Comparison of cultivation and PCR-hybridization for detection of Salmonella in porcine fecal and water samples.

A total of 150 fecal and water samples from four swine farms were tested for the presence of Salmonella enterica using different enrichment techniques as follows: (i) 92 fecal samples from nursery and farrowing barns at three swine farms were preenriched overnight in tryptic soy broth (TSB) at 37 degrees C followed by overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42 degrees C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37 degrees C followed by overnight enrichment in RV10 at 42 degrees C; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42 degrees C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to transfer to Hektoen Enteric agar plates for the recovery of viable Salmonella bacteria. Presumptive Salmonella isolates were biochemically and serologically confirmed. For the PCR detection of Salmonella, a 1-ml portion was removed from each sample after the first overnight enrichment and the DNA was extracted using a Sepharose CL-6B spin column. Amplicons (457 bp) derived from primers to the invA and invE genes were confirmed as Salmonella specific on ethidium bromide-stained agarose gels by Southern hybridization with a 20-mer oligonucleotide probe specific for the Salmonella invA gene. Neither the standard microbiological method nor the molecular method detected all of the 65 samples that tested positive by both methods or either method alone. Salmonella bacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples that were preenriched in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC broth, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreement between Salmonella detection using cultivation with preenrichment and detection by PCR was 76% using the kappa statistic. However, agreement between Salmonella detection using cultivation without preenrichment and detection by PCR was about 6%; the PCR assay detected 80% (20 of 25) of the 25 positive samples, while Salmonella bacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detecting Salmonella in swine feces or water samples from swine farms when using the medium combinations evaluated in this study.

Prevalence and reproductive effects of Ureaplasma diversum in beef replacement heifers and the relationship to blood urea nitrogen level.

A systematic sample of replacement heifers from 5 herds underwent prebreeding vaginal swab cultures for Ureaplasma diversum. Heifers from three of the herds were subsequently sampled at pregnancy examination. Sampled heifers were given a vaginal lesion score (VLS), reproductive tract score (RTS) and body condition score (BCS), and peripheral blood was collected for serum blood urea nitrogen (BUN) estimation. Culture results revealed an overall prevalence of Ureaplasma diversum of 51% (87/171) at prebreeding and 65% (64/98) at pregnancy examination. Within herd prevalence ranged from 36% to 64% at prebreeding and 54% to 76% at pregnancy examination. Prevalence tended to differ between herds (P=0.08). At the prebreeding examination, heifers with a BCS of 5.5 or less were more likely to be culture positive than heifers with a BCS greater than 5.5 (p<0.05). No relationship was noted between BUN, VLS, RTS, or pregnancy status and prebreeding culture status. There was little variability among the heifers for any of these variables, with vaginal lesion scores generally being mild, RTS scores being high and BCS scores being moderate. At pregnancy examination, heifers that were culture negative tended to be more likely to be pregnant (odds 3.7, p=0.10) than culture positive heifers.

Norepinephrine stimulates in vitro growth but does not increase pathogenicity of Salmonella choleraesuis in an in vivo model.

Norepinephrine stimulates growth of Escherichia coli, Yersinia enterocolitica, and Pseudomonas aeruginosa in serum-supplemented media, and in vivo increases in norepinephrine may be important in the pathogenesis of sepsis by gram-negative bacteria. Because salmonellosis often is associated with stress, the effects of norepinephrine on in vitro growth, and in vivo pathogenicity of the swine pathogen Salmonella choleraesuis were investigated. When RPMI 1640 with and without pig serum was inoculated with fewer than 100 S. choleraesuis/ml and incubated overnight, bacterial numbers were 10(4) to 10(6) lower in RPMI containing serum. Norepinephrine restored bacterial growth in RPMI with serum to normal levels, but it did not increase growth in serum-free RPMI. Similar results were obtained with SAPI, a nutrient-poor medium previously used to study the effect of norepinephrine on growth of gram-negative bacteria. Conditioned media were produced by growing S. choleraesuis in RPMI containing serum with and without norepinephrine and filter sterilizing. Conditioned medium produced with norepinephrine stimulated growth of S. choleraesuis but not E. coli, whereas conditioned medium produced without norepinephrine stimulated growth of both bacteria. To determine the in vivo effects of norepinephrine, rats were implanted with tablets that secrete norepinephrine for 20 to 24 hours or with identical tablets without norepinephrine and infected intraperitoneally with graded doses of S. choleraesuis. The LD-50 of S. choleraesuis was the same in both groups, and norepinephrine did not affect the carrier rate at 30 days after infection. We concluded that although norepinephrine stimulates in vitro growth of S. choleraesuis in serum-based media, the increase in norepinephrine levels in the present in vivo system was probably not sufficient to influence the pathogenesis of S. choleraesuis infection.

Conjunctival microbial flora of clinically normal pigs.

Conjunctival swab specimens from healthy pigs were cultured to determine normal microbial population. Four commercial swine operations were selected for study. Pigs of 4 age groups were tested: nursing pigs, nursery pigs, feeder pigs, and sows. Swab specimens were taken from the conjunctival sac of each pig. Bacterial, fungal, and mycoplasmal growth was determined separately. Chlamydia sp was detected by use of an ELISA. Bacteria were recovered from 98% of specimens evaluated. alpha-Streptococcus sp (89%) was the most commonly recovered organism, followed by Staphylococcus epidermidis (39%) and Staphylococcus sp (39%). Mycoplasma sp was not detected in any of the specimens. Chlamydia sp was identified in 28% of all specimens evaluated. These results are similar to reports of normal conjunctival flora in other domestic animals.

Chlamydial infection and perinatal mortality in a swine herd.

Chlamydia psittaci was believed responsible for an episode of high perinatal death loss in a swine herd in which 8.5 pigs per litter normally were weaned. In this episode, 18 sows produced 186 pigs, with 50 survivors. Chlamydia was found in tissue samples, and other bacterial or viral pathogens could not be identified. Chlamydia was diagnosed by isolation (ELISA), histologic examination using immunoperoxidase staining techniques, and electron microscopy. Previously, C psittaci has not been considered in the differential diagnosis of swine perinatal mortality.