Occurrence of Cryptosporidium spp. in Antillean manatees (Trichechus manatus) and Amazonian manatees (Trichechus inunguis) from Brazil

Infections by Cryptosporidium spp. in aquatic mammals is a major concern due to the possibility of the waterborne transmission of oocysts. The aim of the present study was to report the occurrence of Cryptosporidium spp. in Antillean manatees (Trichechus manatus) and Amazonian manatees (Trichechus inunguis) from Brazil. Fecal samples were collected and processed using Kinyoun's method. Positive samples were also submitted to the direct immunofluorescence test. The results revealed the presence of Cryptosporidium spp. oocysts in 12.5% (17/136) of the material obtained from the Antillean manatees and in 4.3% (05/115) of the samples from the Amazonian manatees. Cryptosporidium spp. infection was more prevalent in captive animals than in free-ranging specimens.     

Antimicrobial Photodynamic Therapy: Study of Bacterial Recovery Viability and Potential Development of Resistance after Treatment

Antimicrobial photodynamic therapy (aPDT) has emerged in the clinical field as a potential alternative to antibiotics to treat microbial infections. No cases of microbial viability recovery or any resistance mechanisms against it are yet known. 5,10,15-tris(1-Methylpyridinium-4-yl)-20-(pentafluorophenyl)-porphyrin triiodide (Tri-Py⁺-Me-PF) was used as photosensitizer. Vibrio fischeri and recombinant Escherichia coli were the studied bacteria. To determine the bacterial recovery after treatment, Tri-Py⁺-Me-PF (5.0 μM) was added to bacterial suspensions and the samples were irradiated with white light (40 W m⁻²) for 270 minutes. Then, the samples were protected from light, aliquots collected at different intervals and the bioluminescence measured. To assess the development of resistance after treatment, bacterial suspensions were exposed to white light (25 minutes), in presence of 5.0 μM of Tri-Py⁺-Me-PF (99.99% of inactivation) and plated. After the first irradiation period, surviving colonies were collected from the plate and resuspended in PBS. Then, an identical protocol was used and repeated ten times for each bacterium. The results suggest that aPDT using Tri-Py⁺-Me-PF represents a promising approach to efficiently destroy bacteria since after a single treatment these microorganisms do not recover their viability and after ten generations of partially photosensitized cells neither of the bacteria develop resistance to the photodynamic process.     

Repellent activity of plant-derived compounds against Amblyomma cajennense (Acari: Ixodidae) nymphs

Repellence responses of Amblyomma cajennense nymphs to callicarpenal, intermedeol, Hyptis suaveolens essential oil, extract of Melia azedarach, Cymbopogon nardus, Spiranthera odoratissima, Chenopodium ambrosioides, Ageratum conyzoides, Mentha pulegium, Ruta graveolens, and Memora nodosa were studied. Among these the extract of C. nardus stood out because of the long-lasting repellence, maintaining, in the highest concentration, 35 h of protection against 90% of the nymphs. The essential oil of H. suaveolens and the extracts of C. ambrosioides and A. conyzoides showed good repellence index (66%) when applied in high concentrations. However, greater protection could be obtained at higher concentrations but with a shorter repellence time. Callicarpenal, intermedeol, extract of M. Pulegium, and M. nodosa leaves showed moderate repellence in high concentrations. Extracts from M. azedarach, R. graveolens, S. odoratissima, and M. nodosa roots showed little or no repellent effect. These results show that some plant extracts may represent a promising alternative in the control of infestations by A. cajennense.     

Detection of lymph node micrometastases in malignant mammary tumours in dogs by cytokeratin immunostaining

A series of 131 local and regional lymph nodes from 40 dogs with malignant mammary tumours were evaluated by staining with haematoxylin and eosin and immunohistochemically for antibodies to pancytokeratin (AE1/AE3) and cytokeratin 14. The immunohistochemical tests detected occult micrometastases in 9.2 per cent of the lymph nodes that were negative by haematoxylin and eosin staining. Under the modified TNM classification of canine mammary tumours, these results raised the clinical stage of 12.5 per cent of the affected dogs. However, if the latest TNM classification of human breast cancer had been applied, none of the animals would have been reclassified.     

Short- and intermediate-term effects of site and plant preparation techniques on reforestation of a Mediterranean semiarid ecosystem with <Emphasis Type="Italic">Pinus halepensis</Emphasis> Mill

A 5-year study of the reforestation of a semiarid Mediterranean ecosystem with Pinus halepensis is presented. Reforestation of Mediterranean semiarid ecosystems is challenging as a result of harsh environmental conditions and historical human exploitation leading to degradation. New techniques have to be developed in order to increase survivorship and growth that integrates nursery and field treatments. A total of 60 treatments resulting from 12 site × 5 plant preparation techniques were tested. Plant preparation consists of mycorrhizing in nursery, extra fertilizing in nursery, planting in containers in the nursery, planting in the field with a polypropylene tube, and control plants (bred in plastic bags). Soil preparation consists of hole, subsoiling without amendment, subsoiling with composted organic amendment added in the surface or in the furrow of plantation and subsoiling with fresh organic amendment added in the surface or in the furrow of plantation. Each one of these treatments was tested with a hydrophilic acrylic copolymer and without it. Survivorship was very high (0.91) given the prevailing environmental conditions. Subsoiling promoted higher survival than holes, and plants bred in containers had lower levels of survivorship than control ones. Protecting seedlings with polypropylene tubes had no effect on survivorship. The most effective treatment for promoting growth was organic amendment, the effect of which increased until the fourth year. Protection with tubes was very effective in increasing height, although only in the short-term. Subsoiling also promoted faster growth than holes, although the effect was less noticeable after 3 years and was, in any case, less effective than organic amendment. Other treatments promoting faster growth were the use of fresh organic waste as opposed to composted waste and its incorporation in the plantation furrow rather than as a layer on the surface. The addition of hydrophilic acrylic copolymer reduced growth during the first months of the reforestation. The fertilization of plants in the nursery and inoculation with spores of mycorhyzal fungi did not improve seedling growth. Plants bred in containers performed worse than control plants bred in individual plastic bags. The findings suggests the importance of nutrient supply in semiarid environments, an aspect that has been previously neglected in favor of water supply. Organic amendments reinforce both and can be recommended as long as adequate environmental controls are in place.     

Moment of addition of LH to the culture medium improves in vitro survival and development of secondary goat pre-antral follicles

The present study investigated the effects of time of addition of luteinizing hormone (LH) to culture medium on the in vitro development of caprine pre-antral follicles. Pre-antral follicles (≥ 150 μm) were isolated from fragments of the goat ovarian cortex and individually cultured for 18 days in the absence (control) or presence of 100 ng/ml LH, added on days 0, 6 or 12 of culture. Follicular development was assessed based on antral cavity formation, increased follicular diameter as well as follicular and fully grown oocyte (>110 μm) viability. The results showed that after 18 days of culture, the percentage of surviving follicles in the control treatment was significantly lower when compared to other treatments (p < 0.05). There were no significant differences in antrum formation, follicular diameter and oocyte viability. The addition of LH at D6 of culture significantly increased the rates of oocytes ≥ 110 μm and the resumption of meiosis (p < 0.05). In contrast, when LH was added at the onset of culture, only germinal vesicle oocytes were obtained. In conclusion, the moment of addition of LH to the culture medium affects the performance of in vitro culture of caprine pre-antral follicles. The addition of LH to the medium from day 6 of culture onward improved the rates of follicular survival, as well as the ability of oocytes to resume meiosis. However, prolonged exposure to LH (addition at the onset of culture onward) showed detrimental effects for the meiotic resumption.     

Catalase addition to vitrification solutions maintains goat ovarian preantral follicles stability

The aim of this study was to verify whether the addition of catalase (20 IU/mL) at different steps of goat ovarian tissue vitrification affects ROS levels, follicular morphology and viability, stromal cell density, apoptosis and the expression of proteins related to DNA–damage signaling (γH2AX) and repair (53BP1). Goat ovarian tissues were analyzed fresh (control) or after vitrification: without catalase (VS–/WS–), with catalase in vitrification solutions (VS+/WS–), with catalase in washing solutions (VS–/WS+) or with catalase in both solutions (VS+/WS+). The vitrification without catalase had higher ROS levels than the control. The catalase, regardless the step of addition, maintained ROS levels similar to the control. There were no difference between treatments regarding follicular viability, stromal cell density and detection of γH2AX and 53BP1. There was no difference in follicular morphology and DNA fragmentation between groups vitrified. In conclusion, catalase addition to vitrification solutions prevents ROS formation in cryopreserved goat ovarian tissues.     

Intravenous administration of hypertonic sodium chloride solution with dextran or isotonic sodium chloride solution for treatment of septic shock secondary to pyometra in dogs.

OBJECTIVE: To determine effects of i.v. administration of hypertonic saline (7.5% NaCl) solution with 6% dextran 70 (HSSD) or isotonic saline (0.9% NaCl) solution (ISS) to dogs with septic shock secondary to pyometra. DESIGN: Prospective, randomized, clinical study. ANIMALS: 14 client-owned dogs with septic shock secondary to pyometra. PROCEDURE: Prior to emergency ovariohysterectomy, catheters were placed in pulmonary and femoral arteries of each dog to evaluate hemodynamic and oxygenation status. Immediately prior to surgery, 7 dogs received HSSD (4 ml/kg [1.82 ml/lb] of body weight, i.v.) and 7 dogs received ISS (32 ml/kg [14.54 ml/lb], i.v.) during a 5-minute period. Measurements of hemodynamic and oxygenation variables were obtained before and 5 and 20 minutes after administration of fluids. RESULTS: Mean arterial pressure (MAP) increased significantly 5 and 20 minutes after administration of HSSD, whereas ISS did not affect MAP. However, cardiac output, cardiac index, and oxygen delivery increased and hematocrit decreased after both treatments. Oxygen consumption and extraction rate and degree of acidosis did not improve after either treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Intravenous administration of small volumes of HSSD to dogs with septic shock secondary to pyometra resulted in improvement of hemodynamic and oxygenation status. Although cardiac output, cardiac index, and oxygen delivery improved after administration of a volume of ISS equal to 8 times that of HSSD, MAP increased to > 80 mm Hg only after treatment with HSSD. Administration of HSSD may be an effective treatment for septic shock in dogs.     

Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites

The murine model has been widely used to study the host immune response to Neospora caninum. However, in most studies, the intraperitoneal route was preferentially used to establish infection. Here, C57BL/6 mice were infected with N. caninum tachyzoites by the intragastric route, as it more closely resembles the natural route of infection through the gastrointestinal tract. The elicited T-cell mediated immune response was evaluated in the intestinal epithelium and mesenteric lymph nodes (MLN). Early upon the parasitic challenge, IL-12 production by conventional and plasmacytoid dendritic cells was increased in MLN. Accordingly, increased proportions and numbers of TCRαβ⁺CD8⁺IFN-γ⁺ lymphocytes were detected, not only in the intestinal epithelium and MLN, but also in the spleen of the infected mice. In this organ, IFN-γ-producing TCRαβ⁺CD4⁺ T cells were also found to increase in the infected mice, however later than CD8⁺ T cells. Interestingly, splenic and MLN CD4⁺CD25⁺ T cells sorted from infected mice presented a suppressive activity on in vitro T cell proliferation and cytokine production above that of control counterparts. These results altogether indicate that, by producing IFN-γ, TCRαβ⁺CD8⁺ cells contribute for local and systemic host protection in the earliest days upon infection established through the gastrointestinal tract. Nevertheless, they also provide substantial evidence for a parasite-driven reinforcement of T regulatory cell function which may contribute for parasite persistence in the host and might represent an additional barrier to overcome towards effective vaccination.