Influence of head posture on the respiratory tract of healthy horses DJ RACKLYEFT and DN LOVE Department of Veterinary Pathology, University of Sydney, New South Wales 2006 SUMMARY: Twenty four normal, confined mares were unable to lower their heads for 24 or 48 h. In 21 mares this resulted in increases in the proportion of neutrophils and/or numbers of bacteria in transtracheal aspirates. In eight mares the changes in tracheal washes were accompanied by clinical evidence of mild respiratory disease. In three additional cases respiratory signs were accompanied by systemic illness. These changes reversed once the mares were able to lower their heads. Haematological changes (absolute neutrophilia and/or hyperfibrinogenaemia) were mild and occurred more commonly in horses restrained for 48 h. The results suggest that keeping the heads of healthy horses raised leads to an increased bacterial burden in their tracheobronchial secretions. These changes appeared to be related to head posture effects and not simply confinement in stocks. These findings give further weight to the theory that postural drainage may facilitate clearance of bacteria from the tracheobronchial tree. The possible relevance of such findings to post-transportation pneumonia in horses is discussed. Aust Vet J 67: 402-405
introduction Transportation has been implicated as one of several events that may predispose horses to severe bacterial infections of the lower respiratory tract (Sweeney 1987). It has been speculated that restraint of horses with their heads in an elevated position during long journeys may lead to an increased opportunity for pharyngeal secretions and ingested feed to gain entry into the lower respiratory tract (Sweeney et a1 19891, as well as being an unfavourable posture for the drainage of lower respiratory secretions (Mansmann 1983; Sweeney 1987).The aim of this study was to evaluate the relationship between confinement with elevated head restraint and the clinical health, haematology and tracheal wash characteristics of healthy horses. Materials and Methods Experimental Horses Thirty six Thoroughbred and Standardbred mares between 6 and 20 years were selected from the population at the University of Sydney Horse Unit. They were included if they were clinically normal, (including a normal respiratory rate of less than 20 breaths per min, normal thoracic auscultation at rest and no nasal discharge or cough). In addition, horses were included only if the gross and cytological characteristics of their initial transtracheal aspirate were within normal limits (translucent grey with at most a few fine strands of clear mucus, a majority of columnar ciliated epithelial cells, a small population of macrophages and other mononuclear cells, a few cuboidal epithelial cells and nonciliated columnar cells with foamy cytoplasm, a low proportion of neutrophils and no eosinophils). The study was performed over 2 months during winter (ambient temperature range 5 to 20°C) with horses protected from the weather in a well-ventilated barn. During the investigations horses were kept under the following conditions: Effect of three transtracheal aspirations performed at 24 h intervals - Four horses (Group A) were confined in a yard within the barn. They were able to move about and lower their heads freely and were watered every 2 h and fed lucerne hay from the ground every 6 h. Effect of confinement - Eight horses were confined in stocks (0.85 m x 1.85 m) and were able to lower their heads freely (over a front rail height of 0.9 m). They were watered and fed from the ground as above. Four horses (Group B) were confined thus for 24 h and 4 horses (Group C) for 48 h. Effect of elevated headposition - In total, twenty four horses were confined in stocks and unable to lower their heads (front rail height 1.25 m). Eight horses (Group D) were restrained for 24 h and were watered and fed with their heads in that position, 8 further horses (Group) E were restrained for 24 h but offered water only, and the other 8 horses (Group F) were confined for 48 h and were watered and fed. 402
After the period of restraint all horses were removed from the stocks and kept for 24 h unrestrained in a yard with access to hay and water. Experimental Procedures A clinical examination was performed on each horse before confinement (0 h) and then every 12 h for 72 h. Body temperature, respiratory rate, demeanour and appetite, the presence of any nasal discharge, cough, or abnormal sounds in lung fields or the trachea, were noted. At 0, 24 and 48 h for Groups A, B, C, D, E and F, and also at 72 h for Group C and F, 5 ml of blood was collected by jugular venipuncture into disodium EDTA for haematological examination. Transtracheal aspirates were obtained by a standard technique (Beech 1981) at 0, 24 and 48 h. Ten millilitres of sterile phosphate-buffered saline (PBS) were injected and immediately aspirated, using a 60 ml syringe to obtain a sample of tracheobronchial secretions. Haematology Packed cell volume (CPV) was measured after microhaematocrit centrifugation* and total plasma protein (TPP) using a refractometert. Plasma fibrinogen was measured using the heat precipitation method (Coles 1986). Total leukocyte counts were determined by automated counter$ and differential counts by examination of blood smears stained with a quick-dip stains. Bacteriology Immediately following collection of a tracheal wash, all air was expelled from the syringe which was then capped with a sterile needle. Samples were processed within one h of collection. Colour, opacity and viscosity of the sample were noted. For every sample, 2 drops (approximately 0.1 ml) of undiluted aspirate were spread evenly over 2 sheep blood agar (SBA) plates. To enable counting of individual colonies where the inocula produced very heavy bacterial growth, the samples taken at 24 and 48 h were also diluted 1:1 and 1:3 with sterile PBS, and 2 drops of each of these dilutions were also spread over two SBA plates. One of each pair of respective plates were incubated aerobically and anaerobically at 37 "C. Anaerobiosis for primary incubation was achieved in anaerobic jarsq by gas generating kit# and for subcultured plates by double evacuation of jars and replacement with a special gas mixture (10% hydrogen, 10% carbon dioxide and 80% nitrogen)?* Bacterial colonies on plates incubated Clements Micro Haematocrit, Clements Pty Ud, North Ryde, New South Wales t American Optical Corporation, Buffalo, New York 14215, USA Coulter Electronics Ltd, Dunstable, Bedfordshire, England 5 Diff-QuikO, Lab-Aids, Narrabeen, New South Wales 1 Anaerobic System Code HP 11, Oxoid, Carlton, New South Wales ## Anaerobic System Code BR 38,Oxoid "Commonwealth Industrial Gases, Alexandria, New South Wales
*
Australian Vererinary Journal, Vol. 67, No. 11, November, 1990
TABLE 2 Summary of abnormal clinical, haematologicaland tracheal wash findings in horses unable to lower their heads for 24 h (Groups D and E) or 48 h (Group F)
Abnormal findings at time of observation Horse No.
12 h
24 h
36 h
48 h
60 h
T T T
1,3
72 h
T
H
T,H T 3 4 5 6 7 8
T
H T T,H
1
Group F T
1 2 3 4 5 6 7 8
1 mucoid or mucopurulent nasal discharge 2 abnormal auscultation of lung fields and/or trachea 3 elevated respiratory rate (more than 20 bpm) 4 cough (more than three coughs) 5 depression and loss of appetite 6 fever (body temperature more than 38.5 C) T abnormal tracheal wash characteristics (increased proportion of neutrophils andlor increased number of bacteria) H abnormal haematology (absolute neutrophilia andlor hyperfibrinogenaemia)
TABLE 1 Total viable colony counts in tracheal washes from control horses (Groups A, B and C) and from those with head restraint (Groups D, E and F)
Total viable colony counts in tracheal wash (cfulml) Group No. A (n = 4)
Control B (n = 4) Control C (n = 4)
Control D (n = 8) 24 h Head up E (n = 8) 24 h Head up F (n = 8) 48 h Head up
0 420 f 30 123 f 10 133 f 10 94 f 10 295 f 20 77f 0 -
24 h
465 424 f 596 90 - 1316 1060* 205 155 f 116 430 40 - 310 508 f 214 199 430 190 - 4690 12129 8155 112 50 - 20400 310 414 10080 f 740 1260 110 - 23900 11301 f 12207 103 0 - 26370 260
48 h 323 f 430 20 - 940 73 f 74 0 - 150 2385 f 3932 120 - 8250 427 f 720 20 - 2000 592 f: 754 10 - 1810 13348 f 12584 70 - 29740
counts on 200 cells and for estimation of bacterial numbers, bacterial morphology and Gram reaction. Results Quantitative bacteriological results for the transtracheal washes of all groups are presented in Table 1 and a summary of abnormal clinical, haematological and tracheal wash findings is presented in Table 2.
Effect of Repeated Transtracheal Aspiration (Group A) All horses remained clinically normal and haematological data and transtracheal aspirates remained normal. No bacteria were seen in smears and total viable colony counts remained low (Table 1). The constituent flora did not change with subsequent tracheal washes, consisting of a mixture of predominantly aerobic and/or facultatively anaerobic bacteria with occasional obligately anaerobic isolates.
aerobically were described and counted after 24 and 48 h of incubation. Those on plates incubated anaerobically were. described and counted after 3 and 7 d. Bacterial isolates were subcultured onto SBA plates and identified if possible to genus level, according to criteria and methods described for aerobic and/or facultatively anaerobic bacteria (Cowan 1974; Krieg and Holt 1984; Lennette et a/ 1985; Sneath et a/ 1986) and for anaerobic bacteria (Holdeman et al 1977).
Effect of Confinement (Groups B and C) Seven of 8 horses remained clinically and haematologically normal with normal tracheal wash characteristics. The other horse, confined for 24 h (Group B), developed a cough and a slight mucoid nasal discharge at 24 h and had an opaque, creamy transtracheal aspirate with 80% neutrophils. These changes had partly reversed 24 h following the end of restraint. No bacteria were seen in smears at any time from any of the 8 horses. Total viable colony counts were increased slightly in samples from Group C horses at 24 h and 48 h, but the constituent flora in samples from both Groups Band C did not change and remained similar to Group A.
Cytology One millilitre of each aspirate was centrifuged for 5 min at 500 g, and smears made of the sediment for differential cell
Effect of Inability to Lower Head (Groups 0,E and F) Abnormal clinical, haematological and tracheal wash findings for these groups are summarised in Table 2. Of the 24 horses
* range
Australian Yeterinary Journal, Vol. 67, No. 11, November, 1990
403
in these groups, 9 remained clinically normal. Of the remainder, 6 mares showed the first evidence of clinical abnormality after 12 h of elevated head posture, 7 after 24 h and the 2 remaining mares by 48 h. However, by 24 h after the end of confinement, all mares were clinically normal except the most severely affected mares which, while improved, were not clinically normal for a further 24 to 48 h. Haematological changes were mild. In horses with heads elevated for 24 h, neutrophilia (greater than 5.8 x 109/1) was present in 2 horses at 24 h and in 4 horses 24 h after the end of the head elevation. In horses restrained thus for 48 h, a mild neutrophilia was present in 4 horses at 48 h and, in another horse, while absolute numbers of neutrophils were normal, there were 15% band forms. Hyperfibrinogenaemia (greater than 5.0 g/l) was present in 4 horses. Twenty four hours after head posture restraint, neutrophilia was present in 4 horses and hyperfibrinogenaemia in 2 horses. The gross tracheal wash characteristics of 21 of 24 horses became abnormal at some time during the study. After 24 h of restraint, the tracheal washes of 16 horses were opaque, very rnucoid and flocculent and were grey, cream, yellow or brown in colour. The proportion of neutrophils in aspirates from 18 horses was increased at least 10-fold. Bacteria were observed in tracheal wash smears from 16 horses in numbers ranging from 5 to 70 per HPF. There were large increases in the total viable colony counts in samples from 17 horses (and to at least 100-fold in 14 samples). Twenty four h after the end of head elevation, tracheal washes from horses in Groups D and E had improved in gross appearance and there were decreased proportions of neutrophils and fewer bacteria. When these horses were first released and allowed to lower their heads, in several cases 20 to 100 ml of mucopurulent material drained from their nostrils. In horses with heads elevated for 48 h (Group F), the washes of 6 of 8 horses at this time were opaque with an increased proportion of neutrophils. Bacteria were observed in smears of four of these samples (eight to 150 per HPF). Total viable colony counts were increased in 6 samples. Where large increases in bacterial numbers cultured from tracheal washes occurred, the isolates contributing most frequently were Actinobacillus/Pasteurella sp (18 horses) and Streptococcus egui subsp zooepidemicus ( 5 horses). Other Streptococcus sp were present in large numbers in 4 horses, Escherichia coli and Acinetobacter sp each in 2 horses and Bacillus sp and Aeromonas sp each in one horse. Obligately anaerobic bacteria were not cultured in large numbers, although species such as Peptostreptococcus anaerobius, other anaerobic Gram-positive cocci, killonella sp, Bacteroides sp, Fusobacterium sp, Propionibacterium s p and Clostridium sp were recovered from 12 horses.
Discussion While there is considerable individual variation within the groups of horses in this study, it is evident from the results presented that neither repeated transtracheal washes nor confinement per se had a severe effect on the clinical, haematological, or transtracheal wash characteristics of horses. However, when horses were unable to lower their heads for periods from 24 to 48 h, there was a change in the characteristics measured. This suggests that the changes observed in this experiment were due to elevated head position. This experimentally produced observation is similar to those recorded previously by McClintock et al (1986), where horses were confined with elevated heads in floatation tanks used for the treatment of skeletal injuries. However, it has been shown that in healthy horses and in those with chronic lung disease, there may be little relationship between tracheal wash cytology and the pathological status of the lungs in individual horses (Larson and Busch 1985), and that cell populations in the trachea and lower down in the respiratory tract may differ substantially in such cases (Derksen et al 1989). Nevertheless, since a major objective of the present study was the identification of bacteria in the lower respiratory tract, transtracheal aspiration was chosen as a more suitable collection technique than bronchoalveolar lavage to obtain uncontaminated samples. In addition, samples were not collected from sites more 404
distal in the respiratory tree than the bronchial bifurcation in order to avoid collecting samples from too localised and, perhaps unrepresentative, segments of lung. In those horses whose heads were restrained in an elevated position, the presence of increased numbers of bacteria was probably the result of decreased clearance of contaminating pharyngeal flora and their subsequent multiplication in the lower respiratory tract. lngesta was observed endoscopically in the tracheas of the horses following transportation where they had been fed during the journey and prevented from lowering their heads (Sweeney et a1 1989). Although such obvious contamination of tracheal washes was not seen during the present study, aspiration of even small amounts of oropharyngeal secretions can present the lower respiratory tract with a substantial bacterial challenge (Finegold 1983). Also, elevated head position may have been unfavourable for mucociliary clearance (Turgut and Sasse 1989; Sweeney 1989), and a change in mucus volume and/or flow characteristics may also have decreased the clearance rate. In human beings, it has also been found that mucociliary clearance is delayed in the presence of purulent secretions due to decreased ciliary beat frequency (Wilson et all986). Drainage of respiratory secretions under gravity may also assist in lung clearance under normal circumstances. The reversal of tracheal wash changes, and the disappearance of clinical respiratory disease once mares were again allowed to lower their heads, suggests that postural drainage was certainly an important mechanism of clearing abnormal tracheobronchial secretions. Transtracheal aspirates from healthy horses are usually not sterile as there are a small transient population of predominantly aerobic and/or facultatively anaerobic bacteria in the lower trachea (Mansmann and Strouss 1976; Sweeney et al 1985). During the present study, there was a large increase in numbers of bacteria recovered from tracheal washes. This appeared to be due in nearly all cases to multiplication of the Actinobacillus/ Pasteurelfasp as well as Streptococcusequi subsp zooepidemicus. Interestingly, these speices have been isolated commonly from natural cases of pneumonia in horses (Jang and Hirsch 1987; Jang et a1 1987; Schlater et a1 1989). Many advanced cases of equine pneumonia involve obligately anaerobic bacteria, although large numbers of anaerobic bacteria may be isolated only where the duration of illness has been at least 5 d (Racklyeft unpublished). After postural head restraint for 24 h or 48 h during the present study, while obligately anaerobic bacteria were isolated, tracheal wash flora was still predominantly facultatively anaerobic. It is likely that multiplication and establishment of aerobic and facultative bacteria are necessary to provide appropriate environmental conditions for replication and invasion by the anaerobic components of a n infection. While this study has direct relevance to situations where horses are confined with their heads in an elevated position for prolonged periods of time, they were confined in stocks to which they were accustomed and demonstrated no behavioural evidence of stress (Creiger 1982). Previous studies investigating the association of transportation with equine lower respiratory tract infections have examined changes in aspects of respiratory immunity associated with the stress of transportation. They found mild decreases in the numbers and activity of pulmonary alveolar macrophages and other changes in bronchoalveolar lavage fluid over a one to 4 week period following transportation (Anderson e f al1985; Basaraba e t a / 1986) as well as delayed inflammatory response to a previous bronchoalveolar lavage (Traub-Dargatz et all988). Another study investigating transit stress in horses (Leadon et a1 1989) has documented haematologic abnormalities (neutrophilia and hyperfibrinogenaemia) in horses subjected to long distance journeys by air, which were similar to those which occurred during the present study in those horses restrained for 48 h. This study shows that restraint of horses’ heads in an elevated position for a prolonged period can lead to an increased bacterial load in tracheobronchial secretions. In the face of other stresses, such as transportation, where pulmonary defences may be suppressed, or where there is preexisting respiratory disease, the susceptibility of individual horses to progression to pneumonia may be enhanced. Since such infections frequently have an unfavourable outcome, all measures should be taken to minimise
Australian VeterinaryJournal, Vol. 61,No.
11, November, 1990
bacterial challenge to the lower respiratory tract. Thus, the crosstying of horses with their heads in an elevated position should be avoided, long journeys should be broken u p into 12 h stages, with horses allowed adequate opportunity and encouraged to lower their heads during rest stops, and transport facilities should be designed so horses can lower their heads periodically during long journeys.
Acknowledgments Funding for this study, provided by the New South Wales Racing Research Fund and Jean Walker Fellowship Trust, is gratefully acknowledged. L Patoka provided the bacteriological media.
References Anderson NV, DeBowes RM, Nyrop KA and Dayton AD (1985) - A m J Vet Res 4 6 2272 Basaraba R, Liggitt HD and Bayly WM (1986) - cited in Bayly WM, Liggitt HD, Huston LJ and Laegreid WW (1986) - A m Assoc Equine Pract 32: 253 Beech J (1981) - Equine Vet J 13: 136 Cohrs P (1967) - In Nieberle and Cohrs Textbook of the Special Pathological Anatomy of the Domestic Animal, revised edition, Pergamon Press, Oxford Coles EH (1986) - Veterinary Clinical Pathology, fourth edition, WB Saunders, Philadelphia Cowan ST and Steel KJ (1974) - Cowan and Steel’s Manual for the Identification of Medical Bacteria, second edition, Cambridge University Press; Cambridge Creiger S (1982) - Equine Vet Sci 2: 187 Derksen FJ, Brown CM, Sonea I, Darien BJ and Robinson NE (1989) - Equine Vet J 21: 23
Finegold SM (1983) - In Respiratory Infections: Diagnosis and Management, edited by Pennington JE, Raven Press, New York, p191 Holdeman LV, Cato EP and Moore WEC (1977) -Anaerobe Laboratory Manual, fourth edition, Virginia Polytechnic Institute and State University, Blacksburg, Virginia Jang SS, Biberstein EL and Hirsch DC (1987) - A m J Vet Res 4 8 1036 Jang SS and Hirsh DC (1987) - Vet Clinics of North Am: Equine Pract 3: 181 Kreig NR and Holt JG (1984) - In Bergey’s Manual of Systematic Bacteriology, Vol 1, William and Wilkins Co, Baltimore Larson VL and Busch RH (1985) - Am J Vet Res 46. 144 LennetteEH, Balows A, Hausler WJ and Shadomy HJ (1985) -Manual of Clinical Microbiology, fourth edition, American Society for Microbiology, Washington DC Mansmann RA (1983) - A m Assoc Equine Pract 2 9 71 Mansmann RA and Strous AA (1976) - J A m Vet Med Assoc 169 631 McClintock SA, Hutchins DR, Laing EA and Brownlow MA (1986) Equine Vet J 18: 462 Schlater LK, Brenner DJ, Steigerwalt AG, Moss CW, Lambert MA and Packer RA (1989) - J CIin Microbiol27: 2169 Sneath PHA, Mair NS, Sharpe ME and Holt JG (1986) - In Bergey’s Manual of Systematic Bacteriology, Vol2, Williams and Wilkins Co, Baltimore Sweeney CR (1987) - In Current Therapy in Equine Medicine 2, edited by Robinson NE, WB Saunders Co, Philadelphia, p592 Sweeney CR (1989) - A m J Vet Res 5 0 2135 Sweeney CR, Arthur RM, Merriam JG, Page BT and Wilson WD (1989) - Equine Pract 11: 29 Traub-Dargatz JL, McKinnon AO, Bruyninckx WJ, Thrall MA, Jones RL and Blancquaert AMB (1988) - A m J Vet Res 4 9 1026 Turgut K and Sasse HHL (1989) - Vet Rec 125: 526 Wilson R, Sykes DA, Currie D and Cole PJ (1986) - Thorax 41: 453 (Accepted for publication 13 June 1990)
BOOK REV1EWS Magrane’s Canine Ophthalmology.LC Helpec Lea and Febigec 1989. 287 pp. $89.75.
-
Pyrrolizidine Alkaloids Environmental Health Criteria 80. International Program on Chemical Safew WHO Geneva. 1988. 345 pp. $2100.
This historic text on the canine eye, first written in the 1960’s, has been revised by an eminent ophthalmologist, Lloyd C. Helper of the Universityof Illinois, a charter Diplomateof the American College of Veterinary Ophthalmologistsand now Associate Dean for Academic Affairs in the College of Veterinary Medicine. During his distinguished career Professor Helper, always helpful to his colleagues, has found time to visit Australia more than once. Veterinarians who are Magrane devotees can be reassured; this is a gentle revision very much in the original style. It presents a traditional approach to eye diseases with chapter headings on the normal eye, examination of the eye and its adnexa, ocular therapeutics, diseases and surgery of the lids and lacrimal apparatus, diseases and surgery of the conjunctive, diseases and surgery of the cornea and sclera, diseases and surgery of the uveal tract, diseases and surgery of the orbit, glaucoma, diseases and surgery of the lens, diseases of the vitreous, retina and optic nerve and ocular manifestations of systemic disease. While some excellent photographs,drawings and problem-oriented listings in the index are helpful, the reader presentedwith an ocular problem needs to know some ophthalmology before being able to find which parts of this book are the most relevant. A book of this size cannot and indeed should not “say it all” and does not pretend to be an exhaustive text on ophthalmic practice. Nevertheless it is an excellent first text to be read from cover to cover by practitioners who want to know more and develop an interest in the eye. The cover-to-cover reader, however, will need caution. You cannot always read-and-do. Some of the procedures described are best left to the ophthalmologist, for example, paracentesis of the anterior chamber and vitreocentesis.The easy how-to-do-itstyle of the text could leave the reader not sufficiently aware of the considerable hazard of these procedures in unskilled hands. New graduates and all those who realize they must keep vitally interested in clinical practice to avoid professionalburn-out would do well to buy this book. It is reassuring to find Australian techniques included and acknowledged in this text.
This is the latest and most comprehensivecompilation of information concerningpyrrolizidinealkaloids (PA’s). The publicationwas the work of a WHO expert committee which was chaired jointly by Dr CCJ Culvenor, late of the Division of Animal Health, CSIRO, Parkville, Victoria. The production of this monograph was stimulated by the serious and large-scaleoutbreaks of human pyrrolizidinosis, which have occurred in recent times in various parts of Asia. The book begins with a chapter on PA’s and human health, followed by others on properties and analytical methods, sources, and pathways of exposure, metabolism, mechanisms of toxicity and biological actions, effects on animals, effects on man, biological control of PAcontaining plants and finally a section on human health risks and effects on the environment. There are 3 very useful appendixes, namely a list of the PA’s and their plant sources, a list of plants containing hepatotoxic PA’s and a further list of plants that contain PA’s that are not hepatotoxic. There is a comprehensive list of key references to this field. The main plant groups in which PASoccur, namely the Crotalaria spp, Senecio spp and Boraginaceaeare well represented and widely distributed in Australia, both as native and naturalised introduced plants, and there would hardly be any major region of the country in which this type of toxicity would not at some time occur. This book would certainly be of value in the library of any of our veterinary diagnostic laboratories and at the price would be worth having by practitioners and government veterinary offices in whose area this type of toxicity is likely to be prevalent. In this respect the chapter on the Effects on Animals is of particular value, as it describesthe patterns of disease caused by the different plant genera in the various domestic species. This is an authorativestatement on our knowledgeof the alkaloids and the toxicity they cause and is highly recommended to all veterinarians and animal scientists concerned with nutritional toxicity of livestock.
R Blogg
AA SeawrigM
Australian Veterinary Journal, Vol. 67, No. 11, November, 1990
405
introduction Transportation has been implicated as one of several events that may predispose horses to severe bacterial infections of the lower respiratory tract (Sweeney 1987). It has been speculated that restraint of horses with their heads in an elevated position during long journeys may lead to an increased opportunity for pharyngeal secretions and ingested feed to gain entry into the lower respiratory tract (Sweeney et a1 19891, as well as being an unfavourable posture for the drainage of lower respiratory secretions (Mansmann 1983; Sweeney 1987).The aim of this study was to evaluate the relationship between confinement with elevated head restraint and the clinical health, haematology and tracheal wash characteristics of healthy horses. Materials and Methods Experimental Horses Thirty six Thoroughbred and Standardbred mares between 6 and 20 years were selected from the population at the University of Sydney Horse Unit. They were included if they were clinically normal, (including a normal respiratory rate of less than 20 breaths per min, normal thoracic auscultation at rest and no nasal discharge or cough). In addition, horses were included only if the gross and cytological characteristics of their initial transtracheal aspirate were within normal limits (translucent grey with at most a few fine strands of clear mucus, a majority of columnar ciliated epithelial cells, a small population of macrophages and other mononuclear cells, a few cuboidal epithelial cells and nonciliated columnar cells with foamy cytoplasm, a low proportion of neutrophils and no eosinophils). The study was performed over 2 months during winter (ambient temperature range 5 to 20°C) with horses protected from the weather in a well-ventilated barn. During the investigations horses were kept under the following conditions: Effect of three transtracheal aspirations performed at 24 h intervals - Four horses (Group A) were confined in a yard within the barn. They were able to move about and lower their heads freely and were watered every 2 h and fed lucerne hay from the ground every 6 h. Effect of confinement - Eight horses were confined in stocks (0.85 m x 1.85 m) and were able to lower their heads freely (over a front rail height of 0.9 m). They were watered and fed from the ground as above. Four horses (Group B) were confined thus for 24 h and 4 horses (Group C) for 48 h. Effect of elevated headposition - In total, twenty four horses were confined in stocks and unable to lower their heads (front rail height 1.25 m). Eight horses (Group D) were restrained for 24 h and were watered and fed with their heads in that position, 8 further horses (Group) E were restrained for 24 h but offered water only, and the other 8 horses (Group F) were confined for 48 h and were watered and fed. 402
After the period of restraint all horses were removed from the stocks and kept for 24 h unrestrained in a yard with access to hay and water. Experimental Procedures A clinical examination was performed on each horse before confinement (0 h) and then every 12 h for 72 h. Body temperature, respiratory rate, demeanour and appetite, the presence of any nasal discharge, cough, or abnormal sounds in lung fields or the trachea, were noted. At 0, 24 and 48 h for Groups A, B, C, D, E and F, and also at 72 h for Group C and F, 5 ml of blood was collected by jugular venipuncture into disodium EDTA for haematological examination. Transtracheal aspirates were obtained by a standard technique (Beech 1981) at 0, 24 and 48 h. Ten millilitres of sterile phosphate-buffered saline (PBS) were injected and immediately aspirated, using a 60 ml syringe to obtain a sample of tracheobronchial secretions. Haematology Packed cell volume (CPV) was measured after microhaematocrit centrifugation* and total plasma protein (TPP) using a refractometert. Plasma fibrinogen was measured using the heat precipitation method (Coles 1986). Total leukocyte counts were determined by automated counter$ and differential counts by examination of blood smears stained with a quick-dip stains. Bacteriology Immediately following collection of a tracheal wash, all air was expelled from the syringe which was then capped with a sterile needle. Samples were processed within one h of collection. Colour, opacity and viscosity of the sample were noted. For every sample, 2 drops (approximately 0.1 ml) of undiluted aspirate were spread evenly over 2 sheep blood agar (SBA) plates. To enable counting of individual colonies where the inocula produced very heavy bacterial growth, the samples taken at 24 and 48 h were also diluted 1:1 and 1:3 with sterile PBS, and 2 drops of each of these dilutions were also spread over two SBA plates. One of each pair of respective plates were incubated aerobically and anaerobically at 37 "C. Anaerobiosis for primary incubation was achieved in anaerobic jarsq by gas generating kit# and for subcultured plates by double evacuation of jars and replacement with a special gas mixture (10% hydrogen, 10% carbon dioxide and 80% nitrogen)?* Bacterial colonies on plates incubated Clements Micro Haematocrit, Clements Pty Ud, North Ryde, New South Wales t American Optical Corporation, Buffalo, New York 14215, USA Coulter Electronics Ltd, Dunstable, Bedfordshire, England 5 Diff-QuikO, Lab-Aids, Narrabeen, New South Wales 1 Anaerobic System Code HP 11, Oxoid, Carlton, New South Wales ## Anaerobic System Code BR 38,Oxoid "Commonwealth Industrial Gases, Alexandria, New South Wales
*
Australian Vererinary Journal, Vol. 67, No. 11, November, 1990
TABLE 2 Summary of abnormal clinical, haematologicaland tracheal wash findings in horses unable to lower their heads for 24 h (Groups D and E) or 48 h (Group F)
Abnormal findings at time of observation Horse No.
12 h
24 h
36 h
48 h
60 h
T T T
1,3
72 h
T
H
T,H T 3 4 5 6 7 8
T
H T T,H
1
Group F T
1 2 3 4 5 6 7 8
1 mucoid or mucopurulent nasal discharge 2 abnormal auscultation of lung fields and/or trachea 3 elevated respiratory rate (more than 20 bpm) 4 cough (more than three coughs) 5 depression and loss of appetite 6 fever (body temperature more than 38.5 C) T abnormal tracheal wash characteristics (increased proportion of neutrophils andlor increased number of bacteria) H abnormal haematology (absolute neutrophilia andlor hyperfibrinogenaemia)
TABLE 1 Total viable colony counts in tracheal washes from control horses (Groups A, B and C) and from those with head restraint (Groups D, E and F)
Total viable colony counts in tracheal wash (cfulml) Group No. A (n = 4)
Control B (n = 4) Control C (n = 4)
Control D (n = 8) 24 h Head up E (n = 8) 24 h Head up F (n = 8) 48 h Head up
0 420 f 30 123 f 10 133 f 10 94 f 10 295 f 20 77f 0 -
24 h
465 424 f 596 90 - 1316 1060* 205 155 f 116 430 40 - 310 508 f 214 199 430 190 - 4690 12129 8155 112 50 - 20400 310 414 10080 f 740 1260 110 - 23900 11301 f 12207 103 0 - 26370 260
48 h 323 f 430 20 - 940 73 f 74 0 - 150 2385 f 3932 120 - 8250 427 f 720 20 - 2000 592 f: 754 10 - 1810 13348 f 12584 70 - 29740
counts on 200 cells and for estimation of bacterial numbers, bacterial morphology and Gram reaction. Results Quantitative bacteriological results for the transtracheal washes of all groups are presented in Table 1 and a summary of abnormal clinical, haematological and tracheal wash findings is presented in Table 2.
Effect of Repeated Transtracheal Aspiration (Group A) All horses remained clinically normal and haematological data and transtracheal aspirates remained normal. No bacteria were seen in smears and total viable colony counts remained low (Table 1). The constituent flora did not change with subsequent tracheal washes, consisting of a mixture of predominantly aerobic and/or facultatively anaerobic bacteria with occasional obligately anaerobic isolates.
aerobically were described and counted after 24 and 48 h of incubation. Those on plates incubated anaerobically were. described and counted after 3 and 7 d. Bacterial isolates were subcultured onto SBA plates and identified if possible to genus level, according to criteria and methods described for aerobic and/or facultatively anaerobic bacteria (Cowan 1974; Krieg and Holt 1984; Lennette et a/ 1985; Sneath et a/ 1986) and for anaerobic bacteria (Holdeman et al 1977).
Effect of Confinement (Groups B and C) Seven of 8 horses remained clinically and haematologically normal with normal tracheal wash characteristics. The other horse, confined for 24 h (Group B), developed a cough and a slight mucoid nasal discharge at 24 h and had an opaque, creamy transtracheal aspirate with 80% neutrophils. These changes had partly reversed 24 h following the end of restraint. No bacteria were seen in smears at any time from any of the 8 horses. Total viable colony counts were increased slightly in samples from Group C horses at 24 h and 48 h, but the constituent flora in samples from both Groups Band C did not change and remained similar to Group A.
Cytology One millilitre of each aspirate was centrifuged for 5 min at 500 g, and smears made of the sediment for differential cell
Effect of Inability to Lower Head (Groups 0,E and F) Abnormal clinical, haematological and tracheal wash findings for these groups are summarised in Table 2. Of the 24 horses
* range
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in these groups, 9 remained clinically normal. Of the remainder, 6 mares showed the first evidence of clinical abnormality after 12 h of elevated head posture, 7 after 24 h and the 2 remaining mares by 48 h. However, by 24 h after the end of confinement, all mares were clinically normal except the most severely affected mares which, while improved, were not clinically normal for a further 24 to 48 h. Haematological changes were mild. In horses with heads elevated for 24 h, neutrophilia (greater than 5.8 x 109/1) was present in 2 horses at 24 h and in 4 horses 24 h after the end of the head elevation. In horses restrained thus for 48 h, a mild neutrophilia was present in 4 horses at 48 h and, in another horse, while absolute numbers of neutrophils were normal, there were 15% band forms. Hyperfibrinogenaemia (greater than 5.0 g/l) was present in 4 horses. Twenty four hours after head posture restraint, neutrophilia was present in 4 horses and hyperfibrinogenaemia in 2 horses. The gross tracheal wash characteristics of 21 of 24 horses became abnormal at some time during the study. After 24 h of restraint, the tracheal washes of 16 horses were opaque, very rnucoid and flocculent and were grey, cream, yellow or brown in colour. The proportion of neutrophils in aspirates from 18 horses was increased at least 10-fold. Bacteria were observed in tracheal wash smears from 16 horses in numbers ranging from 5 to 70 per HPF. There were large increases in the total viable colony counts in samples from 17 horses (and to at least 100-fold in 14 samples). Twenty four h after the end of head elevation, tracheal washes from horses in Groups D and E had improved in gross appearance and there were decreased proportions of neutrophils and fewer bacteria. When these horses were first released and allowed to lower their heads, in several cases 20 to 100 ml of mucopurulent material drained from their nostrils. In horses with heads elevated for 48 h (Group F), the washes of 6 of 8 horses at this time were opaque with an increased proportion of neutrophils. Bacteria were observed in smears of four of these samples (eight to 150 per HPF). Total viable colony counts were increased in 6 samples. Where large increases in bacterial numbers cultured from tracheal washes occurred, the isolates contributing most frequently were Actinobacillus/Pasteurella sp (18 horses) and Streptococcus egui subsp zooepidemicus ( 5 horses). Other Streptococcus sp were present in large numbers in 4 horses, Escherichia coli and Acinetobacter sp each in 2 horses and Bacillus sp and Aeromonas sp each in one horse. Obligately anaerobic bacteria were not cultured in large numbers, although species such as Peptostreptococcus anaerobius, other anaerobic Gram-positive cocci, killonella sp, Bacteroides sp, Fusobacterium sp, Propionibacterium s p and Clostridium sp were recovered from 12 horses.
Discussion While there is considerable individual variation within the groups of horses in this study, it is evident from the results presented that neither repeated transtracheal washes nor confinement per se had a severe effect on the clinical, haematological, or transtracheal wash characteristics of horses. However, when horses were unable to lower their heads for periods from 24 to 48 h, there was a change in the characteristics measured. This suggests that the changes observed in this experiment were due to elevated head position. This experimentally produced observation is similar to those recorded previously by McClintock et al (1986), where horses were confined with elevated heads in floatation tanks used for the treatment of skeletal injuries. However, it has been shown that in healthy horses and in those with chronic lung disease, there may be little relationship between tracheal wash cytology and the pathological status of the lungs in individual horses (Larson and Busch 1985), and that cell populations in the trachea and lower down in the respiratory tract may differ substantially in such cases (Derksen et al 1989). Nevertheless, since a major objective of the present study was the identification of bacteria in the lower respiratory tract, transtracheal aspiration was chosen as a more suitable collection technique than bronchoalveolar lavage to obtain uncontaminated samples. In addition, samples were not collected from sites more 404
distal in the respiratory tree than the bronchial bifurcation in order to avoid collecting samples from too localised and, perhaps unrepresentative, segments of lung. In those horses whose heads were restrained in an elevated position, the presence of increased numbers of bacteria was probably the result of decreased clearance of contaminating pharyngeal flora and their subsequent multiplication in the lower respiratory tract. lngesta was observed endoscopically in the tracheas of the horses following transportation where they had been fed during the journey and prevented from lowering their heads (Sweeney et a1 1989). Although such obvious contamination of tracheal washes was not seen during the present study, aspiration of even small amounts of oropharyngeal secretions can present the lower respiratory tract with a substantial bacterial challenge (Finegold 1983). Also, elevated head position may have been unfavourable for mucociliary clearance (Turgut and Sasse 1989; Sweeney 1989), and a change in mucus volume and/or flow characteristics may also have decreased the clearance rate. In human beings, it has also been found that mucociliary clearance is delayed in the presence of purulent secretions due to decreased ciliary beat frequency (Wilson et all986). Drainage of respiratory secretions under gravity may also assist in lung clearance under normal circumstances. The reversal of tracheal wash changes, and the disappearance of clinical respiratory disease once mares were again allowed to lower their heads, suggests that postural drainage was certainly an important mechanism of clearing abnormal tracheobronchial secretions. Transtracheal aspirates from healthy horses are usually not sterile as there are a small transient population of predominantly aerobic and/or facultatively anaerobic bacteria in the lower trachea (Mansmann and Strouss 1976; Sweeney et al 1985). During the present study, there was a large increase in numbers of bacteria recovered from tracheal washes. This appeared to be due in nearly all cases to multiplication of the Actinobacillus/ Pasteurelfasp as well as Streptococcusequi subsp zooepidemicus. Interestingly, these speices have been isolated commonly from natural cases of pneumonia in horses (Jang and Hirsch 1987; Jang et a1 1987; Schlater et a1 1989). Many advanced cases of equine pneumonia involve obligately anaerobic bacteria, although large numbers of anaerobic bacteria may be isolated only where the duration of illness has been at least 5 d (Racklyeft unpublished). After postural head restraint for 24 h or 48 h during the present study, while obligately anaerobic bacteria were isolated, tracheal wash flora was still predominantly facultatively anaerobic. It is likely that multiplication and establishment of aerobic and facultative bacteria are necessary to provide appropriate environmental conditions for replication and invasion by the anaerobic components of a n infection. While this study has direct relevance to situations where horses are confined with their heads in an elevated position for prolonged periods of time, they were confined in stocks to which they were accustomed and demonstrated no behavioural evidence of stress (Creiger 1982). Previous studies investigating the association of transportation with equine lower respiratory tract infections have examined changes in aspects of respiratory immunity associated with the stress of transportation. They found mild decreases in the numbers and activity of pulmonary alveolar macrophages and other changes in bronchoalveolar lavage fluid over a one to 4 week period following transportation (Anderson e f al1985; Basaraba e t a / 1986) as well as delayed inflammatory response to a previous bronchoalveolar lavage (Traub-Dargatz et all988). Another study investigating transit stress in horses (Leadon et a1 1989) has documented haematologic abnormalities (neutrophilia and hyperfibrinogenaemia) in horses subjected to long distance journeys by air, which were similar to those which occurred during the present study in those horses restrained for 48 h. This study shows that restraint of horses’ heads in an elevated position for a prolonged period can lead to an increased bacterial load in tracheobronchial secretions. In the face of other stresses, such as transportation, where pulmonary defences may be suppressed, or where there is preexisting respiratory disease, the susceptibility of individual horses to progression to pneumonia may be enhanced. Since such infections frequently have an unfavourable outcome, all measures should be taken to minimise
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bacterial challenge to the lower respiratory tract. Thus, the crosstying of horses with their heads in an elevated position should be avoided, long journeys should be broken u p into 12 h stages, with horses allowed adequate opportunity and encouraged to lower their heads during rest stops, and transport facilities should be designed so horses can lower their heads periodically during long journeys.
Acknowledgments Funding for this study, provided by the New South Wales Racing Research Fund and Jean Walker Fellowship Trust, is gratefully acknowledged. L Patoka provided the bacteriological media.
References Anderson NV, DeBowes RM, Nyrop KA and Dayton AD (1985) - A m J Vet Res 4 6 2272 Basaraba R, Liggitt HD and Bayly WM (1986) - cited in Bayly WM, Liggitt HD, Huston LJ and Laegreid WW (1986) - A m Assoc Equine Pract 32: 253 Beech J (1981) - Equine Vet J 13: 136 Cohrs P (1967) - In Nieberle and Cohrs Textbook of the Special Pathological Anatomy of the Domestic Animal, revised edition, Pergamon Press, Oxford Coles EH (1986) - Veterinary Clinical Pathology, fourth edition, WB Saunders, Philadelphia Cowan ST and Steel KJ (1974) - Cowan and Steel’s Manual for the Identification of Medical Bacteria, second edition, Cambridge University Press; Cambridge Creiger S (1982) - Equine Vet Sci 2: 187 Derksen FJ, Brown CM, Sonea I, Darien BJ and Robinson NE (1989) - Equine Vet J 21: 23
Finegold SM (1983) - In Respiratory Infections: Diagnosis and Management, edited by Pennington JE, Raven Press, New York, p191 Holdeman LV, Cato EP and Moore WEC (1977) -Anaerobe Laboratory Manual, fourth edition, Virginia Polytechnic Institute and State University, Blacksburg, Virginia Jang SS, Biberstein EL and Hirsch DC (1987) - A m J Vet Res 4 8 1036 Jang SS and Hirsh DC (1987) - Vet Clinics of North Am: Equine Pract 3: 181 Kreig NR and Holt JG (1984) - In Bergey’s Manual of Systematic Bacteriology, Vol 1, William and Wilkins Co, Baltimore Larson VL and Busch RH (1985) - Am J Vet Res 46. 144 LennetteEH, Balows A, Hausler WJ and Shadomy HJ (1985) -Manual of Clinical Microbiology, fourth edition, American Society for Microbiology, Washington DC Mansmann RA (1983) - A m Assoc Equine Pract 2 9 71 Mansmann RA and Strous AA (1976) - J A m Vet Med Assoc 169 631 McClintock SA, Hutchins DR, Laing EA and Brownlow MA (1986) Equine Vet J 18: 462 Schlater LK, Brenner DJ, Steigerwalt AG, Moss CW, Lambert MA and Packer RA (1989) - J CIin Microbiol27: 2169 Sneath PHA, Mair NS, Sharpe ME and Holt JG (1986) - In Bergey’s Manual of Systematic Bacteriology, Vol2, Williams and Wilkins Co, Baltimore Sweeney CR (1987) - In Current Therapy in Equine Medicine 2, edited by Robinson NE, WB Saunders Co, Philadelphia, p592 Sweeney CR (1989) - A m J Vet Res 5 0 2135 Sweeney CR, Arthur RM, Merriam JG, Page BT and Wilson WD (1989) - Equine Pract 11: 29 Traub-Dargatz JL, McKinnon AO, Bruyninckx WJ, Thrall MA, Jones RL and Blancquaert AMB (1988) - A m J Vet Res 4 9 1026 Turgut K and Sasse HHL (1989) - Vet Rec 125: 526 Wilson R, Sykes DA, Currie D and Cole PJ (1986) - Thorax 41: 453 (Accepted for publication 13 June 1990)
BOOK REV1EWS Magrane’s Canine Ophthalmology.LC Helpec Lea and Febigec 1989. 287 pp. $89.75.
-
Pyrrolizidine Alkaloids Environmental Health Criteria 80. International Program on Chemical Safew WHO Geneva. 1988. 345 pp. $2100.
This historic text on the canine eye, first written in the 1960’s, has been revised by an eminent ophthalmologist, Lloyd C. Helper of the Universityof Illinois, a charter Diplomateof the American College of Veterinary Ophthalmologistsand now Associate Dean for Academic Affairs in the College of Veterinary Medicine. During his distinguished career Professor Helper, always helpful to his colleagues, has found time to visit Australia more than once. Veterinarians who are Magrane devotees can be reassured; this is a gentle revision very much in the original style. It presents a traditional approach to eye diseases with chapter headings on the normal eye, examination of the eye and its adnexa, ocular therapeutics, diseases and surgery of the lids and lacrimal apparatus, diseases and surgery of the conjunctive, diseases and surgery of the cornea and sclera, diseases and surgery of the uveal tract, diseases and surgery of the orbit, glaucoma, diseases and surgery of the lens, diseases of the vitreous, retina and optic nerve and ocular manifestations of systemic disease. While some excellent photographs,drawings and problem-oriented listings in the index are helpful, the reader presentedwith an ocular problem needs to know some ophthalmology before being able to find which parts of this book are the most relevant. A book of this size cannot and indeed should not “say it all” and does not pretend to be an exhaustive text on ophthalmic practice. Nevertheless it is an excellent first text to be read from cover to cover by practitioners who want to know more and develop an interest in the eye. The cover-to-cover reader, however, will need caution. You cannot always read-and-do. Some of the procedures described are best left to the ophthalmologist, for example, paracentesis of the anterior chamber and vitreocentesis.The easy how-to-do-itstyle of the text could leave the reader not sufficiently aware of the considerable hazard of these procedures in unskilled hands. New graduates and all those who realize they must keep vitally interested in clinical practice to avoid professionalburn-out would do well to buy this book. It is reassuring to find Australian techniques included and acknowledged in this text.
This is the latest and most comprehensivecompilation of information concerningpyrrolizidinealkaloids (PA’s). The publicationwas the work of a WHO expert committee which was chaired jointly by Dr CCJ Culvenor, late of the Division of Animal Health, CSIRO, Parkville, Victoria. The production of this monograph was stimulated by the serious and large-scaleoutbreaks of human pyrrolizidinosis, which have occurred in recent times in various parts of Asia. The book begins with a chapter on PA’s and human health, followed by others on properties and analytical methods, sources, and pathways of exposure, metabolism, mechanisms of toxicity and biological actions, effects on animals, effects on man, biological control of PAcontaining plants and finally a section on human health risks and effects on the environment. There are 3 very useful appendixes, namely a list of the PA’s and their plant sources, a list of plants containing hepatotoxic PA’s and a further list of plants that contain PA’s that are not hepatotoxic. There is a comprehensive list of key references to this field. The main plant groups in which PASoccur, namely the Crotalaria spp, Senecio spp and Boraginaceaeare well represented and widely distributed in Australia, both as native and naturalised introduced plants, and there would hardly be any major region of the country in which this type of toxicity would not at some time occur. This book would certainly be of value in the library of any of our veterinary diagnostic laboratories and at the price would be worth having by practitioners and government veterinary offices in whose area this type of toxicity is likely to be prevalent. In this respect the chapter on the Effects on Animals is of particular value, as it describesthe patterns of disease caused by the different plant genera in the various domestic species. This is an authorativestatement on our knowledgeof the alkaloids and the toxicity they cause and is highly recommended to all veterinarians and animal scientists concerned with nutritional toxicity of livestock.
R Blogg
AA SeawrigM
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