Effect of passive antibodies derived from rotavirus-like particles on neonatal calf diarrhea caused by rotavirus in an oral challenge model.

Our objective was to evaluate the efficacy of bovine rotavirus antigen-specific passive antibody for reducing the duration of diarrhea induced by oral challenge with bovine rotavirus in a neonatal calf model. The bovine rotavirus-specific passive antibodies were produced before the study by hyperimmunization of pregnant cows during the dry period with an adjuvanted vaccine containing recombinantly-expressed rotavirus virus-like particles. Eighty-three calves were cleanly collected at birth and randomly assigned to 1 of 3 groups as follows: (1) control group that was colostrum deprived and fed milk replacer for first feeding, (2) group that was colostrum deprived and fed milk replacer mixed with antirotavirus antibodies for first feeding, or (3) group that was fed colostrum replacer mixed with antirotavirus antibodies and a product approved by the US Department of Agriculture containing antibodies against Escherichia coli K99 and bovine coronavirus for first feeding. One of the 3 treatments was administered within 6 h of birth to each calf, followed by oral challenge with bovine rotavirus 3 h later. Calves were observed through 7 d of age and scored according to a standardized scale for clinical signs of diarrhea, change in appetite, depression, and dehydration. Twice daily, measurements of rectal temperature and collection of feces were performed. Fecal samples were assessed for infectious agents commonly associated with diarrhea, and bovine rotavirus shedding was quantified. There were 24 of 28 (86%) calves in the control group that received no antibodies that had signs of severe diarrhea, whereas 57% of the calves that received antirotavirus in milk replacer experienced severe diarrhea, and 7% of calves that received colostrum replacer mixed with antigen-specific bovine rotavirus antibodies showed signs of severe diarrhea. Calves that received colostrum replacer mixed with antigen-specific bovine rotavirus antibodies had a mean duration of 0.9 d of diarrhea compared with 2.7 d in the control group. Calves in the group that was colostrum deprived and fed milk replacer with antirotavirus antibodies had a mean duration of diarrhea of 1.7 d. Rotavirus peak fecal shedding was 3.5 d in the group with milk replacer only, 5.5 d in the milk replacer with antibody group, and 6.5 d in calves in the colostrum replacer group. When bovine rotavirus antigen-specific antibody was fed in milk replacer to colostrum-deprived calves or in conjunction with colostrum replacer that also contained supplemental antibodies against Escherichia coli K99 and bovine coronavirus, those calves were observed to have reduced the onset, duration, and severity of diarrhea when compared with milk replacer placebo.

Intracerebral Astrocytoma in a Horse.

An 8-year-old Anglo-European gelding with progressive neurological signs was humanely destroyed and submitted for necropsy examination. The right parietal cortex was disrupted by a well-demarcated, intraparenchymal, 1.5 cm diameter, tan, homogeneous, dense mass. Microscopical examination was consistent with an astrocytoma, which was confirmed on the basis of strong immunohistochemical labelling for glial fibrillary acidic protein. The neoplastic population lacked immunolabelling for oligodendrocyte transcription factor 2. Labelling for ionized calcium binding adaptor molecule 1 highlighted large numbers of reactive microglia throughout the proliferation and in the adjacent neuroparenchyma. While rare, primary brain tumours should be considered as a differential in horses presenting with progressive neurological signs.

Ki67 Labelling Index of Neoplastic Epithelial Cells Differentiates Canine Acanthomatous Ameloblastoma from Oral Squamous Cell Carcinoma.

Canine acanthomatous ameloblastoma (CAA) and oral squamous cell carcinoma (OSCC) are the most common oral tumours of epithelial origin in dogs. Overlapping clinical, radiographical and histological features can make distinction between CAA and OSCC difficult. The ability to distinguish tumour identity is critical due to their different biological behaviour and recommended treatment modalities, as well as respective comparative and translational applications as potential models of human disease. Based on marked differences in biological behaviour (i.e. benign versus malignant), it is reasonable to predict that the tumour cell proliferation activity is lower in CAA than in OSCC. However, to our knowledge, the epithelial cell proliferation activity of CAA has not been studied or compared with that of OSCC. Therefore, the aims of this study were to (1) compare the neoplastic epithelial cell proliferation activity of CAA and OSCC based on conventional mitotic index (MI) and Ki67 labelling index (LI), and (2) correlate these findings with clinical parameters including patient signalment, anatomical tumour location and degree of local invasion at the time of diagnosis as determined by computed tomography. We found that (1) the Ki67 LI of OSSC (n = 14) was significantly higher than that of CAA (n = 25), (2) the Ki67 LI correlated with a more aggressive locally invasive behaviour, and (3) the MI was not associated with tumour type. We conclude that the Ki67 LI, but not the MI, is a useful differential marker of CAA from OSCC, and that the epithelial cell proliferation activities of OSCC and CAA correlate with their known differences in biological behaviour.

Small Intestinal Lymphatic Hypoplasia in Three Dogs with Clinical Signs of Protein-losing Enteropathy.

Intestinal lymphatic hypoplasia (ILH) is a rare but well-documented cause of protein-losing enteropathy (PLE) in human infants. To our knowledge, this condition has not been reported previously in veterinary medicine. Here we report the clinical and histopathological findings in three dogs that presented with clinical signs of PLE. The onset of PLE was early in an 18-month-old Great Pyrenees, while the other two dogs, a pug and a Tibetan terrier, had a later onset at 4 and 12 years of age, respectively. The presence of intestinal lymphatic and blood vessels was assessed by immunohistochemistry for human prospero homeobox 1 (prox-1), a lymphatic endothelial nuclear transcription factor and human von Willebrand factor (vWf), a marker of vascular endothelial cells, respectively. Small intestinal specimens taken from each dog showed severe mucosal oedema with a lack of prox-1 labelling of villous lacteals, dilated and tortuous vWf immunoreactive villous arterial and capillary blood vessels, and variable lamina propria mixed inflammatory cell infiltrates. Other histological features of ILH included club-shaped villi that were lined by low cuboidal epithelium or epithelial cells with cytoplasmic pallor and microvacuolar change, extrusion zone epithelial inversion and thin and inconspicuous villous longitudinal smooth muscles. While ILH is an uncommon diagnosis, it should be considered as a differential in dogs with clinical signs of PLE. The cause of canine ILH is unknown; however, a congenital abnormality with early or late onset of clinical signs is suspected. Diagnosis of ILH can be challenging; however, immunohistochemical labelling of lymphatic endothelial cells with prox-1 is essential for making this diagnosis.

Cecal spirochetosis caused by Brachyspira pilosicoli in commercial turkeys.

Spirochetes that were identified as Brachyspira pilosicoli were present in the ceca of 7.5- to 18-wk-old turkeys with cecal spirochetosis and typhlitis. The identity of B. pilosicoli was confirmed on the basis of ultrastructural morphology of the cecal epithelium adherent microbes, immunohistochemical staining with a Brachyspira genus-specific monoclonal antibody, and amplification of a B. pilosicoli species-specific 16S ribosomal RNA (rrs gene) sequence by using the polymerase chain reaction and DNA obtained by laser-capture microdissection of the epithelium-adherent microbial fringe. To the author's knowledge, this is the first report of B. pilosicoli in the ceca of turkeys.

Outer membrane-associated serine protease of intestinal spirochetes.

Pathogenic intestinal spirochetes cause damage to the intestinal mucosa of humans and animals by an unknown mechanism. The purpose of this study was to assess the pathogenic intestinal spirochetes Serpulina hyodysenteriae, Serpulina pilosicoli, and Brachyspira aalborgi and the non-pathogenic commensal intestinal spirochetes Serpulina innocens and Treponema succinifaciens for protease activity. A partially heat stable, subtilisin-like, serine protease was identified in the outer membrane of all spirochetes and thus may be essential for survival in the intestinal environment. The outer membrane protease may indirectly contribute to intestinal damage caused by pathogenic spirochetes during association with the mucosal surface of the host.

Genome diversity among regional populations of Francisella tularensis subspecies tularensis and Francisella tularensis subspecies holarctica isolated from the US.

Francisella tularensis is a highly infectious facultative intracellular pathogen that is considered a potential agent of bioterrorism. Four different F. tularensis subspecies have been identified and they appear to display different ecological and virulence characteristics as well as differences in geographical distribution. One simple explanation for the variation in ecological and virulence characteristics is that they are conferred by differences in genome content. To characterize genome content among stains isolated from United States, we have used a DNA microarray designed from a shotgun library of a reference strain. Polymorphisms distributed among polyphyletic sets of strains was the most common pattern of genome alteration observed, indicating that strain-specific genome variability is significant. Nonetheless, 13 different contiguous segments of the genome were found to be missing exclusively in each of the subsp. holarctica strains tested. All 13 are associated with repeat sequences or transposases that could promote insertion/deletion events. Comparison of the live vaccine strain to other holarctica strains also identified three regions that are absent exclusively in the live vaccine strain derived from holarctica.

Identification and partial characterization of a group of weakly beta-hemolytic intestinal spirochetes of swine distinct from Serpulina innocens isolate B256.

Comparative analyses of a group of 16 weakly beta-hemolytic spirochetes isolated from feces and mucosal scrapings of intestines of swine in the midwestern United States, and eastern Canada revealed the existence of a phenotypically and genotypically related group of 7 isolates. Although isolates in this group differed from all known reference isolates of intestinal spirochetes of swine, partial similarity was detected with S. joneseae isolate 16, a newly identified weakly beta-hemolytic intestinal spirochete of human beings. In addition to producing weak beta-hemolysis on blood agar plates, S. innocens isolates B256 and 4/71, S. joneseae isolate 16, and the 16 field isolates lacked the characteristic ring phenomenon described for Serpulina hyodysenteriae, an enteropathogenic spirochete of swine. All but one of the field isolates of weakly beta-hemolytic intestinal spirochetes gave negative results for indole production. The same isolates yielded variable results for alpha-galactosidase production. By transmission electron microscopic examination of negatively-stained cross-sections of spirochetes, the isolates segregated into groups containing either 4 to 7 or 9 to 16 profiles of axial filaments per cell cross-section. Analyses of genomic DNA of selected isolates using whole-genome cross-hybridization revealed a single genetic type consisting of 7 field isolates of weakly beta-hemolytic intestinal spirochetes. The 7 field isolates were distinct from the reference isolates S. innocens isolates B256 and 4/71, S. hyodysenteriae isolates B78 and B204, and Treponema succinifaciens isolate 6091 based on the number of axial filaments per cell cross-section and lack of cross-hybridization signal. S. joneseae isolate 16, had the same number of axial filaments per cell cross-section and produced a weak hybridization signal with a representative isolate of the 7 weakly beta-hemolytic field isolates from swine. This report suggests the existence of a widely distributed group of closely related weakly beta-hemolytic intestinal spirochetes of swine with genotypic characteristics distinct from S. innocens isolate B256.

Serological and genotypic characterization of group A rotavirus reassortants from diarrheic calves born to dams vaccinated against rotavirus.

Two strains of bovine rotavirus (BRV), designated strain Nebraska Scottsbluff-1 (NS-1) and NS-2, were isolated from 2 neighboring cow-calf beef cattle ranches where dams had been vaccinated with a commercial vaccine containing group A BRV strain Neonatal Calf Diarrhea Virus (NCDV)-Lincoln (P1:G6). Northern blot hybridizations using whole genomic RNA probes indicated that strains NS-1 and NS-2 had identical group A RNA electrophoretic patterns and were homologous at all gene segments. Strain NS-1 was compared with reference group A BRV strains using serological and genotypic methods. In vitro virus neutralization assays indicated that strain NS-1 was neutralized by a G6-specific neutralizing monoclonal antibody (mAb) and guinea pig hyperimmune serum (GPHS) raised against BRV strain B641 (P5:G6), but not by G10-specific neutralizing mAb or GPHS raised against BRV strain B223 (P11:G10). Nucleic acid hybridization experiments using whole-genomic RNA probes revealed that gene segment 4 of strain NS-1 differed from BRV strains NCDV-Lincoln and B223, but hybridized with strain B641. Conversely, gene segment 5 of strain NS-1 hybridized with BRV strain B223, but not with BRV strains NCDV-Lincoln and B641. A G-specific cDNA probe produced by reverse transcription polymerase chain reaction (RT-PCR) amplification of strain NS-1 hybridized specifically only with G6 strains NCDV-Lincoln and B641, but not with G10 strain B223. Co-electrophoresis experiments using strains NS-1, B641, and B223 further confirmed these results, suggesting that strain NS-1 was a naturally-occurring reassortant BRV between strains B641 and B223. Taken together these results indicated that a naturally-occurring group A BRV reassortant with a P gene different from the vaccine virus was responsible for the diarrheal syndrome observed on both ranches. Results from this study also indicate the existence of at least 2 different gene segments 5 among group A BRV infecting cattle.

Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper.

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolysin that may be an important virulence factor in the pathogenesis of swine dysentery. We examined the effect of Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+ on the hemolytic activity of cell-free supernatant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from late logarithmic phase cultures were incubated in phosphate-buffered saline containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min incubation and washing cycles was determined. The addition of either ZnSO4 or CuSO4 to the PBS-GR caused complete inhibition of hemolytic activity after 3 cycles; other cations gave results similar to control extracts. Reduction in the concentration of Zn2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn2+ by EDTA indicated that Zn2+ was associated with the cell fraction, and inhibition of hemolysin activity was specifically mediated by Zn2+. When the spirochetes were washed after incubation in the presence of ZnSO4 for 2 cycles and incubated in fresh PBS-GR without Zn2+, inhibition of hemolysin activity remained unchanged, indicating that the inhibitory effect of ZnSO4 was due to a direct action of ZnSO4 on the spirochetes. Since neither the viability of the spirochetes nor the activity of pre-formed hemolysin were affected by the presence of ZnSO4, the inhibitory effect of Zn2+ cations was attributed to reduced biosynthesis by viable S. hyodysenteriae cells rather than interference of Zn2+ cations with lysis of erythrocytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO4 revealed clumping of ribosomes and clearing of cell cytoplasm.

Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA.

The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.

Cell membrane permeability and mitochondrial dysfunction-inducing activities in cell-free supernatants from Serpulina hyodysenteriae serotypes 1 and 2.

Membrane permeability (MP) and mitochondrial dysfunction-inducing (MDI) activities were detected in cell-free supernatants (CFS) of Serpulina hyodysenteriae, using either hemoglobin release from porcine red blood cells (RBC) or cytoplasmic lactate dehydrogenase release from porcine peripheral blood lymphocytes (PBL), and reduction of the 3-(4,5-dimethylthiazoyl-2-y1)-2,5-diphenyltetrazolium bromide dye by porcine PBL. The MP and MDI activities of CFS correlated with each other for serotype 1 and 2 isolates taken at different population densities; however, the kinetics of toxin production varied between each serotype. The loss of enteropathogenicity of two field isolates with nonpathogenic phenotypes and pathogenic isolates passaged up to 45 times in vitro was not attributable to a loss of either membrane permeability or mitochondrial dysfunction-inducing activity of cell-free supernatants. Results from this study suggested the potential for two separate toxins being involved in the pathogenesis of swine dysentery, with the MDI activity correlating with age susceptibility to clinical disease.

Distribution of T and B lymphocytes in mammary dry secretions, colostrum and blood of adult dairy cattle.

The distribution of lymphocyte subpopulations from dry secretions, colostrum and blood from 10 healthy adult Hostein-Fresian cows was studied using the TH21A and B26A mouse monoclonal antibodies (MAb) to adult bovine B and T lymphocytes, respectively. The mammary gland lymphocytes (MGL) were isolated from composite sample of all four quarters by density centrifugation over discontinuous gradient of ficoll-diatrizoate. The peripheral blood lymphocytes (PBL) were purified using the ficoll-thrombin method. Isolated PBL and MGL were analyzed using the two fluorochromes method (TFM) and laser flow cytometry (LFC). The mean viability of isolated PBL and MGL from dry secretions and colostrum after the TFM and LFC were 92.4% +/- 3.2%, 91.4% +/- 6.0% and 87.1% +/- 6.1%, respectively. There was a good correlation between the two MAbs and the percentage of surface immunoglobulin (SIg) positive cells in the peripheral blood using the TFM. The PBL yielded a mean percentage of 21.2% B cells, 66.4% T cells and 9.4% "Null cells" (TH21A+; SIg-). The TFM on MGL from dry secretions and colostrum indicated two distinct patterns (group I and II) of SIg and reactivity to MAb markers (p less than 0.001). The MGL data included in group I and group II were gathered from both colostral and dry secretions. In comparison to the distribution of lymphocyte subsets within peripheral blood the mean percentages of B cells, T cells and "Null cells" in the mammary gland were respectively, 2.8%, 88.1% and 5.4% for group I and 3.5%, 89.0% and 15.1% for group II. In the mammary secretions, the use of SIg alone was not considered to be a good marker for B cells; in four animals a mean percentage of 15.6% (13.9/89.0 X 100) of the mammary gland T lymphocytes were also SIg+. Of the TH21A+ MGL, only 18.8% were SIg+ in group II compared with 34.1% for MGL from group I and 69.3% for the PBL. Marked differences in cell size distribution and cell surface antigen density were found when PBL and MGL from dry secretions were compared by LFC using the B26A MAb. The results of this study demonstrate a difference in the percentages of peripheral blood and mammary gland B and T lymphocytes and confirm previous findings in which the T lymphocytes were found to represent the major subpopulation of lymphocytes in bovine mammary secretions. This may represent an essential event in the adoptive transfer of cellular immunity through the colostrum in cattle.

Comparative pathology and pathogenesis of naturally acquired and experimentally induced colonic spirochetosis.

Research in the past decade has led to the recognition of Brachyspira (formerly Serpulina) pilosicoli as the primary etiologic agent of colonic spirochetosis (CS), an emerging cause of colitis in humans and animals. Attachment of spirochetes to the epithelial surface of the lower intestine is considered to be the hallmark of CS. However, because B. pilosicoli, B. aalborgi and unclassified flagellated bacteria are found singly or together in humans and non-human primates with CS lesions, attachment of spiral-shaped bacteria may not represent the same etiopathogenetic entity in all hosts. Moreover, North American opossums with CS are infected with B. aalborgi-like spirochetes together with flagellated bacteria, whereas B. pilosicoli is found alone in dogs, pigs, chickens and other species of birds with CS. Conversely, guinea-pigs with CS have unidentified spirochetes that may be B. pilosicoli or B. aalborgi. The pig model of CS suggests that attachment of B. pilosicoli to epithelial cells may be transient. By contrast, persistence of B. pilosicoli in the cecal and colonic crypt lumina, chronic inflammation caused by spirochetal invasion into the subepithelial lamina propria and translocation to extraintestinal sites may be more important than previously thought. This review describes the lesions seen in naturally occurring and experimentally induced CS of animals, and it sets the stage for future research into the pathogenic mechanisms of infection and colitis caused by B. pilosicoli.

Minimal prophylactic concentration of dietary zinc compounds in a mouse model of swine dysentery.

Dietary supplementation with 6000 mg of Zn2+/kg of feed has been shown to modify the clinicopathologic expression of Brachyspira hyodysenteriae infection in a laboratory mouse model of swine dysentery. However, this concentration impaired the body weight gain of the mice. The purpose of the present study was to determine a minimal prophylactic concentration of feed-grade zinc compounds that would not affect the growth of mice challenge-exposed with B. hyodysenteriae. A total of 440, 6- to 8-week-old, C3H/HeN mice were allocated randomly to groups and fed either a defined diet or a defined diet containing either 1000, 2000 or 4000 mg/kg ZnO, ZnSO4 or zinc-methionine for 7 days before intragastric inoculation with B. hyodysenteriae. From days 7 to 35 after inoculation, mice in each group were necropsied at weekly intervals for determination of body weight, presence of B. hyodysenteriae in the cecum, and histological assessment of cecal lesions. Only ZnO fed at 2000 mg/kg had a prophylactic effect against B. hyodysenteriae infection without affecting the body weight gain of the mice. The prophylactic effect of Zn2+ against infection with B. hyodysenteriae was also affected by the relative concentration of Fe2+ and Zn2+/Fe2+ ratio of the diet.

Laboratory identification and enteropathogenicity testing of Serpulina pilosicoli associated with porcine colonic spirochetosis.

Pathogenic intestinal spirochetes of swine include Serpulina hyodysenteriae, a strongly beta-hemolytic spirochete that causes swine dysentery, and S. pilosicoli, a weakly beta-hemolytic intestinal spirochete (WBHIS) that causes porcine colonic spirochetosis. Because of the existence of nonpathogenic WBHIS in the normal swine colon, it is important to develop laboratory procedures for accurate identification of S. pilosicoli. The purpose of the present study was to assess hippurate hydrolysis and polymerase chain reaction (PCR) amplification of specific 16S ribosomal RNA (rRNA) sequences for identification of porcine S. pilosicoli. Additionally, the enteropathogenicity of 8 field isolates of porcine S. pilosicoli was determined by challenge exposure of 1-day-old chicks and sequential histologic examination of the cecal mucosa. The field isolates of porcine S. pilosicoli hydrolyzed hippurate and yielded S. pilosicoli-specific products by PCR amplification of 16S rRNA sequences. Although all of the field isolates of porcine S. pilosicoli attached to the cecal epithelium of challenge-exposed chicks by day 21 postinoculation, some isolates had locally invasive phenotypes. We concluded that identification of porcine S. pilosicoli could be made on the basis of results of hippurate hydrolysis and 16S rRNA PCR amplification. Challenge inoculation of 1-day-old chicks followed by histologic examination of the cecal mucosa demonstrated the enteropathogenicity of porcine S. pilosicoli.

In vitro activity of four antimicrobial agents against North American isolates of porcine Serpulina pilosicoli.

Porcine colonic spirochetosis is a nonfatal diarrheal disease that affects pigs during the growing and finishing stages of production. The disease is caused by Serpulina pilosicoli, a newly recognized species of pathogenic intestinal spirochete. Antimicrobial therapy aimed at reducing the infection may be helpful in controlling spirochetal diarrhea. In this study, the in vitro antimicrobial susceptibilities of the reference isolate S. pilosicoli P43/6/78 from the United Kingdom and 19 field isolates obtained from pigs in Canada (n = 5) and the United States (n = 14) were determined against the antimicrobial agents carbadox, gentamicin, lincomycin, and tiamulin, all of which are commonly used for control of the related pathogenic intestinal spirochete S. hyodysenteriae. Additionally, the susceptibility or resistance of each isolate against each antimicrobial agent was estimated on the basis of available data on the in vitro antimicrobial susceptibility breakpoints of S. hyodysenteriae. Each isolate was identified on the basis of phenotypic and genotypic markers, and the minimum inhibitory concentration of each antimicrobial agent was determined by the agar-dilution method. All the isolates were susceptible to carbadox and tiamulin. The percentages of isolates susceptible, intermediate, and resistant to lincomycin were 42.1%, 42.1%, and 15.8%, respectively. Slightly less than half of the isolates (47.4%) were susceptible to gentamicin, and the remainder (52.6%) were resistant. Implementation of rational control measures to reduce infection by S. pilosicoli should improve overall health and productivity in swine herds.

Comparison of six commercially available transport media for maintenance of Serpulina (Treponema) hyodysenteriae.

Two anaerobic (A1 and A2), 1 selective (S1), and 3 conventional (C1, C2, and C3) transport media formulations were compared for their capacity to maintain the viability of Serpulina (Treponema) hyodysenteriae. Initial experiments compared the recovery of S. hyodysenteriae from pure cultures held in each transport medium for 0.5, 1, 2, 3, 5, and 7 days at -40 C, 4 C, 25 C, and 36 C. Subsequent experiments compared each transport medium for maintenance of S. hyodysenteriae in fecal specimens obtained from experimentally infected pigs after holding for up to 7 days at 25 C. In each experiment, the viability of S. hyodysenteriae in each transport medium incubated at each temperature and for each period was determined by inoculating the transport medium onto either trypticase soy agar with 5% sheep blood or selective BJ agar and incubating at 42 C anaerobically. Viability and fecal flora contamination were evaluated blindly after 2-, 4-, and 6-day incubation periods. At -40 C, recovery of viable S. hyodysenteriae from pure culture did not differ among the transport media from 0.5 to 7 days, and all of the transport media consistently maintained the viability of the spirochetes for 7 days. At 4 C, the anaerobic and selective transport media maintained the viability of pure cultures of S. hyodysenteriae significantly better than did the conventional transport media group at day 7 (P = 0.019). At the same temperature, the anaerobic media maintained viability better than did the conventional media at 5 days (P less than 0.042).(ABSTRACT TRUNCATED AT 250 WORDS)

Multiplex polymerase chain reaction for simultaneous detection of Lawsonia intracellularis, Serpulina hyodysenteriae, and salmonellae in porcine intestinal specimens.

Proliferative enteritis, swine dysentery, and porcine salmonellosis are the most common enteric bacterial diseases affecting pigs in the growing and finishing stages of production. Currently, diagnoses of these diseases by standard cultural techniques of intestinal specimens can be laborious, time consuming, and expensive (swine dysentery, porcine salmonellosis) or impossible (proliferative enteritis). Amplification by polymerase chain reaction (PCR) of DNA sequences specific for each bacterial agent is a highly sensitive and specific method that overcomes the limitations associated with standard detection methods. A multiplex PCR (M-PCR) assay was developed for simultaneous detection and identification of the etiologic agents associated with proliferative enteritis, swine dysentery, and porcine salmonellosis in a single reaction using total DNA obtained directly from intestinal specimens. Purified DNA obtained from pure cultures of each bacterial agent alone or mixed in different combinations and concentrations and total DNA from intestinal specimens were amplified using the Lawsonia intracellularis-, Serpulina hyodysenteriae-, and salmonellae-specific M-PCR assay. Intestinal specimens consisted of feces and mucosal scrapings obtained from field cases of each disease alone or in combinations and feces obtained from pigs challenged with S. hyodysenteriae. The banding pattern of the amplified PCR products, after agarose gel electrophoresis and staining, indicated the presence of individual or combinations of etiologic agents in each specimen. Results from this study indicated that simultaneous amplification of L. intracellularis-, S. hyodysenteriae-, and salmonellae-specific DNA sequences by M-PCR can be used for specific detection and identification of three major enteric bacterial pathogens associated with proliferative enteritis, swine dysentery, and porcine salmonellosis occurring alone or in combinations. Also, the M-PCR assay can be done using DNA obtained directly from intestinal specimens submitted for diagnostic investigation.

Cecal spirochetosis associated with Serpulina pilosicoli in captive juvenile ring-necked pheasants.

Spirochetes similar to those described in the ceca of broilers with diarrhea and in laying hens with decreased egg production and growth were identified in the ceca of captive-raised juvenile ring-necked pheasants (Phasianus colchicus). The birds were submitted for diagnostic investigation of an illness characterized by a seromucoid ocular discharge, sneezing, swollen infraorbital sinuses, and weight loss. In addition to cecal spirochetosis, the birds had mild enteric coccidiosis, trichomoniasis, and nematodiasis (Heterakis spp.); esophageal capillariasis; and respiratory mycoplasmosis. Weakly beta-hemolytic spirochetes isolated from the ceca of one pheasant were identified as Serpulina pilosicoli with the use of a 16S rRNA sequence-specific polymerase chain reaction amplification assay. Diffuse cecal enterocyte attachment was reproduced in a 1-day-old chick challenged with the pheasant S. pilosicoli isolate. Immunohistochemical staining of sections of ceca from the pheasant and challenged chick with a Serpulina spp. flagellar antigen-specific monoclonal antibody confirmed spirochetal attachment to cecal enterocytes. The etiologic significance of the spirochete infection is unknown because respiratory signs and multiple gastroenteric pathogens dominated the clinicopathologic manifestations and an intestinal disorder was not a clinical complaint.