Humane Endpoints in Swiss Webster Mice Infected with Toxoplasma gondii RH Strain.

The highly virulent Toxoplasma gondii RH strain is maintained through successive passages in mice, but there is still a lack of studies that refine these procedures from a 3Rs perspective, where humanitarian ideals aim to minimize the stress, pain, or suffering of the animals used in the research without the loss of results. The aim of this study was to establish humane endpoints in Swiss Webster mice inoculated with the T. gondii RH strain. A total of 52 mice were infected with 5 × 106 tachyzoites/mL and monitored for periods of up to 5 days. The parameters body weight; hair condition; higher than normal body temperature; hypothermia; respiratory function; pain; soft stools or diarrhea; bloody diarrhea; tense, nervous, or in distress during handling; and ascites were recorded daily in score tables. The results showed that prominent piloerection, respiratory function, pain parameters, and ascites are important clinical signs to be used as a cut-off point for implementing euthanasia. The application of this refinement method helped to avoid animal suffering and pain without compromising the number of parasites recovered. We therefore suggest adopting these parameters in research protocols that require the maintenance of the T. gondii RH strain in murine models to avoid and reduce animal suffering.

Effect of benznidazole on cerebral microcirculation during acute Trypanosoma cruzi infection in mice.

Central nervous system alterations was described in Chagas disease in both human and experimental models, leading to meningoencephalitis, stroke and cognitive impairment. Recently, our group demonstrated that acute infection by Trypanossoma cruzi leads to cerebral microvasculophaty in mice with endothelial dysfunction, capillary rarefaction, increased rolling and leukocyte adhesion. Only benznidazole and nifurtimox are available for clinical treatment, they have an efficiency of 80% in the acute phase and less than 20% in chronic phase. However, the effect of these drugs on brain microcirculation has not yet been evaluated. We hypothesized that early treatment with benznidazole could protect brain microcirculation during acute experimental Chagas disease. Swiss Webster mice were inoculated with 104 trypomastigotes forms of T. cruzi, and after 24 h they were treated with 50 or 100 mg/kg/day of benznidazole for 14 consecutive days. In untreated infected mice, we observed cerebral microvascular rarefaction, increase in leukocyte rolling and adhesion, reduced cerebral blood flow, and increased CD3+ and F4-80+ cells in brain tissue. Early treatment with benznidazole at 100 mg/kg/day and 50 mg/kg/day prevented the occurrence of the alterations mentioned. Here, we show that BZ is able to protect the microcirculation and reduced brain inflammation in acute experimental Chagas disease.

Housing Density and Aggression in Syrian Hamsters.

The Syrian hamster (Mesocricetus auratus) is a solitary and naturally territorial animal, with female hamsters being more aggressive than males. This behavior makes handling difficult because they are usually housed in groups, which can lead to aggressive behavior. The objective of this study was to refine the management of Syrian hamsters in order to minimize aggressiveness, reduce the animal injuries, and lessen the risk of accidents among laboratory animal technicians due to the hamster aggression during handling. The experiment was conducted at the Center for Animals Experimentation, Oswaldo Cruz Institute. Four groups of hamsters were observed by video recording: group 1 (group-housed males, 6 to 8 wk of age), group 2 (group-housed females 6 to 8 wk of age), group 3 (group-housed female, 3 to 4 wk of age), and group 4 (individually housed females, 6 to 8 wk of age). Group 1 animals were less aggressive and agitated both during housing and during handling by the animal technician as compared with groups 2 and 3. Groups 2 and 3 showed greater agitation and aggression. Marked reduction in the level of aggressiveness and agitation was observed in group 4 as compared with all other groups evaluated during handling by the animal technician. Male hamsters housed in groups of 4 and females housed individually have reduced risks of accident during handling, thereby averting distress and consequent physiologic alterations. Avoiding these risks is essential to obtaining reliable experimental results.

Higher sensitivity of immunohistochemistry for bona fide diagnosis of dog Leishmania (Viannia) braziliensis-driven American tegumentary leishmaniasis: description of an optimized immunohistochemistry method.

BACKGROUND: The in situ detection of parasite antigens in tissue sections by immunohistochemistry (IHC) is a diagnostic alternative for human American tegumentary leishmaniasis (ATL), but has not been used for the diagnosis of cutaneous lesions in dogs with ATL. This study describes the results of IHC for the detection of amastigote forms and other Leishmania sp. antigen-positive cells and compares the results of IHC, histopathology and cytopathology for the diagnosis of canine ATL. In addition, possible cross-reactivity with sporotrichosis is analyzed. METHODS: Forty paraffin-embedded biopsies and 40 smears of cutaneous lesions from dogs with ATL, confirmed by isolation and characterization of Leishmania (Viannia) braziliensis, and 40 paraffin-embedded biopsies of cutaneous lesions from dogs with sporotrichosis, confirmed by isolation of Sporothrix schenckii in culture (control group), were studied. RESULTS: Immunohistochemistry was more sensitive in detecting amastigote forms than cytopathology and histopathology, with a positivity rate of 70% (n=28) versus 37.5% and 22.5% for histopathology and cytopathology, respectively. Cytoplasmic staining of mononuclear and endothelial cells was detected by IHC, which was highly specific since no cytoplasmic staining of these cells or staining of fungal structures was observed in sporotrichosis fragments. CONCLUSIONS: In view of the higher sensitivity of IHC in detecting Leishmania sp. antigen and patterns of positivity for Leishmania sp. antigen compared to histopathology or cytopathology and the absence of cross-reactions with sporotrichosis, we recommend this technique for the diagnosis of canine tegumentary leishmaniasis.

Atypical feline sporotrichosis resembling vaccine-induced sarcoma: clinical and histopathological aspects.

A 7-year-old Siamese cat presenting with three ulcerated cutaneous nodules in the lumbosacral region was seen at the Laboratory for Clinical Research on Dermatozoonoses in Domestic Animals in Rio de Janeiro, Brazil. Histopathological analysis showed that the lesions consisted of polyhedral and spindle-shaped voluminous mononuclear cells with loose chromatin and clearly visible nucleoli, few giant cells, and foci of coagulative and caseous necrosis -- findings suggestive of a vaccine-induced sarcoma. No significant mitotic rate, cytological atypias or asteroid bodies were observed. Special histopathological staining with periodic acid-Schiff and Grocott's silver stain demonstrated the presence of small yeast cells characterized by simple and narrow-base budding compatible with Sporothrix schenckii. Mycological culture grew S schenckii. Cytopathology was negative for yeast cells. These atypical clinical and histopathological signs support the importance of histopathological analysis with special staining techniques, in addition to mycological culture in the diagnosis of feline sporotrichosis.

Sporotrichosis: the main differential diagnosis with tegumentary leishmaniosis in dogs from Rio de Janeiro, Brazil.

Seventy-four dogs from the State of Rio de Janeiro with ulcerated cutaneous lesions were submitted to clinical, dermatological, parasitological, mycological, histopathological and cytopathological exams, a leishmanin skin test, an indirect immunofluorescence (IIF) test for leishmaniosis, and nonspecific laboratory tests such as blood count and serum biochemistry. Sporothrix schenckii was isolated from 41 dogs and Leishmania (Viannia) braziliensis was isolated from 33 animals. Most dogs with sporotrichosis were from the municipality of Rio de Janeiro (53.7%) and presented ulcerated cutaneous lesions on the head (68.3%). Laboratory alterations in these animals included anemia (58.5%), hypoalbuminemia (83%) and hyperglobulinemia (75.6%). Histopathology revealed the predominance of a chronic granulomatous inflammatory infiltrate (70.7%), and yeast-like structures were detected in 17% of the histopathological exams and in 32% of the cytological exams. Three of 41 dogs with sporotrichosis were seropositive by IIF for leishmaniosis and 2 of 20 animals tested within this group had a positive leishmanin skin test. Similarly, most of the 33 dogs with leishmaniosis were from the municipality of Rio de Janeiro (69.7%) and had ulcerated cutaneous lesions on the head (84.8%). Laboratory alterations in these animals included anemia (66.7%), hypoalbuminemia (100%) and hyperglobulinemia (91%). Histopathology showed the predominance of a chronic granulomatous inflammatory infiltrate (63.6%) and amastigote forms were detected in 30.3% of the histopathological exams and in 31.8% of the 22 cytological exams performed. About 72.7% of the dogs were seropositive by IIF and five of seven animals had a positive skin test. Due to the clinical similarities, histopathological and nonspecific laboratory results similarities, the serological and skin tests for leishmaniosis positive in dogs with sporotrichosis, and the overlapping endemic areas in Rio de Janeiro, the differential diagnosis between the two diseases requires the demonstration of their respective etiological agents.