Implementation of a New Solution for the Preservation of Anatomical Specimens Made of Non-Toxic Substances

The preservation of anatomical pieces in Veterinary Anatomy is essential since it is not possible to dissect all domestic species. Most techniques use reagents with high levels of toxicity such as formaldehyde. The objective of this work was to develop a new preservation technique that uses reagents with zero toxicity and that allows obtaining preserved pieces suitable for anatomical studies. The alcohol propylene glycol technique was developed, the method of which uses a fixation step with alcohol, sodium chloride, commercial vinegar and subsequently the impregnation of the preservation solution made from propylene glycol and commercial vinegar, which are non-toxic. As a result of this work, adequately preserved sheep hearts were obtained that preserved their morphology with slight changes in size and weight, which did not affect their external and internal anatomical structure. Its coloration was not substantially affected, remaining a little lighter. The pieces obtained showed flexibility which allowed dissections to be carried out. The time to develop the technique was 20 days. A comparative study was carried out with the phenolated glycerin technique that uses toxic reagents (formaldehyde and phenol) and the pieces obtained with the alcohol propylene glycol technique were of better quality, observing that the pieces with phenolated glycerin tend to darken and are more rigid. And the time to develop the technique is 24 days. In conclusion, a preservation technique for anatomical pieces was developed that allowed the preservation of the organs under study, which allow their use for anatomical studies, and which have been preserved without changes until the time of this publication (8 months) and there are pieces preserved with this technique for 2 years.

Stress Biomarkers in the Giant Manta Mobula birostris Associated to Tourism in the Revillagigedo National Park, Mexico

A constant increase in dive tourism over the past years in the Revillagigedo National Park, Mexico, could result in a stressful scenario for giant mantas (Mobula birostris). The purpose of this study was to determine the degree of oxidative stress in terms of changes in catalase units (CAT) and muscle glycogen concentration in this species during two periods of different tourism intensity in this protected area. A total of 21 muscle biopsies were collected in March (peak tourism) and November (lower tourism), 2019. Stress biomarkers were analysed by commercial kits from the company Cayman Chemical. Oxidative stress (catalase activity) was significantly higher during the period with lower tourism (p = 0.002), compared to the period with more tourism, suggesting the presence of the general adaptation syndrome. In males, there was a significant difference (p = 0.0005) in oxidative stress between periods of different tourism intensity, suggesting that the reproductive season may be a stressor. Morphotypes showed different oxidative stress (p = 0.031);however, the reason is unknown. No statistical differences were detected in glycogen concentrations between the tourism periods (p = 0.123), probably because this polysaccharide is not a proper indicator of chronic stress in giant mantas. Based on these findings, giant mantas may have an adequate response in terms of oxidative stress due to an increase in tourism;however the observed increase in catalase suggests that it is within the tolerance range of these organisms.

Hepatitis C virus molecular evolution:Transmission,disease progression and antiviral therapy

Hepatitis C virus(HCV)infection represents an important public health problem worldwide.Reduction of HCV morbidity and mortality is a current challenge owned to several viral and host factors.Virus molecular evolution plays an important role in HCV transmission,disease progression and therapy outcome.The high degree of genetic heterogeneity characteristic of HCV is a key element for the rapid adaptation of the intrahost viral population to different selection pressures(e.g.,host immune responses and antiviral therapy).HCV molecular evolution is shaped by different mechanisms including a high mutation rate,genetic bottlenecks,genetic drift,recombination,temporal variations and compartmentalization.These evolutionary processes constantly rearrange the composition of the HCV intrahost population in a staging manner.Remarkable advances in the understanding of the molecular mechanism controlling HCV replication have facilitated the development of a plethora of direct-acting antiviral agents against HCV.As a result,superior sustained viral responses have been attained.The rapidly evolving field of anti-HCV therapy is expected to broad its landscape even further with newer,more potent antivirals,bringing us one step closer to the interferon-free era.

Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC): Occlusive Effect and Penetration Enhancement Ability

Objective: This work compares the occlusive effect and the penetration enhancement ability of solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), through in vitro skin. Methods: SLN and NLC were prepared by high shear homogenization and characterized by size, polydispersity index, zeta potential, morphology and physical stability. Occlusive effect was assessed by an in vitro test and by measuring TEWL using pig skin. Skin treated with the lipid carriers was visualized by SEM. A penetration test through skin, followed by tape stripping, was carried out using Nile red as a marker. Results: SLN (200 ± 6 nm) and NLC (192 ± 11 nm) were obtained. An occlusion factor of 36% - 39% was observed for both systems, while a reduction in TEWL of 34.3% ± 14.8% and 26.2% ± 6.5% was seen after treatment with SLN and NLC, respectively. SEM images showed a film formed by the lipid carriers, responsible for the occlusion observed. No differences were found between the occlusive effect produced by SLN and NLC in both tests. NLC allowed the penetration of a greater amount of Nile red than SLN: 4.7 ± 1.3 μg and 1.7 ± 0.4 μg, respectively. Conclusion: Both carriers form a film on the skin, providing an occlusive effect with no differences between these two systems. The penetration of a marker (Nile red) into the stratum corneum was quite higher for NLC than for SLN, suggesting an influence of the composition of these particles on their penetration enhancing ability.

Evaluation of the Antiviral Activity of Propolis from Native Bees (<i>Plebeia frontalis</i>) against Canine Distemper Virus

Propolis is a natural substance made from resins collected from trees and plants, and which bees combine with pollen, wax, and their own enzymes. It has a complex chemical composition that varies with the harvest season, vegetation type, bee species, and geographical region. Thanks to its components, it has valuable biological properties such as antifungal, antibacterial, anticancer, antiviral, and immunomodulatory activity. For this study, a sample of propolis harvested in April 2019 was used, which came from a bee native to Mexico (Plebeia frontalis) in whose geographical environment there are seven other native species. Canine distemper virus is an RNA virus that causes a systemic infection with high fatality rates in guests without protective immunity. In this work, the antiviral effect of Plebeia frontalis propolis on canine distemper virus was tested, administering it one hour before and simultaneously to infection. The antiviral effect was evaluated by determining cellular viability with the MTT assay. The results obtained show that this propolis has a statistically significant antiviral effect on both treatments, although it is slightly better when applied one hour before viral infection, so we can recommend it as an antiviral treatment in both domestic animals and human beings. There are currently few studies of the antiviral effect of propolis, this being the first study of a melliponium propolis in veterinary medicine.

A Study on the Influence of Luminance L* in the L*a*b* Color Space during Color Segmentation

In this paper an evaluation of the influence of luminance L* at the L*a*b* color space during color segmentation is presented. A comparative study is made between the behavior of segmentation in color images using only the Euclidean metric of a* and b* and an adaptive color similarity function defined as a product of Gaussian functions in a modified HSI color space. For the evaluation synthetic images were particularly designed to accurately assess the performance of the color segmentation. The testing system can be used either to explore the behavior of a similarity function (or metric) in different color spaces or to explore different metrics (or similarity functions) in the same color space. From the results is obtained that the color parameters a* and b* are not independent of the luminance parameter L* as one might initially assume.

Evaluation Preliminary of a Dry Emulsion System as a <i>Pasteurella multocida</i>Oral Carrier for Pigs

Background: This work evaluated the capacity of a dry emulsion as a carrier of viable microorganisms with potential use as prophylaxis of infectious diseases. Methods: The aqueous phase containing P. multocida not viable in PBS was emulsified in mineral oil to obtain a w/o emulsion. The microorganisms remained stable and only in two cases (n = 6) did the bacterial concentration decrease. Scanning Electron Microscopy (SEM) revealed a structure of a system with the organized association of particles with cubic symmetry. Using two ex vivo bioadhesion systems, it was demonstrated that the disperse-adsorbed system is capable of adhering to the intestinal mucosa and remains adhered for long periods of time. Results: The no viability of the bacteria in the dry emulsion and the possibility of controlled release were confirmed. In vivo trial was conducted in pigs. It was possible to locate the emulsion and the bacteria attached to the gut of the living animal. An ELISA kit was used to monitor the mean antibody titer of treated pigs over a 2-week period, and a classic primary response curve occurred when the titer was plotted against time. Conclusion: We propose the disperse-adsorbed system as an alternative to commonly used vehicles for immunogens in the oral vaccines.

Evaluation of Changes in Actin Filaments of RK13 Cells Infected with <i>Malassezia pachydermatis</i>

Background: Malassezia pachydermatis is the main causative agent of canine otitis and also of a myriad of dermatological problems in companion animals;its interaction mechanisms with host cells are still unclear. Objectives: To establish an in vitro infection model of M. pachydermatis-exposed RK13 cells in order to evaluate cell morphological changes as well as changes in the structure of actin filaments. Methods: Cultures of RK13 cells were infected with M. pachydermatis, alterations caused by the yeast were evaluated by optical and fluorescence microscopy. Results: M. pachydermatis adheres itself to the cell and produces the formation of multiple agglomerates that cause changes in cell morphology, formation of cell aggregates in overlays, presence of syncytia and destruction of cell culture structure. The damaged cells presented changes in the actin filaments consisting of thickening of the cell cortex and loss of stress fibers. On the other hand, the formation of perinuclear actin rings in the yeasts was observed. Conclusions: An in vitro infection model was established with M. pachydermatis and alterations in cell morphology were observed consisting of changes in the structure of the actin filaments, overgrowth of the cells and the presence of syncytia.

Determination of mRNA Expression of Typical Proteins of Tight Junctions in the Intestinal Mucosa of Broilers (<i>Gallus gallus domesticus</i>)

Tight junctions are mainly formed by two types of proteins;claudins and occludin, both of which are fundamental to maintain the integrity and barrier function of the intestinal epithelium. This barrier function allows for the absorption of nutrients, mainly by transcytosis;however, in birds, 90% of the substances are absorbed by paracellular mechanisms. Despite this, claudins present in the different parts of the intestinal tract of adult chickens are not known, much less their functional role. This study aimed to determine the presence of mRNA of claudins 1, 2, 3, 5, 10, 12, 16 and occludin, in the different regions of the intestine (duodenum, jejunum, ileum, cecum, and rectum) in chickens (Gallus gallus domesticus) through RT-PCR. To meet this goal, 7 weeks old roosters destined for slaughter and chicken embryos of 16 days of incubation (positive control) were used. For all the processed samples, amplicons of the expected size were obtained;claudin 1 (662 pb), claudin 2 (162 pb), claudin 3 (185 pb), claudin 5 (224 pb), claudin 10 (687 pb), claudin 12 (738 pb), claudin 16 (191 pb) and occludin (430 pb). To corroborate these findings, obtained amplicons were sequenced and, subsequently, a basic alignment was performed on the NCBI, obtaining a correlation of 100% with the original sequences in all analyzed samples. To our knowledge, the present work represents the first written report regarding the presence of mRNA of the main proteins involved in tight junction formation throughout the intestinal tract of domestic chickens of 7 weeks of age. These findings will allow elucidating the specific function of each of the reported proteins in the process of paracellular absorption in chickens.

Propolis in Dogs: Clinical Experiences and Perspectives (A Brief Review)

In light of the scarcity of novel therapeutic agents that are effective, the pharmaceutical industry has found a newer source of therapeutic compounds in natural products and herbal medicine to address the current health problems in humans and animals. What is particularly promising about these agents is that they produce fewer side effects and are more cost effective than synthetic compounds. This means greater availability of these treatments particularly for less developed countries who can’t afford expensive treatments. The reduced side effects also mean greater patient tolerance and increased compliance thereby yielding maximal therapeutic effect without negatively impacting on quality of life. Among the natural products more frequently employed nowadays is propolis, a resin that is routinely collected by bees (Apis mellifera). Propolis contains flavonoids, caffeic acid esters and diterpenic acids, which provide the bactericidal, antiviral and antifungal properties to this product. The use of propolis to address a variety of conditions in small animal species is beginning to play an important role in the currently available treatments. Its use appears to be an effective treatment with no side effects at low cost. This paper reviews the different applications of this compound to treat diseases in dogs.

Effect Found in the Combination of Kitasamycin with Other Antibiotics Used against <i>Actinobacillus pleuropneumoniae</i>

Background: Combinations of antibiotics are used to increase the therapeutic options via the simultaneous activities of two compounds. The goals of combination therapy are to resolve the bacterial infection while reducing the treatment cost. The use of kitasamycin is used frequently and in addition to other antibiotics, the desired combinations were not found. Methods: Fields strains: we use serotypes 1, 3, 5 y 7. Microbial culturing: The serotypes 1, 3, 5 y 7 was identified. Antibiotics and Reagents: The antibiotics used were kitasamycin;enrofloxacin and oxolinic acid. The following combinations were tested: a) kitasamycin with enrofloxacin;b) kitasamycin with norfloxacin;and c) kitasamycin with oxolinic acid. Preparation of bacterial suspensions: The cultures were adjusted to a concentration of 5 colony-forming units per ml (CFU/ml) using the same sterile culture medium as the diluent. Determination of the fractional inhibitory concentrations (FICs): To test each combination and their interactions and to calculate the fractional inhibitory concentration (FIC) of each antibiotic. Calculation of the index of the fractional minimum inhibitory concentration (FMIC): The FMIC index was calculated by applying the following formula: FIC index = FIC of antibiotic A/FIC of antibiotic B. Results: What was found showed that: A combination of kitasamycin and norfloxacin has been shown to have an indifferent effect. A combination of kitasamycin and enrofloxacin had antagonistic effects against all of the Actinobacillus pleuropneumoniae serotypes studied. Finally, a combination of kitasamycin and oxolinic acid had antagonist effects against serotypes 1, 3 and 5 and a synergistic effect against serotype 7. Conclusion: The “in vitro” combination Kitasamycin-Norfloxacin showed an indifferent effect, and combination Kitasamycin and Enrofloxacin demonstrated an antagonistic interaction between these two antibiotics. A combination of Kitasamycin and Oxolinic Acid had synergistic effect against serotype 7.

Use of Mexican Propolis for the Topical Treatment of Dermatomycosis in Horses

Five horses with lesions compatible with dermatomycosis were selected and skin scrapings were taken from all affected areas. Samples were then treated with 10% KOH and observed by direct microscopic examination;fungal cultures using Mycobiotic Agar and Borelli Agar were also preformed;Trichophyton mentagrophytes was identified in two cases, whereas Candida albicans was identified in the other three. Skin lesions in cases 1 and 2 were treated topically with ketoconazol ointment, while in the remaining cases a weekly bath with propolis-based shampoo and application of propolis-based ointment on the lesions 2 to 3 times a week were implemented. Treatment in all cases lasted 4 weeks. Pictures were taken before and after treatment and, making use of the Image Pro-Express® software, therapy efficacy was determined by measurement of lesion area diameter and a significant reduction in lesion size was observed by the end of the 4 weeks of treatment with ketoconazole and propolis-based products. Thus proving the effectiveness of such propolis-based products, making them a natural therapeutic alternative for the treatment of cutaneous mycoses in horses, and carrying none of the toxic side effects of conventional pharmaceuticals.

Canine Herpesvirus Seroprevalence and Associated Factors in Dogs of Mexico

Canine herpesvirus (CHV-1) causes disease associated with high mortality in infect-ed puppies, which represents large financial losses for dog breeders. Since CHV-1 at the time of the study he had not been reported in Mexico, the main objective of this study was to determine the prevalence of antibodies against CHV-1 in canine kennels in the metropolitan area of Mexico City. A commercial enzyme-linked immuno-sorbent assay (ELISA) was used, and the results were compared to those of a viral neutralization test. The ELISA kit uses the complete viral particle as the antigen. The plaque reduction neutralization test was combined with the immunoperoxidase technique because of the low cytopathic effect of CHV-1. Neutralizing antibodies were also detected in 20 randomly selected samples. The prevalence of CHV-1 with ELISA was 87%. The concordance between ELISA and serum neutralization (SN) was 0.1129, the sensitivity of the ELISA against SN was 1.0 (100%), the positive predic-tive value was 0.39 (39%), and the negative predictive value was 1 (100%). These results show that ELISA is useful for monitoring the dog population for CHV-1;a positive test result requires confirmation with an SN test, and a negative ELISA result indicates a high probability of being SN-negative. The only variables that were sta-tistically associated with CHV-1 prevalence were breed and kennel. A statistically significant relationship between the degree of ELISA and SN titer was obtained, with a confidence level of 95%. None of the clinical presentation factors was statistically significant. These results suggest that most of the canine population studied in Mex-ico is in a herpesvirus latency state.

Pathology Isolation and Identification of Canine Herpesvirus (CHV-1) in Mexico

This work presents the pathology description, isolation and identification of canine herpesvirus (CHV-1) in Mexico, a virus that causes a generalized hemorrhagic infection in puppies from the canidae family. Methods: Isolates were obtained from puppies that died within the first four weeks of life and had lesions consistent with canine herpesvirus. Results: The main gross lesions were petechial and ecchymotic hemorrhages in kidneys, liver and lungs;proliferative interstitial nephritis;multifocal necrosis in liver and kidneys;and encephalitis with intranuclear inclusion bodies. Herpesvirus was confirmed through direct immunofluorescence, electron microscopy and polymerase chain reaction for DNA polymerase and glycoprotein B genes. Discussion: Eight strains were isolated and identified as canine herpesvirus corresponding to three of the working cases with gross and microscopic lesions very similar to those described in the literature;then, isolates were confirmed by PCR gene amplification, positive reactions on immunofluorescence and observations from electron microscopy. This work represents the first report of this disease, including gross and histological lesions, and confirmation by isolation and identification of the canine herpesvirus in Mexico.

Maize seed coatings and seedling sprayings with chitosan and hydrogen peroxide:their influence on some phenological and biochemical behaviors

Objective： To evaluate the effect of chitosan （CH） and hydrogen peroxide （H2O） seed coatings and seedling sprinklings on two different maize varieties by measuring their phenology, the H202 presence, the catalase （CAT） activity, and the protein quantity. Methods： Seven groups of ten seeds for each maize variety were treated with CH （2% （20 g/L） and 0.2% （2 g/L）） or H2O2 （8 mmol/L） by coating, sprinkling, or both. Germination and seedling growth were measured. One month after germination, the presence of H202 in seedlings in the coated seed treatments was evaluated. Protein content and CAT activity were determined under all treatments. Results： H2O2 seed coating en- hanced the germination rate and increased seedling and stem length in the quality protein maize （QPM） variety. Seedlings had a higher emergence velocity under this treatment in both varieties. CH and H2O2 sprinklings did not have an effect on seedling phenology. Exogenous application of H2O2 promoted an increase of endogenousH2O2. OH and H2O2seedling sprinkling increased the protein content in both maize varieties, while there was no significant effect on the CAT activity of treated seeds and seedlings. Conclusions： CH and H2O2 enhance some phenological and biochemical features of maize depending on their method of application.