Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor.

Sera from 112 mares from 5 horse-breeding farms was examined for the presence of antibodies to Neospora caninum and Toxoplasma gondii by an indirect fluorescent antibody test (IFAT), as well as from dogs and cattle present on these properties for the presence of antibodies to N. caninum. Among the 112 mares, 35 had a history of reproductive problems in the last breeding season and 77 had no reproductive problems. The rates of seroprevalence of N. caninum in mares with and without a history of reproductive problems were 25.71% and 6.49% and from T. gondii 2.85% and 1.29%, respectively. In dogs and cattle, the rates of seroprevalence of N. caninum were 10.52% and 15.55%, respectively. A positive correlation was found between the presence of antibodies against N. caninum (p=0.010) in mares and the occurrence of reproductive problems using the Fisher's exact test. Significantly higher seroprevalence for N. caninum in mares was observed on the farm that had seropositive dogs (p=0.018). Cattle on this farm were also seropositive. No significant differences in seropositivity were found on farms where dogs were seronegative, or absent. This result suggests, for the first time, the presence of seropositive dogs as a risk factor for N. caninum in mares and the necessity for further investigation of the epidemiology of this parasite in horse-breeding farms with reproductive problems and the presence of cattle and dogs. This is the first report on the occurrence of antibodies against N. caninum in horses from the state of Santa Catarina, Brazil.

A proposal for an alternative quality control test procedure for inactivated vaccines against food-and-mouth disease virus.

Foot-and-mouth disease (FMD) control in Brazil includes a strict mandatory vaccination program with vaccines produced in certified laboratories subject to inspection by the Brazilian Ministry of Agriculture, Livestock, and Food Supply (MAPA). The FMD vaccine's potency is tested through antibodies titration against structural viral proteins in sera from cattle that have not had any exposure to food-and-mouth disease virus (FMDV), at 28 days post-vaccination. Biological product testing using large animals is expensive and unwieldy. Thus, alternative testing procedures using laboratory animals have been proposed for quality control of these products. Such biological methods for vaccine evaluation using animals from vivarium facilities can have a significant impact through reduced costs, easier handling, and shorter testing times. The present study was designed to access Balb/C mice's humoral immune responses to a FMDV experimental vaccine, the composition of which contains three virus serotypes of FMDV (O1 Campos, A24 Cruzeiro, and C3 Indaial). Balb/C mice were immunized at doses that were 5% and 10% of the vaccine volume administered in cattle. Immunized mice had their antibody titers probed at 14, 21, and 28 DPV (days post vaccination). The results obtained were compared to those previously known from cattle's immune responses to the FMDV vaccine. An adequate immune response to the vaccine was seen with 10% formulation at 21 DPV. The study results are encouraging and indicate that the mouse model can be used for quality control in experimental vaccine testing.

Influence of drying methods over in vitro antitumoral effects of exopolysaccharides produced by Agaricus blazei LPB 03 on submerged fermentation.

Agaricus blazei is a mushroom that belongs to the Brazilian biodiversity and is considered as an important producer of bioactive compounds beneficial to human health. Studies have demonstrated that these compounds present immuno-modulatory, antioxidant and antitumor properties. In order to compare the most used method for fungal polysaccharide drying, lyophilization with other industrial-scale methods, the aim of this work was to submit A. blazei LPB 03 polysaccharide extracts to vaucum, spray and freeze drying, and evaluate the maintenance of its antitumoral effects in vitro. Exopolysaccharides produced by A. blazei LPB 03 on submerged fermentation were extracted with ethanol and submitted to drying processes. The efficiency represents the water content that was removed during the drying process. The resultant dried products showed water content around 3% and water activity less than 0.380, preventing therefore the growth of microorganisms and reactions of chemical degradation. Exopolysaccharide extracts dried by vacuum and spray dryer did not showed any significant cytotoxic effect on cell viability of Wistar mice macrophages. Content of total sugars and protein decrease after drying, nevertheless, 20 mg/ml of exopolysaccharides dried by spray dryer reached 33% of inhibition rate over Ehrlich tumor cells in vitro.

Prion protein gene polymorphisms in sheep in the state of Paraná, Brazil.

To determine the polymorphisms of the prion protein gene in sheep from the state of Paraná, Brazil, 323 animals of meat breeds (Suffolk, Hampshire Down, Texel, Ile de France, Dorper, Dorset, Santa Inês and crossbreds) were genotyped by restriction fragment length polymorphism (RFLP) analysis. The most frequent allele was ARQ, with a frequency of 0.61, followed by ARR (0.30). VRQ and AHQ alleles were present at very low frequencies (0.13 and 0.05 respectively), and the ARH allele was not found. Seven genotypes were identified (ARR/ARR, ARR/ARQ, ARQ/ARQ, ARR/VRQ, ARR/AHQ, ARQ/VRQ and ARQ/AHQ), of which ARQ/ARQ was the most frequent (0.41). The Santa Inês breed and crossbred animals showed the highest genotypic variability.

Investigation of Neospora sp. and Toxoplasma gondii antibodies in mares and in precolostral foals from Parana State, Southern Brazil.

Antibodies to Neospora sp. and Toxoplasma gondii were measured in mares and precolostral foals from a farm in Parana State, Brazil. An indirect fluorescent antibody test (IFAT) was used to determine specific antibodies. Three sampling points, 2003, 2004 and at parturition were included in the study, but not all horses are represented at a parturition time point. In 2003, antibodies to Neospora were detected in 17 mares (47%) at 1:50 dilution and in 5 mares (13.8%) at 1:100 dilution. In 2004, antibodies to Neospora were found in 11/36 (30%) horses with titers of 1:50 and in 6 mares (16.6%) at 1:100 dilution. The prevalence of antibodies against T. gondii was 2.7% in mares, either in 2003 and 2004. Evidence for the role of Neospora sp. in equine reproduction failure was not observed in the farm. Immunoglobulin G antibodies to Neospora were found in two of the nine precolostrum foals. Four seronegative foals were born from seronegative mothers, and three seronegative foals were born from seropositive mothers (1:50). Two seropositive precolostrum foals were born from seropositive mothers (1:50). The foals were born clinically normal, and T. gondii antibodies were not detected in their serum samples. The total immunoglobulins values detected on seronegative precolostrum foals were lower than the levels observed in the seropositive foals. This data warrants additional studies to differentiate N. hughesi and N. caninum serologically and determination if these parasites were associated with equine neurological disease and reproductive failure.

Characterization of human infection by Leishmania spp. in the Northwest of Argentina: immune response, double infection with Trypanosoma cruzi and species of Leishmania involved.

The aims of this study were to characterize human American tegumentary leishmaniasis, which includes cutaneous, mucocutaneous and mucosal leishmaniasis, in Northwest Argentina, to determine the prevalence of double infection with Trypanosoma cruzi and to identify the species of Leishmania in this area. Most of the 330 leishmaniasis patients presented cutaneous ulcers (96.1%), 2.4% mucocutaneous and 1.5% the mucosal form ('espundia'). The aetiological agents, determined by isoenzyme electrophoresis, were identified as Leishmania (Viannia) braziliensis in 16 out of 20 isolates and in the remaining 4 as Leishmania (Leishimania) amazonensis, the first ever-documented in Argentina. Sera analysed by ELISA and IFA using complex antigen from both T. cruzi and L. braziliensis showed a very high percentage of positives (66.3-78.2%). When antigens for specific diagnosis of Chagas' disease were used, 40.9% of the leishmaniasis patients were also found to be infected by T. cruzi. These results indicate that the strong immune response against T. cruzi gave no protection to Leishmania, in spite of the serological cross-reaction between these parasites. In addition, we showed that more than 40% of the patients would be misdiagnosed as chagasic if complex antigens, as epimastigotes or soluble fraction from epimastigotes, were used in IFA or ELISA. This is of paramount importance not only because patients' treatment would be associated to misdiagnosis but the fact that in many countries in Central and South America, a positive test for Chagas' disease means a rejection for those seeking employment.

Trypanosoma cruzi: isoenzyme analysis suggests the presence of an active Chagas sylvatic cycle of recent origin in Paraná State, Brazil.

Thirty-one trypanosomatid stocks were isolated from various sylvatic hosts and vectors in two different regions from the Paraná State of Brazil. The stocks were analyzed by multilocus enzyme electrophoresis (MLEE) on cellulose acetate plates (22 genetic loci). All stocks were unambiguously attributed to Trypanosoma cruzi, and were found to be closely related to the formerly described zymodeme I (TC1 subgroup of T. cruzi). By comparison with other sylvatic cycles with similar sample sizes (Southern USA, Colombia, French Guiana), genetic variability among these stocks was very limited, with only two variable loci out of 22, and only three different multilocus genotypes. Population structure of T. cruzi in these cycles appears to correspond to a set of very closely related clonal genotypes. This very limited genetic variability could be due to a recent foundation of these populations (founder effect). The implications of an active Chagas sylvatic cycle in this area are discussed.

[Sludge anaerobic treatment and its efficiency in reducing the viability of helminth eggs]. / Tratamento anaeróbio de esgoto e sua eficiência na redução da viabilidade de ovos de helmintos.

This study evaluates the prevalence and viability of helminth eggs and protozoan cysts in sludge obtained by anaerobic treatment in four treatment stations in Curitiba, Parana State, Brazil. The parasites observed were helminths: Ascaris sp (85%), Toxocara sp (5.5%), Trichuris sp (4.5%), Hymenolepis diminuta (3.7%), H. nana (1%) and Taenia sp (0.4%), protozoan: Isospora sp, Entamoeba coli, Entamoeba histolytica, Giardia lamblia, Endolimax nana. In biological treatment based on the anaerobic digestion the effectiveness depends on the duration and temperature. The treatment showed efficiency for pathogen reduction of between 59.7 to 93%. However, the number of helminth eggs found in treatment stations was still high and new higher performance treatment is necessary for land application or for other objectives, seeking to reduce the risks for human and animal health.

Lutzomyia whitmani (Diptera: Psychodidae) as vector of Leishmania (V.) braziliensis in Paraná state, southern Brazil.

The phlebotomine sandflies in the northern areas of the state of Paraná, Brazil, particularly those in the '16a' health region, were investigated over a 3-year period. Using CDC light traps (with and without hamster bait) and Shannon traps (with lights and horse or human bait), 16 species were collected from seven municipal districts which were known foci for cutaneous leishmaniasis: Arapongas; Apucarana; Cambira; Marumbi; Faxinal; Florestópolis; and Sabáudia. Although the frequency at which each species was collected varied with the collection site, Lutzomyia whitmani predominated (62.0% of all the sandflies collected), followed by Lu. fischeri (13.3%), Lu. pessoai (10.8%), Lu. migonei (8.2%) and Lu. intermedia (2.8%). Lutzomyia monticola, Lu. shanonni, Lu. firmatoi, Lu. lanei, Lu. alphabetica, Lu. misionensis, Lu. correalimai, Lu. cortellezzii, Lu. longipenis, Brumptomyia brumpti and B. nitzulescui together represented the remaining 3.0% of the collected sandflies. Three of the 1961 female sandflies collected and dissected in the municipal district of Cambira, where a recent case of cutaneous leishmaniasis had been registered, were found to have flagellates in their guts. All three were Lu. whitmani. The parasites from each of these infections were successfully isolated in NNN and 'Tobie and Evans' media and/or by inoculation into a hind foot of a golden hamster. The results of isoenzyme electrophoresis indicated that all three isolates were of Leishmania (Viannia) braziliensis.

Enzymatic polymorphism and phylogenetic relationships in Leishmania Ross, 1903 (Sarcomastigophora: Kinetoplastida): a case study in Colombia.

Leishmaniasis is widespread in Colombia and is found in 30 of 32 Departments. More than 200 infection zones have been reported from different regions, which vary from sea-level to an altitude of 2,300 m along the Atlantic Coast, Pacific coast, Amazon basin, Cauca and Magdalena valleys. We report 76 Leishmania stocks isolated from humans, dogs and phlebotomine hosts. Isoenzyme electrophoresis revealed 16 zymodemes, which could be divided into four phylogenetic complexes, i.e., L. braziliensis, L. amazonensis, L. guyanensis/panamensis and L. infantum. Three zymodemes became integrated into the subgenus Leishmania and the other zymodemes into the subgenus Viannia. Cutaneous infections were due to the L. braziliensis (9.2%) and L. guyanensis/panamensis (85.54%) complexes. Mucous secondary involvement was due to the L. braziliensis and L. guyanensis/panamensis complexes. In this work the specific status of L. (V.) guyanensis and L. (V.) panamensis is discussed.

Influence of peri-domiciliar environmental conditions in the dispersion and proliferation of phlebotomine sandflies in Parana state, southern Brazil

The results of phlebotomine collections with Falcäo light traps are described. The collections were made in an area of the Mururê farm, in the Terra Boa County, Paraná State, south of Brazil. One trap was installed at the border of a modified primitive forest and seven in animal shelters (poultry sheds and pigsties) distant 10 to 600m from this forest. Of a total of 5,122 phlebotomine 4,046 (79 per cent) were caught in an pigsty 10m from the border of the forest. The dominant species was Lutzomya whitmani. Dispersion of phlebotomine was near to 600m from the border of the forest and suggests that good environmental conditions for effective breeding in peridomiciliary areas in Mururê farm were not found, what explains the rare occurrence of tegumentary leishmaniasis there

Helminth eggs viability in sewage and biosolids sludge in Curitiba, Paraná, Brazil

Despite some benefits, municipal sludge poses hazards to human-kind and domestic animals because of the presence of some pathogenic agents in it. Bacteria, viruses, protozoan cysts, and helminth eggs may be present in the sludge. In Paraná State, SANEPAR, the responsible agency for collection and treatment of sewage sludge, is attempting to use the biosolids in agriculture. However, the risk to human and animal infection by the pathogenics should be considered before its use. An attempt was made to study the occurrence, viability and survival of helminth eggs and larva and protozoan cysts in the sludge. Parasitological analyses of twelve sludge biosolids and ten sewage sludge samples were collected and analyzed, from the treatment station ETE Belem (Curitiba, Brazil) for a period of one year. Four of the ten sewage sludge samples examined revealed the presence of helminth eggs as high as up to 20 eggs/l. In the biosolids sludge, the average number of helminth eggs, per 1 g of dry matter, was 4.85. However, the aerobic treatment reduced the viability of helminth egg to 56.67 per cent, and the total number of viable eggs was 1.85/g of dry matter. Ascaris sp. was the prevalent parasite (75.7 per cent), followed by H. diminuta (0.93 per cent), Trichuris sp. (7.4 per cent), Hymenolepis nana (3.6 per cent), Toxocara sp. (2.7 per cent), and Taenia sp. (1.3 per cent). Thus treatments will be necessary to hygienize the sludge for use in agriculture

New isolation of Leishmania enriettii Muniz and Medina, 1948 in Paranástate, Brazil, 50 years after the first description, and isoenzymatic polymorphism of the L. enriettii taxon.

Three cases of cutaneous leishmaniasis in guinea-pigs from a rural area near Curitiba (Paraná State, Brazil) are reported. The three parasite isolates were characterized by isoenzyme electrophoresis as Leishmania enriettii, of which two distinct zymodemes were observed.