Comparison of four polymerase chain reaction assays for the detection of Brucella spp. in clinical samples from dogs.

AIM: This study aimed to compare the sensitivity (S), specificity (Sp), and positive likelihood ratios (LR+) of four polymerase chain reaction (PCR) assays for the detection of Brucella spp. in dog's clinical samples. MATERIALS AND METHODS: A total of 595 samples of whole blood, urine, and genital fluids were evaluated between October 2014 and November 2016. To compare PCR assays, the gold standard was defined using a combination of different serological and microbiological test. Bacterial isolation from urine and blood cultures was carried out. Serological methods such as rapid slide agglutination test, indirect enzyme-linked immunosorbent assay, agar gel immunodiffusion test, and buffered plate antigen test were performed. Four genes were evaluated: (i) The gene coding for the BCSP31 protein, (ii) the ribosomal gene coding for the 16S-23S intergenic spacer region, (iii) the gene coding for porins omp2a/omp2b, and (iv) the gene coding for the insertion sequence IS711. RESULTS: The results obtained were as follows: (1) For the primers that amplify the gene coding for the BCSP31 protein: S: 45.64% (confidence interval [CI] 39.81-51.46), Sp: 95.62% (CI 93.13-98.12), and LR+: 10.43 (CI 6.04-18); (2) for the primers that amplify the ribosomal gene of the 16S-23S rDNA intergenic spacer region: S: 69.80% (CI 64.42-75.18), Sp: 95.62 % (CI 93.13-98.12), and LR+: 11.52 (CI 7.31-18.13); (3) for the primers that amplify the omp2a and omp2b genes: S: 39.26% (CI 33.55-44.97), Sp: 97.31% (CI 95.30-99.32), and LR+ 14.58 (CI 7.25-29.29); and (4) for the primers that amplify the insertion sequence IS711: S: 22.82% (CI 17.89 - 27.75), Sp: 99.66% (CI 98.84-100), and LR+ 67.77 (CI 9.47-484.89). CONCLUSION: We concluded that the gene coding for the 16S-23S rDNA intergenic spacer region was the one that best detected Brucella spp. in canine clinical samples.

Serum insulin, glucose and non esterified fatty acids after administration of follicle-stimulating and luteinizing hormones in bitches.

This paper reports the effect of the simultaneous administration of follicle-stimulating (FSH) and luteinizing hormones (LH) on serum glucose, insulin and nonesterified fatty acid responses after glucose or insulin challenge. The animals were originally at anestrous. FSH (dose 2.5 U/kg body wt.) and LH (0.27 U/kg body wt.) were s.c. injected on days 1, 4, 8 and 11. Vaginal smears were obtained daily. Six untreated controls at anestrous and six treated bitches reaching proestrous were used. Glucose tolerance tests were done with a dose of 1 g of glucose per kg of body weight. Bovine insulin was administered at the dose of 0.25 U/kg body wt. During these tests, neither serum glucose and nonesterified fatty acids nor glucose distribution space and glucose clearance were affected by the treatment. The serum insulin response to hyperglycemia was greatly increased. The distribution space and clearance rate of this hormone were not affected by FSH + LH treatment. We conclude that, in the bitch, FSH + LH treatment, at doses that trigger "sex seasons", increases the serum insulin response to glucose load and produces a moderate resistance to the hypoglycemic, lipogenic and antilipolytic insulin actions. These phenomena are evident during hyperglycemia.

Serum insulin, glucose and non esterified fatty acids after administration of follicle-stimulating and luteinizing hormones in bitches

This paper reports the effect of the simultaneous administration of follicle-stimulating (FSH) and luteinizing hormones (LH) on serum glucose, insulin and nonesterified fatty acid responses after glucose or insulin challenge. The animals were originally at anestrous. FSH (dose 2.5 U/kg body wt.) and LH (0.27 U/kg body wt.) were s.c. injected on days 1, 4, 8 and 11. Vaginal smears were obtained daily. Six untreated controls at anestrous and six treated bitches reaching proestrous were used. Glucose tolerance tests were done with a dose of 1 g of glucose per kg of body weight. Bovine insulin was administered at the dose of 0.25 U/kg body wt. During these tests, neither serum glucose and nonesterified fatty acids nor glucose distribution space and glucose clearance were affected by the treatment. The serum insulin response to hyperglycemia was greatly increased. The distribution space and clearance rate of this hormone were not affected by FSH + LH treatment. We conclude that, in the bitch, FSH + LH treatment, at doses that trigger "sex seasons", increases the serum insulin response to glucose load and produces a moderate resistance to the hypoglycemic, lipogenic and antilipolytic insulin actions. These phenomena are evident during hyperglycemia

Serum insulin, glucose and non esterified fatty acids after administration of follicle-stimulating and luteinizing hormones in bitches.

This paper reports the effect of the simultaneous administration of follicle-stimulating (FSH) and luteinizing hormones (LH) on serum glucose, insulin and nonesterified fatty acid responses after glucose or insulin challenge. The animals were originally at anestrous. FSH (dose 2.5 U/kg body wt.) and LH (0.27 U/kg body wt.) were s.c. injected on days 1, 4, 8 and 11. Vaginal smears were obtained daily. Six untreated controls at anestrous and six treated bitches reaching proestrous were used. Glucose tolerance tests were done with a dose of 1 g of glucose per kg of body weight. Bovine insulin was administered at the dose of 0.25 U/kg body wt. During these tests, neither serum glucose and nonesterified fatty acids nor glucose distribution space and glucose clearance were affected by the treatment. The serum insulin response to hyperglycemia was greatly increased. The distribution space and clearance rate of this hormone were not affected by FSH + LH treatment. We conclude that, in the bitch, FSH + LH treatment, at doses that trigger [quot ]sex seasons[quot ], increases the serum insulin response to glucose load and produces a moderate resistance to the hypoglycemic, lipogenic and antilipolytic insulin actions. These phenomena are evident during hyperglycemia.

Serum insulin, glucose and non esterified fatty acids after administration of follicle-stimulating and luteinizing hormones in bitches

This paper reports the effect of the simultaneous administration of follicle-stimulating (FSH) and luteinizing hormones (LH) on serum glucose, insulin and nonesterified fatty acid responses after glucose or insulin challenge. The animals were originally at anestrous. FSH (dose 2.5 U/kg body wt.) and LH (0.27 U/kg body wt.) were s.c. injected on days 1, 4, 8 and 11. Vaginal smears were obtained daily. Six untreated controls at anestrous and six treated bitches reaching proestrous were used. Glucose tolerance tests were done with a dose of 1 g of glucose per kg of body weight. Bovine insulin was administered at the dose of 0.25 U/kg body wt. During these tests, neither serum glucose and nonesterified fatty acids nor glucose distribution space and glucose clearance were affected by the treatment. The serum insulin response to hyperglycemia was greatly increased. The distribution space and clearance rate of this hormone were not affected by FSH + LH treatment. We conclude that, in the bitch, FSH + LH treatment, at doses that trigger "sex seasons", increases the serum insulin response to glucose load and produces a moderate resistance to the hypoglycemic, lipogenic and antilipolytic insulin actions. These phenomena are evident during hyperglycemia (AU)

Comparative performance of tests using cytosolic or outer membrane antigens of Brucella for the serodiagnosis of canine brucellosis.

Although some ELISA tests using cytoplasmic or outer membrane antigens of Brucella have been developed to improve the diagnosis of canine brucellosis, the performance of these assays has not been compared. In the present study three ELISA tests using lipopolysaccharide (LPS)-free cytoplasmic proteins (CPs) of Brucella abortus, the lumazine synthase (LS) of Brucella spp. or a hot-saline (HS) extract of Brucella canis containing outer membrane antigens were used to test sera from dogs with suspected or confirmed brucellosis (n=36) and from dogs with pathological conditions other than brucellosis (n=212). In the first group the proportion of positive results was 92, 92 and 81% for the ELISAs with HS, CP and LS, respectively, and 94% of the samples were positive by at least one ELISA test. Three dogs that were negative by agglutination (2ME-RSAT) had a positive result by at least one ELISA, and this discrepancy was attributed to the lower analytical sensitivity of agglutination tests. This hypothesis was confirmed by a serological follow-up of seven dogs recently infected with B. canis in three of which the illness was diagnosed earlier by one or more ELISA tests than by 2ME-RSAT. Among dogs having pathological conditions other than brucellosis, specificities were 94.3, 96.7 and 96.7% for the ELISAs with HS, CP and LS, respectively. This study shows that HS-ELISA and CP-ELISA are highly specific and sensitive for the diagnosis of canine brucellosis and can detect the infection by B. canis shortly after the exposure to the pathogen.

Natural estrous cycle in normal and diabetic bitches. Basal serum total lipids and cholesterol. Serum triglycerides profiles during glucose and insulin tests

All mean basal serum, total, cholesterol and lipids (L) levels in both fasted, normal bitches and in bitches with natural diabetes mellitus (DM) at anestrous (A) and during estrous cycle were measured. Mean serum, total triglycerides (TG) concentration in these animals at the same sex, stages, fasted and during intravenous glucose (IVGTT) and insulin (ITT) tolerance tests, were studied. In normal and in diabetic bitches serum cholesterol mean basal level differed significantly; the occurrence of estrous cycles (either phase) failed to affect these levels; DM and estrous cycles did not interact significantly. As for L, the influences of group and phase of estrous cycle on this variable significantly interacted. DM raised the mean basal level of this variable, in the normal group, "sex seasons" occurrence did not affect it whereas in the diabetic animals "in seasons" (either phase) it was above as compared with that found in respective controls at A. Estrogenic and luteal phases (EP, LP) did not differ in this concern. DM raised the mean serum TG levels in the bitches in the fasting condition and also during both tests; sex cycles action is variable. During IVGTT and ITT, the mean serum TG levels were influenced by sex stages and also by time elapsed either from glucose or insulin load. Thus, in the normal group, sex cycling did not vary significantly the TG profile during IVGTT. In the normal bitches "in season" (either phase), serum TG profile at the end of ITT increased more intensely than in the dogs at sex rest. During IVGTT, in the diabetic bitches, this profile was below base line from 15 min after glucose load till the test was over. DM intensely increased the serum TG response to insulin load in the bitches at A whereas such response was moderately decreasing at the end of ITT in the diabetic bitches at LP. All these results are discussed on the bases of the current know ledge on action of endocrine and metabolic products on these variables in normal animals, and the unability of these products to explain themselves the acute, severe, diabetic chryses observed during the LP of estral cycle in diabetic bitches or even in certain normal dogs at this moment of their "season", when diabetic outset uses to occur.

Natural estrous cycle in normal and diabetic bitches. Basal serum total lipids and cholesterol. Serum triglycerides profiles during glucose and insulin tests

All mean basal serum, total, cholesterol and lipids (L) levels in both fasted, normal bitches and in bitches with natural diabetes mellitus (DM) at anestrous (A) and during estrous cycle were measured. Mean serum, total triglycerides (TG) concentration in these animals at the same sex, stages, fasted and during intravenous glucose (IVGTT) and insulin (ITT) tolerance tests, were studied. In normal and in diabetic bitches serum cholesterol mean basal level differed significantly; the occurrence of estrous cycles (either phase) failed to affect these levels; DM and estrous cycles did not interact significantly. As for L, the influences of group and phase of estrous cycle on this variable significantly interacted. DM raised the mean basal level of this variable, in the normal group, "sex seasons" occurrence did not affect it whereas in the diabetic animals "in seasons" (either phase) it was above as compared with that found in respective controls at A. Estrogenic and luteal phases (EP, LP) did not differ in this concern. DM raised the mean serum TG levels in the bitches in the fasting condition and also during both tests; sex cycles action is variable. During IVGTT and ITT, the mean serum TG levels were influenced by sex stages and also by time elapsed either from glucose or insulin load. Thus, in the normal group, sex cycling did not vary significantly the TG profile during IVGTT. In the normal bitches "in season" (either phase), serum TG profile at the end of ITT increased more intensely than in the dogs at sex rest. During IVGTT, in the diabetic bitches, this profile was below base line from 15 min after glucose load till the test was over. DM intensely increased the serum TG response to insulin load in the bitches at A whereas such response was moderately decreasing at the end of ITT in the diabetic bitches at LP. All these results are discussed on the bases of the current know ledge on action of endocrine and metabolic products on these variables in normal animals, and the unability of these products to explain themselves the acute, severe, diabetic chryses observed during the LP of estral cycle in diabetic bitches or even in certain normal dogs at this moment of their "season", when diabetic outset uses to occur. (AU)

Natural estrous cycle in normal and diabetic bitches in relation to glucose and insulin tests

The influence of spontaneous "sex seasons" on blood sugar (BS) and serum insulin levels was studied in bitches with natural diabetes mellitus (DM) and normal controls, in the basal condition and during glucose and insulin tests, was studied. DM increased basal BS, reduced glucose tolerance, distribution space (DS) and clearance from blood, and induced resistance to insulin hypoglycemic action. In normals, occurrence of "seasons", inconsistently modified basal BS, increased glucose tolerance and DS; during estrogenic phase (EP), these variables were above those during luteal phase (LP). In diabetics at LP, BS found in fasting condition and during glucose test were higher than in diabetic biches at EP (respective values at anestrous (A) in between) and glucose DS was smaller. Rate of glucose clearance from blood remained unaffected by "seasons" in both dog groups. Basal serum IRI was not modified by DM or "seasons". In normals, serum IRI response to glucose load was nonsignificant during A and increased during the "seasons"; either insulin DS or the rate of insulin clearance from blood stream remained unchanged under the circumstances the increase being mediated by insulin secretion. During EP, the increase was particulary intense and mean insulinogenic index (MII) rose. During LP, MII returned to A value, whereby diabetic states might be manifest. Serum IRI profiles during insulin test were not modified by "seasons" in normal bitches; such response in diabetic bitches was intense during A, then decreased (EP) or was later abolished (LP). Either in normal or diabetic bitches, the sensitivity to exogenous insulin hypoglycemic action remained unchanged in spite of "seasons". In diabetic bitches at A, serum IRI after glucose challenge peaked higher than in respective normal controls (insulin clearance and insulin DS were similar): They exhibited relative insulin shortage and resistance to insulin hypolgycemic action partly compensated by prometed insulin secretion. Along with "season", abolished serum IRI response to glucose load in diabetics was observed. During EP, extrapancreatic factors regulating serum IRI concentration and MII did not change in respect to A, whereby abolishment appears mediated by depressed insulin secretion. During LP, insulin anatogonism in conjunction with 1) absolute insulin deficiency and 2) intense decrease in MII appears as a powerful factor exposing diabetic bitches to a severe or fatal derangement in diabetic disease.

Natural estrous cycle in normal and diabetic bitches in relation to glucose and insulin tests

The influence of spontaneous "sex seasons" on blood sugar (BS) and serum insulin levels was studied in bitches with natural diabetes mellitus (DM) and normal controls, in the basal condition and during glucose and insulin tests, was studied. DM increased basal BS, reduced glucose tolerance, distribution space (DS) and clearance from blood, and induced resistance to insulin hypoglycemic action. In normals, occurrence of "seasons", inconsistently modified basal BS, increased glucose tolerance and DS; during estrogenic phase (EP), these variables were above those during luteal phase (LP). In diabetics at LP, BS found in fasting condition and during glucose test were higher than in diabetic biches at EP (respective values at anestrous (A) in between) and glucose DS was smaller. Rate of glucose clearance from blood remained unaffected by "seasons" in both dog groups. Basal serum IRI was not modified by DM or "seasons". In normals, serum IRI response to glucose load was nonsignificant during A and increased during the "seasons"; either insulin DS or the rate of insulin clearance from blood stream remained unchanged under the circumstances the increase being mediated by insulin secretion. During EP, the increase was particulary intense and mean insulinogenic index (MII) rose. During LP, MII returned to A value, whereby diabetic states might be manifest. Serum IRI profiles during insulin test were not modified by "seasons" in normal bitches; such response in diabetic bitches was intense during A, then decreased (EP) or was later abolished (LP). Either in normal or diabetic bitches, the sensitivity to exogenous insulin hypoglycemic action remained unchanged in spite of "seasons". In diabetic bitches at A, serum IRI after glucose challenge peaked higher than in respective normal controls (insulin clearance and insulin DS were similar): They exhibited relative insulin shortage and resistance to insulin hypolgycemic action partly compensated by prometed insulin secretion. Along with "season", abolished serum IRI response to glucose load in diabetics was observed. During EP, extrapancreatic factors regulating serum IRI concentration and MII did not change in respect to A, whereby abolishment appears mediated by depressed insulin secretion. During LP, insulin anatogonism in conjunction with 1) absolute insulin deficiency and 2) intense decrease in MII appears as a powerful factor exposing diabetic bitches to a severe or fatal derangement in diabetic disease. (AU)