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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 IS 711 ., 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 IS 711 : 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.

VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The hypo-osmotic (HOS) test has been used in other species as an indicator of the fertilising capacity of spermatozoa. The aims of this study were to assess the response of domestic cat spermatozoa to the hypo-osmotic test, to determine the type of solution, concentration and time of incubation needed to obtain a maximum percentage of swelling, to correlate the selected combination with the percentages of progressive motility and to evaluate whether dilution of the ejaculate alters the results. Incubation for 30 and 45 min in solutions of fructose and of citrate of 50 and 100 mOsmol kg⁻¹ was evaluated. The highest percentage of swelling was obtained using the 50 mOsmol kg⁻¹ solution, and no significant differences were observed between the times of exposure to the solutions. A positive correlation was observed between the percentage of individual progressive motility and the percentage of sperm swelling in a 50 mOsmol kg⁻¹ fructose solution, with no significant differences being observed between raw and diluted semen samples. The results of this study suggest that the HOS test could be useful for evaluating membrane function in domestic cat spermatozoa, both in raw semen and in samples diluted in the EZ Mixin® commercial extender, and thus could be incorporated into routine semen evaluation protocols., (© 2012 Blackwell Verlag GmbH.)

To date, no totally effective antibiotic for the eradication of canine brucellosis has been found. The purpose of this study was to evaluate the efficacy of enrofloxacin in a kennel infected with Brucella canis. Twelve dogs, 2 males and 10 females (including 1 in estrus, 3 pregnant, and 6 in anestrus) infected with B. canis were given 5 mg/kg of enrofloxacin orally every 12 h for 30 days. Females received additional courses of enrofloxacin during the estral and luteal phases of the subsequent cycles (0-2 cycles). They were repeatedly mated by infected males. A serological follow-up was carried out for 38 months. The clinical, serological and bacteriological findings were recorded. In a trial carried out 14 months after the beginning of this study, all dogs were negative on the Rapid Slide Agglutination Test (RSAT). No abortions were observed. All mated female dogs conceived and gave birth to healthy puppies. Cultures of postpartum vaginal discharges (lochia) were negative for B. canis. Similar to other treatments, although enrofloxacin was not completely efficacious in treating canine brucellosis, it maintained fertility and avoided the recurrence of abortions, transmission of the disease to the puppies and dissemination of microorganisms during parturition. We inferred that enrofloxacin could be used as an alternative drug for the treatment of canine brucellosis.

The purpose of this study was to investigate if the suppression of estrus by the administration of a synthetic progestin, megestrol acetate or clormadinone acetate, could be an effective treatment to infertility in bitches with shortened interestrus periods and previous infertility. Ten bitches of different breeds and ages, with history of infertility and presenting repeated interestrus intervals of less than 4 months, were treated daily either with megestrol acetate (2 mg/kg, n = 8) or clormadinone acetate (0.5 mg/kg, n = 2) orally for 8 days. The treatments were begun within a maximum of 3 days after the onset of clinical signs of proestrus. Estrus was prevented in all animals and appearance of the following proestrus cycle was observed within 2.7 +/- 0.6 months (mean +/- S.D.) after the beginning of the treatment. When mated during the first post-treatment estrous cycle, bitches became pregnant and whelped normal healthy offspring. No negative side effects were clinically detected over the study period. Our results show that, in bitches with shortened interestrus intervals and previous infertility, suppression of one estrus with synthetic progestins administered at recommended doses, allows fertile breedings on the subsequent cycle, producing litter sizes within the normal range.

This review discusses the prevalence, etiology, pathogenesis, clinical findings, diagnostic methods, therapy, management and public health considerations of Brucella canis infection in dogs. Canine brucellosis is a contagious infection produced by a gram-negative coccobacilus called Brucella canis. The main sources of infection are vaginal fluids of infected females and urine in males. Routes of entry are venereal, oronasal, conjunctivae mucosa and placenta. The most significant symptoms are late abortions in bitches, epididymitis in males and infertility in both sexes, as well as generalized lymphadenitis, discospondylitis and uveitis. Diagnosis is complex because serology can give false positive results and chronic cases can give negative results, needing to be complemented with bacteriological studies. No antibiotic treatment is 100% effective and the infection often recurs in animals apparently treated successfully. Infected animals must be removed from the kennels and no longer used for breeding. Preferably, males should be castrated and females spayed. Human contagion is not frequent, although it has been reported, and is easily treated.

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.

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.

The detection of fetal pathology is important for the care of puppies in the postnatal period. The objective of this study was to identify parameters of fetal distress by determining fetal heart rate and bowel movements, and also to detect early pathology by means of fetal biometry, which involves determination of the biparietal and abdominal diameters. Ultrasonographic examinations were carried out on 30 pregnant bitches of different breeds and ages at least three times, including examination at days 40-47, days 48-57, and days 58-62 after coitus, and subsequently every 12-14 h for 3 days before parturition. Fetuses that had biparietal:abdominal diameter ratios of < 2 at the second and third series of ultrasonography, or after birth were considered to have intrauterine growth retardation. The average weight of each litter was calculated and puppies that weighed < 20% of the average weight were also considered to have experienced intrauterine growth retardation. Intrauterine growth retardation occurred in 39.6% of the bitches studied. Fetuses were considered to be normal when heart rate, determined by a Doppler flowmeter was > 220 beats min-1; suffering from slight fetal distress when heart rate was between 180 and 220 beats min-1; suffering from severe fetal distress when heart rate was < 180 beats min-1. Some level of distress was observed in 33.0% of puppies. In 86.7% of bitches, bowel movements were observed in at least one puppy, and appeared to be correlated with fetal distress. Bowel movements were observed in all of the puppies that had severe fetal distress (heart rate < 180 beats min-1), whereas bowel movements were observed in only 40.0% of puppies with slight fetal distress (heart rate 180-200 beats min-1). In conclusion, ultrasonographic evaluation allowed identification of fetuses with intrauterine growth retardation, and bowel movements were a reliable indicator of fetal distress; these conditions are presumed to indicate a greater perinatal risk.

Actions and interactions of spontaneous diabetes mellitus (DM) and natural estrous cycles (sex seasons) on the regulation of serum nonesterified fatty acids (NEFAs) and free glycerol (FG) levels in bitches in the fasting condition and during i.v. glucose (IVGTT) and insulin (ITT) tolerance tests, were studied. DM increased serum NEFAs concentration both in the basal condition and during IVGTT; it provoked a fall response to glucose load which is absent in normal controls. Estrous cycles did not modify these observations. Serum NEFAs levels during ITT were unresponsive in normal and diabetic bitches at every sex stage; flat, overlapped serum NEFAs profiles were then observed except for the diabetic group at A, which showed an early abrupt fall response of this variable from its high base line. DM increased also serum FG concentration in the fasting condition and during IVGTT. In the normal controls, serum FG base line was not affected by sex status; similarly shaped, increasing, overlapped curves during the test were observed. In the diabetic bitches "in season" (either phase), serum FG basal value was hardly above in respect to anestrous, but during IVGTT their flat profiles coincided. DM increased serum FG concentration in the basal condition and during ITT, and modified the profiles of this variable. In normal dogs in the basal condition, serum FG concentration remained unaffected by sex status; this variable hard, transiently increased during ITT, which was not influenced by "sex seasons"; therefore, similarly shaped, overlapped serum FG profiles were then observed. In the normal and diabetic bitches, serum-FG base line was not changed by "sex seasons". During ITT, serum FG mean profile in the diabetic bitches at EP was modestly above that observed in those at LP; differences for any other comparisons in normals or diabetic bitches were nonsignificant. As reported by us elsewhere, impaired glucose metabolism and absolute insulin deficiency induced ketose-prone, acidotic, insulin-dependent diabetic chryses in certain normal and diabetic beaches "in season" studied here. The unability of these animals for hydrolizing glyceride-glycerol via lipoproteinlipase (IVGTT) or via hormone sensitive fractions of lipase (ITT) and the abolished serum NEFAs suppressibility during modest hiperinsulinemia (ITT) appear to contribute to the production of such chryses. Results are discussed on the basis of interactions of serum NEFAs and FG with respective blood sugar and serum immunoreactive insulin levels as influenced by DM and estrous cycle.

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 cycle 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 knowledge 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.

An antigenic capture ELISA was developed to measure serum antibodies against an 18 kDa cytoplasmic protein of Brucella. This assay was used to detect anti-18 kDa reactivity in sera from 30 dogs having confirmed or suspected brucellosis. Antibodies against the 18 kDa protein were found in 26 of them, which were also positive by the slide agglutination test (2ME-RSAT). The overall correlation (positive and negative results) between the ELISA and 2ME-RSAT tests was 93.3%. Additionally, these sera were assayed by an indirect ELISA using a whole extract of cytoplasmic proteins of B. abortus (LPS-free CYT). The results of both ELISAs were coincident in 28 of 30 (93.3%) dogs having confirmed or suspected brucellosis. When a serological follow-up was performed on some dogs having confirmed brucellosis, antibody titers measured by both ELISAs showed a parallel progression. On the other hand, the capture ELISA showed good specificity, since a positive result was obtained only in 2 of 103 sera from healthy dogs. These preliminary results show that the ELISA for detecting serum antibodies against the 18 kDa cytoplasmic protein of Brucella could be useful for the diagnosis of canine brucellosis. This study also shows that the results obtained with this single protein of Brucella are equivalent to those obtained with the whole extract of cytoplasmic proteins.

Human menopausal gonadotropin (hMG) was administered intramuscularly to 10 bitches during apparently normal anestrus (n = 7) or persistent anestrus (n = 3). Each dog received a 75-IU dose of hMG (75 IU LH and 75 IU FSH; 1 to 7 units/kg) daily for nine days. Nine bitches responded with obvious signs of proestrus within 3 to 9 days. Of these, 3 bitches exhibited a weak proestrus while 2 exhibited a normal estrus and ovulation but failed to become pregnant The remaining 4 bitches became pregnant at the induced cycle and produced normal litters at 72 to 85 d after the start of treatment, including 1 bitch that had been treated at 24 mo after the last estrus. In 2 cases, treatment resulted in ovulation following 25 or 34 mo of chronic pubertal anestrus, 1 of which became pregnant. The results suggest that hMG can be a useful gonadotropin preparation for inducing estrus in dogs.

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 lasting condition and during glucose test were higher than in diabetic bitches 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 particularly 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 hypoglycemic action partly compensated by promoted 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 antagonism 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.

A preparation of Brucella abortus cytoplasmic proteins was depleted of lipopolysaccharide (LPS) by immunoadsorption with a monoclonal antibody (BC68) specific for the O antigen of B. abortus smooth LPS. This extract was used as antigen in an indirect ELISA for measuring antiprotein humoral immune response in dogs suffering from brucellosis and in healthy controls. All of the affected dogs studied showed IgG antiprotein response, while 2% (2 of 103) of the healthy dogs used as controls gave a positive result. All sera found to be positive by ELISA were also positive by the rapid slide agglutination test. These preliminary results show that the ELISA using B. abortus cytoplasmic proteins could be useful for the specific diagnosis of canine brucellosis.