Characterization of microbiota and histology of cultured sea cucumber Isostichopus badionotus juveniles during an outbreak of skin ulceration syndrome.

Due to the dramatic reduction of sea cucumber Isostichopus badionotus populations in the Yucatan Peninsula by overfishing and poaching, aquaculture has been encouraged as an alternative to commercial catching and restoring wild populations. However, the scarcity of broodstock, the emergence of a new disease in the auricularia larvae stage, and the development of skin ulceration syndrome (SUS) in the culture have limited aquaculture development. This study presents the changes in the intestine and skin microbiota observed in early and advanced stages of SUS disease in cultured juvenile I. badionotus obtained during an outbreak in experimental culture through 16S rRNA gene sequencing and histological evidence. Our results showed inflammation in the intestines of juveniles at both stages of SUS. However, more severe tissue damage and the presence of bacterial clusters were detected only in the advanced stages of SUS. Differences in the composition and structure of the intestinal and skin bacterial community from early and advanced stages of SUS were detected, with more evident changes in the intestinal microbial communities. These findings suggest that SUS was not induced by a single pathogenic bacterium. Nevertheless, a decrease in the abundance of Vibrio and an increase in Halarcobacter (syn. Arcobacter) was observed, suggesting that these two bacterial groups could be keystone genera involved in SUS disease.

The digestive tract sections of the sea cucumber Isostichopus badionotus reveal differences in composition, diversity, and functionality of the gut microbiota.

For the first time, this study analyses the composition and diversity of the gut microbiota of Isostichopus badionotus in captivity, using high-throughput 16S rRNA sequencing, and predicts the metagenomic functions of the microbiota. The results revealed a different composition of the gut microbiota for the foregut (FG) and midgut (MG) compared to the hindgut (HG), with a predominance of Proteobacteria, followed by Actinobacteria, Bacteroidetes, and Firmicutes. The FG and MG demonstrated a greater bacterial diversity compared to the HG. In addition, a complex network of interactions was observed at the genus level and identified some strains with probiotic and bioremediation potentials, such as Acinetobacter, Ruegeria, Streptococcus, Lactobacillus, Pseudomonas, Enterobacter, Aeromonas, Rhodopseudomonas, Agarivorans, Bacillus, Enterococcus, Micrococcus, Bifidobacterium, and Shewanella. Predicting metabolic pathways revealed that the bacterial composition in each section of the intestine participates in different physiological processes such as metabolism, genetic and environmental information processing, organismal systems, and cellular processes. Understanding and manipulating microbe--host-environment interactions and their associated functional capacity could substantially contribute to achieving more sustainable aquaculture systems for I. badionotus.

A One Health Approach Molecular Analysis of Staphylococcus aureus Reveals Distinct Lineages in Isolates from Miranda Donkeys (Equus asinus) and Their Handlers.

Donkeys (Equus asinus) are in decline in Europe. Occupational exposure to farm animals has been associated with increased staphylococci carriage. We aimed to isolate S. aureus and coagulase-negative staphylococci (CoNS) from donkeys and handlers and characterize the antimicrobial resistance profiles and genetic lineages of S. aureus strains. Oral and nasal swab samples were collected from 49 Miranda donkeys and 23 handlers from 15 different farms. Staphylococci species were identified by MALDI-TOF MS. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Molecular typing was performed in S. aureus isolates. From the 49 donkey samples, 4 S. aureus (8.2%) and 21 CoNS (42.9%) were isolated. Ten handlers (43.5%) were carriers of S. aureus and 4 (17.4%) carried CoNS. The CoNS isolates showed resistance to several classes of antimicrobials encoded by the mecA, aph (3')-IIIa, ant (4')-Ia, tetM, tetK, lnuA, ermB, ermC, dfrA and dfrG genes. S. aureus isolates were resistant to penicillin, aminoglicosides and tetracycline harboring the blaZ, aph (3')-IIIa, tetL, tetM and tetK genes. All S. aureus isolates from donkeys belonged to ST49 and spa-type t208 while the strains isolated from the handlers were ascribed to 3 STs and 7 spa-types. However, human isolates were from different STs than the donkey isolates. Donkeys are mainly colonized by methicillin-resistant S. sciuri. S. aureus transmission between donkeys and their handlers appears not to have occurred since the isolates belonged to different genetic lineages.

Follicular dynamics during the non-reproductive season in Miranda jennies.

Jennies' follicular wave patterns have not yet been addressed during the non-breeding and transition seasons in anoestrous jennies. Twelve non-pregnant females of the Miranda donkey breed were followed to describe follicular wave characteristics during the non-reproductive season and determine the anoestrous effect in follicular wave patterns. Five jennies enrolled in this study experienced anoestrus during the non-breeding season, but all retained the continuous emergence of follicular waves. The average duration of the waves from emergence to peak was 11.2 ± 0.021 days (3-29 days). The duration of the different type of waves was 9.91 ± 0.034 days for minor waves,12.5 ± 0.232 days for major secondary waves and 12.5 ± 0.057 for major primary waves. The major waves were significantly longer than the minor waves (p < .001). Older jennies presented longer waves (p = .021). In the jennies presenting anoestrus, the wave duration during anoestrus (11.2 ± 0.125 days, n = 31) was not different from the waves detected in the preceding and subsequent ovulatory cycles (11.3 ± 0.084 days, n = 43) (p = .978). The number of follicular waves emerging in each ovulatory cycle (n = 59) was 2.36 ± 0.011, varied from 1 to 4. Only in a small proportion of cycles, one wave (0.8%) was recorded, with 41 cycles (67.2%) presenting two waves; fourteen cycles presenting three waves (24.6%); and three cycles (6.6%) showing 4 waves. It was concluded that follicular waves do occur in anoestrous jennies during the non-breeding season with two-wave cycles being common.

Selenium and Vitamin E Concentrations in Miranda Jennies and Foals (Equus asinus) in Northeast Portugal.

The increase in donkeys treated by practitioners in recent years has led to an increased interest in finding more information on basic biochemical preliminary reference values. The aims of this study were to measure Se and Vit E levels in plasma from Miranda jennies peripartum and postpartum and in their foals to compare blood profiles of the jenny and foal related to the overall foal's health. Twenty-two healthy peripartum and postpartum Miranda donkeys were sampled (12 jennies and 10 foals) in the northeast of Portugal (Atenor and Paradela) from May to November, 2018. Amounts of selenium in soil were significantly correlated (0.97) to concentrations of selenium in jennies (42.412 µg/L in Atenor and 9.612 µg/L in Paradela) and foals (19.378 µg/L in Atenor and 6.430 µg/L in Paradela). Selenium levels were lower in foals than adults and in males than females. Vitamin E was associated with overall foal health. Foals with a mean vitamin E of 3.585-5.307 mg/L showed signs of weakness, but carpal flexural deformities were observed when the average vitamin E was 11.520 mg/L. Low vitamin E levels (5.307 mg/L) in jennies were related to foal mortality. Diets, location, parity, and age affect blood profiles of jennies and, ultimately, foal health.

Comparative Transcriptomes of the Body Wall of Wild and Farmed Sea Cucumber Isostichopus badionotus.

Overfishing of sea cucumber Isostichopus badionotus from Yucatan has led to a major population decline. They are being captured as an alternative to traditional species despite a paucity of information about their health-promoting properties. The transcriptome of the body wall of wild and farmed I. badionotus has now been studied for the first time by an RNA-Seq approach. The functional profile of wild I. badionotus was comparable with data in the literature for other regularly captured species. In contrast, the metabolism of first generation farmed I. badionotus was impaired. This had multiple possible causes including a sub-optimal growth environment and impaired nutrient utilization. Several key metabolic pathways that are important in effective handling and accretion of nutrients and energy, or clearance of harmful cellular metabolites, were disrupted or dysregulated. For instance, collagen mRNAs were greatly reduced and deposition of collagen proteins impaired. Wild I. badionotus is, therefore, a suitable alternative to other widely used species but, at present, the potential of farmed I. badionotus is unclear. The environmental or nutritional factors responsible for their impaired function in culture remain unknown, but the present data gives useful pointers to the underlying problems associated with their aquaculture.

Gastrointestinal Parasitism in Miranda Donkeys: Epidemiology and Selective Control of Strongyles Infection in the Northeast of Portugal.

In Portugal, equine parasitism in pasture animals is characterized by high parasitic burden and high helminthic biodiversity; both factors are potentially pathogenic for their hosts. The decrease in the number of donkeys over the last years in Portugal, their importance in rural lowland and mountain ecosystems and pastures and the scarce information regarding their parasitism led to this research, which aimed to evaluate the parasitological status of a Miranda donkey breed population, a native breed mainly located in the northeast of Portugal. This study provides better knowledge of their gastrointestinal parasitism, particularly strongyles, and the assessment of a targeted selective treatment (TST) as an alternative control approach of their parasitism. A longitudinal observational study was developed during a period of five years in a population of 62 Miranda donkeys. At first, strategic deworming of these animals was performed every semester, but this was progressively replaced by a TST approach according to the levels of Eggs per Gram (EPG). This new deworming strategy was conducted in association with a regular parasitological monitoring of the animals every three months, being dewormed with ivermectin when egg shedding was higher than 500 EPG. Over the study period, a decrease of the annual prevalence rate of infection by gastrointestinal strongyles was observed, from 35.5% to 19.4%, as well as a negative binomial distribution of parasitic strongyles in donkeys submitted to selective anthelminthic control. The prevalence rate of infection was higher in females (39.5%), in individuals under four years (46.7%) and in those presenting a lower body condition (40.8%). The egg output was higher in animals younger than four years (589.3 EPG) than in those older than 10 years (533.6 EPG) (p < 0.05). However, no differences were observed according to sex during the study period. Results from this study allowed to note the influence of swampy pastures and of the weather changes in the epidemiology of strongylosis in Miranda donkeys. Moreover, it was possible to establish the annual epidemiological curve of strongyle egg shedding, with June being the month with the highest EPG, December having the lowest EPG and March and September showing intermediate numbers. Overall, a lower biodiversity of gastrointestinal parasites was observed. Cyathostomum sensu lato was the most prevalent genus and Strongylus vulgaris was the most observed large strongyle of the Strongylidae family. Trichostrongylus axei and Parascaris sp. were other nematodes with a minor frequency. The higher prevalence of strongyles at the beginning of the study showed a progressive decrease throughout the research period, and also for parasite biodiversity. Therefore, a targeted selective treatment seems to be a rational anthelminthic control approach in Miranda donkey strongyle infection and in other gastrointestinal parasites, since it reduces the antiparasitic treatments, the parasite' prevalence and the EPG level. However, a loss of parasite biodiversity was noted at the end of the study period, as Cyathostominae were the only isolated strongyles. This can be a challenging situation in the long run, taking the ability of these nematodes to adapt easily to any deworming program, meaning that fecal EPG monitoring should be kept as a rule to a rational parasite control program.

Hastening Time to Ejaculation in Donkey Jacks Treated with the PGF2α Analog, Cloprostenol Sodium.

Due to the long courtship needed to attain excitation and erection, donkey semen collection can take up to 90 min. ProstaglandinF2α (PGF2α) has been reported to hasten the onset of erection and ejaculation in domesticated mammals, presumably by inducing smooth muscle contractions in the internal genitalia. However, while it has been anecdotally used in donkeys, it has yet to be critically evaluated. This study aimed to compare behavioral and semen parameters in Catalan, Balearic, Amiata, and Miranda jacks treated with the PGF2α analogue cloprostenol sodium immediately prior to exposure to an estrus jenny. Nineteen donkeys were assigned in a crossover design to receive cloprostenol sodium (125µg, i.m.; n = 53 collections) or saline (1 mL, i.m.; n = 53 collections). There were no differences for erection (52/53 vs. 52/53) or ejaculation (52/53 vs. 48/53) for collection attempts assigned to saline or cloprostenol sodium, respectively. Cloprostenol sodium significantly hastened treatment-to-erection and treatment-to-ejaculation times from 12.0 ± 1.6 to 6.0 ± 1.6 min and from 14.0 ± 1.4 to 9.6 ± 1.4 min, respectively. Significant effects of breed and age were observed in behavioral and parameters, but there were no effects of cloprostenol sodium administration on semen parameters. In conclusion, cloprostenol sodium administration immediately prior to semen collection hastened time to collect semen in donkeys with no detrimental effects on semen quality and can be used by practitioners to circumvent long delays in donkey semen collection.

A Glycosaminoglycan-Rich Fraction from Sea Cucumber Isostichopus badionotus Has Potent Anti-Inflammatory Properties In Vitro and In Vivo.

Sea cucumber body wall contains several naturally occurring bioactive components that possess health-promoting properties. Isostichopus badionotus from Yucatan, Mexico is heavily fished, but little is known about its bioactive constituents. We previously established that I. badionotus meal had potent anti-inflammatory properties in vivo. We have now screened some of its constituents for anti-inflammatory activity in vitro. Glycosaminoglycan and soluble protein preparations reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory responses in HaCaT cells while an ethanol extract had a limited effect. The primary glycosaminoglycan (fucosylated chondroitin sulfate; FCS) was purified and tested for anti-inflammatory activity in vivo. FCS modulated the expression of critical genes, including NF-ĸB, TNFα, iNOS, and COX-2, and attenuated inflammation and tissue damage caused by TPA in a mouse ear inflammation model. It also mitigated colonic colitis caused in mice by dextran sodium sulfate. FCS from I. badionotus of the Yucatan Peninsula thus had strong anti-inflammatory properties in vivo.

Evaluation of two independent protocols for the extraction of DNA and RNA from different tissues of sea cucumber Isostichopus badionotus.

Isostichopus badionotus is a sea cucumber species of great ecological and economic relevance for Mexico and Central American and Caribbean countries; however, the protocols for the extraction of the nucleic acids have not yet been published. In this study, we describe the first protocols to obtain DNA and RNA from different tissues of I. badionotus, which include the respiratory tree, gonad, longitudinal muscle bands, anterior intestine and cloaca. The extraction of high-quality DNA was performed using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA) with minor modifications in different points of the protocol. Concerning the RNA, the method of TRIzol was used. This method is particularly advantageous in situations where cells or tissues are enriched for endogenous RNases or when the separation of cytoplasmic RNA from nuclear RNA is impractical. The methodologies used in this study allowed us to obtain DNA and RNA of high quality and integrity in the different tissues of I. badionotus, which will be the basis for future genomic and transcriptomic studies. •The successful extraction of DNA and RNA was achieved in the different tissues of I. badionotus.•The concentrations of DNA and RNA obtained were adequate for a diversity of analyses at a molecular level.

Seroprevalence of Toxoplasma gondii and Leishmania spp. in domestic donkeys from Portugal.

Toxoplasma gondii and Leishmania infantum are zoonotic protozoal parasites. Serum samples were obtained from 186 donkeys (Equus africanus asinus) from Portugal and assessed for antibodies to T. gondii by the modified agglutination test (MAT). For titration of antibodies to Leishmania spp. the direct agglutination test was used (DAT). Eleven donkeys were seropositive for T. gondii with titres of 20 (n = 7), 80 (n = 2), 640 (n = 1) and ≥ 2560 (n = 1). One donkey was seropositive for Leishmania spp. (titre of 800). Donkeys in Portugal are exposed to and can be infected with T. gondii and Leishmania spp.

Seroprevalence of Toxoplasma gondii and Leishmania spp. in domestic donkeys from Portugal / Soroprevalência de Toxoplasma gondii e Leishmania spp. em jumentos domésticos de Portugal

Abstract Toxoplasma gondii and Leishmania infantum are zoonotic protozoal parasites. Serum samples were obtained from 186 donkeys (Equus africanus asinus) from Portugal and assessed for antibodies to T. gondii by the modified agglutination test (MAT). For titration of antibodies to Leishmania spp. the direct agglutination test was used (DAT). Eleven donkeys were seropositive for T. gondii with titres of 20 (n = 7), 80 (n = 2), 640 (n = 1) and ≥ 2560 (n = 1). One donkey was seropositive for Leishmania spp. (titre of 800). Donkeys in Portugal are exposed to and can be infected with T. gondii and Leishmania spp.

Resumo Toxoplasma gondii e Leishmania infantum são protozoários parasitas com potencial zoonótico. Foram obtidas amostras de soro de 186 jumentos (Equus africanus asinus) e avaliadas para anticorpos anti-T. gondii pelo teste de aglutinação direta modificada (TADM), em Portugal. Para a titulação de anticorpos anti-Leishmania spp. foi usado o teste de aglutinação direta (TAD). Onze jumentos foram soropositivos para T. gondii com títulos de 20 (n = 7), 80 (n = 2), 640 (n = 1) e ≥ 2560 (n = 1). Um jumento foi soropositivo para Leishmania spp. (título de 800). Os jumentos em Portugal estão expostos e podem ser infectados com T. gondii e Leishmania spp.

Antimicrobial-resistant Escherichia coli and Enterococcus spp. isolated from Miranda donkey (Equus asinus): an old problem from a new source with a different approach.

PURPOSE: The Miranda donkey (Equus asinus) is an endangeredasinine from Miranda do Douro region, located in the north east of Portugal. We studied the antimicrobial resistance and virulence genes in Escherichia coli and Enterococcus spp. isolates from these animals. METHODOLOGY: In March 2014, a total of 66 faecal samples were recovered from independent animals. Antibiotic resistance was determined by the disc diffusion method. Carriage of genes coding for antibiotic-resistant and virulent factors was analysed by PCR. RESULTS: A total of 66 E. coli and 41 enterococcal isolates were detected, with Enterococcus faecium (61 %) and Enterococcus hirae (24 %) being the most prevalent species. For enterococcal isolates, high percentages of resistance rates to tetracycline (68.3 %), quinupristin/dalfopristin (51.2 %) and ciprofloxacin (48.8 %) were observed. The genes erm(A) and/or erm(B), tet(M) and/or tet(L), vat(D) and/or vat(E) and aph(3')-IIIa were also found. The most frequent virulence gene detected was gel(E), followed by ace, cpd and hyl. Escherichia coli isolates were highly resistant to streptomycin (78 %), whereas 39 % of them exhibited resistance to aminoglycosides and tetracycline. Genes sul1 and/or sul2 were detected in 66.7 % of trimethoprim/sulfamethoxazole-resistant isolates. The virulence genes detected were fim(A) (46 %) and cnf1 (27 %). CONCLUSION: To the best of our knowledge, this is the first report showing antibiotic resistance among Escherichiacoli and Enterococcus spp. isolates from the Miranda donkey in Portugal, indicating possible antibiotic-resistant bacterial reservoirs. However, the detection of these resistances presents a low risk for other animals and human beings in that rural area.

Diets containing sea cucumber (Isostichopus badionotus) meals are hypocholesterolemic in young rats.

Sea cucumber is widely consumed as a putative functional food. It contains many biologically-active substances, but only limited research on its properties in vivo has been done. The effects of different meals containing Isostichopus badionotus, a sea cucumber from southeast Mexico, on growth performance and body lipid profile in young rats were analyzed. Sea cucumber body wall was either lyophilized, cooked (100 °C, 1 h in water) and lyophilized, or oven-dried (70 °C for 12 h). It was then ground and incorporated into cholesterol-containing diets. I. badionotus meals supported growth and improved lipid profile in rats. In particular, serum cholesterol, low density lipoproteins, triglycerides concentration and atherogenic index values were greatly reduced by some I. badionotus containing diets. Liver total lipids, triglycerides and cholesterol were also reduced. Cooking or heat-treatment of the meals lowered but did not abolish their hypolipidemic potency. Gene expression analysis of several key genes involved in cholesterol and lipid metabolism in liver showed that diets containing I. badionotus repressed the induction of key genes associated with dyslipidemia exerted by cholesterol supplementation. Consumption of I. badionotus from the Yucatan Peninsula is beneficial for dyslipidemia, although biological effect is clearly dependent on preparation method.

Ballast water as a vector of coral pathogens in the Gulf of Mexico: the case of the Cayo Arcas coral reef.

The discharge of nutrients, phytoplankton and pathogenic bacteria through ballast water may threaten the Cayo Arcas reef system. To assess this threat, the quality of ballast water and presence of coral reef pathogenic bacteria in 30 oil tankers loaded at the PEMEX Cayo Arcas crude oil terminal were determined. The water transported in the ships originated from coastal, oceanic or riverine regions. Statistical associations among quality parameters and bacteria were tested using redundancy analysis (RDA). In contrast with coastal or oceanic water, the riverine water had high concentrations of coliforms, including Vibrio cholerae 01 and, Serratia marcescens and Sphingomona spp., which are frequently associated with "white pox" and "white plague type II" coral diseases. There were also high nutrient concentrations and low water quality index values (WQI and TRIX). The presence of V. cholerae 01 highlights the need for testing ballast water coming from endemic regions into Mexican ports.

Caracterizacion de la comunidad de macroinvertebradosde la quebrada paloblanco de la cuenca del rio otun(risaralda colombia) / Characterization of the Macroinvertebrate Community of Paloblanco Gulch, Otun River Basin (Risaralda, Colombia)

Se realizó un estudio de las comunidades de macroinvertebrados presentes en la quebrada Paloblanco, ubicada en la cuenca del río Otún (Risaralda, Colombia). Los puntos de muestreo seleccionados corresponden a la cabecera y la zona cercana a la desembocadura en el río Otún, ambos con características geomorfológicas de corrientes de primer orden. En cada punto se colectaron los macroinvertebrados de las unidades funcionales hojarasca, epiliton, musgo y grava, junto con una muestra de deriva y fauna general (sin discriminar unidad funcional). Se encontraron 42 familias de Insecta, además de Acari, Annelida, Crustacea e Hirudinea. Igualmente se encontraron familias de presencia exclusiva en cada punto, mostrando diferencias en la composición de las comunidades. Los índices ecológicos empleados mostraron una mayor diversidad en punto bajo y diferencias entre algunas de las unidades funcionales, relacionados con el medio y los hábitos presentes. Para el análisis se tuvieron en cuenta los conceptos de continuo y zonación de los ríos. A pesar de tener condiciones de primer orden, existen variaciones a pequeña escala entre los puntos de muestreo, que están relacionadas con la intervención antrópica en la zona aledaña a punto bajo.

Nutritional and physiological responses of young growing rats to diets containing raw cowpea seed meal, protein isolate (globulins), or starch.

The nutritional and physiological effects of raw cowpea (Vigna unguiculata (L) Walp.) seed meal, protein isolate (globulins), or starch on the metabolism of young growing rats have been evaluated in 14-day trials. Wet and dry weight gain, feed conversion efficiency, and lipid and protein accretion were significantly reduced as a result of inclusion of seed meal, globulins, or starch in the diet, with growth retardation being most marked with the seed meal. The proportional weights of the small intestine and pancreas were increased by meal diets, and serum cholesterol levels were slightly reduced. The globulins and raw starch also increased relative small intestine weights but had no effect on the pancreas or serum constituents. The effects of cowpeas on rats appeared to be due primarily to the combined actions of globulins, resistant starches, protease inhibitors, and possibly fiber and non-starch polysaccharides on intestinal and systemic metabolism.