Spleen and lung virome analysis of South American fur seals (Arctocephalus australis) collected on the southern Brazilian coast.

South American fur seals (Arctocephalus australis) are believed to reach the coast of Rio Grande do Sul (RS) through sea currents. They live in colonies and are frequently found resting on the beach. However, it is also common to find dead pinnipeds on beaches, sharing the environment with humans, domestic animals and other wild species on the coast and facilitating the transmission of pathogens. In the present study, a metagenomic approach was applied to evaluate the viral diversity in organs of fur seals found deceased along the coast of the state of RS, southern Brazil. The lungs and spleens of 29 animals were collected, macerated individually, pooled separately (one pool for lungs and another for spleens) and sequenced using the Illumina MiSeq platform. Sequences more closely related to members of the Anelloviridae and Circoviridae families were detected. Nine putative new species of anellovirus and one putative new genus, named Nitorquevirus, were described. Additionally, the circovirus sequences found in the lungs of A. australis have a common ancestor with PCV3, a proposed swine pathogen. Our study expanded the knowledge about viral communities in pinnipeds and could be useful for monitoring new viruses and potential viral sharing among wildlife, domestic animals, and humans.

Risk factors associated with Toxoplasma gondii infection in captive Sapajus spp.

The aim of this study was to identify risk factors associated with prevalence of Toxoplasma gondii infection in captive capuchin monkeys at a facility in the northeastern Brazil. Serum samples from 116 bearded capuchin (Sapajus libidinosus), nine blonde capuchin (Sapajus flavius), five black-capped capuchin (Sapajus apella), and four capuchin monkeys (Sapajus spp.) were tested for T. gondii antibodies using the modified agglutination test (MAT, cut-off ≥25); antibodies were found in 85.3% (99/116) of S. libidinosus, 55.6% (5/9) of S. flavius, 80.0% (4/5) of S. apella, and 75.0% (3/4) of S. spp. The risk factors associated with T. gondii seropositivity were ingestion of raw meat [OR = 4.13 (1.26; 13.50)] and old age [OR = 4.90 (1.70; 14.13)]. Results indicate a very high T. gondii seropositivity in these primate populations. To minimize exposure to T. gondii raw meat should not be fed to these animals.

Gross anatomical features of the gastrointestinal tract (GIT) of blue-and-yellow macaws (Ara ararauna) - oesophagus to cloaca.

Morphological studies of the gastrointestinal tract of blue-and-yellow macaws (Ara ararauna) are scarce. In view of the paucity of information regarding the digestive tract of macaws, this study aims to describe the gross anatomical features (oesophagus to cloaca) as part of a broad study of the gastrointestinal tract (GIT) of these birds. Three animals (two males and one female) adult macaws were anatomically dissected from the oropharynx to the cloaca to expose the GIT. The oesophagus was identified as a muscle-membranous tube continuous with the crop, which was intimately attached to the skin. The internal longitudinal folds of the cervical oesophagus were sparser cranial to the crop and less evident compared to the portion caudal to the crop. The duodenum began in the pylorus and was grey-coloured exhibiting a large lumen. The jejunum was formed by loops in a spiral-fashion model supported by mesojejunum. The ileum was also composed by small loops and was continuous with the colo-rectum forming the large intestine, because the caeca were absent. The large intestine was short, median in position, suspended in the dorsal wall of the abdominal cavity by mesentery and ended in the cloaca. The GIT was similar to the basic patterns in birds, in general, and also presented new unreported morphological data that might be important when studying nutrition and health of the macaws.

Gross anatomical features of the gastrointestinal tract (GIT) of blue and yellow macaws (Ara ararauna) - oral cavity and pharynx.

Morphological studies of the gastrointestinal tract (GIT) of blue and yellow macaws (Ara ararauna) are scarce. Therefore, this study aims to describe the macaw's oropharyngeal cavity in order to supply the deficiency of anatomical data and as part of a broad study of the GIT of these birds. Two male and one female adult blue and yellow macaws were anatomically dissected to expose the oropharynx. The macaw oropharynx was 'V-shaped' and flattened laterally being composed of maxillary and mandibular rhamphotheca of the beak. The tongue, lingual frenulum and laryngeal mound (containing 'spindle-shaped' glottis and prominent mucosal papillae) formed the floor of the oropharynx. The roof revealed two distinct regions separated by a 'step-like depression', whereas in the floor, the mandibular rhamphotheca was separated from the oral cavity mucosa by a large vestibulum enclosing the lingual frenulum. The palate was hard without any signs of rugae nor palatine raphe. A smooth ridge extended caudally from the choana to the common opening of the Eustachian tubes. This study, in addition to confirming the basic features of the oropharynx previously described for birds in general, provided new, unreported morphological data, some of which may be important when studying nutrition and health of these birds.

Modulation of stimulus-secretion coupling in porcine adrenal chromaffin cells by receptor-mediated increases in protein kinase C activity.

Catecholamine (CAT) secretion by adrenal chromaffin cells is primarily triggered by nicotinic receptor-dependent increases in cytosolic Ca(2+). The principal aim of the present study was to determine whether pituitary adenylate cyclase activating peptide (PACAP), which is coreleased with acetylcholine from the splanchnic nerve, can modulate nicotinic receptor-dependent Ca(2+) signaling and catecholamine secretion in porcine adrenal medullary chromaffin (PAMC) cells. Activation of protein kinase C (PKC) with phorbol myristate acetate (PMA) dose- and time-dependently inhibited nicotine (NIC)-induced Ca(2+) transients. At 100 nM PMA, peak Ca(2+) levels were reduced by 27% +/- 2% (P < 0.05) and 41% +/- 3% (P < 0. 05) after 10 and 20 min exposure, respectively. The inhibitory effects of PMA were significantly reduced by preincubation with the PKC inhibitor staurosporine. KCl-induced Ca(2+) transients were also reduced by 20 min PMA treatment (Delta -27% +/- 4%; P < 0.05), suggesting that PKC affects voltage-gated Ca(2+) channel activity. Pretreatment with PACAP also resulted in both time- and concentration-dependent suppression of Ca(2+) transients. After 20 min exposure to 1 microM PACAP, NIC- and KCl-induced transients were reduced by 36% +/- 5% (P < 0.05) and 51% +/- 6% (P < 0.05), respectively. These effects could also be prevented by staurosporine pretreatment. NIC-induced CAT secretion was significantly reduced by pretreatment with both PMA (Delta -56% +/- 2%; P < 0.05) and PACAP (Delta-53% +/- 7%; P < 0.05). This suppressive effect on secretion could be prevented by pretreatment with staurosporine. These data suggest that, in addition to having direct stimulatory effects on catecholamine synthesis and secretion, PACAP can also negatively modulate nicotinic receptor-dependent Ca(2+) signaling and secretion in PAMC cells.

Stimulus-secretion coupling in porcine adrenal chromaffin cells: acute effects of glucocorticoids.

Recent studies from this laboratory have established that long-term exposure (48 hr) to glucocorticoids can modulate voltage-gated Ca(2+) channel activity and subsequent intracellular Ca(2+) transients in porcine adrenal medullary chromaffin (PAMC) cells maintained in primary culture. Consistent with many steroid hormone-mediated responses, this chronic effect of glucocorticoids probably involves increased gene expression and protein synthesis. However, there is now considerable evidence to suggest that steroids can also elicit acute, non-genomic effects. The aim of the present study was to determine whether acute exposure to glucocorticoids also affects nicotinic receptor-dependent catecholamine (CAT) secretion and Ca(2+) signaling in PAMC cells. Acute exposure to dexamethasone (DEX) dose-dependently attenuated the degree of nicotine (NIC)-induced CAT secretion, as well as the amplitude of NIC-induced intracellular Ca(2+) transients. Significant inhibition of CAT secretion occurred immediately upon addition of DEX, reached maximal levels within 5 min of exposure to DEX, and was rapidly reversible after steroid washout. The endogenous porcine glucocorticoid cortisol elicited similar effects. In contrast, DEX had no significant effect on KCl-induced CAT secretion or intracellular Ca(2+) transients. These data demonstrate that acute exposure to glucocorticoids can modulate stimulus-secretion coupling in PAMC cells and suggest that the primary site of action is the nicotinic receptor.

cAMP modulates an S-type K+ channel coupled to GABAB receptors in mammalian respiratory neurons.

Premotor respiratory neurons from neonatal rats express a Ba(2+)-insensitive outward rectifying K+ channel (KOR) which is activated by gamma-aminobutyric acid acting at its beta receptor. The biophysical properties of KOR are similar to those described for the S-channel (KS) which underlies simple forms of non-associative learning in the marine mollusc Aplysia. We show here that KOR, like the S-channel, is inhibited by cAMP. In addition, we demonstrate that this inhibition is due to a change in closed time kinetics. Our data suggests that the ionic and biochemical substrates underlying synaptic plasticity in Aplysia have been phylogenetically conserved in mammalian motor circuits such as that controlling rhythmic breathing movements.

GABAb receptors are coupled to a barium-insensitive outward rectifying potassium conductance in premotor respiratory neurons.

1. Labeled premotor respiratory neurons from neonatal rats in culture were used to study the effects of (+/-)baclofen, a selective gamma-aminobutyric acid (GABA)b receptor agonist known to inhibit rhythmic breathing movements in mammals. Bath application of (+/-)baclofen-activated outward currents in cell-attached patches, suggesting that a second messenger system linked the (+/-)baclofen-activated conductance (GBac) to the GABAb receptor. 2.GBac channels exhibited outward rectification and were insensitive to blockade by Ba2+ and Cs+. The single-channel conductance was 100 pS in symmetrical K+ solutions and decreased as [K+]o was reduced. The reversal potential for the GBac channel shifted 45 mV/decade when [K+]o was changed indicating that it was predominantly selective for K+ ions. These properties were similar to those of the S-channel in Aplysia sensory neurons. 3. The properties of GBac channels were distinct from those associated with the GABAb mediated slow inhibitory postsynaptic potential (IPSP), indicating that GABAb receptors can be associated with more than one type of K+ channel. We propose that GBac channels modulate the repetitive firing activity of premotor respiratory neurons and may also participate in presynaptic inhibition.

Respiratory-associated thalamic activity is related to level of respiratory drive.

We recorded phrenic nerve activity and thalamic single unit firing in unanesthetized, suprathalamically decerebrated, paralyzed and ventilated cats, in which vagi and carotid sinus nerves (CSN) had been ablated. Seventy-six (14%) of 545 neurons in regions of the thalamus related to the ascending reticular system, which had been tonically firing at low respiratory drives, developed rhythmic increases of firing associated with each respiration when drive had been increased by CSN stimulation or hypercapnia. The increases of neuronal firing occurred in late inspiration/post-inspiration but sometimes lasted into expiration; the magnitude of change was graded according to the magnitude of respiratory activity. Thalamic neurons also fired with a rhythm related to ventilator-induced chest expansion, some units showing both the respiratory-associated and the ventilator-related rhythms. Simultaneously recorded mesencephalic and thalamic neurons developed similar rhythms when drive was increased. We suggest that these neuronal activities reflect the conveyance of information about respiration to the cortex, where it may lead to the sensation of dyspnea and perhaps to arousal.

Neural respiratory responses to cortically induced seizures in cats.

Seizure activity can lead to profound respiratory stimulation in spontaneously breathing animals with intact respiratory feedback mechanisms (Paydarfar et al., Am. J. Physiol. 260, R934, 1991). The present study was designed to test the hypothesis that peripheral respiratory feedback mechanisms are not important for the genesis of seizure-induced hyperpnea. Studies were performed in 16 anesthetized, vagotomized, glomectomized cats whose end-tidal PCO2 (PETCO2) was kept constant. Integrated phrenic nerve activity was used to represent respiration. Seizures were induced by injection of penicillin into the parietal cortex and electrocorticographic (ECoG) and biceps femoris nerve activities, arterial pressure, airway PCO2 and brain temperature were recorded continuously. Progressive seizure activity was associated with progressive increases of respiratory frequency and peak phrenic activity, despite constancy of PETCO2 and brain temperature. Patterns of entrainment were identified among ECoG spikes, biceps femoris nerve and phrenic nerve activities. Phrenic nerve activity became highly irregular during generalized ictal seizures and ceased to respond to changes of PETCO2. Acute intercollicular decerebration in all experiments resulted in normalization of respiratory rhythm even while ictal ECoG activity continued. We conclude that stimulation of breathing during seizures occurs in the absence of respiratory feedback mechanisms. The findings suggest that an important cause of the respiratory response is a feedforward mechanism, whereby activation of subcortical structures above medulla and pons results in stimulation of breathing.

Anesthesia affects respiratory and sympathetic nerve activities differentially.

Phrenic and cervical sympathetic nerve responses to hypercapnia were examined before and after anesthesia in twelve midcollicularly decerebrated, vagotomized, glomectomized, paralyzed and ventilated cats. We measured responses of integrated phrenic and cervical sympathetic nerve activities to increases in end-tidal PCO2 (PETCO2) from apneic threshold to approximately 30 torr above threshold. All cats were studied first in the unanesthetized state. Six cats were then restudied after a quarter of a usual dose of chloralose/urethane (10 mg/kg and 62.5 mg/kg, respectively) and then after half the usual dose of chloralose/urethane (20 mg/kg and 125 mg/kg). The other six animals were restudied after quarter of a standard dose of pentobarbital (9 mg/kg), after half the standard dose (18 mg/kg) and then after the full (35 mg/kg) dose. Both anesthetic agents led to significant increases in apneic thresholds for both phrenic and sympathetic nerve activities. These agents also caused dose-dependent decreases in peak, tonic and respiratory-related sympathetic nerve activities. Peak (tidal) phrenic nerve activities, in comparison, were much less affected by the anesthetic agents. CO2 response curves showed that both of these anesthetic agents depressed, at any given level of PETCO2, respiratory-related sympathetic nerve responses more than the responses found in the phrenic nerve. We conclude that the relations between peak, tonic (i.e. between phasic bursts) and respiratory-related sympathetic nerve activities and phrenic nerve activity can be altered by anesthesia.

Respiratory-associated rhythmic firing of midbrain neurones in cats: relation to level of respiratory drive.

1. We recorded phrenic nerve activities and single unit firing of mesencephalic neurones in unanaesthetized supracollicularly decerebrated, paralysed and ventilated cats, in which vagi and carotid sinus nerves had been ablated. We made these measurements first at low levels of respiratory drive associated with normal PCO2 levels, then with increased respiratory drive and levels of phrenic activity produced by hypercapnia or by carotid sinus nerve stimulation. 2. We found that at least a quarter of the neurones in the central tegmental field of the mesencephalon, which were irregularly tonic or silent at low respiratory drives, developed a rhythmic increase of firing associated with each respiration. There appeared to be a threshold at about 50% of maximum respiratory activity, below which the respiratory-associated rhythm did not occur. Above this level, neuronal firing increased in graded fashion with increasing magnitude of respiratory activity. The latency from onset of phrenic activity to onset of increased neuronal firing was quite long (1.0 s) at drives just above the threshold but shortened to as little as 0.3 s as drive increased towards its maximum. 3. Cutting the spinal cord at C1-C2 had no effect on the ability of increased respiratory activity to generate a respiratory-associated rhythm in mesencephalic neurones. 4. Short-lasting anaesthesia with the agent Saffan caused mesencephalic neurones to lose the respiratory-associated rhythm with little change in phrenic activity and no change in respiratory cycle timing. 5. We also found a mesencephalic response to ventilator-induced chest expansion. The latency of the response from onset of expansion, indexed by fall of airway PCO2, to onset of neurone firing was shorter (0.2 s) than that found with the respiratory-associated rhythm. In seventeen neurones we found both the respiratory-associated rhythm and the independent ventilator-associated rhythm. 6. We interpret our findings to show that the respiratory-associated rhythmic firing of midbrain neurones is not primarily involved in generation or modulation of the motor function of the respiratory oscillator. We believe, instead, that these neurones are part of a sensory pathway conveying information about the magnitude of central neural respiratory drive, as well as spinally transmitted information from receptors in the chest wall, to thalamus and cortex. We suggest that the sensation ultimately generated may be that of 'air hunger' or dyspnoea.

Respiratory responses to focal and generalized seizures in cats.

We studied the effects on breathing of seizures induced by focal injection of penicillin G into the parietal cortex in 13 anesthetized cats. Electrocorticograms, ventilation, end-tidal PCO2, and intrapleural and arterial pressures were monitored; changes of these variables were related to the stages of motor seizure. The first respiratory responses, tachypnea and hyperpnea, usually occurred before any peripheral muscular contractions developed. Progression of the seizure was always accompanied by further tachypnea and hyperpnea. The hyperpnea associated with all stages of seizure activity resulted in hypocapnia, which was sustained even during prolonged tonic-clonic motor convulsions that caused a threefold increase of metabolic rate. The extreme tachypnea of tonic generalized convulsions led to increased end-expiratory lung volume because of dynamic hyperinflation associated with very short expiratory durations in the tonic phase. We suggest that the profound effects of seizures on respiration are by feedforward mechanisms from the cortical focus itself and from subcortical circuits, such as hypothalamus, that become involved during seizure propagation and generalization. Peripheral respiratory feedback mechanisms are not important for the genesis of seizure-induced hyperpnea.

Development of short-term potentiation of respiration.

Development of short-term potentiation (STP) of respiration, which leads to the respiratory 'afterdischarge', was studied in anesthetized, paralyzed, vagotomized and glomectomized cats. Phrenic nerve activity was used as an index of respiratory output. Respiratory output was increased and the potentiating mechanism activated by electrical stimulation of a carotid sinus nerve (CSN). Development of STP was determined from the magnitude of potentiation after various durations (0 to 60 sec) of stimulation. The average time constant (TC) for the development of the potentiation was 9 sec, whereas the TC for its decay (afterdischarge) was 46.1 +/- 3.9 sec. The magnitude of potentiation is dependent upon the number of pulses in the stimulus train. We conclude that the development of short-term potentiation of respiration is relatively slow but much faster than the decay, or afterdischarge. We suggest that the slow increase of respiration during a stimulation and the decay afterwards are due to a common mechanism, short-term potentiation of neural activity in respiratory control pathways.

Phase resetting of respiratory rhythm: effect of changing respiratory "drive".

We studied the effect of changing drive on resetting of respiratory rhythm in anesthetized cats and in a model (Van der Pol) of a limit-cycle oscillator. In cats, rhythm was perturbed by brief mesencephalic stimuli. Stimulus time in the cycle (old phases) and times of onset of rescheduled breaths (cophases) were measured. Previous study [Paydarfar and Eldridge, Am. J. Physiol. 252 (Regulatory Integrative Comp. Physiol. 21): R55-R62, 1987] showed distinct types of phase resetting that depended on strength of stimuli. In this study, stimulus strength was kept constant, but respiratory drive was changed by increasing PCO2, by stimulating carotid sinus nerve, or by cooling intermediate areas of ventral medulla. Type 0 (strong) resetting occurred when respiratory drive was low, type 1 (weak) resetting when drive was high, and a phase singularity when drive was intermediate. Phase-resetting patterns generated by the model showed the same behavior when a drive parameter was changed. The findings support the idea that continuous limit-cycle dynamics underlie generation of respiratory rhythm. Increased respiratory drive, by increasing size of the limit cycle, reduces functional effectiveness of the same perturbing stimulus in causing phase resetting.