Integrating comparative expression profiling data and association of SNPs with Salmonella shedding for improved food safety and porcine disease resistance.

Salmonella in swine is a major food safety problem, as the majority of US swine herds are Salmonella-positive. Salmonella can be shed from colonized swine and contaminate (i) neighbouring pigs; (ii) slaughter plants and pork products; (iii) edible crops when swine manure is used as a fertilizer; and (iv) water supplies if manure used as crop fertilizer runs off into streams and waterways. A potentially powerful method of addressing pre-harvest food safety at the farm level is through genetic improvement of disease resistance in animals. In this research, we describe a successful strategy for discovering genetic variation at candidate genes associated with disease resistance in pigs. This involves integrating our recent global gene expression analysis of the porcine response to Salmonella with information from the literature about important candidate genes. We identified single-nucleotide polymorphisms (SNPs) in these functional candidate genes and genotyped three independent pig populations that had data on Salmonella faecal shedding or internal burden (total n = 377) at these loci. Of 31 SNPs genotyped, 21 SNPs segregated in at least two populations with a minor allele frequency of 15% or greater. Statistical analysis revealed thirteen SNPs associated with Salmonella faecal shedding or tissue colonization, with an estimated proportion of false positives (PFP) ≤0.2. The genes with associated SNPs included GNG3, NCF2, TAP1, VCL, AMT, CCR1, CD163, CCT7, EMP1 and ACP2. These associations provide new information about the mechanisms of porcine host response to Salmonella and may be useful in improving genetic resistance to this bacterium.

Use of bioinformatic SNP predictions in differentially expressed genes to find SNPs associated with Salmonella colonization in swine.

Asymptomatic Salmonella-carrier pigs present a major problem in preharvest food safety, with a recent survey indicating >50% of swine herds in the United States have Salmonella-positive animals. Salmonella-carrier pigs serve as a reservoir for contamination of neighbouring pigs, abattoir pens and pork products. In addition, fresh produce as well as water can be contaminated with Salmonella from manure used as fertilizer. Control of Salmonella at the farm level could be through genetic improvement of porcine disease resistance, a potentially powerful method of addressing preharvest pork safety. In this research, we integrate gene expression profiling data and sequence alignment-based prediction of single nucleotide polymorphisms (SNPs) to successfully identify SNPs in functional candidate genes to test for the associations with swine response to Salmonella. A list of 2527 genes that were differentially regulated in porcine whole blood in response to infection with Salmonella enterica serovar Typhimurium were selected. In those genes, SNPs were predicted using ANEXdb alignments based on stringent clustering of all publically available porcine cDNA and expressed sequence tag (EST) sequences. A set of 30 mostly non-synonymous SNPs were selected for genotype analysis of four independent populations (n = 750) with Salmonella faecal shedding or tissue colonization phenotypes. Nine SNPs segregated with minor allele frequency ≥15% in at least two populations. Statistical analysis revealed SNPs associated with Salmonella shedding, such as haptoglobin (HP, p = 0.001, q = 0.01), neutrophil cytosolic factor 2 (NCF2 #2, p = 0.04, q = 0.21) and phosphogluconate dehydrogenase (p = 0.066, q = 0.21). These associations may be useful in identifying and selecting pigs with improved resistance to this bacterium.

A descriptive survey of lesions from cull sows harvested at two Midwestern U.S. facilities.

Physical and reproductive conditions of cull sows (3158) from two U.S. Midwestern harvest plants were assessed. Body condition, feet, shoulders, teeth, lungs, and reproductive tracts were visually evaluated for gross lesions on harvested sows. PROC FREQ (SAS, Cary, NC) was used to calculate the frequency of each binary trait event. Pearson chi-square tests were used to test the alternative hypothesis that a linear association existed between binary traits and body condition score (BCS). The most common foot lesions observed were rear (n=2064, 67.5%) and front (n=1024, 32.9%) heel lesions. Cracked hooves were found on the front feet of 703 (22.6%) and rear feet of 552 (18.1%) sows. Rear digital overgrowth was observed in 644 (21.1%) sows. The most common reproductive gross lesion observed among harvested cull sows was acyclic ovaries (n=277, 9.0%). Presence of acyclic ovaries increased (p<0.01) as BCS decreased. Cystic ovaries were found in 192 (6.3%) sows, which increased (p<0.01) as BCS increased. Pneumonia was observed in 298 (9.7%) sows, and increased in frequency as BCS decreased (p<0.01). The most frequently observed shoulder lesion among harvested cull sows was shoulder abrasions (n=394, 12.5%). The presence of shoulder abrasions increased (p<0.01) as BCS decreased. The prevalence of reproductive lesions detected in the present study was less than the reported percentage of sows culled for reproductive failure from previous studies based on record keeping summaries.

Quantity and distribution of Salmonella recovered from three swine lairage pens.

The quantity of Salmonella recoverable from three lairage pens in a swine abattoir was determined. Using dry four-ply cotton gauze pads measuring 10 by 10 cm, 100 fecal slurry samples were collected from each of the three pens. Salmonella recovery was expressed as the log CFU per milliliter of sample. Mean values were 2.5 log CFU/ml in pen A, 2.7 log CFU/ ml in pen B, and 0.89 log CFU/ml in pen C. Median values were 2.6 log CFU/ml in pen A, 2.0 log CFU/ml in pen B, and below the detectable limit in pen C. In pen C, Salmonella was not recoverable from a high number of samples. Pen B results suggested spatial dependency, i.e., samples close together were more similar than samples farther apart. These results indicate that Salmonella concentrations vary within and between lairage pens. Because of the limited number of pens assessed, it was not possible to identify factors that were associated with the observed variation in Salmonella concentrations within and between pens. However, this variation suggests that numeroussamples are required to adequately describe the concentration of Salmonella in a lairage pen.

Development of a large-animal human brain tumor xenograft model in immunosuppressed cats.

A large-animal model was developed to facilitate the noninvasive investigation of the effect on the human glioma-derived D-54 MG (glioblastoma multiforme) continuous cell line of a variety of therapeutic regimens. Twenty random-bred male cats were inoculated intracerebrally with 1 x 10(7) D-54 MG tumor cells after being initiated on one of three preparatory regimens of cyclosporin A p.o. Reproducible success of D-54 MG xenotransplantation (100%, 6 of 6 cats) was achieved only after pretreatment with 120 mg cyclosporin A p.o. (24-30 mg/kg) daily for greater than or equal to 10 days prior to tumor implantation. High-performance liquid chromatography-derived whole blood cyclosporin A 12-h trough levels of greater than or equal to 640 ng/ml were seen in successful implants. Lesions ranging from 2 to 20 mm in diameter were seen in cats sacrificed 27-44 days after implantation with no growth seen in control animals. Histopathological examination revealed the tumors to be well-circumscribed anaplastic intracerebral tumors with some invasion into surrounding host parenchyma. Perivascular lymphocytic cuffing was observed, but intratumoral lymphocytic infiltration was minimal. Gadolinium-EDTA-enhanced nuclear magnetic resonance imaging provided accurate tumor localization in T1-weighted images (TE 26 ms; TR 600 ms). Biochemical tests of kidney, liver, and hematological function were within normal limits, although 10% (2 of 20) of the animals developed gingival hyperplasia, and 5% (1 of 20) developed intussusception. The reproducible growth of the D-54 MG human glioblastoma cell line in a large-animal model eliminates many of the limitations associated with the standard nude mouse/rat model, thereby providing a novel test bed for a variety of imaging modalities as well as for drug immunoconjugate localization and toxicity studies.

Absence of IFNA and IFNB genes from human malignant glioma cell lines and lack of correlation with cellular sensitivity to interferons.

We report that 5 of 19 human malignant glioma cell lines have neither interferon alpha (IFNA) nor interferon beta (IFNB) genes that are detectable by Southern blotting. Of 5 other of these malignant glioma lines that have a single IFNB gene copy, 3 lack the IFNA genes entirely and two have one copy. One of the lines that lacks the IFNA genes entirely but has one copy of the IFNB gene has a rearrangement near the IFNB gene that is most easily interpreted as an insertion of a large segment of DNA (at least 50 kilobases) the 3' end of which is less than 1.3 kilobases 5' to the known regulatory sequences of the IFNB gene. In spite of the rearrangement, IFNB-specific RNA is highly inducible in this line by poly(I)-poly(C). The ability of interferon alpha or interferon beta to inhibit cell growth does not depend upon the presence or absence of the respective gene. This finding adds solid tumors to those tumor cell lines (acute lymphocytic leukemia, chronic myelogeneous leukemia) previously determined to lack the IFNA and IFNB genes (Diaz et al., Proc. Natl. Acad. Sci. USA, 85:5259-5263, 1988).

In vitro photoradiation therapy of the rat 9L gliosarcoma.

The authors have investigated various factors involved in the photoradiation treatment of 9L glioma cells. The cells were grown in tissue culture and exposed to light from a laser source that allowed accurate quantitation of the light energy. Cell death was determined following treatment using the trypan blue exclusion test. It was shown that the treatment is very wavelength-dependent following the absorption spectrum of hematoporphyrin derivative (HPD). The absorption peaks in the lower part of the spectrum are more efficient than those of higher wavelengths. Photoradiation therapy is more effective the higher the concentration of HPD. Intensity of light is a very important factor in calculating the total dose of light necessary for this treatment.

New long-wavelength Nd:YAG laser at 1.44 micron: effect on brain.

A wavelength-shifted Nd:YAG laser, tuned to coincide with the infrared absorption peak of water at 1.44 microns, was used to make lesions in normal rabbit brain. A total of 48 lesions were made with power up to 20 W, with energy up to 40 joules, and with two different spot sizes. These lesions were compared to lesions made with 1.06 microns radiation from an Nd:YAG laser under identical operating conditions. Measurements of blood-brain barrier damage and width, depth, and volume of tissue affected were obtained 30 minutes after placement of the lesions. It was found that 1.44-microns lesions produced photoevaporative tissue loss at the highest intensities used. The layer of coagulated tissue remaining after photovaporization had a mean thickness of 0.6 mm irrespective of the volume of tissue removed. There was no photovaporization in the 1.06-microns lesions. In addition, the amount of peripheral edema per unit volume of tissue coagulated was approximately half at the 1.44-microns wavelength. These findings suggest that the 1.44-microns Nd:YAG laser may be a useful surgical instrument since it combines the photoevaporative effect of the CO2 laser while maintaining the advantages of the conventional Nd:YAG laser (quartz fiber delivery and effective hemostasis).

Effect of short-term lairage on the prevalence of Salmonella enterica in cull sows.

This study was designed to compare Salmonella enterica prevalence in sows held in a holding pen at the abattoir for approximately 2 h (hold sows) with sows slaughtered immediately after transport to the abattoir (no-hold sows). Cull sows (n = 160) were sampled from four sampling periods over 8 weeks (February to March 2002) at the abattoir. Sows originated from an integrated swine farm and were sent to a live-hog market and then to the slaughter facility. Before testing, sows entered the abattoir pen and four 100-cm2 four-ply gauze squares were placed randomly on the pen floor for S. enterica culture. Sows were alternatively assigned to the hold or no-hold group. Samples collected from sows during slaughter were ileocecal lymph node, cecal contents, transverse colon contents, subiliac lymph node, sponge swabs of the left and right carcass section (300 cm2), and chopped meat. Overall, S. enterica was isolated from 44% (35 of 80) of the no-hold sows, which was significantly less (P < 0.05) than 59% (47 of 80) of the held sows. Also, no-hold sows had a lower cecal content prevalence (39%, 31 of 80) compared with that (55%, 44 of 80) of held sows (P < 0.05). S. enterica serovars isolated from no-hold sows were Brandenburg (n = 16), Derby (n = 12), Hadar (n = 8), Infantis (n = 6), Johannesburg (n = 3), 6,7:z10-monophasic (n = 3), and Typhimurium (n = 1). S. enterica serovars isolated from held sows (n = 61 isolates) were Derby (n = 19), 6,7: z10-monophasic (n = 15), Brandenburg (n = 10), Infantis (n = 6), Hadar (n = 5), Johannesburg (n = 4), and Tennessee (n = 2). Serovars recovered from the pen were Reading (n = 6), Derby (n = 4), Uganda (n = 2), and Manhattan (n = 2). Results of this study suggest that holding pens contribute to increased S. enterica carriage in cull sows. Abattoir holding pens might be an important control point for S. enterica in the ground pork production chain.

The effect of lairage on Salmonella isolation from market swine.

The objective of this paper was to evaluate the effect of lairage (holding >12 h during transport to slaughter) in clean facilities on Salmonella isolation from market swine. We tested 30 market-bound pigs (about 240 lb [110 kg]) on each of 10 occasions from an Iowa farrow-to-finish operation with about 600 sows. All pigs were slaughtered, and samples were collected at a large Midwest abattoir. On the farm, fecal samples were collected for culture of Salmonella. Pigs were alternately assigned to a lairage treatment (holding in a clean, disinfected facility at the National Animal Disease Center) group or a control group (remaining on the farm). After about 18 h, both groups were transported (about 137 km) to a large Midwest abattoir, commingled, and slaughtered. After slaughter, samples were collected for culture of Salmonella (feces from the distal colon, ileocecal lymph nodes, cecal contents, ventral thoracic lymph nodes, subiliac lymph nodes, and carcass swabs). Diaphragm sections were collected for serum ELISA. Salmonella enterica Derby was the only serotype isolated from farm fecal samples (3.4%, 10 of 290). Multiple serotypes (n = 17) were isolated from 71.8% (196 of 273) of the pigs when abattoir-collected samples were cultured: cecal contents (21.2%. 58 of 273), distal colon contents (52%, 142 of 273), and ileocecal lymph nodes (43.6%, 119 of 273). There were lower Salmonella isolation rates from the lairaged pigs (P < 0.05). The predominant serotype isolated at the abattoir varied by week of the study. This study suggests that pigs became internally contaminated with Salmonella after leaving the farm, possibly while in the abattoir holding pens, and that 18 h lairage, in clean facilities, does not increase shedding.

Impact of commercial preharvest transportation and holding on the prevalence of Salmonella enterica in cull sows.

The objective of this study was to examine the prevalence of Salmonella enterica in cull sows at various stages from the farm to the abattoir. Cull sows (n=181) were sampled over 10 weeks. Fecal samples (10 g each) were collected on the farm ca. 24 h before loading and at the live-hog market ca. 3 h before loading. Samples (ileocecal lymph nodes, cecal contents, feces from the transverse colon, ventral thoracic lymph nodes, subiliac lymph nodes, sponge swabs of the left and right carcass sections, and chopped meat) were collected at the abattoir. The percentages of positive fecal samples on the farm and at the live-hog market were 3% (5 of 181 samples) and 2% (3 of 181 samples), respectively. After transport from the live-hog market (10 h) and holding at the abattoir (6 h), 41% (74 of 180) of cull sows yielded S. enterica in one or more sampled tissues. The isolation rate for total cecal contents (33%; 60 of 180 samples) was significantly (P<0.05) higher than those for ileocecal lymph nodes (7%; 12 of 181 samples), feces (11%; 20 of 181 samples), and ventral thoracic and subiliac lymph nodes (2%; 4 of 181 samples). Before a 2% lactic acid carcass wash (lasting 8 to 9 s), 14% (25 of 180) of carcasses were positive, compared with 7% (12 of 179) after the wash (P<0.05). Two S. enterica serotypes, Derby and Infantis, were found on the farm and at the live-hog market. At the abattoir, 12 serotypes that had not previously been found on the farm or at the live-hog market were recovered. The results of this study demonstrate that transport and holding practices may contribute to an increase in S. enterica infection prior to slaughter to levels much higher than those found on the farm.

The importance of traceability for public health and consumer protection.

Since the 1980s, concerns about the safety and quality of food have increased at both governmental and consumer levels. The importance of traceability of animals and animal products has grown as food production and marketing have been removed from direct consumer control. Product traceability, which requires a transparent chain of custody to maintain credibility and to complete information transfer functions, has two components, namely: a unique identification system, and a credible and verifiable mechanism for identity preservation. Traceability systems can be subdivided into the following four categories: country of origin; retail; processor; and farm-to-retail identity. Although the availability of computers and electronic data devices can enhance the speed and accuracy of data acquisition and manipulation, a common set of developmental criteria exists, irrespective of data-handling processes. As data management technologies become more powerful and less costly, product traceability requirements will multiply. Public and private sectors should seize these opportunities to improve public health and quality parameters, or risk a narrowing of their markets.

Prevalence of coronavirus antibodies in Iowa swine.

Three hundred and forty-seven serum samples from 22 Iowa swine herds were screened for TGEV/PRCV neutralizing antibody. Ninety-one percent of the sera and all 22 herds were positive. These sera were then tested by the blocking ELISA test to distinguish TGEV and PRCV antibody. The ELISA test confirmed the high percentage of TGEV/PRCV positive sera. By the blocking ELISA test, 12 herds were PRCV positive, 6 herds were TGEV positive and 4 herds were mixed with sera either positive for TGEV or PRCV antibody. The results suggest a recent increase in TGEV/PRCV seroprevalence in Iowa swine most likely due to subclinical PRCV infections.

Rapid infection in market-weight swine following exposure to a Salmonella typhimurium-contaminated environment.

OBJECTIVE: To evaluate the possibility of swine becoming infected with Salmonella Typhimurium when housed for 2 to 6 hours in an environment contaminated with Salmonella, similar to a lairage situation prior to slaughter. ANIMALS: 40 crossbred market pigs with an approximate body weight of 92 kg. PROCEDURE: Five trials were conducted (8 pigs/trial) in simulated lairage conditions. Superficial inguinal, ileocecal, and mandibular lymph nodes, cecal contents, distal portion of the ileum, and fecal samples were obtained from each pig after 2 (n = 10), 3 (10), and 6 (5) hours of exposure to an environment contaminated with feces defecated by 10 pigs intranasally inoculated with nalidixic acid-resistant Salmonella Typhimurium (chi4232). In addition, 5 control pigs that were not exposed were also evaluated in the same manner. RESULTS: Feces deposited on the floor by intranasally inoculated swine were mixed with water to form slurry with a resulting load of approximately 10(3) colony-forming units of Salmonella Typhimurium/g of material. Eight of 10, 6 of 10, and 6 of 6 pigs exposed to the slurry for 2, 3, or 6 hours, respectively, had positive results for at least 1 sample when tested for the specific strain of Salmonella Typhimurium. CONCLUSIONS AND CLINICAL RELEVANCE: Pigs can become infected during routine resting or holding periods during marketing when exposed to relatively low amounts of Salmonella organisms in the preslaughter environment. Intervention at this step of the production process may have a major impact on the safety of pork products.

Evaluation of diagnostic procedures for detection of mycoplasmal pneumonia of swine.

The complement-fixation test (CFT), a latex-agglutination test (LAT), and Mycoplasma hyopneumoniae isolation procedures were compared with gross and microscopic lung evaluations for the routine diagnosis of mycoplasmal pneumonia (MP) of swine. The difficulty and variability of M hyopneumoniae isolation rendered this technique unacceptable. In market-weight swine, the finding of typical gross lesions was correlated with CFT results (P less than 0.005). The MP status of a herd could not be determined by use of these techniques in 4- to 9-week-old pigs. No technique could be used alone to establish a diagnosis. Gross and microscopic examination of all slaughter specimens, CFT, and clinical evaluation of the herd, used in combination, were the most useful means of determining the MP status of the herd.

Survey of veterinary extension in the United States.

OBJECTIVE: To assess veterinary extension in the United States as perceived by veterinary extension personnel. DESIGN: Cross-sectional survey. SAMPLE POPULATION: Extension veterinarians in the United States. PROCEDURE: 2 surveys were designed and mailed to extension veterinarians listed by the USDA and the American Association of Extension Veterinarians. RESULTS: 34 states had > or = 1 extension veterinarian. The majority (> 60%) of extension veterinarians did not commit time to resident education and were not involved in research activities. Paradoxically, 23% of responding extension veterinarians did not report extension work. Programs for food animal producers, horse owners, and companion animal owners were provided by 100, 63, and 37% of states, respectively. Continuing education (CE) programs were provided for food animal veterinarians, equine veterinarians, and companion animal veterinarians by 96, 63, and 52% of states, respectively. Challenges facing veterinary extension included limited recognition of veterinary extension activities by universities, lack of university personnel to support CE programs, and decreased support for companion animal extension programs. CONCLUSIONS AND CLINICAL RELEVANCE: Extension veterinarians need to identify and clearly articulate the mission of veterinary extension, develop more collaborative programs across regions, and continue to serve as catalysts to bring diverse constituents together. Extension veterinarians must distinguish their mission not solely as information transfer, which can be accomplished in a variety of ways outside of extension, but as a coherent and consistent program of education and policy developed on a national level and distributed locally.