Evaluation of an air ionizer unit and its effects on cellular respiratory defense in racing thoroughbreds

Reviewed

EVALUATIONOF AN AIR IONIZER UNITAND ITS EFFECTSON CELLULAR RESPIRATORYDEFENSEIN RACINGTHOROUGHBREDS Ursula Fogarty MVB, Phd1, MRCVS; Thomas Buckley MSc, FIMLS1 and John A. Scott, MSc, Phd, CPhys2

SUMMARY A double crossover trial, consisting of two three-week periods, was conducted to evaluate an air ionizer unit and its effects on cellular respiratory defenses in racing Thoroughbreds. Each week, samples of the stable air were taken and horses were sampled for hematological and blood biochemical analyses. At the end of both three-week periods, blood neutrophil and bronchopulmonary lavage immune cellular responses were assayed and bronchopulmonary lavage samples were cultured aerobically and anaerobically. The air ionizer unit had no significant effects on the parameters assayed.

INTRODUCTION In the majority of racing stables, reduction of airborne contamination is dependenton natural ventilation methods. Ventilation of this nature can be difficult to regulate with variable weather conditions, existing stable location and structure, the air space per horse, and the number of horses per air space. 14Additional or alternative methods of reducing aerogenous challenge would therefore be beneficial in the management of respiratory disease. One such alternative is air ionizers. Air ionizers pro~tpUthom'addresses: 1Irish Equine Center, Johnstown, Naas, Co. Kildare. hysics Department, University College Dublin, Belfield, Dublin 4. Acknowledgement:The authors wish tothankMr. Phillip McCartan, MRCVS, Ms. Jean Burke, Ms. Anna Collins, for their help with the various aspects of this work, and Ms. Geraldine Barrett for typing the manuscript.

IOVEMBER/DECEMBER 1990

duce both positive and negative ions, and their action is attributable to precipitation of airborne particles, a direct microbicidal action and a biological action through alteration of serotonin levels: ,9.~2 This study was undertaken to investigate a commercially available air ionizer unit and assess its effects on cellular respiratory responses in racing Thoroughbreds.

MATERIALS AND METHODS Eight Thoroughbred racehorses with clinical respiratory disease and showing poor racing performances were used in this study. Clinical signs were variable and included unexpectedly poor performance, occasional coughing, inappetance and poor coat condition. On a few occasions this was accompanied by a serous nasal discharge and occasionally mild and transient pyrexia. The double crossover trial consisted of two consecutive three-week periods. During the first period the air ionizer was used in five of the eight stables, with three stables as controls; during the second period the order was reversed. The air ionizer~ unit used in this study produced ions from corona discharge tubes, and was marketed as being effective in areas up to 100m3. The ion output of the units was measured in a closed laboratory, of volume approximately 100m3, using a Gerdien cylindrical counter and solid state electrometers. For the purposes of this work the ionizers were centrally located in each stable suspended at a heightof 2.8m. Stables 1-5 and 6-8 had air space capacities aGcrmazap, Unithcrm Ltd., Dublin

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•

-~.~.

Table 1. Bacterial and fungal species isolated and their percentage occurence in air samples recovered from eight stables sampled at weekly intervals over a six week period using an Anderson air sampler. Bacteria

%

Fungi

%

Staphylococci

82.5

Mucor

76

(coagulase positive)

Bacillussubtilis

10.5

Penicillum

E. Coli

4.7

Rhizopus3

Streptococci

1.6

Aspergillus

20

(non hemolytic) Micrococci

0.5

of 50m 3 and 60m 3, respectively, with stables 2, 3, 4, 5 and 6,7 and 8 having common air spaces. Blood samples were collected by jugular venepuncture once a week from each horse, into vacutainer EDTA, citrate and plain tubes for hematological and blood biochemical analysis. Blood (15ml) was also collected into a 30ml sterile container containing 100pl of preservative free sodium heparin (5,000 iu/ml) to assay neutrophil function at the end of each three-week period. Bronchoalveolar lavage (BAL)5 samples were collected from each horse at the end of both three-week periods. BAL was carried out using two lengths of sterile siliconized polypropylene tubingu, four 60 ml syringes and preservative and pyrogen free saline or phosphate buffered saline as the lavaging fluid. The tubing was sterilized by autoclaving at 120°C and 15 lbs for 15 minutes. BAL was carded out as previously described.5 Briefly, the horses were restrained using a bridle/headcollar and twitch and some horses tolerated the procedure even without a twitch. The nostril (usually the left) was cleaned, and the BAL tubes positioned for lavage? A total of 180 ml of lavage fluid, at room temperature, was introduced using three 60 ml syringes applying constant but not excessive pressure over approximately a 90-second period. The last syringe was left in bCerdixLtd.,availablefromthe IrishEquineCenter

Table 2. Bacterial species isolated and their percentage occurrence in cultrues of bronchopulmonary lavage fluid recovered from eitht racehorses sampled on two o c c a s i o n s at a three week interval. Bacteria

%

Staphylococci

66

- Coagulase positive - Coagulase negative

-26

-74

Streptococci

30.6

- Non hemolytic - Hemolytic

-Haemolytic

-10 -45 -45

Bacillus subtilis

3

E. Coil

0.4

position and used for the immediate withdrawal of the lavage fluid. Fluid was withdrawn relatively slowly to facilitate collection of the fluid and to minimize airway mucosal and cellular trauma. A lavage was only successful if the volume recovered exceeded the volume of the tubing (45 ml) and contained frothy material denoting the presence of surfactant, u If the lavage was unsuccessful it was possible to repeat the procedure by withdrawing Tube B by one third of its' total length, repositioning it, and introducing a further two-thirds of the initial lavage fluid volume. A hematological and blood biochemical profile was carried out to assess red blood cell and white blood cell parameters and to quantify protein, electrolyte and enzyme (amino aspartate transferase, creatinine kinase and gamma glumatyl transferase) changes. 6 Hematological and blood biochemical values outside the normal range2.3and associated with poor performance8 were scored for statistical analysis on an increasing scale, as the number of parameters outside the normal range increased. Blood neutrophils were separated from plasma on a Ficoll-paque density gradient 4and their chemotactic 1°chemiluminescence, phagocytic and intracellular killing properties assayed. 13 Cells recovered by centrifugation at 300g from bronchopulmonary lavage fluid were identified, and

Table 3. Numbers (Mean + SD) of bacteria and fungi recovered from the lungs and stables of eight Thoroughbred racehorses with and without air ionizer units positioned in their stables for two three consecutive week periods. Stables were samples at weekly intervals and bronchopulmonary lavage samples taken after each three week period.

With ionizer unit Without ionizer unit Significance

Colonyforming units + Score O, 1+, 2+, 3+.

IS(

Stable bacteria

+Stable fungi

Lung bacteria

40 -+.44 58 + 47 N.S.

2+ 1 2+ 1 N.S.

14 -+ 13 17 + 14 N.S.

,,.~ . . . . . . . . . . ......~e~ ;s~~ %. ~ , - ' . :~:::.

.....~'~;~!~,~,:~i:~:~ * ~:~!~!~,:¢:~

~::i~.":~.

Table 4. Celltypes (Mean _+SD) recovered by bronchopulmonary lavage from eitht Thoroug hb red racehorses with and without air !onizer units positioned in their stables for two three consecutive week periods. Bronchopulmonary lavage samples were taken at the end of each three week period.

With ionizer unit

Macrophage % 70_+4

Neutrophil % 4_+2

Lumphocyte % 26_+6

Eosinophil % 1 +4

Without ionizer unit

57 _+18

11 + 13

33 + 11

0.3 + 0.7

their phagocytic andintraceUular killing abilities assayed as above. The cell free portion of the bronchoalveolar lavage fluid was centrifuged at 1500g for 15 minutes and the deposit cultured under aerobic and anaerobic conditions. Colony forming units were identified and counted after 24 hours aerobic incubation and 72 hours anaerobic incubation. Samples of the stable air were taken on a weekly basis using an Anderson air sampler positioned approximately 0.8m above bedding level in a closed stable and air drawn through at a rate of 1 cubic ft/minute. The sampler was loaded with blood agar, McConkey agar, nutrient agar and Sabouraud agar plates. All plates, except the Sabouraud plates, which were left at room temperature, were incubated at 37°C for 24 hours. Colony forming units were identified and counted and fungi grown were identified and scored on a 1+ to 3+ scale. Results were analyzed using a paired Student's"t" test (P<.05) comparing results obtained with and without the presence of an air ionizer.

R ESU LTS

Ion output from the ionizer used was calculated to be in the order of 4 x 103/cm~with equal numbers of positive and negative ions produced. The bacterial and fungal species recovered from the stable air are listed in Table 1. Staphylococci were the predominant bacterial species cultured from the stable air, and were isolated from all stables at each weekly sampling. Mucor sp. constituted 76% of fungal species recovered; it was recovered in 80% of samples and was found in all stables over the study period. Anaerobic bacteria and fungi were not cultured from bronchopulmonary lavage fluid. Staphylococci were the predominant bacterial species recovered from the lung (Table 2). The presence of the air ionizer units did not result in any significant reduction in the numbers of bacteria or fungi recovered from the stable air or the lungs of the eight racehorses over the study period (Table 3).

iOVEMBER/DECEMBER 1990

An elevated fibrinogen level was the most frequently recorded abnormality in the hematological and blood biochemical profile. Other changes observed included leucopenia, leucocytosis,reversal of the neutrophil to lymphocyte ratio, monocytosis, eosinophiUa, potassium depletion, and occasionally anemia, reversal of the albumin to globulin ratio, elevation of total bilirubin levels and increased levels of the enzymes AST, CK and GGT. Individual horses had on average 1-2 abnormalities at each sampling point with a range of 0-4. No significant differences were recorded in the hematological and biochemical profile with the ionizers present or absent. Pulmonary alveolar macrophages were the predominant cell type recovered by bronchoalveolar lavage (Table 4). Other cell types recovered included neutrophils, lymphocytes and eosinophils and occasionally epithelial cells. Intracellular cocci were observed in macrophages recovered from six of the horses examined. There were no significant differences between cell types recovered from the lung with and without air ionizer units in position. Blood neutrophil and bronchoalveolar lavage cellular immune responses are shown in Table 5. There was no demonstrable enhancement of the local or systemic immune response in the presence of the air ionizers.

DISCUSSION

In this study, no significant reduction was noted in the bacterial or fungal contamination of the stable air nor was there any clinical or clinical pathological evidence of improvement in horses with air ionizer units in position. A reduction in the incidence of bronchopneumonia among calves following exposure to small air ions, has been reported and a further report has described increased physical fitness with improved hemaglobin levels, globulin levels, neutrophil phagocytosis and bactericidal activity among horses exposed to small air ions. 1.~ Major differencesbetween the above mentioned studies and the work described here were the exposure levels used, the duration of and periods of exposure. Calves were exposed for six hours a day over a two month period while

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o.~.,.

Table 5. Immunefunctional Assays (Mean + SD) carried out on blood neutrophils and bronchopulmonary lavage (BPL) cells recovered from eight Thoroughbred racehorses with and without air ioniser units positioned in their stables for two threeconsecutive week periods. Horses were bloodsampled at weekly intervals and bronchopulmonary lavage samples recovered at the end of both three-week periods. Chemotaxis With ioniser unit Withoutioniser Significance

0.26+0.07 0.26+0.06 N.S.

Blood Neutrophils Chemiluminescence (ram) (rnv) 78+20 78+12 N.S.

7+0.9 7+1.4 N.S.

horses were exposed three times daily for 15-90 minutes over a 45-day period. 1,7Although in this study exposure was continuous, it is likely that the lack of response observed was due to the low ion output of the units. Ion output from units used in this study was in the order of 4 x 103/cm3 whereas work carded out on the microbicidal action of small air ions has shown that ion concentrations from 5 x 104/cm3to 5 x 106/cm3are necessary to inhibit natural aerial microflora or bacteria in artificially created aerosis. ~.7 Under the conditions of this study, the air ionizer used did not have any significant effect on cellular respiratory defenses; this lack of affect appeared to be primarily attributable to their low ion output. It is possible that if commercially available units with higher output were available they could be beneficial in the management of equine respiratory tract disease.

CONCLUSION In a double crossover study to evaluate an air ionizer unit and its effect on cellular respiratory defense, no obvious beneficial effect could be demonstrated. There was no detectable clinical improvement in the horses nor could any improvement be demonstrated in hematological, bloodbiochemical or bronchoalveolar lavage cellular parameters. No reduction in the numbers of bacteriarecovered from the stable air or the horses lungs was observed. The ion output of units used in this study was lower than thatpublished for other ionizers where beneficial effects have been described. Thelow ion outputcouldpossibly bethe main reason for the lack of effect observed.

158

Phacocytosis (rnins)

Intracellular (%)

72_+17 67_+17 N.S.

70+24 72-+13 N.S.

BPL Cells Phacocytosis killing (%) 73_+11 63-+24 N.S.

Intracellular killing (%) 5_+9 4_+6 N.S.

REFERENCES 1. Abramov SS, Pital'tsv ES, Solov'yanov PI: Physical factors in preventing bronchopneumonia in calves (air ionization, ultraviolet rays and infrared ray s). Veterinariya, Moscow, 12: 66-67,1982. 2. Allen BV, Kane CE, PoweU I3(3: Leucocyte counts in healthy English Thoroughbredsintraining.Equine VetJ 16:207-209,1984. 3. BlackmoreDJ:Aconceptofnormalvalues.Proced.Firstlnter. Symposium on Equine Hematology. pp.276-285,1975. 4. Boyum A: Isolation of mononuclear ceUsand granulocytes from human blood. Scand J C lin Lab lnvest 21:77-89,1968. 5. Fogarty U: A bronchoalveolar lavage technique suitable for routine diagnostic purposes in foals and horses. Equine Vet Ed 2:102104,1990. 6. Fogarty U, Leadon D: Poor PefformanceSyndrome: Areview of the literature and some data on the hematological and blood biochemical changes in two groups of Thoroughbred racehorses performing below expectation. Irish Vet J 41:203 -209,1987. 7. Khrenov NM,Kretinin VK, Vorob'evaZhN, Kalinchenko LM, GolovkoPD: Effectof ionized air on horses. Veterinariya Moscow, 12:2425,1972. 8. Kmeger AP, Reed Ei : Biological impact of small airions. Sci 193:1209-1213,1976. 9. Krueger AP, Smith RF: The biological mechanisms of air ion action. 2. Negative ion effects on the concentrations and metabolism of 4, hydroxytryptaminein the mammalian respimtozy tract. J GenPhysio144, 276,1960. 10. Nelson RD, Quie PG, Simmons RL: Chemotaxis under agarose: anew and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclearleukocytes and monocytes. J lmmunol 115:1650-1656,1975. 11. Reynolds HY: Bronchoalveolar lavage. Am Rev Respir Dis 135:250-263,1987. 12. Udenfriend H, Weissbach CT, ClarkCT: The estimation of 5, hydroxytryptamine (serotonin) in biological tis sues. J Biol Chem 215:337344,1955. 13. Van Furth R, Van Zwet TL: In vitro determination ofphagocytos~sandintracellularkilling bypolymorphonuclear and mononuclear phagocytes, lnHandbookofExperimental Immunology pp.36.1:36.24. Ed. D,M. Weir, Blackwell Scientific, Oxford, 1973. 14. Webster AJ: Calf Husbandry, Health and Welfare pp.7179,1984, Granada, London.

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