STK36 splice site variant in an Australian Shepherd dog with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) represents a group of diseases characterized by impaired movement of cilia and subsequent health problems in diverse organ systems, notably the respiratory tract. Almost 50 candidate genes for PCD are known in humans. In this study, we investigated an Australian Shepherd dog with a history of recurrent respiratory infections and nasal discharge. A transmission electron microscopy investigation led to the diagnosis of PCD with central pair defect, in which the normal 9:2 arrangement of respiratory cilia was altered and reduced to a 9:0 arrangement. Whole genome sequencing data from the affected dog was obtained and searched for variants in PCD candidate genes that were not present in 918 control genomes from different breeds. This revealed a homozygous single base pair exchange at a splice site of STK36, XM_038585732.1:c.2868-1G>A. The mutant allele was absent from 281 additionally genotyped Australian Shepherd dogs. RT-PCR confirmed aberrant splicing in the affected dog with the skipping of exon 20 and the insertion of a cryptic exon, which is predicted to lead to a premature stop codon and truncation of 36% of the STK36 wild-type open reading frame, XP_038441660.1:(p.Met957Profs*11). STK36 variants were previously reported to cause PCD in humans and mice. The knowledge from other species together with the absence of the mutant allele in more than 1000 control dogs suggests STK36:c.2868-1G>A as the most likely candidate variant for PCD in the investigated case.

Situs inversus in Dpcd/Poll-/-, Nme7-/- , and Pkd1l1-/- mice.

Situs inversus (SI) is a congenital condition characterized by left-right transposition of thoracic and visceral organs and associated vasculature. The usual asymmetrical positioning of organs is established early in development in a transient structure called the embryonic node. The 2-cilia hypothesis proposes that 2 kinds of primary cilia in the embryonic node determine left-right asymmetry: motile cilia that generate a leftward fluid flow, and immotile mechanosensory cilia that respond to the flow. Here, we describe 3 mouse SI models that provide support for the 2-cilia hypothesis. In addition to having SI, Dpcd/Poll(-/-) mice (for: deleted in a mouse model of primary ciliary dyskinesia) and Nme7(-/-) mice (for: nonmetastatic cells 7) had lesions consistent with deficient ciliary motility: Hydrocephalus, sinusitis, and male infertility developed in Dpcd/Poll(-/-) mice, whereas hydrocephalus and excessive nasal exudates were seen in Nme7(-/-) mice. In contrast, the absence of respiratory tract lesions, hydrocephalus, and male infertility in Pkd1l1(-/-) mice (for: polycystic kidney disease 1 like 1) suggested that dysfunction of motile cilia was not involved in the development of SI in this line. Moreover, the gene Pkd1l1 has considerable sequence similarity with Pkd1 (for: polycystic kidney disease 1), which encodes a protein (polycystin-1) that is essential for the mechanosensory function of immotile primary cilia in the kidney. The markedly reduced viability of Pkd1l1(-/-) mice is somewhat surprising given the absence of any detected abnormalities (other than SI) in surviving Pkd1l1(-/-) mice subjected to a comprehensive battery of phenotype-screening exams. However, the heart and great vessels of Pkd1l1(-/-) mice were not examined, and it is possible that the decreased viability of Pkd1l1(-/-) mice is due to undiagnosed cardiovascular defects associated with heterotaxy.

Primary ciliary dyskinesia in a Staffordshire bull terrier.

Primary ciliary dyskinesia (PCD) is a diverse group of inherited structural and functional abnormalities of the respiratory and other cilia, which results in recurrent respiratory tract infections. Primary ciliary dyskinesia was diagnosed in a 14-week old Staffordshire bull terrier that had a history of respiratory disease from 7 weeks of age. Pneumonia was diagnosed on thoracic radiographs and transtracheal aspirate. Transmission electron microscopy of the bronchi and trachea indicated the presence of both primary and secondary ciliary dyskinesia. The most prominent primary defects consisted of absent inner dyneim arms, absent radial spokes and absence of the central microtubules. These defects accounted for 62% of the total number of cross-sections screened. Non-specific ciliary abnormalities encountered most often were compound cilia, swollen cilia, addition/deletion of peripheral doublets and disorganised axonemes (26%). To the authors' knowledge, this is the first case of PCD described in the Staffordshire bull terrier and the first report of PCD in South Africa.

Use of ciliogenesis in the diagnosis of primary ciliary dyskinesia in a dog.

Primary ciliary dyskinesia is a congenital condition that may cause chronic rhinitis and bronchopneumonia. Primary ciliary dyskinesia may be diagnosed by induction of ciliogenesis by use of in vitro cell culture. Induction of ciliogenesis allows for differentiation between primary and secondary ciliary dyskinesia.

Primary ciliary dyskinesia in Newfoundland dogs.

Primary ciliary dyskinesia was diagnosed in three Newfoundland dogs with histories of chronic rhinitis and bronchopneumonia from an early age. Thoracic radiographs of two of them showed severe, dependent bronchopneumonia and right displacement of the cardiac apex but normal positioning of other organs. Histopathological examination of sections of lung from the other dog showed severe bronchopneumonia. A semen sample from one dog had a high percentage of spermatozoa with abnormal tails and poor progressive motility. Transmission electron microscopy of nasal brushings from all three dogs showed consistent ultrastructural defects in the cilia, including an absence of outer and inner dynein arms, disorganisation of peripheral doublets, occasional supernumerary doublets and singlets, and consistently disorganised basal bodies and foot processes; sections of trachea from one dog also had disorganised basal bodies. Pedigree analysis was consistent with a monogenic autosomal recessive pattern of inheritance for the defect. One dog is still alive, one dog died aged five years two months, and one dog was euthanased aged nine months. This is the first time primary ciliary dyskinesia has been reported in Newfoundland dogs.

Abnormal length of respiratory cilia in a pig. An ultrastructural study.

The authors report the first case of abnormal length of respiratory cilia in a domestic animal (18 microns versus normal length about 5 microns). These cilia lie on the carpet of cilia of normal length. The width of some of these cilia is also abnormal, measuring 0.5-0.6 microns. Other cilia have hook-shaped tips that could be responsible for an effective beat. We suggest that the ineffective ciliary beat could also be due to the undulating movements of abnormally long cilia, and stress the need for further morphological and biochemical studies of respiratory cilia in pigs. Respiratory pathology is a very common finding in pigs and is responsible for remarkable economical losses.

Primary ciliary dyskinesia in the dog.

A disorder caused by congenital ciliary dysfunction occurs in dogs. Most of the clinical signs are directly or indirectly attributable to immotile or dyskinetic cilia and spermflagella. Due to severely impaired mucociliary clearance, a continuous mucoid nasal discharge and intermittent sneezing and coughing are typically observed during the neonatal period. Recurrent bacterial rhinosinusitis and bronchopneumonia usually start within a few weeks of birth. Hypoplastic nasal sinuses and atresia of the frontal sinuses are variable features of the disease that may be caused by neonatal colonization of these structures by specific bacteria. Bronchiectasis is an acquired lesion resulting from chronic inflammation and obstruction of airways. A secretory otitis media is caused by dysfunction of the cilia in the middle ear, and is manifested in some dogs by sclerotic tympanic bullae. Male infertility is caused by live, but immotile to hypomotile spermatozoa; however, unexplained oligospermia and azoospermia have been reported. Hydrocephalus and situs inversus are common but variable features of the disease; the genesis of these lesions has not yet been determined. The probable mode of inheritance is autosomal recessive, but dominant mutations cannot be excluded. The diagnosis can be confirmed by demonstrating the absence or near absence of nasal or tracheal mucociliary clearance and the presence of a specific ultrastructural lesion in a large percentage of cilia from multiple sites (airways, middle ear, or oviduct). The ultrastructure of sperm flagella should mirror that of the cilia. Not all dogs have ultrastructural ciliary lesions, and in these cases, results of in vitro analysis of ciliary activity may be highly suggestive, if not diagnostic. In dogs without mucociliary clearance in which structural and functional analysis of cilia are not diagnostic, confirmation of congenital ciliary dysfunction can be established only by ruling out other diseases with similar signs (e.g., congenital immunodeficiency syndromes). The clinical course in an longevity of affected dogs are highly variable. Appropriate antibiotic treatment and pulmonary physical therapy may result in prolonged survival, although cor pulmonale and reactive systemic amyloidosis are potential sequelae of chronic hypoxia and chronic bacterial infection of the airways, respectively.

Neutrophil function in dogs with congenital ciliary dyskinesia.

In vitro neutrophil function was assessed in two English Springer Spaniel dogs, two Bichon Frise dogs, and one Chow Chow dog with congenital ciliary dyskinesia; three clinically normal English Springer Spaniel dogs that were presumed heterozygous for congenital ciliary dyskinesia; and five control dogs. Chemotaxis and random migration in affected and heterozygous dogs were found to be comparable to those of control dogs. Increased (P less than or equal to 0.05) neutrophil adhesion, antibody dependent cell-mediated cytotoxicity, iodination of proteins, and oxygen radical production in neutrophils from affected dogs were probably the result of chronic bacterial infection in vivo. Bacterial ingestion by neutrophils from the three heterozygous English Springer Spaniel dogs was significantly increased compared to control dogs but was not different from affected English Springer Spaniel dogs, suggesting a breed-related phenomenon. Significant decreases in neutrophil function were not seen in any of the dogs with congenital ciliary dyskinesia, indicating that a defective microtubular system is not shared by respiratory cilia and neutrophils and that defective neutrophil function does not contribute to respiratory infection.

Primary ciliary dyskinesia in pigs.

The first description of a familial immotile cilia syndrome diagnosed through ovario-hysterectomy in six siblings of pigs has been performed. This report may indicate another possible cause of reproductive failure in domestic animals. In fact, the immotile cilia syndrome has not been considered from this point of view in veterinary medicine. This case emphasizes that a study about ciliary motility in the female reproductive tract in domestic animals is absolutely necessary to improve current knowledge about different causes of infertility. Up to date, it remains unknown what degree of ciliary motility is necessary for mammalian fertility in females.

Suspected ciliary dysfunction in Chinese Shar Pei pups with pneumonia.

Chronic pneumonia was investigated in a litter of young Chinese Shar Pei in which 4 of 6 dogs were affected. Serum immunoglobulin concentrations (IgA, IgG, IgM) determined by radial immunodiffusion varied over time, but were not consistently lower in affected dogs, compared with control dogs. Two dogs that died had hydrocephalus and lymphoid depletion, in addition to severe broncho-pneumonia. Evaluation of ciliary ultrastructure in 2 affected dogs revealed random orientation of adjacent respiratory tract or oviductal cilia and a greater number of microtubular disarrangements, compared with control dogs. In vivo tracheal mucociliary clearance of 99mtechnetium macroaggregated albumin was absent in 1 dog examined. The ciliary abnormalities were suspected to have resulted in an inefficient mucociliary transport system predisposing to the development of pneumonia. Further evaluation of 1 Chinese Shar Pei revealed lymphocyte mitogenesis results that were not consistently less than those of a control dog, normal total hemolytic complement values, and normal blood neutrophil chemotaxis.

Familial immotile-cilia syndrome in English springer spaniel dogs.

A laboratory-maintained colony of English springer spaniel dogs heterozygous for a putative autosomal recessive immotile-cilia syndrome (ICS) has been studied. Matings between dogs thought to be heterozygous for ICS resulted in 22 pups, five (three males and two females) of which were homozygous for ICS. Four of the five ICS-affected dogs had chronic rhinitis and bronchopneumonia. The other dog had a serious nasal discharge and died at 10 days. Four dogs had situs inversus totalis (kartagener syndrome), and the two males of reproductive age were azoospermic. In the two ICS dogs studied for ciliary function, in vivo mucociliary clearance was absent, and in vitro ciliary beat was rarely observed and of low frequency. Scanning and transmission electron microscopy disclosed the same lesions in respiratory cilia from all dogs with ICS, including random orientation and partial outer dynein arm deficiency. Four of five dogs with ICS had dilated lateral ventricles. One female pup with neonatal rhinitis and bronchopneumonia, situs solitus, and dilated lateral ventricles was presumed to be homozygous for ICS, but died without functional or structural confirmation of defective respiratory cilia. An autosomal recessive mode of inheritance for the ciliary defects and respiratory signs of ICS in these dogs is proposed.

Assessment of neutrophil function in dogs with primary ciliary dyskinesia.

Compared with neutrophils from healthy dogs, neutrophils from 2 dogs with primary ciliary dyskinesia had increased distance of random migration, but fewer of the neutrophils migrated. The affected dogs had an increase in the numbers of Staphylococcus aureus phagocytized. Lymphocyte blastogenesis in the affected dogs in response to standard mitogens was considered to be normal.

Immotile cilia syndrome in an aged dog.

An 11-year-old Dalmatian was examined and treated for bilateral nasal discharge and cough of 6 months' duration. Response to medical treatment and surgical intervention was unsatisfactory. Histologic examination of lung tissue revealed chronic severe catarrhal bronchitis and bronchiolitis with bronchiectasis. Histologic findings and barium sulfate bronchography indicated abnormal mucociliary clearance in the respiratory tract. Electron microscopy revealed abnormalities or deletions of outer and/or inner dynein arms in 26% of the ciliary profiles from the affected dog. Similar abnormalities were not found in 500 ciliary profiles from age- and gender-matched control dogs.

Primary ciliary dyskinesia in the dog.

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.

Clinical, pathologic, and ultrastructural features of situs inversus and immotile-cilia syndrome in a dog.

Situs inversus, rhinitis, and bronchitis were diagnosed in a 7-month-old male Golden Retriever. Electron microscopic examination of tracheal and bronchial biopsy specimens revealed 9% of the cilia with abnormal axonemal pattern and 1.2% with a fibrous ring between the circle of microtubular doublets and the cell membrane. A permanent response to treatment with antibiotics was not obtained and the dog was euthanatized. At necropsy the thoracic and abdominal organs were found to be in reversed position. Microscopic examination revealed evidence of chronic bronchitis as well as cystic and distended distal tubules of the kidneys. Foci of fibrosis were observed in the renal cortex. The diagnosis was immotile-cilia syndrome.