Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H. heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs.     

Application of long‐chain alcohols as diet‐composition markers in sheep fed on grass–white clover and heather–gorse plant species

Utilization of long‐chain alcohols (LCOH) as diet‐composition markers in sheep consuming six diets composed of improved pasture species (Lolium perenne and Trifolium repens) with heather–gorse components (Erica spp., Calluna vulgaris and Ulex gallii) was evaluated. Twenty‐four adult cross‐bred sheep were housed in individual stalls. Diet composition was estimated from LCOH concentrations, combined or not with alkanes and long‐chain fatty acid (LCFA) data using least‐squares procedures. Prior to calculations, faecal concentrations were corrected using mean treatment (faecal recovery 1, FR1) recoveries and mean recoveries across diets (FR2). Estimates were compared with those obtained without faecal correction (FR0) and known values. Large differences between plant species and plant parts were found in LCOH patterns and total LCOH concentrations. LCOH provided complementary information to that given by alkanes and LCFA. Even‐chain LCOH comprised the largest fraction, representing on average 0·894 of total concentrations. Faecal recovery was incomplete and tended to increase with carbon‐chain length (CCL) in a curvilinear manner (FR = ?7·872 + 0·580 × CCL ? 0·010 × CCL²; P < 0·001; r² = 0·752). Diet composition influenced (P < 0·001) LCOH recovery, although variability within (CV of 9·0%) and between (CV of 13·3%) diets was low. Accuracy of estimates was influenced (P < 0·001) by faecal‐correction method and markers used, and the best estimate was obtained combining LCOH and alkanes. Improvement in estimate accuracy can be achieved if suitable correction of LCOH faecal concentrations is performed prior to calculations. Results indicated that LCOH showed lower dependence than alkanes and LCFA on the use of accurate faecal‐correction data.     

Ultrastructural and cytochemical aspects of silicon-mediated rice blast resistance

ABSTRACT Although exogenous application of silicon (Si) confers efficient control of rice blast, the probable hypothesis underlying this phenomenon has been confined to that of a mechanical barrier resulting from Si polymerization in planta. However, in this study, we provide the first cytological evidence that Si-mediated resistance to Magnaporthe grisea in rice correlates with specific leaf cell reaction that interfered with the development of the fungus. Accumulation of an amorphous material that stained densely with toluidine blue and reacted positively to osmium tetroxide was a typical feature of cell reaction to infection by M. grisea in samples from Si+ plants. As a result, the extent of fungal colonization was markedly reduced in samples from Si+ plants. In samples from Si- plants, M. grisea grew actively and colonized all leaf tissues. Cytochemi-cal labeling of chitin revealed no difference in the pattern of chitin localization over fungal cell walls of either Si+ or Si- plants at 96 h after inoculation, indicating limited production of chitinases by the rice plant as a mechanism of defense response. On the other hand, the occurrence of empty fungal hyphae, surrounded or trapped in amorphous material, in samples from Si+ plants suggests that phenolic-like compounds or phytoalexins played a primary role in rice defense response against infection by M. grisea. This finding brings new insights into the complex role played by Si in the nature of rice blast resistance.     

Variable number of tandem aminoacid repeats in adhesion-related CDS products in Mycoplasma hyopneumoniae strains

The Mycoplasma hyopneumoniae genome contains at least 22 regions with a variable number of tandem nucleotide repeats (VNTRs) within coding DNA sequences (CDSs). In this work, the VNTR-containing CDSs were analysed in order to evaluate their degree of variation, possible correlations with antigenic properties, and their potential to be used as a basis for a strain typing PCR assay. We have analysed the VNTRs in five M. hyopneumoniae strains (J, 7448, 7422, PMS, and 232), based on published genomic sequences and on amplified and sequenced DNA segments. These VNTRs are distributed among 12 genes, most of which encode putative surface proteins, including known adhesins. The number of repeat units in any of the VNTRs is highly variable among the analysed strains, but they are, without exception, translationally in frame, and, therefore, code for a variable number of aminoacid repeats (VNTARs). These VNTARs determine putative structural, physicochemical and antigenic variations in the corresponding proteins, with potential implications for aspects associated to M. hyopneumoniae pathogenicity, such as cell adhesion and interactions with the host immune system. Considering that the characterized VNTARs are relatively stable, at least in vitro, and their sizes are strain-specific, we have developed a VNTR-based PCR assay for M. hyopneumoniae strain identification, useful for enzootic pneumonia (EP) diagnosis, strain typing, and distinction of circulating field isolates from vaccine strains in animals vaccinated against EP.     

Secreted phosphatase activities in trypanosomatid parasites of plants modulated by platelet-activating factor

ABSTRACT The secreted phosphatase activities of two trypanosomatid parasites were characterized and compared with supernatants of living cells. The plant parasite Phytomonas fran?ai and the phytophagous hemipteran parasite Herpetomonas sp. hydrolyzed p-nitrophenylphosphate at a rate of 15.54 and 6.51 nmol Pi/mg of protein per min, respectively. Sodium orthovanadate (N(a)VO(3)) and sodium fluoride (NaF) decreased the phosphatase activities. The phosphatase activity of P. fran?ai was drastically diminished (73% inhibition) in the presence of sodium tartrate, whereas the phosphatase activity of Herpetomonas sp. was inhibited by 23%. Cytochemical analysis showed the localization of these enzymes on the external surface and in the flagellar pocket of the two trypanosomatids. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor modulated the phosphatase activities, inhibiting P. fran?ai activity and stimulating Herpetomonas sp. phosphatase activity.     

Two successful embryo transfers of mini-donkey embryos in Brazilian Northeastern jennies using an alternative method: Case report

Pregnancy rates after embryo transfer (ET) are disappointing in donkey species. This study aims to report two successful ET of mini-donkey embryos using Brazilian Northeastern jennies as recipients. Eighteen embryo flushes were performed 9 days post-ovulation in two non-pregnant mini-donkeys jennies (11 and 7 cycles per jenny). Eleven embryos (61%, 11/18) were collected and transferred to Brazilian Northeastern jennies 4–6 days post-ovulation by conventional (n = 6) or an alternative (n = 5) technique. The alternative method consisted of inserting a Polansky equine vaginal speculum smeared with lubricant in the vagina of the recipient jenny. The arms of the speculum were extended to allow the visualization of the cervix. Then, using an adapted crafted, elongated, toothed tissue grasping forceps, the external cervical os was held, and the cervix was gently pulled backward, aiming to straight the cervical canal. The ET gun was inserted through the vagina and cervix by visual inspection, and the embryo was released into the uterine lumen. All embryos collected were Grade 1 and classified as Expanded Blastocysts. No jennies become pregnant after conventional ET (0/6), whereas two recipient jennies (40%, 2/5) become pregnant and delivered offspring in the following year after ET using the alternative technique. In conclusion, Brazilian Northeastern jennies can be used as embryo recipients using the alternative method proposed in the present study. However, further investigations are needed to improve the knowledge and results of ET in donkey species.     

Acute tolerance and histopathological effects of ammonia on juvenile maroon clownfish Premnas biaculeatus (Block 1790)

This study evaluated the median lethal concentration values (LC₅₀) and the histopathological effects of un‐ionized ammonia (NH₃^‐‐N) on juvenile maroon clownfish Premnas biaculeatus. After 96 h of exposure to different concentrations of ammonia, juveniles were sampled for histopathological evaluation. The 24 and 96 h LC50 values of NH₃^?‐N determined were 1.68 and 0.89 mg L^?1 respectively. Maroon clownfish exposed to different ammonia concentrations displayed histopathological alterations in the gills, kidney, liver and brain. Gill tissue damage included lamellar hyperplasia, lamellar shorting and hyperplasia and hypertrophy of mucous cells. The kidney showed hyperanaemia, enlarged sinusoids within an apparently decreased amount of haematopoietic tissue, oedema on tubular cells and tubular necrosis, and an enlarged Bowman's capsule. The liver presented dilatation of hepatic sinusoids, fatty deposition in hepatocytes and Mallory bodies. Examination of the brain revealed a proliferation of glial cells, and the Virchow‐Robin space indicated a severe perivascular oedema and signs of neuronal suffering with satellitosis. The results of this study indicate that juvenile maroon clownfish are relatively sensitive to ammonia and particular attention must be given to this toxic compound in culture systems.     

Growth attributes,xylem sap composition,and photosynthesis in common bean as affected by nitrogen and phosphorus deficiency

The effects of nitrogen (N‐) and phosphorus (P‐) deficiency, isolatedly or in combination, on growth, nitrogenous fraction, and inorganic phosphate in xylem exudade, and photosynthesis of common bean (Phaseolus vulgaris L. cv. Negrito) were investigated. Plants were grown in nutrient solution adjusted daily to pH 5.5 and aerated continuously. Ten days after emergence mineral deficiency was imposed. Plants were then supplied with high N (7.5 mol m^‐3) or low N (0.5 mol m^‐3), and also with high P (0.5 mol m^‐3) or low P (0.005 mol m^‐3). All sampling and measurements were made 28 days after emergence. N‐ or P‐deprivation brought about large decreases in total leaf area by inhibiting the emergence of new leaves and primarily the expansion of the leaves. The specific leaf area did not change under N‐ but decreased under P‐limitation. The decreased shoot to root ratio in all deficiency treatments was a consequence of a lowering mass of above‐ground organs, especially of leaves.

The content of chlorophylls declined significantly only under N‐deficiency alone; carotenoids declined under both N‐ and combined N‐ and P‐limitation. No alteration in amino acid concentration in xylem exudate occurred in plants experiencing N‐starvation, while ureides increased by 79%, and nitrate and inorganic phosphate decreased greatly. Under P‐deprivation, amino acids and nitrate in xylem sap dropped by about half; ureides were held relatively constant, and phosphate was severely depressed. Total upward translocation of N through xylem was estimated to be about 16% higher in N‐deficient plants than in plants without mineral limitation, but leaf N levels in the former were lower as compared to control plants. The net carbon (C) assimilation decreased similarly regardless of the imposed deficiency treatment. Such a decrease was mainly determined by non‐stomatal factors. In general, no additive effect between N‐ and P‐limitation on any of measured parameters was observed.     

Net requirements of calcium, magnesium, sodium, phosphorus, and potassium for growth of Nellore × Red Angus bulls, steers, and heifers

A comparative slaughter trial was conducted to assess the net requirements for gain of Ca, P, Na, K and Mg of bulls, steers and heifers of Nellore and Red Angus crossbreds. Twenty seven F1 Nellore and Red Angus crossbred calves (9 steers, 9 bulls, and 9 heifers), averaging 274 kg BW, were used. At the beginning of the trial, three animals from each gender were slaughtered to determine the initial body composition. The remaining 18 animals (3 animals of each gender) were randomly assigned to two treatments: fed 0.75 or 1.5% of BW of concentrate. The diets were based on corn silage and were isonitrogenous (2% N, DM). After three growing periods of 28 d, all animals were slaughtered. The cleaned gastrointestinal tract, organs, carcass, head, hide, tail, feet, and tissues were weighed to determine the empty BW (EBW). These parts were ground separately and subsampled for chemical analyses. The log of the contents of each mineral in the empty body was regressed on the log of the EBW to estimate the net requirement for each mineral per kg of empty body gain (EBG). There were no differences (P > 0.05) in the net requirements for growth of all macrominerals among genders. The equations of the pooled data of the net requirements for growth (g/kg EBG) were: 332.6 × EBW ^− 0.6367 for Ca, 112.1 × EBW ^− 0.5615 for P, 10.85 × EBW ^− 0.3992 for Na, 4.01 × EBW ^− 0.153 for K, and 3.589 × EBW ^− 0.462 for Mg. Our findings indicated that retained Ca and retained P were poorly related to the retained protein.