HAEMATOLOGICAL ALTERATIONS IN CALVES WITH ACUTE RESPIRATORY DISTRESS SYNDROME DUE TO ASPIRATION PNEUMONIA: A PROSPECTIVE STUDY

Th e aim of this prospective study was to investigate the arterial blood gas (ABG) analysis, which is considered the gold standard, and complete blood count (CBC) as a complementary test in neonatal calves with acute respiratory distress syndrome (ARDS) due to aspiration pneumonia. Ten healthy and 20 calves with ARDS due to aspiration of milk/colostrum were enrolled in the study. Clinical examinations were performed at admission. ABG analyses were performed to determine the presence of ARDS and investigate the extent of lung ventilation/damage. CBC analysis was performed from venous blood samples. Heart and respiratory rates and body temperature values were higher in diseased calves than healthy ones ( p < 0.000). Arterial pH, partial pressure of oxygen in arterial blood (PaO 2 ) and saturation of oxygen in arterial blood (SaO 2 ) were lower ( p < 0.001) in the diseased calves, while partial pressure of carbon dioxide in arterial blood (PaCO 2 ) and lactate levels were higher ( p < 0.000) than those in healthy ones. Also, leukocyte (WBC), lymphocyte, monocyte, granulocyte, mean corpuscular volume (MCV), and mean corpuscular haemoglobin (MCH) levels were higher in the diseased calves than the healthy calves ( p < 0.032). As a result of the correlation analysis, the only correlation was determined to be between PaO 2 and WBC, which was moderately


INTRODUCTION
One of the most frequent causes of economic loss in livestock is respiratory disease, which has a multifactorial origin and leads to clinical and pathological abnormalities in the respiratory system. In addition to infectious causes, aspiration pneumonia (AP) is caused by the intake of foreign substances such as milk, colostrum, liquid supplements, or medications that damage the lung tissue as a result of inappropriate drenching methods/bottles or forced feeding (Hattab et al., 2022). Due to the irritating properties of the aspirated material, gangrenous bronchopneumonia may develop in cases where substantial amounts of fl uid have been aspirated, albeit death is nearly always immediate (Akyüz et al., 2022). Th e initiation of clinical symptoms is frequently subtle since clinical and pathological characteristics of aspiration pneumonia are highly variable (Lopez and Martinson, 2017;Hattab et al., 2022).
Lung endothelial and epithelial damage develops as a result of aspiration/inhalation of foreign matter. Aft er aspiration of liquid substances, acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) may develop in cases where gas exchange is impaired (Ider et al., 2022). Th e pulmonary alveolar and bronchial epithelium are also disrupted in cases of ARDS (Osaka et al., 2011) which is characterised by hypoxia (PaO 2 < 60 mmHg), respiratory acidosis, hypercapnia (PaCO 2 > 45 mmHg), tachypnea (respiratory rate>45/min), and abdominal respiration with wheezing (Bleul, 2009;Yıldız and Ok, 2017).
Complete blood count (CBC) and arterial and/or venous blood gas measurements are primary clinical diagnostic tools. Although the fi ndings have been reported to be non-specifi c, CBC analysis is recommended in cases of aspiration pneumonia (Kogan et al., 2008). Th e use of arterial blood samples has been suggested in the evaluation of lung ventilation (Bleul et al., 2007). However, the technical diffi culty of obtaining arterial blood samples under farm conditions in veterinary medicine causes the preference for venous blood samples (Nagy et al., 2002). Th erefore, the aim of this prospective study is to investigate arterial blood gas (ABG) analysis, which is considered the gold standard in assessing lung oxygenation and ventilation-perfusion, and venous CBC fi ndings as a complementary test and their correlation with each other in calves with ARDS due to aspiration pneumonia, and to acquire clinical information about the extent of the damage from venous blood samples in cases where arterial blood samples cannot be obtained.

MATERIAL AND METHODS
Th is study was approved by the decision of the Ethics Committee of the Faculty of Veterinary Medicine of Harran University, with session number 2021/004, dated 07.05.2021 and numbered 01-08. All institutional and national guidelines for the care and use of study animals were followed. All calf breeders gave their consent before the commencement of the study.

Animal Selection
Twenty neonatal calves of Holstein breed, whose developed non-specifi c respiratory disease symptoms such as cough, nasal and/or ocular discharge, tachypnea, and respiratory distress aft er being fed on milk/colostrum with inappropriate methods/bottles constituted the Diseased Group. Ten healthy neonatal calves of Holstein breed which were determined to be healthy in clinical and laboratory examinations (Dillane et al., 2018), constituted the Healthy Group. Calves from dystocia, prematurity, congenital abnormalities, and those with diarrhoea and infection suspicion were excluded from the study. In addition, the formation of aspiration pneumonia due to inhalation of diff erent liquid materials such as digestive stimulants or vitamin-mineral complex solutions was not included in the study as the alteration and severity of the investigated parameters may be aff ected by the character and acidity of the aspirated material. All calves included in the study were treated with appropriate treatment protocols (intravenous fl uid therapy, antimicrobial, anti-infl ammatory, mucolytic and intranasal oxygen administrations) following clinical examination and sampling. However, the follow-ups of the diseased calves could not be performed due to communication and/or economic issues.

Clinical examinations
Clinical examinations were performed both to detect the presence of pneumonia and to determine the present medical condition and included evaluation of hydration status, palpable lymph nodes and mucous membranes, measurements of heart and respiratory rate, and heart and lung auscultation. In addition, thoracic ultrasonography was performed to detect the presence of pneumonia in all the diseased calves. Using a 5 MHz micro-convex probe (Mindray Z60, China), the area of interest was wetted with 70% isopropyl alcohol without clipping the hair and scanned from the 10 th intercostal space towards the cranial. Clinical and ultrasonographic examinations were performed by the same veterinary staff applying the same examination protocols.

Blood Sampling
Venous blood samples (5 mL) were taken once from vena jugularis at the time of admission from all the calves included in the study. Th e time of blood sampling, and thus the fi rst admission to the hospital and the onset of the symptoms, was a median of 5 (1 -12) hours aft er inappropriate colostrum/ milk drenching. Arterial blood samples (1 mL) were taken by a puncture of arteria auricularis caudalis, as previously reported (Nagy et al., 2002). Aft er the sampling, it was ensured that there were no air bubbles and inadequate or excessive anticoagulant. Aft er the sampling, the specimen was analysed immediately. For CBC analysis, venous blood samples (using tubes with K 3 EDTA), and for ABG analysis, arterial blood samples were used to determine the presence of ARDS and investigate the lung ventilation/damage.

Arterial Blood Gas Analysis
Arterial blood pH, partial pressure of carbon dioxide in arterial blood (PaCO 2 ), partial pressure of oxygen in arterial blood (PaO 2 ), saturation of oxygen in arterial blood (SaO 2 ), and lactate measurements were performed using an automatic blood gas analyzer (epoc® Blood Analysis System, Siemens, Germany).

Criteria for defi nition of Acute Respiratory Distress Syndrome
Th e criteria for the presence of ARDS were hypoxia (PaO 2 < 60 mmHg), respiratory acidosis, hypercapnia (PaCO 2 > 45 mmHg), tachypnea (respiratory rate > 45/min), and abdominal respiration with wheezing (Bleul, 2009). Among these parameters, the presence of at least two criteria along with PaO 2 < 60 mmHg was taken into consideration.

Statistical Analysis
Statistical soft ware (SPSS 25.00, IBM®, USA) was used to evaluate the data. We used a one-sample Kolmogorov-Smirnov test to determine whether the variables had normal distributions. Non-parametric data were evaluated by Mann-Whitney U test, and presented as median (min, max). A Spearman's correlation was performed to determine the relationship between ABG and venous CBC results. Sizes of correlation were considered 0.5 -0.7 (-0.5 --0.7) moderately positive (negative), 0.7 -0.9 (-0.7 --0.9) highly positive (negative), and 0.9 -1 (-0.9 --1) very highly positive (negative). Statistical signifi cance was considered as p < 0.05 and p < 0.01.

Anamnestic Data
Anamnestic data revealed that all the calves included in the Diseased Group developed respiratory disease-related symptoms aft er forced feeding on milk/colostrum with inappropriate methods/bottles. Th e samples were taken during the calving season, between June and August. Also, all the calves of the present study, which were housed in individual pens, were naturally born (> 280 days gestation) on the farm and were able to stand within 1 hour of life. Th e body weights of the calves in the groups were similar ( Table 1). All the calves were given 2 litres of milk/colostrum within the fi rst 6 hours of their life. Th e time of admission for all calves to the hospital was the day they were born. Considering the fi rst 6 hours of colostrum administration, the mean time to hospital admission was 11 (7 -20) hours aft er delivery. Th e complete history of each diseased calf is visualized (Figure 1.) as the symptom onset is sudden in cases of aspiration pneumonia.  Table 1. Th e heart and respiratory rates and body temperature of the calves with aspiration pneumonia were signifi cantly higher than the healthy calves (p < 0.000).

Arterial Blood Gas Analysis
Th e results of ABG analysis fi ndings of the healthy calves and the calves with aspiration pneumonia are presented in Table 2. Statistically signifi cant diff erences were observed in pH, PaCO 2 , PaO 2 , SaO 2 , and lactate levels. While the pH, PaO 2 , and SaO 2 levels of the calves with aspiration pneumonia were lower than those in the healthy calves (p < 0.001), PaCO 2 and lactate levels were signifi cantly higher (p < 0.000).

Complete Blood Count Analysis
Th e results of the CBC analysis of the healthy calves and the calves with aspiration pneumonia are presented in Table 3. As a result of CBC analysis, it was determined that WBC, lymphocyte, monocyte, granulocyte, MCV, and MCH levels of the calves with aspiration pneumonia were higher than that of the healthy ones (p < 0.000, p < 0.012, p < 0.028, p < 0.000, p < 0.032 and p < 0.025, respectively).

Correlation Between ABG and CBC Findings
The results of Spearman's correlation test are presented in Table 4. The only correlation detected between ABG and CBC variables was between WBC and PaO 2 , which was moderately negative (r = -0.567).

DISCUSSION
Respiratory diseases, which are considered as one of the most critical health problems of the livestock industry, are very signifi cant due to their economic impact, and the foremost cause is pneumonia. Cattle can acquire haematogenous pneumonias; however, most cases are bronchogenic or of inhalation/ aspiration origin (Gülersoy and Şen, 2017). Th e most common respiratory diseases in neonatal calves are reported as postnatal hypoxia-hypercapnia, pulmonary hypertension, aspiration pneumonia, and bacterial and viral pneumonias (Vallés, 2013).
It has been reported that ARDS and ALI may develop due to impaired alveolar gas exchange and hypoxia that develops following incidental aspiration in cases of aspiration pneumonia (Son et al., 2017). Clinically, calves with ARDS due to aspiration pneumonia are characterized by cough, fever, tachypnea, cyanosis, and abnormal lung sounds (Ider et al., 2022). In the present study, the respiratory rate, body temperature, and the heart rate of the calves with ARDS due to aspiration pneumonia were signifi cantly higher than the healthy calves (p < 0.000) ( Table 1). In addition, tachypnea, weakness, lethargy, mild to moderate cyanosis, tachycardia, and fever were present in all the calves with ARDS due to aspiration pneumonia. Th e clinical fi ndings of the diseased calves in this study were due to the combination of factors and septic nature of aspiration pneumonia (McGuirk and Simon, 2008), and the development of respiratory distress (Ider et al., 2022).
Abnormal blood gas alterations such as hypoxia, hypercapnia, and respiratory acidosis are common fi ndings in calves with respiratory distress (Bleul, 2009). In the present study, pH, PaO 2 , and SaO 2 levels of the calves with ARDS due to aspiration pneumonia were signifi cantly lower (p < 0.001), while PaCO 2 and lactate levels were higher (p < 0.000) than that of the healthy calves (Table  2). However, PaCO 2 levels of some of the diseased calves (6 out of 20) were determined to be less than 45 mmHg, and this fi nding was thought to be related to the volume of aspirated milk/colostrum (Marik, 2001). Th ese fi ndings indicate that signifi cant changes in ABG balance and elevated lactate levels in calves with ARDS were due to aspiration pneumonia associated with hypoxia, lung endothelial and epithelial damage, and impaired gas exchange (Yıldız and Ok, 2017; Ider et al., 2022).
Th e primary clinical diagnostic tools are CBC and arterial and/or venous blood gas measurements (Kogan et al., 2008). It was reported that respiratory diseases lead to direct lung function disturbances with alterations in gas exchange and changes in the haematological profi le (Šoltésová et al., 2015). Due to the septic and acute characteristics of aspiration pneumonia, it has been reported that severe infection may develop (Dhillon et al., 2020). In the present study, WBC, lymphocyte, monocyte, granulocyte, MCV, and MCH levels of the calves with ARDS due to aspiration pneumonia were signifi cantly higher than that of the healthy calves (p < 0.032) ( Table 3). Elevated WBC, lymphocyte, monocyte, and granulocyte levels are indicators of an infl ammatory process (Šoltésová et al., 2015). In the previous reports, diff erences in RBC count, haemoglobin and haematocrit levels were reported. Th ese fi ndings could be a consequence of the adaptation and compensatory processes refl ecting an existing pulmonary disease, hypoxaemia, and the stimulation of erythropoiesis (Hanzlicek et al., 2010;Fraser et al., 2014). In a study in rats, hypoxia was determined by fi ve indicators: WBC, granulocyte and RBC counts, reticulocyte count/percent, and MCH levels (Kondashevskaya et al., 2021). However, in the present study, RBC, haematocrit, MCHC and Hb levels of the diseased calves were not statistically diff erent from the healthy ones. Th ese fi ndings may be related to the fact that these compensation mechanisms have not been activated yet. An elevation in MCV level may result from reticulocytosis, which is quite uncommon in chronic respiratory diseases, but in acute ones (Tsantes et al., 2004). Th erefore, higher MCV and MCH levels of the diseased calves in this study may be associated with hypoxia tolerance and acuteness of the cases (Dzhalilova and Makarova, 2020).
Since blood serves as the primary channel for transferring CO 2 in the opposite direction and O 2 from the lungs to tissues, studies aimed at identifying the eff ects and/or refl ections of hypoxia mostly on blood (Kondashevskaya et al., 2021). Th at is why the use of arterial blood samples has been suggested in evaluating lung ventilation/damage (Bleul et al., 2007). During infectious or non-infectious conditions causing hypoxia, erythroid cells continue performing their specifi c functions, changing their numbers, size, or O 2 content. Th us, under hypoxic conditions, WBC counts in small laboratory rodents, mainly neutrophils, are reported to be elevated (Kondashevskaya et al., 2021). As a result of the correlation analysis, which is one of the main topics of the present study, a moderate negative correlation was determined between PaO 2 and WBC count (Table 4). Th is fi nding may demonstrate that leukocytosis developing due to aspiration pneumonia may have a possible negative contribution to the exacerbation of hypoxia and could worsen the clinical manifestation as a consequence of rapid oxygen consumption by the leukocytes (Chillar et al., 1980). Th erefore, since aspiration pneumonia in calves is an oft en-fatal emergency (Hattab et al., 2022), it may be considered to evaluate CBC as a complementary test along with ABG, at least for the initial assessment, and the prediction of the clinical outcome (McKeever et al., 2016).
Th e major limitation of the present study is the limited number of animals that may infl uence the signifi cance and correlations of some investigated blood parameters. Th e authors recommend evaluating the present results with a larger number of animals. In addition, the lack of histopathological examination indicating lung damage and loss to follow-ups due to social or structural reasons can be considered limitations. Although the results of the present study are promising, evaluation of ABG and CBC fi ndings along with serum biochemistry parameters may allow more accurate and faster assessment of emergency aspiration pneumonia cases based on a routine analysis.

CONCLUSION
Aspiration pneumonia, which is oft en caused by inappropriate drenching and/or forced feeding, is a frequent condition that causes hypoxia, lung endothelial and epithelial damage, ARDS, and even sudden death depending on the amount of aspirated liquid. It emerged that signifi cant changes occur in ABG and venous CBC fi ndings of neonatal calves with ARDS due to aspiration pneumonia. It was observed that leukocytosis that develops in respiratory diseases such as aspiration pneumonia which causes a severe infl ammatory process, could potentially exacerbate hypoxia. As a result, it was concluded that CBC analysis with ABG measurement fi ndings could provide valuable clinical data in calves with ARDS due to aspiration pneumonia.

Author's Contribution
EG and CB made contributions to conception, methodology, involved in data collection, formal analysis and draft ing the manuscript. İG and AŞ carried out the data collection and draft ing of the manuscript. All authors revised the manuscript critically and EG together with CB prepared the fi nal draft of the manuscript etc. All authors read and approved the fi nal manuscript.