Echinococcus and Taenia spp. among carnivorous hosts of the Maasai Mara and Samburu National Reserves.
\r\n\tThis book intends to cover a wide range of important areas of heat pump technology starting from fundamentals, theories, performance to advanced applications. It will also present a state of the art in research and development of heat pumps, their performance and impact in various sectors like social, economic and environment.\r\n
\r\n\tThe book will be of great interest to engineers, researchers, graduate students, and manufacturers whose study/work/business involves heat pumps.
Hypoxia produces deep alterations in a living organism at systemic and cellular level. Cells respond to hypoxia by complex metabolic reprogramming and molecular mechanisms aimed to minimize detrimental consequences of the oxygen deprivation. Moderate hypoxia exposure (such as intermittent hypoxia) is supposed to possess a great therapeutic potential, while severe and prolonged hypoxia has pronounced pathophysiological consequences [1, 2]. Adaptive responses to hypoxia primarily involve metabolic and functional alterations in mitochondria. Being the main consumers of cellular oxygen (up to ~90%) and highly sensitive to oxygen shortage [3, 4, 5], mitochondria are first to respond to oxygen deficiency. Hypoxia evokes complex network of the mechanisms aimed to adapt mitochondria, their morphology, functions and metabolism to oxygen deprivation. In agreement with the present knowledge, the first step in the adaptation to hypoxia is the expression, stabilization and activation of hypoxia-inducible factor HIF, which is the transcription factor that triggers metabolic reprogramming resulting in the shift from oxidative to glycolytic metabolism [6, 7, 8]. Multilevel mitochondrial response to oxygen shortage includes modulations at the transcription level [6, 8], morphological changes [2, 9, 10], alterations in the functioning of ETC at the level of respiratory complexes [11, 12], shift of ATP synthesis from oxidative to glycolytic pathway [7, 11, 13], alterations in the mechanisms of ROS production by the respiratory chain [3, 4, 14], triggering of signaling pathways specific for hypoxic conditions [6, 15, 16] and the modulation of ROS control by matrix antioxidants [17, 18, 19]. These mechanisms working separately or together could explain high effectiveness of the moderate exposures to hypoxia in the adaptation of a living organism to severe oxygen deprivation, such as ischemia and anoxia [1, 12].\n
One common feature of the metabolic alterations in mitochondria under hypoxia is the activation of mitochondrial potassium transport, which is thought to be a part of the adaptive responses of a living organism to oxygen deficiency [20, 21, 22]. Mitochondrial system of potassium transport is represented by several types of potassium channels, which are the pathways for potassium uptake (ATP-sensitive K+ channel (KATP channel), large conductance Ca2+-activated K+ channel (BKCa channel), voltage-gated K+ channels and others, reviewed in ), and K+/H+ exchanger, which is the pathway of potassium efflux (reviewed in ). In the literature, hypoxia was shown to increase the activity of both mKATP and BKCa channels [20, 25, 26, 27] and the overall potassium uptake in mitochondria [21, 28]. Potassium transport is an all-round modulator of mitochondrial functions: oxygen consumption, Ca2+ transport, ATP synthesis, ROS production and mitochondrial morphology [10, 24]. But which are the functions of potassium transport and what benefits could be gained by the activation of potassium channels under hypoxia?\n
Mitochondrial ATP-sensitive potassium channel (mKATP channel) is the most abundant of the K+ channels present in the inner mitochondrial membrane, and the functional effects of ATP-sensitive potassium transport are best studied as compared to other types of K+ transport in mitochondria [23, 24]. For this reason, primarily the functions of ATP-sensitive potassium transport under hypoxia and physiological relevance of mKATP channel functioning will be discussed below based on the published data and the results of the author’s research.\n
“Mitochondrial response” to hypoxia, starting from the modulation of mitochondrial morphology and metabolism, is directed at the adaptation of the organelles to the conditions of oxygen shortage. Morphological changes are highly dependent on the duration and severity of oxygen deprivation. Generally, it was reported that short-term and intermittent hypoxia resulted in the increase of the total number of mitochondria and the enrichment of their subsarcolemmal fraction [9, 10], while severe prolonged hypoxia, on the contrary, suppressed mitochondrial biogenesis and dramatically reduced the number of subsarcolemmal organelles [2, 11]. Apparently, “primary” response to oxygen deficiency is to improve oxygen supply to mitochondria, whereas response to severe prolonged hypoxia fits with the decrease of respiratory capacity and metabolic shift from oxidative mitochondrial to glycolytic metabolism [7, 8].\n
Thus, it was observed  that even short-term exposure to hypoxia (a 30-min hypobaric hypoxia) resulted in obvious changes in mitochondrial morphology and their subcellular distribution. As it was shown in cardiac and muscle tissues [9, 10], oxygen deficiency increases localization of mitochondria near the plasma membrane, in the close proximity to capillaries, and the enrichment of subsarcolemmal fraction of mitochondria, while not affecting interfibrillar one. Also, hypoxia exposure resulted in moderate swelling of cardiomyocyte mitochondria (by 25% from the initial diameter) and the formation of vesicular cristae . Adaptive changes after exposure to 10–14% O2 were also observed in cerebral cortex mitochondria . More dense cristae package was found in the animals showing higher adaptive capacities to hypoxia . It was observed too that short-term hypoxia exposure promoted mitochondrial division, thereby greatly increasing the number of mitochondria due to the formation of new microorganelles . These changes were supposed to improve oxygen supply to mitochondria and the effectiveness of the oxygen consumption because of greatly increasing the total surface of mitochondria . Interestingly, the effects similar to short-term hypoxia on mitochondrial morphology were produced by mKATP channel opener diazoxide. Thus, diazoxide administration in vivo enhanced division of mitochondria and increased the number of newly formed organelles while channel blocker 5-HD abolished this effect . It is worth mentioning that higher adaptive capacities of the animals to hypoxia coincided with higher mKATP channel activity [28, 29], which implies the role of mKATP channel in the modulation of mitochondrial dynamics and agrees with supposed role of the channel in the adaptation to hypoxia.\n
In contrast, prolonged exposure to severe hypoxia has pathogenic consequences for the organism  and detrimental consequences for mitochondria and results in the loss of mitochondrial density, depletion of subsarcolemmal mitochondria, suppression of their biogenesis, decrease in the expression of the respiratory complexes, lowered pyruvate oxidation (complex I substrate), decreased respiratory capacity and de-energization [2, 11]. Thus, chronic exposure to hypobaric hypoxia (5000–6000 m) resulted in ~21% loss of total mitochondrial density and 73% loss of subsarcolemmal mitochondria accompanied by decreased expression of the complexes I and IV , which corresponds to the shift of metabolism from oxidative to glycolytic pathway. Thus, it seems reasonable to agree with Ref.  that therapeutic effect of hypoxia is a matter of dose and assumes that increase in oxygen-sensing properties of mitochondria is a first step in the adaptation of a living organism to hypoxia.\n
Hypoxia alters mitochondrial functions and metabolism primarily at the level of the respiratory chain. Thus, hypoxia affects sensitivity of mitochondria to oxygen and the functions of the respiratory complexes, the sources of electron supply to the respiratory chain, pathways of ATP synthesis and the mechanisms of ROS formation and signaling. There we consider the changes in mitochondrial functions, which are supposed to be a part of non-pathophysiological adaptive responses of a living organism to moderate hypoxia exposures.\n
Respiratory chain is the subject of complex modulation under oxygen deficiency. At the level of ETC, mitochondrial response to hypoxia is manifested by elevated ROS production and HIF stabilization [3, 4, 5, 6], downregulation of the respiratory complexes  and ATP synthase, switch from the oxidative to glycolytic ATP production [7, 11, 12, 13] and triggering of the mechanisms aimed to suppress the respiration, e.g. by S-nitrosation of the respiratory complexes, which is supposed to save oxygen from excess consumption and prevent excess HIF activity . Nitric oxide is supposed to be active player in hypoxia sensing by mitochondria [30, 31]. Thus, it was proposed that under low \n\n electrons from the respiratory chain could reduce nitrite (NO2−) to •NO, which then reacts with oxygen producing superoxide (•O2−). The excess NO production in the presence of •O2− results in S-nitrosation of mitochondrial proteins , particularly, complexes (I and IV) thereby suppressing respiration. The elevated production of hydrogen sulfide capable to directly donate electrons to the respiratory chain  opens the pathways of electron supply substituting for oxygen shortage. Also, the impact of hypoxia on the oxygen-sensing properties of mitochondria appears in the modified sensitivity of the electron transport chain to oxygen via the modified kinetic properties and increased oxygen affinity of cytochrome oxidase (complex IV) . All these alterations are supposed to be directed at the adaptation of mitochondria to oxygen deprivation.\n
The primary step in sensing oxygen deficiency and complex reprogramming of mitochondrial functions under hypoxia is the activation of hypoxia-inducible factor HIF, a transcription factor that takes control over a multiplicity of genes . HIF family counts three known at present members, HIF 1α, HIF 2α and HIF 3α. Most studied are the functions and the regulation of HIF 1, which is composed of constitutively expressed HIF 1β and three HIF α subunits (HIF 1α, HIF 2α, HIF 3α) that are highly sensitive to the changes in oxygen concentration. HIF α subunits are unstable under normoxic conditions (21% oxygen) but are stabilized under oxygen deficiency (10–14% oxygen) and assembled with HIF 1β forming functionally active heterodimers . HIF life span is controlled by prolyl hydroxylases, which require oxygen for their activity and are inactivated when oxygen supply is insufficient. Oxygen-dependent hydroxylation of proline residues (402 and 564 in HIF 1α) by hydroxylase PHD2 and asparagine residue by factor inhibiting HIF (FIH) promotes binding of the von Hippel-Lindau tumor-suppressor protein (VHL), which in turn triggers pathway of HIF degradation by proteasome . ROS formation by mitochondrial respiratory chain contributes to inactivation of prolyl hydroxylases and HIF stabilization, thus HIF stability is critically dependent on both oxygen concentration and ROS formation . Silencing or pharmacological inhibition of prolyl hydroxylases enhanced HIF stability [4, 33], whereas ROS scavenging by antioxidants (N-acyl cysteine, and mitochondrial ROS scavenger mitotracker red) abolished HIF stabilization and HIF-dependent signaling even under oxygen shortage .\n
ROS-dependent stabilization and activation of HIF [3, 4, 5] triggers complex metabolic rearrangement resulting in a switch from oxidative to glycolytic metabolism, which is a hallmark of all hypoxic states, including embryonic and tumor cells known to function in highly hypoxic environment. At transcriptional level, adaptive responses of a living organism to hypoxia involve upregulation of proteins and the enzymes along glycolytic pathway: glucose transporter (GLUT), hexokinase 2 (HK2) and lactate dehydrogenase (LDH). HIF-dependent upregulation of genes encoding glucose transporters  results in the enhanced uptake of glucose and the activation of glucose metabolism with eventual formation of the end product lactate.\n
As shown in literary data, under normoxic conditions about 25% of cellular ATP is supplied by glycolysis, which, for example, in dorsal root ganglion neurons constituted ~3.5 nmol/mg protein . Hypoxia sharply changes relative contribution of the oxidative phosphorylation (OxPhos) and glycolysis to ATP production dramatically suppressing OxPhos pathway while simultaneously upregulating glycolysis. Thus, dependent on the conditions, hypoxia was capable of reducing ATP content by ~50% (which in the above example constituted from ~11 to as low as ~6 nmol/mg ), of which about ~5 nmol/mg (i.e. ~80%) was produced by glycolytic pathway. This pattern shows upregulation of glycolysis by ~1.5 times accompanied by nearly complete inhibition of the OxPhos.\n
ATP obtained by glycolysis, as well as ATP of mitochondrial origin, is consumed by several energy-consuming processes, such as maintenance of transmembrane ion gradients and membrane potential by the work of Na+, K+-ATPase, metabolic processes and protein synthesis. Literary data showed not only inhibition of ATP synthase caused by impaired mitochondrial bioenergetics but also its down-regulation along with down-regulation of the respiratory complexes.\n
As it was established in several studies (in embryonic cardiomyocytes , dorsal root ganglion neurons , malignant cell lines ), there was a reciprocal dependence between impaired mitochondrial bioenergetics, compromised ATP synthesis and upregulation of all steps of glycolytic pathway. Especially in tumors, which metabolic phenotype has much in common with that of normal tissues functioning under hypoxia, elevated expression of glycolytic proteins, starting from glucose transporter and ending with lactate dehydrogenase, and simultaneous downregulation of respiratory complexes and ATP synthase were observed in different cell lines .\n
Under oxygen deficiency, glycolysis was shown to be upregulated not only at transcriptional level but also at the level of metabolism. Thus, as it was shown in the early work of Hohachka et al. , insufficient production of ATP and lowering of cellular ATP content result in the elevation of cellular level of ADP that reaches the range of Km (~100 μM) required for ADP-dependent kinases of glycolysis (phosphoglycerate kinase, pyruvate kinase), which is much higher than it is required for mitochondrial oxidative metabolism (~30 μM). Reduced level of ATP is reflected in lowered phosphocreatine/ATP ratio, which is one of the multiple hallmarks of oxygen deficiency [7, 11].\n
Another aspect of downregulation of the OxPhos is the conversion of ADP to AMP, the increase of the level of AMP and the activation of AMP-dependent protein kinase (AMPK), which was shown to afford several cytoprotective effects first established under the conditions of ischemia/reperfusion . Thus, AMPK activation, which takes place when ATP demand exceeds the supply, i.e. under oxygen deprivation and compromised mitochondrial ATP production (ischemia, hypoxia ), was shown to protect tissues of oxidative stress, opening of mitochondrial permeability transition pore (mPTP) and apoptosis induction. Also, it was shown to be indispensable for the activation of glycolysis as a part of adaptive responses to the lack of oxygen aimed to compensate for ATP deficiency [35, 36, 38].\n
At present, it still remains elusive, which is the “molecular link” between metabolic alterations and elevated expression of KATP channels observed under hypoxia in several works. Recently, it was shown  that AMPK activation under exposure to moderate hypoxia increased the level of the receptor SUR2A subunit of cardiac KATP channels, which was supposed to be a part of adaptive response to oxygen deprivation. Increased expression of SUR2A in cardiomyocytes was also observed after application of AMPK activator AICAR . Thus, AMPK activation under hypoxia appears to be one of the mechanisms connecting metabolic rearrangements with KATP channels opening.\n
Thus, oxidative ATP synthesis under hypoxia gives way to glycolysis, and as it was shown in the literature, glycolysis becomes the prevailing source of ATP production, at dramatic diminution of ATP production by OxPhos. Upregulation of glycolytic metabolism primarily occurs because of ROS-dependent stabilization and activation of transcription factor HIF, triggering of HIF-dependent signaling and upregulation of glycolytic enzymes. Increased level of ADP and AMP because of compromised OxPhos and activation of AMP kinase  largely contribute to upregulation of glycolysis and glycolytic ATP production.\n
While metabolic alterations and redistribution of ATP production between OxPhos and glycolysis is one aspect of hypoxia’s impact on mitochondrial and cellular functions, another as well important aspect of the functional rearrangements under hypoxia is triggering of redox signaling specific to the states of oxygen deficiency [5, 6, 14]. Elevated ROS production under hypoxia, resulting in HIF activation, is accompanied by the activation of redox signaling that in the literature was shown to be largely mediated by plasmalemmal and mitochondrial potassium channels and cytosolic Ca2+.\n
Even short-term exposure to hypoxia triggers complex network of cell-specific signaling pathways involving the induction of early genes [6, 15, 39] and activation of signaling pathways: PKC (phosphatydil-inositol-3-kinase (PI3K)/protein kinase B (Akt), mitogen-activated protein kinase (p38MAPK), AMP-activated protein kinase (AMPK) [15, 16, 40, 41]). Under hypoxia, signaling is known to be tightly coupled to mitochondrial ROS production. The major sources of ROS are NADPH oxidase (NOX), which activation was shown under the states of oxygen deficiency [16, 42, 43], and the respiratory chain [4, 14, 44]. Respiratory chain was shown to be a trigger of redox signaling in response to hypoxia [4, 5, 14]. Literary data show critical role of complexes I and III in HIF stabilization and redox signaling under hypoxia [4, 14, 43]. Mitochondrial complexes I, II and III are known to be the main sites of ROS formation in the respiratory chain . Complex I releases ROS to the matrix space, while complex III releases ROS on both sides of mitochondrial membrane—to the matrix and the intermembrane space . Mitochondrial complex III is supposed to play a pivotal role in triggering ROS signaling [3, 4, 14]. Suppression of mitochondrial respiration by the respiratory inhibitors such as rotenone (complex I), myxothiazol and stigmatellin (complex III) ; genetic deletions within the complexes I  and III , and ROS scavenging by mitochondria targeting antioxidants (mitotracker red) have shown that under hypoxia ROS signaling and HIF stability were primarily dependent on the ROS production by the respiratory chain. As it was shown by many authors, under hypoxia, the primary site of ROS formation within the respiratory chain shifts to the complex III, and therein Q-cycle [3, 4] and Rieske protein (FeS cluster)  were shown to be the major sources of ROS responsible for HIF activation. It is worth notion that HIF stabilization by mitochondria-derived ROS exhibited site specificity. Thus, using approaches based on genetic ablation and pharmacological inhibition, it was shown that Qo site facing cytosol and Rieske protein of complex III were critical for HIF stabilization under hypoxia, while ROS formation at Qi site facing matrix space did not contribute to HIF stability . Critical role of Qo site for HIF stabilization allows an assumption that not so “bulk” ROS production or the lack of oxygen is most important for HIF stability and activation, as ROS signaling from outer boundary of mitochondrial membrane . In line with this observation is the interplay between NOX- and mitochondria-derived ROS, which in the recent decades became a subject of keen interest [16, 42, 43, 44, 46, 47]. Voltage-gated K+ channels of plasma membrane (Kv channels) and mKATP channels were shown to be important link mediating interplay between NOX and mitochondrial ROS formation, which triggers redox signaling specific to the states of oxygen deficiency [16, 42, 43, 44, 47, 48, 49].\n
Under hypoxia, the elevation of ROS production in the cell and mitochondria is accompanied by the increase in the level of cytosolic calcium, [Ca2+]c, which was shown to be ROS-dependent [44, 48, 49] and results either from Ca2+ entry via plasma membrane  explained by suppression of Kv channels by mitochondria-derived ROS and sarcolemmal depolarization [43, 46] or, alternatively, Ca2+ release from sarcoplasmic reticulum via ryanodine receptors [42, 44]. The role of ROS in the elevation of [Ca2+]c was shown by its abolition by antioxidants (pyrrolidine dithiocarbamate, N-acetyl cysteine) as well as overexpression of glutathione peroxidase (GPx), catalase (CAT) and matrix Mn-SOD [48, 49], which showed its dependence on ROS formation by mitochondria. The elevation of ROS, activation of PKCε and the rise in [Ca2+]c, could contribute to the inhibition of Kv channels, extracellular Ca2+ influx  or Ca2+ release from intracellular stores [42, 44].\n
As it was shown in several studies, mKATP channels opening was capable to increase mitochondrial ROS production and trigger redox signaling mediated by PKCε [16, 47, 50, 51]. An interplay between NOX and mitochondrial ROS, dependent on mKATP channel opening; the rise in intracellular [Ca2+]c and the activation of PKCε and other signaling pathways under hypoxia (Akt, MAPK, ERK [40, 41, 52, 53]) were shown in several works. Thus, hypoxia-induced NOX activation was shown to be dependent on mitochondrial ROS, and the suppression of ROS production by respiratory inhibitors (rotenone, myxothiazol) abolished NOX activation [42, 47]. As it was found, the role of mKATP channel in mediating NOX-mitochondria interplay was the direct activation of PKCε, resulting from the increase of mitochondrial ROS production following ATP-sensitive K+ uptake [16, 50].\n
Alternatively, mKATP channel activation was supposed to be a part of a feedback loop mechanism, started by NOX activation [42, 47], ROS release and the increase in mKATP channel activity, which in turn triggered PKCε activation both in mitochondria and cytosol by increasing mitochondrial ROS production [16, 51]. The activation of mKATP channel shown under hypoxia by many authors as well could contribute to the increase in [Ca2+]c, because mKATP channel opening and uncoupling of the respiratory chain by potassium transport favors the elevation of [Ca2+]c, by reducing Ca2+ uptake in mitochondria [54, 55, 56, 57]. Thus, an impact of mKATP channel activity on mitochondrial ROS formation and Ca2+ uptake becomes an important modulator of Ca2+/ROS-dependent signaling under hypoxia.\n
Hypoxia evokes specific mechanisms to control ROS overproduction by the upregulation of antioxidant enzymes: SOD, catalase (CAT) and glutathione peroxidase (GPx). Exposure to different hypoxia regimens resulted in the increased expression and activity of SOD, CAT and GPx. Thus, chronic intermittent hypoxia was shown to upregulate the system of matrix antioxidants: SOD and (CAT), which exhibited elevated expression and activity after hypoxia exposure. Higher expression of SOD, CAT and GPx found in myocardium after the exposure to intermittent hypobaric hypoxia afforded preconditioning-like effect explained by the induction of antioxidant defense .The effect was similar to the pretreatment of the hearts with antioxidant mixture containing SOD and CAT, which helped to restore cardiac contractile function after ischemia/reperfusion . Thus, while mitochondrial ROS generated by the respiratory chain are supposed to trigger the response to hypoxia shown by the increase in [Ca2+]c, HIF stabilization and triggering of redox signaling [3, 4, 42, 43, 44], elevated expression and activation of Mn-SOD, CAT and GPx are capable to abolish or attenuate this response [48, 49] and prevent excess lipid peroxidation and depletion of reduced glutathione .\n
Overexpression of the antioxidant enzymes in pulmonary artery smooth muscle cells showed selectivity towards the inhibition of hypoxic increase in ROS and [Ca2+]c . Common to other cell types, in smooth muscle cells hypoxia exposure increased both ROS production and [Ca2+]c. Overexpression of GPx and CAT, both cytosolic and mitochondrial, attenuated the response to hypoxia. Overexpression of cytosolic Cu, Zn-SOD had no effect on both ROS and [Ca2+]c, whereas overexpression of matrix Mn-SOD augmented [Ca2+]c but had no effect on ROS signaling . These data indicated H2O2 to be signaling molecule to trigger the response to hypoxia in smooth muscle cells. The absence of the effects of SOD on ROS signaling could be explained by increased H2O2 production and signaling explained by the increased SOD activity.\n
ROS-dependent stabilization and activation of HIF, downregulation of the OxPhos, lack in cellular ATP, activation of AMPK and other signaling pathways, elevation of ROS production and triggering of ROS-dependent signaling result in the opening and activation of mKATP channels, which is supposed to be a part of the adaptive response to hypoxia. As it will be shown below, multiple mKATP channel functions under hypoxia are aimed at controlling mitochondrial respiration, ATP synthesis and ROS production relevant to the conditions of oxygen deficiency.\n
Cells maintain high transmembrane gradients of sodium and potassium, which support cellular membrane potential built up by the work of Na+, K+-ATPase, plasmalemmal K+ and Na+ channels and transporters (Na+/H+, Na+/Ca2+ and others), in order to maintain cellular functions and metabolism. Potassium is a prevalent cation of cytosol and mitochondrial matrix, where its concentration reaches 120–150 mM, and virtually there is no transmembrane gradient of this cation between the matrix and the cytosol . Possibly, for this reason, K+ transport for decades was not paid attention needed, till the discovery of mKATP channel (1991), and its importance for tissue protection first observed in experimental models of ischemia/reperfusion . Shortly after it appeared that mKATP channel plays an equally important role in the adaptation of a living organism to oxygen deprivation , and later similar effects of the opening of large conductance calcium activated K+ channel (BKCa channel) were observed . These findings served as a powerful stimulus for extensive studies of the properties and functions of mitochondrial K+ channels and cytoprotective mechanisms triggered by K+ channels opening.\n
The system of mitochondrial potassium transport is represented by several types of potassium channels: ATP-sensitive K+ channel (mKATP channel) large conductance Ca2+-activated K+ channel (BKCa channel), intermediate conductance Ca2+-activated K+ channel (IKCa channel), voltage-gated K+ channel (Kv 1.3), twin pore potassium channel and other types of K+ conductance (reviewed in more detail in ). Potassium uptake via K+ channels is opposed by K+/H+ exchanger, which acting coordinately constitute potassium cycle .\n
As it was reported, different regimens of hypoxia exposure (such as intermittent hypobaric hypoxia [10, 21, 41], brief hypoxia exposure (hypoxic preconditioning) , chronic hypoxia [20, 27, 59]) resulted in the activation of potassium transport: mKATP channel [10, 20, 21, 41], BKCa channel  and K+/H+ exchange . According to these data, mitochondrial K+ channel opening and activation are ubiquitous consequence of oxygen shortage, indicating that K+ channel opening is involved in the response of mitochondria to the lack of oxygen. So, in the light of the above metabolic and functional rearrangements caused by the oxygen deficiency, it is reasonable to ask which advantages are gained by the activation of mitochondrial potassium transport under hypoxia. Potassium uptake and potassium cycling are energy-dissipating processes affecting mitochondrial bioenergetics. So, with regard to the purpose of this review, most important is to consider how the modulation of mitochondrial functions by mKATP channels opening might affect oxygen-sensing properties of mitochondria and “mitochondrial response” to oxygen deficiency.\n
KATP channel is an octameric multiprotein complex ubiquitously present in plasma membranes and mitochondria. KATP channels comprise conducting subunit (Kir6.1 and Kir6.2) highly selective towards K+ and receptor subunit SUR (SUR1A, SUR2A and SUR2B) differently distributed in tissues. The channel is specifically blocked by ATP in the presence of Mg2+. Receptor subunit of the channel binds nucleotides and pharmacological modulators: sulfonylureas (glibenclamide, tolbutamide), which are channel blockers, and the openers (pinacidil, chromakalim, nicorandil, diazoxide). The properties and molecular composition of KATP channels are reviewed in detail in [60, 61].\n
In the literature, it was supposed that protection of tissues against the impairments caused by hypoxia afforded by mitochondrial K+ transport is largely based on bioenergetic effects of K+ transport and signaling triggered by K+ channels opening [16, 41, 50, 53]. The impact of ATP-sensitive K+ transport on the oxygen consumption, membrane potential, ATP synthesis, Ca2+ transport and ROS production is largely dependent on the abundance of the channel in mitochondrial membrane. Oxygen deficiency affects functional consequences of mKATP channels opening by modulating channels expression and activity [20, 53, 59]. As it was shown in cardiomyocytes, the activation of cardiac mKATP channels under hypoxia was mediated by the interactions of conducting subunit Kir6.2 with heat shock protein 90, HSP90  and Kir6.1 with gap junction protein connexin 43 and PKCε . Silencing or pharmacological inhibition of HSP90 and connexin 43 abolished protective effects afforded by mKATP channels opening [25, 26]. At present, there are scarce data on such interactions, which could contribute to cell specificity of molecular mechanisms regulating mKATP channels opening and its functional consequences under hypoxia. To assess how mKATP channels can be involved in mitochondrial response to oxygen deprivation, direct bioenergetic and functional effects of mKATP channels opening need to be considered.\n
In energized mitochondria, potential-dependent potassium transport directed to the matrix space takes place at the cost of proton-motive force (ΔμH), a free energy generated by the electron transport chain. As ΔΨm is the main part of ΔμH, K+ uptake, accompanied by the obligatory electroneutral water uptake , occurs at the cost of ΔΨm and thus results in depolarization. Because of its dramatic effect on ΔΨm and matrix swelling, K+ uptake would be detrimental for mitochondria, if there was not the work of respiratory chain and K+/H+ exchange. Thus, the loss of ΔΨm is opposed by the “compensatory” work of respiratory chain , which increases oxygen consumption proportional to the rate of K+ transport in order to restore ΔΨm; on the other hand, matrix swelling is opposed by potassium extrusion via K+/H+-exchanger, which is accompanied by the matrix contraction . Concurrent work of K+ channels and K+/H+ exchanger constitutes mitochondrial K+ cycle , of which potential-dependent component (K+ uptake) dissipates ΔμH and in this way uncouples mitochondria and affects potential-dependent mitochondrial functions: ATP synthesis, Ca2+ transport and ROS production.\n
The impact of mKATP channels opening on mitochondrial bioenergetics greatly depends on the channels’ activity and their abundance in mitochondrial membrane, which is responsible for the effects of mKATP channels opening on mitochondrial energy state and decides for cell specificity of mKATP channel functions . Thus, higher density of mKATP channel distribution in brain results in slight depolarization, which was observed in the literature and in our studies [63, 64], while lower amount of the channels in the heart and liver was of no effect on ΔΨm even at full activation [24, 65, 66]. Elevated expression of mKATP channel and the channel activation that were observed under hypoxia [20, 21, 41, 59] increase the “weight” of ATP-sensitive K+ transport in the regulation of mitochondrial functions and metabolism. This is still more visible in malignant cells functioning in hypoxic environment, in which overexpression of mKATP channel was shown .\n
Unlike protonophoric uncoupling that reduces transmembrane pH (ΔpH), uncoupling of the respiratory chain by mKATP channel opening is accompanied by the elevated ΔpH because of K+ uptake into matrix occurring in exchange for protons. However, the activation of K+/H+-exchanger reduces this minor gain in ΔpH, and besides, simultaneous increase in the rate of oxygen consumption due to K+ uptake dissipates ΔμH, largely at the cost of ΔΨm. Thus, the regulation of ROS production and other potential-dependent functions of mitochondria, dependent on cell type, are largely affected by the effects of ATP-sensitive K+ transport on ΔΨm and the rate of respiration.\n
Generally, it is supposed that cytoprotective effects of mKATP channels opening are primarily based on the modulation of Ca2+ transport and mitochondrial ROS production, which prevent Ca2+ overload [54, 56, 57] and trigger ROS-dependent signaling, thereby preventing the opening of cyclosporine-sensitive pore (mPTP) . However, of all functional effects produced by mKATP channels opening (the modulation of mitochondrial morphology [9, 10], respiration [63, 65], Ca2+ transport [54, 55, 56], potassium cycle [24, 66], ATP synthesis [20, 67, 68] and ROS production [21, 50, 64]), the effects of ATP-sensitive K+ transport on ROS production appear to be the most controversial. This diversity needs to consider the direct effects of mKATP channels opening on ROS production in mitochondria.\n
ROS production in mitochondria is regulated by a number of thermodynamic and kinetic factors . The diverse, and even contrary, effects of mKATP channels opening on ROS production in mitochondria are difficult to evaluate because mitochondrial ROS production depends on a wide variety of conditions, which include mitochondrial energy state (quantitatively represented by ΔμH), redox potential of the main sites of ROS formation in the respiratory chain [69, 70, 71], the source of the electron supply to the respiratory chain, the rate of respiration  and, at last, the concentration of oxygen [3, 4], which is the end electron acceptor in the redox reactions in the respiratory chain.\n
Standard redox potential of one-electron oxygen reduction to superoxide constitutes −160 mV, and on this basis, the respiratory chain in highly energized mitochondria comprises multiple putative sites of ROS formation [45, 69]. At complex I, ROS formation largely occurs in the course of thermodynamically unfavorable reverse electron transport, which requires high ΔμH and critically depends on both ΔΨm and ΔpH [72, 73]. This mechanism of ROS formation is one best studied “classical” example of thermodynamically regulated ROS production in mitochondria. Unlike this, ROS production at complex III is dependent on both thermodynamic (such as the redox state of the ubiquinone pool) and kinetic factors [45, 69, 70, 71], such as the quantity and the life span of free radical intermediates of the redox reactions, which are regulated by the rate of respiration and the relations between the rates of ROS formation and the removal of these species. Q-cycle is supposed to be the main source of ROS in complex III , and ROS formation at this site exhibits a bell-shaped dependence on the redox state of Q-cycle . Partially oxidized Q-cycle was shown to be most favorable for ROS production at complex III , which implies its dependence both on mitochondrial energy state and the rate of respiration.\n
The share of ATP-sensitive K+ transport in the total K+ transport in brain and liver mitochondria by our estimations, which agreed with literary data , was about ~30–35% [64, 66]. However, in spite of the well-defined characteristics of ATP-sensitive K+ transport obtained in mitochondria of different cell types, the effects of mKATP channels opening on ROS production are difficult to quantify because of their dependence on several mutually dependent parameters. Overlay of the moderate alterations in mitochondrial functions caused by ATP-sensitive K+ transport with closely interrelated thermodynamic and kinetic factors regulating ROS formation in mitochondria could explain apparently contradictory effects of mKATP channel opening on ROS production reported in the literature. Interestingly, both the elevation [16, 41] and suppression [21, 40] of ROS production were reported to improve cardiac and cardiomyocyte functions after the exposure to hypoxia in a way dependent on mKATP channel opening. Based on the published data, it is tempting to hypothesize that bidirectional regulation of ROS production by potassium transport observed in the literature could represent a flexible mechanism of the fast response to the elevation of ROS levels generally observed under hypoxia and that, dependent on conditions, could prevent ROS overproduction [57, 75] or trigger ROS-dependent signaling [16, 41, 50, 53], which makes this function of mKATP channel of especial importance under limited oxygen availability.\n
In several works, including our own studies, an inhibition of both ATP synthesis and hydrolysis, ensuing from mKATP channels opening, was reported [67, 68, 76, 77]. Biochemical mechanism of this effect is not well understood, but, based on the published data, its physiological relevance can be considered.\n
As we have observed in our work on liver mitochondria, even full activation of mKATP channel by diazoxide moderately increased the rate of state 4 respiration and resulted in slight mitochondrial uncoupling not accompanied by depolarization . However, these moderate changes in mitochondrial functions apparently suppressed phosphorylation, which could not be explained by the mild uncoupling effect. This was reflected in the decreased rates of state 3 respiration and phosphorylation, which were proved by measuring respective rates of proton transport after ADP addition . It is worth mentioning that mKATP channel opening essentially reduced oxygen consumption in the course of phosphorylation and increased apparent P/O ratio . These effects were coincident with concurrent activation of K+ cycling, which was the cause of stimulation of state 4 respiration . Based on the literature , we assumed that activation of K+ cycling could be the plausible cause for inhibition of F0F1 ATP synthase functioning, not explained by respiratory uncoupling caused by ATP-sensitive K+ transport.\n
Considering that ATP synthesis and hydrolysis are coupled to proton translocation across mitochondrial membrane, we supposed that concurrent K+ cycling could disturb the molecular mechanism of F0F1 ATP synthase both at the stage of ATP synthesis and hydrolysis. Possibly, close mechanism of such molecular uncoupling called “decoupling” was observed in the literature under the action of K+/H+-ionophore gramicidin, which occurred without apparent changes in ΔμН . While the biochemical mechanism of such decoupling is not quite clear, its physiological meaning appears to be more evident and needs to be considered. In agreement with the literature, we suppose that it is the regulation of cellular levels of ATP [67, 77], but what is still more important, the regulation of the oxygen consumption by mitochondria.\n
The functional effects of mKATP channels opening under hypoxia are similar to those observed in normoxic cells. Thus, under oxygen deprivation, mKATP channel activation reduced mitochondrial Ca2+ loading [57, 79], preserved ATP levels [67, 77] and increased cell survival [16, 40, 41, 80] by suppression of apoptosis via targeting glycogen synthase kinase 3β (GSK3β), an enzyme involved in triggering cell death by promotion of the opening of mitochondrial permeability transition pore, mPTP . Suppression of cell death pathways resulted in stabilization of membrane potential [16, 59, 80] and the restoration of ATP synthesis . Increased expression of both Kir6.2  and SUR2A , similar to pharmacological mKATP channels opening, too was shown to improve the viability and the resistance of cardiomyocytes to hypoxia.\n
As one can see from the above examples, cell response to hypoxia was essentially dependent on the bioenergetic effects of mKATP channels opening. This allows assume that cytoprotection afforded by mKATP channels opening is largely based on a synergistic action of bioenergetic effects of mKATP channel functioning (primarily ROS production and ATP synthesis [20, 40, 41, 67, 79]), and the redox signaling critically dependent on ROS formation caused by mKATP channels opening [16, 50].\n
In the literature, it was obtained rather unambiguous evidence of the suppression of the OxPhos by mKATP channels opening [67, 76, 77]. However, it seems to be surprising that under hypoxia, similar to other metabolic stress conditions, cytoprotection was afforded by contrary effects of mKATP channels opening on free radical formation, and both the reduction [40, 80] and the elevation [16, 41, 50] of ROS production were shown to afford cytoprotective effects. To smooth this apparent contradiction, we recently proposed  that, dependent on the direct impact of ATP-sensitive K+ transport on mitochondrial bioenergetics, mKATP channels opening could afford protection at least in two ways: either directly, by the direct reduction of ROS formation under certain conditions [40, 75, 80, 84] or indirectly, by the elevation of ROS production and triggering of ROS-dependent signaling shown to be cytoprotective under oxygen deprivation (ischemia and hypoxia) [16, 41, 50]. To shed more light on physiological role(s) of mKATP channels under hypoxia, functional consequences of mKATP channels opening on ROS production and ATP synthesis should be considered in more detail.\n
With reference to hypoxia, it is generally supposed that mitochondria respond to oxygen deprivation by the generation of ROS and activation of ROS-dependent signaling pathways [3, 4, 5, 14]. mKATP channel was shown to be involved in ROS signaling triggered both upstream (by the activation of kinases PI3K/Akt, PKCε [16, 80]) and downstream (p38MAPK , PKCε, Akt [16, 41]) of mKATP channels opening. This implies the ability of mKATP channel to sense and convey ROS signals, which agrees with the function of the channel as a “ROS sensor” proposed in the literature [75, 84]. The ability of mKATP channel to accept and convey ROS signals is well illustrated by the fast response to hypoxia exposure by NOX/ROS-dependent activation of PKCε via mKATP channel opening and feedback ROS/PKCε-dependent activation of NOX [16, 42, 47], PI3K/Akt and PKC activation upstream and feedback PKCε activation downstream of mKATP channel opening via increase in ROS formation  and ROS-dependent Akt and PKCε activation downstream of mKATP channel opening , which exerted anti-apoptotic effect by the inhibition of GSK3β and mPTP opening. The ability of ATP-sensitive K+ transport to trigger cytoprotective signaling based on the modulation of ROS production has adverse effects in tumors functioning under limited oxygen supply and known to exhibit high mKATP channel activity. Thus, radioresistance of malignant glioma cells overexpressing mKATP channel was shown to be dependent on mKATP channel opening, increasing mitochondrial ROS emission and triggering of MAPK/ERK signaling, which also resulted in suppression of mPTP opening and prevention of tumor cell death .\n
As shown in the above examples, a hypothesis of mKATP channels acting as ROS sensors [75, 84] could be useful in the appraisal of physiological functions of mKATP channel under hypoxia. It is well known that mKATP channel can be activated by ROS , and elevated channel activity in response to excess ROS formation could serve to regulate mitochondrial metabolism and prevent ROS overproduction [57, 79]. This enables us to consider mKATP channel as the trigger of both ROS-dependent signaling and “ROS sensor” involved in the regulation of mitochondrial ROS production via modulation of mitochondrial bioenergetics. Oxidative modification of mKATP channel can represent a feedback mechanism for the regulation of mKATP channel activity. Being at one time a subject of an oxidative modification and a regulator of ROS formation, mKATP channel could be an effective tool in controlling of mitochondrial ROS production under hypoxia.\n
The impact of mKATP channel opening on mitochondrial energy state, dependent on the channel activity, could serve as a regulatory mechanism directed either on triggering of redox signaling or prevention of ROS overproduction. Apparently, controversial data on the regulation of ROS production by mKATP channel opening possibly reflect one integrated mechanism regulating fast response of mitochondria to the changes of ROS levels in the mitochondrial environment.\n
Physiological role of mKATP channel functioning under hypoxia is not limited to the regulation of ROS production and antiapoptotic effects. In our recent work , we proposed that F0F1 ATP synthase can be one of the principal targets of mKATP channels opening. The modulation of ATP synthesis by ATP-sensitive K+ transport can play particularly important role under hypoxia, which is the regulation of cellular ATP and controlling of oxygen consumption:\n
Generally, it is supposed that suppression of ATP hydrolysis by mKATP channels opening is a plausible explanation for the preservation of cellular ATP of excess depletion observed after application of pharmacological mKATP channel openers under pathophysiological conditions [67, 77]. This assumption was supported by the data showing that inhibition of hydrolytic activity of F0F1 ATP synthase by mKATP channel openers helped to preserve cellular ATP levels under ischemic conditions . Possibly, under hypoxia, suppression of ATP hydrolysis would be helpful in saving ATP available from the glycolytic pathway. Besides, the dramatic fall of the total level of ATP under hypoxia and suppression of ATP synthesis by ATP-sensitive K+ transport should keep mKATP channel in functionally active state in order to maintain other physiological functions of the channel. However, we suppose that inhibition of ATP synthesis could be of particular significance under oxygen deprivation.\n
Under hypoxia, controlling of cellular oxygen level becomes important for cell survival [3, 4, 5, 46]. Mitochondria consume most part (up to 90%) of cellular oxygen. With reference to hypoxia, it needs to be considered that ATP synthesis, which continually occurs in a living cell, is a highly oxygen-consuming process. Thus, it is reasonable to suppose that controlling of oxygen consumption by controlling the rate of ATP synthesis and the reduction of oxygen expenses for oxidative phosphorylation is one vitally important function of mKATP channel under hypoxia. This is in line with other mechanisms suppressing mitochondrial respiration and OxPhos reported in the literature: the activation of AMPK and glycolysis, S-nitrosation of the respiratory complexes and downregulation of F0F1 ATP synthase. Possibly, this function of ATP-sensitive K+ transport (and K+ transport on the whole) to reduce oxygen consumption and save oxygen for oxygen-dependent processes by suppression of the oxidative ATP synthesis could move into first place under hypoxia. Concomitant suppression of ATP hydrolysis should prevent excess ATP consumption, which was confirmed by the data showing a preservation of cellular ATP ensuing from the mKATP channel opening.\n
Mitochondria respond to hypoxia by triggering ROS signaling, HIFs activation, controlling of oxygen consumption, ROS production and the level of cellular ATP. Potassium channels of mitochondria and plasma membrane were shown to be both oxygen sensors and ROS sensors and thus are first to respond to the changes of ROS and oxygen levels in the cell. Several published data discussed in this review allow us suppose that activation of potassium transport in mitochondria and controlling the above processes via mKATP channel opening could be one of the key events in the adaptive responses of the organelles to hypoxia.\n
As it was supposed by many authors, mitochondria, being the main oxygen consumers, deprive the rest of the cell of oxygen. Under these conditions, ATP synthesis via OxPhos becomes too oxygen expensive function of mitochondria. So, phosphorylation should be down-regulated in order to rescue the whole cell from severe oxygen deficiency. Thus, under hypoxia, several mechanisms are brought into action in order to reduce oxygen consumption by mitochondria, i.e. by downregulation and nitrosylation of respiratory complexes, by producing H2S as electron donor to the respiratory chain, by downregulation of the OxPhos and by the activation of mitochondrial ATP-sensitive K+ transport to reduce ATP synthesis and oxygen expenses for one of the most oxygen-consuming mitochondrial functions. Thus, hypoxia upregulates glycolysis in order to save oxygen and preserve cellular ATP needed for energy-consuming processes, such as maintenance of membrane potentials, metabolism, protein synthesis and other cell functions. Inhibition of ATP hydrolysis by potassium transport helps to save ATP obtained by glycolytic pathway.\n
The activation of mitochondrial potassium transport is a ubiquitous phenomenon under the limited oxygen availability. The above brief survey of the literature enables us to propose the following important functions of mKATP channels relevant to hypoxia: (1) ability to accept and convey ROS signals, triggering of ROS signaling specific for hypoxia; (2) controlling of mitochondrial ROS production and preventing overproduction; (3) controlling the level of cellular oxygen by oxygen-saving control of OxPhos and ATP production and (4) saving cellular ATP (obtained from both oxidation and glycolysis) by suppression of ATP hydrolysis. Multiple mKATP channel functions under hypoxia discussed in this work can be summarized in the following scheme.\n
Being important for the understanding of physiological role of mKATP channel, these aspects of mKATP channel functions, largely based on bioenergetic effects of ATP-sensitive K+ transport, cannot yet help in appraisal of the specificity of mKATP channel, as compared to other potassium channels present in mitochondria. Possibly, novel concepts of physiological role(s) of mKATP channels based on the molecular and cellular mechanisms regulating mKATP channel functioning are required to extend our understanding of physiological relevance and the mechanisms regulating mKATP channel functions under hypoxia.\n\n
Wildlife conservation and management is the process of caring for wild animal species and their environments from destruction, including preserving rare species from extinction. All this is done to sustain a better balance within an ecosystem as well as maintaining the beauty of mother nature [1, 2, 3]. In cases where the balance is interrupted, for example in communities where wild carnivores are killed due to wildlife-human conflicts, it may lead to overpopulation of wild-herbivores, and consequently translate into overgrazing of the available vegetation and deforestation [4, 5]. For centuries wildlife, has been reported to serve as a source of food, thus sustaining human life through provision of products such as honey and bush meat . Where strict wildlife management procedures are observed, the chances for transmission of zoonotic diseases are reduced and therefore, good health and disease-free populations . Due to improved wildlife conservation and management strategies, the economy of many countries globally has improved due to income generated from tourism attraction . Tourist visits have in turn led to enhanced social and cultural livelihood in different communities, the Maasai and Samburu of Kenya included [3, 8].
In Africa wildlife has faced great challenges, often attributed to human activities including encroachment into wildlife sanctuaries and loss of habitats . Other challenges include poaching and illegal wildlife trade, activities that have led to the declining numbers of wild animals’ overtime [8, 10, 11]. Besides loss of habitats, poaching, pollution, climate change and invasive species, emerging and re-emerging zoonotic diseases are increasingly featuring as a major challenge in wildlife conservation. The current wild pandemic of covid-19 is a major example of such diseases, transferable from animals to human or/and vice versa which often happen when human encroach wildlife sanctuaries, and affect the balance of Nature, for example, deforestation and modification of natural habitats as a result of land use and land cover changes is responsible for outbreak of about 50% of the emerging zoonoses .
Wide range of pollutants affects wildlife health and sometimes lead to animal death. Diseases in wildlife influence several biological factors like reproduction, survival fitness and abundance of wildlife species . Often arthropods and other animal species of wildlife origin have been reported to transmit diseases including Nile fever, Lyme disease, Encephalomyelopathies, COVID-19, Bovine tuberculosis, among other zoonotic diseases. Ben (2014) stated need for humans to refrain from anthropocentric attitudes towards wildlife and learn a need for respect to ecosystems, emphasizing on major benefits that exist when the balance in nature is maintained. In their report, Vila and group of scientists reported endoparasites causing zoonotic diseases in cattle and wild animals in Europe [14, 15]. The Asian tiger mosquito (Aedes albopictus) was reported as a vector that caused over 22 Arboviruses worldwide. The mosquito has been reported to have caused outbreaks of dengue and chikungunya in Northern Italy . During the time, the dengue fever was cited as a major cause of deaths in children in moat of the Asian countries . In the African continent, tsetse fly (genus Glossina) has been reported to cause trypanosomiasis in both humans and livestock [15, 17, 18]. Simwango et al.  linked exposure of the Maasai people to zoonotic diseases, with their frequent interactions with wildlife. A recent emerging zoonotic disease, COVID-19, caused by Corona virus with impacts to over 210 countries worldwide [12, 19], is linked to animal human transmission cycle [20, 21]. The current emergence of viruses, parasites and bacteria as significant pathogens, originate mainly from human encroachment areas . These organisms had the capability of reducing body immunity and causing acute illnesses that could often be fatal. Helminths, trematodes and cestodes are important parasitic human-wildlife diseases. In East Africa most of the diseases are augmented by the closeness of pastoralists with their livestock into wildlife sanctuaries, especially during cattle herding . However, only limited data on interlinks between human and wildlife disease cycles exist. The impact of emerging and re-emerging zoonotic diseases is a nightmare, which continues to cause heavy pandemics worldwide, more effect being felt in developing countries including Kenya . It is worth noting that zoonotic diseases found in human-wildlife interfaces are complex, and thus hard to predict on time.
Cystic echinococcosis (CE) is a zoonotic disease of human and animals (livestock and wildlife), caused by larval stages of tapeworms of dogs and other carnivores. The disease occurs worldwide, but is particularly prevalent under conditions of extensive livestock keeping, uncontrolled slaughter and low levels of hygiene . In sub-Saharan Africa, CE is a serious public health and economic problem in the eastern and southern parts, especially for pastoralists and nomadic communities, but reliable data are limited . Effective control is prevented by inadequate resources and limited knowledge about the epidemiology. Several Hydatid cysts may occupy space on a lung, liver or kidney making it difficult for the person or animal to breath. The parasite exists in two distinct life cycle patterns, namely the domestic and the wildlife cycles .
Humans get cystic echinococcosis after ingestion of Taeniid eggs that may have been shed through feces of domestic dogs (in the domestic cycle) and/or wild carnivores in the wildlife cycle. Echinococcus granulosus s.l. is a cestode parasite of the family Taeniidae. The parasite is made up of at least five species, namely; E. granulosus sensu stricto, E. equinus, E. ortleppi, E. canadensis and E. felidis. Distribution of these cestode taxa vary greatly across the globe. However sub-Saharan Africa is by far the most diverse region with all species of E. granulosus sensu lato found, with exception of the genotypes G8 and G10 of E. canadensis. [26, 27].
Globally. granulosus sensu stricto (s. s). is the most important agent for human CE in both humans and animals [28, 29]. Contributions of E. equinus and E. felidis in human CE is non-existent, that of E. ortleppi is very marginal and E. canadensis G6/7 is only about 11% [29, 27]. The case report of genotype E. granulosus Gomo from an Ethiopian patient by Wassermann et al., reported in 2016 as well as the prevalence of several E. granulosus taxa in countries such as Kenya present aspects of the disease that is not yet fully understood.
In Kenya, it has been unveiled that the two transmission patterns of Echinococcus exist and an initial observation of an interface was reported previously . Global control of CE in domestic settings is very complex and presents a variation of challenging factors in endemic regions such as illiteracy, poor road networks, social cultural beliefs, and poverty [22, 30]. In parts of Africa, control of CE has only been partially achieved despite establishment of long-term control programs [31, 32]. The diversity of species, a wide range of hosts and various cultural practices in sub-Saharan Africa have made control strategies of CE in the region less successful. Therefore, sylvatic-domestic transmission interface presents a new aspect of Echinococcus species that is the least understood. In Africa, where diversity of E. granulosus (sensu lato) is very high, elucidation of the sylvatic-domestic interaction is very essential. A recent study reported E. felidis, a strain well adapted to lions in the wildlife and also a sister species to the global problematic E. granulosus (sensu stricto) in domestic dogs . The pathogenicity of E. felidis to domestic animals remains unknown. In 2014, Kagendo et al., isolated E. granulosus s. s. eggs from lion feces, however the extent of actual transmission in the wildness or how the lions contracted the taxa was a mare speculation, since there were only a few reports showing the taxa to have been isolated in a stool sample from a warthog . The present study aimed to evaluate the interaction of the sylvatic and domestic cycles of this zoonotic disease in areas adjacent to the national reserves in Kenya.
The study was done in two cystic Echinococcosis (CE) endemic areas of Maasai Mara and Samburu National reserves. The Maasai Mara National Reserve, situated in the northern part of Tanzania’s Serengeti National Park occupies 1500 km2 . The Reserves a part of the Greater Serengeti-Mara Ecosystem which is globally popular for unique phenomenon of wildebeest’s migration. The ecosystem has suitable vegetation and climatic conditions supporting a variety of wild animals, livestock and human beings. In this case, co-existence of wild animals with pastoral communities in the area is evident .
Samburu national reserve covers about 165 km2. Human beings, livestock and wild animals in are primarily dependent on the river ‘Ewaso Nyiro’. Human and wildlife interactions are therefore a common phenomenon, with wild carnivores often preying on livestock and humans fighting back and killing the predators.
Fecal samples of wild carnivores were collected from the environment by following signs and tracks . Similarly, freshly dropped fecal samples of domestic dogs were collected within the homesteads in the two areas. Taeniid eggs were isolated from 3 g of the fecal samples using the Zinc floatation method and subsequent microscopy identification  (Figures 1 and 2).
Individual taeniid eggs were picked under the microscope, lysed in 10 μl of 0.02 N NaOH solution. Lysates were used for amplification of the short fragment of NADH dehydrogenase Sub unit 1 gene (nad1) of Echinococcus spp and other Taenia species (Figure 3).
Amplification of a 200 bp long fragment of nad1 was done in a primary PCR using Nadnest A 5’-TGTTTTTGAGATCAGTTCGGTGTG3’ and Nadnest C 5’ CATAATCAAACGGAGTACGATAG −3′ primers in a 25 μl mix that was constituted using 1x dream Taq green buffer (20 nM Tris–HCl pH 8), 0.2 mM dNTPs, 0.25 μM of each forward and reverse primer, 2 mM MgCl2 and 0.625 U of dream Taq green DNA polymerase (Thermo scientific) and 2 μl of the target DNA template. A nested PCR was done using Nadnest 5’_B CAGTTCGGTGTGCTTTTGGGTCTG-3′ and Nadnest D 5’-GAGTACGATTAGTCTCACACAGCA primers in a 25 μ mixture of same constitution as the primary PCR the use of 1 μl of the of the primary PCR amplicon as a source of DNA template . Cycling conditions of primary and nested PCRs were the same; initial denaturation for 5 minutes at 94°C, and a 35-cycle involving denaturation at 94 0Cfor 30 s, elongation at 55°C for 30 s and annealing at 72°C for 1 minutes., and a final extension at 72°C for 5 minutes. Detection of amplicons was done on a 2% gel red stained agarose gel. All nad1positive individual samples were purified using high pure product purification kit (Roche, Germany) and sequenced using the reverse primer at GATC Biotech AG, Germany.
DNA Sequences were viewed and edited using the GENtle software (Manske M. 2003, University of Cologne, Germany). Clean DNA sequences were then compared with existing sequences in the NCBI GenBank using the Basic Local Alignment Search Tool (BLAST).
Host specificity of all taeniid positive samples from the environment of the parks were done by a method previously described . A PCR system using primer pairs forward 5’-TCATTCATTGA(C/T) CT(C/T) CCCAC(C/T) CCA-3’and reverse 5’-ACGGTA(A/G) GACATA(A/T) CC(C/T) ATGAA(G/T) G-3′ for primary reaction and a secondary reaction with primer pairs forward CA(C/T) CCAA(C/T) ATCTCAGCATGAA and reverse 5′-(G/T) GC(G/T) GTAGCTAT(A/T) ACTGTGAA(C/T) A(A/G)-3′ were used to amplify partial fragment of the cob gene. A different primer pair was used for amplification of cob sequence of domestic dogs including lupus for cob 5’-CATCTAACATCTCTGCTTGATG-3’and lupus rev 5’-CTGTGGCTATGGTTGCGAATAA-3′. The subsequent cob PCR amplicons were purified and sequenced, then used in identification of host origin by comparing to earlier gene bank entries including; hyena (NC_020670), leopard (NC_010641), lion (KC495058) and domestic dogs (NC 002008).
A total of 729 fecal samples of wild carnivores from Maasai Mara (387) and Samburu (342) were screened for taeniid eggs and subsequently characterized to the cestode species level. Of these 53 fecal samples contained taeniid eggs, out of which 521 eggs were isolated. Each egg was treated as isolate in the subsequent molecular analysis. All isolated eggs were screened by a PCR test for Taeniidae amplification of a partial fragment of the NADH dehydrogenase subunit 1 (nad1) which yielded 183/521 (35%) taeniid positive from the two parks; Maasai Mara (86/183) and Samburu (97/183).
DNA sequence analysis of the taeniid eggs revealed occurrence of E. granulosus (G1-G3) and E. felidis in Maasai Mara National Reserve. In Samburu National Reserves there were E. granulosus (G1-G3), E. felidis, and E. canadensis G6/7 (Table 1). Three Taenia spp. were identified in the two National Reserves -Taenia multiceps, and T. hydatigena from Maasai Mara and T. hydatigena, T. multiceps and T. saginata in Samburu (Table 1).
|Park||Animal host||n taeniid positive / n samples||n taeniid positive PCR / n eggs screened||Echinococcus and Taenia spp.|
|Samburu||Crocuta crocuta (Spotted hyena)||13/342||51/156||30 E. felidis, 14 E. granulosus (G1-G3), 6 T.hydatigena, 1 T. saginata|
|Panthera leo (Lion)||3/342||11/36||4 E. felidis, 5 E. granulosus (G1-G3), 2 T. hydatigena|
|Canis lupus familiaris (Domestic dog)||6/342||13/72||8 E. felidis, 2 E. granulosus (G1-G3), 1 E. canadensis G6/7, 1 T.hydatigena, 1 T. multiceps|
|Canis adustus (Side striped jackal)||1/342||5/12||2 E. felidis, 3 E. granulosus (G1-G3)|
|Unidentified host||4/342||17/60||9 E. felidis, 7 E. granulosus (G1-G3), 1 E. canadensis G6/7|
|Maasai Mara||Crocuta crocuta (Spotted hyena)||17/387||61/197||41 E. felidis, 18 T. hydatigena, 2 T. multiceps|
|Panthera leo (Lion)||4/387||12/48||9 E. felidis, 1 T. hydatigena, 2 T. multiceps|
|Canis lupus familiaris (Domestic dog)||1/387||5/12||4 E. felidis, 1 T. hydatigena|
|Unidentified host||4/387||8/48||3 E. felidis,1 E. granulosus (G1-G3), 2 T. hydatigena, 2 T. multiceps|
In addition to signs and tracks used in identifying the source of fecal samples in the field, the actual host origin of the 53 taeniid positive samples (26 from Maasai Mara and 27 from Samburu) were confirmed by PCR and DNA sequencing of the cob gene. The cob DNA sequences indicated the involvement of Crocuta crocuta (30/53), Panthera leo (7/53), Canis lupus familiaris (7/53), and Canis adustus (1/53) in the two National Reserves (Table 1). Host origin of 8 taeniid positive samples could not be determined.
In the vicinity of Samburu National Reserve, 406 fecal samples from domestic dogs were collected; from 21 samples, 304 taeniid eggs were isolated. Ninety-two of the 304 eggs were positive on nad1 PCR and revealed E. granulosus (G1-G3) (9) and E. felidis (47), T. hydatigena (10), T. madoquae (10), T. multiceps (7), and undetermined Taenia spp. (9). The domestic dog origin of all E. felidis positive fecal samples were confirmed by PCR and DNA sequencing. An earlier report where 500 domestic dog fecal samples from Maasai Mara National Reserve were screened, 34 samples were found positive for nad 1, of which 92/213 individual taeniid eggs were identified as E. granulosus (G1-G3) (86), E. ortleppi (2), E. felidis (3) and E. canadensis (1) .
Human encroachment into wildlife sanctuaries has augment domestic-wildlife interactions thereby raising the risk margin for transmission of zoonotic diseases. Reduced interactions between human and wild animals by putting in place strict wildlife conservation and management legislations and strategic measures will reduce the burden of zoonotic disease transmission . Population density in wildlife areas may occur in cases where management strategies include introduction of new animal species, which often result into introduction of new strains of zoonotic diseases, amidst improving number of animal population . Wildlife movements often facilitate transfer of different disease strains from one point to the other, with an example of the wildebeest migration occurring every year from Serengeti to Maasai Mara . While the migratory behavior is termed as a big economic gain due to increased tourist attraction for both countries, transmission of new strains that can cause extinction of wild species is possible. This is evidenced by a report in 2014 on existence of Echinococcus granulosus G1–3 in wildebeests . The increased disease predisposition in wildlife sanctuaries has not only led to mortalities, and hence reduced wild animal populations but also diseases transmitted from wildlife to humans, example being the increasing burden of arboviruses and the current world pandemic of COVID-19 [12, 39, 40].
Zoonotic diseases have caused advanced effect especially in low and middle-income countries . On average, up to 40% deaths occur in Africa due to infectious diseases, most of which are zoonotic . These diseases have been reported to not only cause animal or human sickness but have led to deaths and major economic loses [37, 43]. Echinococcosis, a neglected zoonotic disease, has been reported to have highest prevalence in Kenya [30, 44]. It is hypothesized that the disease transmission could be minimized by improved wild conservation management systems in the country, since this has been seen to work well as reported in previous studies [43, 45]. Most wildlife sanctuaries are unfenced, and cattle are observed often at the heart of the protected areas, and wild animals in human homesteads . This is equally interlinked with human bad slaughter behavior, where condemned offal is offered to domestic dogs, and with wild animals marauding at night, they may access and feed on this offal. This, consequently, leads to transmission of zoonotic diseases including Echinococcosis. The only reliable cure for Echinococcosis is a total removal of hydatid cyst, which is an extremely expensive undertaking, which calls for a specialized surgeon. Emergence and re-emergence of Echinococcus spp. has been reported in most countries in the African continent . Until recently, six genotypes/species have been noted in Sub-Saharan Africa; E. granulosus sensu stricto, E. canadensis G6/7, E. ortleppi, E. equinus, E. felidis and E. granulosus genotype G. omo [9, 25, 26, 27, 33, 34]. These genotypes range from domestic origins (E. granulosus sensu stricto, E. canadensis G6/7, E.ortleppi, E. granulosus genotype Gomo), to wild origin spp. i.e. felidis and E. equinus. In this study, we confirmed the existence of the previously suggested overlap between domestic pets and wildlife cycles of Echinococcus species in Kenya.
During material sampling, it was observed that livestock herding inside the Maasai Mara National Reserve, took place at night as such the accompanying dogs might have access to carcasses of preyed animals. In both reserves the ‘lion strain´ E. felidis could be isolated from both wild carnivores and domestic dogs. The lion strain, E. felidis was first promoted to the species status in 2009 where it was described as a lion strain probably confined to sylvatic transmission in sub-Saharan Africa . Subsequently, E. felidis was isolated from wildlife in Kenya  and South Africa . This seem to confirm its adaptation to sylvatic transmission systems. Isolation of E. felidis in both cycles in the present study is, therefore, an indication of active interaction of wild animals and domestic dogs within the Reserve environments. It is also possible that, these domestic dogs were infected in the process of herding of livestock. During other livelihood activities such as collection of firewood, accompanying dog(s) could scavenge on wild herbivores or might have been infected through coprophagy of wild carnivorous host’s fecal matter. On the other hand, wild carnivores were often observed marauding within manyattas (homesteads of the Samburu and Maasai pastoral communities) at night . In 31 Taeniid eggs from fecal samples of wild definitive host from Samburu National Reserve (hyena, lion and side stripped jackals) were found to have E. granulosus s.s. Following the observations/reports of wild carnivores in manyattas and predation on livestock, it is highly likely that such carnivores acquired E. granulosus s.s. infection as a result of preying on livestock.
Transmission overlap of E. granulosus s.s. and E. felidis in the domestic and sylvatic cycles could not be fully explored with data that were available. However, our observation clearly demonstrates the interaction between domestic and wildlife definitive hosts, raising major public health concerns. The genetic proximity between E. granulosus s.s. and ´lion strain´ E. felidis is well understood , but the pathogenic potential of E. felidis in livestock and human, and the importance of E. granulosus s.s. in wildlife intermediate hosts are some of the crucial aspects of Cystic Echinococcosis (CE) that remain unanswered. E. granulosus s.s. is the most infective species to humans in locally , and worldwide . The mere presence of E. granulosus s.s. in wild carnivores broadens their transmission to human and wild ungulates. So far, warthogs are known to be the most suitable intermediate hosts of E. felidis . It is, however, unclear as to whether domestic pigs or any other livestock plays the intermediate host role to sustain the transmission of E. felidis in the domestic setting. Slaughter data of animals from Maasai land could not reveal the occurrence of E. felidis in the domestic intermediate hosts . This, however, does not rule out the possibility perhaps that the disease situation in other domesticated animals in endemic wildlife vicinity might be the catalytic factor.
Echinococcus canadensis G6/7 was found in a dog fecal sample in Samburu National Reserve area. The genotype, is rare in wildlife and its existence in a domestic dog fecal sample collected from the heart of the Reserve cannot guarantee its wildlife origin. . Possibly, the dog acquired the infection from the domestic setting and defecated at the heart of the Reserve during regular human visits to the area such as herding or collection of firewood mostly by Samburu Morans who often visited the heart of the Reserve often accompanied by their dogs . Furthermore, in the absence of wild intermediate host, its existence in wildlife cycle can henceforth be ruled out.
Increased infection of the Echinococcus species and other Taenia species in domestic dogs, especially in Samburu could be due a long-standing tradition in the community where animal lungs are fed to dogs. This was supported by what the local community had to say as quoted ‘specifically lungs are strictly fed to the dogs during home slaughter or/and at the abattoirs. In the course of our study, other parasites observed included three cosmopolitan Taenia spp. including: T. multiceps and T. hydatigena (found in Samburu and Maasai Mara and in both cycles) and T. saginata, which was rare but reported here in hyena feces collected in Samburu National Reserve environment. Existence of T. multiceps in the domestic and wild definitive hosts is rather alarming. The parasite has earlier been reported in dogs and jackals and is known to cause severe neurological disease in animals (coenurosis) when the larva migrates to the brain and spinal cord . It affects sheep and goats, being their major intermediate hosts . The hyena with T. saginata eggs most likely acquired this through feeding on human feces as previously reported [9, 33]. This protracts the range for cattle to be infected since herding dogs interacts more closely with livestock and, therefore, may increase chances of infection for cattle.
Inadequate data on wildlife-human related infectious diseases has reduced preparedness against disease outbreak in Kenya. More studies on problems relating to wildlife diseases, determining the presence of such diseases, their prevalence and their impact on wildlife conservation and management are inevitable. Existing wildlife management systems are deficient of disease surveillance component, and this has led to human deaths and animal loses to zoonotic infectious disease. Disease transmission between the human-wildlife cycle is a generally gray area to most stakeholders, making disease management strategies difficult. Growth in human population is causing great challenges in environmental conservation management. Changes observed include wildlife habitat change, which has adversely caused ecological changes as well as increased emergence and re-emergence of zoonotic infectious diseases. Based on the findings of this study, it can be hypothesized that if proper wildlife management systems including disease surveillance systems are observed in Kenya, wild animal population will increase, the rare species will be free of illnesses, and human mortalities caused by zoonotic diseases will decrease. There is an overlap in occurrence of E. granulosus s.s. and E. felidis in wildlife and domestic settings in Kenya. Active interaction of wild and domestic Echinococcus in definitive hosts has been observed. However, data on importance of intermediate hosts for the ´lion strain´ E. felidis in domestic and E. granulosus s.s. in wildlife would be key in interpreting transmission dynamics of these parasites. Our study provides a base for further analysis of the sylvatic-domestic transmission interface of Echinococcus spp. in sub-Saharan Africa, and suggests improved wildlife conservation and management systems, with possibility of having all wildlife sanctuaries fenced, for the benefit of human and well as animals.
The authors acknowledge the support of Lynn Nkatha who assisted with field sampling and laboratory analysis. The authors further acknowledge financial assistance by cystic Echinococcosis in Sub-Saharan Africa Research Initiative.
Ethical permission to conduct this research was granted by Kenya Medical Research Institute’s Ethical Committee, animal care and use committee and Meru University of Science and Technology Institutional Research Ethics Review committee (MIREC-035-2017). Permission to collect samples from Maasai Mara and Samburu National Reserves was granted by Kenya Wildlife Services.