Isolation, characterization, and quantitative analysis of C-reactive protein from horses

C-reactive protein (CRP) was isolated from equine serum by use of calcium-dependent affinity chromatography conjugated pneumococcal C-polysaccharide, anion exchange chromatography, and gel filtration. It was identified as genuine CRP by its immunochemical cross-reactivity with anti-human CRP, its homology with human CRP in amino acid composition, and its pentameric structure as revealed by electron microscopy. Purified equine CRP had a molecular weight of approximately 118,000 and was composed of 5 identical, nonglycosylated and noncovalently associated subunits with molecular weight of approximately 23,000 each. Equine CRP migrated in the region between beta- and gamma-globulin by results of immunoelectrophoresis, and its isoelectric point was about 7.0. In horses, increased CRP concentration was associated with clinical pneumonitis, enteritis, and arthritis, compared with values obtained in clinically normal horses by use of single radial immunodiffusion method. After IM administration of turpentine oil or castration, serum CRP concentration increased to 6 times higher than baseline values. Results indicate that CRP may be an acute-phase reactant protein in horses.     

Isolation and characterization of alpha 1-acid glycoprotein from horses, and its evaluation as an acute-phase reactive protein in horses.

Equine alpha 1-acid glycoprotein (alpha 1AG) was isolated from equine serum by successive ammonium precipitation, anion- and cation-exchange chromatographies, and gel filtration. Purified equine alpha 1AG had a molecular weight of 46,000 +/- 1,000, and contained 31.4% carbohydrate. Gel isoelectric focusing revealed an isoelectric point range of 2.8 to 3.7. With immunoelectrophoresis, it was found that alpha 1AG migrated to the alpha 1-globulin region. Single radial immunodiffusion was used for quantitative measurement of alpha 1AG in equine serum. In clinically normal foals, serum alpha 1AG was undetectable (less than or equal to 20 micrograms/ml) in less than or equal to 7-day-old foals, but was detected by 14 days. The alpha 1AG concentration (mean +/- SD) increased to reach mean adult values of 99.23 +/- 26.90 micrograms/ml by 1 year of age. The alpha 1AG concentration in pregnant mares decreased at 2 to 3 months before parturition, then gradually increased until 1 day after parturition, when a brief decrease was observed. The concentration increased again at 2 weeks after foaling, then a decrease was observed, after which the alpha 1AG concentration increased again by 2 to 4 months after parturition. The concentration of serum alpha 1AG quickly rose to peak values 2 to 3 days after castration and jejunojejunostomy in adult horses, returning to baseline values by 14 to 28 days after surgery. The alpha 1AG was concluded to be an acute-phase reactive protein in horses.     

Identification and characterization of Streptococcus agalactiae isolated from horses.

Seven group B streptococcal cultures isolated from three horses reacted with group B-specific antiserum, were CAMP positive, pigmented and showed the typical biochemical properties of Streptococcus agalactiae. The identification could be confirmed by PCR amplification of the 16S rRNA gene and a subsequent RsaI restriction pattern typical for S. agalactiae. In addition, the isolates were identified by amplification of species specific parts of the 16S rRNA gene, the 16S-23S rRNA intergenic spacer region and by amplification of the CAMP-factor (cfb) gene. Six isolates could be classified as serotype III/Rib, one isolate as serotype Ia/cbeta. The occurrence of the protein antigens Rib and cbeta could be confirmed by PCR amplification of the respective genes. The six isolates of serotype III/Rib were hyaluronidase negative, had a hylB gene with a size of 4.6 kb and an insertion element IS1548 of 0.98 kb. The isolate of serotype Ia/cbeta was hyaluronidase positive, had a hylB gene with a size of 3.3 kb and no insertion element IS1548. In addition, all seven isolates had the insertion element ISSag2 and the gene lmb encoding the laminin binding surface protein Lmb and the gene scpB encoding C5a peptidase. According to the present results the group B streptococci isolated from horses showed characteristics of human isolates of this species.     

Isolation, characterization, and quantitative measurement of serum alpha 1-acid glycoprotein in cattle

Bovine alpha 1-acid glycoprotein (alpha 1AG) was purified from pooled normal bovine sera by successive ammonium sulfate precipitation, ion-exchange chromatographies and gel filtration. Bovine alpha 1AG had a molecular weight of 42,000 +/- 2,000 and a sedimentation coefficient of 3.4S. It contained 26.6% carbohydrate. Gel isoelectric focusing revealed a microheterogeneity with 7 to 8 bands in a pI range of 3.2 to 3.7. It migrated to the alpha 1-globulin region upon immunoelectrophoresis. Single radial immunodiffusion was developed for the quantitative measurement of bovine alpha 1AG in serum. The mean serum value of alpha 1AG in 152 healthy Holstein cattle (1-12 years old) was 283.2 +/- 82.3 micrograms/ml. Elevated values (cut-off value = 450 micrograms/ml) were observed in cattle with traumatic pericarditis (100%), arthritis (100%), mastitis (91%), pneumonia (70%), and mesenteric liponecrosis (43%).     

Isolation of mycoplasmas from the respiratory tract of horses in Australia.

Mycoplasmas were isolated from two of 43 nasal swabs taken from live horses, and from one of 28 tracheal swabs taken from slaughtered horses. The slaughtered horse that yielded mycoplasmas had no gross pathological changes in the respiratory tract, but the nasal isolations were made from horses with rhinitis. The three mycoplasmas could be distinguished by cultural characteristics, and probably they represent three different species.     

Isolation of a major form of pepsinogen from gastric mucosa of horses.

In mammalian species studied previously, pepsinogen consisted of biochemically different groups of isozymogens. By use of gel filtration chromatography and electrophoresis, we isolated a predominant pepsinogen from the gastric mucosa of a horse. Peptide mapping with V8 protease revealed differences with its porcine homologue. However, porcine and equine pepsinogens, when activated to pepsin, had a similar pattern of activity when hemoglobin was used as substrate. Those results suggest that differences must exist in the primary structure of the pepsinogens of the 2 species.     

Isolation,characterization, and functional analysis of ferret lymphatic endothelial cells

The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases – the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-I ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles LECs might play in health and disease in animal models.     

Isolation of Acholeplasma oculi from diseased horses in Australia

Eleven isolations of A. oculi were made from nasal swabs from horses with acute upper respiratory disease (3), from an equine lung with extensive emphysema (1), from lung, liver and placenta respectively of three aborted foals, from cerebrospinal fluids from two horses with a paralytic syndrome, from synovial from a horse with acute polyarthritis and from a semen sample from a stallion with a breeding problem.     

Isolation of virulent Rhodococcus equi from native Japanese horses

R. equi was isolated from soil samples obtained from the environment of seven native Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, Miyako and Yonaguni) and from fecal samples collected from three native horse breeds (Hokkaido, Kiso and Misaki). Virulent R. equi at various levels (ranging from 0.5 to 12.9%) was isolated from the feces or soil environment of Hokkaido, Kiso and Misaki horses. Isolates were investigated both for the presence of 15- to 17-kDa antigens (virulence-associated protein antigens; VapA) by colony blotting, using the monoclonal antibody 10G5, and the gene of VapA by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases, and the digestion patterns of the plasmids of virulent isolates were divided into three types. Two of the three types (87-kb type II and 90-kb type I) had already been reported in Japanese isolates, and a new type (tentatively designated as 90-kb type II) had been found in isolates from Kiso horses. Six virulent R. equi isolates from the Hokkaido horses contained an 87-kb type II plasmid. Eight of 24 isolates from the Kiso horses contained an 87-kb type II plasmid, and the remaining 16 contained a 90-kb type II (a new type) plasmid. Two isolates from the Misaki horses contained a 90-kb type I plasmid. These results demonstrate the geographic difference in the distribution of virulence plasmids in R. equi isolates among native Japanese horses.     

Isolation of granulocytes and mononuclear cells from the blood of dogs, cats, horses and cattle

A simple discontinuous Percoll density-gradient technique was adapted for isolation of granulocytes and mononuclear cells from cats, dogs, horses and cattle. Separation was accomplished at low speeds using a standard tabletop centrifuge. Cell purity was 100% for both granulocytes and mononuclear cells and cell viability exceeded 95%. Percent recovery of leukocytes ranged from 69 to 83%.     

Isolation and characterization of mu-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps

Evidence has indicated that mu-calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem tenderization. Simple assays of these 3 proteins at different times postmortem, however, has shown that calpastatin and mu-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of mu-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25 degrees C, and ability of calpastatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem storage, but the Ca(2+) requirement of m-calpain is much higher than that reported to exist in postmortem muscle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11-13 d postmortem. The known properties of the calpains and calpastatin in postmortem muscle have important effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the 2 calpains in postmortem muscle. Dot-blot assays were used to detect proteolytically inactive mu-calpain. After 2 column chromatographic steps, 5 fractions can be identified: 1) calpastatin I that does not bind to an anion-exchange matrix, that does not completely inhibit the calpains, and that consists of small polypeptides <60 kDa; 2) calpastatin II that binds weakly to an anion-exchange matrix and that contains polypeptides <60 kDa; all these polypeptides are smaller than the native 115- to 125-kDa skeletal muscle calpastatin; 3) proteolytically active mu-calpain even though very little mu-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this mu-calpain has an autolyzed 76-kDa large subunit but the small subunit consists of 24-, 26- and a small amount of unautolyzed 28-kDa polypeptides; 4) proteolytically active m-calpain that is not autolyzed; and 5) proteolytically inactive mu-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active mu-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of mu-calpain activity remains unknown.     

Isolation of Salmonella organisms from the mesenteric lymph nodes of horses at necropsy.

OBJECTIVES: To determine the prevalence of Salmonella infections in horses at necropsy. DESIGN: Cross-sectional prevalence survey. ANIMALS: 102 horses. PROCEDURE: Mesenteric lymph nodes were collected from horses that were necropsied. Horses had died or were euthanatized because of severe disease or at the request of the owner. Twenty-eight of the horses were racehorses euthantized following acute catastrophic injuries on the racetrack. Mesenteric lymph nodes were submitted for Salmonella culture via direct plating of tissue specimens on MacConkey agar and by use of 4 enrichment culture techniques that used tetrathionate and selenite enrichment broth and brilliant green and Salmonella-Shigella selective plating media. RESULTS: Salmonella typhimurium was isolated from the mesenteric lymph nodes of 2 foals (2/102, 1.96% of the horses). Salmonella organisms were not isolated from the mesenteric lymph nodes of adult horses. CONCLUSIONS AND CLINICAL RELEVANCE: Prevalence of Salmonella infections in horses of our study (1.96%) suggests that the results of cross-sectional surveys, using bacteriologic culture to determine prevalence of Salmonella infection, should be interpreted with caution. Prevalence of Salmonella infections determined in a single facility may not reflect the prevalence of Salmonella-infected horses in the general population; furthermore, obtaining a Salmonella isolate from a horse does not establish that the horse is a chronic Salmonella carrier.     

Isolation of Streptococcus pneumoniae from the respiratory tract of horses

Streptococcus pneumoniae was isolated from nasopharyngeal swabs and tracheal washings taken from Thoroughbred horses in training at three of four separate stables that were sampled during investigations into respiratory disease. The growth of Strep pneumoniae in culture was enhanced by an environment enriched with carbon dioxide. In one stable, five of 15 horses that were sampled repeatedly were found to carry the organism for at least four months. There was an apparent association between lower respiratory tract inflammatory disease and heavy growths (10(6) to 10(8) colony forming units/ml) predominantly of Strep pneumoniae or of that organism together with large numbers of Strep zooepidemicus obtained from tracheal washings. Twelve strains of Strep pneumoniae isolated from three stables were all of capsule Type 3. Only one strain, which was of capsule Type 9, was isolated from nose and throat swabs taken from 32 staff working in one of the stables and suggested an absence of cross infection between horses and their handlers in this instance.     

Isolation and characterization of a Sagiyama virus from domestic pigs.

In 2002, a strain of Sagiyama virus (SAGV) designated ML/Taiwan/02 was isolated from farmed pigs in Taiwan. The nsP1 and E1 gene sequences of the ML/Taiwan/02 strain shared 98.6 and 96.7% homology, respectively, with corresponding genes of a Japanese strain of SAGV. Nucleotide and amino acid sequence comparison revealed this strain of SAGV to be most closely related to Getah virus, as opposed to its current classification as a subtype of Ross River virus. To investigate the seroprevalence of SAGV infection in Taiwan, a total of 586 pig sera collected from 11 of 17 Taiwanese districts were tested for serum neutralizing antibodies (SNA) against SAGV. Results indicated that 51% of the samples had SNA titer > or = 4, and 40% had SNA titer > or = 48, indicative of repeated exposure to SAGV in the field. To study the pathogenicity of the ML/Taiwan/02 strain, this strain was experimentally inoculated into 4-week-old specific-pathogen-free pigs that were seronegative for SAGV. Viremia was detected during postinoculation days (PID) 2-4, when the SNA titer was < or = 16. By PID 7, viremia was no longer detectable, coinciding with the increase of SNA titer to > or = 48. Clinical illnesses or remarkable lesions were not observed. To the authors' knowledge, this is the first reported isolation of a strain of SAGV from pigs in the field. The virus is experimentally nonpathogenic to pigs but is moderately widespread, most likely via repeated exposure to virus-carrying mosquitoes.