Black Headed Mutton sheep
8 Epidermolysis bullosa in sheep: clinical aspects and results of an experimental breeding in German Black results of an experimental breeding in German Black
Headed Mutton sheep
Epidermolysis bullosa beim Schaf: Klinische Aspekte und Ergebnisse eines Zuchtversuchs beim Deutschen Schwarzköpfigen Fleischschaf
Summary
Based on clinical and pathomorphological changes epidermolysis bullosa (EB) was diagnosed in 13 pure- and 1 crossbred German Black Headed Mutton lambs of three flocks. The three farmers reported of further 20 affected lambs in their flocks in past lambing saisons. The affected lambs were progeny of six rams and 17 ewes. Clinical examinations in 14 affected lambs revealed typical symptoms of EB like shedding of claw horn, erosions and ulcers of skin and mucous membranes. Two rams and six ewes with affected offspring from two farms were used for a breeding trial. In the course of these experimental matings, 21 lambs were born, six of which were affected by EB. All lambs born in this trial underwent clinical and haematological examination and the affected lambs were necropsied. The lambs affected by EB had typical clinical and pathological signs of EB. Histopathology revealed subepidermal splitting and blistering with intact basal keratinocytes and made a junctional or dystrophic form of EB most likely. The results of the test matings confirmed the genetic transmission and indicated an autosomal recessive mode of inheritance.
Keywords: German Black Headed Mutton sheep; epidermolysis bullosa;
subepidermal splitting; experimental breeding; inheritance
Zusammenfassung
Bei 13 reinrassigen Lämmern der Rasse Deutsches Schwarzköpfiges Fleischschaf sowie einem Kreuzungstier dieser Rasse wurden in drei Herden klinisch und pathomorphologisch Epidermolyis bullosa (EB) diagnostiziert. Die drei Züchter der
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Herden berichteten von insgesamt ca. 20 weiteren Fällen von EB in früheren Lammzeiten. Die an EB erkrankten Lämmer waren Nachkommen von insgesamt sechs Böcken und 17 Mutterschafen. Die klinischen Symptome der 14 erkrankten Lämmer, wie Ablösungen des Klauenhorns von der darunter liegenden Lederhaut, Erosionen und Ulzerationen der Haut und Schleimhaut, waren typisch für EB. Von zwei Betrieben wurden zwei Böcke sowie sechs Mutterschafe, die in ihrer Nachkommenschaft betroffene Lämmer hatten, erworben und für einen Zuchtversuch eingesetzt. In diesem Zuchtversuch erkrankten von insgesamt 21 geborenen Lämmern sechs an EB. Alle im Zuchtversuch geborenen Lämmer wurden klinisch und hämatologisch sowie die an EB erkrankten Lämmer pathologisch-anatomisch und -histologisch untersucht. Betroffene Lämmer wiesen typische klinische und pathomorphologische Symptome auf. Bei der histopathologischen Untersuchung konnten subepidermale Spalten- und Blasenbildungen und intakte basale Keratinozyten gesehen werden, was für eine junktionale oder dystrophische Form der Epidermolysis bullosa sprach. Der Zuchtversuch bestätigte die Erblichkeit und legte einen autosomal rezessiven Erbgang nahe.
Schlüsselwörter: Deutsches Schwarzköpfiges Fleischschaf, Epidermolysis bullosa, subepidermale Spaltbildung, Zuchtversuch, Erbgang
Introduction
Epidermolysis bullosa (EB) is a heritable heterogeneous group of skin disorders, which are all characterised by skin and mucous membrane blistering and ulceration in response to minor mechanical trauma (Jubb et al., 2007). This mechanobullous disease is known in horses, cattle, sheep, dogs, cats, mice (Jiang and Uitto, 2005), rats (Brenneman et al., 2000) and man (Bruckner-Tuderman et al., 2005).
The skin zone affected in EB is the dermo-epidermal junction, in which different interactive proteins are responsible for anchoring the epidermis to the dermis.
Blisters develop as a result of structural defects at the basement membrane zone due to mutations of genes responsible for the synthesis of a variety of structural components of this zone. Based on the ultrastructural level of skin cleavage, EB is
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divided into three main groups. In EB simplex (EBS) the detachment of the epidermis is due to cytolysis of keratinocytes. Intraepidermal clefting is the result of cytolysis of the basal keratinocytes. In junctional EB (JEB) disrupture occurs within the lamina lucida of the dermoepidermal basement membrane and dystrophic EB (DEB) is characterised by splitting the superficial dermis below the lamina densa at the level of the anchoring fibril network.
Human EB is classified in more than 20 subtypes based on the mode of inheritance, clinical manifestation of skin lesions, extracutaneous abnormalities and ultrastructural features. The clinical presentation in humans may range from circumscript epidermal involvement of hand and feet to severe generalized blistering with extracutaneous involvement of the cornea, teeth, trachea, gastrointestinal, genitourinary and musculoskeletal tract (Abahussein et al., 1993, Bruckner-Tuderman et al., 2005;
Jubb et al., 2007).
In sheep, only the dystrophic form of EB (DEB) was reported in the breeds Weisses Alpenschaf (Ehrensperger et al., 1987; Bruckner-Tuderman et al., 1991), Welsh mountain (Davies, 1988) and Assaf sheep (Pérez et al., 2005). Other reports of ovine skin disorders resembling EB refer to epitheliogenesis imperfecta (EI) or red foot disease. EI was reported for Romney Marsh crossbreds (Munday et al., 1970), the skin disease “similarly to EB in man and EI in calves” for Suffolk and Dorset Down (Alley et al., 1974) and red foot for Scottish Blackface (McTaggart et al., 1974). The differentiation between EB, EI and red foot disease in sheep is often not clear. In contrast to EB, EI-affected animals (sheep, cattle, horse) often show absence of epidermis at the distal two-third of the extremities (Dubielzig et al., 1986) and most areas of the face and ears (Yeruhama et al., 2005). Hair follicles and cutaneus glands in the denuded corium and subcutis are completely or partially absent at birth (Tontis and Hofstetter, 1991). In contrast to the differences of EB and EI, red foot disease seems to be similar or identical to EB (McTaggart et al., 1974; Davies, 1988).
The DEB in the sheep breed Weisses Alpenschaf follows a recessive mode of inheritance (Bruckner-Tuderman et al., 1991), while in the other breeds, the heredity is not established.
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DEB in Golden Retriever dogs (Palazzi et al., 2000), DEB in Brangus calves (Thompson et al., 1985) and DEB in a calf of the Red breeds of West Flanders (Deprez et al., 1993) seem to follow an (autosomal) recessive mode of inheritance.
JEB in Belgian Draft Horses and French draft horse breeds Comtois and Breton (Baird et al., 2003; Milenkovic et al., 2003) and JEB in German Shorthaired Pointer (Olivry et al., 1997) as well. Bovine EBS in the progeny of a Friesian-Jersey crossbred bull was inherited as an autosomal dominant trait (Ford et al., 2005). The mode of inheritance of an EBS-like skin disorder in the progeny of a Simmental bull and cows of the breeds Friesian and Friesian x Hereford followed an autosomal dominant mode of inheritance with incomplete penetrance (Bassett, 1986).
In humans, EBS most often follows simple autosomal dominant patterns, while JEB is transmitted in an autosomal recessive way and DEB in an either autosomal recessive or autosomal dominant pattern (Abahussein et al., 1993). The Mendes da Costa variant of EBS is an X-linked recessive trait (Pegum and Ramsay, 1973). Mutations in ten genes coding for structural proteins of the dermo-epidermal junction zone like keratin intermediate filaments, hemidesmosome associated proteins and anchoring fibrils were shown to be responsible for different forms of EB in man. A loss of function or complete missing of one of these proteins results in separation and blistering of the skin (Bruckner-Tuderman, 2005).
In horses, dogs and cattle, candidate gene approaches were employed for EB affected individuals. JEB in Belgian and French draft horses was associated with a cytosine insertion (1368insC) in the LAMC2 gene (Spirito et al., 2002; Baird et al., 2003; Milenkovic et al., 2003). The mutation responsible for JEB in the German Pointer was a homozygous insertion of repetitive satellite DNA within intron 35 of the gene LAMA3 (Capt et al., 2005). EBS in the progeny of a Friesian-Jersey crossbred bull was ascribed to a dominant single nucleotide mutation in the keratin 5 gene. The sire had an unaffected phenotype, but both his blood and semen contained the mutant allele albeit in lower proportions (approximately 20 %) than observed in the affected heterozygous offspring. Because the sire was a mosaic in these tissues, his progeny was less often affected by EBS than expected in an autosomal dominant trait (Ford et al., 2005).
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Gene therapy represents the only causal therapy for EB but is not established, yet.
However, different animal models for gene therapy are investigated (Jiang and Uitto, 2005; Spirito et al., 2006). In animals, palliative therapy proves limited success. EB-affected lambs, which were treated with antibiotics, corticosteroids and bandaging of the feet showed a resolving of mouth but not of foot lesions. The treated lambs retarded in growth and finally died in an emaciated condition (Alley et al., 1974;
McTaggart et al., 1974; Ehrensperger et al., 1987). Previous attempts to keep less severe affected lambs alive until breeding age failed because of death from secondary infections (McTaggart et al., 1974). In contrast, EBS in the progeny of a Simmental bull and cows of the breeds Friesian and Friesian x Hereford was not lethal and caused moderate lesions as maturity approached (Bassett, 1986).
In this article we report on the occurrence, clinical and pathomorphological aspects of epidermolysis bullosa in pure- and crossbreds of German Black Headed Mutton sheep and its mode of inheritance as the result of a breeding trial.
Material and methods Animals
EB-affected animals were ascertained in three flocks of pure- and crossbred German Black Headed Mutton sheep located in Lower Saxony, Germany.
Farm I
In this herdbook flock with about 230 ewes, a total of five EB-affected lambs (three males, two females) were born in the progeny of two rams and five ewes in the year 2004. One ram, which sired four affected lambs, had also about 170 unaffected offspring in the same flock. In five families with at least one diseased EB-lamb, the proportion of affected to unaffected lambs was 1:1. The pedigree of the ram, which sired four EB-affected lambs, is shown in Fig. 1. Three of these lambs (A1/I-A3/I) were hospitalised for clinical investigation.
Farm II
The owner of this herdbook flock with about 70 ewes reported five lambs with EB-resembling lesions in 2005 and four lambs with corresponding lesions in 2007.
Another five EB-affected lambs had been born in 2004, but no data about sex and
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identity of the parents were available (data not shown in Fig. 1). In 2006, no lamb with EB was observed. In 2005 another ram (BT5/II) was used in the flock as in 2007, whereas all dams of the EB-affected lambs were daughters of one ram (BT1/II) used in the years 2002 and 2003.
Four EB-affected lambs born in 2005 (A7/II-A10/II) and all EB-affected lambs born in 2007 (A2/II, A4/II-A6/II) were hospitalised. Furthermore, five pregnant ewes, which had given birth to affected lambs (A2/II, A4/II, A5/II, A8/II, A9/II) in the prior lambing season were hospitalised to make an early clinical observation of the newborn lambs possible. These ewes were pregnant from the same ram which was the sire of their EB-affected lambs in the previous breeding season. Two of them gave birth to each one EB-affected lamb (A1/II, A3/II).
Excluding the EB-affected lambs of 2004, a total of seven female and four male lambs with EB were born and - to the knowledge of the farmer - the proportion of affected to unaffected lambs was 11:9. Available pedigree data are shown in Fig. 1.
Farm III
In this small hobby flock the first case of EB was noticed by the farmer before 1993.
Until the year 2000, EB-affected lambs were sired by two purebred German Black Headed Mutton rams. Since the year 2000, the flock consisted of a German Black Headed Mutton x German Leineschaf crossbred ram and about ten ewes of different breeds (German Black Headed Mutton, German White Headed Mutton, German Leineschaf and their crossbreds). From 2000 to 2006 only this ram was used for breeding and was also mated to some of his daughters. In the twenty year history of breeding, a total of approximately 12 lambs (0-2 each year) affected by EB were born but pedigree data were only available for six of these lambs. Five of them were female and the sex of the sixth lamb was unknown. According to pedigree data the proportion of affected to unaffected lambs was 3:2. The affected lamb born in 2007 (A1/III; not shown in Fig. 1) was hospitalised.
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Two rams and six ewes from two farms were used for 13 test matings. This breeding trial was officially announced to the state veterinary office and registered under the number 33.42502-05/1023.
Two ewes from farm I (BT1/I, BT2/I), which had given birth to EB-affected lambs, were brought to the Clinic for Swine and Small Ruminants, University of Veterinary Medicine Hannover. They were mated with a ram from farm II (BT1/II), which was suspected to be a carrier for EB, because he was the sire of all dams that had given birth to EB-affected lambs in farm II. This ram was also mated with four of his daughters from farm II (BT2/II-BT4/II, BT6/II) that had affected lambs. One of the ewes from farm I (BT1/I) was also mated with the sire of the affected lambs from farm II in 2005 (BT5/II). Test matings are shown in Fig. 1 and all lambs born in the test matings are not marked by grey boxes.
Clinical examinations
A total of 20 EB-affected lambs (12 females, 8 males) and all available parents of affected lambs underwent clinical examination. These 20 EB-affected lambs include three lambs (A1/I-A3/I), which were born on farm I in 2004, four lambs born 2005 (A7/II-A10/II) and 2007 (A2/II, A4/II-A6/II) respectively on farm II, two affected lambs of the hospitalised dams from farm II in 2007 (A1/II, A3/II) and one lamb from farm III (A1/III, not shown in Fig. 1) in 2007. Lambs, which were born on farms were hospitalised in the clinic at the age of 12 to 38 days and bottle fed with milk replacer.
In the regular clinical examinations special attention was paid to general condition, health of skin and claws.
Haematological examination
EDTA blood of 19 EB-affected lambs could be collected to determine the hematokrit count, concentration of haemoglobin and erythrocytes, total leukocyte and differential blood count. In addition, serum creatine kinase activity of six and serum protein concentration of 12 lambs were analysed.
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A therapy of seven lambs was attempted. Affected claws and skin lesions on the dorsum of the carpal joint were bandaged to save the areas from contamination. For local treatment of the lesions unguent with iodine and inorganic iodine compounds (Vet-Sept Salbe, Albrecht, Aulendorf) and chlortetracycline-spray (CTC-Blauspray, Albrecht) were used. Besides, an immune stimulation (Baypammune, Bayer, Leverkusen), the antibiotics amoxicillin (Duphamox LA, Fort Dodge, Würselen) the analgesic and anti-inflammatory drugs flunixin-meglumin (Finadyne RPS, Essex) and meloxicam (Metacam, Boehringer Ingelheim, Ingelheim) were subcutaneously injected. For prevention of infection with clostridia, tetanus serum was given subcutaneously (Tetanusserum, WdT, Garbsen).
All EB-affected lambs were euthanised with an intravenous injection of pentobarbital (Eutha 77, Essex, Munich). If therapy was attempted, the lambs were euthanised when their general condition became unacceptable (severe lameness, retardation in growth, apathy).
Post mortem examination
All 20 affected lambs except one animal (A3/I), from which only biopsies of the skin were taken, underwent post-mortem examination. At necropsy, tissue samples of skin and mucous membranes were collected from different, macroscopically affected and non-affected areas like the coronary band, skin areas at the carpal and tarsal joints, lateral thorax, oral mucous membrane and tongue and fixed in 10 % formalin.
Formalin-fixed and paraffin-embedded tissue sections were stained with haematoxylin and eosin (HE) for histopathological examination.
Results Test matings
We employed two rams (BT1/II, BT5/II) from farm II, four ewes (BT2/II-BT4/II, BT6/II) from farm II and two ewes (BT1/I, BT2/I) from farm I for our breeding trial (Fig. 1).
Twenty-one lambs were born in 13 test matings, from which six (A1/IV-A6/IV) were affected by EB. The EB-affected lambs (3 male and 3 female) were progeny of ram
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BT1/II and two dams from farm II (BT2/II, BT4/II). The other ewes from farm I (BT1/I, BT2/I), farm II (BT3/II, BT6/II) and ram BT5/II (sire of one lamb) gave birth only to healthy animals. One female lamb died of pneumonia before clinical symptoms of EB were recognised and thus, its phenotype was considered as unknown.
Taking all lambs into consideration which were sired by the ram BT1/II in the breeding trial (half-sib family), the proportion of affected to unaffected lambs is 6:13 (only female progeny: 3:5; only male progeny: 3:8).
Clinical examinations
There were no distinct differences of clinical findings between affected lambs of the three farms. The EB-affected lambs of the farms were often more severely affected than lambs of the experimental breeding. This was attributed to longer duration of the disease whereas lambs of the experimental breeding were already euthanised before reaching this stage.
Lambs showed increased recumbency and lameness because of the affected claws (supportive lameness of one or two extremities, unsteady gait, setting the hind legs more cranial under the body) and often had a kyphotic attitude (Fig. 2). Sometimes they dwelled on the carpal joints. In some lambs the body temperature was increased.
Typical affected regions of the skin were the dorsum of both carpal joints and the coronary band. A first change on the dorsum of the carpal joints was a diffuse and later roundish or circular loss of hair. The hair could be epilated more easily than in unaffected lambs. Later on, in these areas white or brownish scurf and clotted hair could be seen. In more advanced cases, the epidermis peeled off to expose the red corium (Fig. 3). The cleavage between epidermis and dermis allowed the enlargement of the lesion by traction of the epithelial margin. The exposed dermis underwent inflammatory changes with secretion of fluid that ranged from lucent and serous to ichor and was obscured by clotted blood and soil. Even if also lambs un-affected of EB had sometimes sparse hair on the dorsum of the carpal joints, they never showed an excoriation of the dermis. In none of these cases, blisters of the skin were seen.
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Loss of hair and scurf could also be seen along the coronary band. In some cases hairless areas were located bilaterally plantar or palmar abaxial of the medial and lateral claws directly proximal of the digital pad. Other affected lambs had sparse hair along the coronary band or a longitudinal hairless band up to the fetlocks.
One lamb with docked tail, which suffered from EB for approximately 30 days, showed separation of the epidermis at the tip of the tail of 2 cm length with discharging pus. Other lambs in advanced stadium of EB had an extensive loss of sternal epidermis and a beginning loss of surface epithelium at the hocks.
In some EB-affected lambs the wall of the claws could be very easily impressed and seemed to be painful. Horn temperature was elevated in affected claws. In nearly all lambs in one to all claws, most often abaxially, a demarcation line more or less parallel to the coronary band was discerned in the claw horn (Fig. 4). With increasing age, these grooves were pushed more distally and in some cases, further grooves appeared proximal of the first one. In the proximal part just below the coronet the horn was thinner, sometimes perforated by a cavity or a more or less expanded fissure. Bloody or purulent fluid discharged or could be pressed out of these holes (Fig. 5). Secretion could be stopped by crusty material. In a four day old lamb, such a hole was filled with clotted blood. Even though a slight depression of horn between
In some EB-affected lambs the wall of the claws could be very easily impressed and seemed to be painful. Horn temperature was elevated in affected claws. In nearly all lambs in one to all claws, most often abaxially, a demarcation line more or less parallel to the coronary band was discerned in the claw horn (Fig. 4). With increasing age, these grooves were pushed more distally and in some cases, further grooves appeared proximal of the first one. In the proximal part just below the coronet the horn was thinner, sometimes perforated by a cavity or a more or less expanded fissure. Bloody or purulent fluid discharged or could be pressed out of these holes (Fig. 5). Secretion could be stopped by crusty material. In a four day old lamb, such a hole was filled with clotted blood. Even though a slight depression of horn between