Recovery of Salmonella, Listeria monocytogenes, and Mycobacterium bovis from cheese entering the United States through a noncommercial land port of entry.

A joint multiagency project was initiated in response to a Salmonella outbreak in San Diego County, California, in 2004. Samples of cheese were collected during four 1-day operations at the San Ysidro port of entry, along the United States-Mexico border. Surveyed participants were persons crossing the border as pedestrians or in vehicles who had a minimum of 2.27 kg of cheese, which may suggest a potential diversion to illegal marketing. In addition, data were collected about the cheese to identify risk factors for cheese contamination. Two hundred four cheese samples were submitted to the California Animal Health and Food Safety Laboratory System-San Bernardino Branch and analyzed for potential food pathogens. Ninety-four percent (190 of 203) of the samples tested positive for alkaline phosphatase. Salmonella was detected from 13% (27 of 204) of the samples comprising 11 serogroups and 28 serotypes. Pulsed-field gel electrophoresis DNA fingerprinting analysis, performed following standardized methods, determined that an isolate obtained from this study had an indistinguishable pattern from a recent Salmonella enterica serovar Typhimurium var. Copenhagen epidemic in the San Diego County that was linked to 14 illnesses. Listeria spp. were detected from 4% (8 of 204) of the samples, and of these, half were identified as L. monocytogenes. Escherichia coli O157:H7 was not detected from any of the samples. Mycobacterium bovis was detected from one panela-style cheese sample. Nine additional samples yielded Mycobacterium spp.

Association between findings on palmarodorsal radiographic images and detection of a fracture in the proximal sesamoid bones of forelimbs obtained from cadavers of racing Thoroughbreds.

OBJECTIVE: To determine the distribution for limbs and bones in horses with fractures of the proximal sesamoid bones and relationships with findings on palmarodorsal radiographic images. SAMPLE POPULATION: Proximal sesamoid bones obtained from both forelimbs of cadavers of 328 racing Thoroughbreds. PROCEDURE: Osteophytes; large vascular channels; and fracture location, orientation, configuration, and margin distinctness were categorized by use of high-detail contact palmarodorsal radiographs. Distributions of findings were determined. Relationships between radiographic findings and fracture characteristics were examined by use of chi2 and logistic regression techniques. RESULTS: Fractures were detected in 136 (41.5%) horses. Biaxial fractures were evident in 109 (80%) horses with a fracture. Osteophytes and large vascular channels were evident in 266 (81%) and 325 (99%) horses, respectively. Medial bones typically had complete transverse or split transverse simple fractures, indistinct fracture margins, > 1 vascular channel that was > 1 mm in width, and osteophytes in abaxial wing and basilar middle or basilar abaxial locations. Lateral bones typically had an oblique fracture and distinct fracture margins. Odds of proximal sesamoid bone fracture were approximately 2 to 5 times higher in bones without radiographic evidence of osteophytes or large vascular channels, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Biaxial fractures of proximal sesamoid bones were common in cadavers of racing Thoroughbreds. Differences between medial and lateral bones for characteristics associated with fracture may relate to differences in fracture pathogeneses for these bones. Osteophytes and vascular channels were common findings; however, fractures were less likely to occur in bones with these features.

Environmental air sampling to detect exotic Newcastle disease virus in two California commercial poultry flocks.

The 2002--2003 Exotic Newcastle Disease (END) outbreak in Southern California poultry provided an opportunity to evaluate environmental air sampling as an efficient and cost-effective means of sampling flocks for detection of a circulating virus. Exotic Newcastle Disease virus was detected by real-time reverse transcriptase PCR from air samples collected using a wetted-wall cyclone-style air sampler placed within 2 m of birds in 2 commercial flocks suspected of being naturally exposed to END virus during the outbreak. Exotic Newcastle Disease virus was detected after 2 hours of air sampling the poultry-house environments of the 2 naturally infected flocks.

High-throughput real-time RT-PCR assay to detect the exotic Newcastle Disease Virus during the California 2002--2003 outbreak.

During the 2002--2003 Exotic Newcastle Disease (END) outbreak in Southern California, a high-throughput real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) system was developed to respond to the large diagnostic and surveillance sample workload. A 96-well RNA extraction method, using magnetic bead technology, combined with a 96-well RRT-PCR assay, allowed 1 technician to process and test more than 400 samples per day. A 3-technician team could complete testing on approximately 1,900 samples per day. The diagnostic sensitivity of the high-throughput RRT-PCR assay was 0.9967 (95% CI 0.9937-0.9997) based on 926 virus isolation confirmed positive samples. Diagnostic specificity using an initial 434 virus isolation confirmed negative samples was 100%. A diagnostic specificity of 0.9999 (95% CI 0.9999, >0.9999) was subsequently calculated on the basis of 2 false-positive results among 65,343 surveillance samples collected after the final END-positive case was confirmed in May 2003. Assay performance over 500 replicates, including reproducibility of the combined extraction and RRT-PCR amplification steps yielded a standard deviation of 0.70 RRT-PCR cycle thresholds (Ct) and a standard deviation of 0.59 Ct for the RRT-PCR steps alone. The high-throughput RRT-PCR developed for END contributed significantly to the 2002--2003 END control effort, reducing the predicted timeline for eradication from 3 years to just 11 months, primarily because of the large number of samples that could be rapidly tested. The 96-well approach described for high-throughput END RRT-PCR could be adapted to other rapid, high-volume testing needs, as required for potential foreign animal disease responses or intensive surveillance efforts.

Longitudinal monitoring of two commercial layer flocks and their environments for Salmonella enterica serovar enteritidis and other Salmonellae.

Between August 20, 2001, and September 17, 2002, 1429 samples including drag swabs, egg belt or egg rollout swabs, fan-blade swabs, rodent organ and intestinal pools, beetle (Alphitobius diaperinus) pools, housefly (Musca domestica) pools, chicken organ and intestinal pools, and egg pools were obtained for Salmonella culture from two flocks from two different commercial layer ranches. The two ranches were purposefully selected for the study based on their previous status of Salmonella Enteritidis isolation using environmental drag swabs in cooperation with practicing veterinarians. Salmonella sp. was isolated from 337 out of 979 (34.42%) non-egg samples. No Salmonella was isolated from 450 egg pools collected from either ranch. S. enteritidis was isolated from samples obtained from ranch 1 from manure drag swabs, 4/284 (1.4%); rodent organs, 1/24 (4.2%); and housefly pool cultures 1/21 (4.8%). Salmonella Enteritidis was isolated from ranch 2 from mouse organ and intestinal pool samples, 1/24 (4.2%). Salmonella group B was isolated from all sample types except the insects. There was a statistically significant difference in isolation rates among seven serogroups of Salmonella: groups B, C1, C2, D, E, K, and untypeable (Pearson chi-square 18.96, P = 0.002). Overall, statistically significant differences were observed with respect to Salmonella isolation among the types of samples taken (Pearson chi-square 118.54, P < 0.0001). Intensive monitoring for Salmonella Enteritidis can be used to optimize a Salmonella reduction program for an individual poultry biosecurity unit.

Determination of the median toxic dose of type C botulinum toxin in lactating dairy cows.

Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay [ELISA] for type C botulinum toxin). Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined. Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period. Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism. From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method. These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis. The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals.

Descriptive study of California egg layer premises and analysis of risk factors for Salmonella enterica serotype enteritidis as characterized by manure drag swabs.

This cross-sectional, double-blind study reports the prevalence of Salmonella enterica serotype enteritidis (SE) on California egg layer premises using single vs. pooled manure drag swabs and presents a description of egg production and management systems in the state and an initial analysis of risk factors for SE. The study included 91% of all known eligible egg premises in California, representing the majority of eggs produced in the state. The overall prevalence of SE on California egg layer premises was 10.5%, while 1.1% of all rows sampled were positive for SE. The percentage of positive rows for SE on any premises never exceeded 25% of the 16 swabs collected per premises. A description of egg production and management on California egg layer premises is presented. Statistically significant associations for SE were not evident and were limited because of sample size and the low prevalence of SE on California egg layer premises. Several biological and management factors, such as flock health, stage of production, manure management, ventilation, and watering systems, show trend associations with premises positive for SE and require further investigation. Manure drag swabs serve as a useful tool to validate the core components of an egg quality assurance program for SE based on process control principles.

The occurrence and distribution of Salmonella enteritidis and other serovars on California egg laying premises: a comparison of two sampling methods and two culturing techniques.

Between the summer of 1998 and the winter of 2000, Salmonella analysis was performed on 2128 single and 532 pooled manure drag swabs obtained from 133 California commercial egg laying farms. The isolation of Salmonella from all rows and from all flocks using single or pooled swabs was 80% and 92%, respectively. Hence, there was no statistical difference between single vs. pooled swabs in terms of identifying Salmonella on a row or flock basis. A total of 14 serogroups comprising 44 serotypes were isolated from 123 of 133 farms. When the top 10 serotypes were considered, there was no significant difference in the range of serotypes isolated by the two culturing methods. The overall S. enteritidis prevalence for California flocks was 10.5% (14/133). The overall row prevalence for S. enteritidis for all the farms was 1.1% (24/2128), and the overall pool prevalence was 2.4% (13/532). Sixty percent (12/20) of the S. enteritidis isolates from the positive farms were phage type 4, and 40% (8/20) represented five other phage types (1, 6B, 7, 8, and 28).

Sensitivity and specificity of western blot testing of cerebrospinal fluid and serum for diagnosis of equine protozoal myeloencephalitis in horses with and without neurologic abnormalities.

OBJECTIVE: To determine sensitivity and specificity of western blot testing (WBT) of CSF and serum for diagnosis of equine protozoal myeloencephalitis (EPM) in horses with and without neurologic abnormalities. DESIGN: Prospective investigation. ANIMALS: 65 horses with and 169 horses without neurologic abnormalities. PROCEDURE: CSF and serum from horses submitted for necropsy were tested for Sarcocystis neurona-specific antibody with a WBT. Results of postmortem examination were used as the gold standard against which results of the WBT were compared. RESULTS: Sensitivity of WBT of CSF was 87% for horses with and 88% for horses without neurologic abnormalities. Specificity of WBT of CSF was 44% for horses with and 60% for horses without neurologic abnormalities. Regardless of whether horses did or did not have neurologic abnormalities, sensitivity and specificity of WBT of serum were not significantly different from values for WBT of CSF. Ninety-four horses without EPM had histologic evidence of slight CNS inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: The low specificity of WBT of CSF indicated that it is inappropriate to diagnose EPM on the basis of a positive test result alone because of the possibility of false-positive test results. The high sensitivity, however, means that a negative result is useful in ruling out EPM. There was no advantage in testing CSF versus serum in horses without neurologic abnormalities. Slight CNS inflammation was common in horses with and without S neurona-specific antibodies in the CSF and should not be considered an indication of CNS infection with S neurona.

Molecular weapons against agricultural vulnerability and the war on terror.

The multiple reports in this issue of the Journal from the Agenda for Action conference, coupled with the analysis by the National Academy of Sciences, the National Research Council, and the Auditor General (UK) on bioterror preparedness and homeland security, highlight the immediate need for rapid disease detection and advanced diagnostic capabilities to protect the public health, animal agriculture, and the numerous associated economies in the United States. In response to the potentially devastating consequences that could arise, there is an acute need for rapid detection of a variety of the lethal foreign animal diseases, such as foot-and-mouth disease virus (FMDV), highly pathogenic strains of avian influenza, classical swine fever, rinderpest, exotic Newcastle disease virus (END), and domestic, vesicular look-alike diseases that include bluetongue, epizootic hemorrhagic disease, vesicular stomatitis, bovine herpes IBR, contagious ecthyma, bovine herpes mammilitis virus, vesicular exanthema, malignant catarrhal fever, and papular stomatitis. Some striking advances are occurring in the creation of rapid technology, including microfluidics, robotics, miniaturization, and biostabilization that are quickly being applied to the development of rapid microbial detection assays. These are now providing important weapons to combat this agricultural vulnerability.