Epidemiologic study of environmental sources in a Prototheca zopfii outbreak of bovine mastitis

Bovine mastitis represents the main form of occurrence of protothecosis in animals. The detection of mastitis caused by Prototheca sp. indicates a serious problem which can affect an entire herd. The purpose of this study is to explain some aspects of the epidemiology of mastitis due to Prototheca zopfii with the evaluation of the presence of these microorganisms in samples collected from potential sources in the dairy herd. This study was performed during a Prototheca zopfii outbreak of clinical bovine mastitis in the State of São Paulo, Brazil. The following samples were aseptically collected for microbiological examination: milk (n = 211); rectal swabs (from 15 calves and 2 lactating cows); swabs from teat cup rubbers during milking (n = 2); water (n = 6); soil (n = 6). Prototheca zopfii was isolated from 77 (36.49%) of the 211 milk samples; 11 calves and 2 cows showed Prototheca zopfii in faecal samples; both swabs collected from the teat cup rubbers showed viable forms of Prototheca zopfii; this microorganism was also isolated from 2 water samples, and 1 soil sample collected from the dry cow pasture. Prototheca zopfii seemed to be widespread throughout the dairy herd environment where this outbreak of bovine mastitis occurred.This revised version was published online in October 2005 with corrections to the Cover Date.     

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72–75 °C/1 5 seconds, 72–75 °C/20 seconds and 62–65 °C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. asrepresenting a public health risk. This revised version was published online in June 2006 with corrections to the Cover Date.     

First large-scale isolation of Prototheca zopfii from milk produced by dairy herds in Italy

A total of 1045 milk samples collected from infected and non infected quarters of 269 cows were investigated. This study showed that 4.7% of samples possessed cells of Prototheca spp. (10⁶cells/ml). The presence of other pathogenic microorganisms was also monitored. Prototheca spp. isolates were classified on the basis of current taxonomic guidelines and identified as P. zopfii. Susceptibility tests carried out in vitro by using 25 antibiotic compounds revealed that the strains of P. zopfii. were susceptible only to nystatin and amphotericin B (58 and 33% of total strains, respectively). The present study represents the first large-scale investigation carried out in Italy on the isolation of this achlorophyllous yeast-like microalga in milk samples produced by dairy herds.     

Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate,silver nitrate and chlorexidine

One of the most important forms of the occurrence of protothecosis is bovine mastitis. Studies on the in vivo and in vitro susceptibility to antimicrobials have shown that the microorganism is resistant to most of them. Looking for alternative treatments this study aimed to study the susceptibility to copper sulphate (which has an important algicide effect) and silver nitrate (used in dairy cattle breeding for the cauterization of mammary glands) and also to chlorexidine (an important post-dipping anti-septic used in dairy practice), and the effect of these antimicrobials in the ultrastructure of Prototheca zopfii before and after the exposure to these drugs. The in vitro susceptibility tests to chlorexidine, silver nitrate and copper sulphate of the strains of Prototheca zopfii for the determination of their minimal microbicidal concentrations (MMC), were performed using the tube dilution method in Sabouraud dextrose broth and evaluation of colony growth after plating in Sabouraud dextrose agar. The MMCs of chlorexidine, copper sulphate and silver nitrate of the 50 strains tested were 0.01%, 0.1% and 0.3%, respectively. The tubes containing the material used in the antimicrobial susceptibility tests were prepared for the examination in an electron microscope. The untreated controls of P. zopfii showed a similar ultrastructural appearance with the typical characteristics of the microorganism. Cells exposed to silver nitrate showed changes suggesting thickness of the cell wall. Cells exposed to chlorexidine showed changes suggesting degradation of intra-cellular organelles present in the cytoplasm. P. zopfii treated with copper sulphate showed changes suggesting fibrilation of inner layer of cell wall.This revised version was published online in October 2005 with corrections to the Cover Date.     

Epidemiological and Clinical Aspects of the First Outbreak of Bovine Mastitis Caused by Prototheca zopfii in Goiás State, Brazil

The purpose of this survey was to describe the occurrence of bovine mastitis caused by Prototheca zopfii in Goiás State, Brazil. Samples of milk, environment and udder were taken from a herd of 120 Holstein cows. Sabourauds dextrose agar plates were incubated under aerobic conditions at 37 °C/96 h, for microbiological analysis. Somatic cell count and milk composition were also determined. Histological sections from two udders were stained with HE and PAS. Prototheca zopfii was identified in six cows whose milk had a watery appearance. They also showed a pronounced decrease in milk yield, fat and lactose. Pronounced infiltration of mononuclear cells, atrophy of alveoli and fibrosis were observed. The presence of this agent in other herds in the State is highly likely.     

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk

Aims: To develop a PCR‐based assay to detect Prototheca zopfii (P. zopfii) and its mastitis‐related subtype (genotype 2) directly from milk samples. Methods and Results: The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA‐binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10² colony‐forming units (CFU) ml^?1 for P. zopfii and 5 × 10³ CFU ml^?1 for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. Conclusions: The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. Significance and Impact of the Study: The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.     

Two‐dimensional proteome reference map of Prototheca zopfii revealed reduced metabolism and enhanced signal transduction as adaptation to an infectious life style

Biochemical, serological, and genetic analyses have identified two genotypes of Prototheca zopfii, a unicellular microalga belonging to the family Chlorellaceae. The P. zopfii genotype 1, abundantly present in cow barns and environment, remains nonpathogenic, while P. zopfii genotype 2 has been isolated from cows with bovine mastitis. The present study was carried out to identify the protein expression level difference between the pathogenic and nonpathogenic strains of P. zopfii. A total of 782 protein spots were observed on the 2D fluorescence difference gel electrophoresis (2D DIGE) gels among which 63 and 44 proteins were identified to be overexpressed in genotypes 1 and 2, respectively. The limited number of protein entries specific for Prototheca in public repositories resulted mainly in the identification of proteins described in other algae, microorganisms, or plants. Gene ontology (GO) analysis indicated reduced carbohydrate metabolism in genotype 1, while genotype 2 displayed enhanced DNA binding, kinase activity, and signal transduction. These effects point to metabolic and signaling adaptations in the pathogenic strain and provide insights into the evolution of otherwise highly similar strains. All MS data have been deposited in the ProteomeXchange with identifier PXD000126.     

Diagnosis of Clinical Bovine Mastitis by Fine Needle Aspiration Followed by Staining and Scanning Electron Microscopy in a Prototheca zopfii Outbreak

Fruit anthracnose of ugurassa caused by Colletotrichum acutatum is hereby reported for the first time in Sri Lanka and it is proposed that C. acutatum is considered together with C. gloeosporioides, as a causal agent of this disease. C. acutatum was characterised by fusiform conidia and white to orange colonies with slight shades of light mouse grey aerial mycelia. C. gloeosporioides produced grey colonies with a dark mouse grey centre and conidia were cylindrical. The other differences between the ugurassa isolate of C. acutatum and C. gloeosporioides were slower growth at temperatures ranging from 15-30 degrees C and extremely high tolerance of two fungicides, carbendazim and thiophanate methyl.     

Dermatophytosis of pigs by Trichophyton mentagrophytes

We describe the isolation of Trichophyton mentagrophytes in two family-operated farms where the animals were suffering skin ailments characterized by a swelling and a reddening of the back and flanks.This condition affected 2 and 5% respectively of the animals on the farms, the younger ones being more frequently affected.     

Chafing in fishes: occurrence,ontogeny, function and evolution

Synopsis Modal action patterns used in maintenance behavior have often been included in animals' communication repertoires during evolution, yet for fish neither the occurrence nor subsequent ritualization of maintenance behavior has been demonstrated. We review the literature and show that one maintenance behavior, the chafe, occurs in at least 30 families of fish. The chafe thus appears to offer a fruitful tool for the phylogenetic analysis of behavior of fish. We show that chafing in Etroplus maculatus and Xiphophorus helleri functions to remove sources of irritation from their skin. We also show that during ontogeny and when in social groups the frequency of chafing does not change with age in E. maculatus and E. suratensis. Etroplus suratensis have about twice the body surface and chafe twice as often as E. maculatus. Chafe-like behavior in Etroplus is observed during ontogeny when young glance on their parents' body. Young E. maculatus without parents present chafe more frequently than young with parents present suggesting that chafing on the substrate may substitute for parent contacting. Chafing is also included in adult behavior where it occurs during pair-formation, nest relief behavior and as an interspecific signal performed by the cleaner E. maculatus upon the host E. suratensis. The maintenance chafe and glance upon the parent both occur rapidly while the chafe performed during pair-formation is slow suggesting a degree of ritualization.     

Natural occurrence of Azospirillum brasilense in strawberry plants

Azospirillum species are free-living nitrogen-fixing bacteria commonly found in soil and in association with roots of different plant species. For their capacity to stimulate growth they are known as plant growth-promoting bacteria (PGPB). In this work, we demonstrate the natural occurrence and colonization of different parts of strawberry plants by Azospirillum brasilense in the cropping area of Tucumán, Argentina. Although bacteria isolations were carried out from two strawberry cultivars, e.g., Camarosa and Pájaro, attempts were successful only with the cultivar Camarosa. Whereas different strains of Azospirillum were isolated from the root surface and inner tissues of roots and stolons of the cultivar Camarosa, we have not obtained Azospirillum isolates from the cultivar Pájaro. After microbiological and molecular characterization (ARDRA) we determined that the isolates belonged to the species A. brasilense. All isolates showed to have the capacity to fix nitrogen, to produce siderophores and indoles. Local isolates exhibited different yields of indoles production when growing in N-free NFb semisolid media supplemented or not with tryptophan (0.1 mg ml⁻¹). This is the first report on the natural occurrence of A. brasilense in strawberry plants, especially colonizing inner tissues of stolons, as well as roots. The local isolates showed three important characteristics within the PGPB group: N₂-fixation, siderophores, and indoles production.     

Natural occurrence of toxigenic fungi and mycotoxins in rice bran

Thirty four samples of rice bran, of which 9 were from raw (untreated) rice (RR) and 25 from parboiled rice (PbR) were collected from commercial rice mills in and around Madras and analysed for storage mycoflora and mycotoxins. Fungi of the Aspergillus flavus group were present in 29 of the 34 samples (8 from RR and 21 from PbR) in quantities ranging from <1–432 thousand propagules/g, though not always as the dominant mycoflora. Fungal numbers were usually higher in RR than in PbR samples.Five of the 9 RR samples and 6 of the 25 PbR samples were positive for aflatoxins. Among 29 isolates of A. flavus obtained from the bran samples, 16 isolates –6 from RR bran and 10 from PbR bran — were found to be toxigenic in vitro. Some isolates of A. candidus also seemed to produce aflatoxin and other fluorescent substances.     

Natural occurrence of aflatoxin andAlternaria mycotoxins in oilseed rape from Catalonia (Spain): Incidence of toxigenic strains

During an investigation of the mycoflora on oilseed rape, the predominant fungal species present in 20 samples collected from Catalonia (Spain) wereAlternaria alternata (Fries) Keissler,Penicillium spp. andAspergillus flavus. None of the 20 samples analyzed presented contamination byAlternaria mycotoxins (tenuazonic acid, alternariol, alternariol methyl ether, altertoxin I and altertoxin II). Only aflatoxin B₁ was detected in 1 of the 20 samples analyzed, with a concentration of 0.25 ppb. Of the 40Aspergillus flavus strains isolated from oilseed rape samples, only 3 revealed aflatoxigenic capacity. None of thePenicillium spp. isolated from oilseed rape samples revealed mycotoxigenic capacity (citreoviridin, griseofulvin, citrinin, patulin and penicillic acid).     

Natural occurrence of fumonisins and their correlation to Fusarium contamination in commercial corn hybrids growth in Argentina

Fifty commercial corn hybrids with different endosperm characteristics, vegetative cycle length and cross class grown in the same geographical area (Cordoba Province, Argentina) were analysed for fumonisin accumulation. All hybrids analysed showed fumonisin B₁ and B₂ contamination ranging from 185 to 27,050 ng/g for FB₁ and from 40 to 9950 ng/g for FB₂. Although most of the hybrids analysed had flint-type endosperm, two hybrids with dent-type endosperm (e.g. Prozea 10 and AX 746) showed the highest level of fumonisin (37,000 ng/g) and more FB₂ than FB₁ (FB₂/FB₁ ratio 2.42), respectively. There was no correlation between fumonisin concentration and length of the vegetative cycle. Among 18 hybrids examined for Fusarium species contamination there was also no correlation between fumonisin contamination and the level of infection with Fusarium species (Section Liseola). Eighteen hybrids showed fumonisin levels lower than 1000 ng/g. This result suggests that there is some possibility of selecting hybrids resistant or less susceptible to fumonisin and Fusarium contamination.     

Concentrations of copper, iron, and zinc in the major organs of the wistar albino and wild black rats

The concentrations of copper, iron, and zinc in the major organs of Wistar albino (Rattus norvegicus) and wild black rats (Rattus rattus) were measured by means of atomic absorption spectroscopy. The copper levels in the kidneys and liver of the Wistar albino rats (WARs) were significantly higher (p<0.05) than in the wild black rats (WBRs). There were no significant differences in the concentrations of zinc in the liver, lungs, kidneys, and brain between the two study groups, but zinc was significantly higher in the spleen (p<0.05) and lower in the heart (p<0.05) of WAR, compared to WBRs. Iron was significantly higher (p<0.05) in the heart and spleen of WBRs, compared to WARs. There were no extreme differences in the organ concentrations of trace elements between the two species, but, cumulatively, the WARs tend to have higher metallic concentrations in their system than the WBRs. The potential of these differences on the experimental results should not be overlooked and will serve as basis to further consider the complex interrelationships of these animals in their microenvironments and macroenvironments.     

日粮添加褐藻糖胶对断奶仔猪抗炎能力和肠道微生物多样性的影响

【目的】本试验旨在研究日粮添加海带提取物褐藻糖胶对断奶仔猪生长性能、营养物质消化率、机体免疫力和肠道微生物多样性的影响。【方法】试验选用36头初始体重为(7.43±0.12) kg的健康仔猪,按照随机区组设计分为3组,每组12头。日粮处理组分别为不含抗生素的基础日粮组、抗生素组和褐藻糖胶组;试验期为28d。评价褐藻糖胶对仔猪生长性能和营养物质消化率的影响;通过比色法和酶联免疫吸附法检测血清中与免疫相关的指标;通过16S rRNA扩增子高通量测序检测试验第0、14和28天肠道微生物多样性。【结果】日粮添加褐藻糖胶可降低试验0–14 d仔猪耗料增重比(P0.05),但对试验全期仔猪平均日增重和平均日采量无显著影响(P0.05)。与对照组相比,饲喂褐藻糖胶日粮后,仔猪的中性洗涤纤维和酸性洗涤纤维表观消化率显著提高(P0.05);仔猪饲喂抗生素和褐藻糖胶日粮后,血清IL-22含量显著降低。试验第14天,抗生素组和褐藻糖胶处理组中Bacteroidetes数量呈上升趋势(P=0.07);试验第28天,抗生素组和褐藻糖胶处理组Actinobacteria丰度显著高于对照组(P0.05),且褐藻糖胶处理组Bacteroides属的菌群丰度显著高于对照组和抗生素组。【结论】日粮添加褐藻糖胶提高了断奶仔猪纤维养分消化率和拟杆菌属的丰富度和多样性,并且降低了促炎性细胞因子IL-22含量,这有助于缓解仔猪的断奶应激反应,建立稳定健康肠道菌群。     

Impact of Enterococcus faecium on specific activity of disaccharidases in small intestine of gnotobiotic pigs

The aim of the present work was to study the changes in the activity of disaccharidase enzymes (lactase. maltase, saccharase) in the small intestine of gnotobiotic pigs aged 0–35 days and inoculated with Enterococcus faecium. The continual decrease of lactase activity was observed from the 14^th day of age up to the end of the experiment. The most significant decrease of specific lactase activity in the duodenum (2.1 μmol/mg protein/hour) was noted from the 21^st to the 28^th day of age. On the other hand, the specific saccharase activity increased moderately during the post weaning period and maltase activity maintained a constant level. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Natural occurrence of Wolbachia-infected and uninfected Trichogramma species in tomato fields in Portugal

Minute egg parasitoids of the genus Trichogramma (Hymenoptera; Trichogrammatidae) are promising candidates for biological control of lepidopteran pests in tomato in Portugal. This certainly applies to native Trichogramma strains that have thelytokous reproduction, i.e., produce only daughters. In Trichogramma wasps, thelytoky is mostly induced by the intracellular bacterium Wolbachia. In this study, we carried out a field survey of native Trichogramma species in four locations in Ribatejo, the main processing tomato region of Portugal, and determined the prevalence of Wolbachia in those species. Five Trichogramma species were found to emerge from lepidopteran eggs collected in the field, namely Trichogramma bourarache, Trichogramma cordubensis, Trichogramma evanescens, Trichogramma pintoi, and Trichogramma turkestanica. T. evanescens and T. pintoi were by far the dominating species representing, respectively, 64.9 and 26.4% of the trichogrammatids collected. Total natural parasitism rates of the collected lepidopteran eggs by Trichogramma wasps ranged from 28.2 to 64.6%. Three Trichogramma species were found to be infected with Wolbachia, namely T. cordubensis, T. evanescens, and T. turkestanica. All the wasp broods belonging to T. cordubensis were infected, whereas low infection rates were found in T. evanescens (0.9% of the broods) and T. turkestanica (4.5% of the broods). The latter represents the first record of a Wolbachia infection in T. turkestanica. Sequencing of the Wolbachia surface protein, wsp, revealed this Wolbachia infection to be related to other Wolbachia infections in Trichogramma wasps. As Wolbachia-infected thelytokous strains exist for T. evanescens, the most abundant Trichogramma species naturally occurring in the tomato fields of the Ribatejo region, this species offers interesting and powerful options for biological control of lepidopteran pests in processing tomato in this region.     

Natural occurrence of Bacillus thuringiensis in leguminous phylloplanes in the New Delhi region of India

Bacillus thuringiensis (Bt) isolates were present on the phylloplanes of chickpea (Cicer arietinum), pigeon pea (Cajanus cajan), pea (Pisum sativum) and mung bean (Vigna radiata). Bt index (ratio of the number of Bt colonies to the total number of spore-forming colonies per g of leaves) differed significantly among these plants, with the highest (0.20) in the chickpea phylloplane, followed by pigeon pea (0.17). Bt population of the chickpea phylloplane varied with plant age, being maximal in 45-day-old plants. Diversity was observed among Bt isolates for growth (up to 10-fold difference), antibiotic resistance, PCR product profile and toxicity to Helicoverpa armigera. Two isolates with high activity towards H. armigera were found. This revised version was published online in July 2006 with corrections to the Cover Date.