Comparison of bacterial diversity and distribution on the gills of Atlantic salmon (Salmo salar L.): an evaluation of sampling techniques.

AIMS: Assess bacterial diversity and richness in mucus samples from the gills of Atlantic salmon in comparison to preserved or fixed gill filament tissues. Ascertain whether bacterial diversity and richness are homogeneous upon different arches of the gill basket. METHODS AND RESULTS: Bacterial communities contained within gill mucus were profiled using 16S rRNA gene sequencing. No significant difference in taxa richness, alpha (P > 0·05) or beta diversity indices (P > 0·05) were found between the bacterial communities of RNAlater preserved gill tissues and swab-bound mucus. A trend of lower richness and diversity indices were observed in bacterial communities from posterior hemibranchs. CONCLUSIONS: Non-lethal swab sampling of gill mucus provides a robust representation of bacterial communities externally upon the gills. Bacterial communities from the fourth arch appeared to be the least representative overall. SIGNIFICANCE AND IMPACT OF THE STUDY: The external mucosal barriers of teleost fish (e.g. gill surface) play a vital role as a primary defence line against infection. While research effort on the role of microbial communities on health and immunity of aquaculture species continues, the collection and sampling processes to obtain these data require evaluation so methodologies are consistently applied across future studies that aim to evaluate the composition of branchial microbiomes.

Dead or alive: microbial viability treatment reveals both active and inactive bacterial constituents in the fish gut microbiota.

AIMS: This study evaluated the microbial viability of fish gut microbiota in both digesta (faecal) and mucosal samples using a modified propidium monoazide (PMA) protocol, followed by 16S ribosomal RNA (rRNA) gene sequencing. METHODS AND RESULTS: Digesta and gut mucosal samples from farmed yellowtail kingfish (Seriola lalandi) were collected and a modified PMA treatment was applied prior to DNA extraction to differentiate both active and nonviable microbial cells in the samples. All samples were then sequenced using a standard 16S rRNA approach. The digesta and mucosal samples contained significantly different bacterial communities, with a higher diversity observed in digesta samples. In addition, PMA treatment significantly reduced the microbial diversity and richness of digesta and mucosal samples and depleted bacterial constituents typically considered to be important within fish, such as Lactobacillales and Clostridales taxa. CONCLUSIONS: These findings suggest that important bacterial members may not be active in the fish gut microbiota. In particular, several beneficial lactic acid bacteria (LAB) were identified as nonviable bacterial cells, potentially influencing the functional potential of the fish microbiota. SIGNIFICANCE AND IMPACTS OF THE STUDY: Standardizing the methods for characterizing the fish microbiota are paramount in order to compare studies. In this study, we showed that both sample type and PMA treatment influence the bacterial communities found in the fish gut microbiota. Our findings also suggest that several microbes previously described in the fish gut may not be active constituents. As a result, these factors should be considered in future studies to better evaluate the active bacterial communities associated with the host.

Effect of sample type and the use of high or low fishmeal diets on bacterial communities in the gastrointestinal tract of Penaeus monodon.

In shrimp aquaculture, manufactured diets that include various supplements and alternative fishmeal ingredients are increasingly being used and their effect on the gastrointestinal (GI) microbiota studied. However, dietary effects on different shrimp GI samples are not known. We investigated how a high (HFM) or low (LFM) fishmeal diet affects bacterial communities from different sample types collected from Penaeus monodon gastrointestinal tract. Bacterial communities of the stomach, intestine tissue and intestine digesta were assessed using 16s rRNA gene sequencing. The feed pellets were also assessed as a potential source of bacteria in the GI tract. Results showed substantial differences in bacterial communities between the two diets as well as between the different sample types. Within the shrimp GI samples, stomach and digesta communities were most impacted by diet, while the community observed in the intestine tissue was less affected. Proteobacteria, Firmicutes and Bacteroidetes were the main phyla observed in shrimp samples, with enrichment of Bacteroidetes and Firmicutes in the LFM fed shrimp. The feed pellets were dominated by Firmicutes and were largely dissimilar to the shrimp samples. Several key taxa were shared however between the feed pellets and shrimp GI samples, particularly in the LFM fed shrimp, indicating the pellets may be a significant source of bacteria observed in shrimp GI samples. In summary, both diet and sample type influenced the bacterial communities characterised from the shrimp GI tract. Thus, it is important to consider the sample type collected from the GI tract when investigating dietary impacts on gut bacterial communities in shrimp. KEY POINTS: • Shrimp gastrointestinal communities are influenced by diet and sample type. • The low fishmeal diet enriched bacteria that aid in polysaccharide metabolism. • Feed pellets can be a source of bacteria-detected gastrointestinal tract of shrimp.

Searching for the sweet spot of amoebic gill disease of farmed Atlantic salmon: the potential role of glycan-lectin interactions in the adhesion of Neoparamoeba perurans.

One of the first critical steps in the pathogenesis of amoebic gill disease (AGD) of farmed salmon is the adhesion of the causative amoeba to the host. The current study aimed to investigate the potential involvement of glycan-binding proteins expressed on the extracellular surface of Neoparamoeba perurans in gill tissue recognition and binding. The glycan-binding properties of the surface membrane of N. perurans and the carbohydrate binding profile of Atlantic salmon gill-derived epithelial cells were identified through the use of glycan and lectin microarrays, respectively. The occurrence of specific carbohydrate-mediated binding was then further assessed by in vitro attachment assays using microtitre plates pre-coated with the main glycan candidates. Adhesion assays were also performed in the presence of exogenous saccharides with the aim of blocking glycan-specific binding activity. Comparative analysis of the results from both lectin and glycan arrays showed significant overlap, as some glycans to which binding by the amoeba was seen were reflected as being present on the gill epithelial cells. The two main candidates proposed to be involved in amoeba attachment to the gills are mannobiose and N-acetylgalactosamine (GalNAc). Adhesion of amoebae significantly increased by 33.5 and 23% when cells were added to α1,3-Mannobiose-BSA and GalNAc-BSA coated plates. The observed increased in attachment was significantly reduced when the amoebae were incubated with exogenous glycans, further demonstrating the presence of mannobiose- and GalNAc-binding sites on the surfaces of the cells. We believe this study provides the first evidence for the presence of a highly specific carbohydrate recognition and binding system in N. perurans. These preliminary findings could be of extreme importance given that AGD is an external parasitic infestation and much of the current research on the development of alternative treatment strategies relies on either instant amoeba detachment or blocking parasite attachment.

Sleep-disordered breathing and nocturnal oxygen desaturation in postmenopausal women.

Twenty postmenopausal women were monitored for disordered breathing (apnea and hypopnea) and oxygen desaturation during one night's sleep. These women were compared with 18 premenopausal women previously reported to have a low incidence of sleep-disordered breathing and nocturnal oxygen desaturation. Twelve of the 20 postmenopausal women had 102 episodes of sleep-disordered breathing and 118 episodes of oxygen desaturation, compared with only six episodes of apnea in two premenopausal women (P less than 0.01). Premenopausal women did not become desaturated. Of the postmenopausal women, 11 became desaturated; and in five of them saturation decreased to less than 85 per cent. Duration of sleep, and increased age and weight: height ratios, correlated significantly with the incidence of desaturation (P less than 0.01--P less than 0.05). Postmenopausal women resemble men with respect to disordered breathing during sleep and nocturnal oxygen desaturation. Protection from these sleep events in premenopausal women might be afforded by the respiratory stimulant effects of circulating progesterone.

Disordered breathing and oxygen desaturation during sleep in patients with chronic obstructive lung disease (COLD).

Seven patients with chronic obstructive lung disease (COLD) were monitored during their overnight sleep to determine the occurrence of disordered breathing and oxygen desaturation. Nasal and oral airflows were sensed by thermistor probes, chest wall movement by impedance pneumography and arterial oxygen saturation by ear oximetry. These variables were correlated with electroencephalographic and electrooculographic tracings. The subjects had a mean base line oxygen saturation of 89.2 per cent and slept an average of 218 minutes. Six of these seven subjects had one to 30 episodes of oxygen desaturation (decrease more than 4 per cent), 4 seconds to 30 minutes in duration, with declines in saturation as great as 36 per cent. In two subjects, saturation dropped to less than 50 per cent. Breathing was disordered in five of the seven subjects and included apnea and hypopnea. Subjects experienced from nine to 37 episodes of disordered breathing. Disordered breathing caused 42 per cent of the episodes of desaturation, all of which were less than 1 minute in duration. The mean maximum decline in saturation was 7.6 per cent. All episodes of desaturation lasting longer than 5 minutes occurred in rapid eye movement (REM) sleep and were not caused by disordered breathing. The mean maximal decrease in saturation was 22 per cent. This study reveals that disordered breathing is common in subjects with COLD and often causes desaturation but that it cannot explain all episodes of sleep desaturation.

Menopause, medroxyprogesterone and breathing during sleep.

Twenty-one postmenopausal women were monitored for sleep-disordered breathing and nocturnal oxygen desaturation to evaluate the contribution of progestational hormones to the occurrence of these sleep events. For approximately one month 11 subjects received 30 mg of medroxyprogesterone (MPG) daily, and 10 received placebo tablets in a randomized, double-blind controlled study. Respiration, saturation and electroencephalography were monitored during one night of sleep before and one night after therapy. Contrasted with the low incidence of disordered breathing and desaturation in premenopausal women, 71 percent of the postmenopausal women had such events. In the placebo-treated group, all measured variables of sleep and breathing were identical on the two nights, which suggested that the findings of a single night of sleep monitoring may be representative of other nights of sleep. Although several subjects appeared to show improvement with MPG, only the maximum duration of apnea was significantly reduced the second night (p less than 0.03).

PIP: 21 postmenopausal women were monitored for sleep-disordered breathing and nocturnal oxygen desaturation to evaluate the contribution of progestational hormones to the occurrence of these sleep events. For approximately one month, 11 subjects received 30 mg of medroxyprogesterone (MPG) daily, and 10 received placebo tablets in a randomized, double-blind controlled study. Respiration, saturation and electroencephalography were monitored during one night of sleep before and one night after therapy. Contrasted with the low incidence of disordered breathing and desaturation in premenopausal women, 71% of the postmenopausal women had such events. In the placebo-treated group, all measured variables of sleep and breathing were identical on the 2 nights, suggesting that the findings of a single night of sleep monitoring may be representative of other nights of sleep. Although several subjects appeared to show improvement with MPG, only the maximum duration of apnea was significantly reduced the 2nd night (p0.03). The study supports the observation that disordered breathing and desaturation are similarly frequent in postmenopausal women and in men.

Alcohol increases sleep apnea and oxygen desaturation in asymptomatic men.

Using standard sleep techniques, we performed a placebo-controlled and randomized study to assess the effect of alcohol ingestion (2 ml/kg of body weight) on breathing and oxygen saturation during sleep. Twenty asymptomatic men volunteered for the two-night study: 11 were given a placebo on night 1, and alcohol on night 2 (group A); nine were given alcohol on night 1 and a placebo on night 2 (group B). We compared the incidence of sleep events (apnea, hypopnea and arterial oxygen disaturation) during the nights the subjects received alcohol and during the nights they received the placebo. Alcohol was associated with significant increases in the occurrence of the following: the number of sleep events (207 to 383,p less than 0.01), the events of arterial oxygen disaturation (118 to 226, p less than 0.01) and the number of apneic events (20 to 110, p less than 0.01). Alcohol had no significant effects on the number of times hypopnea occurred. Values obtained during sleep on the control night after alcohol ingestion also showed that the episodes of arterial oxygen desaturation remained statistically increased over control values before the ingestion of any alcohol (p = 0.01). These results show that in asymptomatic men alcohol ingestion increases the incidence of arterial oxygen desaturation and disordered breathing during sleep and that the increase in arterial oxygen desaturation persists for an additional night, even when no alcohol is consumed.

Sleep-disordered breathing and oxygen desaturation in obese patients.

Fourteen morbidly obese subjects, referred to our institution for bypass surgery for obesity, were studied with regard to pulmonary function and respiratory patterns during sleep. The seven female patients experienced no episodes of desaturation or disordered breathing during sleep. Six of the seven male patients experienced desaturation or disordered breathing. The one who did not had hypogonadism, suggesting that testosterone may have a role in the regulation of breathing during sleep. The two patients with the most frequent episodes of apnea and lowest oxygen saturation had a clinical picture consistent with the pickwickian syndrome. This supports the relationship previously noted between the degree of hypoxia and the presence of hypersomnolence.

The effect of weight loss on sleep-disordered breathing and oxygen desaturation in morbidly obese men.

Four morbidly obese men who had been found to have significant sleep-disordered breathing and oxygen desaturation were restudied after an average weight loss of 108 kg (range 53-155 kg). In all subjects, weight loss was accompanied by a significant reduction in the number of episodes per hour of sleep-disordered breathing events. In three of the four subjects, there was improvment in the severity of desaturation accompanying abnormal breathing. The two subjects with daytime somnolence and hypercapnia prior to weight loss showed the most dramatic improvement in desaturation. This suggests that obesity is a cause, rather than an effect, of the sleep apnea syndrome.

Nocturnal pulmonary hypertension in patients with chronic obstructive pulmonary disease.

Oxygen desaturation occurs during sleep in some patients with COPD. To investigate the effects of these hypoxemic episodes on the pulmonary vasculature, we studied four patients with our routine polysomnographic techniques and simultaneously recorded pulmonary artery pressure. In all four subjects, nocturnal episodes of desaturation were accompanied by elevations in the pulmonary artery pressure. Low flow oxygen abolished the drops in arterial oxygen saturation (but not the breathing abnormalities) and no elevations in the PA pressure were observed. We postulate that in some COPD patients these initially transient events may lead to sustained pulmonary hypertension and cor pulmonale. Nocturnal oxygen therapy may be indicated in more patients than previously suspected and may prevent the development of cor pulmonale.

The effect of low flow oxygen on sleep-disordered breathing and oxygen desaturation. A study of patients with chronic obstructive lung disease.

Oxygen desaturation occurs during sleep in many patients with chronic obstructive lung disease (COLD) and is often caused by sleep-disordered breathing (SDB). Nocturnal oxygen therapy should improve nighttime hypoxemia, but might also worsen SDB. Using standard polysomnographic techniques, we evaluated the frequency and duration of oxygen desaturation and SDB during sleep in 11 patients with stable COLD. During half of the night the patients breathed air through a nasal cannula and during the other half of the night they breathed oxygen at 2 liters per minute. Five patients had arterial lines inserted for determination of arterial blood gas levels during periods of SDB or desaturation. The ten men and one woman slept 70 minutes (52 percent of time in bed) while on air and 111 minutes (80 percent of time in bed) while on oxygen (p < 0.001). Oxygen therapy reduced the number of episodes of desaturation per hour and the time spent in desaturation. However, there was no difference between air and oxygen in episodes of SDB per hour, the duration of episodes of SDB, baseline sleeping PaCO2 or PaCO2 during episodes of desaturation or SDB. Therefore, in most patients with stable COLD, administration of oxygen at 2 liters per minute improves oxygenation, prolongs sleep, but does not adversely affect SDB.

Oxygen desaturation during sleep as a determinant of the "Blue and Bloated" syndrome.

Certain patients with chronic obstructive pulmonary disease may be classified as blue bloaters or pink puffers. Recent studies suggest that physiologic changes during sleep contribute to the clinical expression of these syndromes. To investigate this, we monitored four blue bloaters and six pink puffers during one night's sleep to determine the incidence of sleep disordered breathing (SDB) and of arterial oxygen desaturation. There were no significant differences between the two groups for sleep period time, awake oxygen saturation, or the number of episodes of SDB. Blue bloaters had lower baseline oxygen saturations, more episodes of arterial oxygen desaturation, and larger falls in oxygen saturation and spent more time at low levels of oxygen saturation while asleep. We propose that the degree and the duration of sleep hypoxemia of blue bloaters but not of pink puffers may contribute to early pulmonary hypertension and cor pulmonale.

Physiological effects of corticosteroids in foodstuff aspiration.

Aspiration of gastric contents that contain small food particles has been shown to cause severe pneumonitis, even when the pH of the aspirate is greater than 2.5. The effects of corticosteroids on the hemodynamic and ventilatory consequences of this type of aspiration have not been investigated. Twenty healthy mongrel dogs were anesthetized and 2 mL/kg of gastric contents (pH 5.9) that contained small food particles were injected into their tracheas. Group 1 animals (controls, N = 10) received no therapy. Group 2 animals (steroid-treated, N = 10) were given methylprednisolone intravenously, 30 mg/kg, three times a day beginning one hour after aspiration. The animals were observed for 14 days, during which time there were no statistically significant differences between the groups in mortality, PaCO2, pH, systemic and pulmonary arterial blood pressures, fractional intrapulmonary shunt, or histological findings. Arterial PO2 was lower at 24 hours in group 2 dogs than in group 1 dogs. We conclude that steroids are of no benefit in the treatment of foodstuff-induced aspiration pneumonia.

The effect of nasal packing on sleep-disordered breathing and nocturnal oxygen desaturation.

Nasal obstruction is known to cause abnormal ventilation during sleep in infants, but its effects on breathing and oxygenation during sleep in adults are unknown. However, in adults, obstruction of the nose by nasal packing has been shown to cause hypoxia, and on occasion, hypercarbia and sudden death. We have investigated the pattern of ventilation and the level of oxygenation during sleep in seven patients who had nasal packs after nasal polypectomy or septoplasty. Using standard polysomnographic techniques, we monitored chest wall motion, nasal and oral airflow, and arterial oxygen saturation and sleep stages. Nasal packing either caused or worsened sleep-disordered breathing in all patients and significantly increased the number, duration, and frequency of episodes for the group as a whole. Several patients also had a greatly increased number and severity of episodes of nocturnal oxygen desaturation. This study shows that obstruction of the nose by packing causes marked alterations in breathing during sleep in adults.

Chronic obstructive pulmonary disease.

The diagnosis of COPD can easily be made on the basis of history, physical findings, and simple laboratory procedures. Treatment is aimed at symptoms and is individualized to the patient's needs. Therapy must be given on a long-term basis. The patient should be educated in the basic nature of his disease and encouraged to return to as normal a life as possible. Patients with COPD often are severely restricted in their earning ability, and treatment can cause a severe financial drain. Thus the physician should carefully consider costs--especially the cost of medications. Finally, the correct choice and use of the many therapeutic measures available can yield marked symptomatic relief and improvement in lifestyle--often allowing the patient to return to productive employment.