Comparison of three diagnostic techniques for the detection of leptospires in the kidneys of wild house mice (Mus musculus)

Forty-one wild house mice (Mus musculus) were trapped in an urban area, near railways, in Santa Fe city, Argentina. Both kidneys from each mouse were removed for bacteriological and histological examination. One kidney was inoculated into Fletcher semi-solid medium and isolates were serologically typed. The other kidney was microscopically examined after hematoxylin-eosin, silver impregnation and immunohistochemical stains. Leptospires, all of them belonging to the Ballum serogroup, were isolated from 16 (39%) out of 41 samples. The presence of the agent was recorded in 18 (44%) and in 19 (46%) out of 41 silver impregnated and immunohistochemically stained samples respectively. Additionally, leptospires were detected in high number on the apical surface of epithelial cells and in the lumen of medullary tubules and they were less frequently seen on the apical surface of epithelial cells or in the lumen of the cortical tubules, which represents an unusual finding in carrier animals. Microscopic lesions consisting of focal mononuclear interstitial nephritis, glomerular shrinkage and desquamation of tubular epithelial cells were observed in 13 of 19 infected and in 10 of 22 non-infected mice; differences in presence of lesions between infected and non-infected animals were not statistically significant (P=0,14). The three techniques, culture, silver impregnation and immunohistochemistry, had a high agreement (k³0.85) and no significant differences between them were detected (P>0.05). In addition, an unusual location of leptospires in kidneys of carrier animals was reported, but a relationship between lesions and presence of leptospires could not be established.

Evaluation of skin sensitivity in dogs bearing allergic dermatitis to standardized allergenic extract of house dust and storage mites

The objective of the study was to evaluate whether allergenic extracts of five house dust and storage mite species standardized for humans might be used for the diagnosis of canine atopic dermatitis (CAD). Extracts of Dermatophagoides pteronyssinus (Pyroglyphidae), D. farinae (Pyroglyphidae), Blomia tropicalis (Glycyphagidae), Lepidoglyphus destructor (Glycyphagidae) and Tyrophagus putrescentiae (Acaridae) were evaluated by intradermal testing in 20 healthy dogs (control) and 25 dogs with allergic dermatitis. A significant difference in the response was observed between the two groups (p<0.05). Only one dog (5%) in the control group reacted to the intradermal test, whereas 14 dogs (56%) in the allergic group were positive for at least one extract (odds ratio = 24.2). Most of the positive reactions observed in the allergic group occurred against the extracts of T. putrescentiae or L. destructor, each inducing reactions in 10 dogs (40%). D. farinae, D. pteronyssinus e B. tropicalis extracts induced reactions in 7 (28%), 3 (12%) and 3 (12%) dogs, respectively. The allergenic extracts standardized for humans evaluated in the present study may be used as a tool to complement the diagnosis of the disease, as well as to select potential allergen candidates for allergen-specific immunotherapy.

Serological identification of house dust mite allergens in dogs with atopic dermatitis

House dust mite antigens have been used for decades to diagnose allergic diseases in humans and animals. The objective of this study was to identify allergens in commercial Dermatophagoides farinae and Blomia tropicalis extracts by immunoblotting using sera from allergic dogs and anti-dog IgE conjugate. The analysis of antigens present in the D. farinae extract (FDA Allergenic) using sera from 10 dogs allergic to D. farinae showed that eight sera recognized a band of approximately 102 kDa, eight recognized two bands of 52 to 76 kDa, five recognized one band of approximately 76 kDa, four recognized one band of 31 to 38 kDa, and two recognized one band of 12 to 17 kDa. Immunoblot assays of the B. tropicalis extract (FDA Allergenic) using sera from 10 animals allergic to B. tropicalis showed that five sera recognized two bands of 52 to 76 kDa. These results demonstrate the importance of the two house dust mite species for the pathogenesis of canine atopic dermatitis in Brazil. In addition, the results indicate which allergens should be present in allergenic extracts used for diagnosis and allergen-specific immunotherapy.

Ocorrência de auto-incompatibilidade homomórfica em Mitracarpus longicalyx E. B. Souza & M. F. Sales (Rubiaceae - Spermacoceae)

O estudo da biologia reprodutiva de Mitracarpus longicalyx E.B. Souza & M.F. Sales foi realizado durante os meses de setembro a dezembro de 2004, em Feira de Santana, BA, Brasil. Esta espécie é uma erva anual ocorrente em áreas de caatinga, florescendo de junho a dezembro. As inflorescências são glomérulos com 90 flores em média, que apresentam corola hipocrateriforme, medindo 4-5 mm de comprimento e coloração branca, sendo visitadas e polinizadas por borboletas da espécie Hemiargus hanno hanno (Stoll, 1790) (Lycaenidae). As flores são protândricas, com duração da fase estaminada de três dias e da fase pistilada de cinco dias. Não ocorre sobreposição das duas fases na mesma flor, mas podem ocorrer flores em diferentes fases na mesma inflorescência. Polinizações experimentais indicaram que M. longicalyx é uma espécie auto-incompatível não agamospérmica, com produção natural de frutos elevada (97,8%) na população estudada. A frutificação também foi elevada nas polinizações cruzadas manuais (82,3%), não ocorrendo frutificação nas polinizações manuais geitonogâmicas. Tubos polínicos provenientes de polinizações cruzadas alcançaram o ovário dentro de 24 horas, enquanto que grãos de pólen provenientes de autopolinização não germinaram ou, quando germinaram, não penetraram as papilas estigmáticas. Os resultados indicam que M. longicalyx possui auto-incompatibilidade homomórfica, possivelmente do tipo esporofítica, atuando em adição à protandria no impedimento da autofertilização. Auto-incompatibilidade homomórfica é muito rara nas Rubiaceae, uma família com abundante ocorrência de auto-incompatibilidade heteromórfica.

Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ß turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

In-house method validation and occurrence of alpha-, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone and trifluralin residues in strawberry

A method for determination of organohalogen pesticides in strawberry by gas chromatography with electron capture detection was validated and applied in a monitoring program. Linearity, matrix effects, and day effect were evaluated for the analytes alpha-endosulfan, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone, and trifluralin. The linear range varied according to the chromatographic response of the analyte. Significant matrix effects were observed. The mean recoveries ranged from 74.6 to 115.4%, with repeatability standard deviations between 1.6 and 21.0% and intermediate precision between 5.9 and 21.0%. Detection, quantification and decision limit, and detection capacity ranged from 0.003 to 0.007 mg/kg, 0.005 to 0.013 mg/kg; 0.003 to 3.128 mg/kg; and 0.005 to 3.266 mg/kg, respectively. The method was fit for the purpose of monitoring organohalogen residues in strawberries. Residues of these pesticides were detected in 124 of the 186 samples analyzed between 2009 and 2011 in the state of Minas Gerais. Nine of them did not comply with the current legislation requirements; among them, seven (3.8%) had residues of unauthorized pesticide for the culture of strawberry, one (0.5%) had residues above the maximum residue limit, and another one (0.5%) exhibited both non-conformities.