Vaccine effectiveness and use of collar impregnated with insecticide for reducing incidence of Leishmania infection in dogs in an endemic region for visceral leishmaniasis, in Brazil.

Although a national programme for control of visceral leishmaniosis (VL) is being run in Brazil, the disease continues to spread. This programme is essentially based on culling infected dogs from endemic regions. Thus, there is an urgent need to develop other control measures against VL to deter its advance. Here, a subunit vaccine, a recombinant vaccine, an insecticide-impregnated collar and the associations between these measures were evaluated for reducing the incidence of Leishmania infection in dogs. This was through a cohort study conducted in an endemic region of Brazil, considering the incidence and time of total exposure over a period of 1 year. The incidence of VL was estimated by means of serological and molecular diagnostic tests, 180 and 360 days after the application of the control measures. The estimates of the effectiveness (EF) were not significant in any cohort. The EF of the subunit vaccine, the recombinant vaccine and the collar were 26.4%, 32.8% and 57.7% and the upper limit of the 95% confidence interval for EF were 63.7%, 67.9% and 82.5%, respectively. In conclusion, under the conditions of this study, none of the immunogens for VL control was sufficiently effective to protect dogs against infection. On the other hand, use of collars impregnated with insecticide seems to constitute a method with better prognosis, corroborating other studies in this field.

Serological and molecular diagnostic tests for canine visceral leishmaniasis in Brazilian endemic area: one out of five seronegative dogs are infected.

Euthanasia of infected dogs is one of the measures adopted in Brazil to control visceral leishmaniasis (VL) in endemic areas. To detect infected dogs, animals are screened with the rapid test DPP® Visceral Canine Leishmaniasis for detection of antibodies against K26/K39 fusion antigens of amastigotes (DPP). DPP-positives are confirmed with an immunoenzymatic assay probing soluble antigens of promastigotes (ELISA), while DPP-negatives are considered free of infection. Here, 975 dogs from an endemic region were surveyed by using DPP, ELISA and real-time PCR (qPCR) for the diagnosis of VL. When DPP-negative dogs were tested by qPCR applied in blood and lymph node aspirates, 174/887 (19·6%) were positive in at least one sample. In a second sampling using 115 cases, the DPP-negative dogs were tested by qPCR in blood, lymph node and conjunctival swab samples, and 36/79 (45·6%) were positive in at least one sample. Low-to-moderate pairwise agreement was observed between all possible pair of tests. In conclusion, the official diagnosis of VL in dogs in Brazilian endemic areas failed to accuse an expressive number of infected animals and the impact of the low accuracy of serological tests in the success of euthanasia-based measure for VL control need to be assessed.

On-line identification of fermentation processes for ethanol production.

A strategy for monitoring fermentation processes, specifically, simultaneous saccharification and fermentation (SSF) of corn mash, was developed. The strategy covered the development and use of first principles, semimechanistic and unstructured process model based on major kinetic phenomena, along with mass and energy balances. The model was then used as a reference model within an identification procedure capable of running on-line. The on-line identification procedure consists on updating the reference model through the estimation of corrective parameters for certain reaction rates using the most recent process measurements. The strategy makes use of standard laboratory measurements for sugars quantification and in situ temperature and liquid level data. The model, along with the on-line identification procedure, has been tested against real industrial data and have been able to accurately predict the main variables of operational interest, i.e., state variables and its dynamics, and key process indicators. The results demonstrate that the strategy is capable of monitoring, in real time, this complex industrial biomass fermentation. This new tool provides a great support for decision-making and opens a new range of opportunities for industrial optimization.

Phenotypic and genotypic characterization of two Toxoplasma gondii isolates in free-range chickens from Uberlândia, Brazil.

The aim of this study was to determine the seroprevalence of Toxoplasma gondii infection in free-range chickens from Uberlândia, Minas Gerais state, Brazil, and characterize the genotypic and phenotypic features of two isolates of this parasite, considering the importance of these hosts in the epidemiology of toxoplasmosis. Serum samples from 108 free-range chickens were obtained from ten different districts, and submitted to the modified agglutination test (MAT) for the presence of anti-T. gondii antibodies, and brain and heart tissue samples from infected chickens were processed for mouse bioassay. An overall seroprevalence of 71·3% was found and antibody titres ranged from 16 to 4096. After confirmation of seropositivity by mouse bioassay, the determination of the T. gondii genotypes of two isolates was performed by PCR-RFLP, using primers for the following markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, new SAG2, Apico and CS3. These T. gondii isolates, designated TgChBrUD1and TgChBrUD2, were obtained from heart samples of free-range chickens. The TgChBrUD1 isolate belonged to ToxoDB PCR-RFLP genotype 11 and the TgChBrUD2 isolate belonged to ToxoDB PCR-RFLP genotype 6. Both isolates demonstrated high virulence in a rodent model, with the TgChBrUD1 isolate able to induce brain cysts, in accord with its pattern of multiplication rates in human fibroblast culture. Taken together, these results reveal high prevalence of T. gondii infection in free-range chickens throughout Uberlândia, indicating an important degree of oocyst environmental contamination and the existence of considerable risk for T. gondii transmission to humans by consumption of free-range chicken as a food source.

Evaluation of an Immunochromatographic Test to the Diagnosis of Canine Brucellosis Caused by Brucella canis.

This study evaluated the performance of an immunochromatographic test (ICT) for the diagnosis of canine brucellosis caused by Brucella canis, comparing its results with that of the rapid slide agglutination test with and without the use of 2-mercaptoethanol and the agar gel immunodiffusion test (AGID). The microbiological culture, PCR and clinical examination were used as reference. According to the results obtained in clinical examination, blood culture, culture of semen and vaginal swab and PCR in blood, semen and vaginal swab, a total of 102 dogs were divided into three groups: B. canis-infected dogs (Group 1), B. canis-non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The diagnostic sensitivity of RSAT, 2ME-RSAT, AGID and ICT in Group 1 was, respectively, 75%, 37.5%, 27.8% and 89.58%. The diagnostic specificity of RSAT, 2ME-RSAT, AGID and ICT in Group 2 was, respectively, 91%, 100%, 100%, and 100%. In dogs with suspected brucellosis, 9.67% were RSAT positive, none was positive by 2ME-RSAT, 3.22% were AGID positive and 6.45% were ICT positive. The main drawback concerning canine brucellosis diagnosis is the lack of a highly sensitive serological assay to be used as a screening test to the rapid identification of infected animals. The ICT showed a high diagnostic specificity and a diagnostic sensitivity value greater than that observed in the RSAT, 2ME-RSAT and AGID. However, 10.41% of infected dogs had negative results by ICT. These dogs were positive by microbiological culture and/or PCR, indicating active infection and consequently a higher potential of spreading Brucella. Although rapid and simple to perform, the ICT lacked sensitivity to be used as a screening test.

Betacoronavirus 1 in alpacas (Vicugna pacos) in the High Peruvian Andes.

Genetic sequences highly related to Bovine coronavirus (BCoV) were detected in fecal samples from Peruvian 1-3 week old alpaca crias located on six farms in Puno department, some of which shared pastures with cattle. A total of 60 samples were screened for coronavirus using a nested PCR amplification of a fragment of the RNA-dependent RNA polymerase (RdRp) gene. Sequences from 11 positive samples were highly similar to the Kakegawa, Quebec and Mebus BCoV strains (99.5-100.0%) and 99.2% identical to an alpaca Coronavirus (CoV) previously detected in the USA. The detection of genetic sequences related to BCoV from Peruvian alpaca crias suggests possible role of this virus on enteric disorders etiology in the High Andes.

In vivo genotoxicity of treated water containing the cylindrospermopsin-producer Cylindrospermopsis raciborskii.

Cylindrospermopsin (CYN) is an alkaloid commonly produced by some cyanobacteria that has been implicated in outbreaks of human illness. The aim of this study was to investigate the genotoxicity of Cylindrospermopsis raciborskii cellular content (including CYN) and its byproducts resulting from chlorination during water treatment. DNA damage in blood and liver cells was analysed by the comet assay and micronucleus test (MN). Mice were injected intraperitoneally with the following treatments: (a) physiological saline, (b) treated water, (c) treated water plus C. raciborskii extract (CYN producer strain, CYPO-011 K), (d) C. raciborskii extract (CYN producer strain, CYPO-011 K), (e) C. raciborskii extract (CYN non producer strain), and (f) treated water plus C. raciborskii extract (CYN non producer strain) extract. After 48 h, samples were taken to perform tests (blood and liver cells to the comet assay and bone marrow to MN test). The CYPO-011 K had a genotoxic and mutagenic effects on liver and bone marrow cells. The group that received chlorine-treated water plus CYPO-011 K also exhibited genotoxic effects in the liver, as well as in the blood, and a mutagenic effect in blood marrow cells. The results emphasise the need of improving CYN monitoring in waters bodies in order to reduce the risk of human exposure.

Bacterial septicemia and herpesvirus infection in Antarctic fur seals (Arctocephalus gazella) stranded in the São Paulo coast, Brazil.

In August 2021, two juvenile male Antarctic fur seals (Arctocephalus gazella) stranded in the southeastern Brazilian coast and were referred to rehabilitation centers. The animals presented increased body temperature, prostration, respiratory distress and despite treatment died. A necropsy following a standardized protocol was performed, and formalin-fixed tissues were processed for microscopic examination. Samples were screened for morbillivirus, herpesvirus, and Brucella spp. by molecular analyses (PCR, RT-PCR). Bacteriological culture was performed in samples collected from the lungs, trachea, and lymph nodes of both cases. The main histopathologic findings were of infectious nature, including multifocal necrotizing and fibrinous mixed interstitial pneumonia, bronchiolitis, and bronchitis, with intralesional myriad bacteria associated with vascular fibrinoid necrosis. Pseudomonas aeruginosa was isolated from tracheal and lung swabs of Case 1, and Klebsiella oxytoca was found in nostril swabs, tracheobronchial lymph nodes, and lung of Case 2. Gammaherpesvirus infection was detected in both cases, and the sequences retrieved were classified into the genus Percavirus. All tested samples were PCR-negative for Brucella spp. and morbillivirus. We hypothesize that the deficient immunological status in association with starvation predisposed the reactivation of herpesvirus and secondary bacterial co-infections. To the authors' knowledge, this is the first molecular detection of herpesvirus in an Antarctic pinniped. These findings reinforce that Otariid gammaherpesvirus circulating in the Southern Hemisphere are likely endemic in the Arctocephalus genus. This report contributes to the current knowledge of health aspects affecting wild pinnipeds, especially in the poorly studied Antarctic species.

Evaluation of the Joypex 5 and Root ZX II: an in vivo and ex vivo study.

AIM: To evaluate in vivo and ex vivo the accuracy of the multifrequency electronic root canal length measurement device (ERCLMD) Joypex 5 and compare it with the Root ZX II. The ex vivo methodology for evaluation of the accuracy of ERCLMDs was also validated by comparison with in vivo results. METHODOLOGY: Twenty single-rooted human teeth that were scheduled for extraction were selected. Working length measurements were performed in vivo with the Joypex 5 and Root ZX II. After the teeth were extracted, each canal was measured with the ERCLMDs ex vivo. Then, a size 15 K-file was used to determine the reference working length (RWL), which was established 0.5 mm from the major foramen by direct observation. The measurements of working length obtained with the different methods and ERCLMDs were compared by analysis of variance and Tukey's test at P < .05. Statistical correlations were also performed. RESULTS: No significant difference was observed between in vivo and ex vivo measurements or the different types of ERCLMDs. The Bland-Altman plot confirmed the agreement of different methods and ERCLMDs to measure canal length. The R(2) coefficient obtained in both situations, comparing the ERCLMDs, was close to 1, denoting a strong agreement between measurements obtained with the Joypex 5 and Root ZX II and between in vivo and ex vivo methodologies. CONCLUSION: The Joypex 5 and Root ZX II had similar accuracy in determining working length of root canals. The ex vivo methodology for evaluation of the accuracy of ERCLMDs was validated when compared with the in vivo results.

Curcumin acts synergistically with fluconazole to sensitize a clinical isolate of Candida albicans showing a MDR phenotype.

The primary objective of this work was to evaluate the capability of curcumin, a natural compound found in the Curcuma longa plant, to sensitize a clinical isolate of Candida albicans, which was found to have a high resistance to fluconazole. In addition, we assessed whether the resistance of this isolate was the result of the existence of efflux pumps, which could confer a multiple drug resistance phenotype. To evaluate azole resistance, we used the Clinical Laboratory Standard Institute (CLSI) MIC assays procedures with minor modifications. For evaluation of synergistic interaction of curcumin and fluconazole, checkerboard experiments were employed. Nile red and Rhodamine 6G accumulation assays were used to evaluate efflux pump activity. Curcumin was found to have a great capability to inhibit fluconazole resistance of the isolate of C. albicans. It was capable of restoring its sensitivity to this azole when used at 11 µM. Analysis with different azoles and the two indicated dyes showed that an efflux pump could be acting and contributing to the resistance of this isolate to fluconazole. The results suggest that a major facilitator superfamily (MFS) transporter might be involved in this process.

Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV-positive and HIV-negative isolates.

OBJECTIVE: This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records. METHODS: The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. RESULTS: Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. CONCLUSION: These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.

Cariogenic and erosive potential of the medication used by HIV-infected children: pH and sugar concentration.

OBJECTIVE: The aim of this work was to analyze pH and sugar concentration in seven antiretroviral and three antibacterial medications frequently prescribed to HIV infected paediatric patients. METHOD: Sugars (sucrose, glucose, lactose and fructose) and pH were measured from every one of ten medications with different serial numbers in two samples. The pH was determined by a previously calibrated digital pHmeter (Beckman). Analysis of free sugars was performed using thin-layer chromatography (TLC). The pH results and the amount of sugar originated from the two samples in each lot were added. The arithmetic mean of these results were computed. RESULTS: Two antiretrovirals (Zidovudin and Abacavir Sulphate) had pH below critical level (3.55 and 3.93, respectively). All three antibacterials analyzed had pH above 5.5, and one of them (Azithromycin) had the highest pH level of the ten medications examined (9.28). Sugar was present in seven out of 10 of the medications analyzed. The antibacterials contained the highest concentration of sucrose, ranging from 40% to 54%. Glucose was found in one of the ten, sucrose was present in seven of them and none showed lactose. Fructose was not observed with the technique used. CONCLUSIONS: A number of medications frequently used by HIV-infected children may cause a significant risk of both caries and dental erosion.

Histatin-5 induces the reversal of Pdr5p mediated fluconazole resistance in Saccharomyces cerevisae.

BACKGROUND: Candidiasis is a major opportunistic fungal infection in humans. The low number of antifungal drugs available to treat Candida infections and the increasing incidence of multidrug resistant (MDR) strains point to an urgent need of identifying new therapeutic options. The role of salivary components can provide insights for the development of new methodologies of control. OBJECTIVE: The aim of this study was to evaluate the ability of histatin-5, a constitutive immunological peptide present in saliva, in reversing fungal MDR phenotype, using a resistant Saccharomyces cerevisiae strain as model of study. RESULTS: A total of 2.5µg and 5µg of histatin-5 revealed to be able to chemosensitize (to revert antifungal resistance) a MDR strain to fluconazole impairing its intrinsic resistance. The presence of histatin-5 decreased the strain growth when associated to fluconazole, and also assisted in the retention of rhodamine 6G within cell cytoplasm. The ATPase activity of Pdr5p, an ABC efflux transporter, was significantly reduced up to 65% within physiological concentration of the peptide. CONCLUSION: Results revealed that histatin-5 is able to revert MDR phenotype and may be considered a potential alternative MDR inhibitor. Since Pdr5p is homologous to Candida albicans CaCdr1p and CaCdr2p, data obtained might be extrapolated to these transporters, inferring that associating fluconazole and histatin-5 may be a useful tool to circumvent failure treatments of infections caused by Candida MDR strains.

ß-lapachone and α-nor-lapachone modulate Candida albicans viability and virulence factors.

BACKGROUND: Candida albicans is the most important fungal pathogen that causes infections in humans, and the search for new therapeutic strategies for its treatment is essential. OBJECTIVE: The aim of this study was to evaluate the activity of seven naphthoquinones (ß-lapachone, ß-nor-lapachone, bromide-ß-lapachone, hydroxy-ß-lapachone, α-lapachone, α-nor-lapachone and α-xyloidone) on the growth of a fluconazole-resistant C. albicans oral clinical isolate and the effects of these compounds on the viability of mammalian cells, on yeast's morphogenesis, biofilm formation and cell wall mannoproteins availability. RESULTS: All the compounds were able to completely inhibit the yeast growth. ß-lapachone and α-nor-lapachone were the less cytotoxic compounds against L929 and RAW 264.7 cells. At IC50, ß-lapachone inhibited morphogenesis in 92%, while the treatment of yeast cells with α-nor-lapachone decreased yeast-to-hyphae transition in 42%. At 50µg/ml, ß-lapachone inhibited biofilm formation by 84%, whereas α-nor-lapachone reduced biofilm formation by 64%. The treatment of yeast cells with ß-lapachone decreased cell wall mannoproteins availability in 28.5%, while α-nor-lapachone was not able to interfere on this virulence factor. Taken together, data show that ß-lapachone and α-nor-lapachone exhibited in vitro cytotoxicity against a fluconazole-resistant C. albicans strain, thus demonstrating to be promising candidates to be used in the treatment of infections caused by this fungus.

Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil.

Neospora caninum was isolated from a naturally infected sheep from Brazil by bioassay in dogs. Approximately 70g of brain from each of two 4-month-old sheep with indirect fluorescent antibodies (>or=1:50) to N. caninum was offered to a different IFAT negative dog (Sheep n. 302, IFAT 1:400-Dog 1 and Sheep n. 342, IFAT 1:50-Dog 2). Parasite DNA was detected in both sheep brains using a PCR targeting the Nc-5 gene of N. caninum. Shedding of Neospora-like oocysts was noticed only in Dog 1, from 10 days post-inoculation (PI) to 25 days PI (a total of approximately 27,600 oocysts). Seventy days after infection, Dog 1 was euthanized and brain/cerebellum and medulla were collected and submitted to molecular methods, as were the oocysts, to confirm the identity of the isolate. Serum samples collected weekly from both dogs from the infection to the end of the experimental period had no antibodies anti-N. caninum by IFAT (<1:50). Oocysts, brain/cerebellum and medulla specimens of Dog 1 proved positive by a PCR assay targeting the Nc-5 gene of N. caninum. In addition, the oocysts have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid ITS1 sequence. The PCR products of ITS1 were sequenced, confirming again the isolate as N. caninum. Oocysts were also orally inoculated in two Swiss white mice two Mongolian gerbils (Meriones ungulatus) and two large vesper mice (Calomys callosus) (10(3)oocysts/animal). The rodents were sacrificed 2 months PI, and fresh preparations of brains showed Neospora thick-walled cysts in gerbil brains, but molecular detection using the Nc-5 PCR assay revealed DNA parasite in gerbil and also C. callosus brains. This is the first report of isolation and sequencing of N. caninum from a Brazilian sheep and the first report of molecular detection of N. caninum from C. callosus.

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs.

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. Of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of non-infected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen.

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches.

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.

Diagnosis of canine brucellosis: comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer.

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Low toxicity superparamagnetic magnetite nanoparticles: One-pot facile green synthesis for biological applications.

Superparamagnetic magnetite nanoparticles have been synthesized by a highly reproducible polyvinyl alcohol (PVA)-based modified sol-gel process using water as the only solvent. The synthesis method has proven to be effective, time and cost saving and environmental friendly, resulting in PVA-coated magnetite nanoparticles as direct product from the synthesis, without any special atmosphere or further thermal treatment. X-ray diffraction and transmission electron microscopy revealed that the biocompatible PVA-coating prevents the nanoparticle agglomeration, giving rise to spherical crystals with sizes of 6.8nm (as-cast) and 9.5nm (heat treated) with great control over size and shape with narrow size distribution. Complementary compositional and magnetic characterizations were employed in order to study the surface chemistry and magnetic behavior of the samples, respectively. Cytotoxicity endpoints including no observed adverse effect concentration (NOAEC), 50% lethal concentration (LC50) and total lethal concentration (TLC) of the tested materials on cell viability were determined after 3, 24 and 48h of exposure. The PVA coating improved the biocompatibility of the synthesized magnetite nanoparticles showing good cell viability and low cytotoxicity effects on the MTT assay performed on BHK cells. Preliminary assessment of nanoparticles in vivo effects, performed after 48h on Balb/c mice, exposed to a range of different sub-lethal doses, showed their capacity to penetrate in liver and kidneys with no significant morphological alterations in both organs.

Brucella canis infection in dogs from commercial breeding kennels in Brazil.

Canine brucellosis caused by Brucella canis is a neglected zoonosis worldwide and is a leading cause of reproductive failure in dogs, often causing substantial economic losses in breeding kennels. This study aimed to investigate the occurrence of B. canis infection in dogs of commercial breeding kennels located in São Paulo State, Brazil. A total of 753 dogs (183 males and 570 females) from 38 commercial kennels were clinically examined, and blood samples were collected for brucellosis diagnosis through blood culture. The association between clinical manifestations suggestive of brucellosis and positive results through blood culture was determined. Of the 753 dogs tested, 166 (22.0%) had at least one clinical sign suggestive of brucellosis and 158 (20.9%) had positive blood cultures. Seventy-two dogs had positive blood culture and had at least one clinical sign suggestive of brucellosis, while 91 dogs showed at least one clinical manifestation suggestive of brucellosis although blood culture was negative. Of the 38 kennels, 16 (42.1%) had at least one positive dog. The prevalence of infection in each kennel varied from 3.8% to 62.6%. Abortion/stillbirth, failure to conceive and enlargement of lymph nodes were significantly associated with brucellosis in female. No association of clinical signs and positive results in blood culture was observed in males. None of the kennels has been carrying out programmes to control brucellosis, and the sale of infected dogs was considered a common practice yielding risks to the public health, in view of the zoonotic potential of the infection.