毒害艾美耳球虫卵囊壁蛋白的SDS-PAGE及Western blot分析

利用聚丙烯酰胺凝胶(SDS-PAGE)电泳技术,分析了毒害艾美耳球虫未孢子化卵囊壁可溶性蛋白。用鼠抗毒害艾美耳球虫重组配子体蛋白rEnGAM56和rEnGAM59多克隆抗体,对卵囊壁可溶性蛋白进行了Western blot分析。结果显示,至少有24条较清晰的电泳条带,其中6条为相对明显的主带,其相对分子质量分别为37 000,35 000,34 000,20 000,14 000和12 000。Western blot分析显示,鼠抗rEnGAM59和rEnGAM56多抗均能识别1条条带,前者的相对分子质量约14 000,后者约30 000。推测30 000和14 000卵囊壁蛋白分别来自毒害艾美耳球虫配子体蛋白EnGAM56和EnGAM59。     

Serum concentrations of acute phase proteins in dogs with leishmaniasis

The concentrations of haptoglobin, C-reactive protein and ceruloplasmin were measured in symptomatic and asymptomatic dogs naturally infected by Leishmania infantum, and in healthy uninfected dogs to determine the potential value of these proteins for the diagnosis and prognosis of leishmaniasis. The concentrations of the acute phase proteins were significantly higher in the dogs with leishmaniasis than in the control dogs, and the concentration of C-reactive protein was significantly higher in the symptomatic dogs than in the asymptomatic dogs. There were no correlations between the acute phase proteins and the gamma globulins, the albumin/globulin ratio or the titre of anti-leishmanial antibodies.     

Western blot analysis of Leishmania infantum antigens using sera from pentamidine-treated dogs

Pentamidine treatment has been used successfully to establish immune cellular responses in recovered dogs. In this paper, we examined the appearance and disappearance of antibodies over time against crude Leishmania antigens and purified gp63 or gp70 proteins in sera from cured dogs using a Western blotting technique.Following the treatment, a pattern of antibody specificities to parasite antigens was observed in the sera of cured dogs. Antibodies to gp63 and gp70 were maintained after cure. In addition, the reaction with a 26 kD band observed during the clinical phase was no longer recognized by sera taken from recovery dogs. Interestingly, two proteins, 85 and 110 kD, not observed during the patent phase were detected by sera taken from treated dogs. Such patterns of antibody specificities to various parasite antigens might represent a useful parameter to determine the actual phase of the disease process.     

Anti-histone antibodies in dogs with leishmaniasis and glomerulonephritis

The association between serum anti-histone antibodies and glomerulonephritis was studied in 43 dogs with leishmaniasis (Leishmania infantum). Dogs with increased serum creatinine levels and urine protein-creatinine ratio >1 were considered to have glomerulonephritis. Moderately elevated anti-histone antibodies were found in 38.89% (7/18) of infected dogs without glomerulonephritis, whereas 88% of dogs with glomerulonephritis (22/25) showed moderate or strongly elevated anti-histone antibodies. Prevalence of positive anti-histone antibodies reactions and mean serum concentration was significantly higher (P < 0.001; P < 0.0001) in infected dogs with glomerulonephritis. Correlation between anti-histone antibodies and urine protein-creatinine ratio was significant when groups were analysed together (P < 0.046). Positive predictive value for glomerulonephritis of positive anti-histone antibodies was 88%. In conclusion, high anti-histone antibodies are significantly associated with glomerulonephritis. Although other factors must be involved, dogs with moderate or strong positive anti-histone antibodies reactions may have a higher probability to develop glomerular lesions in canine leishmaniasis.     

Anti-histone antibodies in dogs with leishmaniasis and glomerulonephritis

The association between serum anti-histone antibodies and glomerulonephritis was studied in 43 dogs with leishmaniasis (Leishmania infantum). Dogs with increased serum creatinine levels and urine protein-creatinine ratio >1 were considered to have glomerulonephritis. Moderately elevated anti-histone antibodies were found in 38.89% (7/18) of infected dogs without glomerulonephritis, whereas 88% of dogs with glomerulonephritis (22/25) showed moderate or strongly elevated anti-histone antibodies. Prevalence of positive anti-histone antibodies reactions and mean serum concentration was significantly higher (P < 0.001; P < 0.0001) in infected dogs with glomerulonephritis. Correlation between anti-histone antibodies and urine protein-creatinine ratio was significant when groups were analysed together (P < 0.046). Positive predictive value for glomerulonephritis of positive anti-histone antibodies was 88%. In conclusion, high anti-histone antibodies are significantly associated with glomerulonephritis. Although other factors must be involved, dogs with moderate or strong positive anti-histone antibodies reactions may have a higher probability to develop glomerular lesions in canine leishmaniasis.     

Evaluation of serum cystatin-C in dogs with visceral leishmaniasis

Serum Cystatin C (sCys-C) is one of the most important serum markers of renal function assessment in dogs. The purpose of this study was to determine the sCys-C concentration in dogs with visceral leishmaniasis (VL). In the study, 16 dogs with VL and 10 clinical healty dogs (control) were used. Mean sCys-C concentration of the infected dogs was significantly higher than that of the control group (p < 0.05). Mean serum creatinine concentration was lower and mean blood urea nitrogen, albumin and globulin concentrations were higher in dogs with VL; however, these changes were not statistically significant. Mean total protein and phosphorus concentrations were found to be higher in dogs with VL than healthy dogs (p < 0.05). No significant correlation had been determined between sCys-C and other variables. Visceral leishmaniasis in dogs has increased sCys-C concentration indicating a possible renal impairment; however, further studies are needed to be performed together with renal biopsies in the investigation sCys-C in dogs with VL.     

Western blot analysis of the humoral response of dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866)

Seven cross-bred dogs were inoculated with Angiostrongylus vasorum and serum samples were analyzed using the enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). ELISA detected specific antibodies anti-A. vasorum, from 14 to 28 days after inoculation (DAI) and persisted throughout the experiment. Using WB, the main antigens detected had molecular weight of approximately 115, 102, 86, 76, 69, 56, 41, 32, 28, 20-22 and 10kDa.     

Presumed brain infarctions in two dogs with systemic leishmaniasis

Clinical signs and magnetic resonance imaging findings of multiple brain infarcts in two dogs infected with Leishmania spp. are reported. Clinical signs of intracranial dysfunction were peracute and there was no further deterioration. Magnetic resonance images of the brain were consistent with multifocal, non-haemorrhagic, ischaemic lesions. Routine serum biochemistry revealed hyperproteinaemia and hyperglobulinaemia. Serum antibody titres were highly positive for Leishmania infantum and Leishmania amastigotes were seen within bone marrow macrophages in both cases. Canine leishmaniasis can cause cerebrovascular alterations, such as vasculitis, that might predispose dogs to brain infarcts.     

Immunoglobulin isotype distribution of antinuclear antibodies in dogs with leishmaniasis

A modified indirect immunofluorescence method, using rat liver as substrate, was developed to determine the immunoglobulin isotypes forming antinuclear antibodies in sera from 12 antinuclear antibody-positive dogs out of 121 dogs with natural Leishmania infection. Immunoglobulin M was found to be the most frequent component of antinuclear antibodies (91·7 per cent), followed by IgG (41·7 per cent) and IgA (33·2 per cent). When these immunoglobulin isotypes were titrated, IgG antinuclear antibodies showed higher titres (1:200) than IgM and IgA antinuclear antibodies (1:50 and 1:20 respectively). Most of the antinuclear antibody-positive dogs simultaneously had two immunoglobulin isotypes, whereas none had all three immunoglobulin isotypes at the same time. Spearman rank correlation analysis showed no significant correlation between antinuclear antibody titres and circulating immune complexes or immunoglobulin levels. The low incidence of antinuclear antibodies and the absence of a clear relationship between isotype titres and clinical signs suggest a minor pathogenic role of antinuclear antibodies in canine leishmaniasis.     

不同型魏氏梭菌菌体蛋白和膜蛋白SDS-PAGE电泳图谱分析

运用ＳＤＳ－ＰＡＧＥ技术分别对Ａ、Ｂ、Ｃ、Ｄ四个型魏氏梭菌的菌体蛋白和细菌膜蛋白进行分析。结果在菌体蛋白图谱中Ａ型缺少５４．９ＫＤ蛋白条带，Ｃ型缺少１０７．８ＫＤ和３６．１ＫＤ蛋白条带，Ｄ型缺少１０７．８ＫＤ和６６．６ＫＤ蛋白条带。在膜蛋白图谱中仅Ｂ型有９４．７ＫＤ蛋白条带但缺少５１ＫＤ蛋白条带，Ｃ型缺少７４．２ＫＤ蛋白条带，Ｄ型缺少３１ＫＤ蛋白条带，但比其它３个型多１个２４．３ＫＤ蛋白条带，且３     

Systemic hypertension in dogs with leishmaniasis: prevalence and clinical consequences

A prospective study was performed (November 1998 to December 2003) to determine the prevalence of systemic hypertension (SH) in dogs with glomerular disease secondary to leishmaniasis. One hundred and five dogs with leishmaniasis were screened and staged for the presence of renal disease (RD) and SH. For the purpose of the study, RD was defined as serum creatinine concentration > or = 1.4 mg/dL, a urine protein/creatinine ratio > or = 0.5, or both. SH was defined as a systolic blood pressure (SBP) > or =180 mm Hg or an SBP between 150 and 179 mm Hg in the presence of clinical manifestations of SH. Fifty-two (49.5%) of the dogs had some degree of RD, and 32 (61.5%) of these dogs were diagnosed with SH. Moreover, SH also was diagnosed in 3 dogs without RD. Left ventricular hypertrophy (LVH), estimated by echocardiography, was the most frequently observed systemic consequence of hypertension, being present in 32 (91.4%) of the hypertensive dogs. Echocardiographic abnormalities were not detected in any of the 33 dogs with leishmaniasis without RD, which were used as controls. Ocular consequences of SH were observed in only 2 (5.7%) of the dogs with hypertension. We conclude that SH is prevalent in dogs with RD secondary to leishmaniasis, not only in the more severe stages but also in the early course of the illness before azotemia becomes apparent. Canine leishmaniasis may be a useful natural model to study SH secondary to glomerular disease.     

Glomerular filtration rate in dogs with leishmaniasis and chronic kidney disease

BACKGROUND: Glomerular filtration rate (GFR) measurement is an indicator of kidney function. However, its usefulness in dogs at early stages of spontaneous chronic kidney disease (CKD) of glomerular origin, where routine laboratory techniques are not sufficiently sensitive, remains unproved. HYPOTHESIS: That GFR is reduced in proteinuric nonazotemic or mildly azotemic dogs with CKD secondary to leishmaniasis. ANIMALS: Twenty-six dogs with CKD secondary to leishmaniasis and 10 healthy dogs (control group). METHODS: CBC, serum biochemistry, and urinalysis (microalbuminuria and urine protein/creatinine ratio [UPC]) were performed in all dogs. GFR was calculated by measuring exogenous creatinine clearance. Based on degree of proteinuria and serum creatinine concentration (SCr), dogs were classified as group A (control; n = 10): UPC < 0.2, SCr < 1.4 mg/dL; group B (n = 8): UPC, 0.2-0.5, SCr < 1.4 mg/dL; group C (n = 10): UPC > 0.5, SCr < 1.4 mg/dL; group D (n = 5): SCr, 1.4-2 mg/dL; group E (n = 3): SCr > 2 mg/dL. Results: GFR (mL/kg/min) was 3.9 +/- 0.29, 4.4 +/- 0.74, 4.5 +/- 1.44, 2.8 +/- 0.97, and 1.5 +/- 0.43 for groups A, B, C, D, and E, respectively. Eleven dogs (1 from group B, 3 from group C, 4 from group D, and all 3 dogs from group E) had an abnormally low GFR. Four dogs from group B and 5 dogs from group C had a GFR above the upper reference range (>4.5 mL/min/kg). CONCLUSION AND CLINICAL RELEVANCE: Some proteinuric nonazotemic or mildly azotemic dogs with leishmaniasis have low GFR, but glomerular hyperfiltration occurs in other dogs.     

Use of allopurinol for maintenance of remission in dogs with leishmaniasis

Current treatments for infected dogs with leishmaniasis do not always provide long-term control of the disease and clinical relapses are common. In this study, the usefulness of long-term allopurinol administration in the maintenance of clinical remission in canine leishmaniasis was evaluated. Fifteen dogs with natural leishmania infection were subjected to an initial treatment based on the simultaneous administration of meglumine antimoniate (100 mg/kg/day) and allopurinol (30 mg/kg/day). Once clinical remission was achieved, a maintenance treatment with allopurinol (20 mg/kg/day) administered for one week a month was instituted. Results were compared with those of a retrospective control group comprising 15 infected dogs which only followed the induction treatment. Relapses occurred in 86 per cent of control dogs within 14 months of discontinuing treatment. In contrast, those dogs on intermittent oral allopurinol administration were successfully maintained in clinical remission for a follow-up period of 10 to 44 months. In this latter group, specific antibody titres decreased or were unchanged, no side effects directly attributable to allopurinol were seen and treatment was well accepted by the owners. It is concluded that long-term intermittent administration of allopurinol is an effective way of maintaining clinical remission in dogs with leishmaniasis.     

Clinical availability of urinary N-acetyl-beta-D-glucosaminidase index in dogs with urinary diseases

Urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) was examined in healthy dogs and dogs with urinary diseases, and its clinical usefulness as an indicator of urinary diseases was discussed. Twenty-eight healthy dogs and 20 dogs with urinary diseases were used. Urinary NAG activity was measured using p-nitrophenyl N-acetyl-beta-D-glucosaminide as substrate, and expressed as units per gram of urinary creatinine (NAG index). Urinary NAG index in urine of healthy dogs was 3.2+/-2.4 U/g, and NAG index in the dogs with chronic renal failure or lower urinary tract infection accompanied by pyelonephritis was higher than that in healthy dogs. However, the dogs with lower urinary tract infection without pyelonephritis showed normal values of NAG index. Some dogs with diabetic mellitus showed elevated values of NAG index when control of blood sugar was not successful. Increase of NAG index was observed in some dogs with pyometra before increases of BUN and serum creatinine concentration. Therefore, NAG index in urine seems to be a good indicator for urinary diseases in dogs.     

Plasma and urinary trypsinogen activation peptide in healthy dogs, dogs with pancreatitis and dogs with other systemic diseases

OBJECTIVE: To determine the specificity and sensitivity of plasma and urinary trypsinogen activation peptide (TAP) concentrations in diagnosing pancreatitis in dogs. DESIGN: Retrospective analysis of clinical cases. PROCEDURE: Dogs were classified into three groups: healthy animals, dogs with confirmed pancreatitis and dogs with nonpancreatic disease, which clinically or biochemically resembled pancreatitis. This last group was further subdivided into dogs with renal and those with nonrenal disease. The plasma and urinary TAP concentration was determined by a competitive enzyme immunoassay. Clinical cases additionally had serum trypsin-like immunoreactivity concentration measured, as well as radiography and ultrasound of the abdomen and further diagnostic procedures. Nonparametric analysis of variance (Kruskal-Wallis test) was performed using Statistix 4.0 program. RESULTS: There was a wide range of urinary TAP concentration in healthy dogs (mean 52.30 nmol/L, standard deviation 55.25) that made interpretation of urinary TAP concentrations difficult in the other groups. There was a narrow reference range for plasma TAP (mean 2.67 nmol/L, standard deviation 0.93). Plasma and urinary TAP concentrations, as well as urinary TAP to creatinine ratio, were all increased in dogs that died with necrotising pancreatitis. Values were not increased in mild, interstitial pancreatitis. Increased plasma TAP concentrations were also present in dogs with severe renal disease. CONCLUSION: Plasma TAP concentration is a good prognostic indicator in naturally occurring pancreatitis in dogs. The failure of TAP to increase in mild pancreatitis, and the increase present in severe renal disease, suggests its measurement has limited application as a sole diagnostic tool for canine pancreatitis. Further investigations are required in order to explain the large variability of urinary TAP concentration and the presence of circulating TAP in healthy dogs.