Shizuo Yamamoto

Changes in serum C-reactive protein levels in dogs with various disorders and surgical traumas.

The serum levels of C-reactive protein (CRP) produced as an inflammatory response in dogs with various disorders and surgical traumas were measured by enzyme-linked immunoabsorbent assay and slide reversed passive latex agglutination test (RPLA). The CRP levels were greatly increased 1–2 days after surgery in most of the dogs (n=29) subjected to surgery. These levels had markedly decreased by the time the sutures were removed. In dogs with various disorders (n=58), the serum CRP levels at first diagnosis were high in infectious diseases. In dogs from which paired serum samples were examined, the serum CRP usually showed a decrease with improvement in the condition (n=11) or a terminal increase (n=4) but, conversely, some showed an increase with improvement in the condition (n=3).

PHYSIOLOGICAL LEVELS OF C-REACTIVE PROTEIN IN NORMAL CANINE SERA

This study was undertaken to investigate whether the level of C-reactive protein (CRP) in the serum of dogs undergoes physiological variation, using 10 normal Beagle dogs (5 males and 5 females), 1–2 years old, maintained in a healthy condition in a controlled environment. The CRP concentration in the sera collected seven times each day at intervals of approximately 3 h ranged from 0.8 to 16.4 µg/ml (mean 5.06±3.60) in one experiment and from 0.8 to 14.0 µg/ml (mean 4.50±2.80) in a second experiment. On examining the 24-h variations in the concentration of CRP in serum, neither consistent changes nor a definite pattern of circadian rhythm was detected. During 28 days observation, only very slight changes, which seemed attributable to analytical errors, were seen in any of the dogs, except one. The concentration of CRP in the serum during the 28 days ranged from 0.8 to 22.6 µg/ml (mean 3.65±1.40). The concentrations underwent no significant variations in individual dogs, but significant differences were found between the dogs (p<0.01).

A comparison of the concentrations of C-reactive protein and α1-acid glycoprotein in the serum of young and adult dogs with acute inflammation

The concentrations of C-reactive protein (CRP) and α1-acid glycoprotein (AAG) were evaluated in 1-, 3- and 18-month-old dogs (four of each age) that had been inoculated with turpentine oil. The CRP and AAG in 3-month-old and younger dogs subjected to surgery or inoculated with either Staphylococcus aureus or a viral vaccine were also evaluated. The average CRP concentration in the sera peaked 2 days after inoculation of turpentine oil. The peak CRP concentrations in 3- and 18-month-old dogs were significantly (p<0.05) greater than those in 1-month-old dogs. The average AAG concentration in the sera peaked 4 days after inoculation of turpentine oil. No significant difference was found in AAG concentrations between any of the age groups. When experimentally inoculated with S. aureus or subjected to oophorohysterectomy, the CRP and AAG concentrations increased in 3-month-old dogs, but they increased little in 1-month-old dogs. The CRP and AAG in dogs inoculated with the viral vaccine did not increase. In dogs with fractures or subjected to percutaneous gastrostomy, the CRP and AAG concentrations correlated with the condition of dogs.

Isolation of canine C-reactive protein and characterization of its properties.

C-reactive protein (CRP) was isolated from the acute phase serum of dogs subjected to surgical stimulation. Its properties were characterized. Canine CRP was isolated by ion-exchange chromatography using DEAE-Sephacel and DEAE-Sephadex A-50 and affinity chromatography using protein A-Sepharose CL 4B in combination with agar-block electrophoresis. In immunoelectrophoresis, canine CRP had the same gamma-mobility as human gamma-type CRP. The molecular weight of purifined canine CRP was estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 157,000 and 155,000 respectively. This CRP was a thermolabile protein which completely lost its antigenicity by heating at 70 degrees C for 15 min. The serum concentration of CRP in normal beagle dogs ranged from 0.198 to 0.826 micrograms ml-1 (0.486 +/- 0.170 micrograms ml-1). The concentration was acutely increased by surgery as it was in man and was rapidly decreased with convalescence. Dogs can be a useful animal model for investigation of the mechanism of CRP production and the function of CRP.

Analysis of 16S rRNA gene sequences of Ehrlichia canis, Anaplasma platys, and Wolbachia species from canine blood in Japan.

Abstract: In the present study, three canine blood samples from Japan, that were suspected to be ehrlichia positive were examined. After sequencing the 16S rRNA genes, each dog was found to be infected either with Ehrlichia canis (Kagoshima 1), Anaplasma platys (Okinawa 1), or Wolbachia sp. (Okinawa 2). Phylogenic analysis was performed on these sequences. The nearly entire 16S rRNA sequence of Kagoshima 1 was found to be most similar to the sequences from Oklahoma and Venezuela E. canis strains (1 base pair difference out of 1,387, 99.9% sequence identity). The 16S rRNA gene sequence of Okinawa 1 showed the closest DNA identity to the French strain of A. platys (1 base deletion out of 1,385 bp, 99.6% sequence identity). The 16S rRNA gene sequence of Okinawa 2 illustrated the closest DNA identity to that of a Wolbachia sp. from Dirofilaria immitis (98.9% sequence similarity). These data imply a low diversity within E. canis strains and within A. platys strains, including those strains reported in this study. This is also the first demonstration of Wolbachia DNA in dog blood, suggesting the involvement of Wolbachia sp. in canine febrile illnesses.

Determination of C‐reactive protein in serum and plasma from healthy dogs and dogs with pneumonia by ELISA and slide reversed passive latex agglutination test

The concentrations of C-reactive protein (CRP) in serum from dogs diagnosed as normal by clinical, haematological, and biochemical examination were determined by enzyme-linked immunosorbent assay (ELISA) and slide reversed passive latex agglutination (RPLA), using IgG antibody isolated from rabbit anti-canine CRP serum. The mean value of CRP in 66 normal dogs kept in private households was 8.4 +/- 4.9 micrograms/ml by ELISA and 8.5 +/- 6.3 micrograms/ml by RPLA. Thus, no significant difference was demonstrated between the values obtained by ELISA and RPLA. No significant age and sex-related differences were found in the CRP values. The mean concentration of CRP in 84 6-month-old Beagle dogs kept in kennels by breeders was 6.2 +/- 3.9 micrograms/ml by ELISA and 8.0 +/- 4.0 micrograms/ml by RPLA. Again no significant difference was found. The CRP values determined by ELISA and RPLA were closely correlated (r = 0.913). The serum and plasma concentrations of CRP measured by RPLA were also closely correlated (r = 0.994). This indicates that plasma can be used as well as serum to determine CRP concentrations by RPLA.

Bovine haptoglobin: Single radial immunodiffusion assay of its polymeric forms and dramatic rise in acute-phase sera

Using purified bovine haptoglobin (Hp) and specific antisera, a single radial immunodiffusion (SRID) assay method has been developed to measure the serum Hp level in cattle. Bovine Hp is a highly polymerized protein showing heterogeneous molecular forms in serum. After treatment with cysteine or glutathione, Hp was partially reduced to a homogeneous form, suitable for SRID assay. This method gives values comparable to those obtained by hemoglobin-binding capacity assay, and has the advantage of being simple and convenient. Although serum Hp was not detectable in healthy cattle, it was found more than 50-fold after invasive surgery, indicating that Hp is a characteristic acute-phase protein in cattle.

C-reactive protein (CRP) measurement in canine serum following experimentally-induced acute gastric mucosal injury

To establish the diagnostic significance of canine C-reactive protein (CRP) in gastrointestinal disorders, the serum canine CRP concentration was measured in dogs with experimentally-induced acute gastric mucosal injury. Gastric injury was induced in one male and one female beagle by a single dose oral administration of acetylsalicylic acid (200 mg/kg body weight) or indomethacin (60 mg/kg body weight), or sodium chloride (1000 mg/kg body weight). CRP was measured prior to dose, and 1, 3, 7, and 14 days after the administration of the drugs, together with the total leucocyte counts and serum iron. Changes in the serum CRP in dogs with gastric injury were similar for the three test compounds, and reflected by the endoscopic findings. CRP values increased from 87 to 390 mg/l within 1 to 3 days after the compound administration but returned nearly to the predose levels within 14 days. Endoscopy revealed haemorrhagic erosion of the gastric mucosa in all dogs one day after dosing, with no evidence of the erosions observed after 7 days in many of the dogs. Changes of the total leucocyte and serum iron also occurred following gastric injury, but these changes were not as marked as those observed for CRP. The results of this study suggest that serum CRP level may be a useful indicator of a gastrointestinal mucosal injury in dogs.

Serum α2-macroglobulin and cytokine measurements in an acute inflammation model in rats

An enzyme-linked immunosorbent assay (ELISA) for rat α2-macroglobulin (α2M) using a monoclonal antibody was developed, and used to measure α2M in sera from rats injected intramuscularly with turpentine oil as an inflammatory agent. The mean concentration of α2M gradually increased and peaked 2 days after the turpentine oil injection. The peak α2M concentration ranged from 2362–8472 µg/ml (mean 4531 µg/ml), which was 50–290 times higher than the pre-dosing levels of 23–61 µg/ml. In addition, interleukins (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, and interferon (IFN)-γ were measured using commercial ELISA reagent kits. IL-6 and IL-8 increased and peaked 12 h after turpentine oil injection, the levels being 5–51 times and 2–38 times the pre-dosing ones, respectively. The concentrations of IL-1, IL-2, IL-4, IL-10 and IFN-γ in rats injected with turpentine oil did not change.

Serum C-reactive protein and immune responses in dogs inoculated withBordetella bronchiseptica (phase I cells)

Eight Beagle dogs were inoculated intrabronchially with 5×109 live, avirulent cells ofBordetella bronchiseptica L-414 strain (phase I cells) (B. bronchiseptica) to investigate the serum levels of their C-reactive protein, the white blood cell counts, the antibody responses toB. bronchiseptica in the sera and tracheal secretions, and the effects of prednisolone given to four of the dogs on C-reactive protein (CRP), white blood cells (WBC) and immune responses. In two Beagle dogs inoculated intrabronchially with sterile physiological saline, the concentrations of CRP and the WBC counts did not increase. CRP was markedly increased one day after inoculation in the dogs inoculated withB. bronchiseptica to 385.0–720.0 µg/ml (mean 498±132 µg/ml) in the group given theB. bronchiseptica inoculation only, and to 372.0–649.0 µg/ml (mean 551±106 µg/ml) in the group treated with prednisolone following inoculation ofB. bronchiseptica, as determined by an enzyme-linked immunosorbent assay (ELISA). The CRP levels were 23–95 times the pre-inoculation values, which indicated that prednisolone had no effect on the production of CRP. In the prednisolone-treated group, the WBC count increased and stayed at an increased level for approximately 12 days. An indirect fluorescent antibody test led to the detection of anti-B. bronchiseptica IgM and IgG antibodies in the sera from 5 days afterB. bronchiseptica inoculation and S-IgA and IgG anti-B. bronchiseptica antibodies in the tracheal secretions on the day after the challenge exposure toB. bronchiseptica. The increase in CRP after challenge exposure toB. bronchiseptica was significantly (p<0.05) smaller than that found after the first inoculation ofB. bronchiseptica.