**2. Experimental area**

The region of Agadir, the district capital of Souss-Massa, is located on the southern Atlantic side of Morocco in the foothills of the Anti-Atlas. The area chosen for our investigations, is situated at the following geographical coordinates: 30°250 North, and 9°360 West. The type of dust targeted were collected from two sites in the plain of Souss-Massa, namely: the site of Adrar AD (30°250 37.6″ N, 9°320 24.1″ W) and the site of Halieutic-Park HP (30°240 48.4″ N, 9°240 04.5″ W) (**Figure 1**).

The meteorological data were acquired from a station located in Agadir, University IBN ZOHR, Department of Geology, for a period of 6 months from 24/02/2021 to 06/ 09/2021. The representative data of irradiance, and ambient temperature are shown in **Figure 2**.

Besides the interesting solar potential ranging from 0.8 to 1.2 kWh/m2 , the study area is hosting intensive industrial, touristic, and agricultural activities. Such activities are likely to contribute to the soiling of PV panels. In **Figure 2**, the average of temperature (green curves) in Agadir is respectively between 10°C and 45°C.


*Edited by Justine Rudner*

The book "Apoptosis", published by InTech and edited by Dr. Justine Rudner, of the Department of Radiation Oncology, University Hospital of Tuebingen, Germany, is comprised of 8 Open Access chapters, covering a wide range of Apoptosis-related scientific research.

ISBN 978-953-51-1133-7

Apoptosis

Photo by rightdx / iStock

## Apoptosis

**APOPTOSIS**


**Abstract**

Artificial intelligence (AI) is substantially changing the world of business. The growth of AI and its impact on business and society are explored in this chapter, including dilemmas and emerging challenges, highlighting the existing gap in the adoption of ethical principles. The elements of human-centered AI (HCAI), such as privacy, explainability, equity, and fairness, are presented, analyzing its relevance in business. and how they need to be addressed to guarantee profitable investments in AI technologies. The aim of this book chapter is to present the essential knowledge needed by business about how to behave more ethically in AI development and deployment. In this regard, we discuss how to increase user confidence and usage of AI devices by presenting a best-practice guide to underscore biases and ensure fairness in AI-based products, exemplified in the financial and healthcare sector. Improving people's understanding of how AI models operate and having a clear HCAI strategy that evaluates negative potential biases of AI systems will increase user trust, spread, and usage of AI devices, thus ensuring the full acceptance of AI in society, thus promoting human, economic, and institutional growth.

**Keywords:** AI ethics, trustworthy AI, AI biases, human-centered AI, innovation, artificial intelligence, business


**1. Introduction**

Calcium titanate (CaTiO3) or perovskite was found by Rose [1] in the Ural Mountains in 1839 and named after the Russian Statesman Count Lev Perovski. Perovskite is a relatively rare mineral, which is a promising material for use as matrices for safe long-term storage of actinides and their rare earth analogs that are present in radioactive waste [2]. It is of particular interest for petrology and cosmochemical research as a mineral which is a part of refractory Ca-Al inclusions often found in carbonaceous chondrites, which are the earliest objects of the solar system with unusual isotopic characteristics [3–5].

In addition to perovskite, two more calcium titanates, Ca3Ti2O7 [6] and Ca4Ti3O10 [7], melting incongruently, were found in the CaO-TiO2 system. The other calcium titanates, Ca4TiO6 [8], Ca3TiO5 [9], Ca8Ti3O14 [8, 10], Ca2TiO4 [9], Ca5Ti4O13 [11], Ca2Ti3O8 [6], CaTi2O5 [12, 13], CaTi3O7 [14], Ca2Ti5O12 [15], and CaTi4O9 [16], are mentioned in the literature. They also seem to be unstable, which does not exclude their possible existence [17]. Compiled in this paper on the basis of data [7, 18], as well as the results of recent studies by Gong et al. [19], the phase diagram of the CaO-TiO2 system in the high-temperature region is shown in **Figure 1**.


**4.1. Liver**

The liver is inspected in the basin. Five hundred milliliter of UW solution (4°C) is perfused to the portal vein system. The common bile duct is flushed with 200 ml of UW solution by using a syringe. The liver is placed into the first sterile bag filled with 700–1000 ml of UW solution, and the bag is tied. The first bag is then placed into the second sterile bag filed with 1 L cold normal saline and tied. The second bag is placed into the third sterile bag and then tied. The liver in the three-layered bag is then placed in the Iskey filled with ice blocks for transportation to the recipient hospital with the document secured in the Iskey (**Figure 10**) [7].

#### **4.2. Pancreas**

The pancreas is inspected to ensure its quality for transplantation without injury from the procurement and then is packed by the same method as for the liver. It is placed in the Iskey with retrieved vessels for transportation to the recipient hospital. Often, the pancreas is allocated with the left kidney to the same recipient for simultaneous pancreas-kidney transplantation (SPKT). Care is taken to avoid manipulation over the pancreas. Perfusion to the pancreas on the back table is not recommended to avoid perfusion injury and pancreatitis.

**Figure 10.** Package of the liver. The first bag filled with UW solution.


mechanisms. Initially, changes in redox status within smooth muscle cell mitochondria mediate alterations in potassium and voltage-gated calcium channel flux, promoting contraction (63). Subsequently, vasoconstriction is maintained by mechanisms that include reduced bioavailability of NO (66), release of endothelial-derived vasoconstrictors (67) and increases in myofilament calcium sensitivity (68). The focus of this article will be on the role of the hypoxia-inducible factors (HIFs) in vascular remodeling due to chronic hypoxic exposure.

# HYPOXIA-INDUCIBLE FACTORS (HIFs) AND PULMONARY VASCULAR REMODELING

HIFs are a family of heterodimeric transcription factors, discovered in 1995 (69), whose alpha subunits are stabilized in hypoxia by the inhibition of oxygen-dependent prolyl hydroxylase (PHD) enzyme activity (70); under normoxic conditions, hydroxylation of HIF-alpha by PHDs targets them for ubiquitination by the VHL complex, resulting in subsequent proteasomal degradation (**Figure 1**) (71). Whilst HIF-1α is expressed ubiquitously throughout body tissues (72), HIF-2α expression is tissue specific with an endothelial bias (73, 74). In hypoxia, stabilization of HIF-α subunits induces transcription of targets with a wide range of functions.

Perhaps unsurprisingly, genetic variation in the HIF pathway has been identified amongst indigenous altitude dwellers. Notably, genome wide association studies in the Tibetan

population have identified single nucleotide polymorphisms (SNPs) in EPAS1 (encoding HIF-2α) and ELGN1 (encoding PHD2) that were not enriched in lowlanders (75–77). EPAS1 variants were associated with lower PAP (78) and a high frequency ELGN1 mutation has been linked to reduced proliferation of erythroid progenitors in response to EPO, thus dampening hypoxia-induced erythrocytosis (79). These findings demonstrate a selection pressure for specific HIF pathway polymorphisms over 25,000 years at altitude that has aided adaptation for the Tibetan population. Interestingly, such variation is not observed in Andean counterparts, a population that has resided at altitude for 15,000 years and who are more susceptible to PH (80, 81) and erythrocytosis (82).

However, the evidence for HIF pathway polymorphisms influencing remodeling processes is weakened by the observation that correction for erythrocytosis reduces mPAP amongst Andean populations to values near those of Tibetans (57). Thus, correcting for erythrocytosis argues against a susceptibility of Andean populations to HIF-mediated remodeling processes. In light of the new PH definition (8), however, Andean corrected mPAP remains consistently above 20 mmHg at rest and the slope of rise in mPAP with cardiac output is steeper than that of lowlanders (57).

Furthermore, evidence from murine models strongly implicates the HIF pathway in hypoxia-induced vascular remodeling. Soon after the discovery of the pathway, early work in both Hif1a and Hif2a heterozygotes revealed a marked reduction in PH and vascular remodeling following chronic exposure to 10% oxygen (83, 84). Conversely, HIF2A (EPAS1) gain-of-function mutations can predispose to PH; a Hif2a variant in high altitude cattle increases susceptibility to brisket disease (85), whilst a HIF2A mutation causing familial erythrocytosis is also associated with elevated systolic PAP in humans (86). A mouse generated to have the same G536W gain-of-function mutation in the Hif2a gene also developed erythrocytosis and PH, providing further evidence of cross-species conservation of this HIF-2α role (87). Additionally, both animal models and patients with Chuvash polycythaemia (CP), characterized by a VHL mutation, exhibit marked erythrocytosis and elevated PAP that could be rescued in mice by Hif2a but not Hif1a deletion (88–90). While the descriptions of elevated PAP in humans with CP did not include right heart catheter data or haematocrit-corrected PVR, it is worth noting that elevations in systolic PAP and vessel muscularisation in young mice with homozygous VHL mutations, preceded the onset of polycythaemia (90).

### TISSUE-SPECIFIC MANIPULATION OF HIF EXPRESSION REVEALS DISTINCT ROLES FOR HIF ISOFORMS IN PULMONARY VASCULAR REMODELING

Evidence is now emerging as to how the HIF isoforms regulate pulmonary vascular cell function, with advances gained through use of murine tissue-specific deletion models, see **Figure 1**. For example, HIF-1α has been implicated in both vasoconstriction and vascular cell proliferation, the two key components of hypoxic pulmonary hypertension. Ball et al. demonstrated that inducible Hif1a deletion in PASMCs reduced right ventricular systolic pressure, arterial wall thickness and vessel muscularisation in chronic hypoxia (91), whilst Shiekh et al. reported a dependence on Hif1a in PASMC progenitors in order to drive distal migration and expansion (92). Proposed mechanisms that could explain these findings include enhanced intracellular calcium via Hif1a dependent downregulation of K+ channels (93) and upregulation of transient receptor potential calcium channels (94), recognized to enhance vasoconstriction, PASMC proliferation and migration (95). HIF-1α also mediates pro-proliferative metabolic changes in PASMCs and fibroblasts that could contribute to hypoxia-induced remodeling. One widely recognized consequence of HIF signaling amongst cancer cells is the favoring of glycolysis over oxidative phosphorylation in aerobic conditions, known as the "Warburg effect," inducing glycolytic enzymes to enhance ATP production and promote tumor growth (96, 97). Interestingly, HIF signaling amongst pulmonary arterial smooth muscle cells (PASMCs) and fibroblasts results in a similar shift to aerobic glycolysis as seen in tumors (98–100), with increased glucose uptake observed in the lungs of rats with hypoxia-induced PH and in PAH patients (101, 102). This metabolic reprogramming of pulmonary vascular cells has proven stable ex vivo with evidence of underlying epigenetic regulation (103, 104). Targeting these mechanisms may limit hypoxia-induced PASMC proliferation in the pulmonary vasculature.

Consistent with its predominantly endothelial expression profile, a growing body of evidence implicates endothelial cell (EC) HIF-2α expression as essential for pulmonary vascular remodeling through varied biological mechanisms, see **Figure 1**. Two studies have demonstrated severe and spontaneous PH following Phd2 knockdown in murine ECs (105, 106). Double knockouts of Phd2 and either HIF isoform revealed that this was a Hif2a-mediated phenotype (105, 106) but the studies highlighted different mechanisms: one associating HIF-2α expression with reduced expression of the potent vasoconstrictor endothelin-1 (ET-1) (106) and the other demonstrating HIF-2α involvement in CXCL12-mediated PASMC proliferation (105). Reduced EC Phd2 expression was also observed amongst occlusive vessels in IPAH (105), implying relevance to human pathology. Notably, these Phd2 knockout mice did not develop polycythaemia prior to the development of PH.

The NO synthesis pathway has also been implicated in EC HIF-2α-mediated remodeling. Cowburn et al. observed a similar level of protection from hypoxia-induced PH as Hif2a knockdown following EC-specific deletion of arginase-1 (Arg1), a downstream HIF-2α target and negative regulator of NO synthesis (107). Additionally, ECs from PH patients demonstrated impaired NO production in vitro, restored on arginase inhibition (107). A further mechanism by which HIF-2α could contribute to remodeling is through regulation of endothelial-mesenchymal transition (EMT), a process implicated in pathogenic remodeling (108). Tang et al. showed that markers of EMT were regulated by HIF-2α in ECs and that while endothelial-specific deletion of Hif2a protected mice from hypoxia-induced PH, deletion of Hif2a in vascular smooth muscle cells did not (109).

There remain notable controversies in the literature surrounding HIF-mediated regulation of remodeling. Whilst Ball et al. demonstrated a role for PASMC Hif1a in chronic hypoxic remodeling using a tamoxifen-inducible conditional deletion (91), Kim et al. reported enhanced pulmonary arterial tone in the absence of arterial muscularisation following constitutive PASMC-specific Hif1a deletion (110). Similarly, constitutive EC Hif1a deletion was found to confer no protection to PH by three authors (105–107), whilst Shiekh et al. could ameliorate PH following tamoxifen-inducible conditional EC Hif1a deletion, which prevented PASMC expansion and distal migration (92). Alongside evidence detailing the importance of embryonic HIF signaling for the developing vasculature (111), these observed differences imply a role of early HIF-1α signaling in pulmonary vessel development.

The crucial role of HIF isoforms in hypoxia-induced PH has identified the inhibition of these molecules as an important strategy for targeting remodeling processes. Whilst efforts to develop HIF pathway inhibitors have previously proven challenging due to poor efficacy, HIF isoform specificity and adverse effects (112, 113), a specific HIF-2α small molecule inhibitor developed to treat renal cancer has demonstrated a favorable safety profile in a recent Phase I trial (114). Encouragingly, the use of another HIF-2α inhibitor, C76, has recently been demonstrated to attenuate remodeling in three murine models of PH, with no notable inhibition of HIF-1α (115).

The pleiotropic nature of HIF signaling has identified several other pathways as possible therapeutic targets. Using congenic linkage analysis, Zhao et al. discovered a dependence on intracellular zinc in hypoxia-induced remodeling. Homozygous deletion of the zinc transporter ZIP12, a target of both HIF-1α and HIF-2α, was found to attenuate PAP, RVH and vascular remodeling in chronic hypoxia (116). Additionally, induction of ZIP12 was also reported in the pulmonary tissue in Brisket disease and highland human populations (116). How intracellular zinc influences hypoxia-induced remodeling remains unclear; however, targeting intracellular zinc homeostasis may represent a further therapeutic strategy.

### CONCLUSIONS

This article has reviewed historical observations connecting hypoxia and pulmonary hypertension and described more recent insights into the molecular mechanisms involved in hypoxia-induced remodeling. Notably, the evidence linking HIF expression to processes involved in vascular remodeling strongly raises the prospect of HIF inhibition, and in particular HIF-2α inhibition, as a strategy in order to ameliorate vascular pathology in the context of chronic hypoxia. The recent success of a HIF-2α inhibitor in several murine models is supportive of such a strategy and may lead to the consideration of clinical trials amongst PH patients in the future. However, the mechanistic links between HIF-pathway activation and PH, notably the development of spontaneous PH following Phd2 deletion, should also raise a note of caution for use of PHD inhibitors which are currently undergoing Phase II/III clinical trials in renal anemia (117).

Genetic insights gained through study of high-altitude populations suggests that a greater appreciation of factors underlying altitude adaptation may highlight further mechanisms involved in the regulation of vascular remodeling. However, while similarities exist between the pathological features of hypoxia-induced PH and other forms of the disease, the extent of overlap in the pathological mechanisms, even for patients with chronic respiratory disease, remains unclear.

#### REFERENCES

Furthermore, the notable lack of correction for haematocrit in previous work reporting PVR at altitude casts some doubt over some of the apparent differences between altitude populations, which may in fact be due to differences in haematocrit. Nonetheless, a reversal of PH on return to sea level provides the tantalizing possibility that exploiting endogenous mechanisms might provide agents that reverse vascular remodeling in hypoxic disease. Therefore, there is still hope that lessons learned from studying hypoxia-induced disease could impact on the search for agents that target pervasive vascular remodeling in other forms of PH.

#### AUTHOR CONTRIBUTIONS

JY wrote the manuscript. DW and AART drafted additional text and edited the manuscript. All authors approved the final version.

#### FUNDING

AART is supported by a British Heart Foundation Intermediate Clinical Fellowship (FS/18/13/33281).

by inducing endothelial-to-mesenchymal transition. Am J Physiol Cell Mol Physiol. (2017) 314:ajplung.00096.2017. doi: 10.1152/ajplung. 00096.2017

with HIF-2α inhibitor. Am J Respir Crit Care Med. (2018) 198:1423–34. doi: 10.1164/rccm.201710-2079OC

**Conflict of Interest Statement:** AART has received funds to attend educational events from Actelion.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Young, Williams and Thompson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Arrhythmic Burden and Outcomes in Pulmonary Arterial Hypertension

Jennifer T. Middleton1,2,3, Angshuman Maulik 2,3, Robert Lewis 1,2, David G. Kiely 1 , Mark Toshner 4,5, Athanasios Charalampopoulos 1 , Andreas Kyriacou3 and Alexander Rothman1,2,3 \*

*1 Sheffield Pulmonary Vascular Disease Unit, Royal Hallamshire Hospital, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom, 2 Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, United Kingdom, 3 Department of Cardiology, Northern General Hospital, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom, 4 Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom, 5 Royal Papworth Hospital NHS Foundation Trust, Cambridgeshire, United Kingdom*

#### Edited by:

*Argyrios Tzouvelekis, Alexander Fleming Biomedical Sciences Research Center, Greece*

#### Reviewed by:

*Iraklis M. Tsangaris, National and Kapodistrian University of Athens, Greece Stylianos Orfanos, National and Kapodistrian University of Athens, Greece*

> \*Correspondence: *Alexander Rothman<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Pulmonary Medicine, a section of the journal Frontiers in Medicine*

Received: *03 April 2019* Accepted: *08 July 2019* Published: *23 July 2019*

#### Citation:

*Middleton JT, Maulik A, Lewis R, Kiely DG, Toshner M, Charalampopoulos A, Kyriacou A and Rothman A (2019) Arrhythmic Burden and Outcomes in Pulmonary Arterial Hypertension. Front. Med. 6:169. doi: 10.3389/fmed.2019.00169* Pulmonary arterial hypertension (PAH) is a devastating, life-limiting disease driven by small vessel vascular remodeling leading to a rise in pulmonary vascular resistance (PVR). Patients present with a range of symptoms including shortness of breath, exercise intolerance, palpitations or syncope. Symptoms may be related to vascular disease progression or arrhythmia secondary to the adaptation of the right heart to pressure overload. Arrhythmic burden is high in patients with left heart disease and guideline-based treatment of arrhythmias improves quality of life and prognosis. In PAH the incidence and prevalence of arrhythmias is less well-defined and there are no PAH-specific guidelines for arrhythmia management. We undertook a literature search identifying 13 relevant papers; detection of arrhythmias was acquired from 12-lead electrocardiogram (ECG) or Holter monitors. In all forms of pulmonary hypertension (PH) the prevalence of supraventricular arrhythmias (SVA) was 26–31%, ventricular arrhythmias (VA) 24% and a 5-year incidence of SVA ∼13.2–25.1%. Prevalence and incidence of arrhythmias in PAH is less clear due to limited study numbers and the heterogenous nature of the patient population studied. For arrhythmia treatment, only single-arm studies of therapeutic strategies were reported using antiarrhythmic drugs (AAD), direct current cardioversion (DCCV) and ablation. Periods between ECG or Holter have not been investigated, highlighting the possibility that significant arrhythmias may be undetected. Advances in monitoring allow long-term surveillance via implanted/non-invasive monitors. Use of such technologies may provide an accurate estimate of incidence and prevalence of arrhythmias in patients with PAH, further defining relationships to adverse outcomes, and therapeutic options.

Keywords: arrhythmia, pulmonary arterial hypertension, right heart failure, atrial fibrillation, atrial flutter, ventricular tachycardia

# INTRODUCTION

The recent World Symposium classifies pulmonary hypertension into five groups based on underlying cause (1). Group one incorporates pulmonary arterial hypertension including familial and idiopathic causes; group two is secondary to left sided heart disease; group three to lung disease and hypoxia; group four is secondary to chronic thromboembolic disease (CTEPH) and group five includes miscellaneous causes including sarcoidosis and hematological disease (1). Increased right ventricular pressure and volume overload of the right heart leads to structural changes that impair left ventricular filling (2) and function which may predispose to the development of arrhythmias. In this review we will discuss the prevalence and incidence of arrhythmia in PAH and the potential treatment options.

# CARDIAC ARRHYTHMIAS: CLASSIFICATION AND MECHANISM

Cardiac arrhythmias are caused by either disruption of the cardiac action potential or structural/functional changes to the heart that result in abnormalities of electrical conduction. Classification is made based on heart rate (bradycardic or tachycardic) and sub-divided based on site of origin (supraventricular or ventricular arrhythmia). In the left and right atrium, dilatation and stretch results in structural, and ion channel adaptations resulting in an increased development of SVA's including atrial fibrillation (AF) and atrial flutter (see **Figures 1A,B**). Subsequent loss of atrial contraction leads to underfilling of the ventricle and reduction in cardiac output (CO). VA's can be precipitated by a dilated, dysfunctional ventricle, or altered ventricular substrate in the form of scar (3), giving rise to pro-arrhythmic myocardium (see **Figures 2A,B**). In left sided heart failure there is an increased risk of arrhythmia, which when present often exacerbates left ventricular failure (3, 4). Impaired left ventricular function results in reduced CO due to inadequate filling and contraction leading to compensatory hypertrophy and dilatation of the left ventricle. These changes increase sarcomere length and alter sensitivity to calcium leading to more forceful ventricular contraction (5), the resultant ion channel remodeling (6) can make myocardium pro-arrhythmic (3). Bradyarrhythmia presents often secondary to changes in heart structure, nodal fibrosis, or rate-limiting medication used to treat heart failure.

Current guidelines for the treatment of arrhythmia specifically relate to patients with left sided heart disease (4–9). Management strategies include AAD's that alter the action of specific ion channels to stabilize myocardium; anticoagulation to reduce stroke risk in AF (6); synchronized DCCV to revert to normal sinus rhythm (NSR); electrophysiology studies (EPS); and/or ablation to identify abnormal heart rhythms using intracardiac catheters and ablation to disrupt abnormal conduction pathways and finally permanent pacemakers or internal defibrillator devices (7, 10). Guidance for the treatment of arrhythmias in patients with PAH is lacking despite the potential harmful/unknown effects of AAD's and or therapies in this cohort (7).

#### Arrhythmias in PAH

Patients with PAH often describe palpitations and/or syncope. The incidence and prevalence of cardiac arrhythmias in PAH is not well-described, however a number of recent studies describe a significant arrhythmic burden in all forms of PH, and a relationship to clinical outcome. These studies are based on "snapshots" in time using 12 lead ECGs or shortterm Holter monitors. We undertook a literature-based review searching PubMed with the search terms "pulmonary arterial hypertension, supraventricular, and ventricular arrhythmias, atrial fibrillation/flutter, ventricular tachycardia, and mode of death," 13 relevant papers were reviewed as well as PAH registry data. A review of the REVEAL PAH registry data demonstrated a relationship between increased heart rate and adverse outcomes (11, 12) and during follow up, Burger et al. found 5.1% of patients were admitted secondary to an arrhythmia, however further details were not provided (13).

### Supraventricular Arrhythmias

#### **Prevalence**

In a multi-center, observational study (14), patients with PAH/inoperable CTEPH attending routine outpatients had their notes reviewed retrospectively for evidence of AF or atrial flutter. 297 patients were identified as fulfilling criteria (PAH 266/CTEPH 31) with 90% on PAH specific medication. An ECG diagnosis of AF or atrial flutter was noted in 79 (26.5%), approximately 50% of those were deemed to be paroxysmal in nature. Patients with AF were as a group, of increased age, male gender with a higher systolic blood pressure (BP), a reduced left ventricular ejection fraction (LVEF), and had a higher rate of chronic obstructive lung disease (COPD). At right heart catheterization (RHC), the AF group had a higher pulmonary capillary wedge pressure (PCWP), and lower pulmonary vascular resistance, potentially suggesting a more elderly population with a greater burden of co-morbid condition. There was no difference found in mean pulmonary artery pressure (PAP) or CO. Results suggest that a diagnosis of permanent or paroxysmal AF/atrial flutter in PAH confers an increased mortality. The use of 12 lead ECGs alone and the unbalanced co-morbidities of patients with and without AF/atrial flutter masked the true burden of arrhythmias in patients with PAH (14).

Rottlaender et al. (15), investigated patients with all forms of PH who developed SVA in a retrospective study of 225 patients. Thirty one percentage of patients recruited were found to have AF (41% paroxysmal) and it was apparent that AF was more likely if secondary to PH-left heart disease and less common in CTEPH. All patients identified as having AF deteriorated clinically at the time of arrhythmia. The true prevalence of SVA was again difficult to determine due to the inclusion of all types of PH. In contrast Ruiz-Cano et al. retrospectively reviewed 282 patients with PAH. In total 28 SVA episodes were detected: AF 12 patients (42.8%); atrial flutter 12 (42.8%); other SVA 4 (14.2%), 82% of SVA episodes resulted in symptoms and clinical deterioration / worsening right heart failure (RHF).

Time from PAH diagnosis to SVA was approximately 60 months and time from SVA diagnosis to death/transplant was 17.8 months suggesting increased morbidity and mortality with the development of SVA.

#### **Incidence**

A number of studies investigated incidence through prospective/retrospective cohort studies (summarized in **Table 1**), with 6-year incidence of SVA in PAH approximately 15.8% and in all forms of PH a 5-year incidence of between 13.2 and 25.1% (17–21). In a retrospective, cohort study, Cannillo et al. examined patients with PAH, lung disease-associated PH, and CTEPH who had NSR at baseline on a 12-lead ECG, patients with previous SVA diagnosis were excluded (18). Retrospective analysis of ECG's was undertaken during follow-up, if admitted or if they developed palpitations. Holter monitors were only

FIGURE 2 | (A) An ECG of ventricular tachycardia. An example of an ECG from a patient with sustained monomorphic ventricular tachycardia. It is characterized by regular, broad QRS complexes of similar morphology often over 100 bpm. (B) An ECG of Ventricular fibrillation. An example of an ECG from a patient with ventricular fibrillation. It is characterized by rapid, erratic ventricular activity as the heart fibrillates rather than pumps effectively. This rhythm quickly degenerates into cardiac arrest. fitted in patients reporting palpitations, not as a screen for asymptomatic SVA's. 77 patients met criteria, 17 patients (22%) were newly diagnosed with SVA during the follow-up period (35 months). Patients were found to have persistent AF in 8 patients (47%), permanent AF 3 patients (17%), paroxysmal SVA 3 patients (17%), atrial ectopic tachycardia 2 patients (12%), right atrial flutter 2 patients (12%) and paroxysmal AF (PAF) in 1 patient (6%), other SVA 1 patient (6%). Diagnosis of SVA's occurred on average 15.1 months after PH diagnosis and was associated with worsening parameters (World health organization (WHO)-functional class/6-min walk test (6MWT)/brain natriuretic protein (BNP), and increased mortality. This suggests SVA to be a manifestation of more severe PAH [as per Tongers (21) and Ruiz-Cano (16)]. Severe PAH resulted in more hospital admissions and closer monitoring by default, potentially introducing detection bias of SVA in these patients. Consistent with these findings, Mercurio et al. (17) again looked at PAH only and specifically idiopathic PAH and systemic sclerosis-associated pulmonary hypertension (SSc-PH). A prospective, single center study recruited 317 patients in total (116 idiopathic PAH) and of these 42 developed SVA. In keeping with Smith et al. (14) these patients had higher baseline PCWP and in this case right atrial pressure (RAP). In 90.1% cases the onset of SVA resulted in clinical worsening and RHF. A 5-year incidence of SVA in idiopathic PAH and SSc-PAH was found to be 13.2%, lower than previously documented by Olsson et al. in a similar PH group (20). Olsson et al. described a 5-year incidence of 25.1% of SVA in PAH and inoperable CTEPH. Patients again had NSR at baseline and diagnosis of arrhythmia was based on 12-lead ECG's only. Forty-Eight patients (20%) had at least one episode of SVA. They also showed patients with persistent AF to have a worse prognosis than those with PAF or NSR (20). This followed on from a previous study by Tongers et al. (21), that retrospectively recruited 231 patients under follow-up for PAH and inoperable CTEPH. Thirty-one episodes of SVA were noted on 12-lead ECG in 27 patients (Atrial flutter 15, AF 13, and other SVA 3). Episodes were again associated with marked clinical deterioration and RHF (84% SVA episodes), improving if NSR was restored. Interestingly this study appears to show an increased mortality in AF patients where NSR could not be restored. All 15 patients with atrial flutter had NSR, restored with medication (1), DCCV (6), overdrive pacing (15) or ablation (5), with one death during follow-up. In the AF group only 2 patients had NSR restored by DCCV. NSR in the remaining 9 was not achieved despite multiple medications and DCCV, with 8 deaths during follow-up. This suggests an increased mortality in AF and PAH if NSR is not achieved. It would also appear that treatment measures in the AF group were less aggressive (ablation was not attempted) but the study was from 2007 so potentially now outdated as ablation is more common place today.

In patients with idiopathic PAH on disease specific therapies Wen et al. (19) undertook a prospective, cohort study with 280 patients recruited specifically with NSR at baseline. Forty patients developed SVA at least once during follow-up, 5-year incidence was calculated as 15.8%, potentially a more realistic reflection in PAH alone. Patients as with previous studies did not tolerate SVA and restoration of NSR correlated with clinical recovery. Studies again only diagnosed SVA on 12-lead ECG, potentially failing to diagnose asymptomatic SVA.

#### **Treatment**

AAD's, overdrive pacing, DCCV, and radiofrequency ablation are all guideline based therapies for SVA (6, 7), (although not PAH-specific). Existing data showed that rhythm control has been the most popular first line strategy and restoration of NSR demonstrated clinical improvement. Cannillo et al. (18) attempted rhythm control in 76% of patients diagnosed with SVA, NSR was restored in 11 (65%) cases but there was a high recurrence rate (∼80%). Olsson et al. (20) diagnosed patients with SVA on 12-lead ECG and largely from patients presenting with deterioration of PAH/RHF symptoms rather than symptoms of arrhythmia [as per Cannillo et al. (18)]. Those with atrial flutter received ablation earlier with the concurrent use of AAD's with or without synchronized DCCV. For stable AF 10–14 days of oral amiodarone was prescribed pre DCCV and continued thereafter. If there were signs of RHF, amiodarone was given intravenously, and DCCV was performed. NSR was restored in 21/24 patients with atrial flutter and 16/24 patients with AF, resulting in clinical improvement. As such this observational study demonstrates that amiodarone was well-tolerated in PAH. Five-year survival in PAH/inoperable CTEPH was 68% with a fall to 58% if the patient developed a transient SVA and reduced further to 47% in permanent SVA. They also found that AF was more resistant to treatment than atrial flutter (similar to non-PAH disease). Consistent with these findings, Ruiz-Cano et al. (16) found that after the first episode of SVA (despite restoration of NSR/adequate rate control) 46.4% patients required an increase in PAH-specific medications secondary to progressive RHF, an interesting observation but not guideline directed. This data may suggest that the onset of SVA can be a prelude to RHF and/or clinical deterioration and that restoration of NSR or escalating PAH-specific therapy should be considered. Zhang et al. (22) specifically reviewed the efficacy and safety of electrophysiology studies and ablation in SVA and PAH. A retrospective study it reviewed 300 patients over 3 years [PAH 100 (observation) and non-PAH 200 (control)] with PAF undergoing ablation for the first time. All patients had a 24 h Holter monitor fitted immediately post ablation and AAD's were stopped 2 months later. EPS and ablation were found to be safe and reasonably effective in this cohort of patients. Bandorski et al. (23) agreed and found that 12/14 patients (all PH types) with a diagnosis of atrial flutter during EPS subsequently had successful ablation. Zhang et al. (22) noted that 11.3% of patients had an early recurrence PAF and 7.3% a late recurrence, with some correlation between a higher RAP and the incidence of late PAF recurrence.

In conclusion, incidence of SVA in PH (including PAH) ranges from 13.2 to 25.1% with a relationship to clinical worsening. As such this may be an indicator for restoration of NSR or alteration of PAH specific therapy. A range of therapeutic strategies have been investigated, however conclusions on efficacy are challenging to substantiate as the majority of studies were single arm. Little evidence of harm was identified. As in left heart disease NSR was easier to restore in atrial flutter vs. AF and all

#### TABLE 1 | Summary of literature reviewing SVA in PH.

studies suggested that restoration of NSR resulted in improved clinical outcomes.

#### Ventricular Arrhythmias

The impact of ventricular arrhythmias in patients with PH is examined in a series of papers by Bandorski et al. An initial retrospective, two-center study (23) analyzed data from 55 PH patients presenting with indications for EPS (14 with group I PH). Fifteen had non-sustained ventricular tachycardia (NSVT) on a Holter monitor, however the prognostic relevance is unclear although likely to infer increased risk and extrapolation of prevalence from such a study is challenging due to small numbers and the large contribution of patients (23 in this study) with left heart disease-associated PH. There were no evidence suggesting EPS or ablation to be unsafe or ineffective in PH patients.

In a larger study, Bandorski et al. (24) sought to determine the incidence of VA in PH. Ninety two patients were enrolled in total (54 Group I, 10 Group 3, 26 Group 4, 2 Group 5), all of whom were on PAH-specific medication and in NSR at the time of enrolment. During 72-h Holter monitoring, 17 patients (18.5%) had a detectable arrhythmia [NSVT (12 patients), second degree atrioventricular block (1), intermittent complete heart block (1), and atrial flutter (1)]. Although small and including patients with all PH types, they undertook Holter monitoring and found arrhythmia in asymptomatic patients highlighting the potential inaccuracy of determining the incidence or prevalence of asymptomatic arrhythmia from 12-lead ECG. It highlighted that the use of Holter monitoring in this cohort was beneficial for arrhythmia diagnosis and this may be a useful tool in PAH patients also. To determine the prognostic significance of NSVT Bandorski et al. (25) examined 78 patients with PAH or inoperable CTEPH. Fifty-Five patients with PAH and 23 CTEPH underwent a clinical review, bloods, Holter monitoring, 6 MWT, echocardiography and RHC (25), of whom 12 had newly detected NSVT.

Relatively little evidence exists defining the prevalence and incidence of VA in PAH. Prevalence is estimated to be ∼27% in all PH types with a considerable proportion of whom have co-existing left heart disease. Longer studies utilizing Holter monitoring rather than 12-lead ECG show higher rates of VA suggesting a bias in methods. The most reliable incidence of VA in all PH was ∼15–18.5% based on Holter monitoring (25) suggesting a significant burden. Therapeutic options are unclear with only single arm studies available.

#### Bradyarrhythmia's

Limited data is available. Whilst determining the incidence of VA in PH Bandorski et al. (24), identified that 4/17 patients with newly diagnosed arrhythmias had intermittent heart block. Two patients, despite not taking rate limiting medication, progressed to complete heart block, and required pacemaker implantation. Studies are small and the relationship to poor outcome is uncertain.

#### Mode of Death

The predominant cause of death in patients with PAH is thought to be RHF or sudden cardiac death (SCD) (26). Hoeper et al. (27) undertook a retrospective, multi-center (17 referral centers in Europe and the United States) looking at patients with PAH who had developed cardiac arrest. 3130 patients with PAH were treated over 3 years and 513 had circulatory arrest. Cardiopulmonary resuscitation (CPR) was attempted in 132 (26%) but despite the majority occurring in a hospital setting only 8 patients (6%) survived to > 90 days. No apparent differences were found preceding cardiac arrest accounting for why a patient did or did not survive. It is difficult to fully assess cause of death, this data showed that 54% of patients were admitted with intercurrent illness; 49% died from progressive RHF; 18% respiratory failure, and 8% from other causes. Seventeen percent died from SCD and it is unclear as to whether this was secondary to PAH progression or potentially treatable arrhythmia.

# CONCLUSION

Present data suggest that patients with PH have increased risk of arrhythmia, however accurate estimates of incidence and prevalence in patients with PAH remain elusive. PAH is a rare disease and as such patient numbers are often limited and the studies undertaken have grouped patients with PAH with other forms of related disease. As such, longitudinal studies with defined enrolment criteria are required to determine the arrhythmic burden of patients with PAH. The enrolment of patients with pre-existing symptoms is a potential source of bias exemplified by the increased rates of asymptomatic SVA and VA (24) identified with prolonged monitoring. Arrhythmias have been demonstrated to precede adverse clinical events and it is therefore of clinical importance to accurately define prevalence and incidence and examine potential therapeutic options. Current evidence highlights gaps in our knowledge as we only have "snapshots" of data from a 12-Lead ECG or Holter monitoring. Advances in technology now allow for long term monitoring of cardiac rhythm and as such a prospective study with continuous monitoring may further inform incidence, prevalence and relationships to adverse outcomes, prior to studies of therapeutic strategies.

# AUTHOR CONTRIBUTIONS

JM and AR wrote the manuscript draft. All authors critically reviewed the paper and approved the final manuscript for submission.

### FUNDING

JM is funded by a Donald Heath research Fellowship. AR is supported by a Wellcome Trust Clinical Research Career Development Fellowship (206632/Z/17/Z).

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Middleton, Maulik, Lewis, Kiely, Toshner, Charalampopoulos, Kyriacou and Rothman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# NOMENCLATURE

**6MWT**: 6-minute walk test **AAD**: Antiarrhythmic drugs **AF**: Atrial fibrillation **AVNRT**: Atrioventricular node re-entry tachycardia **BP**: Blood pressure **BNP**: Brain natriuretic protein **CO**: Cardiac output **COPD**: Chronic obstructive pulmonary disease **CPR**: Cardiopulmonary resuscitation **CTEPH**: Chronic thromboembolic pulmonary hypertension **DCCV**: Direct current cardioversion **ECG**: Electrocardiogram **EPS**: Electrophysiology study **LVEF**: Left ventricular ejection fraction **NSR**: Normal sinus rhythm **NSVT**: Non-sustained ventricular tachycardia **PAF**: Paroxysmal atrial fibrillation **PAH**: Pulmonary arterial hypertension **PAP**: Pulmonary arterial pressure **PCWP**: Pulmonary capillary wedge pressure **PH**: Pulmonary hypertension **PVR**: Pulmonary vascular resistance **RAP**: Right atrial pressure **RHC**: Right heart catheter **RHF**: Right heart failure **SCD**: Sudden cardiac death **SSc-PH**: Systemic sclerosis-associated pulmonary hypertension **SVA**: Supraventricular arrhythmia **VA**: Ventricular arrhythmia **WHO**: World health organization

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Meet the editors

Dr. David Gaze is currently a lecturer in Clinical Biochemistry at the University of Westminster. His academic research interests are in the development and clinical utility of cardiac biomarkers for the detection of cardiovascular disease with a special interest in the cardiorenal population. He has authored and co-authored more than 150 peer-reviewed papers and 200 abstracts. He has contributed five book chapters to cardiovascular-related text-

books as well as is writing a textbook on cardiac troponin. He is a peer reviewer for 25 medical journals. He is the commissioning editor for review articles for the *Annals of Clinical Biochemistry & Laboratory Medicin*e and is Co-editor-in-chief of Practical Laboratory Medicine.

Aleksandar Kibel is an assistant professor at the Department of Physiology and Immunology at the Faculty of Medicine, University of Osijek, as well as an internal medicine specialist and cardiology subspecialist at the Department for Heart and Vascular Diseases, Osijek University Hospital Centre, Croatia. He graduated with a maximum possible grade average from the Faculty of Medicine, University of Osijek, with an MD degree

in 2009, conducting experimental research activities during almost all study years and receiving awards that include the country's top scholarship ("Top Scholarship for Top Students"). Research activities included laboratory practicums and research visits at a number of foreign institutions including the University Clinic Hamburg-Eppendorf (Germany), the Medical College of Wisconsin (USA), Chulalongkorn University in Bangkok (Thailand), etc. He obtained his Ph.D. degree at the Department of Physiology and Immunology with a focus on vascular physiology and pathophysiology and teaches at the medical high school, university, and postgraduate doctoral levels. He served as a mentor on several diploma works. Prior work experience also includes work at the Emergency Medicine Department and an internship at the University Hospital Zagreb, as well as volunteer work at the Red Cross. He has published a number of scientific papers and three book chapters and has actively participated in many international scientific conferences, in research projects at the pre-clinical and clinical levels, and in pharmacological clinical trials as a sub-investigator. He is a guest editor of several special issues and an editorial board member of several international scientific journals.

Contents

**Section 1**

of Myocardial Infarction

*by John G. Kingma*

*by Ivana Škrlec*

of Takotsubo Syndrome

*by Ketrell L. McWhorter*

*by Shogade Taiwo and Akpabio Akpabio*

*Andrei Bojan and Manuela Ciocoiu*

**Preface XIII**

Cardiac Risk **1**

**Chapter 1 3**

**Chapter 2 13**

**Chapter 3 35**

**Chapter 4 53**

**Chapter 5 65**

**Chapter 6 77**

**Chapter 7 101**

Epigenetics of Circadian Rhythm Disruption in Cardiovascular Diseases

Gender Differences in Clinical Outcomes of Patients with Coronary

*by Yaya Guo, YanPing Bai, Yan Gao, Chenxia Wang and Zhilu Wang*

Obesity Acceptance: Body Positivity and Clinical Risk Factors

Genetic Determinant of Familial Dilated Cardiomyopathy

*by Jing Zhong, Li-Ping Li, Jian-Feng Zhou and Yong-He Ding*

and Genotype-Targeted Therapeutic Strategy

Correlations between Inflammation and Thrombosis in the Pathogeny

*by Iris Bararu Bojan, Oana-Viola Badulescu, Maria Vladeanu,*

Acute Myocardial Infarction: Perspectives on Physiopathology

Overview of the Global Prevalence and Diagnostic Criteria

Artery Disease after Percutaneous Coronary Intervention

of Myocardial Injury and Protective Interventions


The Role of Cryptosporidiosis in Sheep Welfare

*María Uxúa Alonso Fresán and Alberto Barbabosa Pliego*

#### **Abstract**

Welfare in animal production has been defined as the optimal mental and physiological state of the animals. It has been recently redefined according to animals' freedoms. As systems, individual sheep and herds are dynamic with constant interaction with each other and the environment. In this interaction, diseases play a fundamental role in welfare. Parasitism is common in sheep, and several management practices have been established to maintain the herds healthy. *Cryptosporidium* represents a special case, because it is a highly resistant environmental parasite, that can easily infect lambs, producing weakening diarrheas and even death. In this chapter, the role of cryptosporidiosis in sheep welfare and economic loss will be analyzed, as means of providing information on how to minimize and deal with the infection.

**Keywords:** *Cryptosporidium*, cryptosporidiosis, sheep, lamb, welfare, diarrhea, economic loss, prevention, control, treatment

#### **1. Introduction**

Animal welfare and health status in herds go hand in hand. When pathogens are present, disease may develop and will be reflected on decreased productivity. Parasites are pathogens which may be found in the environment as well as in the hosts, some of them as opportunists that can cause disease. Such is the case of *Cryptosporidium spp*., a ubiquitous parasite with worldwide distribution, which causes diarrhea in newborn lambs that can lead to death or self-limiting diarrheas in immunocompetent hosts. In this chapter, welfare and cryptosporidiosis is discussed in an attempt to provide information on how to prevent and control the disease.

#### **2. Welfare generalities**

According to the World Organization for Animal Health (OIE), animal health and welfare is defined as "the physical and mental state of an animal in relation to the conditions in which it lives and dies", and covers "the five freedoms": "1) freedom from hunger, malnutrition and thirst; 2) freedom from rear and anxiety: 3) freedom from heat stress or physical discomfort; 4) freedom from pain; and 5) freedom to express normal patterns of behaviour", [1]. These were developed by the UK Animal Welfare Council (FAWC) in 1979, after researching on farmed animals in intensive systems, [2].

Derived from these freedoms and based on [3], Codes of Best Practices for Welfare Establishments have been developed, such as the one from the Welsh Government [4], in which a series of issues are covered, in order to keep the animals healthy and productive. As an example, this one indicates not only how the animals should be managed, but also how staff and volunteers should be managed too. Regarding animal management, a conscientious record of animal admissions, behavior and assessment, housing and environment, cleaning and hygiene among others are defined. Animal health and disease are also included, as well as rehabilitation, rehoming of release, and transportation, all based on the corresponding legislation.

There is a close relationship between animal health and welfare. If they are optimal, they will promote high productivity and the reduction in the use of antimicrobials, as well as reduced risk for foodborne diseases to humans [5].

#### **3. Welfare and disease**

In this sense, the AWIN welfare assessment protocol for sheep [6], defines welfare indicators according to principles and criteria, in which good health is measured by the absence of disease, and according to this protocol and the objective of this chapter, it is indicated by fecal soiling. The European Food Safety Authority (EFSA) has identified gastroenteric disorders among the main welfare consequences in lambs [1]. Regarding gastroenteric disorders, diarrhea is defined as a "complex, multifactorial disease involving the animal, the environment, nutrition, and infectious agents" [7], in which *Cryptosporidium spp.* is among the most common. It is an apicomplexan protozoa that causes profuse diarrhea in neonatal lambs (4–9 days old), with low mortality, increasing concurrent infection and deficiencies in nutrition and husbandry. In well-fed animals, it often persists for 5–7 days, lessens and lambs recover. Diarrhea leads to dehydration, inappetence, abdominal pain and lethargy. Diarrhea is liquid and yellowish, varying from mild and self-limited to severe (when mixed with other pathogens) but relapse is quite common [8]. Cryptosporidiosis produces severe villous atrophy, caused by loss of enterocytes, with crypt hyperplasia by the replacement of epithelial cell loss. This occurs due to the cytotoxic effect of the parasite and apoptosis it causes, resulting in a malabsorptive diarrhea, with prostaglandinmediated anion secretion [9].

Other pathogens might be present in diarrheas caused by Cryptosporidium spp., such as rotaviruses, *E. coli*, *Salmonella spp*. or coronaviruses. In this case, prognosis degenerates and clinical signs and treatment become complicated [10].

#### **4. Species and life cycle**

There are more than 38 species of Cryptosporidium, but only three main species are reported in small ruminants: *C. parvum*, *C. xiaoi* and *C. ubiquitum* [11]. *Cryptosporidium spp.* oocysts size ranges from 4 to 4.5 μm, with spherical to ovoid shape, and four sporozoites per oocyst. They can be identified through Ziehl-Neelsen stain, where oocysts are shown as bright pink round bodies. The transmission of the infection happens by direct ingestion of oocysts present in food and water. Once in the host, oocysts excyst in the gut and sporozoites are released, entering the intestinal epithelial cells through the brush border [12]. After excystation, sporozoites are found in the extracytoplasmatic

**Figure 1.** *Cryptosporidium spp. life cycle [14].*

parasitophorus vacuole, where the parasite reproduces either asexually or by schizogony, leading to 8 merozoites within a type I meront. Merozoites are capable of invading neighboring epithelial cells and other sites of the intestine, where they either reproduce asexually forming thin-wall oocysts that cause autoinfection or a sexually forming type II meronts differentiating into microgametocytes and macrogametocytes, which will unite to form the zygote. This zygote will form 4 sporozoites in thick or thin-walled oocysts, by a process called sporogony. The ones with the thick wall are released to the environment through feces [13] (**Figure 1**). Up to 1X106 -2X106 oocysts/g of feces can be excreted by the host (which are immediately infectious) becoming a source of infection to susceptible lambs [15]. Oocysts can survive in water and in the environment several months, as soon as cool temperatures and suitable moisture exists, and can be easily transported through air and water [16].

#### **5. Prevalence**

Cryptosporidium was incidentally discovered by Tyzzer in 1907, but until 1978 oocyst identification in feces samples was confirmed through microscopy, making it the diagnostic method. Several stains have been proven, but the most common is modified Ziehl-Neelsen. Next came immunological tests which are more sensitive and specific for the detection of antigens, giving rise to immunofluorescent tests. These are specially used in water samples. Finally, genetic tools such as PCR were developed to identify the species [17, 18]. Nevertheless, microscopy using stains still remains as the simplest, cheapest and the most common way for detection.

Ever since it was first detected and confirmed as pathogen, there have been many studies regarding prevalence in sheep from different geographical regions, in which its presence may be attributed to either infectivity or the contamination of the environment [19]. In an attempt trying to explain the wide variation in prevalence, it is shown in tables according to Koeppen's climate classification (**Tables 1-5**).

#### *Sheep Farming - Herds Husbandry, Management System, Reproduction and Improvement…*

#### **Table 1.**

*Cryptosporidium spp. prevalences reported in climate a (tropical).*

#### **Table 2.**

*Cryptosporidium spp. prevalences reported in climate B (dry).*

#### *The Role of Cryptosporidiosis in Sheep Welfare DOI: http://dx.doi.org/10.5772/intechopen.99876*

**Table 3.** *Cryptosporidium spp. prevalences reported in climate C (temperate).*

#### *The Role of Cryptosporidiosis in Sheep Welfare DOI: http://dx.doi.org/10.5772/intechopen.99876*

#### *Sheep Farming - Herds Husbandry, Management System, Reproduction and Improvement…*

*Dfa/Dfb Humid continental; Dfb Warm-summer humid continental; Dsa Hot summer continental; Dwc Monsooninfluenced subartic.*

#### **Table 4.**

*Cryptosporidium spp. prevalences reported in climate D (continental).*

*Am: Tropical monsoon; Aw: Tropical wet savanna; Bsh: Mid-latitude steppe and desert; BSk: Cold semiarid; BWh: Tropical and subtropical desert; Bwk: Tropical and subtropical desert; Dfb: Warm-summer humid continental; Cfa: Humid subtropical; Cfb: Marine West Coast; Csa: Warm summer Mediterranean; Csb: Hot summer Mediterranean; Cwa: Monsoon-influenced humid subtropical; Dfc: Subartic; Dsb: Mediterranean-influenced warm-summer humid continental.*

#### **Table 5.**

*Cryptosporidium spp. prevalences reported in studies from countries in which several regions were sampled.*

#### *The Role of Cryptosporidiosis in Sheep Welfare DOI: http://dx.doi.org/10.5772/intechopen.99876*

Results show that it can be found in all different climates. One important factor is the high resistance of the oocysts when shed to the environment. Whenever there are conditions in which the parasite can survive (protection from desiccation or avoidance of direct sunlight) it will be present, even in zones with low humidity [87]. As cryptosporidiosis is multifactorial, it is also very important to take into account the immune status of the host. Immunocompetency varies with age, therefore making lambs the most susceptible. When sheep are immunocompetent, they develop the disease with weakening diarrhea and excrete high loads of the parasite, but it is self-limiting. They remain as healthy carriers, still excreting infective oocysts to the environment. Therefore, it is also important to analyze the prevalence results according to the health status of the host. In some countries, such as Iran, where studies with different climates have been undertaken (**Table 5**), prevalence has maintained with almost no shifts through time (2014: 11.3%; 2021: 9.9%). Nevertheless, in this same country, in studies where samples were taken in specific zones (dry climate), results show ample variation (2002: 14.28 and 17.69%; 2019: 5.7%), in samples taken from sheep with different ages. In other cases such as Mexico, prevalence was reported in 2005 in a temperate region (34.3%), 2007 in a dry region (100%) and in 2017 (41.58%) and 2018 (19.5%) in a tropical region, where variation in prevalence is wide. India is another example of wide variation in results. In 2013 a prevalence of 1.8% was reported in several climate regions, in 2015, 45% (tropical and temperate climate) and in 2019, 54.05% in continental climate. The studies in Nigeria with only a difference of two years show a prevalence of 43.3% in 2001 and 2.1% in 2003 in tropical zones. Turkey reports in 2004 a prevalence of 79.1% and in 2005 of 2.97% in lambs with wide variation too in the temperate climate zone. With all these examples, it is evident that generalizations cannot be made, neither by country nor continent. These results only prove the parasite's worldwide distribution throughout different climate zones, ages and health status of sheep.

#### **6. Economic loss**

Economic loss is reflected not only in mortality, but in retarded growth rates, decreased feed conversion rate, poor carcass quality, veterinary assistance and increased costs due to extra care. Moreover, healthy sheep can shed oocysts, specially periparturient ewes, with the possibility of maintaining the infection within the flock [88] and contaminate the environment [73].

*Cryptosporidium* causes diarrheas in developing countries, and it has been found that in developed countries it has become a serious problem, due to the limited number of parasitic protozoa in their surveillance programmes, as well as being a neglected disease caused by high confidence in hygiene, municipal sanitization services and good agriculture and livestock practices [18].

The model GloWPa-Crypto was used in [89] and differences were found in oocyst load in extensive and intensive systems, in which intensive ones provided a higher load directly on land.

#### **7. Prevention, control and treatment**

Transmission of *Cryptosporidium spp.* is influenced by reservoirs and environmental characteristics. Oocysts are sensible to high temperature and desication. Direct sunlight for several hours entirely inactivates oocysts [90]. To prevent and protect lambs from cryptosporidiosis, colostrum is fundamental. Hygiene measures are very important to destroy the parasite and prevent its transmission in between

animals and from the environment. Cleaning and disinfecting pens and buildings, the use of clean straw beds, avoiding high number of ewes in the parturition area and separating healthy from ill animals during diarrhea outbreaks, as well as an appropriate and in time administration of colostrum to lambs, aid in the prevention of cryptosporidial outbreaks and decreases the morbidity and mortality in sheep herds [91].

There are three main ways for treating cryptosporidiosis in farms: using drugs (most of them have been proven at laboratory level), fluid therapy and reducing the quantity of oocysts excreted to the environment [92].

There is no specific drug targeted against cryptosporidiosis, although more than 140 active principles have been tested *in vivo* and *in vitro* but none of them have been useful for eliminating cryptospodiosis from the infected animal. This can be explained because of its particular intracellular but extracytoplasmatic location in the intestine, making it resistant to antimicrobials as well as a difficult drug target. The first attempts to cure cryptosporidiosis were based on using the same drugs as the ones for genetically related pathogens (Apicomplexa) such as *Toxoplasma* and *Plasmodium*, but are not suitable against *Cryptosporidium* because of differences in its cell biology and biochemistry: it has no apicoplast organelle, neither citiric acid cycle nor cytochrome respiratory chain. Due to the fact that cryptosporidiosis has had a higher impact in underdeveloped countries, the strategy for treatment has been the use of existing medications [14].

Significant results have been found when using halofuginone lactate [93], which is the only one licensed treatment for cryptosporidiosis in calves, thought to interfere with the merozoite and sporozoite stages of *Cryptosporidium* and proven effective in the control of oocyst shedding [94]. It is an antiprotozoal drug for *Eimeria* and *Theileria*, derivative of quinazolinone [95], used both for prevention and treatment [96]. Other drugs that have also reduced the excreted number of oocysts [40] are: paramomycin, (an aminoglycoside broad-spectrum antibiotic, poorly absorbed in the gut, remaining active in the lumen [97]), metronidazole (a nitroimidazole used against giardiasis [95]), benzoxaborole (a synthetic boronheterocyclic compound that inhibits essential enzymes, used an antimalarial) and occidiofungin (a broad-spectrum glycolipopeptide with antifungal activity) [98, 99]. Nitazoxanide (nitratiazole benzamide derivative, licensed in humans as treatment for cryptosporidiosis [95]), beta-cyclodextrin (a cyclic oligosaccharide made up of glucose residues [100], used as pharmaceutical excipient [101]) as well as colostrum preparations, probiotics and decoquinate (coccidiostat quinolone derivative [102]) have also been used [103]. Therefore, the following sanitary and breeding recommendations are essential: sectioning of age groups, assurance of sufficient colostrum supply and intake, isolation of sick animals from the healthy ones, avoiding over-population, cleanliness and dryness of the environment, disinfection of premises and daily cleaning of equipment [93].

As alternative treatments, experiments in mice using watery and alcoholic extracts from *Curcuma longa* and *Coriandrum sativum* have been effective in reducing the number of excysted oocysts [104]. *Peganum harmala*, *Artemisea herb-alba* and *Olea europea* have shown similar results [105].

The most important measure to lessen the clinical signs of cryptosporidiosis is oral or intravenous fluid therapy. To decrease the spread of the disease, it is imperative to avoid oocyst shedding to the environment. For decontamination of surfaces, over 35 disinfectants have been tested, but only 5 are effective: 50% ammonia, 3% hydrogen peroxide, 10% formalin, Exspor and Oo-cide. Formaldehyde or ammonia gas used for steam heat sterilization and fumigation are also recommended [103]. UV radiation and ethylene oxide have been reported to be effective [106].

#### **Figure 2.**

*Cryptosporidium spp. in fecal smear (Ziehl-Neelsen stain, oil immersion, 100X). Arrows show deep pink oocysts. (photograph by Alonso-Fresán).*

Not only lambs may be infected through the fecal-oral route, but they can also get infected by drinking contaminated water. This may happen due to the high number of oocysts released to the environment and their resistance to chlorine and other conventional disinfectants used in water treatment processes. Oocyst concentrations of more than 150/L of surface water have been found in agricultural run-off water [15]. Therefore, another effective way of preventing Cryptosporidium spread into the environment and watersheds is using vegetation buffers, which aid in the retention of oocysts (**Figure 2**) [107].

Proper composting of manure (>60°C) inactivates oocysts and reduces the risk of their viability. Slurry storage produces ammonia and low pH, and anaerobic digestion using mesophilic and thermophilic bacteria also helps reducing oocyst viability. Fencing sheep away form water sources prevents water contamination with feces [108].

Even though many antigenic target candidates have been characterized, there is still no vaccine with proven effectiveness or ideal cost–benefit ratio [109].

#### **8. Conclusion**

*Cryptosporidium spp.* is a ubiquitous parasite that directly affects animal welfare by producing self-limiting diarrheas in immunocompetent hosts. In lambs, it is capable of producing a weakening diarrhea which may lead to death. The disease may be complicated by the interaction of other pathogens such as *E. coli* or *Salmonella spp.* Asymptomatic sheep may excrete oocysts, becoming a source of infection and contaminating the environment. Prevalence worldwide in sheep herds may be up to 100%. To prevent the disease, hygiene measures should be taken to avoid environmental contamination. Pregnant ewes should be separated from the herd during and after parturition. If lambs get infected, they should be treated in the same way as with any other diarrheic disease, with fluid therapy.

#### *Sheep Farming - Herds Husbandry, Management System, Reproduction and Improvement…*

Many active principles have been tested, as well as alternative natural remedies such as plant extracts in an attempt to cure cryptosporidiosis which have only shown to reduce the number of oocysts excreted, mildening the disease in lambs and reducing environmental contamination, but none of them cures the disease. Vaccines are still in development as means of prevention. Even though there is neither a specific treatment for curing cryptosporidiosis nor a vaccine, manure should be properly composted and to avoid water contamination, vegetation buffers may be used.

#### **Author details**

María Uxúa Alonso Fresán1 \* and Alberto Barbabosa Pliego2

1 Faculty of Veterinary Medicine and Zootechnique, Autonomous University of the State of Mexico, Toluca, Mexico

2 Research Investigation Center on Animal Health, Faculty of Veterinary Medicine and Zootechnique, Autonomous University of the State of Mexico, Toluca, Mexico

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Role of Cryptosporidiosis in Sheep Welfare DOI: http://dx.doi.org/10.5772/intechopen.99876*

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*Edited by Manuel Gonzalez Ronquillo and Carlos Palacios Riocerezo*

This book provides an overview of sheep husbandry in different parts of the world, including information on production and management systems, reproduction, and animal health. Chapters discuss different types of sheep and sheep husbandry in Poland, India, Africa, Spain, and North America, as well as zoonotic diseases such as cryptosporidiosis and their adverse impacts on the economics of sheep herding. This book is a useful resource for producers, veterinarians, animal scientists, researchers, biologists, students, and other interested readers.

Published in London, UK © 2022 IntechOpen © Andrew Goodsell / iStock

Sheep Farming - Herds Husbandry, Management System, Reproduction

and Improvement of Animal Health

Sheep Farming

Herds Husbandry, Management System,

Reproduction and Improvement

of Animal Health

*Edited by Manuel Gonzalez Ronquillo and Carlos Palacios Riocerezo*


*3.1. Densification Analysis*

Being porosity-free is of importance to the mechanical properties of the final products. Thus, it is necessary to investigate the e ffects of the process parameters on the formation of defects in order to obtain high relative density parts. In this section, the relationships between the relative density and process parameters are analyzed. Figure 6 shows the variation of the relative density with di fferent volumetric energy densities. With an increase of VED, the relative density first became higher and tended to be stable. The relative density of the final samples fabricated in this work can were nearly 100% free from cracks. Figure 7 depicts the actual transverse morphology of the samples obtained under di fferent VEDs. With an increase in the value of the VED, the number of defects in the samples gradually decreased. When the input of the VED was insu fficient, the low-melting-point metals (Ni/Fe) were melted, and the liquid phase was formed. The un-melted tungsten particle gaps were filled with the liquid phase. However, a low VED indicated a combination of a low laser power (200 W) and a high scan speed (300 mm/s). This result implies that the residence time of the formed liquid phase was too short to fully fill the gaps, so irregular defects were formed, as shown in Figure 8a. Poor densification was caused by inadequate liquid phase content or a short residence time under the processing conditions. Figure 8b presents the high-magnification transverse morphology of the samples fabricated with a high VED. Nearly full densification could be obtained under the process parameters.

**Figure 6.** Variation of the relative density with the volumetric energy density. The inserted figure shows the different transverse morphologies corresponding to the VEDs.

**Figure 7.** The transverse morphologies of different VEDs. (**a**) 185.19 J/mm3; (**b**) 277.78 J/mm3; (**c**) 396.83 J/mm3; (**d**) 518.52 J/mm3; (**e**) 592.60 J/mm3; (**f**) 617.28 J/mm3; (**g**) 634.92 J/mm3; (**h**) 648.15 J/mm3; (**i**) 777.78 J/mm3.

**Figure 8.** Transverse morphology with a high magnification. (**a**) 253.97 J/mm3; (**b**) 648.15 J/mm3.

The pores were nearly fully filled with the formed liquid phase. A combination of high laser power (350 W) and low scan speed (150 mm/s) produced a longer residence time for the liquid phase. Therefore, the best rearrangement characteristics for the tungsten powder particles were observed under the capillary force of liquid, and the densification of samples was improved. When the laser power was 350 W, the viscosity of the formed molten pool decreased, and partial tungsten powder particles could be melted. In this way, an adequate liquid phase and better fluidity for the molten pool were obtained, and the densification process of the samples could be completed.

It can also be seen that under the same VED, there are di fferent relative densities corresponding to distinct transverse morphologies, as shown in Figure 9. This phenomenon could be ascribed to the varying process parameter combinations and similar results of other materials that have been reported [28,29]. Here, the signal-to-noise (S/N) ratio of Taguchi's method was used to compare the effect of every process parameter on the relative density, which means the sensitivity of the relative density to the selected process parameters. In general, the signal-to-noise ratio in Taguchi's method can be classified into three categories: "lower is better", "nominal is best", and "higher is better". In this study, the highest relative density for the sample was expected. Thus, the "higher is better" category was adopted, which can be calculated as follows [30]:

$$\frac{S}{N} = -10 \log \left( \frac{1}{n} \sum\_{1}^{n} \frac{1}{y\_i^2} \right) \tag{5}$$

where *n* is the number of experiments and *yi* is the tested data of relative density for the *i*th sample.

**Figure 9.** Transverse morphology of the same VED (370.37 J/mm3).

With the tested data for the relative density under different combinations of process parameters, the calculated results for the S/N ratios are presented in Figure 10 and Table 3. It can be seen that the laser power was identified as the most important factor influencing the final relative density of the samples. The scan speed had a relatively insignificant effect on relative density, and the hatching distance showed the least significant effects among all the selected process parameters. Therefore, the sensitivity of the relative density to the three process parameters decreased according to the following order: laser power > scan speed > hatching distance. This result is consistent with previous results and could be ascribed to the special thermophysical properties of tungsten and tungsten heavy alloys [31].

**Figure 10.** Main effect plots of signal-to-noise (S/N).

In this section, the nearly full densification of the 90W-7Ni-3Fe sample fabricated by SLM was realized. The densification behaviors under different process parameters were analyzed and discussed. Laser power is a dominant factor in the SLM process of the 90W-7Ni-3Fe samples. For the 90W-7Ni-3Fe sample with a high relative density in SLM, a higher laser power (≥250 W) was preferred, and the value of volumetric energy density had to be no less than 300 J/mm3. The morphology of the 90W-7Ni-3Fe sample presented similar sintering, characteristic in traditional powder metallurgy [32]. The microstructure is mainly composed of a refractory tungsten particle skeleton and a liquid phase formed by low-melting-point metals (Ni/Fe).

#### *3.2. Phase Identification and Microstructure*

The XRD patterns of the 90W-7Ni-3Fe samples processed using different volumetric energy densities are shown in Figure 11. The main phases of the 90W-7Ni-3Fe samples fabricated by SLM were W and Ni-Fe solid solution phases, although the volumetric energy density varied from 185.19 J/mm3 to 777.78 J/mm3. There seems to have been no significant difference in the phase composition among the samples obtained under different volumetric energy densities. This means that low-melting-point metals (Ni/Fe) still melted, even under low volumetric energy densities.

**Figure 11.** The XRD pattern of different VED.

Figure 12a shows the typical SEM morphology of the 90W-7Ni-3Fe sample fabricated by SLM. Many tungsten particles were distributed in the matrix. The gaps between the tungsten particles were filled with liquid phases formed by low-melting-point metals (Ni/Fe). In addition, partial tungsten particles were contacted under the driving force of the liquid phase during the processes of melting and solidification. Fine tungsten grains can also be found in Figure 12a. The microstructural region can be divided into three main regions: the W particle phase, the fine W dendrite region, and the Ni–Fe matrix region with dissolved W. Scanning electron microscope (SEM) and energy-dispersive spectrometer (EDS) analyses were performed in order to further observe the microstructural constitution and confirm whether the tungsten element had been dissolved into the matrix. EDS analysis results (Figures 12b and 13a) confirmed that the particles were nearly composed of 100% W with little Ni and Fe. EDS analysis results of the Ni–Fe matrix indicated that partial tungsten had been dissolved into the matrix (Figure 13b). This phenomenon could be ascribed to the different solubilities of W, Ni, and Fe in W-Ni-Fe systems. Tungsten has a high solubility in the Ni/Fe matrix, but the solubility of Ni and Fe in tungsten is practically negligible. Moreover, fine W dendrites formed in the matrix when W particles were partially melted and W particles acted as heterogenous nucleation sites. Similar tungsten dendrites were also observed in the WHA samples fabricated by SLM [33,34].

**Figure 12.** (**a**) The microstructure of 90W-7Ni-3Fe under VED = 518.52 J/mm3; (**b**) element distribution.

**Figure 13.** Energy-dispersive spectrometer (EDS) element analysis. (**a**) Point A; (**b**) point B.