Pathologic fracture healing after femoral limb salvage in a dog.

BACKGROUND: Bone sarcomas are a significant cause of pain, disability, and mortality in dogs. A variety of surgical limb salvage options are available to preserve limb function with comparable prognosis to amputation. CASE REPORT: This report describes successful healing after plate fixation of an undifferentiated sarcoma pathologic femoral fracture in a dog. The fracture was treated surgically with curettage of the tumour site, placement of autogenous bone graft, and then stabilized using a locking plate rod construct. The patient regained excellent mobility after surgery and was managed with monthly pamidronate therapy. Serial radiographs demonstrate progressive healing of the pathologic fracture. Ultimately, the patient developed a maxillary fibrosarcoma and was euthanased 1 year after treatment of the femoral fracture. Postmortem histopathological evaluation of the pathologic fracture site demonstrated complete bone healing. CONCLUSION: This case highlights the possibilities of limb salvage by fracture stabilization and bone healing as a viable option in select patients.

Purified deoxynivalenol or feed restriction reduces mortality in rainbow trout, Oncorhynchus mykiss (Walbaum), with experimental bacterial coldwater disease but biologically relevant concentrations of deoxynivalenol do not impair the growth of Flavobacterium psychrophilum.

Diets containing deoxynivalenol (DON) were fed to rainbow trout Oncorhynchus mykiss (Walbaum) for 4 weeks followed by experimental infection (intraperitoneal) with Flavobacterium psychrophilum (4.1 × 10(6) colony-forming units [CFU] mL(-1) ). Mortality of rainbow trout fed either 6.4 mg kg(-1) DON or trout pair-fed the control diet was significantly reduced (P < 0.05) in comparison with trout fed the control diet to apparent satiation (<0.1 mg kg(-1) DON). In a second experiment, trout were fed one of three experimental diets; a control diet, a diet produced with corn naturally contaminated with DON (3.3 mg kg(-1) DON) or a diet containing purified DON (3.8 mg kg(-1) ); however, these fish were not experimentally infected. The presence of DON resulted in significant reduction (P < 0.0001) in feed intake as well as weight gain after 4 weeks. Respiratory burst of head-kidney leucocytes isolated from rainbow trout fed diets containing purified DON (3.8 mg kg(-1) ) was significantly higher (P < 0.05) at 35 day post-exposure compared with controls. The antimicrobial activity of DON was examined by subjecting F. psychrophilum in vitro to serial dilutions of the chemical. Complete inhibition occurred at a concentration of 75 mg L(-1) DON, but no effect was observed below this concentration (0-30 mg L(-1) ).

Commentary: the role of the toxicologic pathologist in academia.

Veterinary pathologists working as toxicologic pathologists in academic settings fill many vital roles, such as diagnosticians, educators, and/or researchers. These individuals have spent years investigating pathology problems that mainly or exclusively focus on the reactions of cells, organs, or systems to toxic materials. Thus, academic toxicologic pathologists are uniquely suited both to help trainees understand toxicity as a cause of pathology responses and also to provide expert consultation on toxicologic pathology. Most toxicologic pathologists in academia are employed at colleges of medicine or veterinary medicine, even though specific toxicologic pathology faculty appointments are uncommon in Europe and North America. Academic toxicologic pathologists typically receive lower financial compensation than do toxicologic pathologists in industry, but academic positions generally provide alternative rewards, such as higher workplace autonomy and scheduling flexibility, professional enrichment through student interactions, and enhanced opportunities for collaborative research and advanced diagnostic investigations. Regular participation by academic toxicologic pathologists in professional training opportunities (eg, as pathology and toxicology instructors in medical and veterinary medical courses, graduate programs, and residencies) offers an important means of engendering interest and inspiring veterinarians to select toxicologic pathology and toxicology as their own areas of future expertise.

Phytase properties and locations in tissues of transgenic pigs secreting phytase in the saliva.

A transgenic Cassie (CA) line of Yorkshire (YK) pigs was developed using a transgene composed of the mouse parotid secretory protein promoter linked to the Escherichia coli phytase gene integrated in chromosome 4. Previous studies documented that salivary secretion of phytase was sufficient to enable efficient digestion of plant feed phytate P. In the present study the catalytic properties and tissue distribution of the phytase in CA pigs were determined by a combination of enzymatic assays, immunohistochemistry, and immunoblots of tissue samples. The E. coli phytase had a mass of 44.82 kDa whereas the phytase secreted in CA saliva had a mass of 52.42 kDa as a result of glycosylation of the enzyme in the parotid gland. Despite the difference in size, the 2 enzymes exhibited similar substrate specificities, and substrate affinity ( K: m) and maximum hydrolytic activity ( V: max) catalytic properties. Phytase assays showed that the enzyme was present at high specific activity in the salivary glands with low activity in the soft palate and essentially none in the kidney, lean (muscle), liver, or skin of CA pigs and none in YK pigs. This conclusion was supported by immunoblot analysis using a polyclonal anti-phytase antibody. Immunohistochemical analysis of 83 different tissue locations of CA and YK pigs confirmed the ubiquitous presence of phytase in serous cells of the salivary glands and the localized presence of phytase in both serous and mixed cell types in the submucosal glands of the oropharynx; in the pharynx, tonsils, and esophagus; in some Bowman's glands in the nasal mucosa and eustachian tube; and in the prostate gland of CA boars. Furthermore, it showed the absence of phytase from the kidney, lean, liver, and skin of CA pigs. Phytase was not detected in any of the conventional YK tissues tested. The phytase was found to be glycosylated with the allergenic galactose-α-1,3-galactose (α-gal) epitope by immunoblotting using α-gal specific monoclonal antibodies. Galactose-α-1,3-galactose glycosylation of proteins is a common feature of pork and other red meats. The α-gal epitope was shown to be associated with a few proteins in muscle and skin but with the greatest number of proteins in kidney and parotid tissues of CA and YK pigs. The absence of phytase from the major food tissues and the displacement of other α-gal glycosylated proteins in the parotid glands by α-gal glycosylated phytase in conjunction with previously published data support the contention that expression of the novel phytase has minimal influence on pork quality and safety.

Hepatic injury correlates with apoptosis, regeneration, and nitric oxide synthase expression in canine chronic liver disease.

Assessment of the clinical severity, pathogenesis, and prognosis of canine chronic liver disease poses significant challenges to clinicians and pathologists, relating in part to a lack of standardized terminology and assessment methods and also to a lack of understanding of the pathogenesis of chronic liver disease in the dog. This study graded the severity of necroinflammatory activity in chronic liver disease in dogs using a modification of Ishak's grading scheme for human chronic liver disease and examined the association of grade score with hepatocellular apoptosis, regeneration, nitric oxide synthase isoform expression, copper and iron accumulation, and indicators of oxidative stress. Formalin-fixed, paraffin-embedded hematoxylin and eosin (HE)-stained liver biopsies from 45 dogs with chronic liver disease and 55 healthy control dogs were graded for various morphologic components of liver injury and response. The cumulative score for grade of necroinflammatory activity was strongly and significantly correlated with immunoreactive labels for hepatocellular proliferation (Ki-67); apoptosis (cleaved caspase-3); inducible nitric oxide synthase (iNOS) in lobular, portal, and septal stromal cells; endothelial nitric oxide synthase (eNOS) in hepatocytes and lobular, portal, and septal stromal cells; and total stainable hepatic iron. A weaker significant correlation was found between grade and accumulation of hepatocellular copper. No significant correlation was found between grade and immunoreactivity for malondialdehyde-protein adducts. These results document a method for grading of the severity of necroinflammatory disease in canine liver biopsies and show an association with increased iNOS and eNOS expression.

Facile production of minor metabolites for drug development using a CYP3A shuffled library.

Metabolic profiling of new drugs is limited by the difficulty in obtaining sufficient quantities of minor metabolites for definitive structural identification. Biocatalytic methods offer the potential to produce metabolites that are difficult to synthesize by traditional medicinal chemistry. We hypothesized that the regioselectivity of the drug metabolizing cytochrome P450s could be altered by directed evolution to produce minor metabolites of drugs in development. A biocatalyst library was constructed by DNA shuffling of four CYP3A forms. The library contained 11 ± 4 (mean ± SD) recombinations and 1 ± 1 spontaneous mutations per mutant. On expression in Escherichia coli, 96% of mutants showed detectable activity to at least one probe substrate. Using testosterone as a model drug-like substrate, mutants were found that preferentially formed metabolites produced in only trace amounts by parental forms. A single 1.6L batch culture of one such mutant enabled the facile isolation of 0.3mg of the minor metabolite 1ß-hydroxytestosterone and its ab initio structural determination by 1D- and 2D-NMR spectroscopy.

Single nucleotide polymorphisms in collagenous lectins and other innate immune genes in pigs with common infectious diseases.

Innate immune recognition of pathogens involves various surface receptors and soluble proteins that precede agglutination, complement activation, phagocytosis, and the adaptive immune response. Mannan-binding lectins (MBLs), ficolins (FCNs) and surfactant protein A (SP-A) are soluble collagenous lectins that bind surface structures of various bacteria, viruses and fungi. Some single nucleotide polymorphisms (SNPs) in collagenous lectin genes of humans and other species, including pigs, have been implicated in variation in susceptibility to infectious and inflammatory diseases. In this study we determined the frequencies of 13 SNP alleles of MBL-A, MBL-C, ficolin-α, ficolin-ß, and SP-A in 1324 healthy pigs and 461 pigs diagnosed with common infectious diseases at necropsy. For comparison, we also analyzed 12 other SNP alleles in several other innate immune genes, including galectins and TLRs. Several SNPs within genes encoding porcine MBL-A, MBL-C and SP-A were more frequent in pigs diagnosed at necropsy with various diseases or pathogens. These findings suggest that several collagenous lectin SNPs are associated with disease susceptibility and therefore might be genetic markers of impaired innate immune function.

Concentration of lipocalin region of collagen XXVII alpha 1 in the serum of dogs with hemangiosarcoma.

BACKGROUND: Hemangiosarcoma (HSA) is a common malignancy of dogs with characteristic early, aggressive metastasis. Diagnosis of HSA is challenging because of lack of sensitive and specific diagnostic tests. HYPOTHESIS: Specific proteins that are increased in serum of dogs with HSA might represent useful biomarkers of the disease. ANIMALS: Thirty-four dogs with HSA and 42 healthy dogs from the Ontario Veterinary College Teaching Hospital. METHODS: This case-control study compared serum proteins in dogs with HSA and healthy dogs. Proteins were separated by 2-dimensional difference gel electrophoresis and identified by liquid chromatography and tandem mass spectrometry. RESULTS: Western blot analysis showed that serum collagen XXVII peptide concentration in serum of dogs with large metastatic HSA burdens (1,488, 231-3,754 DU; median, minimum-maximum); was, on average, 9.5-fold higher than in healthy dogs (156; 46-2,101 DU). While concentrations for dogs with osteosarcomas (678; 124-3,251 DU), lymphomas (423; 92-2,777 DU), carcinomas (1,022; 177-3,448 DU), and inflammatory disease were also increased, values were consistently lower than those for HSA. Receiver operating characteristic curves revealed an estimated area under the curve of 83% for HSA cases whereas areas for other neoplastic and nonneoplastic diseases were nondiscriminatory. Serum collagen XXVII peptide concentration before splenectomy (1,350; 1,156-1,929 DU) was reduced after tumor removal (529; 452-562 DU) and chemotherapy but increased in 2 dogs with tumor recurrence (511-945 DU; 493-650 DU). CONCLUSIONS AND CLINICAL IMPORTANCE: Collagen XXVII peptide might be useful for diagnosis and monitoring of advanced HSA.

Finite element analysis of wall stress in the equine pulmonary artery.

REASONS FOR PERFORMING STUDY: Arterial calcification is found frequently in the pulmonary artery of racehorses, but the aetiology is unknown. Calcification might be associated with increased wall stress due to arterial geometry (shape) and exercise-induced hypertension. HYPOTHESIS: High wall stress levels are found in the regions associated with calcified lesion formation, exacerbated as transluminal pressure increases to levels associated with exercise. METHODS: The pulmonary arteries of 5 horses, unaffected by calcification, were dissected and pressurised to resting and exercising physiological transluminal pressures and scanned with MRI. Arterial geometries were reconstructed to form 3D computer models and finite element analyses performed. Wall stress levels were measured in 4 regions of interest: the arterial trunk and bifurcation, the wall ipsilateral and contralateral to the bifurcation. Measurements were made for arterial transluminal pressures of 25, 50 and 100 mmHg. RESULTS: High wall stress levels were consistently found at the pulmonary artery bifurcation and wall ipsilateral to the bifurcation, where calcified lesions typically form. Lower wall stress levels were found along the trunk and the wall contralateral to the bifurcation where lesions are less frequently found. Wall stress levels increased 5-fold over a 4-fold increase in pressure. The wall stress levels ranged 10 kPa in the wall of the branch contralateral to the bifurcation at 25 mmHg to 400 kPa in the bifurcation at 100 mmHg. CONCLUSIONS: Wall stress from arterial geometry and increased pulmonary artery transluminal pressure are factors that may be associated with calcification of the equine pulmonary artery. POTENTIAL RELEVANCE: Arterial calcification may increase the risk of arterial wall failure in racing horses.

Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss).

In the present study, the pattern of immuno-reactive ladderlectin and intelectin in healthy rainbow trout is compared to rainbow trout infected with a variety of infectious agents. In healthy rainbow trout, both proteins were localized to individual epithelial cells of the gill and intestine and both proteins were clearly demonstrated within cytoplasmic granules of polymorphonuclear leucocytes and macrophages/monocytes found in blood vessels, hepatic sinusoids, renal interstitium, mucosal epithelium and submucosa of normal intestine. In tissue from infected rainbow trout, there was an overall relative increase in both lectins compared to healthy fish and both proteins were detected in extra-cellular spaces surrounding bacteria, fungi and protozoa. Increased distribution and density of both RTLL and RTInt was demonstrated along mucosal surfaces and within inflammatory leucocytes in infected tissues and immune related organs. These findings represent one of the few examples of in vivo association of defence lectins and infectious agents.

Arterial calcification in race horses.

Calcification of large arteries has been sporadically reported in horses. The pathogenesis is still unknown, but recent studies in humans suggest that this is a regulated biomineralizing process. This study surveyed the prevalence, distribution, and severity of vascular calcification in Thoroughbred and Standardbred racehorses. Histopathologic, ultrastructural imaging, and energy dispersive X-ray elemental analyses were used to examine the lesions. Calcification of the tunica media, predominantly the pulmonary artery, was found in 82% of horses (83/101). Young adult horses (mean [SD] age in years, 4.44 +/- 2.17) of both breeds and sexes were similarly affected. Lesions appeared as white-to-yellowish, hard, and gritty plaques of variable size. On microscopic examination, elastic fibers within the tunica media were thinned, fragmented, and calcified, and surrounded by dense collagen matrix. Elemental analysis showed distinct peaks for calcium and phosphorus, consistent with hydroxyapatite mineral. The frequent occurrence of calcification in the tunica media of large pulmonary arteries of young racing horses indicates the need to investigate its pathogenesis and potential clinical implications.

Proteins associated with the early intrauterine equine conceptus.

A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF(2 alpha). Two forms of uteroglobin were distinguished by partial amino acid sequences of approximately 6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF(2 alpha) one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.

Identification, cloning and tissue localization of a rainbow trout (Oncorhynchus mykiss) intelectin-like protein that binds bacteria and chitin.

Intelectins are a recently identified group of animal lectins involved in innate immune surveillance. This paper describes the primary structure, expression and immunohistochemical localization of a rainbow trout plasma intelectin (RTInt). RTInt exhibited calcium-dependent binding to N-acetylglucosamine (GlcNAc) and mannose conjugated Toyopearl Amino 650 M matrices. When GlcNAc eluates from chromatography matrices were analyzed by reducing 1D PAGE and Western blots, the lectin appeared as approximately 37 kDa and approximately 72 kDa bands. Similar analysis of plasma revealed a single 72 kDa band under reducing conditions. MALDI-TOF MS demonstrated five, approximately 37 kDa isoforms (pI 5.3-6.1) separated by 2D-PAGE. A 975 bp cDNA sequence obtained by RT-PCR from liver and spleen tissue encoded a 325 amino acid secretory protein with homology to human and murine intelectins, which bind bacterial components and are induced during parasitic infections. Gene expression and immunohistochemistry detected RTInt in gill, spleen, hepatic sinusoid, renal interstitium, intestine, skin, swim bladder and within leukocytes. Direct binding assays demonstrated the ability of RTInt to bind relevant bacterial and chitinous targets. These findings suggest that RTInt plays a role in innate immune defense against bacterial and chitinous microbial organisms.

Cloning, binding properties, and tissue localization of rainbow trout (Oncorhynchus mykiss) ladderlectin.

The present paper describes the primary structure, expression and immunohistochemical localization of rainbow trout ladderlectin (RTLL), a multimeric serum lectin that binds Sepharose and LPS of Aeromonas salmonicida. Two rainbow trout cDNAs (504 and 546bp) and a genomic sequence (2kb) were amplified using ladderlectin-specific primers. The sequences were identified as group VII mannose-binding C-type lectins from predicted amino acid sequences and showed highest identity with the Atlantic salmon mannose-binding lectin. The two cDNA sequences (RTLL-1 and RTLL-2) had 92% identity and encoded 173 and 187 amino acids, respectively. The genomic sequence of RTLL, obtained by PCR, was found to encompass six exons and five introns, with exon 2 encoding 14 amino acids which were exclusive to RTLL-2. The relative expression of both transcripts was highest in the renal kidney, while the intestine, gill and skin exhibited higher relative RTLL-2 expression than RTLL-1. RTLL was immunohistochemically present within cells of the branchial epithelium, hepatic sinusoids, biliary epithelium, renal interstitium, skin, and sub-mucosal granular layer of the intestine. RTLL bound galactan-based Sepharose 6B and Sepharose CL-6B matrices but did not bind unmodified acrylic resin base Toyopearl AF-Epoxy 650M, Toyopearl AF-Amino 650M matrices or N-acetylated Toyopearl AF-Amino 650M acrylic matrices. Two-dimensional SDS-PAGE and Western blots of whole plasma and plasma proteins which bound chitin and intact bacteria demonstrated multiple electrophoretic isoforms of RTLL ranging in size from 16 to 18kDa and isoelectric points between pH 4.9 and 6.3. These findings show that RTLL is a group VII C-type lectin with multiple isoforms that bind pathogen-associated molecular patterns such as chitin and microbial surfaces.

Changes in major proteins in the embryonic capsule during immobilization (fixation) of the conceptus in the third week of pregnancy in the mare.

During the third week of pregnancy, the equine conceptus is enclosed within a capsule, the glycan composition of which changes at around day 16 (ovulation = day 0) when the conceptus becomes immobilized (fixed) in the uterine lumen. Our objective was to characterize the process of fixation by identifying changes in major capsule-associated proteins. Individual equine conceptuses (n = 55) were collected transcervically by uterine lavage between days 13.5 and 26.5. Major proteins extracted from capsules were compared with those in fluids from the uterus and yolk sac by SDS-PAGE. Until day 14, a major capsule-associated protein that migrated at approximately 10 kDa was identified by N-terminal sequencing as equine beta2 microglobulin (beta2M). During fixation, beta2M in the capsule underwent limited proteolysis to an approximately 8 kDa form lacking nine amino acids from the N terminus, and was subsequently degraded. Expression of beta2M mRNA was detected in the yolk-sac wall tissues and endometrium between days 13.5 and 17.5. During this period, beta2M in the capsule was evidently not part of a complex with major histocompatibility complex class 1 heavy alpha chain bands because these were undetectable in the capsule and uterine lavage. Uterocalin (p19) was detected in uterine lavage and capsule throughout fixation, but in yolk-sac fluid only before fixation. These studies indicate that intact beta2M is a major protein associated with the embryonic capsule before fixation, after which it undergoes limited proteolysis to a truncated approximately 8 kDa form that remains in the capsule after the conceptus is immobilized.

Single nucleotide polymorphisms in mannan-binding lectins and ficolins in various strains of mice.

Mannan-binding lectin (MBL) and ficolin are collagenous lectins produced primarily by the liver and are involved in innate resistance to microbial pathogens. Mice have two MBL genes (Mbl1 and Mbl2) that encode MBL-A and MBL-C, respectively. Similarly, the murine Fcna and Fcnb genes encode ficolin-A and ficolin-B. Several single nucleotide polymorphisms (SNP) in the human MBL2 gene are responsible for various innate immune dysfunctions due to abnormal structure or expression of human MBL-C. In these studies, we identified SNPs in the expressed collagenous lectin genes Mbl1, Mbl2, Fcna, and Fcnb in 10 strains of mice designated high priority Group A strains by the Mouse Phenome Project (129S1/SvImJ, A/J, BALB/cByJ, C3H/HeJ, C57BL/6 J, DBA/2 J, FVB/NJ, SJL/J, CAST/EiJ and SPRET/EiJ) by sequencing gene exons by reverse transcription-polymerase chain reaction (RT-PCR). Sequence comparisons identified a total of 15 structural SNPs in Mbl1 in two strains, 27 SNPs in Mbl2 in five strains, and 19 and 15 SNPs in Fcna and Fcnb, respectively, in two strains. Two non-synonymous SNPs were identified in the collagen-like domain of mouse Fcnb that are similar to the coding polymorphisms in the collagen-like domain of human MBL2. Most of the non-synonymous SNPs identified in Mbl1 and Mbl2 occurred in the carbohydrate-recognition domains (CRDs), and some resulted in altered residues close to known ligand binding sites. Similarly, most non-synonymous SNPs of Fcna and Fcnb were identified in the fibrinogen-like CRD. The miscoding SNPs found in the CRD regions of mouse Mbl1, Mbl2, Fcna and Fcnb may be associated with strain differences in glycan binding avidity and disposition of microbial or host ligands. Furthermore, the non-synonymous mutations in the collagen-like domain of Fcnb may alter the structure of the mature ficolin-B protein leading to functional deficiencies. These differences may be important in the pathogenesis of susceptibility differences between inbred strains to various infectious microorganisms.

Plasma proteomic analysis of the acute phase response of rainbow trout (Oncorhynchus mykiss) to intraperitoneal inflammation and LPS injection.

Few acute phase proteins are known in fish and better knowledge of them would provide a basis for more reliable methods to objectively assess fish health and welfare. An acute phase response was induced in rainbow trout (Oncorhynchus mykiss, Walbaum) by inflammation triggered by intraperitoneal administration of purified Aeromonas salmonicida lipopolysaccharide emulsified in Freund's incomplete adjuvant (LPS/FIA) or a commercial oil-based multivalent vaccine. Acute phase proteins were characterized by comparative densitometry of plasma proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by MALDI-TOF and ESI MS/MS mass spectrometry. In one experiment, plasma samples were compared between treatment and control groups in which fish were terminally bled. In another experiment, individual fish were sampled repeatedly. Proteins scored as increased were those whose normalized value increased three-fold or greater between pre- and post-stimulus. Proteins scored as decreased were those whose normalized values decreased two-fold or greater. Unaltered proteins were those that were not altered or did not meet either of these criteria. Proteins that were absent in pre-stimulus gels but present in post-stimulus profiles were considered to be induced. Only those proteins that were altered in all fish for a given treatment were considered. In both experiments, protein p36 was increased up to 13-fold and several proteins were detected that had not been previously. In all fish treated with LPS/FIA, p9.5 was consistently increased an average of 75-fold in plasma. We have constructed a plasma protein panel of eight increased or induced proteins (p9.5, p10.5, p24a, p24b, p24c, p25a, p36 and p37), one decreased (p16) and two that are unaltered (p28a, p28b) in rainbow trout following inflammation or injection with LPS/FIA. Proteins from this panel that were similar to previously identified proteins were pre-cerebellin-like (p24a), transferrin (p37) and apolipoprotein (p10.5, p24c and p28).

Purification and binding properties of porcine plasma ficolin that binds Actinobacillus pleuropneumoniae.

Previous studies demonstrated that porcine plasma ficolin binds the important pig pathogen Actinobacillus pleuropneumoniae (APP) in an N-acetylglucosamine-dependent manner. In the present study, attempts to characterize the bacterial-binding properties of ficolin indicated ficolin is the major porcine plasma protein that binds directly to epoxy-activated chromatography matrices. We developed an efficient method for purifying ficolin using epoxy-activated Toyopearl and compared these with forms retrieved from other chromatography matrices and from intact APP. Purified ficolins retained their GlcNAc- and bacterial-binding properties, and migrated as two high molecular weight multimers composed of 38, 40 and 42 kDa reduced forms (pI 5.2-6.0). An N-acetylated amine-activated Toyopearl matrix bound ficolin, and ficolin was dissociated from this matrix with acetamide. Acetate, acetamide, and GlcNAc, but not glucose or glucosamine, dissociated plasma ficolin from the surface of intact APP serotype 5b, which contains N-acetylated saccharides in the capsule. These studies indicate that porcine ficolin binds APP 5b and an N-acetylated matrix in a similar manner, supporting the view that N-acetyl groups may be important for binding of porcine plasma ficolin to some microbial surfaces.

Prevalence of hepatitis E virus antibodies in Canadian swine herds and identification of a novel variant of swine hepatitis E virus.

Swine hepatitis E virus is a newly identified potentially zoonotic virus from pigs of particular concern for possible direct transmission to a human xenotransplant recipient by organ transplantation. In the present study, prevalence of serum antibodies to hepatitis E virus was examined in Canadian swine herds. A total of 998 serum samples collected from 6-month-old healthy slaughter hogs were examined by enzyme immunoassay and Western blot analysis for antibodies to the recombinant open reading frame 3 (ORF3) protein of hepatitis E virus expressed in Escherichia coli. These samples represented more than 80 different swine production units from five major swine-producing provinces across Canada. From this study, 594 samples (59.4%) were found to be positive for hepatitis E virus antibody. The seroprevalence was higher in Quebec (88.8%) and Ontario (80.1%) than in Alberta and Saskatchewan (38.3%). By PCR using a pair of oligonucleotide primers deduced from the ORF2 sequence of human hepatitis E virus, a specific hepatitis E virus sequence was recovered from feces of pigs. The nucleotide sequence identity between the U.S. swine hepatitis E virus and the Canadian isolate (SK3) was only 85.8%, suggesting that genotypic variations may exist in swine hepatitis E virus in North America. Among 165 serum samples collected from humans in Saskatchewan, 2.4% were found to be positive for antibodies to the hepatitis E virus ORF3 protein. Our data indicate that hepatitis E virus is highly prevalent in commercial swine populations in Canada and support the suggestion that the swine hepatitis E virus may be an important zoonotic agent for humans.

Avian air sac and plasma proteins that bind surface polysaccharides of Escherichia coli O2.

Some serovars of Escherichia coli, mainly O2 and O78, are responsible for air sac and systemic infections in farm-raised turkeys (Meleagris gallopavo) and chickens (Gallus gallus). We looked in air sac surface fluid from young turkeys to identify proteins that bind surface polysaccharides of pathogenic respiratory E. coli O2. Turkey air sac surface fluid was subjected to affinity chromatography on Toyopearl AF-Epoxy-650M, coupled with either lipopolysaccharide (LPS) or lipid-free polysaccharide (LFP) purified from an avian pathogenic E. coli O2 isolate. A multimeric protein termed lipid-free polysaccharide binding protein-40 (LFPBP-40) composed of six covalently associated subunits of approximately 40 kDa was isolated by elution from LFP by EDTA or L-rhamnose. An analogous protein in air sac fluid proteins bound to intact E. coli O2 and eluted with L-rhamnose or N-acetylglucosamine (GlcNAc). The N-terminal amino acid sequence of LFPBP-40 DINGGGATLPQHLYLTPDV was related to the N-terminus of fragment 3 of a partially characterized human protein possessing T cell stimulation activity in synovial membrane of rheumatoid arthritis patients. However, endogenous amino acid sequences were unrelated to other known proteins. LFPBP-40 was immunoreactively distinct from pulmonary collectins and ficolins. These studies demonstrate a novel avian respiratory soluble lectin that can bind surface polysaccharides of pathogenic E. coli responsible for respiratory disease.