Primary Ciliary Dyskinesia in the Dog

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1 Primary Ciliary Dyskinesia in the Dog Wallace B. Morrison, DVM, Norman J. Wilsman, DVM, PhD, Leslie E. ox, DVM, and Cornelia E. arnum, DVM, PhD Electron microscopy was used to diagnose primary ciliary dyskinesia in a litter of English pointer dogs and in a golden retriever dog. A technique of membrane solubilization, fixation, and negative staining with glutaraldehyde tannic acid identified abnormally constructed central and B microtubules in respiratory cilia from dogs with primary ciliary dyskinesia. Shortened outer dynein arms commonly associated with primary ciliary dyskinesia actually represents the absence of a specific subset of the three most peripheral components of the whole outer dynein arm structure. (Journal of Veterinary Internal Medicine 1987; 1:67-74) PRIMARY ciliary dyshnesia (PCD) is a disease that is characterized by absent or deficient mucociliary clear- The core or axoneme of a motile cilium is composed of nine peripheral microtubule doublet pairs (A and B ance. Rhinosinusitus, bronchitis, bronchiectasis, and microtubules) and two central single microtubules. Each bronchopneumonia in PCD are attributed to abnormal respiratory ciliary function and ineffectual clearance of mucus from the airways. Mucus and mucus-secreting cells from patients with PCD have not been studied. Early in this century, Siewert, and later Kartagener, identified a triad of disorders in man consisting of situs inversus, chronic rhinosinusitis, and bronchiectasis, known today as Kartagener s syndrome. -3 In about 50% of the patients, in whom situs inversus is absent or bronchiectasis fails to develop, the condition has been termed immotile cilia syndrome.. Kartagener s triad represents a subset of patients with immotile cilia syndrome. Cilia from these patients may have random orientation and they may have abnormal motility (functional immotility), but it is rare that a given patient s cilia are actually immotile. The majority of such patients have cilia that are vigorously motile, but because of the configuration of the wave form and the orientation of the cilia, mucus transport is ineffe~tual.~ A better term to describe the abnormal functional characteristics of the A microtubules has a pair of dynein arms, an outer arm and an inner arm, that when viewed from the base of the cilium toward the tip, project clockwise toward the adjacent B microtubule of the next doublet pair (igs. 1, 2). Each dynein arm is composed of multiple polypeptides that assemble into morphologically recognizable subunits. Outer dynein arms are composed of 14 polypeptides that assemble into five morphologic subunits, the proximal foot, distal foot, the head (which is thought to contain the high-molecular weight dynein proteins), the connector, and the stalk, which attaches to the adjacent B tub~le.,~* Nexin links further connect the doublet pairs to one another. Radial spokes connect the peripheral doublet pairs to the central sheath and are thought to hinge on the central sheath at the spoke heads and thus facilitate movement (igs. 1-3).2,3*899 The list of ultrastructural defects claimed to be specific for ciliary functional dyskinesia is extensive, but regardless of the ultrastructural defect, the clinical features of the disease are similar. The defects include of cilia in such instances is primary ciliary dy~kinesia.~-~ shortened or absent dynein arms, absence of radial Congenital ciliary dysfunction slows mucus clearance, spokes, transpositioning of microtubules, and random which results in rhinosinusitus, bronchitis, and bron- orientation of cilia and their basal bodies. I- Only a few chopneumonia. Bronchiectasis results from chronic mucus plugging and inflammation of small airways. of the reports of PCD in the dog have been substantiated with electronmicroscopy (Table l).2,39 0 A variant of rom the Department of Veterinary Clinical Sciences, Iowa State University (Momson), Ames, Iowa; the Departments of Comparative Biosciences (Wilsman, arnum), and Medical Sciences (ox), University of Wisconsin-Madison, Madison, Wisconsin. Reprint requests: Dr. W. B. Momson, Department of Veterinary Clinical Sciences, Veterinary Teaching Hospital, College of Veterinary Medicine, Iowa State University, Ames, IA PCD exists in man, where immotile or dyskinetic cilia occur without ultrastructural defects. People with ultrastructural and or functional defects in their respiratory cilia may have a variety of associated clinical abnormalities that are related to organs with ciliated epithelia. Examples include renal fibrosis, dilated renal tubules, otitis media and hearing loss, male infertility, hydro-

2 68 MORRISON ET AL Journal of Veterinary Internal Medicine Outer dynein arm IG. I. Diagram of a normal cilium in cross-section. cephalus, frontal sinus hypoplasia, nasal polyps, and recurrent pne~monia.~,','~ Other associated conditions in man include defective neutrophil function; abnormalities of vertebrae, sternebrae, and ribs; neonatal respiratory distress; polysplenia; and extrahepatic biliary atresia Abnormalities associated with primary ciliary dyskinesia in dogs include, renal fibrosis, dilated distal renal tubules, abnormal sternebrae, vertebrae, and ribs, and mild hydrocephalu~.~,~,'~ Case Reports our 12-week-old female English pointer littermates were presented to the Iowa State University Veterinary Teaching Hospital, Ames Iowa, for evaluation of bilateral purulent nasal discharge. Three male and one female littermates were unaf- IG. 2. Cilium from a clinically normal dog in cross-section (original magnification X48,OOO). IG. 3. Cilium from an English pointer dog in cross-section showing absent and shortened outer dynein arms (original magnification X48,OOO). fected (a fifth male littermate was stillborn). A deceased female sibling to the dam of this litter was known to have had bilateral nasal discharge all of her life. All of the dogs with nasal discharge had a leukocytosis (17,900-27,000 WBC/pl), and three of four dogs had a monocytosis ( cells/pl). Urinalysis and serum chemistry values were normal. Two of the affected dogs died shortly after the initial examination. One pup died of parvovirus enteritis, and the other died of canine distemper. One pup had situs inversus of the thorax only and one had situs solitus. Both dogs had radiographic evidence of bronchiectasis and thickened tympanic bullae. The last sternal vertebra was malformed in one of these dogs. In both surviving dogs, mucopurulent, bilateral nasal discharge has persisted for more than 2 years despite antimicrobial therapy. The most frequently isolated microorganisms from the nasal exudate of these dogs have been Streptococcus zooepidemicus and Pjeudomonas aeruginosa. Both dogs have radiographic evidence of bronchiectasis. One of these dogs has situs inversus totalis, and one has situs solitus. Specimens of tracheal mucosa were obtained under thiopentol-induced general anesthesia. Inhalation anesthetics were avoided to minimize any potential effects upon ciliary motility. Some of the specimens were placed in Hank's balanced salt solution and observed under an interference contrast microscope. Ciliary motility was approximately one half that of a clinically normal animal of the same breed. Motile cilia had clear multiplanar motion indistinguishable from that of the clinically normal animal. Tracheal mucosal biopsy specimens of the two surviving affected dogs and two clinically normal dogs were processed for electron microscopy using a previously described method of membrane solubilization and fixation and negative staining with glutaraldehyde tannic acid.' ' Tracheal mucosal biopsy

3 VOl. 1. NO. 2 PRIMARY CILIARY DYSKINESIA IN THE DOG 69 TABLE 1. Summary of Clinical and Ultrastructural eatures of Dogs With Primary Ciliary Dyskinesis Age at Situs Breed Diagnosis Sex Inversus Ultrastructural Abnormalities Clinical eatures Radiographic indings Ref. No. Border collie 7 weeks M N.D. Partial cleft of soft palate Abnormal tooth English 8 months N.D. setter Doberman 7 weeks M N.D. pinscher Chihuahua English springer spaniel Golden retriever Old English sheepdog English pointer* Golden retriever* 15 months 4 months 7 months 8 weeks Chronic cough Increased density of 17 nasal cavity and frontal sinus Bronchitis Alveolar and interstitial lung densities (bronchopneumonia) Bronchitis N.D. Random orientation of cilia Chronic cough Agenesis of right 13th rib Hypoplastic turbinates Hypoplastic frontal sinuses Thickened tympanic bullae Hypoplastic rostra1 calvarium Hypoplastic right orbit Malformations of vertebrae and sternebrae Increased bronchial pattern Bronchointerstitial M M Abnormal dynein arms Chronic cough Hydrocephalus () pattern with dependent alveolar consolidation Bronchiectasis () () Increased interstitial 2 Chronic cough pattern Bronchitis M Abnormal dynein arms Absent central microtubules ibrous ring under cell membrane M 12 weeks No No Transposition of microtubules Random orientation of cilia Electron-dense material in basal bodies Dynein arm abnormalities Radial spoke abnormalities Nexin link abnormalities Transposition of microtubules Electron-dense material in basal bodies Dynein arm abnormalities B-microtubule abnormalities Central microtubule pair abnormalities Dynein arm abnormalities B-microtubule abnormalities Central microtubule pair abnormalities Hydrocephalus (M) 14 months M * The dogs of this report. N.D. = not done. Bronchiectasis Bronchiectasis Thickened tympanic bullae Bronchitis Mild bronchiectasis 19 10

4 70 MORRISON ET AL Journal of Veterinary Internal Medicine sence of one or both central microtubules, and absence of both central microtubules combined with a transpositioning of a peripheral microtubular doublet pair to a central position. (ig. 6) In profiles of 300 cilia, 92% of cilia observed in one English pointer and 94% of cilia in the other English pointer were abnormal in one or more A IG. 4. Cilium from an English pointer dog in cross-section showing an absence of both central microtubules and transposition of a peripheral doublet pair to the center (original magnification X48,OOO). specimens of the dog that died of parvovirus enteritis were taken immediately after death and processed for electron microscopy using standard glutaraldehyde fixation. A 14-month-old golden retriever was presented to the University of Wisconsin-Madison Veterinary Teaching Hospital for evaluation of bilateral purulent nasal discharge noted at 10 days of age. Mild azotemia (BUN 47 mg/dl, creatinine 1.8 mg/dl), attributed to 4 weeks of gentamicin therapy prior to evaluation at the University of Wisconsin-Madison, resolved with active diuresis. The urine was isotheuric (specific gravity 1.010) but otherwise normal. (27,600 WBC/pl) and monocytosis (1380 cells/jd) were present. Situs inversus totalis, bronchitis, mild bronchiectasis, and bronchopneumonia were observed radiographically. All sperm from this dog were immotile. Biopsy specimens from the dogs tracheal mucosa were taken as previously described. Microorganisms isolated from nasal exudate and bronchial washings included P. aeruginosa, Clostridium perfringens, and Bacteroides fragilis. Ultrastructural indings Specific ultrastructural defects of cilia in surviving English pointer dogs consisted of complete or partial absence of outer dynein arms, transposition of microtubules, and electron-dense basal bodies (igs. 4, 5B, 5C). All of these ultrastructural characteristics are considered to be specific for PCD. The central microtubule pair and the A microtubule of the microtubular doublets normally contain 13 protofilaments, whereas the B microtubule contains 10 protofilaments. Various protofilaments from the B microtubules were occasionally absent (ig. 6). Abnormalities of the central microtubules consisted of incompletely formed central microtubules, ab- IG. 5. Higher-magnification micrographs illustrating normal outer dynein arms from a clinically normal dog (A), and shortened outer dynein arms (3) and absent outer dynein arms (C) from an affected dog (original magnification X48,OOO).

5 VOI. 1. NO. 2 PRIMARY CILIARY DYSKlNESlA IN THE DOG 71 ultrastructural features (Table 2). Similar ultrastructural defects were observed in the dead English pointer. Identical ultrastructural defects were observed in the golden retriever, and all 300 ciliary profiles were abnormal in one or more ultrastructural features (Table 2). The individual protofilaments that comprise the microtubules and microtubular doublets, the dynein arms, and the radial spokes were easily resolved following TABLE 2. Percentage of 300 Ciliary Profiles With Various Abnormalities English English Golden Abnormality Pointer (I) Pointer (2) Retriever Shorter outer dynein arms only Outer arm plus B-tubule Outer arm plus central tubule Outer arm, central and B-tubule Normal 56% 53% 1% 14% 22% 19% 7% 8% 8% 6% 38% 15% 42% 5% 0% IG. 6. Examples of outer dynein arm abnormalities in combination with other ultrastructural defects. All cilia are from the three surviving dogs and all have shortened or absent dynein arms. A illustrates a cilium with an absent central microtubule and incomplete B microtubules. B illustrates a cilium with a missing central microtubule. The second central microtubule consists of only four protofilaments. C illustrates a cilium with an incompletely formed central microtubule consisting of only seven protofilaments. D illustrates a cilium with extra A and B microtubules. Compound cilia such as these can be normal, but the cilium shown here contains shortened and absent outer dynein arms. E illustrates a compound (giant) cilium containing multiple axonemes. The ciliary axoneme that is in sharp focus has short and absent outer dynein arms (original magnification X48,OOO). membrane solubilization and fixation and negative staining with glutaraldehyde tannic acid in comparison with the appearance of these structures in standard preparations. All of the ultrastructural abnormalities were present in all affected dogs but not in clinically normal animals. Discussion One of the primary defense mechanisms associated with the respiratory system involves the trapping of inhaled particles and microorganisms in a blanket of mucus. The mucus is secreted by goblet cells and submucosal glands, and overlies the respiratory epithelial cells. Respiratory cilia move this blanket of mucus toward the pharynx, where it is disposed of by unconscious swallowing. The normal mucociliary clearance mechanism, therefore, involves the coordinated activities of two complex components (the mucus and the respiratory cilia). Movement of cilia and hence movement of the mucus blanket are effected through a sliding of adjacent microtubular doublets. The energy is provided by hydrolysis of ATP by the high-molecular weight dynein proteins found in the dynein arm^.^,^ Cilia in normal ciliated respiratory epithelium have a constant orientation with respect to the pharynx. The plane of the central microtubular pair is approximately parallel in adjacent cilia and perpendicular to the direction of ciliary beat. The basal feet (lateral projections from the basal bodies) point iir the direction of the effective ciliary beat.3 Abnormal ultrastructure and orientation result in dyskinetic motion or functional immotility. The ultrastructural lesions observed in the two English Pointer dogs included shortened outer dynein arms and abnormalities of the central and B microtubules. Abnormalities of the B microtubules have not been reported previously in either dogs or humans. The shortened outer dynein arm represents the absence of the three most peripheral subunits, including the head subunit thought to be the location of the high-molecular weight dynein proteins. The short arm represents the presence of the proximal foot, the distal foot, or both the proximal and distal feet superimposed on one another (ig. 5). The techniques used in this study are not capable of discriminating among these possibilities.

6 72 MORRISON ET AL Journal of Veterinary Internal Medicine The incidence of ciliary microtubular abnormalities in clinically normal dogs is approximately 2%, whereas isms that have been isolated from dogs with PCD represent a proliferation of normal flora or represent organin clinically normal humans it is 2-5%.63 Abnormal isms that are not removed because of ineffective ciliary axonemal patterns from one dog with PCD were present in 9% of cilia examined.2 This compares with abnormal patterns in % of the cilia from the dogs in this study. It was not possible to quantity ciliary beat frequency in these dogs. However, previous experience in measuring ciliary beat frequency in normal dogs by using a Chawick-Helmuth (Chawick-Helmuth, El Monte, CA) xenon strobelight interfaced into the light path of a interference contrast microscope allowed the observation that the cilia were beating at a rate slower than normal. The beat frequency remained too rapid to be counted in a simple wet mount preparation. Almost all cilia in the English pointer dogs of this study had short outer dynein arms (95%), but only about 40-50% of these cilia were immotile. Several studies have shown that the outer dynein arms of cilia are not required for motility or normal wave form but are necessary for normal ciliary beat frequency.29- Based on these observations, we speculate that the cilia with short outer dynein arms are motile but beat at a slower rate than normal. We also speculate that abnormalities associated with the central microtubules and the B microtubules do result in immotile cilia since Chlamydomonas beating. The most common microorganisms isolated from people with PCD have been Hemophilus influenzae and Diplococcus pneumoniae; whereas, in dogs, we and others have found a variety of Streptococcus organisms and gram-negative rods.35 Mycoplasma species have been previously reported in dogs with PCD, and they were also cultured from the surviving dogs of this Canine antisera used to identify the species of mycoplasmas isolated from the dogs of this study included M. bovigenitalium, M. canis, M. maculosum, M. spumans, and Acholeplasma laidlawii. Only M. maculosum was positively identified in one English pointer. Although Mycoplasma organisms may attach to respiratory epithelial cells, cause cell injury, and disrupt normal ciliary function, their significance in these patients is ~nknown.~~.~ The groups of microorganisms in man and dog are different, but these groups parallel the normal flora in the respective specie^.^*-^' The mode of inheritance of PCD in dogs is unclear. In man, it is reported to be an autosomal recessive trait that is not ~ex-linked.~ However, transmission based upon a single defective allele that results in the multiple ultrastructural abnormalities associated with PCD seems unlikely. Recent biochemical studies of the ciliary axomutants that lack central microtubules are imm~tile.~~ neme (core) have indicated that the ciliary axoneme is Our observation of 40-50% immotility in cilia from these dogs fits closely with the actual incidence of combined B and central microtubule abnormalities observed. One of the significant aspects of this study of dogs is the variation of microorganisms which have been isolated over time. While primary ciliary dyskinesia is not curable, this study documents that it is manageable. A key element in its management is the monitoring of the infecting microorganisms and occasional use of antibicomposed of polypeptides and that each polypeptide is probably coded for by a different gene.,32 Nevertheless, PCD does have some genetic basis. Sperm tails have the same arrangement of microtubules as the axonemes of respiratory cilia. In human beings affected with PCD, the same ultrastructural defects can be found in respiratory cilia and spermatozoan tails. Although the sperm are alive, infertility frequently results from poor m~tility.~,~, ~,~~ On rare occasions, persons with dyskinetic respiratory cilia have structurotics to which these microorganisms are sensitive. The ally normal and fully motile spermato~oa.~~ Spermatolong-term prognosis of primary ciliary dyskinesia in zoa from dogs with PCD have been poorly character- - man is good.33 Even in patients with little mucociliary transport, coughing is an effective method of mucus removal and affected human patients can have normal lifespans. Most of the dogs in the previous reports of primary ciliary dyskinesia have been euthanatized or have died at an early age.2~3, 0~15, ne group of dogs with PCD has been maintained for at least 5 years.34 requently, multiple microorganisms were cultured simultaneously from the trachea or from the nasal exudate. Whenever bacterial isolates from tracheal washings and nasal exudates were compared, the same microorganisms were isolated from both sites. This allows more confidence in monitoring the respiratory flora in these patients from nasal exudate alone. It is interesting to speculate whether the microorgan- Hearing loss from otitis media is attributed to chronic obstruction of the ciliated auditory (eustachian) tube. Hearing loss is difficult to quantitate in dogs, but abnormal thickening of the tympanic bulla suggesting otitis media has been observed in two dogs of this report and in one previous report.i3 Hydrocephalus in association with PCD may be due to ineffective ependymal cilia lining the ventricular system of the brain which impairs normal flow of cerebrospinal fluid. Hydrocephalus has been reported previously in dogs with PCD.3,10 Normal adult human kidneys usually contain single or occasionally two primary cilia per cell throughout the nephron. The renal tubular cilia differ from respiratory

7 VOl. 1. NO. 2 PRIMARY CILIARY DYSKlNESlA IN THE DOG 73 cilia in that they usually lack a central pair of microtubules.26 The renal disorders accompanying some cases of PCD may be related to ultrastructural and functional abnormalities of these renal cilia. The relationship of situs inversus to PCD is unknown. Situs inversus by itself is asymptomatic, but every fourth or fifth person with situs inversus has rhinosinusitis and bronchiectasis. In human beings of North American (White Anglo-Saxon) or of northern European ancestry, the estimated incidence of situs inversus is 1 in 10,000.2 Several hypotheses have been advanced to explain the association of situs inversus and ciliary motility. One hypothesis states that normal coordinated beating of cilia on embryonic epithelia is necessary for normal visceral ~rientation.~ In the absence of this normal coordinated motility, visceral orientation is determined by chance, so that 50% of the time visceral orientation would be to the left and 50% of the time it would be the right. If this hypothesis is correct, it means that the situs inversus observed in humans and in dogs with PCD may not have a genetic basis. Another hypothesis regarding visceral orientation is based on observations in mice. The hypothesis in this model of situs inversus suggests that normal visceral rotation is controlled by a dominant gene that, when absent, allows for the determination of visceral orientation that is based on chance. In either case, approximately half of patients with PCD have situs inversus and hence Kartagener s syndrome. In the reports of PCD in dogs, more than half have had situs inversus. The population of dogs with PCD that has been reported is probably biased toward animals with Kartagener s syndrome because situs inversus is an easily recognized feature. An interesting aspect of situs inversus of the thorax is the electrocardiographic diagnosis of dextrocardia that is reflected by inversion of lead I and transposition of leads I1 and 111. As a clinical feature associated with PCD, situs inversus remains an important finding. Confirmation of a diagnosis of PCD, at present, requires ultrastructural observations, which are not always available. However, PCD should be considered whenever chronic respiratory disease is observed, and with increasing certainty when in combination with bronchiectasis and situs inversus or in combination with immotile or abnormal sperm in dogs. The incidence of PCD in domestic animals is unknown, but it may be more prevalent than is generally realized. or example, PCD has never been diagnosed in cats, horses, cows, or pigs despite the high incidence of respiratory disease in these species. Dogs with PCD may also have bronchopneumonia and the associated clinical signs of fever, and ocular and nasal discharge can mimic canine distemper. The true incidence of PCD in domestic animals will never be known unless it is included in the differential diagnosis of chronic respiratory disease in all species. Summary Primary ciliary dyskinesia is a very heterogenous syndrome. Clinical signs are apparent early in life. The prognosis for dogs with primary ciliary dyskinesia is good, although continual medical monitoring is necessary to prevent bronchopneumonia. Short outer dynein arms were the most common ultrastructural abnormality found in the dogs of this study. We speculate that cilia with short outer dynein arms are motile but beat at a slower rate than normal. We also speculate that abnormalities of the central microtubules and the B microtubules do result in immotile cilia. References 1. Afzelius BA. Genetical and ultrastructural aspects of the immotile-cilia syndrome. Am J Hum Genet 1981; 33: Afzelius BA, Carlsten J, Karlsson S. Clinical, pathologic, and ultrastructural features of situs inversus and immotile-cilia syndrome in a dog. J Am Vet Med Assoc 1984; Edwards D, Patton CS, Bemis DA, et al. Immotile cilia syndrome in three dogs from a litter. J. Am Vet Med Assoc 1983; 183: Newhouse MT. Immotile-cilia: syndrome and ciliary abnormalities induced by infection and injury. Am Rev Respir Dis 1982; 3: Sleigh MA. Primary ciliary dyskinesia. Lancet 1981; 2: Rossman CM, Lee RM, orrest JB, et al. 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