Characterization of the DNA polymerase loci of the novel porcine lymphotropic herpesviruses 1 and 2 in domestic and feral pigs.
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Two novel porcine gammaherpesviruses, porcine lymphotropic herpesviruses 1 and 2 (PLHV-1 and -2), have been detected by amplification of short DNA polymerase (DPOL) sequences from blood and spleen of domestic pigs while searching for unknown herpesviruses in pigs as possible risk factors in xenotransplantation. In the present study, the DPOL genes of the two viruses and the open reading frames (ORFs) that follow in the downstream direction were amplified by PCR-based genome walking from adaptor-ligated restriction fragment libraries of porcine spleen samples. The sequences determined for the two PLHVs exhibited a very low G+C content (37 mol%) and a marked suppression of the CpG dinucleotide frequency. The DPOL proteins encoded were 95% identical and showed a close relationship (60% identity) to the DPOL protein of a ruminant gammaherpesvirus, alcelaphine herpesvirus 1 (AlHV-1). This was confirmed by phylogenetic analyses of the conserved regions of the two PLHV DPOL proteins. The PLHV ORFs downstream of DPOL exhibited 83% identity to each other and >>50% similarity to ORF A5, the position equivalent of AlHV-1. From these data, the PLHVs can be firmly classified to the subfamily Gammaherpesvirinae: To find a natural reservoir for the PLHVs, organs of feral pigs were screened with five different PCR assays, targetting either the DPOL gene or 3'-flanking sequences. In all samples, PLHV sequences were detected that originated predominantly from PLHV-2, suggesting the possibility of virus transfer between feral and domestic pig populations. (+info)
Cloning, expression, sequence analysis, and characterization of streptokinases secreted by porcine and equine isolates of Streptococcus equisimilis.
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Streptokinases secreted by nonhuman isolates of group C streptococci (Streptococcus equi, S. equisimilis, and S. zooepidemicus) have been shown to bind to different mammalian plasminogens but exhibit preferential plasminogen activity. The streptokinase genes from S. equisimilis strains which activated either equine or porcine plasminogen were cloned, sequenced, and expressed in Escherichia coli. The streptokinase secreted by the equine isolate had little similarity to any known streptokinases secreted by either human or porcine isolates. The streptokinase secreted by the porcine isolate had limited structural and functional similarities to streptokinases secreted by human isolates. Plasminogen activation studies with immobilized (His)(6)-tagged recombinant streptokinases indicated that these recombinant streptokinases interacted with plasminogen in a manner similar to that observed when streptokinase and plasminogen interact in the fluid phase. Analysis of the cleavage products of the streptokinase-plasminogen interaction indicated that human, equine, and porcine plasminogens were all cleaved at the same highly conserved site. The site at which streptokinase was cleaved to form altered streptokinase (Sk*) was also determined. This study confirmed not only the presence of streptokinases in nonhuman S. equisimilis isolates but also that these proteins belong to a family of plasminogen activators more diverse than previously thought. (+info)
A descriptive study of the frequency and characteristics of proliferative enteropathy in swine in Ontario by analyzing routine animal health surveillance data.
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Routine surveillance data, collected on pathology submissions at the Animal Health Laboratory in Guelph between 1992 and 1997, were analyzed to determine demographic, clinical, and pathologic characteristics of cases of proliferative enteropathy and the frequency of this condition relative to other infectious enteric diseases in swine in Ontario. The most commonly reported disease was Escherichia coli enteritis (average cases/year = 70.0). Among infectious enteropathies that occur typically in neonatal pigs, coccidiosis (28.4 cases/year) and rotaviral enteritis (5.6 cases/year) were reported. Among infectious enteropathies generally associated with diarrhea in weaner and grower/finisher pigs, the most frequently reported was proliferative enteropathy (27.6 cases/year), followed by swine dysentery (23.3 cases/year), transmissible gastroenteritis (19.6 cases/year), and salmonellosis (8.4 cases/year). Diarrhea and bloody diarrhea were reported in 29% and 31%, respectively, of herds diagnosed with proliferative enteropathy. Important gross intestinal lesions included mucosal hypertrophy (62% of cases), hemorrhage (47%), and mucosal necrosis (34%). Histologic intestinal lesions included epithelial hyperplasia (90% of cases), mucosal necrosis (59%), and inflammation (49%). Our results suggest that proliferative enteropathy is a major infectious enteric disease in grower/finisher pigs in Ontario. (+info)
Oxidation and reduction of pig skeletal muscle ryanodine receptors.
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Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615)) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents 4,4'-dithiodipyridine (4, 4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all reagents. DTNB (1 mM), either cytoplasmic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB, inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTDP and DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys(615) in RyR(MH) is functionally unimportant in redox cycling. (+info)
Analysis of porcine cytomegalovirus DNA polymerase by consensus primer PCR.
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We used a consensus primer PCR method to amplify a region of herpesviral DNA-directed DNA polymerase gene using degenerate primers for initial characterization of the porcine cytomegalovirus (PCMV) genome. The sequence of the PCR product from PCMV DNA template and its alignment with other herpesvirus DNA polymerase counterparts showed that both conserved amino acid residues and conservative amino acid substitutions are in parallel. Phylogenetic analysis revealed that PCMV should be included in the clade comprising human herpesvirus 6 and 7, rather than human and mouse cytomegaloviruses, in Betaherpesvirus subfamily. (+info)
Characterization of PaxA and its operon: a cohemolytic RTX toxin determinant from pathogenic Pasteurella aerogenes.
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Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times. (+info)
Scanning electron microscopy and fluorescent in situ hybridization of experimental Brachyspira (Serpulina) pilosicoli infection in growing pigs.
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Two groups of six 8-week-old pigs were challenged with 1x10(9) cfu Brachyspira (Serpulina) pilosicoli or Serpulina intermedia daily for 3 consecutive days to study the pathology of porcine colonic spirochetosis by scanning electron microscopy (SEM) and fluorescent in situ hybridization (FISH) with oligonucleotide probes targeting ribosomal RNA specific for B. pilosicoli and the genus Brachyspira/Serpulina. Six pigs served as noninoculated controls. The animals were euthanatized successively between postinoculation days 14 and 24. B. pilosicoli was reisolated in feces from all of the inoculated pigs; however, only two pigs developed transient watery diarrhea. S. intermedia was reisolated from four of the inoculated pigs, but clinical signs were not observed. Gross examination of the B. pilosicoli-infected pigs revealed dilated large intestines with a hyperemic mucosa, whereas the large intestines of the S. intermedia-inoculated pigs and the control pigs appeared normal. SEM examination of B. pilosicoli-infected pigs revealed degenerated epithelial cells and spirochetal colonization of the colonic mucosa in four pigs. By FISH, B. pilosicoli cells were found colonizing and invading the surface epithelium and the crypts in all the pigs. Spirochetal crypt colonization markedly exceeded the occurrence of spirochetes on the mucosal surface. SEM examination of S. intermedia-inoculated pigs revealed no abnormalities, and Serpulina cells were detected only sporadically in the otherwise normal-appearing mucosa of four pigs by FISH. The results provide further evidence that B. pilosicoli is associated with colitis in pigs, although the gross lesions are mild. The spirochete is capable of colonizing the large intestine, inducing mucosal damage, invasion of the crypt and surface epithelium, and focal infiltration of the lamina propria. In addition, the study shows the applicability of FISH for specific identification of B. pilosicoli in formalin-fixed tissue. (+info)
In situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets.
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Detection and localization of porcine epidemic diarrhea virus (PEDV) was studied by in situ hybridization with a nonradioactive digoxigenin-labeled probe in formalin-fixed, paraffin-embedded tissues from 10 naturally infected piglets. A 377-base pair cDNA probe for viral RNA encoding the membrane proteins of PEDV cell-culture-adapted strain V215/78 was generated by the reverse transcription polymerase chain reaction. In the retrospective study of pigs from herds with diarrhea, the 10 piglets naturally infected with PEDV had positive signals for PEDV by in situ hybridization. When intestinal tissues were hybridized with the PEDV probe, a strong signal was seen in the villus enterocytes of jejunum and ileum but not in the cecum and colon. Positive cells typically had dark brown reaction products in the cytoplasm. Scattered epithelial cells along the ileal Peyer's patches dome areas contained viral RNA. In one piglet, hybridization signal was also found in the duodenum. PEDV was not demonstrated in tissues outside of the intestinal tract. These findings indicate that jejunal and ileal villus enterocytes are the main target of PEDV replication during epizootic outbreaks of the disease. (+info)