among dogs. While influenza virus infection was likely responsible for the disease outbreaks, the contribution that S. zooepidemicus might have made to the disease and the severity of clinical manifestations remains to be further evaluated since the bacterium has been implicated in respiratory disease and septicemia-associated problems in many different animal species (8,9).

A Multilaboratory Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Test for the Detection of Antibodies against Mycobacterium Avium Subsp. Paratuberculosis in Cattle

Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturer's directions. The S/ P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA results with kit lot being a primary contributor. Similar data for other ELISA tests for antibodies to M. avium subsp. paratuberculosis or other antigens also should be developed.

USDA Licenses Rapid Assays For BSE

The Department of Agriculture has licensed two rapid tests for use in detecting mad cow disease, known technically as bovine spongiform encephalopathy (BSE). The tests, made by Bio-Rad Laboratories in Hercules, Calif., and Idexx Laboratories in Westbrook, Maine, produce results in four to five hours. USDA had announced in mid-March that it would for the first time use rapid assays in its BSE testing program, which aims to examine brain tissue from 201,000 to 268,000 slaughtered cattle over a 12- to 18-month period (C&EN, March 22, page 7). USDA's National Veterinary Services Laboratories in Ames, Iowa, as well as NVSL facilities in 25 states, will use the Bio-Rad and Idexx kits for the testing. The program is expected to start in June. The Bio-Rad kit is the most widely used BSE assay in Europe and is used almost exclusively in Japan to test all its slaughtered animals. The Idexx kit has been submitted to ...

Investigation of H7N2 avian influenza outbreaks in two broiler breeder flocks in Pennsylvania, 2001-02.

An avian influenza (AI) outbreak occurred in meat-type chickens in central Pennsylvania from December 2001 to January 2002. Two broiler breeder flocks were initially infected almost simultaneously in early December. Avian influenza virus (AIV), H7N2 subtype, was isolated from the two premises in our laboratory. The H7N2 isolates were characterized as a low pathogenic strain at the National Veterinary Services Laboratories based on molecular sequencing of the virus hemagglutinin cleavage site and virus challenge studies in specific-pathogen-free leghorn chickens. However, clinical observations and pathologic findings indicated that this H7N2 virus appeared to be significantly pathogenic in meat-type chickens under field conditions. Follow-up investigation indicated that this H7N2 virus spread rapidly within each flock. Within 7 days of the recognized start of the outbreak, over 90% seroconversion was observed in the birds by the hemagglutination inhibition test. A diagnosis of AI was made within 24 hr of bird submission during this outbreak using a combination of virus detection by a same-day dot-enzyme-linked immunosorbent assay and virus isolation in embryonating chicken eggs. Follow-up investigation revealed that heavy virus shedding (90%-100% of birds shedding AIV) occurred between 4 and 7 days after disease onset, and a few birds (15%) continued to shed virus at 13 days post-disease onset, as detected by virus isolation on tracheal and cloacal swabs. AIV was not detected in or on eggs laid by the breeders during the testing phase of the outbreak. The two flocks were depopulated at 14 days after disease onset, and AIV was not detected on the two premises 23 days after depopulation.

A prolonged outbreak of polioencephalomyelitis due to infection with a group I porcine enterovirus.

A commercial swineherd in Indiana experienced high death loss of nursery pigs with neurologic disorders for a prolonged period. Polioencephalomyelitis was the consistent histopathological lesion in affected animals. A porcine enterovirus (PEV) classified to group I was isolated from spinal cords and brains collected from the affected animals. The isolate (ISUVDL 200103183) was determined to belong to either serogroup 5 or 6 at the National Veterinary Services Laboratories, Ames, IA. No other significant viral or bacterial agents were isolated from or detected in the animals. A prospective longitudinal serological monitoring of pigs in the index herd for the PEV isolate revealed that colostrum-derived neutralizing antibodies to the virus rapidly declined, and by the age of 21 days the majority of piglets had no or minimal neutralizing antibody against the virus. Seroconversion to the virus then coincided with increased mortality in the herd. Results of diagnostic and cohort observations supported a diagnosis of PEV infection as the cause of the prolonged outbreak of "polio." Investigation into factors that may be contributing to the prolonged problem is currently in progress.

A field investigation of Bacillus anthracis contamination of U.S. Department of Agriculture and other Washington, D.C., buildings during the anthrax attack of October 2001.

In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples. Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents. Surface swabs and air samples were also cultured for B. anthracis at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa. From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings. None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002. DNA extracted from these suspected B. anthracis colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for capA and vrr genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of B. anthracis, suggesting they arose from cross-contamination during the attack through the mail. The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates.

Minimum bacteriostatic and bactericidal concentrations of household sanitizers for Escherichia coli strains in tryptic soy broth

The minimum bacteriostatic and bactericidal concentrations of various household sanitizers were determined for four different strains of Escherichia coli grown in tryptic soy broth. The sanitizers included apple cider vinegar, white vinegar, bleach, hydrogen peroxide, and lemon and lime juices. The strains included American Type Culture Collection (ATCC) 8739 (a strain used for the assay of antimicrobial preservatives), Center for Disease Control (CDC) 1932 (a nalidixic acid-resistant strain), National Veterinary Services Laboratory (NVSL) A940-1-5 (an animal isolate of O157:H7) and NVSL H8302 (human isolate of O157:H7). The minimum bacteriostatic concentrations for the strains tested ranged from 1·2–2·0% for bleach, 0·3–0·4% for commercial hydrogen peroxide solution, and 11·7–15% for white vinegar. The minimum bactericidal concentrations were 1·7–2·5% for bleach; 0·4% for commercial hydrogen peroxide solution, 16·7–20% for white vinegar, 2·7% for apple cider vinegar, and 6·7% for both lemon and lime juices. The various sanitizers tested differed significantly ( P<0·05) in their inhibitory action. The only significant differences ( P<0·05) among the E. coli strains tested were for ATCC 8739, which was more resistant to bleach, and CDC 1932, which was more sensitive to white vinegar.

Failure of porcine reproductive and respiratory syndrome virus to replicate in porcine endothelial cell cultures.

Clinical, gross, and microscopic pathologic and immunohistochemical findings in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) suggest that PRRSV may replicate in endothelial cells. Endothelial cell cultures from porcine aorta and pulmonary artery were tested for susceptibility to various strains of PRRSV. Cultures were identified as endothelium by light microscopy and immunohistochemical staining for P-selectin and von Willebrand factor. Five strains of PRRSV, i.e., the National Veterinary Services Laboratories, Ames, IA PRRSV strain 130-PDV and 4 field strains isolated from pneumonic lungs, failed to replicate in these porcine large-vessel endothelial cell cultures.

Diversity of Salmonella serotypes in cull (market) dairy cows at slaughter

To determine the diversity of Salmonella serotypes isolated from a large population of cull (market) dairy cows at slaughter. Cross-sectional study. Salmonella organisms isolated from the cecal-colon contents of 5,087 market dairy cows. During winter and summer 1996, cecal-colon contents of cull dairy cows at slaughter were obtained from 5 US slaughter establishments. Specimens were subjected to microbiologic culturing for Salmonella spp at 1 laboratory. Identified isolates were compared with Salmonella isolation lists published by the Centers for Disease Control and Prevention (CDC) and the National Veterinary Services Laboratory (NVSL) for approximately the same period. The Simpson diversity index was used to calculate the likelihood that Salmonella isolates selected randomly by establishment were different. Of 58 Salmonella serotypes identified, Salmonella ser. Montevideo was the most prevalent. Two of the top 10 CDC serotypes identified from in 1996, Salmonella ser. Typhimurium and S Montevideo, appeared on our top 10 list; 8 of the top 10 were found on NVSL listings. Thirty-one of 59 S. Typhimurium isolates were identified as DT104 and found at a west slaughter establishment, 30 during the winter and 1 during the summer. The greatest diversity of serotypes was at a southeast establishment during the summer; the least diversity was at a central establishment in the winter. 58 Salmonella serotypes were isolated from market dairy cows at slaughter and could pose a threat for food-borne illness. Salmonella Montevideo was the most frequently isolated serotype and may contribute substantially to salmonellosis in dairy cattle.

The Isolation of Edwardsiella ictaluri with a Limited Tolerance for Aerobic Growth from Channel Catfish

While culturing trunk kidneys from channel catfish Ictalurus punctatus with deep ulcerative lesions, we discovered strains of Edwardsiella ictaluri that could not be isolated under aerobic conditions. After passage of these bacterial isolates in an aerobic environment, minimal growth was observed. In thioglycolate broth, the passaged isolates had growth characteristics of bacteria with a limited tolerance for oxygen. Isolate S97-133, from May 1997, was identified as E. ictaluri by the National Veterinary Services Laboratories, Diagnostic Bacteriology Laboratory, Ames, Iowa. The S97-133 isolate and three others from 1994 were also definitively identified as E. ictaluri by the use of the monoclonal immuno dot-blot technique. In all, eight isolates were obtained from channel catfish over an 8-year period. All isolates were obtained during the months of May, June, July, and November.