**4.4 Lymphedema**

About 20−25% of women after axillary lymph node dissection [27] and about 5% after sentinel lymph node biopsy [28] develop lymphedema (LE), most of them within the first 2−3 years [29]. As an incurable condition, LE has grave consequences

**Figure 7.** *Combined squats and resisted elbow flexion.*

for patients' physical and psychic quality of life. LE is divided into four stages: 0 = subclinical; I = pitting edema; II = non-pitting edema, and III = elephantiasis. LE can possibly be preceded by an axillary web syndrome, also called cording, with visible and palpable cords in the middle of the axilla and the upper arm. Other early factors proved to contribute to LE formation are high BMI (≥26), skin puncture, mastectomy, RT, or wound infection [30–32]. Taxan-based CT can furthermore compromise lymphangiomotoric function, that is, lymphatic contractions [33]. To capture LE early, a preoperative circumference or volume assessment is recommended. For high-risk patients, post-surgery indocyanine green lymph scintigraphy can be indicated. Women should be educated on early symptoms and how to perform regular self-measurements (pitting test, stemmer's test, and arm circumference with

#### *Physiotherapeutic Management in Breast Cancer Patients DOI: http://dx.doi.org/10.5772/intechopen.108946*

a one-hand tape measure). Measurements should be taken for at least one, better three-year post-surgery [34]. The gold standard for measuring and capturing LE is perometry or the water displacement method. Overall, arm edema incidence is reclining, whereas breast edema incidence is inclining [35]. Therefore, medical staff should be familiar with the corresponding symptoms and signs and be attentive.

The standard treatment for LE is complex decongestional therapy (CDT), which consists of manual lymphatic drainage (MLD) [36], compression therapy (CT), exercises, skin maintenance, patient education, and if necessary, dietary programs. CDT is divided into two phases, that is, intensive and maintenance phase. If primary edema volume exceeds 40%, in comparison with the healthy arm, patients are asked to complete first an inpatient rehabilitation continued afterward with phase II at home. MLD is a very gentle kind of massage, with the intention to redirect lymphatic fluid to non-compromised body quadrants by using accessory lymph paths. These are the non-affected axilla *via* ventral and dorsal, the neck and the ipsilateral inguinal region by improving lymphangiomotoricity. A thorough MLD includes treatment of the neck, the unaffected breast and back, the affected breast and back, and the whole of the arm, including the fingers. Afterward, in phase I, the patient is provided with a multi-layer compression bandaging inclusive of extended padding. In phase II, a customized compression sleeve and/or bra is prescribed to be worn daily from morning till evening. The most prescribed upper limb compression is class II, which ranges from 23 to 32 mmHg. If a glove is also necessary for finger or hand edema, this should be separated from the sleeve for easier donning. In lymphedema stage I, the patient is instructed to adopt elevated arm positions while resting; later on, this has no effect on the now chronic edema. Patient's adherence to CT is crucial for therapy success [37], which makes patient information and education even more important.

Exercises consist of a) endurance exercises and b) resistance exercises. The compression must be worn during these exercises to avoid lymphatic backflow. Possibilities are fist-pumping exercises or squeezing a little ball with the hand (30 repetitions, 3 series) [38] or moving a hand ergometer at 10−25 Watts [15], or any other exercise which incorporates parts of the arm musculature. If possible, the arm exercises should be combined with high arm positioning. Nordic walking [39] is also recommended as a whole-body endurance therapy and for achieving an upright walking posture. Enhanced breathing during this endurance training leads to a suction of the lymph fluid from the thoracic duct into the central veins (subclavian, jugular internal vein).

For skin maintenance, the patient applies suitable moisturizers or lotions on their arm after doffing the compression sleeve and before going to bed. While doing this, she should always distribute the lotion by stroking from the distal hand to the proximal arm. Keeping healthy and well-nourished skin is paramount for erysipelas prophylaxis. Patients should further carry along a little bottle of disinfectant at all times. In case of little wounds, for example, insect stings, thorn injury, the disinfectant should be applied immediately to protect against bacteria and secondly, erysipelas.

Patient education can be administered single-wise or group-wise [40]. Patients are learning to interpret early symptoms [41], do their self-assessments as mentioned before, and learn about arm mobility, everyday behavior, hygiene, erysipelas and lymphedema prophylaxis, wound-healing, and scar formation [42]. All these measures improve patients' health literacy [43] and facilitate ongoing shared decision-making.

Dietary measures are important if patients are overweight, that is, have a BMI exceeding 26. A correlation has been established between this high BMI and the lymphedema stage, so patients are advised to maintain or reach a healthy body weight [31]. This is achieved by a) diet and b) sports therapy.

#### **4.5 Sports therapy**

Women should be encouraged to start or re-start sporting activities. This is rewarded by easier ADLs, a better quality of life, better coping with the disease, and a better adherence to ongoing cancer therapies. Furthermore, women can reach a reduction of overall and disease-specific mortality as well as an extended recurrence-free time [44].

In the beginning, an oncologic physician's assessment to evaluate heart and kidney condition by taking the blood pressure, ECG, echocardiography, and fitness level is indispensable. A 6-minute-walk-test supports in assessing the patient's actual fitness level and is afterward used to define her walking speed during walking exercises. Lastly, a multiple repetition maximum test determines the possible repetitions for resistance exercises within different muscles/muscle groups. Sports therapy can be conducted during and after oncologic treatment with certain adaptations and precautionary measures. All in all, 150 minutes of activities per week with a level of 3−6 METs (metabolic equivalent of tasks) are recommended [45]. For example, brisk walking takes place at an intensity of approximately 5 METs. To ensure safety, patients should wear a portable heart rate monitor or watch. If dyspnea has to be taken into consideration, also, oxygen saturation should be measured during the training session and be constant above 90%. During all kinds of exercises, the compression sleeve should be worn [46]. The most popular sports after breast cancer surgery are gymnastics, walking, Nordic walking, and swimming. Women should be discouraged from dangerous and arm-exhausting sports such as judo or climbing. Other precautions are necessary in case of a port-a-cath or a PICC (peripherally inserted central catheter), which should not be under local mechanical pressure, or get dislocated or wet.

No sports therapy can be executed on the day of CT and the following day, even if patients are feeling well. There is too high risk of cardiotoxicity and nephrotoxicity. Afterward, the highest possible heart rate should be set between 40 and 60% maximal heart rate if patients still are in between chemo-cycles. Training should of course be postponed if patients have fever, are dizzy, or feel otherwise unwell.

It is possible to start with sports therapy about 4−6 weeks after BCS and 6−8 weeks after mastectomy. In the beginning, types of movement, which focus motoric load on lower limbs, such as walking or stationary cycling, are to be preferred. A combination of endurance training and resistance training promises optimized outcomes in terms of patient-reported outcomes and overall fitness [47].

In the case of bone metastases, training is only possible supervised in not metastasis-affected body regions, For example, the patient can train with the lower limbs if metastases were detected in the humerus. Moreover, the radiologist or oncologist has to give her/his approval if the bones are robust enough for the training. The training focus should have lain on activities of daily life promoting exercises. The potential benefits and harms must be weighed for each individual patient [48]. Manual techniques that rely on heavy stretching or give lots of pressure, resistance, or vibrations on the tissue are contraindicated for these patients because of possible pathological fractures.

#### *4.5.1 Endurance training*

Endurance training (ET) takes place if a minimum of 1/6−1/7 of the whole-body musculature is involved in the training. Breast cancer patients are advised to perform ET because their average activity level is about 30% less than that of age-wise comparable inactive women without breast cancer [12]. ET can have positive effects on the cardiopulmonary capacity, for example, heart muscle contractility, quality

of life (QoL), and sleeping quality, and can be a means to control the patients' BMI. Furthermore, it was suggested that ET fosters cerebral plasticity in the case of "chemobrain" [12], and therefore, cognitive functions can improve.

Training is possible during as well as after CT, many training regimes are combined interventions of ET and strength training (ST) [49]. Outdoor activities are recommended for better mental health and an increase in vitamin D levels [50]. As previous or ongoing CT can change heart rate, heart rhythm, and cardial function, the usage of the heart rate as an intensity parameter has to be interpreted with caution. Alternatively, the VO2-max or lactate levels can be determined.

The ideal training type is interval endurance training, as it turns out to be less exhausting and is also feasible in patients with other co-morbidities. One- to twominute intervals interchange with 30−60 seconds active breaks. The whole training session should last 10−15 minutes at a minimum (training and break minutes added) with an intended increase of 30 minutes over time. Training heart rate is set between 60−80% of maximum heart rate. As the training proceeds, training intervals are intensified, that is, prolonged, as breaks are shortened or even omitted. Recent research was able to demonstrate that even high-intensity interval training, a time-efficient method for improving cardiovascular capacity, is possible in breast cancer patient cohorts, but should be supervised [51]. In high intensity interval training (HIIT) patients are exercising short periods at high intensity (i.e. > 75% VO2 max), followed by low-to-moderate intensity recovery periods (40–50% VO2 max). However, HIIT did not improve outcome when compared with regular ET [52]. While exercising, patients should determine their subjective exhausting level, that is, rate of perceived exertion or BORG Scale with 4−5.5/10 (i.e., moderate). To enhance patient's motivation, the type of ET, for example, walking, running, cycling, and cross-country-skiing, needs to be matched with her preferences and experiences. Overweight women should possibly select a training style with reduced body weight for the lower limbs, that is, (stationary) cycling or swimming to protect the limbs' joints.

#### *4.5.2 Strength training*

Strength training is recommended because inactivity, fatigue, cancer cachexia, and CT side effects on protein synthesis deteriorate muscle strength [53]. ST is capable to increase fast twitch muscle fibers type II, improving posture, ROM, coordination, and by facilitating ADL, it secondly optimizes QoL [54]. It can be performed in supervised and unsupervised settings, provided that patients are instructed properly. In former years, strength training was mainly avoided out of concern of triggering lymphedema, a hypothesis that was falsified [55]. Strength training should involve both upper and lower limbs, and a great effect size (ES = 0.99) can be expected [56]. Low-intensity strength training implies an intensity of 30−40% 1-RM (repetition maximum) with 15−30 repetitions or 40−60% with 10−15 repetitions. An increase to 50−80% 1-RM with 8−12 repetitions, that is, moderate intensity is possible [55]. Two or three sets of each exercise incorporating various muscle groups for an overall 30−45 minutes are recommended. The multiple repetition maximum test defines the exact individual training capacity while avoiding the hazards of a real 1-RM test.

A warm-up phase (5−10 minutes light endurance exercise on the treadmill or stationary bike) in advance as well as a cool-down phase (stretching exercises, relaxation) afterward is mandatory. Strength training is recommended two times a week with a minimum of 48 hours in between to recover. If adherence to this training frequency is problematic, even a once-a-week ST could show an increase in muscle

strength and could therefore be an alternative for frail patients [57]. ST can be performed with free weights or rubber bands, own body weight, or strength-training machines. Changing body positions can facilitate or aggravate the training intensity by implementing or eliminating gravity and/or balance and coordination. ST should be postponed if thrombocyte counting is less than 20.000, if hemoglobin falls short of 8 g/dl or if the patient is suffering from arterial hypertension as well as on the days of CT and the following day.

Specific exercises, including arm flexion, above head height should not be implemented for three-month post-surgery. The patient is supposed to maintain steady breathing through all kinds of exercise to avoid a Valsalva mechanism and therefore high intrathoracic pressure. Common exercises are the seated row, leg extension, chest press, or the latissimus pull-down, when exercising with machines. Body weight-driven exercises are squats or lunges, combined with shoulder press, arm abduction, or resisted upper arm curls (cp. **Figure 7**).

Most efficient exercises involve muscles with trunk-associated insertion to furthermore improve spine bone density. This is of special interest in the case of (hormone therapy-associated) osteoporosis. To enhance this effect, patients are exercising while standing, also combined with an unstable surface, such as a balance pad or a tightly rolled-up gymnastic mat.

#### **4.6 Cancer-related fatigue syndrome**

Cancer-related fatigue syndrome (CRF) is one of the most troublesome symptoms after cancer and/or its treatments and is often present even after years [58]. Firstly, the patient should keep the so-called "energy-diary" for at least 14 days. Therein, low- and high-exhausting activities and the state of exhaustion should be noted with a numeric rating scale (1–10). This serves to identify energy-robbing activities, which should be avoided, if possible, or split up into smaller segments.

Exercise has been proven to be an efficient means of CRF, and it enhances QoL and depression [59, 60]. Low endurance training [61], that is, 40−60% of the heart rate maximum is recommended, that is, (treadmill) walking at a speed of 4−5 km/h or stationary cycling. Additionally, low-level resistance training with incorporating great muscle groups is indicated. Patients start with only a 5-minute sequence, subsequently increasing the session up to 30 minutes over weeks and months. Concerning endurance training, interval training, that is, alternating training and resting intervals suit these patients' demands better. They start with a 30−60 seconds training period, followed by 60−120 seconds resting period, representing a 1:2 frequency. Training time can be divided into two sessions per day. Patients should assess their personal rate of perceived exertion (BORG Scale) with "very easy" to "easy" (1−2/10). Strength exercises should be chosen with particular consideration of functional ADLs.

Relaxation training is another important part of CRF treatment. It aims at diminishing fear, depression, or sleeping problems. Jacobson's progressive muscle relaxation [62] or very gentle yoga exercises [63], for example, the "Dead Men's Position," "Shavasana," or the "The lying Butterfly," "Supta Baddha Konasana" are recommended in these circumstances. In each of these, the patient is concentrating on her own body, its' functions, and her personal breathing cycle. The positions can be practiced for up to 7−10 minutes. Alternatively, the patient can try a verbally guided relaxation exercise, which takes her to a virtually preferred environment, such as a gentle tropical beach or a lush-green forest surrounding.

#### **4.7 Chemotherapy-induced polyneuropathy**

Chemotherapy-induced polyneuropathy (CIPN) affects approximately 30−60% of patients. It affects sensory, motoric, and autonomous nervous system and deteriorates QoL. As a consequence, patients suffer from pain, paresthesia, and tingling sensations. They are furthermore at a high risk of falling [64]. Upper limb ADL dexterity problems, for example, closing buttons, counting coins, or holding cutlery are equally common. Training is even more effective if it is launched in advance of the symptom onset of CIPN [65]. Patients are advised to start walking training [66] and balance training on even and uneven grounds, for example, a rolled-up gymnastic mat, a balance pad, gravel underground, or a soft forest surface. Different standing positions with normal to small-positioned feet, semi-tandem and tandem stance as well as squats or lunges are practiced. If necessary and to ensure safety, patients can grasp a wall or a table. To enhance the exercises' difficulty, patients can close their eyes during exercise or add other motor or cognitive tasks simultaneously [67], for example, open or close a zip while walking, counting while stepping, etc. Furthermore, electric vibration plates can be used combined with different positions, for example, standing, sitting, or bridging the tool with the feet while lying supine with bent legs.

For upper limb and hand, sensory functions patients can exercise using a "hedgehog ball" or a spiked acupuncture massage ball, which they move around in their palm. They practice finger dexterity by picking up small objects such as coins, pens, and marbles. Therapists can give patients a hand massage to relieve numbness and pain by increasing blood circulation.

#### **4.8 Scar therapy**

Scarring in breast cancer patients is not only a cosmetic issue but can affect patients' physical as well as psychological well-being [68]. Scars can induce pain and additionally impede shoulder joint and thoracic mobility. Furthermore, they can be an obstacle for lymphatic flow [69]. Scar formation in deeper layers can even lead to muscle weakness. Patients can report pruritus and feelings of disfigurement. A well-healed scar is thin and flat, showing a similar color to the surrounding skin. On the contrary, hypertrophic scars are red or pink and raised, while keloids are beyond the original scar region. After the wound-healing process is completed, the scar tissue is manually mobilized, massaged, and stretched by both the PT and the educated patient. The patient cares for the scar by applying special moisturizers. This is even important after RT, which induces skin and tissue fibrosis [70], followed by a deteriorated ROM. Starting too early, on the other hand, would have deleterious effects, for example, aberrant wound healing, such as hypertrophic scarring or even keloids, because of an ongoing inflammation process [71].

Different physiotherapeutic techniques, such as massage, manual lymphatic drainage, connective tissue massage, acupuncture massage, or compression therapy, can be employed to improve the before-mentioned symptoms [72]. Silicone-based wound dressings are recommended for scar management. They ensure hydration and reduce inflammation. Additionally, patients should avoid excessive sun exposure. Scar therapy is possible as a self-treatment after accordingly instruction for five to ten minutes daily. This aims to loosen agglutinated connective fibers, to improve the scar pliability, the itching sensations, the cosmetic outcome, the aberrant color, and the pain [72]. Lymph vessel growth should be induced by stretching the skin during MLD, which is supposed to release vascular endothelial growth factor VEGF-c [73]. Evidence suggests that the activation of lymphatic vessels is correlated with anti-inflammatory

mechanisms [74]. Therefore, it can be hypothesized that regenerated lymphatic function in scar tissue can avoid excessive scarring and scar-associated lymphedema.

It should be mentioned that clearly defined therapy regimes concerning duration, direction, frequency, or intensity are lacking until now [75]. In addition to that, robust scar treatment trials and therapy should utilize validated scar measurement tools.

#### **4.9 Radiation-induced lung injury**

Radiation-induced lung fibrosis is a long-term side effect after RT with an incidence of 1−5% in breast cancer patients. RT-induced tissue damages are followed by inflammation processes of the alveoli and lung fibrosis at the final stages. The patient suffers from dyspnea, chest pain or tightness, a dry cough, and low cardiopulmonary function [76]. All in all, this represents a restrictive lung syndrome with reduced lung volumes, lung compliance, gas diffusion, and decreased mucociliary clearance. The patient's dyspnea can be disproportionately in comparison with the radiation dose she was exposed to. The diaphragm's workload during inspiration is intensified because the lung's expansion is more difficult than in healthy conditions.

The compromised oxygen uptake capacity can be enhanced through endurance training (cp. chapter 4.5.1). The patient is educated the learn active diaphragmatic breathing techniques and different lung stretching positions, for example, the "moon position" (the body is forming a "C-shape" in a supine position, arms stretched over the head). Diaphragmatic breathing is facilitated by gravity if patients exercise in a vertical position, that is, sitting [77] and is guided by the hands lying on the abdomen. Additional oxygen can be prescribed for symptomatic relief. Self-mobilizing techniques for the diaphragm while lying supine with one's own fingers reaching up to the diaphragm *via* the short ribs can be taught. Airway secretion mobilization is crucial to avoid high bacterial load and concurrent infections. This includes postural drainage, the instruction of effective coughing techniques, and chest percussions by the therapist's cupped hand or massage machines to induce vibration [78].

## **5. Conclusion**

Breast cancer patients are present with a multitude of complaints and symptoms according to their type of surgery, oncologic treatment, and ongoing behavior. Physiotherapy, as a body- and patient-oriented approach, offers a wide range of hands-on and hands-off treatment modalities and techniques to enhance patients´ physical and psychological well-being. It is the therapists' task to a) assess the patient thoroughly, b) evolve a suitable therapy plan, c) implement, and d) evaluate this plan for effectiveness. Physiotherapy guides the patient from early postoperative mobilization back to daily independence, social participation, and better awareness of a healthy lifestyle and their own bodies. Physiotherapy is a means to complete state-of-the-art medical cancer treatment.


of view, larger matrix size, and smaller focal spot. Through plane z-axis resolution is improved by having a greater number of smaller detectors. High isotropic spatial resolution with modern MDCTA has regular square-shaped voxels of 0.3–0.4 mm or less, allowing full reconstruction of the entire 3D dataset in any arbitrary orientation, making this technique arguably more powerful than DSA [9]. Noise, caused by random fluctuations in radiation exposure, is another important factor affecting image quality in MDCTA, reducing overall image contrast, and can be remedied by increasing tube current/voltage or increasing voxel size. Finally, image contrast which is the difference in intensity between one tissue and another can be improved by lower kVp but is also amplified in MDCTA by use of an iodinated contrast agent.

## **20.4.2 Scanner Design**

Modern CT scanners have a wide array of detectors, with as many as 500–600 in a single row, which absorb the X-ray exposure from the X-ray tube during a single rotation. This allows greater anatomic coverage at high spatial resolution. This is combined with spiral CT mode, where the table and patient are moved steadily through the scanner gantry, as the tube is rotated around the patient. The relationship between patient and gantry motion is referred to as pitch, which is defined as table movement (mm) per tube rotation divided by collimator width (mm). A higher pitch, which is frequently used with CTA of the peripheral arteries, results in lower z-axis resolution but faster coverage and lower radiation exposure. Dual source CT technology consists of two X-ray tubes orientated at right angles to each other and two corresponding sets of detectors. This design improves temporal resolution and overall image quality. Dual-energy CT is when each of the two X-ray tubes on the dual source CT scanner emits X-rays at different energy levels and is detected by the corresponding detector array. The unique absorptive spectra allow discrimination of different tissues. In peripheral CTA, this may have a role in improved characterization of pathology in the vessel wall. Other manufacturers offer alternative scanner designs with rapid kV switching or dual-layer detector technology. Apart from the increased temporal resolution, the improved capability to discriminate tissues is similar.

## **20.4.3 Radiation Dose**

CT is responsible for a significant share of overall radiation dosage to the patient population in medical imaging and MDCTA of the peripheral vasculature, with its extensive anatomic coverage, may result in higher than normal radiation exposure [10]. Radiation dose is dependent on tube current (mAs) and tube voltage (kV). Increasing mAs may improve image quality by increasing contrast to noise and lowering overall noise; however, radiation dose doubles with a doubling of mAs. Lower kV results in lower radiation exposure, but higher kV may be required to attain adequate penetration of the X-ray beam in larger patients. Adaptive scanning, whereby tube current is either reduced or switched off during rotations over sensitive body parts, such as the breast, can result in significant decreases in radiation dose. It has also been shown that peripheral CTA at tube currents as low as low as 50 mAs results in decreased radiation dose without a compromise in diagnostic efficacy [11].

## **20.4.4 Contrast Administration**

With the more rapid acquisition speeds of MDCTA, contrast administration becomes challenging, and timing takes on added importance [12, 13]. High-quality CTA typically necessitates contrast densities of greater than 200 HU in vessels of interest. There are several factors affecting contrast enhancement and time to peak following contrast administration including iodine content [14], injection rate, and patient's cardiac status. In general, higher injection rates will produce a higher peak of enhancement with a tighter bolus; injection rates of 4–5 mL/s are typical with CTA. Similarly, higher iodine content contrast medium will produce a greater enhancement peak, although when adjusted for iodine delivery rate differences are minimized. One disadvantage of modern scanners is that the scanner may "overshoot" the contrast bolus so that for peripheral CTA it may be necessary to slow down the acquisition by slowing the table speed [15].

Accurate contrast timing is typically achieved with either bolus tracking or test bolus techniques. With bolus tracking, scan acquisition is triggered when contrast density reaches a predefined threshold (e.g., 150 HU in abdominal aorta for peripheral CTA), as defined by operator (Fig. 20.3). The test bolus technique involves injecting a small dose of contrast (e.g., 20–30 mL) first to accurately measure the contrast transit time beforehand.

## **20.4.5 Lower Extremity CTA Technique**

A scout view is typically carried out initially. A pre-contrast scan from the upper abdomen to the feet is optional and may be desirable in patients who may have extensive calcifications (e.g., diabetics, dialysis-dependent chronic renal failure) or those with metal in soft tissues (e.g., prior surgery, trauma with metallic foreign body) [16]. Bolus tracking is usually used for contrast timing in CTA of lower extremities. A region of interest (ROI) is placed in the abdominal aorta at the level of the celiac origin, and the scan acquisition is triggered when the density within the vascular lumen reaches more than 150 HU. Contrast agent is injected at 5 mL/s via a large bore (i.e., 18G) intravenous cannula. Images are acquired in a cranial to caudal direction with slice thickness of 1 mm or less and collimator thickness of 0.4–0.6 mm.

Typically, a slower scan time is preferable for lower extremity CTA so as not to overshoot the contrast bolus. Table pitch of 1.1, gantry rotation of 0.37 s, table increment of 21.1 mm/360° rotation, and table speed of 63 mm/s will have a scan time of 23 s; a fast contrast injection is preferred. If there is suspected severe disease with slow flow, then a slower contrast injection bolus and longer scan time may be preferred. This can be achieved by reducing the table speed

**Fig. 20.3** Scout image of the abdomen and lower extremities is used for planning the CT scan (**a**). Source CT images from CTA study of the abdomen, pelvis, and lower extremities following intravenous injection

of iodinated contrast (**b**). Bolus tracking technique where region of interest placed in abdominal aorta detects contrast arrival; scan is automatically triggered when density rises above a predefined threshold (**c**)

through the scanner, i.e., the pitch, and by slowing the gantry rotation time.

Dual-energy MDCTA may have advantages at separating calcium from contrast in the vessel lumen. Subtraction of both datasets will produce a contrast only luminal image. Additionally, the calculation of a "virtual" non-contrast dataset will avoid the need for a separate acquisition and may reduce overall radiation exposure. Dual-energy MDCTA has shown high sensitivity for detecting stenosis compared to the gold standard DSA [17].

Dynamic CT, whereby multiphase CT acquisitions are acquired to follow contrast filling and drainage in a timeresolved manner, may have benefits for evaluating the infrapopliteal vessels, particularly in patients with slow or asymmetric flow. Dynamic CTA has been shown to have higher contrast and diagnostic confidence compared to conventional CTA [18]. Limitations of this technique include higher radiation dose and increased volumes of contrast.

#### **Key Point**

• In CTA of the peripheral arteries, images are acquired during continuous infusion of contrast agent. Acquisition is initiated by bolus tracking software. The exact CTA protocol for imaging the peripheral arteries depends on the hardware used.

# **20.4.6 Image Processing**

With fully isotropic submillimeter spatial resolution, it is possible to reconstruct the acquired CTA 3D dataset in any anatomic orientation. The 3D data is reconstructed with multiplanar reformatted (MPR), maximum intensity projection (MIP) and volume rendered (VR) algorithms (Fig. 20.4). Basic axial, coronal, and sagittal MPR images are reconimages with positive (**a**) and negative (**b**) contrast from a patient with significant infra-popliteal peripheral vascular disease. Note incomplete bone removal in the pelvis, which is a limitation of automatic image post processing. In this case bone needed to be manually edited away

structed automatically at the scanner immediately following the CT acquisition. Image data is then transferred to dedicated post-processing software where specific MIP and VR images are created. Curved MPR images of the vessel may be useful to accurately quantify the degree of stenosis and interrogate the vessel wall (Fig. 20.5).

**Fig. 20.5** Curved MPR displays the vessel along its entire length and allows accurate measure of stenosis and depiction of vessel wall, if needed

# **20.4.7 Diagnostic Accuracy**

The diagnostic accuracy of CTA for detection and grading of peripheral arterial disease is high and similar to that of MRA. Several meta-analyses have been performed, and all of these report uniformly high values for sensitivity, specificity, and accuracy in patients with intermittent claudication [19–21]. There is a relative lack of data on the diagnostic accuracy of CTA in patients with CCI.

# **20.5 Clinical Applications**

# **20.5.1 Atherosclerotic Peripheral Arterial Disease**

In patients with suspected or known PAD, it is essential to determine and describe the exact location of arterial and extent of arterial lesions. Almost invariably, PAD affects the aorta and iliac and lower extremity arteries. In patients with diabetes mellitus, PAD typically presents with lower leg arterial involvement, while the proximal arteries are relatively spared. Apart from the aortic arch branch vessel origins, the upper extremity is almost never affected. The degree of stenosis is usually described as more or less than 50% luminal narrowing, and occlusions need to be mentioned separately. There is no consensus on how stenosis should be measured (i.e., percentage reduction of luminal diameter or percentage reduction in cross-sectional area); therefore, most observers use visual analysis to estimate the former. Both methods are highly accurate if images of good quality are available. Regardless of which method is used, sensitivity, specificity, and accuracy for detection of angiographically proven stenosis are generally in the range of 90–100% [3, 4, 19–21] with MRA and CTA.

In patients with known stents or metal implants, image quality may be degraded with both MRA and CTA. For pMRA examinations are especially important that the requesting physicians note this on the study request in order to avoid false impression of stenosis. An example of stent artifacts is shown in Fig. 20.6.

**Key Point**

• Both MRA and CTA have high diagnostic accuracy for detection of location and extent of atherosclerotic narrowing.

## **20.5.2 Aneurysmal Disease**

Aneurysm is defined as a focal enlargement of an artery to more than 1.5 times its normal diameter. For the aorta the general cutoff value that warrants follow-up is 3.0 cm and for the iliac arteries 1.8 cm. Measurements of aneurysms should always be performed perpendicular to the center lumen line and should include any mural thrombus as well as the arterial wall (Fig. 20.7). *True aneurysms* involve the intima, media, and adventitial layers of the vessel. Aneurysm is categorized as *false* when fewer than three layers are involved.

# **20.5.3 Non-atherosclerotic Peripheral Arterial Occlusive Disease**

Although atherosclerosis is by far the most common cause of PAD, there are other causes as well. Any disease process that leads to narrowing or occlusion of peripheral arteries may lead to the typical history and complaints of IC or CCI. Nonatherosclerotic causes of PAD should be considered in young patients or patients without typical risk factors. Depending on the underlying disease process, involvement of specific anatomic sites or characteristic angiographic findings may be seen.

#### **Key Point**

• Both MRA and CTA can help identify nonatherosclerotic causes of peripheral arterial disease. These modalities are the technique of choice in patients with incongruencies between symptoms and risk factors for atherosclerotic peripheral arterial disease.

#### **<IP_ADDRESS> Vasculitis**

Vasculitis refers to the process of acute or chronic inflammatory changes of small, medium, or large arteries as well as veins. In addition to signs of vascular narrowing or occlusion, patients typically present with systemic signs such as fever,

**Fig. 20.6** There is signal loss in the right common iliac artery due to the presence of a stainless steel stent (arrow). Artifacts can be recognized by abrupt caliber change of arterial lumen. In this patient it is not possible to assess the degree of stenosis with certainty due to the signal loss

**Fig. 20.7** Partial volume maximum intensity projection of bilateral common iliac artery aneurysms in a 68-year-old male (**a**). Diameter measurements of aneurysms should always be performed perpendicular

to the center lumen line to avoid over- or underestimation of aneurysm diameters (**b**)

malaise, weight loss, and abnormal laboratory tests such high erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), as well as leukocytosis with granulocytosis, thrombocytosis, and normochromic/normocytic anemia. The latter reflect the acute phase response. It is important to understand that there are no universally accepted diagnostic criteria for large-vessel vasculitides, including giant cell arteritis and Takayasu arteritis. More information about the diagnostic aspects can be found in the excellent review by Keser and Aksu [22]. Both CTA and MRA can suggest the diagnosis of vasculitis, but nowadays whole-body PET imaging is often used in the diagnostic workup. There is a large list of inflammatory arteriopathies that may lead to symptoms of PAD. Below, we discuss the commonly encountered diseases.

#### **<IP_ADDRESS> Takayasu Arteritis**

Takayasu arteritis (TA) is a large vessel vasculitis that may affect the aorta, its main branches, as well as the upper extremities. Occasionally, the iliac arteries are involved. The disease mainly affects younger women and can go unrecognized for a prolonged period due to its relatively low prevalence. Besides the typical history of vague, non-specific complaints, TA is characterized by a specific morphological pattern of arterial narrowing; stenoses typically have an elongated "hourglass" aspect (Fig. 20.8). This is in contrast to the more serrated and

**Fig. 20.8** Coronal (**a**) and sagittal (**b**) maximum intensity projections of thoracic MR angiogram show typical smooth elongated "hour-glass" narrowing of the descending thoracic aorta in a 22-year-old female patient with Takayasu disease. PET scanning confirmed active disease in the narrowed segment (**c** and **d**)

abrupt appearance of atherosclerotic stenoses. Both MRA and CTA are excellent diagnostic tools for diagnosis of TA [23].

#### **<IP_ADDRESS> Thromboangiitis Obliterans**

Thromboangiitis obliterans (TAO) is an uncommon segmental inflammatory arteriopathy that affects medium- and small-sized arteries, veins, and nerves of the arms and legs. The typical clinical scenario is that of a young male heavy smoker [24]. The main differential diagnosis in case of distal lower extremity involvement is atherosclerotic PAD in diabetes mellitus. However, the presence of diabetes rules out the diagnosis of TAO. Angiographically, TAO is characterized by short segmental occlusions of arteries or veins by inflammatory thrombi. Often, these occlusions are bridged by corkscrew collaterals. This is known as *Martorell's* sign (Fig. 20.9).

#### **<IP_ADDRESS> Fibromuscular Dysplasia**

Fibromuscular dysplasia (FMD) is a noninflammatory arteriopathy that usually affects the medium-sized arteries such as the renal, carotid, and iliac arteries in young Caucasian women. Several subtypes are distinguished [25]. The characteristic angiographic appearance is the socalled string of beads which refers to the presence of multiple stenoses interspersed with aneurysmal dilatations (Fig. 20.10). **Fig. 20.10** A 37-year-old female patient with bilateral intermittent

claudication. In both external iliac arteries, typical "string-of-beads" arterial narrowing can be seen (arrows). This finding is highly suggestive of fibromuscular dysplasia

**Fig. 20.9** MR angiogram of a 29-year-old male patient with symptoms of critical ischemia in the left lower leg. The patient was a heavy smoker. Left panel (**a**) shows maximum intensity projection with impression of distal lower leg arterial occlusion and moderate to severe venous enhancement. Source images (**b**) show multiple thrombi in pos-

terior tibial artery (arrows). Transverse multiplanar reconstructions at two different levels (**c**) show arterial thombi in posterior tibial and fibular arteries (arrows). *Image courtesy of Roland Bezooijen, MD PhD, Medisch Spectrum Twente, Enschede, The Netherlands*

#### **<IP_ADDRESS> Popliteal Entrapment**

Popliteal artery entrapment refers to abnormalities of the popliteal fossa anatomy, whereby the popliteal artery is displaced medially by the medial head of the gastrocnemius muscle or when it courses anterior to the popliteus muscle [26]. The popliteal artery can be dynamically compressed when the gastrocnemius muscle is actively used. There are four types of popliteal entrapment that may be encountered. Types I–III refer to various degrees of medial displacement by the medial head of the gastrocnemius muscle, and in type IV, both the popliteal artery and vein course anterior to the popliteus muscle. Because the diagnosis of popliteal entrapment relies on precise anatomical knowledge of the popliteal fossa, it is necessary to supplement the MR angiography acquisition with high spatial resolution T2-TSE anatomical images of the popliteal fossa to visualize the relationship between the muscles and the vascular structures (Fig. 20.11).

#### **<IP_ADDRESS> Cystic Adventitial Disease**

Another non-atherosclerotic cause of PAD is cystic adventitial disease (CAD). In CAD, a mucoid cyst develops in the arterial wall which can lead to compression of the lumen and symptoms of PAD. The disease leads to smooth arterial narrowing of the affected artery. The cyst can easily be diagnosed by ultrasonography, CT, or MRI (Fig. 20.12), although MRI is the technique of choice in case of suspected CAD due to its ability to definitively identify the mucoid cystic source of the arterial narrowing. Although infrequently encountered, treatment consists of simple percutaneous aspiration of the mucoid material which results in immediate relief of symptoms [27].

**Fig. 20.11** A 39-year-old male patient with complaints of intermittent claudication of the right leg. MR angiogram in neutral position shows normal caliber of popliteal artery on both sides. Supplemental T2-TSE images in neutral position (left) and during plantar flexion (right) show

dynamic compression of right popliteal artery (arrows) due to crowding of the right popliteal fossa. Note patent left popliteal artery at rest and during plantar flexion

**Fig. 20.12** A 41-year-old male patient with intermittent claudication of the right leg. MR angiogram shows smooth luminal narrowing of the right popliteal artery. The age of the patient in combination with the

# **References**

aspect of the stenosis is highly suggestive for non-atherosclerotic peripheral arterial disease. Presence of arterial wall cyst is confirmed on fat-suppressed T2-weighted images (arrows)

choice of imaging parameters for each field of view. J Magn Reson Imaging. 2000;11:368–77.

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**Author details**

Sevcan Aydin, Nalan Tavşanli\*, Ahmet Arıhan Erözden and Mahmut Çalişkan Faculty of Science, Department of Biology, Istanbul University, Istanbul, Turkey

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 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.


*Article* **Preventive Potential of Dipeptide Enterocin A/P on Rabbit Health and Its Effect on Growth, Microbiota, and Immune Response**

**Monika Pogány Simonová 1,\*, L'ubica Chrastinová 2, Jana Ўcerbová 1, Valentína Focková 1, Iveta Plachá 1, Zuzana Formelová 2, Mária Chrenková 2 and Andrea Lauková 1**

**Simple Summary:** Rabbits are animals sensitive to alimentary disturbances and various spoilage agents, mostly during the weaning period. For this reason, the use of natural feed additives has become an area of research in rabbit nutrition, mainly with a focus on prevention. The "in vivo" administration of bacteriocins/enterocins shows an increasing potential in the prevention/treatment of animals' diseases. Therefore, our study focused on the preventive potential of the dipeptide enterocin (Ent) A/P against the methicillin-resistant (MR) *Staphylococcus epidermidis* SE P3/Tr2a strain in rabbit model, determining its effect on the growth performance, phagocytic activity, secretory (s) IgA, and gut microbial composition of rabbits. Ent A/P increased the weight gain of rabbits and its antibacterial effect showed a tendency to stabilize and improve gut microbiota due to reduction of MR staphylococci, total bacteria, and coliforms. The immune-stimulatory effect of Ent A/P was noted due to increased phagocytic activity. Achieved results showed the great potential of Ent A/P application as a feed additive in rabbit nutrition to improve the health and productivity of animals.

**Abstract:** The present study investigated the effect of the dipeptide enterocin (Ent) A/P on growth, immune response, and intestinal microbiota in rabbits. Eighty-eight rabbits (aged five weeks, M91 meat line, both sexes) were divided into three experimental groups: E (Ent A/P; 50 μL/animal/day for 14 days; between 0–14 days); S (methicillin-resistant *Staphylococcus epidermidis* SE P3/Tr2a strain; 500 μL/animal/day for 7 days starting at day 14 to day 21); andE+S (Ent A/P between 0–14 days and SE P3/Tr2a strain between 14–21 days) groups, and the control group (C). The additives were administered in drinking water. Administration of Ent A/P lead to an increase in weight gain, reduction of feed conversion; phagocytic activity was stimulated and gut microbiota were optimized due to reduction of coliforms, total bacterial count, and methicillin-resistant staphylococci. Good health and increased weight gain also showed that methicillin-resistant *S. epidermidis* SE P3/Tr2a strain did not have any pathogenic effect on rabbits' health status.

**Keywords:** enterocin; rabbit; health; immunity; microbiota; prevention

#### **1. Introduction**

The routine and indiscriminate use of conventional antibiotics in agriculture presents a growing threat, leading to an increase in drug-resistant bacteria in animals and their transmission to humans. Current research is focusing on alternative antimicrobial compounds for use in animal production and veterinary medicine. Bacteriocins are antimicrobial proteins with a broad antibacterial spectrum produced by Gram-positive and Gram-negative bacteria, mostly by lactic acid bacteria (LAB), including enterococci (producing bacteriocins

**Citation:** Pogány Simonová, M.; Chrastinová, L'.; Ўcerbová, J.; Focková, V.; Plachá, I.; Formelová, Z.; Chrenková, M.; Lauková, A. Preventive Potential of Dipeptide Enterocin A/P on Rabbit Health and Its Effect on Growth, Microbiota, and Immune Response. *Animals* **2022**, *12*, 1108. https://doi.org/10.3390/ ani12091108

Academic Editors: João Freire and Raffaella Rossi

Received: 8 March 2022 Accepted: 22 April 2022 Published: 26 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

named mostly enterocins; [1]), currently indicated for use in food preservation, but they also have great potential in the prevention and treatment of animals' diseases. Bacteriocins/enterocins have a low tendency to develop resistance compared to conventional antibiotics and they are characterized by biosafety and several bioactive roles–they possess antimicrobial, anticancer, antioxidant, and immunomodulatory effects [2–5]. Therefore, bacteriocins could be a promising alternative as feed additives in livestock farms to improve the health and productivity of food animals. Regarding their success to eliminate multi-drug resistant (MDR) bacteria, they can also be applied in several areas of veterinary medicine with preventive effects and/or to treat bacterial infections [4,6,7].

MDR bacteria represent a public health problem worldwide, with the focus mainly on vancomycin-resistant enterococci, carbapenem-resistant enterobacteria, and pseudomonads. There is also a focus on methicillin-resistant *Staphylococcus aureus* (MRSA) and other coagulase-positive (MRCoPS) and coagulase-negative staphylococci (MRCoNS) [8]. While MRSA in human medicine is detected mostly as hospital-acquired MRSA (HA-MRSA), in veterinary medicine methicillin-resistant staphylococci (MRS) are causative agents of cows' mastitis, chickens' lameness, pigs' exudative epidermitis, canine pyoderma, skin lesions in dogs and cats, and abscesses, mastitis, and septicemia in rabbits [9,10]. Because of a great diversity of MRS host species–pets, food, and wild animals [11]–a special attention is required regarding MRS' co-resistance to other antibiotics, influencing the antimicrobial therapeutic effect and offering a source of resistance genes for other staphylococcal species. For this reason, new antimicrobial agents directed against antibiotic-resistant bacteria, such as bacteriocins, are requested. In vitro bacteriocins testing against MRS, MRSA, and multi-drug resistant staphylococci (MDRS) showed promising results, including their antimicrobial and antibiofilm effect [12–14], confirmed also in several animal models, mostly in the murine model [3]. While the anti-MRSA resp. anti-MRS activity of bacteriocins was presented in a number of papers, mostly during lantibiotics' testing, the effect of enterocins on MRS isolates are documented only in some studies [15–17]. Moreover, most of the mentioned in vivo experiments present the therapeutic effect of bacteriocins tested in animal models [3], but the prevention of pathogenic and resistant bacteria multiplication using bacteriocins as feed additives is also important.

Therefore, the aim of this study was to test the preventive effect of a dipeptide enterocin (Ent) A/P (previously named Ent EK13, produced by *Enterococcus faecium* EK13 strain deponed to the Czech Collection of Microorganisms in Brno, Czech Republic with no. CCM7419 [18]) against methicillin-resistant *Staphylococcus epidermidis* SE P3/Tr2a strain in a rabbit model. The effect on growth performance, immune response, *Eimeria* spp. oocysts' occurrence, and the intestinal microbiota composition of rabbits was controlled.

#### **2. Materials and Methods**

#### *2.1. Animals Care and Use*

The experiment was performed in cooperation with our colleagues at the experimental rabbit facility of the National Agricultural and Food Centre, Research Institute for Animal Production, Nitra, Slovakia. All applicable international, national and/or institutional guidelines for the care and use of animals were followed appropriately, and all experimental procedures were approved by the Institutional Ethic Committee (permission code: SK CH 17016 and SK U 18016) and the State Veterinary and Food Administration of the Slovak Republic (4047/16-221).

#### *2.2. Experiment Schedule, and Diet*

A total of 88 post-weaned hybrid rabbits, (meat lines M91 and P91, weaned at the age of 35 days, both sexes, equal male to female ratio per treatment), were used in this experiment. Rabbits were divided into three experimental groups (E, S, E + S) and one control group (CG), with 22 animals in each group. The average live weight of rabbits at the start of the experiment was 1091.8 g ± 149.3. The rabbits were kept in standard metal cages (61 × 34 × 33 cm, Kovobel company, Domažlice, Czech Republic), two animals per cage. A cycle of 16 h of light and 8 h of dark was used throughout the experiment. Temperature (20 ± 4 ◦C) and humidity (70 ± 5%) were maintained throughout the experiment by heating and ventilation systems, and data were recorded continuously with a digital thermograph positioned at the same level as the cages. Animals were fed a commercial pelleted diet for growing rabbits (KV, Tekro-Nitra, Ltd., Nitra, Slovakia; Table 1) during the whole experiment with access to water *ad libitum*. The ingredients and chemical composition of this diet are presented in Table 1. The chemical analyses were conducted according to AOAC [19] and Van Soest et al. [20]. The animals in group E were administered Ent A/P (prepared according to Mareková et al. [21]), a dose of 50 μL/animal/day, with activity of 25,600 AU/mL during the first 14 days of the treatment period (between days 0/1 and 14), to control the preventive effect of tested enterocin A/P (Figure 1). The activity of Ent A/P was tested by the agar spot test according to De Vuyst et al. [22] against the principal indicator strain *E. avium* EA5 (isolated from the feces of piglets, our laboratory). Rabbits in group S received only the methicillin-resistant *Staphylococcus epidermidis* SE P3/Tr2a strain (1.0 × 105 CFU/mL; Pogány Simonová et al. [17]) in their drinking water at a dose of 500μL/animal/day for 7 days, between 14 and 21 days. The strain was marked by rifampicin to differentiate it from the total staphylococci and prepared as described previously by Strompfová et al. [23]. Rabbits in the E + S group firstly consumed the Ent A/P for 14 days (between 0–14 days) and after it the SE P3/Tr2a strain was applied to animals for 7 days, starting at day 14 (when the Ent A/P application was ceased), and finishing at day 21 of the trial. Based on our previous experiments, that these additives can be dissolved in distilled water [21], the additives were applied firstly to 100 mL of drinking water in all cages to ensure that rabbits received the applied additives, and after consuming this volume the rabbits had access to water ad libitum. Control rabbits (group C) had the same conditions, but without additives being applied to their drinking water, and they were fed a commercial diet. Drinking water was provided through nipple drinkers. The experiment lasted for 21 days.

**Table 1.** Nutrient content of commercial granulated diet for growing rabbits.

\* mg·kg−1 feed.

**Figure 1.** Scheme of the Ent A/P and S. epidermidis SE P3/Tr2a strain application.

#### *2.3. Growth Performance*

Body weight (BW) and feed consumption were measured every week during the experiment; average daily weight gain (ADWG) and feed conversion ratio (FCR) were calculated mathematically. Mortality was recorded daily throughout the whole experiment.

#### *2.4. Phagocytic Activity in Blood*

For phagocytic activity (PA), blood (*n* = 8) was sampled from the marginal ear vein (*Vena auricularis*) into Eppendorf tubes containing micro-spheric hydrophilic (MSH) particles and heparin at days 0, 14, and 21. Briefly, 50 μL of MSH particle suspension (ARTIM, Prague, Czech Republic) was mixed with 100 μL of blood in an Eppendorf-type test tube and incubated at 37 ◦C for 1 h. Blood smears were then prepared and stained in accordance with May–Grünwald and Giemsa–Romanowski, and the direct microscopic counting procedure, calculating the number of white cells containing at least three engulfed particles per 100 white cells (monocytes/granulocytes), was used for PA analysis [24].

#### *2.5. Immunoglobulin A in Small Intestinal Wall*

Eight rabbits in each group were killed at 21 days of the trial (56 d of age) using electronarcosis (50 Hz, 0.3 A/rabbit for 5 s), immediately hung by the hind legs on the processing line and quickly bled by cutting the jugular veins and the carotid arteries. Samples (*n* = 8) of the small intestine were collected for IgA analyses, and the appendix and cecum contents for microbial analysis.

The concentration of immunoglobulin A (IgA) in the intestinal wall was measured using the competitive inhibition enzyme immunoassay technique (Rabbit Immunoglobulin A, IgA ELISA kit, Cusabio, Houston, TX, USA). Samples of intestinal wall were prepared according to Nikawa et al. [25] and analyzed using the Multireader Synergy HTX (Biotek, Shoreline, WA, USA), at the wavelength 450 nm according to the manufacturer of Cusabio kit.

#### *2.6. Microbial Analyses and Eimeria spp. Oocysts Detection*

Freshly-voided feces were collected using nets mounted under the cages, five nets under 11 cages. Because there were two animals housed in each cage, and in some places the feces were mixed, we decided to collect mixed samples, one mixed sample per net, i.e., five mixture samples per group. Feces were sampled at day 0/1 (at the start of the experiment and Ent A/P application; 10 mixture samples from all rabbits, respectively, from all groups together–initial microbial background, at day 14 (2 weeks after the start of the experiment; the end of Ent A/P and the start of the *S. epidermidis* SE P3/Tr2a strain application; five mixture samples from each group) and at day 21 (56 days of age; the end of SE P3/Tr2a strain application). For microbial testing, samples of feces, cecum, and appendix contents (1 g) were treated using the standard microbiological dilution method (International Organization for Standardization (ISO)). The appropriate dilutions in Ringer solution (pH 7.0; Oxoid Ltd., Basingstoke, Hampshire, England) were plated onto the following media: M-Enterococus Agar (NF-V04503, Difco Laboratories, Detroit, MI, USA) for enterococci, M17 Agar (Difco) for streptococci, MacConkey agar (ISO 7402, Oxoid) for coliforms, Mannitol Salt Agar for coagulase-negative staphylococci (CoNS, ISO 6888), Plate Count Agar (Biomark Laboratories, Pune, India) for total bacterial growth, Oxacillin Resistance Screening Agar (Oxoid) to confirm methicillin-resistant staphylococci. Brain-Heart Infusion agar (Difco) enriched with rifampicin (Rifasynt, Medochemie Ltd., Limassol, Cyprus) was used to determine *S. epidermidis* P3/Tr2a. Bacteria were cultivated at 30 ◦C and/or 37 ◦C for 24–48 h depending on the bacterial genera and their counts were expressed in log 10 of colony forming units per gram (log 10 CFU/g ± SD). Randomly picked up representatives of selected bacterial groups were confirmed by MALDI-TOF identification system (Bruker Daltonics, Billerica, MA, USA).

Fecal samples were stored at 4 ◦C and examined for the *Eimeria* spp. oocysts by the flotation technique, according to McMaster [26]. Oocysts were not differentiated at the species level but designated as *Eimeria* spp.; they were counted microscopically, and the intensity of infection was expressed as oocysts per gram of feces (OPG).

#### *2.7. Statistical Analysis*

Treatment effects on the growth parameters, microbiota in fecal samples, and phagocytic activity (PA) were analyzed using two-way analysis of variance (ANOVA), followed by Bonferroni post-hoc test for pair-wise comparisons, where appropriate. Fixed effects for the model included period and treatment, and the interaction between them. Statistical analysis of microbiota from cecal and appendix samples, feed conversion and secretory IgA was performed with one-way analysis of variance (ANOVA), followed by Tukey's post hoc test or pair-wise comparisons, where appropriate. The statistical model included the time, treatment effects and their interaction. When the interaction was significant, Fisher's Least Significant Difference test (Fisher's LSD) was applied post hoc to determine significant differences among the means. Statistical analysis of the applied SE P3/Tr2a strain in fecal, cecal and appendix samples was performed using unpaired Student *t*-test. All statistical analyses were performed by the GraphPad Prism statistical software (GraphPad Prism version 9.3.1., GraphPad Software, San Diego, CA, USA). Differences between the mean values of the different dietary treatments were considered statistically significant at *p* < 0.05. Data are expressed as means and standard deviations of the mean (SD).

#### **3. Results**

#### *3.1. Growth Performance of Rabbits*

The animals were in good health throughout the experiment. Mortality was noted in groups S, E + S, and C (Table 2). Higher BW and ADWG were recorded in all experimental groups during additives' application compared to the control data (E: by 20.5%; S: by 17.1%; E + S: by 25.6%; Table 2). The highest BW and ADWG (E; *p* < 0.0001) and the lowest FCR (E: by 10.2%) were noted during Ent A/P application. Lower FCR (by 1.5%) was also noted in the E + S group, compared to C.

**Table 2.** Growth performance, phagocytic activity, and secretory IgA of rabbits.

E–Ent A/P application between 0–14 days; S–SE P3/Tr2a application between 14–21 days; E + S–Ent A/P preventive application for 2 weeks (between 0–14 days) before SE P3Tr2a strain addition for 1 week (between 14–21 days); C–control group (without additives); Data are expressed as means and standard deviations (SD). a, b, c, d Means within lines with different superscript letters are significantly different (*p* < 0.05) using Bonferroni post hoc test; A, B, C, D Means within lines with different superscript letters are significantly different (*p* < 0.05) using Fisher's LSD post hoc test. The bold letters mean significant changes.

#### *3.2. Immunoglobulin A in Small Intestinal Wall and Phagocytic Activity in Blood*

The time and treatment effect was noted on the PA in this study (*p* < 0.0001; Table 2). Ent A/P addition increased the PA values compared to C (day 14; E, E + S vs. C: *p* < 0.001); higher PA was noted one week after its withdrawal only in group E (day 21; E vs. E + S, C: *p* < 0.001). The SE P3/Tr2a strain application to rabbits decreased PA in the E + S group after 2 weeks of Ent A/P addition (day 21; E + S vs. E, S and C: *p* < 0.001), but, on the contrary, the PA value was elevated in group S, without Ent A/P preventive application (S vs. E, E + S, C: *p* < 0.001). The interaction effect was noted at day 14 (E vs. S, C: *p* < 0.0001; E + S vs. E, C: *p* < 0.0001), and at day 2 (E vs. S, E + S, C: *p* < 0.0001; S vs. E + S, C: *p* < 0.0001; E + S vs. C: *p* < 0.01).

IgA levels were similar among the experimental groups, except the E + S group, showing the highest IgA value (*p* = 0.0220; Table 2).

#### *3.3. Microbial Population*

In feces, most bacteria were influenced by time, treatment, and their interaction (except amylolytic streptococci; Table 3). At day 14, lower counts of all tested bacterial groups, except MRS, were recorded compared to the initial data (day 0/1) and the highest count of coliforms, amylolytic streptococci, staphylococci, MRS, and total bacteria was noted in the S group on this day. At the end of SE P3/Tr2a strain application in the E + S group (day 21), a significant reduction of enterococci, coliforms, MRS (E + S vs. E, S, C: *p* < 0.001), amylolytic streptococci, and total bacterial count (E + S vs. E, S: *p* < 0.001) was detected. The time and treatment interaction was noted on coliforms already at day 14 (S vs. E: *p* < 0.0001; S vs. E + S: *p* < 0.001; S vs. C: *p* < 0.01). At the end of the experiment, enterococci (E + S vs. E, C: *p*< 0.0001; E + S vs. S: *p* < 0.001), staphylococci, and total bacteria (E vs. E + S: *p* < 0.05; S vs. E + S: *p* < 0.001; S vs. C: *p* < 0.01), MRS (E vs. S: *p* < 0.01; E + S vs. E, C: *p* < 0.0001; E + S vs. S: *p* < 0.001), and coliforms (E vs. E + S: *p* < 0.05; S vs. E + S: *p* < 0.01) were influenced by the time and treatment interaction.

#### **Table 3.** Bacterial counts (log 10 CFU/g ± SD) in feces of rabbits.

E–Ent A/P application between 0–14 days; S–SE P3/Tr2a application between 14–21 days; E + S–Ent A/P preventive application for 2 weeks (between 0–14 days) before SE P3Tr2a strain addition for 1 week (between 14–21 days); C–control group (without additives); Data are expressed as means and standard deviations (SD). a, b, c, d Means within lines with different superscript letters are significantly different (*p* < 0.05) using Bonferroni post hoc test; A, B, C Means within lines with different superscript letters are significantly different (*p* < 0.05) using Fisher's LSD post hoc test. The bold letters mean significant changes.

The SE P3/Tr2a strain was able to colonize the digestive tract of rabbits after its oneweek application, reaching counts in the range 1.20–1.66 log cycle in the feces (Table 3) and also in the range 0.90–1.00 log cycle in the cecum and appendix (Table 4).

**Table 4.** Bacterial counts (log 10 CFU/g ± SD) in cecum and appendix of rabbits.

E–Ent A/P application between 0–14 days; S–SE P3/Tr2a application between 14–21 days; E + S–Ent A/P preventive application for 2 weeks (between 0–14 days) before SE P3Tr2a strain addition for 1 week (between 14–21 days); C–control group (without additives); Data are expressed as means and standard deviations (SD). a, b Means within lines with different superscript letters are significantly different (*p* < 0.05) using Tukey's post hoc test. The bold letters mean significant changes.

Most of the tested bacteria were enumerated in lower counts in the cecum and appendix (except cecal MRS), than in fecal samples. In the cecum, counts of enterococci and coliforms were reduced significantly (*p* < 0.001, Table 4) compared with C, while MRS and total bacteria were increased in the E + S group (E + S vs. E, C: *p* < 0.001).

Fecal samples in all groups (experimental and control) were *Eimeria* spp. oocytes absent.

#### **4. Discussion**

Good health status, higher BW, and ADWG of rabbits recorded in all experimental groups during additives' application indicated the beneficial effect of Ent A/P on the one hand, and on the other hand no negative impact of the SE P3/Tr2a strain on animals' growth was noted. However, reduced FCR in the E and E + S groups reconfirms the positive influence of Ent A/P on rabbits' growth performance, which can be explained by better feed intake and consumption; this finding is important from the economic point of view (less feed for faster growth). Faster growth and higher weight gain in rabbits were described in many studies during probiotics and their metabolites' application [27–30]. The beneficial effect of bacteriocins on the ADWG and FCR in rabbit experiments was repeatedly confirmed in this study, according to previous results [30–33]. The preventive effect of Ent A/P before SE P3Tr2a strain application to rabbits is reflected in the ADWG elevation and the FCR reduction compared to data from animals receiving only the SE P3/Tr2a strain, without enterocin treatment.

In addition to the positive impact of beneficial strains and/or their bacteriocins on animals' growth, their immunostimulatory effect is also important. These bioactive compounds can enhance immunity in several ways, including intestinal microbiota balance and the ability to modulate the host's innate and specific immune response. In general, stimulation of non-specific immunity is demonstrated by increased phagocytosis and modified cytokine production, while elevated immunoglobulins reflect the specific means of stimulation [34]. However, whilst several works have been published on the immune indices in healthy rabbits, existing knowledge on probiotics and bacteriocins' effect on phagocytic activity (PA) is still limited and needs to be expanded [35–37]. A significant increase of PA after 14 days of Ent A/P administration showed the non-specific immunity enhancement. Higher PA values noted one week after its cessation repeatedly confirmed the prolonged immuno-stimulative effect of Ent A/P, as it was previously described during several bacteriocins/enterocins treatment in rabbits [30,38]. The long term immuno-moderating effect of

enterocins can be explained either by the maintenance of intestinal health, microbiota, and immunity via supporting the gut-associated lymphoid tissue (GALT), or by the adopting of animals on them. This finding also confirms the use of bacteriocins with a preventive effect in animals, compared to beneficial bacteriocin-producing strains, possessing immunoactivity already during their application and demonstrating more of the therapeutic effect of probiotics. The protective effect of Ent A/P was noted in the E + S group, as no increase in PA was noted after the SE P3/Tr2a strain application, while the strain itself without Ent A/P stimulated the rabbit immune system.

Several authors presented improved cell-mediated immunity [37] and an increased number of mast cells in the cecum, as well as higher IgM and IgG levels in serum [39] after probiotic administration to rabbits. It is well known that natural feed additives can stimulate immunity via enhancing rabbits' gut health. The predominant isotype of the mucosal immune system is the secretory IgA (sIgA), which can keep the mucosal immunity balance between commensals' microbiota and pathogens' defenses on the mucosal surface [40]. SIgA is synthesized by plasma cells in the lamina propria and translocated through intestinal epithelial cells, serving as the first line of defense to protect the intestinal epithelium from enteric toxins and pathogenic microorganisms by immune exclusion. Liu et al. [41] presented maintained intestinal barrier function and increased sIgA production after *Clostridium butyricum* probiotic administration to weaning rabbits, similarly to Plachá et al. [42] who detected a higher amount of IgA in the intestinal wall after thymol application to rabbits. In contrast to Chen et al. [43], who reported increased bile IgA in bacteriocin-treated broiler chicks, our results showed only slight (not significant) sIgA secretion after Ent A/P and SE P3/Tr2a strain application separately. On the other hand, SE P3/Tr2a application after preventive administration of Ent A/P significantly elevated the sIgA concentration. From this result, we assume that the higher amount of IgA detected in the intestinal wall is the result of the primary immuno-stimulation that is evoked by Ent A/P, and the secondary induced by methicillin-resistant SE P3/Tr2a strain as a possible agent. Given these initial results regarding the enterocin-protective effect, further studies on IgA secretion and mucosal immunity in rabbits are needed.

In addition to healthy intestinal immunity, a stable gut microbiome also contributes to the formation of animals' gut health. This microbial and immunological stability is often negatively influenced by several exogenous factors (stress, dietary and climate changes, etc.), but also can be improved mostly by natural feed additives, via strengthening the intestinal barriers, optimizing microbial balance, and supporting enzymatic activities and nutrient absorption in the gut. The in vivo antimicrobial effect of bacteriocins/enterocins in rabbits' gastrointestinal tract has been presented in several works [29,30,44–46], when mostly the counts of enterococci, clostridia, coliforms, and staphylococci were reduced. In rabbits, colibacillosis and clostridiosis are the main infections, particularly around the weaning period, when the animals are the most susceptible to these bacteria. Staphylococci as a part of the skin and mucosal barriers, and digestive tract as well, are also often detected as the main causative agents of skin lesions and abscesses and/or as associated pathogens in other multifactorial/multiorgan infections. Several works present the anti-staphylococcal activity of bacteriocins, including also MRS isolates [7,15,17]. While enterococci, coliforms and total bacteria were reduced at the end of Ent A/P application (14 days of the trial) compared to their initial data (day 0/1), balanced counts of tested bacteria within the experimental and control groups indicated no significant antibacterial activity of Ent A/P, including its anti-staphylococcal activity. These findings are contradictory to previously observed results during in vitro and in vivo enterocins' administration [15,30–33,45]. At day 21, at the end of SE P3/Tr2a strain application to rabbits, enterococci and coliforms significantly decreased in the E + S group, while staphylococci were noted in the highest counts; this fact may also indicate a possible competitive effect between the microbiota established in the gut and the applied SE P3/Tr2a strain. For this reason, we also expected a multiplication of the MRS bacteria, but, surprisingly, the lowest MRS counts were noted in the E + S group. On the other hand, this significant MRS reduction in the E + S group

compared to negative (group C) and positive (group S) control at day 21 (after one week SE P3/Tr2a strain application) indicated the anti-MRS effect of Ent A/P, previously confirmed under enterocins in vitro testing against methicillin-resistant staphylococcal isolates [17]. The inhibitory effect on enterococci and coliforms was repeatedly confirmed in cecal samples of rabbits, similar to previous studies [30–33,45]. A significant increase of MRS and the total bacteria in the cecum, and their higher counts compared to fecal samples at day 21, can be explained as result of re-colonization with SE P3/Tr2a strain due to cecotrophy in the rabbits. To expand these results and already known facts concerning rabbits' health, immunity, and microbial balance by additives' supplementation is necessary through further investigations.

Coccidiosis is the most frequent parasitic disease in rabbit farms, affecting all ages, especially young, weaned rabbits, and causing high morbidity and mortality rates [46]. The oocysts are always present in the intestines of rabbits, and they cannot be eliminated even by the use of coccidiostat. Because the EU has banned antibiotics as feed additives and growth promoters, they are replaced with alternative anticoccidials, including prebiotics and probiotics, based on their bactericidal and/or bacteriostatic activities. Although *Eimeria* spp. oocytes were not detected in the feces of rabbits, the anticoccidial effect of Ent A/P can be expected, similarly to the anticoccidial effect of Ent 7420 noted in rabbits [30]; however, further studies are needed.

#### **5. Conclusions**

Good health and increased weight gain reflect the beneficial effects of Ent A/P on the growth performance of rabbits. These results also showed that the methicillin-resistant S. epidermidis SE P3/Tr2a strain did not have any pathogenic effect on rabbits' health status. The preventive effect of Ent A/P was recorded due to improved zootechnical parameters (elevated ADWG, reduced FCR), stimulated non-specific immunity (higher PA), and stabilized intestinal microbial environment (stable sIgA, reduced counts of coliforms, staphylococci, amylolytic streptococci, and total bacterial count as well) of rabbits. Reduced MRS and total bacteria after one week of SE P3/Tr2a strain application also point to the antibacterial effect of dipeptide Ent A/P and its anti-MRS potential. This study has an impact on basic research, confirming the in vivo anti-MRS activity of enterocin and also helping to spread knowledge regarding the interaction between intestinal microbiota and immunity.

**Author Contributions:** Conceptualization, M.P.S. and A.L.; methodology, L'.C., J.Š., V.F., I.P., Z.F. and M.C.; validation, M.P.S.; investigation, M.P.S., L'.C. and A.L.; resources, M.P.S.; data curation, L'.C. and M.P.S.; writing—original draft preparation, M.P.S.; writing—review and editing, M.P.S.; visualization, M.P.S.; supervision, M.P.S. and A.L.; project administration, M.P.S.; funding acquisition, M.P.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Scientific Grant Agency of the Ministry for Education, Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences VEGA, grant number 2/0005/21.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the State Veterinary and Food Administration of the Slovak Republic on 1 December 2016 (approval numbers SK CH 17016 and SK U 18016).

**Data Availability Statement:** Data available upon reasonable request to the corresponding author.

**Acknowledgments:** Part of the preliminary results regarding the growth performance was presented in the Slovak Journal of Animal Science, 2021, 54(2), pp. 57–65. We are grateful to G. Štrkolcová for *Eimeria* spp. oocysts analysis, to D. Melišová and P. Jerga for their skillful technical assistance and to J. Pecho for slaughtering. We are grateful for English language editing.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


*3.3.2 Bacterial pneumonitis*

*Special Considerations in Human Airway Management*

**3.2 Pathophysiology of pulmonary aspiration**

**3.3 Types of aspiration-related pulmonary complications**

*Summary of pathophysiology of pulmonary aspiration, modified from Dal Santo et al. [8].*

1.Aspiration pneumonitis or chemical pneumonitis

*3.3.1 Aspiration pneumonitis or chemical pneumonitis*

Chemical pneumonitis, the most common type of pulmonary aspiration, was first described by Curtis Lester Mendelson in 1946 as inflammation of the lung parenchyma resulting from aspiration of sterile gastric contents. The morbidity and mortality associated with aspiration pneumonitis can be attributed to the acidity and volume of the aspirate. If the pH of the aspirate is less than 2.5 and the volume is >0.3 ml/kg (20–25 ml in adults), it can lead to fatal

The acidity of the aspirate can cause chemical injury to the tracheobronchial tree and the lung parenchyma which can trigger a series of immune responses. The direct corrosive effect of gastric acid on the alveolar-capillary epithelium peaks in 1–2 h. This is followed by an inflammatory response which peaks in 4–6 h which involves neutrophilic invasion of the alveoli and lung parenchyma. This phase is also characterized by the involvement of a spectrum of inflammatory mediators, inflammatory cells, adhesion molecules, and enzymes, including tumor necrosis factor α, interleukin-8, cyclooxygenase and lipoxygenase products, and reactive

2.Bacterial pneumonia

**Figure 1.**

pneumonitis [9].

3.Particle-associated aspiration

**106**

A contaminated aspirate can lead to bacterial infection of the lung parenchyma, with a course of complete recovery or progress to lung abscess formation, exogenous lipoid pneumonia or chronic interstitial fibrosis. The most common pathogens are *Staphylococcus aureus*, *Pseudomonas aeruginosa*, Enterobacter species, Klebsiella species and *Escherichia coli*. The major anaerobes that have been isolated from pulmonary infections include Pepto streptococcus, Fusobacterium nucleatum, Fusobacterium necrophorum, Prevotella, Bacteroides melaninogenicus [11].


**5. Virginia tech shootings**

Cho Seung-Hui the mass killer at Virginia Tech also had a diagnosis of Autism. He was a shy, withdrawn child who had problems with social know how and social cop on. He hated hugging as a child and was very violent. He was clingy to his mother. He showed poor communication and often gave one word answers to questions. He great aunt [2] stated that "when others called his name he just answered yes or no but never showed any feelings or emotions. We started to worry he was autistic". Later he was formally diagnosed with Autism. He was bullied within the educational system.

A Professor Lucinda Roy complained to the campus police about him and she also gave him "individual lessons" and said to him: "You seem so lonely"-"Do you have any friends?". "I am lonely". Cho replied. "I don't have any friends". A student at Virginia Tech Karen Grewal [17] noted that "he was so quiet, it was almost as if he wasn't there and was invisible. He must have been worried that he'd would be found out".

[3] noted that there were "college girls who reported him to the police for stalking and got him carted off to a mental hospital after he sent them shy love messages full of mean‐ ing". Cho [3] also wrote "by a name, I know not how to tell who I am". This is typical of the identity diffusion of autism. He was unable to get a girlfriend and "he had to make up with a fantasy girlfriend". [3]. He was a loner as a child. [2] notes that a fellow student noted he was "obsessed with violence and with serious personal problems". He admired the Columbine killers. Professor Nikki Giovanni one of his teachers at Virginia Tech was concerned about his writing i.e. "your bra is torn and I am looking at your flesh". [3]. He'd never speak but he frightened everyone. According to [17] "he insisted on wearing sunglasses and pulling his baseball cap low on his forehead" and that neighbours "descri‐ bed him as a surly youth who did not communicate and ignored them in the street". He was a major mathematical talent like many with Autism. [9]. Professor Louis Schlesging‐ er a Professor of Forensic Psychology states that "mass killers tend to be aggrieved, hurt, clinically depressed, socially isolated and above all paranoid". (Begley 2007).

In the psychiatric hospital in 2007 a psychiatrist noted his "affect is flat and mood is depressed". (Begley 2007).