Situs Inversus Totalis and Primary Ciliary Dyskinesia (Kartagener's Syndrome) in a Horse

A female Standardbred yearling was presented at the Faculty of Veterinary Medicine, Ghent University, with a history of recurrent respiratory problems characterized by tachypnea, nasal discharge, and intermittent coughing since birth. During 1st year of life, the horse had been examined on 4 occasions in a veterinary hospital. On each occasion, tracheal aspirates revealed numerous neutrophils and bacteriological culture yielded 3 times Streptococcus equi subsp. zooepidemicus and once Streptococcus equi subsp. equi. After treatment with antibiotics and bronchodilators, the foal improved, but relapsed when medication was stopped. At the time of examination the horse had bilateral mucopurulent nasal discharge and was in poor body condition (body condition score 3/9). Rectal temperature was normal, respiratory rate was slightly increased, and on auscultation generalized “moist” lung sounds were heard. Hematology and serum biochemical values were within normal limits. Endoscopic examination showed mucopurulent exudate in the trachea and in the large bronchi, but no other upper or lower respiratory tract abnormalities were seen. An aspirate of the tracheal mucus revealed a leukocyte count of 52,000/mm3 with a differential count of 90% neutrophils and 10% macrophages. On bacteriological examination of the aspirate, Streptococcus equi subsp. zooepidemicus was isolated. Differential cell count of bronchoalveolar lavage fluid revealed 45% macrophages, 38% lymphocytes, 16% neutrophils, and 1% eosinophils. Ultrasonographic examination of the lungs revealed a relatively smooth visceral pleural surface, but in the ventral part of the lungs radiating comet-tail artifacts were seen. Because of the recurrent respiratory infections without obvious underlying causes, the possibility of an immune deficiency was investigated. No major dysfunction of the immune system could be detected. Serum concentrations of IgA, IgM, and IgG were within reference range, and analysis of the numbers of circulating IgM+, CD2+, CD4+, CD8+, or CD11a+ cells did not reveal significant imbalances in these cell populations. The horse continued to suffer from recurrent respiratory disorders and remained in a poor body condition. Therefore a problem in mucus composition, mucociliary clearing, or both was included in the differential diagnosis and the horse was re examined. An ultrasound examination of the thorax revealed a laterally reversed heart without any other cardiac malformations. On abdominal ultrasound, all abdominal organs were found in a complete mirror image of the normal situs. Rectal palpation confirmed the location of the spleen and a kidney on the right side, the caecum on the left side, whereas on the left side no kidney could be palpated. Nasogastric intubation showed the esophagus to be located on the right side of the trachea. The diagnosis of situs inversus totalis was made. Because of the coexistence of situs inversus totalis and mucus accumulation in the respiratory tract, the latter was supposed to be caused by a defective mucociliary clearance caused by primary ciliary dyskinesia (PCD). Because of the unfavorable long-term prognosis, the horse was euthanized and necropsy was performed. At necropsy a total left-right inversion of all the thoracic and abdominal organs was found. Macroscopically, the organs were morphologically normal with normal anatomical relations and ligaments adapted to the laterally reversed position. Inspection of the respiratory system revealed mucopurulent rhinitis, catarrhal sinusitis, and chronic tracheitis. Microscopically, there was mucopurulent bronchitis with infiltration of plasma cells, lymphocytes, and fibroblasts, focal chronic purulent bronchopneumonia, and chronic fibrotic pneumonia. Samples from epithelium of the nasal cavity, the trachea, and the bronchi were taken for light microscopic, transmission, and scanning electron microscopic (TEM-SEM) examination and for cell culture. With light microscopy almost no cilia were seen on the nasal epithelium and only few cilia were detected in the trachea. Therefore, ciliary functional analysis with the determination of the ciliary beating frequency (CBF) and the coordination of ciliary activity could not be performed. This analysis of the ciliary function can contribute to the diagnosis of PCD, especially when no ultrastructural abnormalities are found.1 Cell culture of nasal and tracheal epithelium was performed following the procedure described by Jorissen et al 2 for human nasal epithelial cell culture. The ciliogenesis in culture did not succeed because of overgrowth of yeasts and molds. For this reason, the ultrastructural analyses could be performed only on biopsy material and not on cultured cells. With SEM almost no cilia were seen on the biopsy of the nasal epithelium, few cilia were seen in the trachea (Fig 1), and also a reduced number of cilia were seen in the biopsy of the bronchial epithelium. Scanning electron microscopy of a biopsy of the trachea, which clearly shows the general lack of cilia. On the majority of the cell surfaces only microvilli and no cilia are seen. TEM was performed on biopsies of 9 areas of the trachea. Because of the lack of cilia in the nasal cavity, TEM of the nasal epithelium could not be performed. Analyses of 46 ciliary cross sections of the trachea revealed significant ultrastructural abnormalities in 31 of them. Outer and inner dynein arms could be counted in 3 cilia, but no deficiency of either of the dynein arms was noticed (Fig 2). In 26 cilia (56%) the central pair had an eccentric location (Fig 3), which is caused by defective radial spokes,3 and 23 of these cilia (50%) also had transposition of peripheral microtubules (Fig 4). In 4 cilia (9%), 2 central pairs were seen instead and in 1 cilium there was a complete disorganization of the microtubules. In conclusion, 67% abnormal cilia and 65% central pair defects were found, which is indicative of the presence of PCD.3 Drawing of the ciliary ultrastructure (9 + 2). P = central pair; A,B = peripheral microtubules; I,O = inner and outer dynein arm; S = radial spoke; C = central sheet; N = nexin. Transmission electron microscopy of the trachea demonstrates an eccentric location of the central pair without transposition of peripheral microtubules (arrow head). Transmission electron microscopy of the trachea, indicating ciliae with a normal axonemal ultrastructure (white arrow head), with an eccentric location of the central pair and a transposition of peripheral microtubules (black arrow) and with a double central pair (black arrow head). Like all vertebrates, horses have an anatomical left-right asymmetry, established during embryogenesis. Failure to establish this normal organ position (situs solitus) results in randomization (situs ambiguous) or a complete mirror image (situs inversus) of all internal organs.4 The incidence of situs inversus in humans is 1/8,000 to 1/25,000 live births.5 These figures may underestimate the true incidence of situs inversus because, in general, a mirror-image reversal of the organs poses no threat to the health of the patient.4 However, every 4th or 5th person with this condition also has PCD, formerly called immotile cilia syndrome (ICS).6 Moreover, 50% of all cases with PCD also present situs inversus. The combination of these 2 congenital defects is defined as the Kartagener's syndrome.7 PCD is an inherited disorder, characterized by the complete absence of mucociliary clearance caused by decreased or absent motility of the cilia.8, 9 In these patients, the development of the situs inversus might be a result of the ciliary dyskinesia because the left-right asymmetry of the body is presumably determined by the movement of the monocilia in the embryonic Hensen's node.10 Clinically the impaired mucociliary clearance leads to respiratory symptoms that are typically present at birth and predispose the affected individuals to recurrent respiratory infections and bronchiectasy.8 Most cases with PCD have ultrastructural defects of the cilia. Exceptionally there are PCD cases with ultrastructurally normal cilia. In humans there is a variety of ultrastructural abnormalities in PCD. Changes considered specific to PCD are complete or partial lack of dynein arms, defective radial spokes, and an absent central pair of microtubules.3, 11, 12 Healthy control individuals can also have ultrastructural altered cilia, but in those cases less than 4% of their cilia are abnormal.3 A variety of respiratory diseases may alter ciliary ultrastructure resulting in secondary ciliary dyskinesia (SCD). SCD is the acquired form, mainly correlated with other ultrastructural defects such as microtubular abnormalities and compound cilia.3 The normal ultrastructure of cilia of the equine respiratory epithelium has been examined by Galati et al13 in 9 healthy horses. Their research shows that only 5.2% of tracheal cilia are atypical in healthy horses. By analogy with published data on human cases, the ultrastructural anomalies seen in those healthy horses can be considered as typical secondary, acquired abnormalities.3, 12 However, the differentiation between PCD- and SCD-altered cilia is not always straightforward, which can complicate the diagnosis of PCD.12, 14 In domestic animals, reports of deviations from the normal situs solitus are rather rare. Situs inversus has been reported in dogs,6, 15 cats,16 sheep,17 pigs,18 and calves.19 There are only 5 reports of horses with situs inversus.20-22 Only twice has the diagnosis been made antemortem.21, 22 All dogs that were reported to have situs inversus presented Kartagener's syndrome6, 15 whereas, to the authors' knowledge, Kartagener's syndrome has never been reported in horses. Although the triad of Kartagener includes bronchiectasis,7 the latter was not found in the filly in this case. This could be explained by the young age of the horse, because, at least in humans, bronchiectasis becomes more prominent with increasing age.14 In this horse, the diagnosis of Kartagener's syndrome was based on the combination of situs inversus and the presence of ultrastructurally abnormal cilia. According to the literature, dynein deficiency is the most frequent cause of PCD and this is found in 80% of all human cases.12 According to Jorissen and Cassiman,3 deficiency of the spokes, with the eccentric location of the central pair as its hallmark, has a prevalence of 6% in human PCD cases. In the same article they also state that spoke defects can be considered as a reliable cause of PCD only if 2 of 3 cilia are affected.3 In our case only a slightly lower number of the cilia (56%) had an eccentric location of the central pair. Therefore, it could be questioned if this really is a case of PCD or if the changes are secondary and acquired by chronic respiratory problems. However, as stated by Bush et al23 the diagnosis of PCD should be made by a combination of signs and symptoms and not by 1 single feature on its own. In our case the combination of situs inversus, respiratory problems since birth, and 56% of the cilia with a radial spoke defect is highly suggestive for PCD.23 The shortage of altered cilia could also be explained by the fact that the percentage of central pair anomalies seems to change with the level at which the cilia are cross-sectioned (Jorissen, personal communication). Moreover, recent research shows that the percentage of defective cilia, as mentioned by Jorissen and Cassiman,3 most likely is an overestimation because on an average, only 40.3% defective cilia were found in cases where the eccentric location of the central pair is confirmed to be the possible cause of PCD (Jorissen, personal communication). The difficult differentiation between SCD- and PCD-altered cilia is a frequently recurring problem in the diagnosis of PCD. Moreover, PCD and SCD are frequently associated, because most PCD cases suffer from respiratory infections and inflammations, also resulting in SCD. These secondary, acquired abnormalities can then superimpose on the primary, inherited abnormality.1, 14 For this reason in vitro culture of the respiratory epithelium of the horse was attempted, because ciliogenesis allows distinguishing primary from secondary abnormalities.12 It has been proven that ciliogenesis decreases SCD 10-fold and that in the majority of the cases the total secondary altered cilia is less than 5%, which can be considered normal.12 On the contrary, primary, inherited abnormalities are completely expressed after ciliogenesis in culture.12 In general, the cell culture technique of human epithelium is successful in 75% of the samples, but infection, as in our case, is one of the main causes of culture failure.24 Analyses of the ciliary ultrastructure and the ciliary function after ciliogenesis could have confirmed the diagnosis of PCD.25 However, TEM is considered as a trustworthy diagnostic test, especially when, as in our case, a sufficient percentage of ciliary deformations is found.3, 12 In our case the tissues were taken postmortem, but it is possible to diagnose PCD antemortem on samples collected by nasal brush or on biopsies of the respiratory epithelium collected by endoscopy. Despite the significant morbidity and discomfort associated with PCD, human PCD cases can have a fair to normal life. In terms of prognosis it is very important that the diagnosis is made in an early stage, so that necessary precautions can be made to prevent any permanent damage. Some of these precautions include methods to improve clearance, such as physiotherapy and medication, but also preventive vaccination and prompt treatment of infections are important to prevent further progression to bronchiectasis.9 For animals, it is more difficult to obtain a reasonable quality of life because the measures mentioned above are often too intensive and too expensive to be continued during the whole life span of the animal. Furthermore, it is questionable whether such intensive care would improve the condition of an affected horse in such a way that it would allow any type of performance. For these reasons, all the reported dogs with PCD were euthanized in an earlier or later stage of the illness.6, 15 This case report shows that in horses, PCD should be included in the differential diagnosis of chronic, recurrent respiratory problems, especially in young animals. Organ position can be easily checked by ultrasound to diagnose situs inversus. However, one should be aware that, similar to humans, PCD might also occur with normal organ position.