PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P > 0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P < 0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P > 0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P < 0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P < 0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar. © 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Asymptomatic carriers of Plasmodium spp. as infection source for malaria vector mosquitoes in the Brazilian Amazon

We have described the existence of asymptomatic carriers of Plasmodium vivax and Plasmodium falciparum infections in native Amazon populations. Most of them had low parasitemias, detected only by polymerase chain reaction (PCR). Because they remain symptomless and untreated, we wanted to determine whether they could infect Anopheles darlingi Root, the main Brazilian vector, and act as disease reservoirs. Fifteen adult asymptomatic patients (PCR positive only) were selected, and experimental infections of mosquitoes were performed by direct feeding and by a membrane-feeding system. Seventeen adult symptomatic patients with high parasitemias were used as controls. We found an infection rate in An. darlingi of 1.2% for the asymptomatic carriers and 22% for the symptomatic carriers. Although the asymptomatic group infected mosquitoes at a much lower rate, these patients remain infective longer than treated, symptomatic patients. Also, the prevalence of asymptomatic infections is 4 to 5 times higher than symptomatic infections among natives. These results have implications for the malaria control program in Brazil, which focuses essentially on the treatment of symptomatic patients. © 2005 Entomological Society of America.

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus)

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful. © American Society of Parasitologists 2005.

Distribuição sazonal de parasitos branquiasis em diferentes especies de piexes em pesque-pague do municipio de Guariba-SP, Brasil.

This study was accomplished with the objective of knowing the seasonal distribution of the main species of parasites causes of branchial damages to the fish from fee-fishing at Guariba, State of São Paulo. This city is situated at 21 degrees 15'22'' S, 48 degrees 18'58'' W, in altitude 595 meters. This research was conducted between April, 1997 to March, 1999. From 408 fishes examined, 29.1% of them were infested by the protozoans e metazoans. Leporinus macrocephalus showed the highest parasitism by Trichodina sp., although Piaractus mesopotamicus and Cyprinus carpio were also parasited. Piscinoodinium pillulare infestation were found in L. macrocephalus, P. mesopotamicus, C. carpio e Colossoma macropomum. Monogenetics metazoan have largely been observed in L. macrocephalus, P. mesopotamicus, C. carpio, C. macropomum e cross-bred tambacu. The incidence of this parasite was elevated in the summer for L. macrocephalus, spring for P. mesopotamicus and C. macropomum, and both seasons for cross-bred tambacu. Lernaea cyprinacea copepodes were observed in L. macrocephalus, P. mesopotamicus, C. carpio and cross- breeding tambacu, in spite of the fact that C. carpio has demonstrated high parasitism of adult form. The fish L. macrocephalus, P. mesopotamicus and C. carpio were sponged intensely by Dolops carvalhoi, in situation of high population density. Piaractus mesopotamicus was considered the species more parasited by myxosporídeos, that were also observed in L. macrocephalus and cross-breeding tambacu.

Diphyllobothriasis, Brazil

Cases of human diphyllobothriasis have been reported worldwide. Only 1 case in Brazil was diagnosed by our institution from January 1998 to December 2003. By comparison, 18 cases were diagnosed from March 2004 to January 2005. All patients who became infected ate raw fish in sushi or sashimi.

Comparação entre técnicas coproparasitológicas no diagnóstico de ovos de helmintos e oocistos de protozoários em cães.

In this study we evaluated the frequency of enteroparasites in pet dogs and their association with age, sex and breed, as well as the efficiency of the Willis-Mollay, Faust, Sedimentation and Direct exam methods. By these methods we processed 401 fecal samples. The samples were positive in the following percentages: Ancylostoma spp. (53.1%), Toxocara canis (20.7%), Cystoisospora ohioensis (15.7%), Trichuris vulpis (3.7%), Dipylidium caninum (2.5%) and Taenia spp. (1.0%). Toxocara canis (67.3%) and C. ohioensis (47.3%) showed higher positivity in the puppies. The Willis-Mollay technique was more efficient in the diagnosis of Ancylostoma spp. and T. canis eggs. The Direct method was the least efficient. It was found that the majority of the cases of D. caninum were diagnosed by the Sedimentation method (8=2.0%), while for T. vulpis Willis-Mollay (12=3.0%) and Sedimentation (13=3.2%) were more efficient. In view of these results, we can recommend the association of Willis-Mollay and Sedimentation methods for the diagnosis of gastrointestinal helminths. Due to the elevated occurrence of Ancylostoma spp. and T. canis, which are involved in zoonotic diseases, it becomes necessary to apply more efficient prophylaxis of canine intestinal parasitosis at the City of Araçatuba, state of São Paulo.

Espécies do gênero Eimeria (Apicomplexa: Eimeriidae) em Tamanduás-bandeira (Myrmecophaga tridactyla Linnaeus, 1758) em cativeiro.

The parasitism of the two giant anteaters adults (Myrmecophaga tridactyla), one male and one female, infected naturally with Eimeria escomeli, E. tamanduae e E. marajoensis was related in the present research. In E. escomeli oocysts were 23.9 +/- 1.89 by 19.7 +/- 1.60 microm and its sporocysts were 11.47 +/- 1.25 by 6.48 +/- 0.80 microm. In E. tamanduae oocysts were 23.52 +/- 0.95 by 20.59 +/- 0.92 microm and its sporocysts were 12.19 +/- 0.65 by 7.15 +/- 0.55 microm. In E. marajoensis oocysts were 13.5 +/- 1.7 by 13.1 +/- 1.8 microm and its sporocysts were 7.4 +/- 0.58 by 5.4 +/- 0.8 microm. Eimeria escomeli was described before parasitizing giants anteater from Bolivia, and it was point out as the first time in Brazil. The presence of E. tamanduae and E. marajoensis parasitizing giant anteaters indicate the possibility of having co-infection of them among animals of the family Myrmecophagidae.

Effect of air humidity on the egg viability of predatory mites (Acari: Phytoseiidae, Stigmaeidae) common on rubber trees in Brazil

Rubber pest mites, Calacarus heveae and Tenuipalpus heveae, reach economic damage levels at the end of the rainy season and the beginning of the dry season in Brazil. Therefore, low humidity adaptation might be an important characteristic for predatory mites to successfully control pest organisms. This study determined the effect of the relative humidity (RH) levels of 30-100% on the hatching of larvae of Amblyseius acalyphus, Euseius citrifolius, Iphiseiodes zuluagai, Metaseiulus camelliae, Agistemus floridanus and Zetzellia malvinae at 25 ± 0.5°C. These predatory mites are common on rubber trees in the state of São Paulo and might be used for introduction in the major rubber tree production regions in the state of Mato Grosso. At 70% RH or higher, viability was 70% or higher for all species, indicating that their performance might be higher during the rainy season than during the dry season. Eggs of E. citrifolius and M. camelliae presented higher viability at the lower relative humidity levels than those of other species, indicating that these species might have higher chance to persist in the dry season. It is suggested that M. camelliae should be further evaluated for introduction in the state of Mato Grosso, considering that this mite is not yet present in that area. © Springer 2006.

Efficacy of homeopathic treatment against natural infection of sheep by gastrointestinal nematodes.

The efficacy of the homeopathic treatment with the Fator Vermes, administered according to the manufacturer's recommendations, was evaluated against gastrointestinal nematodes infections in sheep. The experiment was divided into two phases: in the first phase (January/06/2004 to April/30/2004), the animals of the treated (n=10) and control (n=10) groups were treated individually with conventional anthelmintics to avoid deaths. In the second phase (April/30/2004 to July/06/2004), the sheep from the group that received the Fator Vermes were treated as they had been in the previous phase, while the control group animals were treated with conventional anthelmintics at 14 day intervals. In the first phase of the experiment, there was no significant difference (P>0.05) between group means regarding egg counts in feces (EPG), weight gain, or packed cell volume (PCV). Meanwhile, in the second phase, the control group sheep had a significantly higher weight gain, higher PCV values, and lower EPG counts. Infective larvae of Haemonchus spp., Trichostrongylus spp., Cooperia spp., and Oesophagostomum spp. were identified in the fecal cultures. After six months of daily treatment with the Fator Vermes, it was not possible to substantiate the product's benefits in both sheep health and productivity or in the prophylaxis of gastrointestinal nematode infections.

Um surto de coccidiose em perdizes (Rhynchotus rufescens), criadas em cativeiro, por Eimeria rhynchoti Reis e Nóbrega, 1936 (Apicomplexa: Eimeriidae).

Eimeria rhynchoti is redescribed parasitizing partridge (Rhynchotus rufescens), reared in captivity, from Jaboticabal City, São Paulo State, Brazil. Sporulation takes place in 48 hours, the shape of oocysts found vary from spherical to elliptic with 23.01 micro +/- 1.57 of length by 21.0 micro +/- 1.78 of width. The microple, polar cap and residuum of the oocysts were absent. The oocyst wall, measures 2.2 micro +/- 0.31 of thickness, is composed by two smooth layers; the polar granule is present. The sporocysts length was 15.03 mm +/- 2.12 by 8.08 mm +/- 0.84 of width vary from elliptic to elongate. Sporocyst wall slender with is fine and Stieda body; the residue found in form of several smaller granules spherical compacts. The sporozoites are contrary extending along the sporocysts wall possessing refracts body of easy visualization.

Toxoplasma gondii: Comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7 × 107 viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10 = 2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as gold standard and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISA × IFAT (κ = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 ± 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs. © 2005 Elsevier Inc. All rights reserved.

Efeitos do parasitismo sobre a estrutura branquial de Leporinus macrocephalus Garavello e Britsk, 1988 (Anastomidae) e Piaractus mesopotamicus Holmberg, 1887 (Osteichthyes:Characidae).

This work described the lesions caused in different species of fish by gill parasites from fee-fishing at Guariba, State of São Paulo. The research was developed from april, 1997 to march to 1999, seeking to verified the kind tissues lesions from fish. Of these, forty and seven were Leporinus macrocephalus and fifty and five Piaractus mesopotamicus. About 87.2% of the L. macrocephalus, and 58.1% of the P. mesopotamicus were sponged by several species of parasites. The parasite most abundant in L. macrocephalus was Piscinoodinium pillulare, while monogenean, Trichodina sp and myxosporidian infected P. mesopotamicus. Severe gill lesions have been observed in L. macrocephalus and P. mesopotamicus caused by monogenean, P. pillulare e Trichodina sp. parasitism, such as intersticial hemorrhage, sub-epithelium edema, inflammation, epitelial hiperplasy in filaments and lamina, proliferation of mucosal cells and laminar fusion.

Toxoplasma gondii: Detection by mouse bioassay, histopathology, and polymerase chain reaction in tissues from experimentally infected pigs

In the present study, we evaluated three techniques, mouse bioassay, histopathology, and polymerase chain reaction (PCR) to detect Toxoplasma gondii infection in tissues from experimentally infected pigs. Twelve mixed breed pigs, seronegative for T. gondii using an indirect immunofluorescent antibody test (IFAT), were used. Ten pigs were infected with 4 × 104 VEG strain oocysts, and two were maintained as uninfected controls. Animals were killed 60 days pos infection. Muscle (heart, tongue, diaphragm, and masseter) and brain samples were collected to investigate the presence of T. gondii tissue cysts by the different assay methods. For the bioassay, samples of brain (50 g) and pool of muscle samples (12.5 g of tongue, masseter, diaphragm, and heart) were used. PCR was performed using Tox4 and Tox5 primers which amplified a 529 bp fragment. The DNA extraction and PCR were performed three times, and all tissue samples were tested individually (brain, tongue, masseter, diaphragm, and heart). For histopathology, fragments of tissues were fixed in 10% of buffered formal saline and stained with HE. Histopathological results were all negative. PCR showed 25/150 (16.6%) positive samples, being 17/120 (14.1%) and 8/30 (26.6%) from muscle, and brain tissues, respectively. Tissue cysts of T. gondii were identified by mouse bioassay in 54/98 (55.1%) samples, being 31/48 (64.6%) from muscle samples, and 23/50 (46.0%) from brain samples. Toxoplasma gondii isolation in muscle samples by mouse bioassay was higher than in PCR (P < 0.01). Results indicate that DNA from pig tissues interfered with 529-bp-PCR sensitivity, and mouse bioassay was better than PCR in detecting T. gondii in tissues from pigs. © 2006 Elsevier Inc. All rights reserved.

The taxonomic and phylogenetic relationships of Trypanosoma vivax from South America and Africa

The taxonomic and phylogenetic relationships of Trypanosoma vivax are controversial. It is generally suggested that South American, and East and West African isolates could be classified as subspecies or species allied to T. vivax. This is the first phylogenetic study to compare South American isolates (Brazil and Venezuela) with West/East African T. vivax isolates. Phylogeny using ribosomal sequences positioned all T. vivax isolates tightly together on the periphery of the clade containing all Salivarian trypanosomes. The same branching of isolates within T. vivax clade was observed in all inferred phylogenies using different data sets of sequences (SSU, SSU plus 5.8S or whole ITS rDNA). T. vivax from Brazil, Venezuela and West Africa (Nigeria) were closely related corroborating the West African origin of South American T. vivax, whereas a large genetic distance separated these isolates from the East African isolate (Kenya) analysed. Brazilian isolates from cattle asymptomatic or showing distinct pathology were highly homogeneous. This study did not disclose significant polymorphism to separate West African and South American isolates into different species/subspecies and indicate that the complexity of T. vivax in Africa and of the whole subgenus Trypanosoma (Duttonella) might be higher than previously believed. © 2006 Cambridge University Press.

Electrophysiological responses of Atta sexdens rubropilosa workers to essential oils of Eucalyptus and its chemical composition

The leaf-cutting ant Atta sexdens rubropilosa Forel, 1908 is the most harmful of the Eucalyptus pests, causing severe losses in wood production through defoliation. Various strategies have been tried and effort spent on the development of methods to control this pest, however no practical and environmentally acceptable one currently exists. In this work the chemical composition of the essential oil of seven Eucalyptus species was identified and the selectivity and sensitivity of antennal receptors of A. sexdens rubropilosa workers to the volatile compounds were determined using the electroantennographic technique (EAG and GC-EAD). Analysis by GC-EAD showed in E. cloesiana and E. maculata, respectively, seventeen and sixteen terpenes that elicited responses in ant workers' antennae, indicating the potential role of the essential oils as allelochemicals that determine the choice of the foraging material. © 2006 Verlag der Zeitschrift für Naturforschung.