Silvia Cristina Osaki

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

INTRODUCTION Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. METHODS First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. RESULTS In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. CONCLUSIONS The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

INTRODUCTION The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

Neospora caninum in properties in the west region of Paraná, Brazil: prevalence and risk factors

Neospora caninum is a heteroxenous protozoa, whose definitive hosts are canids and intermediate hosts are herbivores, and is of great importance in cattle. The objectives of this study were to determine the prevalence of N. caninum in dairy cattle and dogs, to detect the presence of the protozoa at the molecular level in aborted fetuses, and to identify the risk factors associated with infection in properties in the western region of the state of Paraná. For this study, 600 bovine serum samples from 60 properties, 163 canine serum samples from 52 properties and 17 bovine fetuses from nine properties were collected. Data were collected using an epidemiological questionnaire to verify the risk factors. Serum samples were analyzed using the indirect fluorescent antibody test. Fetal tissues were analyzed using polymerase chain reaction and subsequent DNA sequencing. Of the bovine samples, 23.67% were positive for N. caninum. Among the canine samples, 11.66% were positive for N. caninum. Risk factors in cattle were history of abortion, low milk production, extensive breeding, and Jersey breed (p<0.05). Protozoan DNA was detected in 52.94% of the 17 fetuses and the sequencing presented high similarity with N. caninum.

Antimicrobial Resistance Phenotypic Profile of Isolates from Clinical Infections in Dogs

Background: Antimicrobial resistance is described as a condition in which a micro-organism is able to survive when exposed to an antimicrobial agent. The resistance rates to antimicrobials in companion animals have risen considerably. Studies of local antimicrobial susceptibility profiles are needed as well as education and warning about the use of tests for the identification and susceptibility of pathogenic bacterial strains. The aim of this study was to identify the main antimicrobial resistance in clinical samples of dogs, and to detect multidrug-resistant strains of importance to public health. Materials, Methods & Results: Bacterial pathogens of 77 dog infections were isolated and their sensitivity profile to antimicrobials was determined. One hundred bacterial isolates were identified. Of these, 61 were Gram-positive (55 Staphylococcus spp., 4 Enterococcus spp. and 2 Streptococcus spp.) and 39 Gram-negative (36 fermenters and 3 non-fermenters). Seventy-nine isolates were considered multiresistant following individual assessment of drugs, and 85 following the evaluation of classes. Only 3 were sensitive to all drugs. Four isolates were resistant to all classes and only sensitive to some antibiotics. Of the 55 samples of Staphylococcus spp., 36 (65.45%) were identified as phenotypically MRS. Two isolates of Enterococcus spp. were resistant to vancomycin (VRE). Also 66.67% (26/39) of the samples were positive for the presumptive test for ESBL. For the MRS-positive isolates detected in this study, chloramphenicol was the antimicrobial that showed superior sensitivity in 74.29% of the cases (27/36); therefore it is considered the most appropriate for treatment of this type of micro-organism. In case of aminoglycosides, when their resistance was checked in MRS isolates, all resistance percentages increased, implying a limited use of this class for such a type of multi-resistant micro-organism. Contrarily, in case of ESBL, a superior sensitivity was observed towards MRS isolates, thus making them a prime treatment choice for the infection caused by these micro-organisms. Discussion: Literature have reported a gradual increase in multidrug resistance towards antimicrobial agents in veterinary medicine over the past decades. In this study, 64% of multiresistant strains were considered of significant importance, notably MRS (36), VRE (2) and ESBL (26). The early identification of pathogens in animals has become an important step in order to minimize the transmission of antibacterial resistance. The increase in the number of multidrug-resistant bacteria in animals and humans demonstrates the need to develop and implement measures in order to monitor and control the spread of this resistance. It is possible that the increased drug resistance is linked to the constant exposure to these drugs and the subsequent selective pressure, causing the transfer of resistant genes between strains. Carbapenems and glycopeptides should be used with caution in veterinary medicine in order to prevent such processes of selection that develop resistance in micro-organisms to these two classes, which can result in cross-resistance between animals and humans and create obstacles in the treatment of patients, especially for the two drugs mentioned, since they are important for the treatment of nosocomial infections in humans. The resistance percentage towards fluoroquinolones was identified to be higher in Gram-positive isolates, particularly in MRS, which showed 75% resistance against this class (according to the CLSI, resistance to one fluoroquinolone antimicrobial agent provides resistance to other antimicrobials of this class). For ESBL isolates, the resistance was shown to be 50%. The resistance towards the fluoroquinolones and aminoglycosides class can be associated with the expression of the genes that produce ESBL.

Occurrence of anti-Neospora caninum antibodies in beef cattle of microrregion of Guarapuava, Paraná State, Brazil

Occurrence of anti-Neospora caninum antibodies in blood samples of 250 bovine beef cattle of the microrregion of Guarapuava, Parana State was verified by Indirect Fluorescent Antibody Test (IFAT) (> 1:200) and correlated to age, sex and breed of animals. The statistical analysis was carried out through Fishers Exact and qui-square tests (p < 0.05) to associate the results of the serology with the analyzed variables. From 250 evaluated samples, 33 (13.2 %) were positive for N. caninum. The titles obtained for N. caninum were 1:200 (8), 1:400 (14) and 1:800 (11). Seropositives animals were present in 40% (10/25) of the evaluated properties. These results demonstrate wide distribution of the protozoa among the beef cattle in the region of Guarapuava, PR. Animals without defined breed showed higher rates of seropositives for N. caninum (P = 0.002). The age had positive association with the incidence of antibodies against N. caninum (P = 0.02), indicating that horizontal transmission plays an important role in the epidemiology of this coccidia.