Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals

Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone) has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL). The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.

Community structures of soil animals and survival of land snails on an island of the Ogasawara Archipelago

On Chichijima, one of the Ogasawara (Bonin) Islands located in the Western Pacific Ocean, land snails have declined, the suggested cause being predation pressure by an invasive flatworm (Platydemus manokwari). Soil fauna were investigated in areas where the snail survives, and where it has become extinct. Much of the fauna, dominated by introduced earthworms and ants, was undiminished, however, one undescribed but endemic carabid (Badister sp.), which selectively feeds on land snails, was absent in snail-extinct areas. The invasive flatworm P. manokwari has been reported to feed also on the carcasses of earthworms, as well as on live snails, and is therefore expected to occur in most parts of Chichijima Island. Among other groups, the density of isopods (also dominated by exotic species) was very low, in comparison with the reported ones 30 years ago. Community structure is currently reflected by dominance of earthworms and ants, decline of endemic isopods, and a high frequency of introduced or alien species.

Exogenous induction of ovarian activity and ovulation and transfer of fresh embryos of domestic cat (Felis catus)

The objective of the present study was the exogenous stimulation of ovarian activity and definition of embryo collection, and transfer protocols, in the domestic cat for potential application in non-domestic endangered species. Sixteen adult queens and two adult male reproducers kept in the experimental cat house at the Morphology sector at the Veterinary Department (DVT), UFV, were used in this study. All the queens received a single application of 150 IU Equine Chorionic Gonadotropin (eCG) in the post estrus to induce ovarian activity and 80 to 84 hours later, received a single application of 100 UI Human Chorionic Gonadotropin (hCG) to induce ovulation. After hCG application, only the donor queens were naturally mated. The receptor queens received extra stimulus for induction of ovulation through manipulation of an intravaginal swab. Five to six days after hCG application, the donor queens were subjected to a laparotomy for embryo collection that was performed by trans-horn uterine washing. On average, six embryos were surgically inovulated. They were classified as type I and III compact morula and blastocysts in four receptor queens. Three animals presented pregnancy confirmed by ultrasound at day 36 and two of these animals gave birth to litters of two and four offsprings, respectively, at 66 and 63 days after induction of ovulation. Except for one still birth, all the offspring developed normally.

Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

The objective of this study was to compare the histopathological changes and expression of CR3 and CR4 in the liver and spleen of dogs naturally and experimentally infected with L. chagasi. The basic histopathological lesions observed mainly in naturally infected dogs were: epithelioid hepatic granulomas, hyperplasia and hypertrophy of Kupffer cells, Malpigui follicles and mononucleated cells of the red pulp of the spleen. Sections from the liver and spleen by immunocytochemistry technique showed the presence of CD11b,c\CD 18 antigens in the control and infected animals and no qualitative or quantitative differences in the liver. Nevertheless, CD18 was always increased in the spleen of naturally and experimentally infected dogs. These results indicate that there is a difference in the activaton of CD 18 in both experimental and natural cases of canine visceral leishmaniasis that should play an important role in the immunological response to Leishmania chagasi infection.

Serologic survey for hantavirus infections among wild animals in rural areas of São Paulo State, Brazil

A serosurvey was conducted in wild animals captured close to two areas where hantavirus cardiopulmonary syndrome (HCPS) occurred in São Paulo State, Brazil. Serum samples from a total of 43 mammals were tested for antibodies reactive with Sin Nombre (SN) hantavirus using a strip immunoblot assay. RNAs from the blood clots of the positive samples were submitted to reverse transcriptase-polymerase chain reaction (RT-PCR). Two rodents of the genus Oligoryzomys were positive for hantavirus antibodies. These animals were captured in the Iguape region and represented 16.7% (2/12) of the sera from rodents and 100.0% (2/2) of the Oligoryzomys captured in that area. RT-PCR failed to amplify any viral cDNA. These results are in agreement with other data that suggest that members of this genus are important reservoirs of hantaviruses in Brazil.

PATHOGENICITY OF Blastocystis sp. TO THE GASTROINTESTINAL TRACT OF MICE: RELATIONSHIP BETWEEN INOCULUM SIZE AND PERIOD OF INFECTION

The pathogenic potential of Blastocystis sp. in experimental models requires further investigation. In this work, the pathogenicity of this parasite in the gastrointestinal tract of male Swiss mice was evaluated according to the inoculum size and period of infection. Animals were infected intragastrically, with 100, 500, 1,000, 5,000 and 10,000 Blastocystis sp. vacuolar forms obtained from a mixture of eight human isolates cultured axenically in Jones' medium. After seven, 14, 21, 28 and 60 days of infection, the animals were sacrificed and fragments of the small intestine (duodenum), large intestine, and cecum were subjected to histopathological analysis. Blastocystis sp. triggered an inflammatory response in the different tissues analyzed, with a predominance of mononuclear cells. The parasite was found in the muscular layer of the cecum, showing its invasive character. Larger inocula triggered inflammatory processes earlier (seven days) than smaller ones (from 21 days). We conclude that, in the proposed model, the pathogenicity of Blastocystis sp. isolates that were studied is related to inoculum size and period of infection.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Vaccination of C57BL/10 mice against cutaneous leishmaniasis using killed promastigotes of different strains and species of Leishmania

Antigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacin® and PH8 monovalent vaccine yielded the highest IFN-gamma levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania) amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L.) amazonensis infection.

Tityus stigmurus (Thorell, 1876) (Scorpiones; Buthidae): response to chemical control and understanding of scorpionism among the population

In this study, the events following application of the insecticideDemand 2.5 concentrated solution (CS) in the field, to control Tityus stigmurus, were investigated. Data on attitudes and practices relating to scorpionism were collected using a questionnaire. During the months of May to July 2005, 69 premises were monitored on different days following insecticide treatment, focusing on scorpion frequency and mortality. According to the results, 42% of the premises showed scorpion incidence, with an average of three specimens per house. The highest incidence was recorded during the first week following the treatment. Only 7% of the specimens were found dead. Most (72%) of the population showed knowledge about prevention and control measures. Despite this, 100% of the premises presented breeding sites, mainly in debris (79.7%). These results indicate that the scorpion control method used by health agents during this investigation was not efficient, and the results suggest that the method may have had a dispersive effect on these animals.

Seroprevalence and seroincidence of Leptospira infection in dogs during a one-year period in an endemic urban area in Southern Brazil

INTRODUCTION: Leptospirosis is a zoonosis that affects both humans and animals. Dogs may serve as sentinels and indicators of environmental contamination as well as potential carriers for Leptospira. This study aimed to evaluate the seroprevalence and seroincidence of leptospirosis infection in dogs in an urban low-income community in southern Brazil where human leptospirosis is endemic. METHODS: A prospective cohort study was designed that consisted of sampling at recruitment and four consecutive trimestral follow-up sampling trials. All households in the area were visited, and those that owned dogs were invited to participate in the study. The seroprevalence (MAT titers ≥100) of Leptospira infection in dogs was calculated for each visit, the seroincidence (seroconversion or four-fold increase in serogroup-specific MAT titer) density rate was calculated for each follow-up, and a global seroincidence density rate was calculated for the overall period. RESULTS: A total of 378 dogs and 902.7 dog-trimesters were recruited and followed, respectively. The seroprevalence of infection ranged from 9.3% (95% CI; 6.7 - 12.6) to 19% (14.1 - 25.2), the seroincidence density rate of infection ranged from 6% (3.3 - 10.6) to 15.3% (10.8 - 21.2), and the global seroincidence density rate of infection was 11% (9.1 - 13.2) per dog-trimester. Canicola and Icterohaemorraghiae were the most frequent incident serogroups observed in all follow-ups. CONCLUSIONS: Follow-ups with mean trimester intervals were incapable of detecting any increase in seroprevalence due to seroincident cases of canine leptospirosis, suggesting that antibody titers may fall within three months. Further studies on incident infections, disease burden or risk factors for incident Leptospira cases should take into account the detectable lifespan of the antibody.

Developmental morphology of mouthparts and foregut of the larvae and postlarvae of Lepidophthalmus siriboia Felder & Rodrigues, 1993 (Decapoda: Callianassidae)

In this study, the gross morphology of the mouthparts and foregut of the ghost shrimp Lepidophthalmus siriboia were investigated from larvae and postlarvae reared in the laboratory. The mouthparts (maxillae and maxillipeds) of the zoeae have a reduced number of setae and spines (or is absent in some individuals), and the foregut, under developed, have few minute setae in the cardiac and pyloric chambers. In contrast, after the metamorphosis into megalopa stage, all feeding appendages have many setae and, the foregut shows a well-developed gastric mill with strong lateral teeth. In the juvenile stage occurs an increase of setae and spines in the mouthparts and the foregut becomes more specialized. These observations strongly suggest that a lecithotrophic development occurs during all zoeal stages but the megalopa and juvenile stages are feeding animals. The functional morphology of the feeding structures of L. siriboia and other decapods will be briefly discussed.

Growth, molt and survival of Palaemonetes argentinus (Decapoda, Caridea) under different light-dark conditions

Growth, survival and molting rate in Palaemonetes argentinus Nobili, 1901 were compared under different light-dark conditions. During 80 days, 150 immatures of both sexes (initial mean weight 0.09±0.002g), from Los Padres lagoon, Mar del Plata, Argentina, were maintained in aquaria at 19±0.4°C under three light conditions: 0:24, 10:14 and 13:11 (L-D). They were fed daily on an artificial diet (45% proteins, 17.2% lipids, 7% water, 7% ash). Good weight increment was obtained with the three treatments, finding a positive linear correlation between mean weight and time (0:24, r=0.97; 10:14, r=0.99; 13:11, r=0.98). There were no significant differences in the percentage increment in mean weight among the treatments (0:24, 19.3%; 10:14, 29.3% and 13:11, 26.5%) (p<0.05). Molting rate was significantly higher at a long-day photoperiod (MR=1.7) than at a short-day (MR=0.6) or continuous dark condition (MR=0.3) (p<0.05). The lowest survival was found in animals maintained under 13:11 L-D conditions (77%), being statistically different of the other two treatments (92% and 89% at 10:14 and 0:24, respectively) (p<0.05). These results suggest that the best growth and survival in P. argentinus result with a 10:14 L-D cycle, and that the growth is less affected by photoperiod than molting rate and survival.

Behavior of triatomines (Hemiptera, Reduviidae) vectors of Chagas' disease. II. Influence of feeding, lighting and time of day on the number of mating, mating speed and duration of copulation of Panstrongylus megistus (Burm, 1835) under laboratory conditions

To determine in influence of feeding, lighting and time of day on the copulating behavior of Panstrongylus megistus, 480 insect pairs were divided into four groups of 120 each and tested in the following respective situations: without food deprivation (F.D.), with five days of F.D., with ten days of F.D., and with 20 days of F. D. The tests were performed between 9:00 a.m. to 12:00a.m. and 7:00 p.m. to 10:00 p.m., with light (700-1400 lux) and in the dark (1.4-2.8 lux) and behavior was recorded by the time sampling technique. Mating spped (MS) and duration of copulation (DC) were also calculated for each situation. The maximum frequency of copulation was observed after five days of F.D., at night, in the dark (n = 16), and the minimum was observed for recently-fed pairs, at night, with light (n = 4). Males approached females more often than females approached males. MS was lowest in pairs with twenty days of F.D., at night, with light (X = 23.0 ± 16.0 minutes), and highest in recently-fed pairs, during the day, with light (X = 2.9 ± 2.5 minutes). DC was shortest in recently-fed insects, during the day, in the dark (X = 23.5 ± 6.7 minutes), and longest in recently-fed animals, at night, in the dark (X = 38.3 ± 6.9 minutes).