Postinspiratory and preBötzinger complexes contribute to respiratory-sympathetic coupling in mice before and after chronic intermittent hypoxia.

The sympathetic nervous system modulates arterial blood pressure. Individuals with obstructive sleep apnea (OSA) experience numerous nightly hypoxic episodes and exhibit elevated sympathetic activity to the cardiovascular system leading to hypertension. This suggests that OSA disrupts normal respiratory-sympathetic coupling. This study investigates the role of the postinspiratory complex (PiCo) and preBötzinger complex (preBötC) in respiratory-sympathetic coupling under control conditions and following exposure to chronic intermittent hypoxia (CIH) for 21 days (5% O2-80 bouts/day). The surface of the ventral brainstem was exposed in urethane (1.5 g/kg) anesthetized, spontaneously breathing adult mice. Cholinergic (ChAT), glutamatergic (Vglut2), and neurons that co-express ChAT and Vglut2 at PiCo, as well as Dbx1 and Vglut2 neurons at preBötC, were optogenetically stimulated while recording activity from the diaphragm (DIA), vagus nerve (cVN), and cervical sympathetic nerve (cSN). Following CIH exposure, baseline cSN activity increased, breathing frequency increased, and expiratory time decreased. In control mice, stimulating PiCo specific cholinergic-glutamatergic neurons caused a sympathetic burst during all phases of the respiratory cycle, whereas optogenetic activation of cholinergic-glutamatergic PiCo neurons in CIH mice increased sympathetic activity only during postinspiration and late expiration. Stimulation of glutamatergic PiCo neurons increased cSN activity during the postinspiratory phase in control and CIH mice. Optogenetic stimulation of ChAT containing neurons in the PiCo area did not affect sympathetic activity under control or CIH conditions. Stimulating Dbx1 or Vglut2 neurons in preBötC evoked an inspiration and a concomitant cSN burst under control and CIH conditions. Taken together, these results suggest that PiCo and preBötC contribute to respiratory-sympathetic coupling, which is altered by CIH, and may contribute to the hypertension observed in patients with OSA.

Chronic intermittent hypoxia reveals role of the Postinspiratory Complex in the mediation of normal swallow production.

Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder that results in multiple bouts of intermittent hypoxia. OSA has many neurological and systemic comorbidities, including dysphagia, or disordered swallow, and discoordination with breathing. However, the mechanism in which chronic intermittent hypoxia (CIH) causes dysphagia is unknown. Recently, we showed the postinspiratory complex (PiCo) acts as an interface between the swallow pattern generator (SPG) and the inspiratory rhythm generator, the preBötzinger complex, to regulate proper swallow-breathing coordination (Huff et al., 2023). PiCo is characterized by interneurons co-expressing transporters for glutamate (Vglut2) and acetylcholine (ChAT). Here we show that optogenetic stimulation of ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2 mice exposed to CIH does not alter swallow-breathing coordination, but unexpectedly disrupts swallow behavior via triggering variable swallow motor patterns. This suggests that glutamatergic-cholinergic neurons in PiCo are not only critical for the regulation of swallow-breathing coordination, but also play an important role in the modulation of swallow motor patterning. Our study also suggests that swallow disruption, as seen in OSA, involves central nervous mechanisms interfering with swallow motor patterning and laryngeal activation. These findings are crucial for understanding the mechanisms underlying dysphagia, both in OSA and other breathing and neurological disorders.

Chronic Intermittent Hypoxia reveals role of the Postinspiratory Complex in the mediation of normal swallow production.

Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder that results in multiple bouts of intermittent hypoxia. OSA has many neurologic and systemic comorbidities including dysphagia, or disordered swallow, and discoordination with breathing. However, the mechanism in which chronic intermittent hypoxia (CIH) causes dysphagia is unknown. Recently we showed the Postinspiratory complex (PiCo) acts as an interface between the swallow pattern generator (SPG) and the inspiratory rhythm generator, the preBötzinger Complex, to regulate proper swallow-breathing coordination (Huff et al., 2023). PiCo is characterized by interneurons co-expressing transporters for glutamate (Vglut2) and acetylcholine (ChAT). Here we show that optogenetic stimulation of ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2 mice exposed to CIH does not alter swallow-breathing coordination, but unexpectedly disrupts swallow behavior via triggering variable swallow motor patterns. This suggests, glutamatergic-cholinergic neurons in PiCo are not only critical for the regulation of swallow-breathing coordination, but also play an important role in the modulation of swallow motor patterning. Our study also suggests that swallow disruption, as seen in OSA, involves central nervous mechanisms interfering with swallow motor patterning and laryngeal activation. These findings are crucial for understanding the mechanisms underlying dysphagia, both in OSA and other breathing and neurological disorders.

Role of the postinspiratory complex in regulating swallow-breathing coordination and other laryngeal behaviors.

Breathing needs to be tightly coordinated with upper airway behaviors, such as swallowing. Discoordination leads to aspiration pneumonia, the leading cause of death in neurodegenerative disease. Here, we study the role of the postinspiratory complex (PiCo) in coordinating breathing and swallowing. Using optogenetic approaches in freely breathing anesthetized ChATcre:Ai32, Vglut2cre:Ai32 and intersectional recombination of ChATcre:Vglut2FlpO:ChR2 mice reveals PiCo mediates airway protective behaviors. Activation of PiCo during inspiration or the beginning of postinspiration triggers swallow behavior in an all-or-nothing manner, while there is a higher probability for stimulating only laryngeal activation when activated further into expiration. Laryngeal activation is dependent on stimulation duration. Sufficient bilateral PiCo activation is necessary for preserving the physiological swallow motor sequence since activation of only a few PiCo neurons or unilateral activation leads to blurred upper airway behavioral responses. We believe PiCo acts as an interface between the swallow pattern generator and the preBötzinger complex to coordinate swallow and breathing. Investigating PiCo's role in swallow and laryngeal coordination will aid in understanding discoordination with breathing in neurological diseases.

Postinspiratory complex acts as a gating mechanism regulating swallow-breathing coordination and other laryngeal behaviors.

Breathing needs to be tightly coordinated with upper airway behaviors, such as swallowing. Discoordination leads to aspiration pneumonia, the leading cause of death in neurodegenerative diseases. Here we study the role of the postinspiratory complex, (PiCo) in coordinating breathing and swallowing. Using optogenetic approaches in freely breathing-anesthetized ChATcre, Vglut2cre and co-transmission of ChATcre/Vglut2FlpO mice reveals this small brainstem microcircuit acts as a central gating mechanism for airway protective behaviors. Activation of PiCo during inspiration or the beginning of postinspiration triggers swallow behavior, while there is a higher probability for stimulating laryngeal activation when activated further into expiration, suggesting PiCo's role in swallow-breathing coordination. PiCo triggers consistent swallow behavior and preserves physiologic swallow motor sequence, while stimulates laryngeal activation variable to stimulation duration. Sufficient bilateral PiCo activation is necessary for gating function since activation of only a few PiCo neurons or unilateral activation leads to blurred behavioral response. Viral tracing experiments reveal projections from the caudal nucleus of the solitary tract (cNTS), the presumed swallow pattern generator (SPG), to PiCo and vice versa. However, PiCo does not directly connect to laryngeal muscles. Investigating PiCo's role in swallow and laryngeal coordination will aid in understanding discoordination in breathing and neurological diseases.

Breathing disturbances in Rett syndrome.

Rett Syndrome is an X-linked neurological disorder characterized by behavioral and neurological regression, seizures, motor deficits, and dysautonomia. A particularly prominent presentation includes breathing abnormalities characterized by breathing irregularities, hyperventilation, repetitive breathholding during wakefulness, obstructive and central apneas during sleep, and abnormal responses to hypoxia and hypercapnia. The condition and pathology of the respiratory system is further complicated by dysfunctions of breathing-motor coordination, which is reflected in dysphagia. The discovery of the X-linked mutations in the MECP2 gene has transformed our understanding of the cellular and molecular mechanisms that are at the root of various clinical phenotypes. However, the genotype-phenotype relationship is complicated by various factors which include not only X-inactivation but also consequences of the intermittent hypoxia and oxidative stress associated with the breathing abnormalities.

Optogenetic stimulation of pre-Bötzinger complex reveals novel circuit interactions in swallowing-breathing coordination.

The coordination of swallowing with breathing, in particular inspiration, is essential for homeostasis in most organisms. While much has been learned about the neuronal network critical for inspiration in mammals, the pre-Bötzinger complex (preBötC), little is known about how this network interacts with swallowing. Here we activate within the preBötC excitatory neurons (defined as Vglut2 and Sst neurons) and inhibitory neurons (defined as Vgat neurons) and inhibit and activate neurons defined by the transcription factor Dbx1 to gain an understanding of the coordination between the preBötC and swallow behavior. We found that stimulating inhibitory preBötC neurons did not mimic the premature shutdown of inspiratory activity caused by water swallows, suggesting that swallow-induced suppression of inspiratory activity is not directly mediated by the inhibitory neurons in the preBötC. By contrast, stimulation of preBötC Dbx1 neurons delayed laryngeal closure of the swallow sequence. Inhibition of Dbx1 neurons increased laryngeal closure duration and stimulation of Sst neurons pushed swallow occurrence to later in the respiratory cycle, suggesting that excitatory neurons from the preBötC connect to the laryngeal motoneurons and contribute to the timing of swallowing. Interestingly, the delayed swallow sequence was also caused by chronic intermittent hypoxia (CIH), a model for sleep apnea, which is 1) known to destabilize inspiratory activity and 2) associated with dysphagia. This delay was not present when inhibiting Dbx1 neurons. We propose that a stable preBötC is essential for normal swallow pattern generation and disruption may contribute to the dysphagia seen in obstructive sleep apnea.

Laryngeal and swallow dysregulation following acute cervical spinal cord injury.

Laryngeal function is vital to airway protection. Although swallow is mediated by the brainstem, the mechanism underlying the increased risk of dysphagia after cervical spinal cord injury (SCI) is unknown. We hypothesized that: 1) loss of descending phrenic drive affects swallow and breathing differently, and 2) loss of ascending spinal afferent information alters swallow regulation. We recorded electromyograms (EMGs) from upper airway and chest wall muscles in freely breathing pentobarbital-anesthetized cats and rats. Laryngeal abductor activity during inspiration increased about twofold following C2 lateral hemisection. Ipsilateral to the injury, the crural diaphragm EMG amplitude was reduced during breathing (62 ± 25% change postinjury), but no animal had complete termination of activity; 75% of animals had increased contralateral diaphragm recruitment, but this did not reach significance. During swallow, laryngeal adductor and pharyngeal constrictor muscles increased activity, and diaphragm activity was bilaterally suppressed. This was unexpected because of the ipsilateral-specific response during breathing. Swallow-breathing coordination was disrupted by injury, and more swallows occurred during early expiration. Finally, to determine if the chest wall is a major source of feedback for laryngeal regulation, we performed T1 total transections in rats. As in the C2 lateral hemisection, inspiratory laryngeal recruitment was the first feature noted after injury. In contrast to the C2 lateral hemisection, diaphragmatic drive increased after T1 transection. Overall, we found that SCI alters laryngeal drive during swallow and breathing, and alters swallow-related diaphragm activity. Our results show behavior-specific effects, suggesting that swallow is affected more than breathing is by SCI, and emphasizing the need for additional studies on the effect of ascending afferents from the spinal cord on laryngeal function.NEW & NOTEWORTHY This is the first manuscript to determine the impact of cSCI on laryngeal and swallow function, and to describe a possible mechanism for dysphagia and altered airway protection after injury.

Evidence of intermediate reticular formation involvement in swallow pattern generation, recorded optically in the neonate rat sagittally sectioned hindbrain.

Swallow is a primitive behavior regulated by medullary networks, responsible for movement of food/liquid from the oral cavity to the esophagus. To investigate how functionally heterogeneous networks along the medullary intermediate reticular formation (IRt) and ventral respiratory column (VRC) control swallow, we electrically stimulated the nucleus tractus solitarius to induce fictive swallow between inspiratory bursts, with concurrent optical recordings using a synthetic Ca2+ indicator in the neonatal sagittally sectioned rat hindbrain (SSRH) preparation. Simultaneous recordings from hypoglossal nerve rootlet (XIIn) and ventral cervical spinal root C1-C2 enabled identification of the system-level correlates of 1) swallow (identified as activation of the XIIn but not the cervical root) and 2) Breuer-Hering expiratory reflex (BHE; lengthened expiration in response to stimuli during expiration). Optical recording revealed reconfiguration of respiration-modulated networks in the ventrolateral medulla during swallow and the BHE reflex. Recordings identified novel spatially compact networks in the IRt near the facial nucleus (VIIn) that were active during fictive swallow, suggesting that the swallow network is not restricted to the caudal medulla. These findings also establish the utility of using this in vitro preparation to investigate how functionally heterogeneous medullary networks interact and reconfigure to enable a repertoire of orofacial behaviors.NEW & NOTEWORTHY For the first time, medullary networks that control breathing and swallow are recorded optically. Episodic swallows are induced via electrical stimulation along the dorsal medulla, in and near the NTS, during spontaneously occurring fictive respiration. These findings establish that networks regulating both orofacial behaviors and breathing are accessible for optical recording at the surface of the sagittally sectioned rodent hindbrain preparation.

The Pathophysiology of Rett Syndrome With a Focus on Breathing Dysfunctions.

Rett syndrome (RTT), an X-chromosome-linked neurological disorder, is characterized by serious pathophysiology, including breathing and feeding dysfunctions, and alteration of cardiorespiratory coupling, a consequence of multiple interrelated disturbances in the genetic and homeostatic regulation of central and peripheral neuronal networks, redox state, and control of inflammation. Characteristic breath-holds, obstructive sleep apnea, and aerophagia result in intermittent hypoxia, which, combined with mitochondrial dysfunction, causes oxidative stress-an important driver of the clinical presentation of RTT.

Sex-specific vagal and spinal modulation of breathing with chest compression.

Lung volume is modulated by sensory afferent feedback via vagal and spinal pathways. The purpose of this study was to systematically alter afferent feedback with and without a mechanical challenge (chest compression). We hypothesized that manipulation of afferent feedback by nebulization of lidocaine, extra-thoracic vagotomy, or lidocaine administration to the pleural space would produce differential effects on the motor pattern of breathing during chest compression in sodium pentobarbital anesthetized rats (N = 43). Our results suggest that: 1) pulmonary stretch receptors are not the sole contributor to breathing feedback in adult male and female rats; 2) of our manipulations, chest compression had the largest effect on early expiratory diaphragm activity ("yield"); 3) reduction of spinally-mediated afferent feedback modulates breathing patterns most likely via inhibition; and 4) breathing parameters demonstrate large sex differences. Compared to males, female animals had lower respiratory rates (RR), which were further depressed by vagotomy, while chest compression increased RR in males, and decreased yield in females without changing RR. Collectively, our results suggest that balance between tonic vagal inhibition and spinal afferent feedback maintains breathing characteristics, and that it is important to specifically evaluate sex differences when studying control of breathing.

Sex-specific vagal and spinal modulation of swallow and its coordination with breathing.

Swallow-breathing coordination is influenced by changes in lung volume, which is modulated by feedback from both vagal and spinal sensory afferents. The purpose of this study was to manipulate feedback from these afferents, with and without a simultaneous mechanical challenge (chest compression), in order to assess the influence of each sensory pathway on swallow in rats. We hypothesized that manipulation of afferent feedback would shift the occurrence of swallow toward the inspiratory phase of breathing. Afferent feedback was perturbed by lidocaine nebulization, extra-thoracic vagotomy, or lidocaine administration to the pleural space in sodium pentobarbital anesthetized rats (N = 43). These different afferent perturbations were performed both in control conditions (no chest compression), and with chest compression. Manipulating pulmonary stretch receptor-mediated volume feedback in male animals decreased swallow occurrence. Female rats appear to rely more on spinal afferent feedback, as swallow occurrence shifted to late expiration with chest compression and vagotomy or lidocaine injections. Results suggest that sex-specific mechanisms modulate swallow-breathing coordination, and that vagal feedback is inhibitory to swallow-related muscles, while spinal feedback from pleural afferents has excitatory effects. This study supports the theory that a balance of vagal and spinal afferent feedback is necessary to maintain an optimal swallow pattern and swallow-breathing coordination.

Swallow Motor Pattern Is Modulated by Fixed or Stochastic Alterations in Afferent Feedback.

Afferent feedback can appreciably alter the pharyngeal phase of swallow. In order to measure the stability of the swallow motor pattern during several types of alterations in afferent feedback, we assessed swallow during a conventional water challenge in four anesthetized cats, and compared that to swallows induced by fixed (20 Hz) and stochastic (1-20Hz) electrical stimulation applied to the superior laryngeal nerve. The swallow motor patterns were evaluated by electromyographic activity (EMG) of eight muscles, based on their functional significance: laryngeal elevators (mylohyoid, geniohyoid, and thyrohyoid); laryngeal adductor (thyroarytenoid); inferior pharyngeal constrictor (thyropharyngeus); upper esophageal sphincter (cricopharyngeus); and inspiratory activity (parasternal and costal diaphragm). Both the fixed and stochastic electrical stimulation paradigms increased activity of the laryngeal elevators, produced short-term facilitation evidenced by increasing swallow durations over the stimulus period, and conversely inhibited swallow-related diaphragm activity. Both the fixed and stochastic stimulus conditions also increased specific EMG amplitudes, which never occurred with the water challenges. Stochastic stimulation increased swallow excitability, as measured by an increase in the number of swallows produced. Consistent with our previous results, changes in the swallow motor pattern for pairs of muscles were only sometimes correlated with each other. We conclude that alterations in afferent feedback produced particular variations of the swallow motor pattern. We hypothesize that specific SLN feedback might modulate the swallow central pattern generator during aberrant feeding conditions (food/liquid entering the airway), which may protect the airway and serve as potentially important clinical diagnostic indicators.

The Role of the Cerebellum in Control of Swallow: Evidence of Inspiratory Activity During Swallow.

Anatomical connections are reported between the cerebellum and brainstem nuclei involved in swallow such as the nucleus tractus solitarius, nucleus ambiguus, and Kölliker-fuse nuclei. Despite these connections, a functional role of the cerebellum during swallow has not been elucidated. Therefore, we examined the effects of cerebellectomy on swallow muscle recruitment and swallow-breathing coordination in anesthetized freely breathing cats. Electromyograms were recorded from upper airway, pharyngeal, laryngeal, diaphragm, and chest wall muscles before and after complete cerebellectomy. Removal of the cerebellum reduced the excitability of swallow (i.e., swallow number), and muscle recruitment of the geniohyoid, thyroarytenoid, parasternal (chestwall), and diaphragm muscles, but did not disrupt swallow-breathing coordination. Additionally, diaphragm and parasternal muscle activity during swallow is reduced after cerebellectomy, while no changes were observed during breathing. These findings suggest the cerebellum modulates muscle excitability during recruitment, but not pattern or coordination of swallow with breathing.

Swallow-breathing coordination during incremental ascent to altitude.

Swallow and breathing are highly coordinated behaviors reliant on shared anatomical space and neural pathways. Incremental ascent to high altitudes results in hypoxia/hypocapnic conditions altering respiratory drive, however it is not known whether these changes also alter swallow. We examined the effect of incremental ascent (1045 m, 3440 m and 4371 m) on swallow motor pattern and swallow-breathing coordination in seven healthy adults. Submental surface electromyograms (sEMG) and spirometry were used to evaluate swallow triggered by saliva and water infusion. Swallow-breathing phase preference was different between swallows initiated by saliva versus water. With ascent, saliva swallows changed to a dominate pattern of occurrence during the transition from inspiration to expiration. Additionally, water swallows demonstrated a significant decrease in submental sEMG duration and a shift in submental activity to earlier in the apnea period, especially at 4371 m. Our results suggest that there are changes in swallow-breathing coordination and swallow production that likely increase airway protection with incremental ascent to high altitude. The adaptive changes in swallow were likely due to the exposure to hypoxia and hypocapnia, along with airway irritation.

Cough Effectiveness and Pulmonary Hygiene Practices in Patients with Pompe Disease.

PURPOSE: While factors leading to hypoventilation have been well studied in Pompe disease, cough effectiveness and airway clearance practices are less understood. We aimed to identify significant factors that influence peak cough flow (PCF) in Pompe, and to detect whether pulmonary hygiene practices were reflective of reduced PCF. METHODS: This is a prospective observational study of 20 subjects with Pompe disease (infantile-onset: 7, juvenile-onset: 6, adult-onset: 14). Subjects performed spirometry, maximal respiratory pressures, and cough (voluntary: n = 24, spontaneous: n = 3). Subjects or their parents reported airway clearance and secretion management practices. Relationships between disease variables, pulmonary function, and cough parameters as well as group differences in cough parameters were evaluated. RESULTS: Subjects with infantile-onset disease had significantly lower PCF (p < 0.05) and tended to require more external ventilatory support (p = 0.07). In juvenile- and adult-onset disease, PCF differed according to external ventilatory requirement [daytime: 83.6 L/min (95% CI 41.2-126.0); nighttime: 224.6 L/min (95% CI 139.1-310.2); none: 340.2 L/min (95% CI 193.3-487.6), p < 0.005]. Cough inspiratory volume also differed significantly by ventilatory requirement [daytime: 5.5 mL/kg (95% CI 3.0-8.0); nighttime: 16.0 mL/kg (95% CI 11.8-20.2); none: 26.8 mL/kg (95% CI 11.9-41.7), p < 0.001]. However, routine airway clearance or secretion management practices were only consistently reported among patients with infantile-onset disease (infantile: 86%, juvenile: 0%, adult: 14%, p < 0.005). CONCLUSIONS: Cough weakness was detected in the majority of patients with Pompe disease and was influenced by both inspiratory and expiratory muscle function. Patients at risk for problems or with ineffective PCF should be urged to complete routine pulmonary hygiene.

Strategies for the Integration of Cough and Swallow to Maintain Airway Protection in Humans.

PURPOSE: Airway protective behaviors, like cough and swallow, deteriorate in many populations suffering from neurologic disorders. While coordination of these behaviors has been investigated in an animal model, it has not been tested in humans. METHODS: We used a novel protocol, adapted from previous work in the cat, to assess cough and swallow independently and their coordination strategies in seven healthy males (26 ± 6 years). Surface electromyograms of the submental complex and external oblique complex, spirometry, and thoracic and abdominal wall kinematics, were used to evaluate the timing of swallow, cough, and breathing as well as lung volume (LV) during these behaviors. RESULTS: Unlike the cat, there was significant variability in the cough-swallow phase preference; however, there was a targeted LV range in which swallow occurred. CONCLUSION: These results give insight into the differences between the cat and human models in airway protective strategies related to the coordination of cough and swallow behaviors, allowing for better understanding of dystussia and dysphagia.

Effect of Hops Beta Acids on the Survival of Unstressed- or Acid-Stress-Adapted-Listeria Monocytogenes and on the Quality and Sensory Attributes of Commercially Cured Ham Slices.

This study evaluated the antilisterial activity of hops beta acids (HBA) and their impact on the quality and sensory attributes of ham. Commercially cured ham slices were inoculated with unstressed- and acid-stress-adapted (ASA)-L. monocytogenes (2.2 to 2.5 log CFU/cm(2) ), followed by no dipping (control), dipping in deionized (DI) water, or dipping in a 0.11% HBA solution. This was followed by vacuum or aerobic packaging and storage (7.2 °C, 35 or 20 d). Samples were taken periodically during storage to check for pH changes and analyze the microbial populations. Color measurements were obtained by dipping noninoculated ham slices in a 0.11% HBA solution, followed by vacuum packaging and storage (4.0 °C, 42 d). Sensory evaluations were performed on ham slices treated with 0.05% to 0.23% HBA solutions, followed by vacuum packaging and storage (4.0 °C, 30 d). HBA caused immediate reductions of 1.2 to 1.5 log CFU/cm(2) (P < 0.05) in unstressed- and ASA-L. monocytogenes populations on ham slices. During storage, the unstressed-L. monocytogenes populations on HBA-treated samples were 0.5 to 2.0 log CFU/cm(2) lower (P < 0.05) than control samples and those dipped in DI water. The lag-phase of the unstressed-L. monocytogenes population was extended from 3.396 to 7.125 d (control) to 7.194 to 10.920 d in the HBA-treated samples. However, the ASA-L. monocytogenes population showed resistance to HBA because they had a higher growth rate than control samples and had similar growth variables to DI water-treated samples during storage. Dipping in HBA solution did not adversely affect the color or sensory attributes of the ham slices stored in vacuum packages. These results are useful for helping ready-to-eat meat processors develop operational procedures for applying HBA on ham slices.