Ana Carolina Alves

Molecular typing and differences in biofilm formation and antibiotic susceptibilities among Prototheca strains isolated in Italy and Brazil

Bovine mastitis caused by Prototheca is a serious and complex problem that accounts for high economic losses in the dairy industry. The main objective of this study was to identify and characterize at genetic level different Prototheca strains and provide the most complete data about protothecal antibiotic resistance. The study involves 46 isolates from Italian (13 strains) and Brazilian (33 strains) mastitic milk. These strains were identified by multiplex PCR and single strand conformation polymorphism analysis and characterized by randomly amplified polymorphic DNA (RAPD)-PCR. Moreover, biofilm production and antibiotic susceptibility were evaluated. Forty-two strains resulted as Prototheca zopfii genotype 2, whereas 4 isolates could belong to a potential new Prototheca species. The RAPD-PCR, performed with 3 primers (M13, OPA-4, and OPA-18), showed a notable heterogeneity among isolates and grouped the strains according to the species and geographical origin. Biofilm production was species-dependent and P. zopfii genotype 2 strains were classified as strong biofilm producers. In vitro antibiotic susceptibility tests indicated that Prototheca strains were susceptible to antibacterial drugs belonging to aminoglycosides group; the highest activity against Prototheca strains was observed in the case of colistin sulfate, gentamicin, and netilmicin (100% of susceptible strains). It is interesting to note that all the Italian P. zopfii genotype 2 strains showed lower minimum inhibitory concentration values than the Brazilian ones. Nisin showed more efficacy than lysozyme and potassium sorbate, inhibiting 31% of the strains. Results obtained in this study confirmed that RAPD-PCR is a rapid, inexpensive, and highly discriminating tool for Prototheca strains characterization and could give a good scientific contribution for better understanding the protothecal mastitis in dairy herd.

Efeitos de dietas com níveis crescentes de milho no metabolismo ruminal de energia e proteína em bubalinos

The effects of diets with increasing ground corn grain levels (0, 22, 37 and 49% in the dry matter) substituting coast-cross hay, with different protein:non-fibrous carbohydrate ratios (CP:NFC = 1.01; 0.39; 0.33 and 0.27) were assessed on buffalo ruminal metabolism. Four rumen fistulated buffaloes were used kept in a 4 × 4 latin square design, to collect liquid ruminal samples, collected in each experimental period (28 days) at 0, 2, 4 and 8 hours after feeding. In general, the buffalo presented good buffering capacity in the rumen, with high mean pH (6.70) and increase in ground corn intake. The increase in the corn levels in the diet resulted in increased butyric acid production. Only the diet with 49% corn showed better ruminal fermentation, with a small proportion of acetic:propionic acids. The CP:NFC ratio of 1.01 indicated energy deficiency in the diet available for microorganisms in the rumen throughout the day, although the diets with CP:NFC between 0.39 and 0.27 resulted in similar ruminal fermentations, that indicated synchronization in nitrogen and energy use by the rumen microorganisms under these conditions.

Virulence markers associated with Trueperella pyogenes infections in livestock and companion animals

Trueperella pyogenes is an opportunistic pathogen that causes diverse pyogenic infections in livestock. The genes that encode the exotoxin pyolysin (plo) and other putative factors that promote adhesion of pathogen to host cells (fimbriae fimA, fimC, fimE, fimG, neuraminidases nanH, nanP, and collagen‐binding protein cbpA) have been associated with virulence, particularly in mastitis and uterus infections of dairy cows. However, the role of these virulence markers in the pathogenicity of the agent in domestic animals infections still is incompletely understood. The genes plo, fimA, fimC, fimE, fimG, nanH, nanP, and cbpA were investigated in 71 T. pyogenes strains recovered from cattle, sheep, goats, dogs, equines, and a pig, recovered from mastitis (n = 35), and non‐mastitis (n = 36) cases (abscesses, reproductive tract diseases, pneumonia, lymphadenitis, encephalitis). The most common genes harboured by the isolates were: plo (71/71 = 100·0%), fimA (70/71 = 98·6%), nanP (56/71 = 78·9%), fimE (53/71 = 74·6%), fimC (46/71 = 64·8%) and nanH (45/71 = 63·4%), whereas cbpA (6/71 = 8·4%) and fimG (4/71 = 5·6%) were uncommon. The most frequent genotypes were plo/fimA/fimE/fimC/nanH/nanP (17/71 = 23·9%), plo/fimA/fimE/nanH/nanP (13/71 = 18·3%), and plo/fimA/fimE/fimC/nanP (11/71 = 15·5%). No association was observed between the presence of genes vs clinical signs or host species. To the best of our knowledge, this is the first report on aforementioned virulence factors of pathogen detected in diseased horses and dogs.

Identification of Mycobacterium species and Rhodococcus equi in peccary lymph nodes

Mycobacterium species and the virulence-associated proteins (vapA, vapB, and vapN genes) of Rhodococcus equi isolated from 330 lymph nodes of collared peccaries (Tayassu tajacu) and white-lipped peccaries (Tayassu pecari) intended for human consumption were investigated. Thirty-six (10.9%) R. equi strains were isolated; 3.3% (n = 11/330) were from white-lipped peccary lymph nodes, and 7.6% (25/330) were from collared peccary lymph nodes. Among the 11 isolates of R. equi from the white-lipped peccaries, 90.9% (n = 10/11) were obtained from the mesenteric lymph nodes, and only 9.1% (n = 1/10) were obtained from the mediastinal lymph nodes. In the 25 isolates of R. equi obtained from the collared peccaries, 40.0% (n = 10/25) were recovered from the mesenteric lymph nodes, 36% (n = 9/25) from the submandibular lymph nodes, and 24.0% (n = 6/25) from the mediastinal lymph nodes. No vapA, vapB, or vapN genes (plasmidless) or three host-associated types (pVAPA, pVAPB, and pVAPN) were identified among the R. equi isolates. Mycobacterium species were isolated in 3.03% (n = 10/330) of all the lymph nodes analyzed. Among the 10 mycobacterial isolates, 60% (n = 6/10) were from the white-lipped peccary lymph nodes, and 40% (n = 4/10) were from the collared peccary lymph nodes. Ten Mycobacterium species were detected by PCR-PRA with a predominance of M. avium type 1. Sequencing of the hsp65 and rpob genes revealed mycobacteria that were saprophytic (M. sinense and M. kumamotonense) and potentially pathogenic (M. colombiense and M. intracellulare) to humans and animals. To our knowledge, this is the first description of R. equi and/or mycobacterial species identified in the lymph nodes of peccary specimens. R. equi (plasmidless) and the mycobacterial species described here have been reported as causes of pulmonary and extrapulmonary infections in both immunocompetent and immunocompromised humans.

Prototheca blaschkeae subsp. brasiliensis subsp. nov., isolated from cow milk

A strain of an achlorophyllic alga, named PR24T, was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Protothecablaschkeae SAG2064T. However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24T (=DSM 103592T=IHEM 26958T).

Substituição parcial de silagem de milho por farelo de glúten de milho desidratado na alimentação de vacas holandesas em lactação

The objective was to study the effects of partial replacing of corn silage by corn gluten feed (CGF-21) dehydrated on milk production and composition of milk and in the blood parameters of dairy cows, beyond the economic evaluation. Inclusion levels of 0, 8 and 16% of CGF-21 (% DM) to the diets were evaluated. Thirty cows were used for study of milk production and composition and fifteen cows for blood parameters study. A 3 x 3 Latin Square experimental design with ten replications was used. There was difference among diets for milk yield and for 3.5% fat correct milk. No difference among diets was observed for the other studied characteristics. The results obtained for milk yield were: 22.44, 23.69 and 23.88 kg/anim.d and for 3.5% fat correct milk were: 23.25, 24.71 and 24.44 kg/anim.d, respectively, for the inclusionj levels of CGF-21 for the inclusion levels of 0, 8 and 16% of CGF-21 in the diet. The results obtained for the composition of milk were: percentage of fat, 3.69, 3.76 and 3.65%; milk protein, 3.22; 3.20 and 3.25%; lactose, 4.29, 4.32 and 4.31%; total solids, 12.12, 12.23 and 12.14%; N-ureic milk, 16.57, 16.50 and 14.96mg/dL, and for blood parameters were: plasma N ureic, 109.92, 21.17 and 20.21 mg/dL, seric glucose, 55.69, 56.50 and 54.78 mg/dL, respectively, for the inclusion levels of 0, 8 and 16% of CGF-21 in the diet. The diet with the inclusion of 16% of CGF-21 result on a greater profit.

Successful therapy in unusual generalized Dermatophilus congolensis infection in a calf based on modified in vitro disk diffusion test

Arq. Inst. Biol., v.84, 1-7, e0382017, 2017 RESUMO: A dermatofilose bovina é uma dermatite caracterizada por lesões focais ou localizadas com aspecto de “pincel” e, ocasionalmente, como lesão cutânea disseminada. Relata-se o caso de uma fêmea bovina de um ano de idade, que foi atendida apresentando história de lesões cutâneas crônicas imediatamente após um período de alta pluviosidade. Ao exame clínico, lesões serosas a purulentas, com hiperqueratose, coalescentes, não pruriginosas, ressecadas, de coloração amarelada à acinzentada foram observadas distribuídas de modo generalizado pelo animal. A remoção das crostas revelou áreas ulceradas ou hemorrágicas, com crostas irregulares e elevadas semelhantes a “pincel”. O diagnóstico microbiológico possibilitou a identificação do micro-organismo Dermatophilus congolensis. Apesar das lesões disseminadas e crônicas, a cura do animal foi obtida com tratamento parenteral usando oxitetraciclina de longa duração, baseado em teste in vitro de sensibilidade microbiana modificado. O presente relato ressalta o sucesso no tratamento de caso incomum de lesões generalizadas de dermatofilose bovina com respaldo de teste in vitro de sensibilidade modificado, bem como a necessidade do uso responsável de antimicrobianos em animais de produção.