Nebulization as complementary therapy for dogs with respiratory tract infections

Respiratory tract infections in dogs are usually contagious and are caused by bacterial and viral agents [1,2]. Infectious and confounding agents include mycoplasma, streptococci, staphylococci and viruses (canine parain luenza virus, canine herpesvirus, canine adenovirus type 2). Compromised immunity, infections with commensal pathogens, stress associated with sudden change of environment, parasitic and metabolic diseases, and nutritional de iciencies contribute to respiratory infections [3].


Introduction
Respiratory tract infections in dogs are usually contagious and are caused by bacterial and viral agents [1,2]. Infectious and confounding agents include mycoplasma, streptococci, staphylococci and viruses (canine parain luenza virus, canine herpesvirus, canine adenovirus type 2). Compromised immunity, infections with commensal pathogens, stress associated with sudden change of environment, parasitic and metabolic diseases, and nutritional de iciencies contribute to respiratory infections [3].
Respiratory infections provoke pathological mechanisms in lung tissue [4,5], including increased secretion of respiratory mucus and edema caused by in lammatory cell in iltration. These processes produce adverse symptoms such as cough (wet and dry, subject to the type of in lammation) and dyspnea caused by airway constriction, mucosal edema and increased secretion of respiratory mucus. The above can increase the thickness of the blood-air barrier in pulmonary alveoli (between capillaries and the alveolar lumen). According to Eleanor, [6], impaired gas exchange increases carbon dioxide concentration and decreases oxygen concentration in the Abstract Respiratory tract infections in dogs pose a signifi cant problem and often require prolonged treatment. The effectiveness of pharmacological therapy can be improved through the administration of nebulized compounds to liquidize mucus and promote its evacuation from the respiratory tract. The aim of this study was to evaluate the effectiveness of nebulized 0.9% NaCl in dogs with respiratory tract infections. Respiratory functions were assessed based on the results of arterial blood gas analyses, and the patients' clinical status was determined by evaluating the severity of symptoms on a point grading scale. Inhalation of nebulized 0.9% NaCl signifi cantly accelerated improvement in the patents' clinical status (normalization of body temperature, decreased cough frequency, decrease/elimination of nasal and ocular discharge, improved appetite) and improved pulmonary gas exchange by reducing partial pressure and total content of carbon dioxide and increasing partial pressure and total content of oxygen in blood.
body, which can disrupt metabolic processes and decrease the ef icacy of treatment [7]. Kiers, et al. [8] demonstrated that hypoxia also affects the immune system, which can undermine positive treatment outcomes. There are various approaches to improving gas exchange. Steroidal anti-in lammatory drugs are effective, but they deliver immunosuppressive effects that have to be managed with antibacterial drugs, and they disrupt vaccination schedules in young animals. Mucolytic drugs and expectorants cause mucus thinning and increase the output of bronchial secretions. They facilitate the secretion of the luid fraction of bronchial mucus and water, and they increase mucus volume, which stimulates the response of ciliated epithelium and promotes the evacuation of secretions from the bronchi. However, excessive amounts of mucus can partially " lood" the lungs and compromise gas exchange. Acetylcysteine is one of the few mucolytic drugs that do not increase mucus volume, but only decrease its viscosity [9]. The drug breaks down disul ide bridges in mucopolysaccharides, reduces mucus viscosity and facilitates its evacuation. The administration of nebulized sodium chloride solution at physiological or higher concentrations does not involve the use of drugs with potential adverse side effects, such as digestive disturbances, that are not always well tolerated by the patient and are not approved by the owners [10][11][12]. Inhalation of saline aerosol combined with physical therapy (percussion, encouraging physical exercise) can speed up therapy and reduce pharmacological interventions [13,14]. Sudo, et al. [2] reported a decrease in the viscosity of bronchial mucus in dogs administered nebulized dextran-Ringer solution. According to Fernandes and Vanbever [15], and Chow, et al. [16], nebulization is a promising non-invasive method of delivering pharmacological treatments with general systemic effects. In "human medicine" it was used to treat not only pulmonary diseases [17][18][19][20] but in pain or neoplasm therapy too [1,21].

Objective of the study
The objective of this study was to evaluate the ef icacy of nebulized sodium chloride solution in dogs with mixed respiratory tract infections as complementary therapy that speeds up convalescence.

Animals
The experiment was performed on dogs of various breeds, both sexes (21 females and 25 males), aged 3 months to 9 years, with body weight of 4 to 35 kg, kept under similar conditions (indoor pets), which were patients of the Department of Internal Diseases at the Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn. All of the dogs were treated according to Good Clinical Practice rules and regulations of the local ethical committee of animal welfare.

Inclusion criteria
The inclusion criteria were symptoms of respiratory tract infection: cough (minimum 4 episodes per day), body temperature higher than 39 °C, nasal and/or ocular discharge, dyspnea, decreased appetite, and presence of pathogenic microorganisms in throat and tracheal aspirate cultures (determined by PCR).

Laboratory tests (genetic and blood gas analysis)
Viral DNA or RNA and bacterial DNA were isolated with the QIAamp cador Pathogen Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The genetic material of the canine parain luenza virus (CPiV) was detected with the Transcriptor One-Step RT-PCR Kit (Roche Diagnostics, Meylan, France) in the Mastercycler nexus gradient thermal cycler (Eppendorf, Hamburg, Germany). The genetic material of the canine herpesvirus (CHV), canine adenovirus type 2 (CAV2) and Bordetella bronchiseptica was analyzed with the HotStarTaq plus Master Mix Kit (Qiagen, Hilden, Germany) in the Mastercycler nexus gradient thermal cycler (Eppendorf, Hamburg, Germany). Before the collection of respiratory specimens, arterial blood was sampled from the femoral vein into gas-tight glass capillary tubes coated with heparin for blood gas analysis with the use of the Rapidlab 348 analyzer, including body temperature, blood pH, partial pressure of carbon dioxide (pCO 2 ), partial pressure of oxygen (pO 2 ), concentration of bicarbonate ions (HCO 3 -), base excess/de icit (BE), oxygen saturation (O 2 SAT) and total carbon dioxide content (ctCO 2 ) in blood on the irst day of the experiment and on experimental days 7 and 15.

Patient's treatment
All patients were administered amoxicillin with clavulanic acid (subcutaneous dose of 7.0 mg of amoxicillin and 1.75 mg of clavulanic acid per 20 kg BW, once daily for 5 days), metamizole (intramuscular injection of 35 mg/kg BW, administered once daily until fever reduction) and vitamin C (oral dose of 50 mg/kg BW, twice a day for 5 days). Chest percussion was performed twice daily to facilitate mucus evacuation. In randomly selected dogs (every other case) that constituted group II, nebulized 0.9% NaCl solution was administered with the use of the Tajfun2 MU2 ultrasonic nebulizer. Partly sealed transport cages of the appropriate size were used as nebulization chambers. Nebulization therapy was administered twice a day for 14 days. Every treatment lasted minimum 20-40 minutes, and 50-150 ml of 0.9% NaCl was nebulized, subject to the animal's size. Group I animals did not receive inhalation therapy.

Score health evaluation
The animals' clinical status was evaluated (on the irst day of the experiment and on days 3, 7, 10 and 15) based on body temperature, frequency and severity of cough, nasal/ocular discharge and appetite with the use of a point grading scale: body temperature up to 38.6 °C -0 points, 38.7-38.9 °C -1 point, 39.0-39.3 °C -2 points, above 39.4°C -3 points; cough episode -none -0 points, 1-2 episodes per day -1 point, 3-4 episodes per day -2 points, 5 and more episodes per day -3 points; cough accompanied by a gag re lex -additional 1 point; nasal/ocular discharge -none -0 points, clear, serous nasal discharge -1 point, clear, serous nasal and ocular discharge -2 points, purulent nasal discharge -3 points, purulent nasal and ocular discharge -4 points; appetite -normal -3 points, decreased -2 point, decreased by 50% from baseline -1 points, loss of appetite or very weak appetite -0 points. The results were analyzed statistically by Student's t-test.

Results
Symptoms of respiratory tract infection were observed in Group I and Group II dogs on the irst day of the experiment. According to the information provided by the owners, infection symptoms had persisted for more than 6 days.

Pathogens
The genetic material of pathogens responsible for respiratory tract infections was detected in throat and tracheal aspirate swabs. Mixed viral and bacterial infections were identi ied in most patients (Table 1). Infections with exclusively viral etiology were diagnosed in 2 dogs, and infections caused only by bacteria were noted in 3 animals.

Clinical symptoms -body temperature
The severity of clinical symptoms in the evaluated patients is presented in Graph 1 and

Clinical symptoms -cough
On day 1, more than 4 cough episodes per day with an accompanying gag re lex were observed in some dogs, and the average point score for this parameter was 3.894 points in Group I and 3.829 points in Group II. Cough severity decreased on successive days of the experiment and scored 3.725 points in Group I and 3.544 points in Group II on day 3. Cough severity and the gag re lex were signi icantly alleviated in Group II patients on days 7, 10 and 15 relative to Group I dogs, and the average score was 2.788 points in Group I and 2.106 points in Group II on day 7, 2.274 points in Group I and 0.857 points in Group II on day 10, and 1.208 points in Group I and 0.211 points in Group II on day 15.

Clinical symptoms -nasal/ocular discharge
On day 1, the average score for purulent nasal discharge and, in most cases, purulent ocular discharge was 3.657 points in Group 1 dogs and 3.709 points in Group II animals. On day Mixed viral and bacterial infections were identi ied in 18 dogs from Group I and in 17 animals from Group II. Infections caused by two or three of the identi ied viruses were diagnosed in the remaining animals. The canine parain luenza virus was detected in 23 dogs (11 dogs from Group I and 12 dogs from Group II), the canine herpesvirus -in 35 dogs (10 and 15, respectively) and the canine adenovirus -in 4 dogs (2 and 2, respectively). Bordetella bronchiseptica infections were observed in 38 animals.

Clinical symptoms -appetite
On day 1, the average score for appetite was 0,281 points in Group I dogs and 0,236 points in Group II animals. On day 3, appetite levels were similar in both groups at 0,569 points in Group I and 0,593 points in Group II. Signi icant differences in appetite were observed between groups beginning on day 7 when the average score was 0,851 points in Group I, but 1.492 points in Group II. The most signi icant differences between groups were noted on day 10 at 1.338 points in Group I and 2,334 points in Group II. On day 15, the differences between groups were signi icant, at 2,385 points in Group I and 3,847 points in Group II.

Blood gas parameters
The results of arterial blood gas analyses are presented in Table 3. On day 1, the measured parameters were similar and outside the normal range in both groups. Average blood pH was determined at 7.302 in Group I and 7.311 in Group II. On days 7 and 15, blood pH was closer to the normal range with an average of 7.334 in Group I and 7.358 in Group II on day 7, and 7.352 and 7.361, respectively, on day 15. On day 1, partial pressure of carbon dioxide (pCO 2 ) was determined at 54.784 mmHg in Group I and 53.284 mmHg in Group II (signi icant difference relative to successive measurements). On day 7, pCO 2 differed signi icantly between groups at 51.018 mmHg in Group I and 41.108 mmHg in Group II. On day 15, pCO 2 in Group I was signi icantly lowest relative to the previous measurements at 42.364 mmHg. In Group II, the value of the above parameter reached 39.819 mmHg on day 15.

Discussion
The severity of symptoms associated with respiratory tract infection, including elevated body temperature, frequent cough with an accompanying gag re lex, serous or serous-purulent nasal and ocular discharge, decrease or loss of appetite, was similar in all dogs at the beginning of the experiment. The above symptoms were caused by mixed infections with CPiV, CHV, CaV2 and Bordetella bronchiseptica, which were also identi ied as causative agents of canine respiratory tract infections in a study conducted by Schulz, et al. [22] in southern Germany.
Wet cough and abundant respiratory discharge point to the accumulation of respiratory secretions which, when accompanied by mucosal edema and in lammatory cell in iltration, lead to airway constriction and thickening of the blood-air barrier in capillaries that surround pulmonary alveoli. According to Eleanor, [6], Orellana, et al. [4] and Rahman and Mac Nee, [5], the above decreases the amount of air entering the lungs and inhibits gas exchange in pulmonary alveoli. In our study, higher carbon dioxide levels and oxygen de iciency contributed to respiratory acidosis which was further stimulated by intense metabolic activity during fever. Acidosis and hypoxia intensify the depressive effect of fever and together with respiratory obstruction, they contribute to a decrease or complete loss of appetite, general fatigue and apathy. Such symptoms were observed in all dogs on the irst day of the experiment. They were also reported by the owners. These processes disrupt metabolic activity [23] and if persistent, they can lead to emaciation and negative treatment outcomes [7]. Similar results were reported by Nagy, et al. [24], in cattle with bronchopneumonia.
Conventional treatments for respiratory tract infections rely on drugs with anti-in lammatory, anti-infective and immuno stimulating properties which do not directly resolve gas exchange abnormalities. Lung ventilation can be improved through nebulization which promotes the liquidation of mucus and its evacuation from the bronchial tree. In this study, nebulization therapy was applied in Group II, where it reduced the severity of respiratory infection symptoms and improved the overall condition of all animals. Beginning on day 7, a signi icant improvement in the following clinical parameters was observed in Group II dogs relative to Group I animals: restoration of normal body temperature, lower cough frequency, decrease or elimination of nasal and/or ocular discharge, improved appetite, and improvement in the patients' overall condition. The above changes were mirrored by the results of arterial blood gas tests. A greater decrease in the partial pressure of carbon dioxide and total content of carbon dioxide, and a greater increase in the partial pressure of oxygen and oxygen saturation were observed in dogs receiving nebulization treatment relative to Group I dogs. Normal BE values were restored more rapidly in Group II patients. These changes testify to improved gas exchange [25], which promotes the liquidation of mucus and its evacuation from the respiratory tract. The bene icial effects of nebulization treatment in human patients were described by Bennett, et al. [13] and Main, et al. [14] who combined nebulization with physical therapy to facilitate mucus clearance from the bronchi. In our experiment, percussion was performed to loosen and evacuate mucus. Adverse responses to nebulization were not observed during or after treatment. Percussion intensi ied mucus clearance in Group II dogs, and it improved the quality of breathing and the patients' overall condition.
Our research were carry out on dogs of various breeds, including brachycephalic (2 mix breed and 3 pugs), but we don't estimate important differences between this and others dogs. According to Aroma, et al. Darcy,et al. Davs,et al. Fawcett, brachycephalic dogs suffer for BOAS, pneumonia, soft palatine diseases, thermoregulation problems etc, and that should be aim of the future investigation.
The objective of this study was to evaluate the ef icacy of nebulized only sodium chloride solution in dogs. In animal clinic treatment [31,32] and human medicine [1,17,[19][20][21] there are used many different pharmacological substances, for example steroids, antibiotics, mucolytic etc. Using this kind of drugs by nebulization should be potential future directions of investigations.

Conclusion
A comparison of the patients' clinical status and the results of arterial blood gas tests in both groups indicates that the administration of nebulized 0.9% NaCl speeds up convalescence and improves the wellbeing of animals undergoing treatment for respiratory tract infections. Our indings also indicate that arterial blood gas tests are highly useful for monitoring the condition of the canine respiratory system.