- Case report
- Open Access
Situs ambiguus in a Brown Swiss cow with polysplenia: case report
- Alois Boos†1Email author,
- Hans Geyer†1,
- Urs Müller1,
- Jeanne Peter1,
- Tanja Schmid2,
- Christian Gerspach2,
- Matteo Previtali2,
- Maja Rütten3,
- Titus Sydler3,
- Colin C Schwarzwald4,
- Elisabeth M Schraner1 and
- Ueli Braun2
© Boos et al; licensee BioMed Central Ltd. 2013
Received: 27 June 2011
Accepted: 11 February 2013
Published: 20 February 2013
Laterality defects are rare in cattle and usually manifest as asplenia orpolysplenia syndrome. These syndromes may be associated with situs ambiguus,which is a dislocation of some but not all internal organs. The objective ofthis report was to describe the clinical and post-mortem findings includingthe macroscopic and microscopic anatomy of selected organs in a cow withpolysplenia and situs ambiguus.
A 3.5-year-old Brown Swiss cow was referred to the Department of FarmAnimals, Vetsuisse Faculty, University of Zurich, because of poor appetiteand recurrent indigestion. A diagnosis of situs ambiguus was based on theresults of physical examination, ultrasonography, exploratory laparotomy andpost-mortem examination. The latter revealed that the rumen was on the rightside and lacked compartmentalisation. There were two spleens, one on theleft (26.5 x 12.0 cm) and one on the right (20.5 x 5.5 cm), and the omasumwas located craniolateral to the ruminoreticulum on the left. The abomasumwas located on the right, although it had initially been displaced to theleft. The three-lobed liver occupied the left and central cranioventralaspect of the abdominal cavity (cavum abdominis). Only the right and lefthepatic veins (vena hepatica dextra and sinistra) drained into the thoracicsegment of the caudal vena cava (vena cava caudalis), and histologicalchanges in the liver were indicative of impaired haemodynamics. Themesojejunum was not fused with the mesentery of the spiral loop (ansaspiralis) of the ascending colon (colon ascendens). The latter was foldedand the transverse colon (colon transversum) ran caudal to the cranialmesenteric artery (arteria mesenteria cranialis). Fibrotic constrictionswere seen in the lumen of the caecum and proximal loop (ansa proximalis) ofthe ascending colon. Both kidneys were positioned retroperitoneally in alumbar position. The lumbar segment of the caudal vena cava did not descendto the liver and instead drained into the right azygous vein (vena azygosdextra).
Recurrent digestive problems and poor production in this patient may havebeen caused by a lack of rumen compartmentalisation, abnormal abomasalmotility, constrictions in the large intestine (intestinum crassum) andfibrosis of the liver. The abomasum had abnormal motility most likelybecause it was anchored inadequately and only at its cranial aspect to theliver by the lesser omentum (omentum minus) and to the dorsal abdominal walland rumen by a short greater omentum (omentum majus).
Laterality defects are very rare in domestic animals. Situs inversus is the mostcommon laterality defect in dogs [1–4] and horses . Primary ciliary dyskinesia (PCD) also known as Kartagener’ssyndrome [3, 5, 6], is thought to be the primary cause of this condition, which is oftenassociated with recurrent chronic respiratory problems including bronchiectasia andchronic rhinitis. The few reports on laterality defects in ruminants deal withpost-mortem findings in cattle and sheep [7–11]. There have been only two cases of heterotaxy(“heteros” = other and“taxis” = arrangement) described in cattle and they werereferred to as situs inversus [7, 8], although at least one of these reports was restricted to intra-thoracicorgans, and both were not well documented. The anatomical findings in most of thedocumented cases [9–11] can be best characterised as situs ambiguus, which can be divided intoasplenia (right isomerism) and polysplenia (left isomerism). These conditions arealso assumed to be morphological correlates of PCD in humans [12–15]. It has been shown that several gene defects are correlated with PCD andthus with laterality defects [12, 16–19]. In humans, PCD is a genetically heterogeneous disorder with an autosomalrecessive mode of inheritance in most cases [14, 15, 19].
To the authors’ knowledge, this is the first detailed case report of a situsambiguus in a cow. Because not all organs were reversed from their normal positions,and some organs had specific deviations from normal in addition to heterotaxy, thiscase was not diagnosed as situs inversus. The findings serve to expand our knowledgeabout the clinical signs, functional implications and topography of internal organsin cattle with situs ambiguus.
Animal and methods
A three-year-old, non-pregnant primiparous Brown Swiss cow, which had previouslyproduced a live calf was referred to the Department of Farm Animals, VetsuisseFaculty, University of Zurich, because of poor body condition and a tentativediagnosis of caecal dilatation. The cow was small for its age and therefore bredlate, resulting in an age at first calving of 39 months. The cow was often seenruminating but had a history of poor appetite, and production during earlylactation was two thirds of the herd average. The cow resumed ovarian cyclicityand was inseminated twice post partum but did not conceive. The animal underwentclinical and ultrasonographic examinations and blood was collected forhaematological and biochemical analyses. Haematocrit, total leukocyte count andthe concentrations of fibrinogen and total protein from EDTA blood samples weredetermined on an automated blood analyser (CELL-Dyn 3500, Abbott DiagnosticsDivision, Baar). The concentrations of serum bilirubin, urea nitrogen, sodium,chloride, potassium, calcium, inorganic phosphorus and magnesium were determinedat 37°C using an automated analyser (Cobas-Integra-800-Analyser, RocheDiagnostics, Basel) and the manufacturer’s reagents (Roche-Reagents)according to the International Federation of Clinical Chemistry and LaboratoryMedicine (IFCC). Rumen fluid was collected using a stomach tube and the chlorideconcentration measured with an MK-II-Chloride Analyser 9265 (Sherwood,Cambridge). Results were compared with reference values established at thisclinic.
A standing right-flank exploratory laparotomy was done because of apparentabnormalities in the topography of the abdominal organs, suspected caecaldilatation and left displacement of the abomasum. During laparotomy it wasconfirmed that the abdominal organs were arranged in a mirror image reversal ofthe normal positioning. The left dorsal displacement of the abomasum wascorrected and the general condition of the patient improved and the cow wasdischarged five days postoperatively.
The cow was re-admitted to the clinic 2.5 months later because she failed to gainweight and milk production remained poor. Because of a grave prognosis, the cowwas euthanised, exsanguinated, fixed in standing position with 2.8% formaldehydeadministered through the common carotid artery (arteria carotis communis), andnecropsied. Samples of the mucosa of nasal conchae (conchae nasales), frontalsinus (sinus frontalis) and trachea were collected and routinely processed forscanning and transmission electron microscopy for the examination of thepresence and structural integrity of ciliated cells. Samples of liver tissue andof two control livers of non-liver-diseased cows ageing five and six years werecollected, fixed in 10% formalin, embedded in paraffin and routinely processedfor light microscopy and immunohistochemistry. Staining with haematoxylin andeosin, Gomori’s blue trichrome (Artisan™, Dako) andreticulin-nuclear fast red stains (Foot, Artisan™, Dako), histochemicaldetection of bilirubin (according to Hall), copper (rhodamine method for copper)and ferric iron pigments (Iron stain, Artisan™, Dako) andimmunohistochemical detection of α-smooth muscle actin (monoclonal mouseanti-human alpha muscle actin, clone 1A4, Dako) and desmin (monoclonal mouseanti-human desmin, clone D33, Dako) were carried out at the Institute ofVeterinary Pathology, University of Zurich using standard protocols. Cytokeratin(mouse anti-human cytokeratin, MNF1166, Dako) and von Willebrand factorimmunohistochemistry (polyclonal rabbit anti-human von Willebrand factor, Dako)were done to distinguish vessels from bile ducts. The size of 20 liver lobulesfrom this cow as well as from two healthy controls, aged five and six years, wasmeasured histomorphometrically using AxioVison software (Release 4.63, Zeiss),means and standard deviations were calculated, and differences analysed using anunpaired t-test (Statview 5.0 for Windows).
Clinical findings during the first hospitalisation
Auscultation on the left revealed no ruminal contractions and showed that thecontent of the digestive tract exhibited no layered arrangement at this site.Faeces had normal consistency and colour.
Ultrasonographic examination revealed that the heart had a normal size and shapeand the large vessels were positioned normally within the thoracic cavity (cavumthoracis). The rumen was large and gas-filled and only a few loops of the smallintestine (intestinum tenue) could be seen on the right side of the abdominalcavity. There were three reticular contractions during a two-minute period. Theliver was visible ventrally on the left side of the abdominal cavity and thegall bladder (vesica fellea) was moderately enlarged.
Exploratory laparotomy revealed a heterotaxy of abdominal organs. The rumen wasalmost empty and situated on the right side of the abdominal cavity. Thereticulum was attached to the rumen cranioventrally and contained sand. Theomasum was to the left of the rumen and had a firm consistency. The abomasum hada normal size, was on the left side and displaced dorsally. It contained a smallamount of firm and doughy ingesta. The intestines were to the left of the rumen,almost empty and had good peristalsis. The distal half and the tip of the caecum(apex caeci) had a normal size and shape. The greater omentum, which normallyforms the supraomental recess (recessus supraomentalis) containing theintestines in ruminants, was not detected during surgery (see post-mortemfindings). The abomasum was repositioned ventrally in the abdomen before closureof the abdominal wall.
Biochemical analysis of selected variables in serum and ruminalfluid
Chloride (ruminal fluid)
The general condition of the cow improved postoperatively, and one to two ruminalcontractions per minute could be ausculatated one day after surgery. The cow wasdischarged five days postoperatively, but was readmitted 2.5 months laterbecause of failure to gain weight and poor milk production.
Clinical findings during the second hospitalisation
The findings after re-admission to the clinic were similar to those of theinitial physical examination with regard to the forestomach, abomasum, whichstill had a ventral position, and intestines. Percussion and ultrasonographyrevealed that the liver was located in the ventral median part of the abdominalcavity directly caudal to the diaphragm.
The anatomical features and position of the heart were normal. The abdominalcavity demonstrated one striking abnormality of the venous drainage of thetrunk, pelvic limbs, kidneys, and pelvic organs. This large venous trunk(caudal vena cava) did not descend to the dorsal margin of the liver, drainthe liver, pass through the diaphragm and end in the right atrium of theheart. Instead it connected with the right azygous vein as it passed betweenthe crura of the diaphragm, below the vertebral column (columna vertebralis)on the right surface of the descending aorta (aorta descendens). Thisenlarged right azygous vein drained into the right atrium of the heart(Figures 10 and 12, seeAdditional file 11: Figure S11).
Both ruminal arteries (Aa. ruminales dextrae et sinistrae) run along thecraniodorsal wall of the rumen to the right and left side of this organ andproceeded caudally giving rise to dorsal and ventral branches (Figure 5).
In ruminants, the most common manifestation of heterotaxy of internal organs appearsto be situs ambiguus, which is divided into two primary subtypes: Asplenia syndromeor right isomerism, and Polysplenia syndrome or left isomerism [9–11]. Situs inversus, however, describes a situation in which all visceralorgans are reversed or mirrored from their normal position (referred to as situssolitus ) and is considered extremely rare in ruminants [7, 8]. To our knowledge, there have been three cases of asplenia, six cases ofhypoplastic spleens, 15 cases of two spleens and only two cases of situs inversusdescribed in cattle [7, 8, 10, 11]. The main findings in our patient (continuation of the caudal vena cavainto the right azygos vein, polysplenia, continuation of a common hepatic vein intothe right atrium, heterotaxia of the digestive tract, tri-lobed liver,retroperitoneal position of both kidneys, positioning of the left kidney cranial tothe right kidney and normal anatomy and topography of heart and lungs) were ingeneral agreement with other reports of polysplenia syndrome in cattle [10, 11] und thus strongly indicate the presence of this syndrome in our cow.Characteristics of the polysplenia syndrome in cattle - as reported in literatureand detected in this animal - are: continuation of the vena cava caudalis into thevena azygos dextra (n = 6; number of cases) or sinistra(n = 3), continuation of a common hepatic vein into the atrium dextrum(n = 4), situs inversus (n = 2) and left isomerism(n = 4) of the liver, situs inversus of the stomach (n = 2),retroperitoneal position of both kidneys (n = 1), positioning of theleft kidney cranial to the right kidney (n = 1) and normal anatomy andtopography of the lungs [10, 11].
Primary ciliary dyskinesia has been associated with chronic airway diseases andimpaired fertility in dogs, horses, and humans [1–3, 5, 13–15, 20]. It is an unlikely cause of heterotaxy in cattle because in the patientdescribed in this report, there was no chronic respiratory disease and theultrastructure of the cilia was normal. Although fertility was severely reduced inthis cow, this does not support an etiological role of PCD since the cow wasinseminated late because of small size and did therefore produce a calf at 39 monthsof age. Because genetic and functional analyses were not carried out, PCD cannot beruled out in this case.
In humans, neonates with polysplenia syndrome have a high incidence of severe heartdisease and therefore high juvenile mortality. Only children without severecardiovascular defects and those in which cardiovascular defects are correctedimmediately after birth survive and reach adulthood. Therefore, in adults,polysplenia syndrome is clinically characterised by gastrointestinal heterotaxia andnon-life-threatening vascular abnormalities [13, 15, 18, 21, 22], which is similar to findings in our patient and in accordance withreports in the literature [10, 11]. Further investigations in neonate calves are needed to determine whethersimilar cardiovascular defects occur in cattle with polysplenia or aspleniasyndrome; unfortunately most calves that die shortly after birth because ofpostnatal asphyxia or cyanosis are not necropsied.
Many of the topographical abnormalities observed in the present case can be explainedby specific developmental stages of organs during embryogenesis. The topography ofthe liver in our patient was characteristic for the position of this organ in theperinatal period in cattle, indicating that the ruminant-specific shift to the rightside and anti-clockwise quarter-turn of the organ during the postnatal life had notoccurred . The caudal vena cava is usually composed of a sequence of severalsagittal embryonic vessels . The lack of connection or a faulty connection between the intra-hepaticor immediate post-hepatic parts of these sagittal veins results in a dorsalanastomosis between the right or left azygous vein and the abdominal segment of thecaudal vena cava. The latter vein therefore does not descend to the dorsal margin ofthe liver. This modification of vasculogenesis leads to a continuation of the caudalvena cava into the right azygous vein, which occurred in our patient and also inseveral other cattle . The only function of the final segment of the caudal vena cava, whichran from the liver and passed through the diaphragm to join the right atrium of theheart, was drainage of the liver. From a functional point of view, it served as acommon hepatic vein.
The asymmetrical morphology of the tri-lobed liver was reflected by the specificdrainage of the two large lobes by two large hepatic veins, one on the right and oneon the left. This finding was also in agreement with the very small size of a liverlobe that possibly represent the quadrate lobe and the complete absence of thecaudate lobe (lobus caudatus), which are normally drained by the large middlehepatic vein (vena hepatica media) . The specific anatomy of the liver and its midline position in theabdominal cavity support the concept of a polysplenia syndrome/left isomerism inthis animal.
The histological characteristics of the hepatic lobules were indicative of abnormalhaemodynamics. However, different types of haemodynamic changes may cause identicalstereotypical abnormalities in the portal triad (pattern of portal veinhypoperfusion) similar to those described in our patient. Causes of portal veinhypoperfusion include congenital portosystemic shunts, arterioportal fistulas andobstruction or hypoplasia of the portal vein. The macroscopic and histologicalchanges in the vasculature of our patient represented primary pre- and intra-hepatichypoplasia of the portal vein . The changes in liver microstructure may also have reflected or causedthe poor nutritional status of this cow. Normal liver function is a prerequisite formany catabolic and anabolic processes, and abnormalities in serum enzyme activitiesand bilirubin concentration in this cow were characteristic of chronic impairment ofliver function . There have been no previous reports of the histological changes in theliver of ruminants with situs ambiguus.
In the present case report, there were striking abnormalities in the anatomy,topography, fixation and thus mobility of the abdominal parts of the digestivetract, which resulted in obvious digestive malfunction, small body seize, poor bodycondition and low milk production of the cow.
These findings seen in our patient may have been caused by a number of factorsincluding the loose connection of the abomasum to the cranially situated abdominalorgans and wall and thus rendering susceptibility to dislocation and bloating,constrictions of the large intestine at the level of the caecum and the proximalloop of the ascending colon, and lack of normal ruminal compartmentalisation. Thelaboratory findings suggested reduced feed intake caused by mild abomasal refluxsyndrome and/or prolonged periods of recumbency. Whether liver abnormalitiescontributed to poor body condition is not clear. However, marked fibrosis of theliver reflected poor nutritional status and possibly chronically impairedmetabolism. The absence of inflammation and cholestasis and the presence ofarteriolar proliferation, hypoplastic venules, portal fibrosis and bile ductproliferation were indicative of abnormal haemodynamics.
It was surprising that the cow was able to produce a live calf, given that fertilitywas obviously impaired. Equally surprising was that the cow had ruminal contractionsand was seen by the owner to ruminate given the ruminal anomalies observed. Thenormal consistency of the faeces was an indication of a certain level of normalcy ofdigestive tract function.
Consent was obtained from the owner of the cow for publication of this casereport and any accompanying images.
The authors thank Mrs. E Högger-Manser for her excellent technicalassistance.
- Reichler IM, Hoerauf A, Guscetti F, Gardelle O, Stoffel MH, Jentsch B, Walt H, Arnold S: Primary ciliary dyskinesia with situs inversus totalis, hydrocephalusinternus and cardiac malformations in a dog. J Small Anim Pract. 2001, 42: 345-348. 10.1111/j.1748-5827.2001.tb02471.x.PubMedView ArticleGoogle Scholar
- Cavrenne R, De Busscher V, Bolen G, Billen F, Clercx C, Snaps F: Primary ciliary dyskinesia and situs inversus in a young dog. Vet Rec. 2008, 163: 54-55. 10.1136/vr.163.2.54.PubMedView ArticleGoogle Scholar
- Witsberger TH, Dismukes DI, Kelmer EY: Situs inversus totalis in a dog with a chronic diaphragmatic hernia. J Am Anim Hosp Assoc. 2009, 45: 245-248.PubMedView ArticleGoogle Scholar
- Kayanuma H, Suganuma T, Shida T, Sato S: A canine case of partial heterotaxia detected by radiography andultrasound. J Vet Med Sci. 2000, 62: 897-899. 10.1292/jvms.62.897.PubMedView ArticleGoogle Scholar
- Palmers K, van Loon G, Jorissen M, Verdonck F, Chiers K, Picavet MT, Deprez P: Situs inversus totalis and primary ciliary dyskinesia (Kartagener’ssyndrome) in a horse. J Vet Intern Med. 2008, 22: 491-494. 10.1111/j.1939-1676.2008.0069.x.PubMedView ArticleGoogle Scholar
- Watson PJ, Herrtage ME, Peacock MA, Sargan DR: Primary ciliary dyskinesia in Newfoundland dogs. Vet Rec. 1999, 144: 718-725. 10.1136/vr.144.26.718.PubMedView ArticleGoogle Scholar
- Ries R, König HE: Situs inversus von Lunge, Herz und Leber bei einem Rind. Tierarztl Prax. 1988, 16: 251-252.PubMedGoogle Scholar
- Murakami T, Hagio M, Kaizo S: Situs inversus in a calf. J Jpn Vet Med Assoc. 2008, 61: 55-58.View ArticleGoogle Scholar
- Larsen C, Kirk EJ: Abdominal situs inversus in a sheep. N Z Vet J. 1987, 35: 113-114. 10.1080/00480169.1987.35401.PubMedView ArticleGoogle Scholar
- Fisher KR, Wilson MS, Partlow GD: Abdominal situs inversus in a Holstein calf. Anat Rec. 2002, 267: 47-51. 10.1002/ar.10086.PubMedView ArticleGoogle Scholar
- Okada K, Kuroshima T, Murakami T: Asplenia and polysplenia in cattle. Adv Anim Cardiol. 2007, 40: 39-47.Google Scholar
- El Zein L, Omran H, Bouvagnet P: Lateralization defects and ciliary dyskinesia: lessons from algae. Trends Genet. 2003, 19: 162-167. 10.1016/S0168-9525(03)00026-X.PubMedView ArticleGoogle Scholar
- Cohen MS, Anderson RH, Cohen MI, Atz AM, Fogel M, Gruber PJ, Lopez L, Rome JJ, Weinberg PM: Controversies, genetics, diagnostic assessment, and outcomes relating to theheterotaxy syndrome. Cardiol Young. 2007, 17 (Suppl 2): 29-43.PubMedGoogle Scholar
- Morillas HN, Zariwala M, Knowles MR: Genetic causes of bronchiectasis: primary ciliary dyskinesia. Respiration. 2007, 74: 252-263. 10.1159/000101783.PubMedView ArticleGoogle Scholar
- Kennedy MP, Omran H, Leigh MW, Dell S, Morgan L, Molina PL, Robinson BV, Minnix SL, Olbrich H, Severin T, Ahrens P, Lange L, Morillas HN, Noone PG, Zariwala MA, Knowles MR: Congenital heart disease and other heterotaxic defects in a large cohort ofpatients with primary ciliary dyskinesia. Circulation. 2007, 115: 2814-2821. 10.1161/CIRCULATIONAHA.106.649038.PubMedView ArticleGoogle Scholar
- Chodhari R, Mitchison HM, Meeks M: Cilia, primary ciliary dyskinesia and molecular genetics. Paediatr Respir Rev. 2004, 5: 69-76. 10.1016/j.prrv.2003.09.005.PubMedView ArticleGoogle Scholar
- Geremek M, Witt M: Primary ciliary dyskinesia: genes, candidate genes and chromosomalregions. J Appl Genet. 2004, 45: 347-361.PubMedGoogle Scholar
- Bush A, Chodhari R, Collins N, Copeland F, Hall P, Harcourt J, Hariri M, Hogg C, Lucas J, Mitchison HM, O’Callaghan C, Phillips G: Primary ciliary dyskinesia: current state of the art. Arch Dis Child. 2007, 92: 1136-1140. 10.1136/adc.2006.096958.PubMedPubMed CentralView ArticleGoogle Scholar
- Escudier E, Duquesnoy P, Papon JF, Amselem S: Ciliary defects and genetics of primary ciliary dyskinesia. Paediatr Respir Rev. 2009, 10: 51-54. 10.1016/j.prrv.2009.02.001.PubMedView ArticleGoogle Scholar
- Halbert SA, Patton DL, Zarutskie PW, Soules MR: Function and structure of cilia in the fallopian tube of an infertile womanwith Kartagener’s syndrome. Hum Reprod. 1997, 12: 55-58. 10.1093/humrep/12.1.55.PubMedView ArticleGoogle Scholar
- Ticho BS, Goldstein AM, Van Praagh R: Extracardiac anomalies in the heterotaxy syndromes with focus on anomalies ofmidline-associated structures. Am J Cardiol. 2000, 85: 729-734. 10.1016/S0002-9149(99)00849-8.PubMedView ArticleGoogle Scholar
- Fulcher AS, Turner MA: Abdominal manifestations of situs anomalies in adults. Radiographics. 2002, 22: 1439-1456. 10.1148/rg.226025016.PubMedView ArticleGoogle Scholar
- Geyer H, Aberger G, Wissdorf H: Beitrag zur Anatomie der Leber beim neugeborenen Kalb. TopographischeUntersuchungen mit Darstellung der Gallenwege und der intrahepatischenVenen. Schweiz Arch Tierheilkd. 1971, 113: 577-586.PubMedGoogle Scholar
- Cornillie P, Simoens P: Prenatal development of the caudal vena cava in mammals: review of thedifferent theories with special reference to the dog. Anat Histol Embryol. 2005, 34: 364-372. 10.1111/j.1439-0264.2005.00625.x.PubMedView ArticleGoogle Scholar
- Shirai W, Sato T, Shibuya H, Naito K, Tsukise A: Three-dimensional vasculature of the bovine liver. Anat Histol Embryol. 2005, 34: 354-363. 10.1111/j.1439-0264.2005.00623.x.PubMedView ArticleGoogle Scholar
- Cullen JM, van den Ingh TSGAM, Bunch SE, Rothuizen J, Washabau RJ,Desmet VJ: Morpholocical classification of circulatory disorders of the canine and feline liver. In WSAVA liver standardization group: Standards for clinical and histological diagnosis of canine and feline liver disease.Philadelphia: Saunders; 2006:41–59View ArticleGoogle Scholar
- Bain PJ: Liver. In Veterinary Laboratory Medicine. Clinical Pathology. 4th edition. Edited by Latimer KS, Mahaffey EA, Prasse KW. Iowa: Blackwell Publishing; 2003:193–214.Google Scholar
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