Establishment of a microplate assay for flow cytometric assessment and it is use for the evaluation of age-related phenotypic changes in canine whole blood leukocytes

The effectiveness of flow cytometric assays for canine use is still requiring standardization. Despite several studies using purified mononuclear cells, no methodology or reference ranges are available for immunophenotyping of whole blood leukocytes (WBL). Fresh and pre-fixed WBL were used to identify cell-subsets, (Thy-1(+)/CD5(+)/CD4(+)/CD8(+)/CD21(+) and CD14(+)) and measure MHC-II, CD45RA/CD45RB expression. We described here an efficient method for fast quantification of canine-WBL, using pre-fix in a microplate assay, which allows long-term sample storage prior to phenotyping. Decreased percentage of CD5(+)-T-cells within the lymphocyte-gate and increased percentage of CD21(+)-B-cells were observed in young animals, which led to higher T/B cell ratios in middle-aged dogs. Lower numerical counts of Thy-1(+), CD4(+), CD8(+) and CD21(+) lymphocyte were observed when compared to young animals. In addition, we identified an age-related decline of MHC-II/CD45RA expression by lymphocytes. We proposed an improved method for phenotyping of canine peripheral blood mononuclear cells (PBMC) that has significant use for researchers and veterinary clinicians. The hematological changes of senescence previously identified on PBMC could be adequately reproduced on features identified by whole blood. Furthermore, this study supplies normal range references as baseline standards for clinical purposes, besides specific immunological parameters to monitor canine aging process.     

Sub-clinical infection as an effective protocol for obtaining anti-Leishmania chagasi amastigote antibodies of different animal species

This work aims at identifying an effective protocol to raise anti-Leishmania chagasi amastigote antibodies in different animal species. Protocols of immunization by subcutaneous injections of L. chagasi promastigote and amastigote lysates or by either intravenous or subcutaneous inoculation of live metacyclic promastigotes were assessed in mice, rabbits, and dogs. The immunization with live promastigotes produced a strong humoral immune response against L. chagasi amastigotes in all three animal species. The sera from animals immunized with the promastigote lysate did not react with amastigotes and, conversely, the sera from mice immunized with the amastigote lysate did not react with promastigotes. Taken all data together, the immunization through infection with metacyclic promastigotes was considered the most satisfactory way to immunize animals for obtaining anti-amastigote and anti-promastigote antibodies, since it did not only allowed the obtention of antibody against the two forms of the parasite, but it is also cheap, less laborious than carrying out the purification of amastigotes from infected tissues and avoid the use of a large number of hamsters for obtention the amastigotes, necessary to produce the immunogenic lysates. Furthermore, this immunization protocol was comparable to the amastigote lysate immunization protocol for the obtaining of mouse monoclonal antibodies (mAbs).     

Mycobacterium szulgai infection in a captive population of African clawed frogs (Xenopus tropicalis).

A colony of captive Xenopus tropicalis became infected with Mycobacterium szulgai. Clinical signs, when observed, were lethargy, weight loss, and emaciation. Visceral granulomas were common findings at laparoscopy and necropsy. The diagnosis of mycobacteriosis was based on histologic appearance and Ziehl-Neelsen staining of tissues. The identification of M. szulgai organisms was based on comparison of the 16S rRNA gene sequence with several GenBank databases. There have been no reports of this mycobacterial species as the causative agent of naturally occurring disease in amphibians.     

Current knowledge and future perspectives of the use of seaweeds for livestock production and meat quality: a systematic review

The effects of dietary macroalgae, or seaweeds, on growth performance and meat quality of livestock animal species are here reviewed. Macroalgae are classified into Phaeophyceae (brown algae), Rhodophyceae (red algae) and Chlorophyceae (green algae). The most common macroalga genera used as livestock feedstuffs are: Ascophyllum, Laminaria and Undaria for brown algae; Ulva, Codium and Cladophora for green algae; and Pyropia, Chondrus and Palmaria for red algae. Macroalgae are rich in many nutrients, including bioactive compounds, such as soluble polysaccharides, with some species being good sources of n-3 and n-6 polyunsaturated fatty acids. To date, the incorporation of macroalgae in livestock animal diets was shown to improve growth and meat quality, depending on the alga species, dietary level and animal growth stage. Generally, Ascophyllum nodosum can increase average daily gain (ADG) in ruminant and pig mostly due to its prebiotic activity in animal's gut. A. nodosum also enhances marbling score, colour uniformity and redness, and can decrease saturated fatty acids in ruminant meats. Laminaria sp., mainly Laminaria digitata, increases ADG and feed efficiency, and improves the antioxidant potential of pork. Ulva sp., and its mixture with Codium sp., was shown to improve poultry growth at up to 10% feed. Therefore, seaweeds are promising sustainable alternatives to corn and soybean as feed ingredients, thus attenuating the current competition among food-feed-biofuel industries. In addition, macroalgae can hinder eutrophication and participate in bioremediation. However, some challenges need to be overcome, such as the development of large-scale and cost-effective algae production methods and the improvement of algae digestibility by monogastric animals. The dietary inclusion of Carbohydrate-Active enZymes (CAZymes) could allow for the degradation of recalcitrant macroalga cell walls, with an increase of nutrients bioavailability. Overall, the use of macroalgae as feedstuffs is a promising strategy for the development of a more sustainable livestock production.     

Carbohydrate reserves as a competing sink: evidence from tapping rubber trees

Carbohydrate reserve storage in trees is usually considered a passive function, essentially buffering temporary discrepancies between carbon availability and demand in the annual cycle. Recently, however, the concept has emerged that storage might be a process that competes with other active sinks for assimilate. We tested the validity of this concept in Hevea brasiliensis Müll. Arg. (rubber) trees, a species in which carbon availability can be manipulated by tapping, which induces latex regeneration, a high carbon-cost activity. The annual dynamics of carbohydrate reserves were followed during three situations of decreasing carbon availability: control (no tapping), tapped and tapped with Ethephon stimulation. In untapped control trees, starch and sucrose were the main carbohydrate compounds. Total nonstructural carbohydrates (TNC), particularly starch, were depleted following bud break and re-foliation, resulting in an acropetal gradient of decreasing starch concentration in the stem wood. During the vegetative season, TNC concentration increased. At the end of the vegetative season, there were almost no differences in TNC concentration along the trunk. In tapped trees, the vertical gradient of starch concentration was locally disturbed by the presence of the tapping cut. However, the main effect of tapping was a dramatic increase in TNC concentration, particularly starch, throughout the trunk and in the root. The difference in TNC concentration between tapped and untapped trees was highest when latex production was highest (October); the difference was noticeable even in areas of the trees that are unlikely to be directly involved in latex regeneration, and it was enhanced by Ethephon stimulation, which is known to increase latex metabolism and flow duration. Thus, contrary to what could be expected if reserves serve as a passive buffer, a decrease in carbohydrate availability resulted in a net increase in carbohydrate reserves at the trunk scale. Such behavior supports the view that trees tend to adjust the amount of carbohydrate reserves stored to the level of metabolic demand, at the possible expense of growth.     

The animal's microbiome and cancer: A translational perspective

Cancer is a substantial global health problem both in humans and animals with a consistent increase in mortality and incidence rate. The commensal microbiota has been involved in the regulation of several physiological and pathological processes, both within the gastrointestinal system and at distant tissue locations. Cancer is not an exception, and different aspects of the microbiome have been described to have anti- or pro-tumour effects. Using new techniques, for example high-throughput DNA sequencing, microbial populations of the human body have been largely described and, in the last years, studies more focused on companions' animals have emerged. In general, the recent investigations of faecal microbial phylogeny and functional capacity of the canine and feline gut have shown similarities with human gut. In this translational study we will review and summarize the relation between the microbiota and cancer, in humans and companion animals, and compare their resemblance in the type of neoplasms already studied in veterinary medicine: multicentric and intestinal lymphoma, colorectal tumours, nasal neoplasia and mast cell tumours. In the context of One Health, microbiota and microbiome integrative studies may contribute to the understanding of the tumourigenesis process, besides offering an opportunity to develop new diagnostics and therapeutic biomarkers both for veterinary and human oncology.     

An eye on the Shih Tzu dog: Ophthalmic examination findings and ocular surface diagnostics

Objective

To characterize the ocular surface parameters and determine the prevalence of ocular pathology in Shih Tzu dogs.

Animal Studied

Fifty Shih Tzu dogs (28 male, 22 female).

Procedures

Each dog underwent a complete ophthalmic examination (recording any pathology) and a series of diagnostics, allowing for a 10 min-interval between tests: intraocular pressure (IOP), blink rate, palpebral fissure length (PFL), corneal tactile sensation (CTS), Schirmer tear test and nasolacrimal reflex without (STT-1, NL-STT1) and with topical anesthesia (STT-2, NL-STT2), tear ferning, strip meniscometry test (SMT), tear film breakup time (TFBUT), and punctate fluorescein staining (PFS) of the cornea.

Results

Mean ± SD test values were as follows: IOP (17.9 ± 3.7 mmHg), blink rate (2.4 ± 1.4 blinks/min), PFL (23.8 ± 1.8 mm), CTS (1.8 ± 0.7 cm), STT-1 (22.0 ± 5.5 mm/min), NL-STT1 (24.2 ± 4.7 mm/min), STT-2 (16.9 ± 6.5 mm/min), NL-STT2 (18.5 ± 7.5 mm/min), SMT (7.5 ± 3.5 mm/5 s), TFBUT (5.3 ± 2.4 s), tear ferning (1.3 ± 0.7), and PFS (1.6 ± 0.6). PFL was significantly greater in male vs. female Shih Tzus (p< .001). Age was negatively correlated with TFBUT results (r = −0.31, p = .027). Lagophthalmos was observed in 82% eyes. Ocular surface pathology was common, including adnexal abnormalities (100% eyes with caruncular trichiasis and medial lower lid entropion) and corneal opacification (27% pigmentation, 20% fibrosis, 12% neovascularization).

Conclusions

Qualitative tear film deficiency (low TFBUT), along with several anatomical abnormalities that promote ocular irritation and reduce globe protection, together help explain the concerningly high prevalence of ocular surface disease in the Shih Tzu breed. Prophylactic measures (e.g., medial canthoplasty, topical lubrication) could be considered to improve ocular health in Shih Tzus.     

Food safety in raw milk production: risk factors associated to bacterial DNA contamination

While human illness from milkborne pathogens may be linked to contamination of the product after pasteurization or improper pasteurization, such diseases are usually associated with consumption of raw milk or its by-products. Molecular biology tools were applied to investigate contamination by Listeria monocytogenes, Salmonella spp., some pathogenic strains of Escherichia coli, and Campylobacter jejuni in 548 raw milk samples from 125 dairy farms established in two regions from southern Brazil. Moreover, 15 variables were evaluated for their association with raw milk contamination levels, and the risk factors were determined by multiple regression analysis. Salmonella spp. were more frequently detected, followed by pathogenic E. coli. There was difference in contamination index between the regions, in which risk factors such as temporary cattle confinement, low milk production, low milking machine cleaning frequency, and milk storage area without tile walls were identified. The risk factors were specific to each region studied. Nevertheless, the data can be used to improve milk quality of dairy farms/herds with similar management practices.     

Grapevine trunk diseases: complex and still poorly understood

This review presents an overview of eutypa dieback, esca and botryosphaeria dieback, the predominant grapevine trunk diseases worldwide. It covers their symptomatologies in the trunk, leaves and berries; the characteristics of the different fungal species associated with them; and host–pathogen interactions. Here, the host–pathogen relationship is defined at the cytological, physiological and molecular levels. Currently available experimental tools for studying these diseases, both in vitro and in the field, are discussed. Finally, a progress report on their control, which, since the ban of sodium arsenite, comprises chemical, biological and/or sanitation methods, is presented.     

Pathogenic diversity of Phytophthora sojae pathotypes from Brazil

Phytophthora root and stem rot has developed in commercial soybean fields since 2006 in Brazil, and cultivars with resistance to this disease have not been targeted for this region. Thus, the Phytophthora sojae pathotypes are expected to have virulence to few if any of the Rps genes. The objectives of this study were to characterize the pathotype diversity of P. sojae in Brazil, determine the distribution of the pathogen and predict which Rps genes will be effective and should be used in breeding programs. Isolates were collected in six states (Rio Grande do Sul, Santa Catarina, Paraná, Mato Grosso do Sul, Minas Gerais, and Goiás). The virulence formulae were based on the response of a differential set with 14 Rps genes (1a, 1b, 1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6, 7, and 8). None of the 17 pathotypes found was reported previously. The most common virulence formulas were: 1d, 2, 3c, 4, 5, 6, 7 (octal code 05471, representing 24 % of the occurrences); 1d, 2, 3b, 3c, 4, 5, 6, 7 (05671, 13 %); 1b, 1d, 2, 3a, 3c, 4, 5, 6, 7 (25571, 8 %); and 1d, 3a, 5, 7, 8 (01123, 8 %). Percentages of isolates with a susceptible interaction with each Rps gene was Rps1a (3 %), Rps1b (11 %), Rps1c (3 %), Rps1d (100 %), Rps1k (3 %), Rps2 (86 %), Rps3a (32 %), Rps3b (19 %), Rps3c (73 %), Rps4 (70 %), Rps5 (89 %), Rps6 (59 %), Rps7 (100 %), and Rps8 (22 %). There was apparently no relationship between pathotypes and origin. Stacking resistance genes Rps1a, Rps1b, Rps1c, and Rps1k with Rps3b or Rps8 would be highly effective for soybean cultivars targeted for Brazil.