Hannover 2016

Home , Northern cassowary, North African ostrich, Ostrich, Ratite, Somali ostrich

University of Veterinary Medicine Hannover Department of Pathology

Aimara Bello

Casuistic evaluation of necropsied ratites (Struthioniformes) in northwestern Germany - a retrospective study -

Hannover 2016

Attachment 4a: § 9 paragraph 1 of the Doctoral Regulations

University of Veterinary Medicine Hannover

Casuistic evaluation of necropsied ratites (Struthioniformes) in northwestern Germany - a retrospective study -

INAUGURAL –DISSERTATION in partial fulfillment of the requirements of the degree of Doctor of Veterinary Medicine -Doctor medicinae veterinariae – (Dr. med. vet.)

Submitted by Aimara Bello Caracas, Venezuela

Hannover 2016

Academic supervision: 1. Prof. Dr. med.vet. W. Baumgärtner. Department of Pathology TiHo

1. Referee: Prof. Dr. med.vet. W. Baumgärtner

2. Referee: Prof. Dr. med. vet. S. Rautenschlein

Day of the oral examination: 31.10.2016

Results of the Doctoral Thesis were partially presented in the following events:

Authors: A. Bello, S. Lapp, P. Wohlsein, W. Baumgärtner Title (German): Retrospektive Untersuchung der Erkrankungs- und Todesursachen von 66 Laufvögeln (Struthioniformes). Format: Oral presentation Event: 57. Jahrestagung der Fachgruppe Pathologie der Deutschen Veterinärmedizinischen Gesellschaft, 08. – 09.03.2014. Fulda, Germany. Published in: Tierärztliche Praxis G 42 (2014) S. A9

To my family

“It is only with the heart that one can see rightly; what is essential is invisible to the eye.”

― Antoine de Saint-Exupéry, The Little Prince Table of contents

Table of Contents

Chapter 1: Introduction 9 Chapter 2: Literature overview 10 2.1 Zoological classification 10 2.2 Most common diseases in ratites: overview 12 2.2.1 Diseases of the skin 12 2.2.2 Diseases of the musculoskeletal system 13 2.2.3 Diseases of the lymphoid and hematopoietic system 13 2.2.4 Diseases of the cardiovascular system 14 2.2.5 Diseases of the respiratory system 14 2.2.6 Diseases of the alimentary system 15 2.2.7 Diseases of liver and pancreas 16 2.2.8 Diseases of the urogenital system 17 2.2.9 Diseases of nervous system 17 2.2.10 Diseases of eyes and ocular adnexae 18 2.2.11 Systemic diseases 18 2.2.12 Diseases of the yolk sac 19 Chapter 3: Materials and Methods 20 3.1 Study design 20 3.2 Histopathology 20 3.3 Immunohistochemistry 21 Chapter 4: Results 22 Spontaneous diseases of captive ratites (Struthioniformes) in northwestern Germany: a retrospective study 22 Chapter 5: Discussion 5.1 General aspects 62 5.2 Musculoskeletal system 62 5.3 Alimentary system 64 5.4 Cardiovascular system 66 5.5 Nervous system 68 Table of contents

5.6 Respiratory system 69 5.7 Urogenital system 70 5.8 Eyes and ocular adnexae 70 5.9 Systemic diseases 70 5.10 Conclusion 71 Chapter 6: Summary 72 Chapter 7: Zusammenfassung 73 Chapter 8: References 75 Chapter 9: Acknowledgements 102

List of abbreviations

A. Aspergillus

ABC Avidin–biotin-peroxidase complex

BDV Borna disease virus

C. Clostridium

CNS Central nervous system

DNA Deoxyribonucleic acid

EEE Eastern Equine Encephalitis

DIC Disseminated intravascular coagulopathy

E. Escherichia

EHV Equine herpesvirus

FOS Fading ostrich syndrome

GI Gastro-intestinal

M. Mycobacterium

ND Newcastle disease

PCR Polymerase Chain Reaction

PrP Prion protein

S. Struthiolipeurus

TBE Tick-borne encephalitis

TSE Transmissible spongiform degeneration

WEE Western Equine Encephalitis

Chapter 1: Introduction

Ratites belong to the order Struthioniformes, and common ratite species include ostriches (e. g. North African ostrich [Struthio camelus]), emus (Dromaius novaehollandiae), rheas (e. g. Rhea americana), cassowaries (Casuarius casuarius), and kiwis (Apteryx australis) (FOLCH 1992; STEWART 1994; TULLY 2009). In zoological collections, ratites are very popular animals but in recent decades there has been a worldwide increase in farming ostriches, emus and rheas because of their assumed healthy red meat or their versatile use of the skin in the leather industry (TULLY 2009). Ratites represent an alternative livestock in some countries (COOPER 2004) with the potential to expand in small areas that are unproductive for other farm work (STEWART 1994). Ratite producers around the world are currently developing a niche for the meat, hide and byproducts of these avian species. Among the ratites, diseases of ostriches are best documented because of the development of a breeder industry (KUMMROW 2015). However, compared to poultry, there are differences in disease susceptibilities and relative prevalences. In addition, clinical and morphological characteristics of many infectious diseases are also shared by psittacines, waterfowl as well as other common companion and aviary birds (STEWART 1994). Due to the increase and development of the ostrich and other ratites farming in Germany since the end of the 1980s and the scarcity of literature on ratites disorders in Germany, this retrospective study focuses on the analysis of gross and histopathological findings and highlights rare salient lesions in different age groups of ratites that originated from zoos and farm in northwestern Germany.

Chapter 2: Literature overview

2.1 Zoological classification Ratites are classified into four suborders within the order Struthioniformes regarding the different geographic distributions: Struthiones (Africa), Rhea (South America), Cassuarii (Australia, New Guinea), and Apteryges (New Zealand) (FOLCH 1992). Struthiones include a single family, Struthionidae, with one species, Struthio, and four recognized subspecies, the North African ostrich (Struthio camelus [S. c.] camelus) (Fig. 1), the Somali ostrich (S. c. domesticus), the Massai ostrich (S. c. massaicus), and the South African ostrich (S. c. australis).

Figure 1. North African ostrich (Struthio camelus camelus) (Courtesy of I. Hoppe)

Chapter 2: Literature overview

There are two species in the family of Rheaidae within the suborder Rheae including the common or greater rhea (Rhea americana) (Fig. 2) and the Darwin´s or Lesser rhea (Pterocnemia pennata) (FOLCH 1992).

Figure 2. Common or greater rhea (Rhea Americana) (Courtesy of I. Hoppe)

The families Dromaiidae (emus) and Casuariidae (cassowaries) are closely related and pooled in the common suborder Casuarii. Whereas emus (Dromaius novaehollandiae) (Fig. 3) represent the only species, cassowaries constitute three distinct species (Southern cassowary, Casuarius [C.] casuarius; Dwarf cassowary, C. bennetty; Northern cassowary, C. unappendiculatus), with numerous subspecies (FOLCH 1992). Apteryges includes one family, Apterygidae, with three species (Brown Kiwi, Apteryx [A.] australis, Little Spotted Kiwi, A. owenii and Great Spotted Kiwi, A. haastii) (KUMMROW 2015).

Chapter 2: Literature overview

Figure 3. Emu (Dromaius novaehollandiae) (Courtesy of I. Hoppe)

2.2 Most common diseases in ratites: overview Diseases of ratites, especially ostriches, are reviewed by several authors (STEWART 1994; TULLY and SHANE, 1996; HUCHZERMEYER 1998; SHANE 1998; HUCHZERMEYER 1999; VERWOERD 2000; HUCHZERMEYER 2002; COOPER et al. 2004; TULLY 2009; BLACK and GLATZ, 2011; KUMMROW 2015).

2.2.1 Diseases of the skin Skin peeling, loss of feathers and dermolytic blistering similar to a genetically related mechanobullous skin disease like epidermolysis bullosa are described in juvenile ostriches (PERELMAN et al. 1995). Feather picking occurs commonly in adult ostriches and emus kept in small, barren paddocks possibly caused by malnutrition or environmental stress (STEWART 1994; SAMSON 1997). Ingestion of parsley may result in photosensitivity (KUMMROW, 2015). Viral diseases of the skin include ostrich pox virus infection that is transmitted by direct contact or via mosquitos (COOPER et al. 2004) and affects preferentially juvenile individuals (ALLWRIGHT et al. 1994; RAIDAL et al. 1996; PERELMAN et al. 1988). Mycotic infections of the skin with Trichophyton sp. and Microsporum sp. are observed occasionally (ONDERKA and DOORNENBAL 1992; HUCHZERMEYER 1998). Several ectoparasites affect ratites including feather lice (Struthiolipeurus [S.] struthionis, S. rheae, S. nandu, S. stresemanni, Meinertzhageniella lata, Meinertzhageniella

Chapter 2: Literature overview schubarti, Dahlemhornia asymmetrica), thribs (Limothrips denticornis), and quill mites (Gabucinia sp., Struthiopterolichus sp., Dermoglyphus parchycnemis) causing pruritus, feather and skin damage (PONCE GORDO et al. 2002; COOPER 2005; NEMEJC and LUKESOVA 2012). Hippoboscid flies (Struthiobosca struthionis) may irritate the animals and act as disease vector (NEMEJC and LUKESOVA 2012). Various species of ticks (Hyalomma sp., Amblyomma sp., Haemaphysalis sp., Rhipicephalus sp., Argas sp., Otobius sp.) are known to infect ratites (CRAIG and DIAMOND 1996; KUMMROW 2015). A fibroma is described in the skin of an ostrich (COOPER et al. 2010).

2.2.2 Diseases of the musculoskeletal system Degenerative myopathy occurs particularly in young ratites and includes exertional myopathy, selenium or vitamin E deficiency, furazolidone and ionophore intoxications (VAN HEERDEN et al. 1983; VORSTER 1984; GREGORY et al. 1992; SPEER 1996; BAIRD et al. 1997; MUSHI et al. 1998; WOTTON and HEWITT 1999; SMITH et al. 2005; MENON et al. 2014). Prolonged recumbency may lead to muscle necrosis and tendon contraction (STEWART 1994). Intramuscular injection of irritant substances, particularly certain antibiotics, may result in severe myositis (HUCHZERMEYER 1999). Deformities of legs appear at different stages of growth and include tibiotarsal or tarso-metatarsal rotation, rolled toes, slipped tendons (perosis), and bowed legs (TERZICH and VANHOOSER 1993; SPEER 1996; SAMSON 1997; SQUIRE and MORE 1998; HUCHZERMEYER 2002; KUMMROW 2015). Retarded growth may be caused by vitamin B (pantothenic acid) and A deficiency (SAMSON 1997; KUMMROW 2015). Disturbances of the calcium, phosphorus and vitamin D3 metabolism may lead to insufficiently mineralized bones, rickets (COOPER and GIMBI, 1994; SPEER 1996; MORROW et al. 1997). In contrast to wing fractures, fractures of the legs occur commonly in ratites and are associated with muscular degeneration, laceration, haemorrhages, local circulatory disturbances and result occasionally in internal exsanguination (STEWART 1994). Additional skeletal disorders include degenerative joint disease in ostriches and emus (SPEER 1996), subchondral cysts, bone sequestration, and arthritis (BURBA et al. 1996, TULLY et al. 1996).

2.2.3 Diseases of the lymphatic and haematopoietic systems Infectious bursal disease virus causes severe destruction of the lymphatic tissue of the bursa of Fabricius (SPEER 1996). Severe depletion of lymphatic tissues is also observed in adenovirus infection in ostriches (RAINES et al. 1997), Eastern Equine Encephalitis (EEE)

Chapter 2: Literature overview virus infection in emus (VEAZEY et al. 1994) and the fading ostrich syndrome (TERZICH and VANHOOSER 1993). Lymphoid leukosis with multiple organ involvement is reported in an ostrich (GARCÍA-FERNANDEZ et al. 2000). Various protozoal parasites are reported in ratites affecting cells of the circulatory system including Plasmodium sp. (FOX et al. 1996; COOPER 2005), Leukocytozoon sp. (HUCHZERMEYER 1998), Babesia sp. (JEFFERIES et al. 2008) and Hepatozoon sp. (BLACK and GLATZ 2011).

2.2.4 Diseases of the cardiovascular system Endocardiosis of the mitral valves with congestive heart failure is documented in an adult captive ostrich (KUBBA and AL-AZREG 2013). Myocardial degeneration due to avocado (Persea americana var. guatemalensis) poisoning is described in ostriches (BURGER et al. 1994). A Turlock-like bunyavirus may cause lympho-histiocytic and plasmacytic myocarditis in ostriches (SHIVAPRASAD et al. 2002). Non-suppurative myocarditis is seen after Western equine encephalitis (WEE) virus infection (SHANE 1998). Dissecting aneurysms with spontaneous rupture are often located at the aortic arch occurring especially in overweight yearling ostriches, but also in emus (MITCHINSON and KEYMER 1977; STEWART 1994; VANHOOSER et al. 1994; BAPTISTE et al. 1997; FERRERAS et al. 2001). Vasculitis occurs in emus infected with WEE and EEE viruses (AYERS et al. 1994; TULLY et al. 1992). Increased vascular permeability with widespread haemorrhages occurs after Senecio sceleratus poisoning (COOPER 2007a). An intrathoracic haemangiosarcoma is recorded in an ostrich (HEADLEY 2005).

2.2.5 Diseases of the respiratory system Various subtypes of avian influenza A virus (AIV) are described in ratites (ALEXANDER 2000). AIV infection in ratites may cause respiratory (e. g. ocular and nasal discharge) and gastro-intestinal (e. g. diarrhea) clinical signs. The pathogenicity depends especially on the particular virus subtype. Morphological findings include mucoid to pyogranulomatous sinusitis, purulent laryngitis, muco-purulent to necrotising tracheitis, necrotising bronchitis and bronchiolitis, pyogranulomatous pneumonia, fibrinous air sacculitis, necro-suppurative hepatitis, necrotising enteritis, splenomegaly and nephritis with greenish coloured urine (STEWART 1994; SWAYNE et al. 1996; HUCHZERMEYER 1999; WOOLCOCK et al. 2000). Paramyxovirus type 1 (Newcastle Disease [ND] virus) causes pneumonia and air sacculitis in ostriches and emus (JØRGENSEN et al. 1998).

Chapter 2: Literature overview

Bacterial infections of the respiratory tract occur predominantly under predisposing conditions (hypothermia, stress, overcrowding, poor air quality) and may be caused by Klebsiella sp., Escherischia (E.) coli, Pseudomonas sp., Corynebacterium sp., Pasteurella sp., Bordetella sp., Haemophilus sp. [infectious coryza], Staphylococcus sp., Chlamydophila psittaci, Streptococcus sp. and Mycoplasma sp. (SAMSON 1997; BLACK and GLATZ 2011). Morphological findings are variable depending on the manifestation of the infection and may include rhinitis, sinusitis, laryngo-tracheitis, pneumonia and air sacculitis (MOMOTANI et al. 1995; SHANE and TULLY 1996). Respiratory mycosis occurs in ratites kept in enclosed, dusty environments and is mainly caused by Aspergillus (A.) fumigatus, but also A. niger and A. flavus. Infection results in granulomatous air sacculitis and pneumonia (ASOK KUMAR et al. 2014; PERELMAN and KUTTIN 1992; SHANE 1998; CHANG REISSIG et al. 2002; PÉREZ et al. 2003; COPETTI et al. 2004; YOKATA et al. 2004; COOPER 2005; KHOSRAVI et al. 2008). Parasitic infestations of the respiratory tract include Syngamus trachea (ostrich, emu), Paronchocerca struthionus (ostrich), and Cyathostoma variegatum (emu) (STEWART 1994; CRAIG and DIAMOND 1996; SHANE 1998; NEMEJC and LUKESOVA 2012).

2.2.6 Diseases of the alimentary system Congenital malformations of the beak are seen in ostrich chicks (HUCHZERMEYER, 1998). Partial or complete impactions of the proventriculus, mainly occurring in animals less than six months, may be acute (within a few days) or chronic (within weeks to months), and comprise soft (plant fibers) or hard (sand, rocks) materials (STEWART 1994; SPEER 1996; SAMSON 1997; HUCHZERMEYER 1999; KUMMROW 2015). Stasis of gizzard contractions may secondary lead to proventricular food accumulation (HUCHZERMEYER 1999; OCAL et al. 2006). Ingestion of sharp foreign bodies is a common problem in ratites possibly leading to perforations of the alimentary tract (SAMSON 1997). Displacements of the intestinal tract comprise intussusception, intestinal torsion, and cloacal prolapse, mainly in chicks; all disorders are mostly associated with other enteric disorders (STEWART 1994; SHWALUK and FINLEY 1995; SPEER 1996; FRASCA and KHAN 1997; SAMSON 1997; BERRIDGE et al. 1998). Proventriculitis and ventriculitis may be caused by gastric impaction, Macrorrhabdus ornithogaster (HUCHZERMEYER et al. 1993), or fungal infection such as zygomycetes or Candida sp., the latter may also responsible for fibrinous pharyngitis and oesophagitis

Chapter 2: Literature overview

(JEFFREY et al. 1994; STEWART 1994; SHANE and TULLY 1996; SHANE 1998; HUCHZERMEYER 1999; GULBAHAR et al. 2000; COOPER 2005). Enteritis is mainly observed in chicks and juvenile ratites caused by a multitude of micro-organisms resulting in various forms of inflammation such as catarrhal, fibrinous, ulcerative, necrotizing, granulomatous, haemorrhagic and proliferative. Avian influenza, EEE virus, ND, adeno-, rota- and coronavirus represent viral causes for enteritis in ratites (HINES et al. 1995; RAINES et al. 1997; JØRGENSEN et al. 1998; SHANE 1998). Pathogenic bacterial species comprise E. fergusonii, E. coli, Salmonella sp., Klebsiella sp., Aeromonas sp., Pseudomonas sp., Campylobacter jejuni, Clostridium (C.) perfringens, Serpulina-like spirochetes now termed Brachyspira sp., Lawsonia intracellularis, Chlamydia sp. (SAGARTZ et al. 1992; KOLB et al. 1993; BUCKLES et al. 1997; LEMARCHAND et al. 1997; SAMSON 1997; SHANE 1998; VERWOERD 2000; KWON et al. 2004; HERRÁEZ et al. 2005; FRANҪA et al. 2009; MAPPLEY et al. 2014; KEOKILWE et al. 2015). Protozoal organisms isolated from the gastro-intestinal (GI) tract of ratites include coccidia (e. g. Cryptosporidium sp., Isosporum sp. and/or Eimeria sp.), amoebae (e. g. Entamoeba sp.), ciliates (e. g. Balatidium struthionis), flagellates (e. g. Histomonas melagridis, Giardia sp., Trichomonas sp.), Blastocystis sp. and microsporidia (CRAIG and DIAMOND 1996; GRAY et al. 1998; SHANE 1998; PONCE GORDO et al. 2002; COOPER 2005; NEMEJC and LUKESOVA 2012; QI et al. 2014). Ratites may harbour also numerous GI metazoic parasites comprising the trichostrongylid nematodes Libyostrongylus sp. found in the crypts of the glandular portion of the proventricus and gizzard wall of ostriches, Odontospiruria cetiopenis and Sicarius uncinipenis in rheas. Furthermore, Trichostrongylus tenuis causing haemorrhagic typhlitis in emus, Codiostomum struthionis and Heterakis dispar found in ostriches, Capillaria sp., Paradeletrocephalus and Deletrocephalus sp. in rheas have been documented. The principal cestode of ostriches and rheas is Houttuynia struthionis (STEWART 1994; SHANE 1998; HUCHZERMEYER 1998, 1999; CRAIG and DIAMOND 1996; HUCHZERMEYER 2002; PONCE GORDO et al. 2002; COOPER 2005; ZETTERMANN et al. 2005; SCHULZE et al. 2006; NEMEJC and LUKESOVA 2012)

2.2.7 Diseases of liver and pancreas Necrotizing hepatitis is described following infections with adenovirus (RAINES et al. 1997), avian influenza virus (HUCHZERMEYER 2002; COOPER 2004), EEE virus (VEAZEY et al. 1994), ND virus (JØRGENSEN et al. 1998), E. coli, C. sordellii, C. difficile, C. perfringens, Campylobacter coli and Campylobacter jejuni subsp. jejuni (POST et al.

Chapter 2: Literature overview

1992; POONACHA and DONAHUE 1997; STEPHENS et al. 1998; SHIVAPRASAD 2003; COOPER 2005;). Fasciola hepatica causes chronic cholangitis in emus and rheas (VAUGHAN et al. 1997; NEMEJC and LUKESOVA 2012). In rheas, cases of biliary cystadenoma and carcinoma are described (DELL´ARMELINA ROCHA et al. 2013; DELL´ARMELINA ROCHA et al. 2015). Pancreatic atrophy, lympho-plasmacytic inflammation and ductal epithelial necrosis associated with Cryptosporidium sp. are documented in poorly growth ostriches (JARDINE and VERWOERD 1997).

2.2.8 Diseases of the urogenital system Nephrocalcinosis may be associated with high dietary calcium intake in ostrich chicks (MORROW et al. 1997). Malformations of the phallus may result in infertility (HICKS-ALLDREDGE 1998). Intersex in ostriches is common and due to the lack of oestrogen the male´s feather are black coloured (STEWART 1994). Damage, infection and prolapse of the phallus may occur after prolonged eversion, however, phallus prolapse is described in association with cloacal and bursal infection with Cryptosporidium sp. (PENRITH et al. 1994). Testicular tumours occur rarely in ratites (HICKS-ALLDREDGE 1998). Conformational defects of the oviductal infundibulum may result in internal ovulation with subsequent infertility. Egg binding may be caused by genetic factors, malnutrition, cold weather or lack of exercise (HICKS-ALLDREDGE 1998). Ovarian torsion with necrosis and aseptic peritonitis is reported in an ostrich (SUÁREZ-BONNET et al. 2012). Ratites may suffer from oviduct infections (salpingitis), metritis, egg retention, vaginal and uterine prolapse, and egg-related peritonitis. Peritoneal hernias located in the caudal abdominal cavity allow the intestines and uterus to prolapse into the pericloacal region (STEWART 1994). Common bacterial pathogens infecting the reproductive tract include E. coli, Pseudomonas sp., Acinetobacter sp., and Salmonella sp. The latter may be vertically transmitted to eggs. Cystic ovarian changes may occur when eggs are continuously removed from the hen resulting in permanent gonadotropin stimulation (HICKS-ALLDREDGE 1998).

2.2.9 Diseases of the nervous system A hereditary lysosomal storage disease is identified in emus as mucopolysaccharidosis type IIIB resembling human Sanfilippo syndrome type B (BERMUDEZ et al. 1995; KIM et al. 1996; BERMUDEZ et al. 1997; FREISCHÜTZ et al. 1997; GIGER et al. 1997;

Chapter 2: Literature overview

ARONOVICH et al. 2001). A spongiform degeneration of brain tissue causes neurological signs in three ostriches kept in German zoos (SCHOON et al. 1991 a; b). Encephalomalacia in emus may be associated with hypovitaminosis E (AYE et al. 1998). Viruses affecting the central nervous system of ratites causing non-suppurative inflammation of meninges and/or neuroparenchyma include avian paramyxovirus (Newcastle Disease), WEE virus, Borna disease virus and Turlock-like bunyavirus (RANDOLPH et al. 1994; ASHASH et al. 1996; SHANE and TULLY 1996; SHANE 1998; HUCHZERMEYER 1999; HUCHZERMEYER 2000; SHIVAPRASAD et al. 2002; COOPER 2004). Botulism in ostriches comprises total paralysis, paresis and ataxia caused by an ostrich carcass in the camp in which the birds were kept (ALLWRIGHT et al. 1994). Verminous encephalitis in ratites is caused by Baylisascaris procyonis (KAZACOS et al. 1982, KAZACOS et al. 1991) and Chandlerella quiscali (LAW et al. 1992).

2.2.10 Diseases of the eyes and ocular adnexae Disorders of the eye may be caused by laceration, abrasion and ulceration due to direct contact to objects (STEWART 1994), viral infections such as avian influenza and pox virus (PERELMAN et al. 1988; ALLWRIGHT et al., 1993), bacterial infections including Moraxella phenylpyruvica (SAROGLU et al. 2003; GÜREL et al. 2004), Mycobacterium avium complex (POCKNELL et al. 1996; ŠEVČİKOVÁ et al. 1999; GARCÍA et al. 2001), Staphylococcus hyicus subsp. hyicus (CHIRINO-TREJO and WHELER 1989), Staphylococcus aureus and E. coli (SAHINDURAN 2004), Chlamydia psittaci (KÖSTERS et al. 1996), as well as parasitic infestations in ostriches and rheas with the trematode Philophthalmus gralli (GREVE and HARRISON 1980; MUKARATIRWA et al. 2005; VEROCAI et al. 2009; NEMEJC and LUKESOVA 2012; CHURCH et al. 2013) and the thribs Limothrips denticornis (COOPER 2007b). Ingestion of the toxic plant Lantana camara caused severe conjunctivitis (COOPER 2007a). Cataracts commonly occur in older ostriches (STEWART 1994).

2.2.11 Systemic diseases High mortality in ostrich fetuses may be caused by malpositioning and severe subcutaneous oedema during artificial incubation (STEWART 1994; BROWN et al. 1996; SPEER 1996). The “fading ostrich syndrome” (FOS; Syn.: “chick fading syndrome”) that also occurs in rheas (FERNÁNDEZ et al. 2013) is a multifactorial disease complex characterized by growth retardation, weight loss, anasarca, ascites, delayed or inappropriate intake of food,

Chapter 2: Literature overview gastric stasis with accumulated ingesta, absence of faeces, yolk sac retention and increased mortality in 1 to 3-week-old chicks (PHILBEY et al. 1991; STEWART 1994; HUCHZERMEYER 2002). It is associated with improper management, stress and possibly infectious pathogens such as circovirus (EISENBERG et al. 2003) or adenovirus (EL- ATTRACHE et al. 2001). The ostrich is the only avian host susceptible to Bacillus anthracis (HUCHZERMEYER 1998). Infection results in septicaemia followed by sudden death, splenomegaly, hepatomegaly, vascular congestion and tarry blood exuding from the body orifices (SHANE 1998; THEILER 1912; VERWOERD 2000; COOPER 2005). Infection with Erysipelothrix rhusiopathiae, described in emus (GRIFFITHS and BULLER 1991; MORGAN et al. 1994) and ostriches (SHANE 1998), is characterized by serosal petechiae, venous congestion, splenomegaly and hepatomegaly. Mycobacteriosis is commonly caused by Mycobacterium (M.) avium, rarely by M. bovis (KELLY et al. 2013), and may result either in localized lesions, e. g. granulomatous inflammation of the cloaca, eye, phallus, or in a generalized infection with granulomas in the GI tract, liver and spleen (SHANE et al. 1993; SHANE 1998; DONELEY et al. 1999; HUCHZERMEYER 1998, 1999; GARCÍA et al. 2001; KELLY et al. 2013). Paralysis with intestinal haemorrhages, multifocal hepatic necroses and splenomegaly is described in ostriches associated with C. chauvoei infection (LUBLIN et al. 1993). Septicaemia caused by Pasteurella multocida resulting in hepato-splenomegaly and vascular congestion is rarely documented in ratites (OKOH 1980). Chlamydia psittaci infection may cause generalized disease with high mortality rates in ostriches (HUCHZERMEYER 1999).

2.2.12 Diseases of the yolk sac Omphalitis is seen in chicks less than 1 week of age, often as a result of incorrect working conditions of the incubator and/or hatcher. Ascending bacterial infection may lead to yolk sac infection. A persistent yolk sac occurs commonly in artificially raised ratites associated with incorrect management conditions and poor hygiene leading to insufficient uptake of nutrients and antibodies and, after decomposition of the yolk sac, to absorption of bacterial toxins (SPEER 1996; HUCHZERMEYER 1998, 1999; STEWART 1994; BLACK and GLATZ 2011). Occasionally exposure of small or larger parts of the yolk sac is encountered which predisposes for suppurative bacterial infections (STEWART 1994; SPEER 1996; BLACK and GLATZ 2011). Transovarian or transoviductal infection with Salmonella sp. may result in embryonic infection (SPEER 1996).

Chapter 3: Material and Methods

Chapter 3: Materials and Methods

3.1 Study design Data records and histological preparations of 71 captive ratites originating from German zoological gardens and private farms in northwestern Germany were reviewed retrospectively. Animals included 54 ostriches (Struthio camelus), 5 emus (Dromaius novaehollandiae) and 12 rheas (Rhea americana) comprising 37 adults, 23 juveniles and 11 neonates and eggs. The animals were necropsied between 1968 and 2014 at the Department of Pathology, University of Veterinary Medicine Hannover, and at the Chemisches und Staatliches Veterinäruntersuchungsamt Arnsberg, North Rhine-Westphalia. The body condition had been scored as good, moderate, poor and cachectic. Animals in a good body condition revealed vast amounts of fat tissue within the furcular and abdominal regions whereas animals with a moderate body condition showed reduced amounts of corporal fat tissue. Animals in a poor body condition possessed only low amounts of fat reserves frequently associated with mild pectoral muscle atrophy. In contrast cachectic animals lacked fat reserves, displayed a serous atrophy of the coronal myocardial cardiac coronary fat tissue and moderate to severe atrophy of the pectoral muscles. Detailed information about the investigated animals including species, gender, age, nutritional status, and pathologic findings is given in Annex Table 1. This study was carried out in accordance to the German animal welfare act. All animals were dead at the time of submission for necropsy and post-mortem examinations were carried out by order of the operators of the zoological gardens and private farms to investigate the causes of illness or death.

3.2 Histopathology Samples for histopathology had been taken from the majority of animals and included skin, musculoskeletal system, respiratory system, alimentary system, reproductive system, urinary system, cardiovascular system, endocrine and lymphatic organs, nervous system and eye. Samples had been fixed in 10% neutral buffered formalin, dehydrated and embedded in paraffin wax according to standard laboratory procedures. Tissue sections of 3 μm thickness had been mounted on glass slides (Engelbrecht Medizin- und Labortechnik GmbH, Edermünde, Germany) and stained with haematoxylin and eosin (HE). Applied histopathological stainings were performed according to standard procedures as described (RIEDELSHEIMER and WELSCH 2010). In selected cases additional sections were stained 20

Chapter 3: Material and Methods with HE, periodic acid-Schiff-(PAS) reaction, Gram, Congo red, von Kossa´s, Ziehl- Neelsen´s, and Heidenhain's Azan trichrome stain, Grocott's methenamine silver, or Warthin- Starry´s silver impregnation according to standard laboratory protocols (RIEDELSHEIMER and WELSCH 2010).

3.3 Immunohistochemistry For the detection of tick-borne encephalitis (TBE) virus, Borna disease virus (BDV), equines herpesvirus (EHV), rabies virus, avian influenza virus (AIV), Lawsonia intracellularis and Listeria monocytogenes pathogen-specific antibodies and the avidin– biotin-peroxidase complex (ABC) method (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, California) was performed as previously described (GUEDES and GEBHART 2003; SCHWAB et al. 2007; BRANDT et al. 2010; WOHLSEIN et al. 2011; NICHOLLS et al. 2012).

C h a p t e r 4 : R e s u l t s

Chapter 4: Results

Spontaneous Diseases in Captive Ratites (Struthioniformes) in Northwestern Germany: A Retrospective Study

Aimara Bello1¶, Samuel Frei1, 2¶, Martin Peters3, Anne Balkema-Buschmann4,

Wolfgang Baumgärtner1, Peter Wohlsein1*

1Department of Pathology, University of Veterinary Medicine Hannover Foundation, Hannover, Lower Saxony, Germany. 2Zoo Wuppertal, Wuppertal, North Rhine-Westphalia, Germany 3Chemisches und Veterinäruntersuchungsamt Westfalen, Arnsberg, North Rhine- Westphalia, Germany 4Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institute, Greifswald, Insel Riems, Mecklenburg-Western Pomerania, Germany.

¶ These authors contributed equally to this work.

* [email protected]

Running head: Spontaneous Diseases in Captive Ratites

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Abstract A retrospective study was carried out to define the spectrum of spontaneous diseases in captive ratites, order Struthioniformes, in northwestern Germany. The investigation included 71 ratites necropsied between 1968 and 2014. They consisted of 54 ostriches, 5 emus, and 12 rheas with 37 adults, 23 juveniles and 11 neonates and eggs. Necropsy reports were reviewed, histologic preparations were re- examined and additional histochemical and immunohistochemical stains were carried out in selected cases. In many animals more than one morphologic diagnosis attributable to different disease processes was found. In adult animals (n = 37), the most commonly altered organ systems were the musculoskeletal system (49%), the digestive system (46%), and the cardiovascular system (46%) affected by traumatic lesions, inflammatory and degenerative changes, respectively. Lesions of the nervous system (35%) included spongy degeneration; however, immunohistochemistry and western blotting failed to detect accumulations of pathological prion protein. In juvenile animals (n = 23), the musculoskeletal system (44%) and the digestive system (43%) were predominantly affected by traumatic and inflammatory lesions, respectively. In eggs and neonates (n = 11) the major cause of death was the fading ostrich syndrome (64%). Summarized, most of the lesions observed in adult and juvenile ratites living in northwestern Germany are related to trauma, inflammatory and degenerative disorders, whereas in eggs and neonates the fading ostrich syndrome was found predominantly. Surprisingly, a degenerative encephalopathy with spongy degeneration of unknown cause and pathogenesis that await further studies was found in adult animals.

Key words: emu, ostrich, pathology, ratites, retrospective investigation, rhea

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Introduction ―Ratite‖ is not a strict taxonomic term, but it is used to refer to flightless birds that lack a keel at their sternum and have a rather flat ―raft-like‖ breast. In general, ratites belong to the order Struthioniformes, and common ratite species include ostriches (e. g. North African ostrich [Struthio camelus camelus]), emus (Dromaius novaehollandiae), rheas (e. g. the Common or Greater Rhea [Rhea Americana]), cassowaries (e. g. Southern cassowary [Casuarius casuarius]), and kiwis (e. g. Brown kiwi [Apteryx australis]) [1]. Interestingly, they are not a homogenous group and show numerous anatomical and physiological differences [2, 3]. The natural habitat of ratites is the southern hemisphere, i. e. ostriches in Africa, rheas in South America, emus in Australia, kiwis in New Zeeland, and cassowaries in Australia and New Guinea [3]. Historically, ratites have been very popular animals in zoological collections. In addition, there has been a worldwide interest in farming ostriches, emus and rheas in recent decades, because of the assumed healthy red meat and versatile use of the skin in the leather industry, and recently they are appreciated as an alternative livestock in some countries [3]. A concern was raised in the European Union (EU) that ostriches, which are adapted to hot dry climates, would experience difficulties to live under cold winter conditions [4]. The EU Council Directive 98/58 ―Concerning the protection of animals kept for farming purposes‖ states that breeding of animals is prohibited if it causes adverse effects on their health or welfare [4]. The EU also recommended that the environmental conditions in areas where ostrich farms are located should allow the birds to be kept outside most of the day in any season, to satisfy their need for exercise and grazing [4]. Frequent infectious and non-infectious spontaneous diseases in ostriches and other ratites have been reviewed [1, 3, 5-13], describing diseases that occur mostly in ratites farmed in countries in and outside of their areas of origin. It was noted that commercialization of ratite production goes along with increased disease frequency and parasitism[8]. This increment was related at least partially to improper management of farmed ratites in most cases [4]. Surveys about spontaneous diseases of ratites in Europe are scarce [14]. Due to the increase and development

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of ostrich and other ratites farming in Germany since the late 1980s, this retrospective analysis focuses on the characterization of frequently observed gross and histopathological findings and will highlight rare salient lesions in different age groups of ratites held in captivity in northwestern Germany.

Materials and Methods Cases records The study was conducted on 71 ratites of the order Struthioniformes including 54 ostriches (Struthio camelus), 5 emus (Dromaius novaehollandiae) and 12 rheas (Rhea americana) kept in northwestern Germany that had been submitted for necropsy between 1968 and 2014. The records on medical history, animal data (gender, age at time of death), macroscopic and histological findings were analyzed. Furthermore, available results of additional investigations, such as immunohistological, parasitological, microbiological and virological investigations were included. The body condition had been scored as good, moderate, poor and cachectic. Animals in a good body condition revealed vast amounts of fat tissue within the furcular and abdominal regions whereas animals with a moderate body condition showed reduced amounts of corporal fat tissue. Animals in a poor body condition possessed only low amounts of fat reserves frequently associated with mild pectoral muscle atrophy. Cachectic animals lacked fat reserves, displayed serous atrophy of the coronary fatty tissue and moderate to severe atrophy of the pectoral muscles. Detailed information about the investigated animals including case number, animal identification number, species, age group, sex, nutritional status, husbandry, manner of death and major pathologic findings is given in table 1. This study was carried out in accordance to the German animal welfare act. All animals were dead at the time of submission and the authors confirm that no animal was sacrificed for the purpose of this study. Postmortem examinations were carried out to investigate the cause of illness or death.

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Histopathology Each case was reviewed retrospectively, except case Nos. 7, 8 and 12, because paraffin blocks and sections were not available any longer. Histologic sections, stained with hematoxylin and eosin, were re-evaluated. In selected cases additional sections were stained with hematoxylin and eosin (HE) or special stains including periodic acid-Schiff (PAS) reaction, Gram, Congo red, von Kossa´s, Heidenhain's Azan, Ziehl-Neelsen´s stain, Grocott's methenamine and Warthin-Starry silver impregnation according to standard laboratory protocols [15].

Immunohistochemistry For the detection of tick-borne encephalitis virus, Borna disease virus, equine herpesvirus, rabies virus, influenza A virus, Lawsonia intracellularis and Listeria monocytogenes, pathogen-specific antibodies were applied as previously described [16-20], and the avidin–biotin-peroxidase complex method was used according to the manufacturers´ instructions (VECTASTAIN Elite ABC Kit, Vector Laboratories, Burlingame, California). For the detection of Brachyspira sp., a direct immunofluorescence test using a polyclonal rabbit antibody against Brachyspira hyodysenteriae was applied (kindly provided by Prof. G. Amtsberg, Institute of Microbiology, University of Veterinary Medicine Hannover, Germany). For the visualization of accumulated pathologic prion protein (PrP), paraffin sections were autoclaved at 121°C for 20 min in citrate buffer, pH 6.0 after dewaxing and incubation for 30 min in 3% H2O2 in methanol. The PrP-specific primary monoclonal antibodies (mabs) F99/97.6.1 (VMRD, Inc.; Pullman, WA, USA)[21] or mab 6C2 (Central Veterinary Institute Wageningen, NL) [22] were applied in Tris- buffered saline with 10% goat serum and 0.03% sodium azide, at a concentration of 0.2 µg/ml and 0.7 µg/ml and incubated for 2 hours at room temperature. As negative control, sections were incubated with 0.2 µg/ml of a monoclonal antibody directed against glycoprotein GP5 of the porcine reproductive and respiratory syndrome virus (PRRSV) used as a null-mab. As a conjugate, the avidin-biotin-peroxidase complex method was used as described above. For both methods 3, 3′-diaminobenzidine-

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tetrahydrochloride (DAB, Sigma-Aldrich, Munich, Germany) was used as chromogen. Sections were counterstained with Mayer`s hematoxylin, and mounted. Polymerase chain reaction For the detection of genome fragments of Brachyspira hyodysenteriae and Lawsonia intracellularis polymerase chain reactions (PCRs) were performed as previously described [23].

Western Blot In order to test the binding of mammalian PrP-specific antibodies to ratite-associated PrP, 10% cattle, ostrich and rhea brain homogenates were prepared in Sucrose- DOC-NP40 (0.42 M Sucrose, 0.5% deoxycholic acid sodium, 0.5% Nonidet P40). Samples were mixed with gel loading buffer containing 6% sodium dodecyl sulphate (SDS) and analyzed in a SDS-PAGE as described previously [24]. Mabs F99/97.6.1 and 6C2 were applied as detection antibodies at a concentration of 0.05 µg/ml and 0.07 µg/ml respectively, which are well established for the diagnostic of transmissible spongiform encephalopathy in ruminants. Both antibodies bind to conserved regions with little variation in the prion protein sequence between ruminants and ratites.

Results Between 1968 and 2014, a total of 71 ratite including 54 ostriches, 5 emus and 12 rheas were submitted for pathological examination. Spontaneous death was recorded in 19 adults, 17 juveniles and 10 neonates and eggs, whereas 8 adults, 5 juveniles and one neonate had been euthanized for humane reasons. For the remaining 11 cases the mode of death was not recorded. 83% of the animals (n = 59) were kept in zoological institutions and 17% (n = 12) in private farms in northwestern Germany. 52% (n = 37) of the ratites were adults (older than 1.5 years), 32% (n = 23) were juvenile birds (older than 5 days and under 1.5 years of age), and 15% (n = 11) were neonates (under 5 days of age) or eggs. The body condition was regarded as good in 57% (n = 41), as moderate in 10% (n = 7), and as poor in 17% (n = 15). In 6% (n = 4) of the ratites a cachectic body condition was noted and in 10% (n = 7) the body condition was not mentioned.

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In many animals more than one morphologic change attributing to a different disease processes was found.

Musculoskeletal system Adult ratites Diseases of the musculoskeletal system were diagnosed in 18 (49%) animals (case Nos. 3, 5, 6, 8, 13, 21, 23, 32, 47, 48, 52 - 55, 61, 62, 70, 71). Traumatic injuries to the legs and trunk associated with muscular degeneration, lacerations, hemorrhage and local circulatory disturbances characterized by edema and vascular thrombosis were present in 11 cases (case Nos. 3, 5, 13, 21, 23, 32, 48, 52, 54, 70, 71). Two animals (case Nos. 32, 53) had fractured legs. One of these (emu No. 53) suffered from an intraosseous fibrosarcoma (Fig. 1). Grossly, the left limb showed a focal oblique pathologic fracture of the distal tarsometatarsus with concurrent lacerations and hemorrhage in the adjacent muscles and skin. In the marrow cavity, a fibrosarcoma with a proximo-distal extension of 9 cm was found. The tumor was composed of interlacing bundles of moderately pleomorphic spindle-shaped cells embedded in a collagen fiber-rich extracellular matrix and rare mitotic figures (Fig. 2). Tumor-associated osteolysis of trabecular bone was multifocally observed. Other changes comprised mild focal fibrino-purulent tarsitis and hip arthrosis in one ostrich (case No. 8), articular gout in a rhea (case No. 48), and mild hyaline muscular degeneration (case Nos. 11, 61, 62). Juvenile ratites Ten (44%) out of 23 juvenile animals presented morphological lesions in the musculoskeletal system. Various forms of trauma were observed in six animals (case Nos. 4, 15, 16, 28, 45, 50). Two ratites (case Nos. 4, 16) presented a fractured vertebral column. One ostrich (case No. 15) showed multiple rib fractures and swellings in the costochondral areas suggestive of rickets. Furthermore, slipped tendons (perosis) with lateral displacement of the gastrocnemius tendon and deformation of the distal tibiotarsal and tarsometatarsal bones were observed in two ostriches (case Nos. 12, 14). In addition, two animals exhibited hyaline muscular degeneration (case Nos. 26, 28) of unknown cause. Ostrich No. 60 showed

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widespread hyaline degenerations of the musculus gastrocnemius, musculus extensor metacarpi radialis, musculus iliotibialis lateralis and musculus elevator caudalis as well as a degenerative cardiomyopathy associated clinically with severe depression after transportation. The findings were indicative of capture myopathy. Eggs and neonates One neonatal ostrich (case No. 37) showed a traumatic fracture of the femur.

Digestive system Adult ratites Disorders of the alimentary system were observed in 17 (46%) out of 37 animals (case Nos. 1, 3, 6, 17, 18, 21, 22, 32, 41, 48, 51 - 53, 56, 59, 61, 69). In one adult rhea (case No. 41), proliferative enteritis with marked thickening of the mucosa (Fig. 3, 4), liquid dark brown intestinal content, infiltration of heterophils, macrophages, lymphocytes and plasmacells, as well as severe hyperplasia of intestinal crypts was found (Fig. 5, 6). Although Warthin-Starry silver impregnation and immunohistochemistry for Lawsonia intracellularis antigen failed to detect the pathogen, PCR identified genome fragments of Lawsonia intracellularis in samples taken from the small intestine. In addition, Clostridium (C.) perfringens, C. sordellii, Bacillus lentus, and Enterococcus durans were isolated from the large intestine of the same animal. Moreover, severe enteritis was diagnosed in ostrich No. 6. In case Nos. 48, 52, 56 and 59 intraluminal nematodes (not further identified) without inflammatory reactions were observed. Hepatic lesions comprised moderate to severe randomly distributed lympho- histiocytic and lympho-plasmacytic hepatitis (case Nos. 21, 51, 53) as well as mild to severe diffuse hepatic lipidosis (case Nos. 3, 32, 59, 61, 69). Juvenile ratites Disorders of the alimentary system were observed in 10 (43%) out of 23 animals (case Nos. 9, 11, 31, 38, 58, 64 - 68). Non-purulent and purulent inflammation of the glandular stomach was observed in two cases. One rhea (case No. 66) showed multifocal mineralization of the mucosa with associated lympho-plasmacytic infiltrates and one ostrich (case No. 68) had a

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mild to moderate purulent proventriculitis associated with numerous intralesional nematodes (not further identified) in the lamina propria mucosae. Inflammatory changes of the intestine were found in four animals. In two cases catarrhal and fibrinous enteritis (case No. 65, 67) and catarrhal typhlocolitis (case No. 38) were diagnosed. One rhea (case No. 31) suffered from fibrino-necrotizing typhlocolitis characterized by numerous erosions with accumulation of cellular debris as well as infiltration of heterophils and macrophages (Fig. 7, 8). Warthin-Starry silver impregnation applied on the tissue sections and a Brachyspira-immunofluorescence assay applied on feces revealed countless tortuous bacteria of up to 25 µm in length (Fig. 8). Genome fragments of Brachyspira (B.) sp. were detected by PCR. An intestinal intussusception with associated necrotizing and ulcerative inflammatory changes and intralesional bacterial colonies were observed in an ostrich (case No. 64). One ostrich (case No. 58) showed severe large intestinal impaction of unknown cause with subsequent fibrinous serositis. Eggs and neonates Perforation of the muscular stomach wall due to ingestion of a sharp foreign body associated with severe purulent and necrotizing inflammation of the gastric wall and serosa was detected in one neonatal rhea (case No. 24).

Cardiovascular system Adult ratites Diseases of the cardiovascular system were diagnosed in 17 animals (46%). Myocardial lesions were found in 7 and vascular changes in 10 animals, respectively. In two ostriches and one rhea (case Nos. 2, 47, 60) a degenerative cardiomyopathy with extensive hyaline degeneration of cardiomyocytes and histiocytic inflammation was observed. In one ostrich (case No. 51), a severe chronic valvular endocarditis with thrombus formation and intralesional Gram-positive bacteria was detected. Ostrich No. 61 showed a focal moderate purulent epi- and myocarditis. A female ostrich (case No. 57), weighing 80 kg, suffered from a severe left ventricular hypertrophic cardiomyopathy with an absolute heart weight of 1.3 kg. Streak-like changes were seen in the aortic intima consisting of fibrous tissue that

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was interpreted as arteriosclerotic change. Morphological findings indicating decompensation such as left atrial hypertrophy and dilatation, chronic pulmonary congestion or effusions were not recorded. A 15-year-old female ostrich (case No. 56), weighing 146 kg, died of hypovolemic shock after rupture of a dissecting abdominal aortic aneurysm. Adjacent to the rupture rims, the aortic wall showed multifocal mural calcifications and necrosis. Perivascular hematomas, extensive granulation tissue and thrombosis of periaortic blood vessels were additionally observed. Moreover, a moderate focal granulomatous myocarditis was diagnosed in the same animal, but special stains (Ziehl-Neelsen´s stain, PAS-reaction) failed to detect intralesional pathogens. In eight cases (case Nos. 6 - 8, 19, 20, 22, 23, 33) a gradually variable hypertrophy and hyperplasia of the tunica media and proliferation of smooth muscle cells in the tunica intima of arterial vessel walls was diagnosed in various organs (Fig. 9). In one ostrich disseminated intravascular coagulopathy associated with multifocal intravascular fibrin thrombi of undetermined cause was seen (case No. 6). Juvenile ratites In one ostrich (case No. 9) disseminated intravascular coagulopathy associated with meningoencephalitis and focal cortical encephalomalacia most likely caused by hypoxia was found.

Nervous system Adult ratites In 13 (35%) out of 37 adult ratites lesions of the brain were detected. In 6 ostriches (case Nos. 7, 8, 17, 19, 20, 34) and two emus (case Nos. 18 and 55) a degenerative encephalopathy with spongy degeneration of various regions of the brain was found. Reported clinical signs in ostriches Nos. 7, 8, 17 and 19 that were necropsied between 1986 and 1993 comprised slowly progressive ataxia with disturbances of balance, depression and anorexia. Ostrich No. 20 showed mild ataxia and possible blindness. In addition, ostriches Nos. 17 and 19 exhibited unusual twisting of the neck and increasing sternal recumbency. In animals Nos. 34 and 55 no nervous signs were noticed. Histological findings in ostriches Nos. 17 and 19 consisted of

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mild to moderate numbers of round to oval clear vacuoles in the perikaryon of neurons of the substantia grisea centralis, nucleus nervi oculomotorii, and nucleus reticularis pontis oralis and in the neuropil of the brain stem (Fig. 10). Spongy changes were distributed bilaterally and symmetrically. The lesions of ostriches Nos. 7 and 8 have been described previously in detail [25, 26]. Briefly, severe vacuolation was observed in the perikaryon of neurons of the nucleus ruber, nucleus vestibularis and nucleus reticularis, and in the neuropil of the brain stem. In addition, a fine granular, under UV light autofluorescent, PAS-positive and acid-fast pigment was observed in the perikaryon of numerous neurons in the same animals interpreted as lipofuscin. In animals Nos. 34 and 55 single vacuoles admixed with a fine granular, golden- brown, PAS-positive pigment interpreted as lipofuscin were present in the perikaryon of some neurons of the brain stem (Fig. 11, 12). The immunohistochemical evaluation of ostriches Nos. 17, 19, 34 and emu No. 55 failed to demonstrate specific PrP accumulations. Signals observed after incubation of the sections with a null-mab directed against GP5 of PRRSV were indistinguishable from those observed after incubation with both PrP-specific mabs. Analysis of the binding affinity of the two PrP-specific mabs generally used for the transmissible spongy encephalopathy diagnostics in ruminants (F99/97.6.1 and 6C2) did not reveal PrP-specific bands to verify the binding of both antibodies to ratite PrP, although especially in the case of mab 6C2, the binding epitope within the PrP displays no mutation between ruminant and ratite PrP. In contrast, PrP-associated bands were visible for the cattle brain sample loaded as a control (data not shown). One emu (case No. 53) and one ostrich (case No. 54) with a clinical history of acute trauma suffered from inflammatory lesions in the brain. In emu No. 53 moderate multifocal perivascularly accentuated granulomatous encephalitis was detected predominantly in the telencephalon. However, Ziehl-Neelsen´s stain failed to detect intralesional pathogens. In ostrich No. 54 moderate multifocal perivascular lympho-histiocytic and plasmacytic encephalitis predominantly in the cerebellum and in the brain stem was recorded. Immunohistochemical evaluations for tick-borne

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encephalitis virus, Borna disease virus, equine herpesvirus, rabies virus, influenza A virus and Listeria monocytogenes antigens remained negative in this case. In ostriches Nos. 49, 52 and 59, mild to moderate golden-brown pigment accumulations interpreted as lipofuscin were observed in the perikaryon of some neurons of the brain stem. Juvenile ratites In 5 (22%) out of 23 juvenile ratites lesions of the brain were detected. Spongy degeneration was found in three juvenile male ostriches (case Nos. 12, 29, 30). According to a previous report [26], ostrich No. 12 that died in 1989 showed clinically slowly progressive ataxia, including disturbances of balance and loss of appetite. Lesions were characterized by severe vacuolation of the neuropil and few neurons of the brain stem and medulla oblongata [26]. Ostriches no. 29 and 30 presented with clinical signs of apathy, somnolence and opisthotonos. Mild intraneuronal vacuolizations in Purkinje cells, the cortex cerebri and neuropil of the brain stem were found. In addition, both animals were in a cachectic nutritional status. Immunohistochemical evaluation failed to demonstrate specific PrP accumulations. In ostrich No. 9 multifocal meningoencephalitis and focal cortical encephalomalacia was found in association with disseminated intravascular coagulopathy. One rhea (case No. 27) displayed severe diffuse granulomatous inflammation of the choroid plexus without demonstrable pathogens using PAS- reaction and Ziehl-Neelsen´s stain.

Respiratory system Adult ratites Seven (19%) out of 37 adult ratites showed significant changes of the respiratory system. Findings included one case of mycotic rhinitis with numerous vascular thrombi in an ostrich due to Aspergillus sp. (case No. 57) and one case of purulent to necrotizing rhinitis in an ostrich (case No. 61). A rhea (case No. 62) with a history of respiratory distress showed purulent laryngitis, multifocal to coalescing suppurative to necrotizing laryngo-tracheitis (Fig. 13, 14) and lympho-plasmacytic parabronchitis. Immunohistochemically intralesional influenza A virus nucleoprotein (Fig. 15) was

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detected in trachea and lung. Molecular analysis performed at the Veterinary State Reference Laboratory revealed subtype H7N7 of influenza A virus. Furthermore, three ostriches (case Nos. 1, 34, 59) suffering from severe granulomatous pneumonia with intralesional septated fungal hyphae indicative of Aspergillus sp. were also recorded. One ostrich (case No. 17) showed a purulent aerosacculitis. Juvenile ratites In ostrich No. 68 nematodes (not further specified) were detected in the nasal cavity associated with a severe diffuse ulcerative and purulent inflammation caused by a secondary bacterial infection. Another ostrich (case No. 30) had a severe granulomatous and necrotizing rhinitis with associated fungal hyphae, most likely Aspergillus sp. Eggs and neonates In one neonatal ostriches (case Nos. 37) interstitial lympho-histiocytic pneumonia of unknown etiology was observed.

Urogenital system Adult ratites Morphological changes of the urogenital system were present in 7 (19%) out of 37 adult ratites and included mild multifocal lympho-histiocytic interstitial nephritis (case Nos. 21, 53, 55), multifocal tubulonephrosis and tubulonecrosis (case No. 2), proliferative glomerulonephritis (case No. 62), and a renal adenoma in an adult rhea (case No. 48). Cystic lesions in the testicle without other associated lesions were detected in an ostrich (case No. 3). Juvenile ratites Mild multifocal tubulonephrosis was diagnosed in one ostrich (case No. 29.).

Systemic diseases Adult ratites In 3 (8%) adult animals (case Nos. 34, 49, 63) systemic diseases characterized by granulomatous inflammations were observed. Using Ziehl-Neelsen´s stain intralesional acid-fast bacterial rods were found in all cases. In ostrich No. 34,

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granulomas were present in conjunctiva, lung, spleen, liver, ovaries and serosal surface of the body cavity. In ostrich No. 49 granulomas were observed in spleen and thymus. Microbiologic examination failed to isolate Mycobacterium (M.) sp. in both cases. In emu No. 63, housed together with wallabies, M. avium subsp. avium was identified in granulomatous lesions of trachea, lung, spleen and liver (Fig. 16, 17). One of the wallabies was also reported to be infected with the same bacterium. In two animals (no 34, 63) an additional granulomatous mycotic infection in lung, air sacs and serosal surface of the body cavity characterized by intralesional septated hyphae was observed. Juvenile ratites In ostrich No. 30, granulomatous inflammation with intralesional septated hyphae most probably Aspergillus sp. was observed in nasal cavity, esophagus and body cavity. Eggs and neonates Seven (64%; case Nos. 35, 36, 39, 40, 42 – 44) out of 11 ostriches with generalized subcutaneous edema were diagnosed with the fading ostrich syndrome. In three animals there was also a moderate to severe edema of the musculature present. Additionally, purulent omphalitis (Fig. 18) most likely due to a bacterial infection was found in two ostriches (case Nos. 35, 36). In ostriches Nos. 39 and 40, mild mineralized concrements in renal tubuli were observed, and in ostriches Nos. 43 and 44 mild mineralizations were detected in the proventricular mucosa.

Miscellaneous disorders Adult ratites Thyroidal adenoma was found in an adult rhea (case No. 47). Granulomatous dermatitis occurred in an ostrich (case No. 57), however, Ziehl- Neelsen´s stain and PAS-reaction failed to detect intralesional pathogens. In two ratites (case Nos. 34, 55) a granulomatous and proliferative conjunctivitis with intralesional acid-fast bacterial rods and a lympho-plasmacytic conjunctivitis were documented respectively. In one emu (case No. 18), a fibrino-purulent inflammation of the body cavity was found and Haemophilus sp. was isolated from the lesion. One

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emu (case No. 55) showed a bilateral retinal detachment associated with lympho- plasmacytic uveitis of unknown etiology.

Discussion In this retrospective study pathological findings and their frequency related to different age groups of necropsied ratites in northwestern Germany are described. The survey was conducted in order to elucidate the principal causes of disease in various species including ostriches, emus and rheas kept in captivity.

Musculoskeletal system Lesions of the legs and trunk with associated hemorrhage, muscular laceration and degeneration most likely caused by traumatic impacts was the most prominent cause of disease in adult ratites. The high number of traumatic injuries detected in adult animals, supported by clinical evidence of trauma, may be caused by housing situations on farms and in zoological institutions. Femoral and tibiotarsal fractures frequently resulted in severe hemorrhages followed by hypovolemic shock and death. The tibiotarsus is probably the most trauma-exposed bone of the ratite skeleton [27]. In addition, tarsometatarsal fractures are usually open with the risk of infection and prolonged recumbency often resulting in muscular degeneration [7]. Injuries most often occur when ratites are either chased or become frightened and run into fences and other obstacles [6]. With the exception of one emu (case No. 53), evidence of predisposing conditions for pathological fractures was not observed in adult ratites in the current study. However other mechanisms or underlying causes due to metabolic changes cannot be ruled out completely and would require additional investigations including determination of calcium and phosphor values in the blood and bones. In emu No. 53, a spontaneous pathological fracture due to an intraosseous fibrosarcoma was found. Skeletal fibrosarcomas arise from connective tissue stroma in either the medullary cavity, as in this case, or the periosteum. Fibrosarcomas of different locations are among the most frequently observed neoplasm in pet birds [28]. However, in ratites this variant has not been described before. Histologically, this malignant tumor shares features with fibrosarcomas of other body sites and

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species and is characterized by interlacing bundles of fusiform cells with vesicular nuclei. In ostrich No. 60, hyaline muscular degeneration (Zenker´s degeneration) of various skeletal muscles was observed. With respect to the clinical history of depression after transportation, this finding is suggestive of capture myopathy as possible cause of disease and death. Capture myopathy, selenium or vitamin E deficiency, furazolidone and ionophore intoxications are some of several etiologies which reportedly cause degenerative myopathy in ratites [1, 7, 29, 30]. Clinical signs in ratites include depression, reluctance to rise or move, and a rapid progression to death. Transportation has been considered to be one of the most stressful procedures in managing ratites [29, 31]. These bipedal animals with a height of up to approximately 3 m in adult ostriches have their center of gravidity rather high above the ground [29]. This causes difficulties in achieving a balance, particularly during shipping on trucks. In addition, ratites are very nervous birds and highly susceptible to capture myopathy, making their transportation on trucks more complex and stressful than for food and companion animals like cattle and horses [29, 31]. However, in ostrich No. 60 an additional degenerative cardiomyopathy was present. As exertional myopathy caused by transportation is reported to be restricted to skeletal muscles [6] differential diagnoses for degenerative skeletal and cardiac muscular disorders have to be considered including selenium deficiency. Although a variety of drugs and chemicals are potentially myotoxic and cardiotoxic to birds, there are very few documented cases. Severe cardiomyopathy in ostriches due to avocado (Persea americana var. guatemalensis) intoxication was reported in South Africa [32]. Moreover, in many animal species, ionophore toxicity characterized by focal degenerative cardiomyopathy and skeletal muscle degeneration was reported as an important cause for cardiomyopathies [33], however, myocardial damage has not been reported in ostriches [34]. Further laboratory investigations were not performed in the described case. In juvenile ratites, two cases of slipped tendon were encountered. Slipped tendon occurs in all ratite species, but ostriches and emus are most frequently affected [8]. Slipped tendon is a lay term used to describe the lateral displacement of the

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gastrocnemius tendon from the normal anatomical location between the condyles of the distal tibiotarsal and proximal tarsometatarsal bones [8]. Causes include genetic predisposition and malnutrition (manganese, biotin, pantothenic acid, folic acid and choline deficiency) as possible predisposing factors [8, 35]. Elevated selenium levels as well as a lack of copper and calcium were suggested to play a role in the development of slipped tendon in ostrich chicks [36]. In the cases presented here, no essential nutrients or mineral values were determined. There are additional metabolic conditions that can affect the bone quality especially in juvenile ratites. In the current study, one juvenile ostrich (case No. 15) showed lesions suggestive of rickets and several rib fractures that were most likely the result of trauma and inadequate mineralized bone. Similar lesions have been described in rheas [37]. Fibrous osteodystrophy caused by secondary hyperparathyroidism has been described in birds and can also lead to weakening of bones with subsequent fractures [38-40].

Digestive system In this study, gastro-intestinal disorders were the major cause of disease in juvenile ratites. Common factors that can cause enteritis in chicks and juveniles are failure to establish a balanced gut flora, the use of antibiotics, lack of fibers in the diet, hypothermia, excessive coprophagy, and poor hygiene [1, 41]. Bacterial pathogens causing enteritis include E. coli, Salmonella sp. Klebsiella sp., Aeromonas sp., Pseudomonas sp., certain strains of Campylobacter jejuni and Clostridium (C.) perfringens may also be involved [1, 3, 8, 41-43]. In the current study, Lawsonia intracellularis, C. perfringens, C. sordellii, Bacillus lentus and Enterococcus durans (case No. 41), as well as Brachyspira sp. (case No. 31) were identified in diseased ratites. Lawsonia intracellularis is the sole species in the bacterial genus Lawsonia and is often found in pigs, where it causes a proliferative enteropathy [44]. Other affected species include hamsters, rabbits, ferrets, foxes, dogs, rats, sheep, deer, emus, ostriches, nonhuman primates, and guinea pigs [12]. This disease is characterized macroscopically by thickening of the intestinal mucosa due to enterocyte

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proliferation. As a result, mild to severe diarrhea represents the major clinical sign described in affected animals [12]. Histologically, thickened villi, crypt hyperplasia, dilated glands filled with mucus and heterophils, and a dense infiltration of heterophils, lymphocytes, macrophages and plasma cells in the lamina propria are morphological findings reported in emus [12]. The intestinal changes in rhea No. 41 of this study correspond to the described lesions and PCR confirmed the presence of Lawsonia intracellularis, which represents the first case in this ratite species. In a juvenile rhea (case No. 31) a severe fibrino-necrotizing typhlocolitis with intralesional spirochetes was found. Immunofluorescence and molecular analyses confirmed the presence of Brachyspira sp. which is regarded as the causative agent of disease in this animal. The fastidious, anaerobic spirochete Brachyspira is capable of causing enteric disease in avian, porcine and human hosts, amongst others, with a potential for zoonotic transmission [45]. Rheas naturally colonized with B. hyodysenteriae commonly show severe typhlitis and mortality can be as high as 80% [46, 47]. Severe disease is characterized by dilation of the caeca with thickened walls and ulceration, severe mucosal necrosis, crypt elongation and goblet cell hyperplasia [45]. Lesions were most common in rheas less than 3 months of age. Stress after shipping was proposed as a contributing factor [46]. The rhea in this case was 3 months old which corresponds well with published findings [46]. Intussusception is often seen in ostrich chicks and juveniles following gastric impaction. Impaction with foreign body material is associated with abnormal compensatory picking (disorientation stress, desertion stress, or frustration caused by finding food from change of location or diet) [1]. Other causes of intestinal intussusception are chronic diarrhea, frequently associated with other intestinal disorders such as gastro-intestinal displacement, torsion and volvulus, and abnormal peristaltic contractions due to unknown reasons [8]. In the described ostrich (case No. 64), no underlying cause could be found for the intussusception. One case of poor body condition associated with mild to moderate purulent inflammation of the glandular stomach associated with infestation of numerous nematodes was diagnosed in one ostrich (case No. 68). A considerable number of endoparasites can infect ratites and gastro-intestinal nematodes are of highest

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importance as they cause economic losses in ratite farming [8, 48]. Nematodes such as Libyostrongylus (L.) douglassii, L. dentatus and L. magnus are found in glands of the proventriculus and under the coilin layer of the gizzard of ostriches [42, 48]. Only the former two have been reported outside Africa [49] with L. douglassi also infecting ostriches in Germany [50] and The Netherlands [51]. The resulting inflammation inhibits gastric secretions and hinders digestion. The disease is called ―vrootmag‖ or ―rotten stomach‖ as food decays within the stomach and can lead to weight loss and high mortality in ostrich chicks and juveniles [7, 8, 48]. Rheas can also be infected, although the parasite seems to be rather host specific [1]. In one young rhea (case No. 24) perforation of the muscular stomach was diagnosed, presumably due to ingestion of a foreign body. Ingestion of foreign bodies is a common problem in ratites [8, 41], because these animals will swallow anything that fits into their mouths, and their curiosity and enquiring eye ensure that they will find many unusual items in their enclosure [8].

Cardiovascular system Reports on cardiovascular disease in ratites are scarce and some of the vascular changes described here were most likely incidental findings with no associated clinical signs. One of the well described vascular disorders in ostriches is aortic aneurysm. One adult ostrich in this study (case No. 56) was diagnosed with a dissecting aortic aneurysm with degenerative changes of the aortic wall which probably was a predisposing factor along with assumed hypertension. Several cases of aortic aneurysm have been described in the literature so far in which no atherosclerotic changes of the aorta were associated with the rupture site, however, copper deficiency that predisposes to defective elastin was suggested as a possible cause [52-55] which is also described in turkeys [56]. Other causes might be systemic hypertension and a genetic predisposition [53]. In the presented case, no copper values were determined. Atherosclerosis is the most important vascular lesion in birds especially in parrots and lesions can be found within the aorta, brachiocephalic trunks, and pectoral and carotid arteries [57]. Atherosclerotic lesions in psittacines are characterized by

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progressive accumulation of inflammatory cells, cholesterol, fat and cellular debris in the intima and luminal side of the vascular media. These changes are classified into seven lesion types of which the atheroma (type IV lesion) covered by a fibrous cap (type V lesion) are both features of advanced lesions and are often associated with clinical signs [58]. Complications of plaques include mineralization, ulceration, superimposed thrombosis, intraplaque hemorrhages and aneurysmal dilation [33]. The vascular lesions identified in this study (case Nos. 6 - 8, 19, 20, 22, 23 and 33) were characterized by gradually variable thickening of the tunica media of arteriolar vessel walls of parenchymatous organs like kidney and spleen. These changes did not resemble atherosclerosis and were regarded most likely as incidental findings. In ostrich No. 57, a considerable hypertrophy of the myocardium was found with a relative heart weight of 1.63%. The reference range of the absolute heart weight in ostriches of about 122 kg body weight is reported to be 1.054 g +/- 172 g corresponding to an absolute heart weight of 0.86% (+/- 0.14%). The cause of the cardiac hypertrophy in the study case remains undetermined, however, evidence of decompensation was not found. In the same animal streaks of fibrous tissue were present in the aortic intima. Whether these vascular changes were pathogenetically related to the cardiac disorder or represent a separate disease process remains undetermined. Abnormal jets of blood flow or turbulences have to be considered as possible causes.

Nervous system The principal change encountered in the central nervous system was a spongy degeneration in six adult ostriches (case Nos. 7, 8, 17, 19, 20, 34), two emus (case Nos. 18, 55) and three juvenile ostriches (case nos. 12, 29, 30). The clinical and morphological findings of three of these animals (case Nos. 7, 8, 12) from two different zoological collections in Germany have already been described [25, 26]. In case Nos. 17 and 19 the lesions were observed bilaterally in the brain stem, specifically affecting the substantia grisea centralis, nucleus nervi oculomotorii, and nucleus reticularis pontis oralis. The lesions are regarded most likely as cause of the clinical nervous signs.

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Spongy degeneration in domestic animals may affect either gray or white matter, or both, in a bilateral and symmetrical fashion and is caused by cytotoxic edema resulting in intracellular fluid accumulation due to disrupted osmoregulation [59]. Possible underlying mechanisms include toxins, gene defects and infectious as well as metabolic processes. The cause of the spongy degeneration observed in the present study remained undetermined. In case Nos. 7, 8 and 12 material was not available for additional investigations on the role of pathologic prion proteins. The temporal coincidence of case Nos. 7, 8, 12, 17 and 19 during the years 1986 to 1993 with the emergence of bovine spongiform encephalopathy in the United Kingdom and the subsequent occurrence throughout many European countries since 1990 raised the possibility of a causal link between both diseases. So far all investigations failed to proof a link between transmissible spongy encephalopathy and the central nervous system lesions in ratites [60]. Although the presented cases share clinical and morphological features described for transmissible spongiform encephalopathies in mammalian species, the application of two different PrP-specific antibodies that are routinely used for the diagnostics of ruminant transmissible spongiform encephalopathy revealed no accumulation of pathological PrP. However, the binding affinity of the applied antibodies to ratite PrP could not be irrevocably demonstrated. Spongy degeneration of unknown cause involving the gray matter has been described in domestic animals [61]. However, there is little information concerning similar changes in the central nervous system of birds. In addition, no indications for inherited, toxin-related, dietary-related or metabolic disturbances that could result in similar nervous lesions were found in the present cases. In ostriches Nos. 34 and 55 the observation of marked lipofuscin deposits in neurons and the absence of nervous signs suggest an age-related spongy degeneration. Neuronal vacuolation in case No. 29 may possibly be related to the poor nutritional condition that might be caused, at least in theory, by nutritional deficiencies, metabolic disorders, and intoxications [60]. In one emu and an ostrich (case Nos. 53 and 54), inflammatory lesions of the brain were found to be associated with multiple hemorrhages on the head and neck most likely leading to circulatory failure. Presumably, acute neurologic disturbance

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was the cause of the polytrauma in ostrich No. 54. The cause of the encephalitis remained undetermined in both cases, but an infectious agent, most likely a virus, has to be considered in these animals. Ratites are susceptible to a number of viruses that affect the central nervous system such as Borna disease virus and avian paramyxovirus [8]. Newcastle disease has been reported as an important contagious notifiable viral infection of ostriches characterized by neurological clinical signs [6]. However, microscopic changes are non-specific and include hemorrhages of the heart and an enlarged liver [5]. None of these morphological changes were observed in the current case, and due to the lack of neurological clinical signs in this bird, an infection with Newcastle disease virus was regarded as unlikely.

Respiratory system Respiratory diseases were recorded in adult ostriches and rheas including two cases (case Nos. 57, 61) with rhinitis, one case (case No. 62) with tracheitis, laryngitis and parabronchitis due to influenza A virus infection, and three cases (case Nos. 1, 34, 59) with mycotic granulomatous pneumonia. Fungal infections of the respiratory organs occur most frequently in neonates and juveniles housed in enclosed facilities and exposed to dust or hay which is alternatively wet or dry [42]. Aspergillus fumigatus and other Aspergillus sp. are commonly isolated and can also affect older birds, commonly in conjunction with immunosuppression or concurrent disease [1, 42]. Grossly, yellow to white granulomas are observed most frequently in the lung and/or air sacs, rarely also in viscera [42]. Fungal granulomatous rhinitis restricted to the nose as in case No. 57 has been described seldom [62]. Ostriches, emus and rheas are highly susceptible to avian influenza, and various subtypes have been identified including H7N1, H5N9, H5N2, H9N2 and H10N7 [1]. Natural infection with the subtype H7N7 has not been documented in ratites before. Avian influenza A virus causes respiratory (e. g. ocular and nasal discharge) and gastro-intestinal (e. g. diarrhea) clinical disease [63]. Other signs are ruffled feathers, and complications arising from secondary infections [63]. However, there are no specific clinical signs linked with avian influenza infection in ratite species.

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Characteristic postmortem findings include fibrinous aerosacculitis, mucoid sinusitis, multifocal hepatic necrosis, splenomegaly and nephritis [3]. Rhea No. 62 showed suppurative to necrotizing tracheitis, laryngitis, parabronchitis, and purulent hepatitis most likely caused by secondary bacterial infection. The first influenza A virus isolated from ratites was the highly pathogenic avian influenza A strain H7N1. It had caused an epizootic in ostriches in South Africa in 1991 in which high mortality was seen among young birds [63]. Young ostriches infected with this strain showed multifocal hepatic necrosis, splenomegaly and, in advanced cases, nephrosis and fibrinous aerosacculitis [8]. The same strain of avian influenza A virus was also isolated from ostriches in The Netherlands, Denmark, Italy, and from emus and cassowaries in The Netherlands [8, 63, 64]. The appearance of viruses of the same strain in various European countries and in South Africa may be caused by movement of infected domestic poultry and/or products as well as by wild and migratory birds. Furthermore, the increase in international trade in ostriches and other ratites may have contributed to the spread of influenza A virus [63]. For these reasons, the observation of avian influenza A virus infection in a rhea from Northwestern Germany is not surprising.

Ocular disorders In ostrich No. 34, conjunctival manifestation as part of a systemic avian tuberculosis was found which has been observed in ratites [65] [66]. In case of an isolated mycobacterial lesion of the conjunctiva an entry of the organism through an eyelid defect has to be considered. Systemic spread after primary conjunctival infection cannot be excluded.

Urogenital system and miscellaneous disorders in other organs Alterations observed in the urogenital system, endocrine glands and skin in animals of this study were diagnosed as secondary or incidental findings without great clinical importance.

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Systemic disease Three adult ratites showed granulomatous lesions in multiple organs with intralesional acid-fast positive bacteria indicative of mycobacteriosis, presumably an infection with M. avium subsp. avium. The susceptibility of ratites to avian tuberculosis remains unknown. It has been rarely reported in ratites as compared to other avian species [67]. The disease has been described mainly in emus and rheas and occasionally in ostriches [67]. Tuberculosis has great potential economic significance in emus, first due to loss of valuable birds and second due to the risk of transmission to other avian and mammalian species [65]. Infections with other species of mycobacteria are rarely reported in ratites. In one case, M. bovis was isolated from granulomatous lesions in an ostrich [67]. In ratites, the disease occurs more commonly in multispecies exhibition places like zoos than on single species farms [6]. One emu (case No. 63) was affected by granulomatous lesions of the trachea, spleen and liver. The distribution of granulomatous lesions suggests an oronasal infection as the possible portal of entry. This animal was housed in a zoo together with wallabies, one of which was diagnosed with mycobacteriosis caused by M. avium subsp. avium. Based on this finding a spill-over infection of mycobacteria is likely. In eggs and neonates, the most common cause of disease and death was the fading ostrich syndrome (FOS). FOS occurs in animals of less than 3 weeks of age and is a multifactorial disease complex that relates to improper incubation and rearing conditions, and possibly viral infections. Affected chicks often hatch with increased generalized edema, are weak, reluctant or unable to exercise, and show delayed or inappropriate intake of food [68]. FOS is also characterized by depression, anorexia, and death 3 to 5 days after onset of clinical signs in ostriches of less than 3 weeks old [69]. Except for the interstitial edema in the musculature the animals in this study showed no morphological changes of the musculature which is in contrast to mild, degenerative myopathy reported in another study [70]. Additionally, depletion of fat reserves is also reported [71]. However, it is possible that this syndrome may refer to more than one clinical condition or cause [68]. The causes of FOS are not known. Inadequate management practices and husbandry may act as contributing factors of

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these conditions. Vertically transmitted viral agents, such as adeno- or circoviruses, which cause a similar syndrome in turkeys, may act as primary pathogens [72, 73]. Two ostrich adenovirus isolates (isolate 1781 and B492) have been found to be noninfectious in inoculated ostrich chicks less than 8 weeks of age [71]. In addition, E. coli and/or Klebsiella pneumoniae were isolated from various organs of affected neonatal ostriches [68]. Chicks from the same study also showed a severe lymphocytic depletion of the bursa of Fabricius [68], which was not a feature in this study. Circovirus that possibly is transmitted vertically, has been suspected to be involved in FOS on a farm in the Netherlands and a vertical transmission was suspected [72]. As accidental findings, mineralized tubular concrements in the kidney were observed in two neonate ostriches (case Nos. 39, 40). Mineralized tubular depositions are frequently observed in embryos and unhatched eggs [8]. Mineral or crystalline deposits comprise either calcium or uric acid. In low amounts these deposits are regarded as clinically irrelevant [8]. The commonly known causes in chicks include hypervitaminosis D3, and in embryos incubation-related dehydration with subsequent urate accumulation in the tubules [8]. Multifocal mineralization of the proventriculus was detected in two other ostriches (case Nos. 43 and 44). Similar deposits may occur in cases with hypercalcemia and renal failure. However, widespread calcium deposits, e. g. pulmonary elastic fibers or renal tubular basement membranes were not present in both cases and the cause of the gastric mucosal mineralization remains unclear.

Conclusion The high frequencies of trauma including fractures of the long bones, as well as traumatic injuries of the neck of juvenile and adult captive ratites living in northwestern Germany underline the susceptibility of ratites to mechanical injuries. The cause of spongy changes of the central nervous system observed in adults and juvenile animals in association with central nervous signs remains obscure. A similar disorder is not known in wild ratites, and hereditary or exogenous toxic causes have to be considered.

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In juvenile animals, bacterial infections of the alimentary tract were an important cause of disease in the present study. Whereas these disorders occur only very rarely in wild ratites, they are often reported in ratites of this age group held in captivity, even in their natural habitat. Fading ostrich syndrome is a worldwide disease occurring in farm husbandry and in zoological institutions. Among various factors particularly inadequate management practices such as improper incubation conditions for eggs contribute to this disorder. In addition, some salient lesions like intraosseous fibrosarcoma, isolated mycotic rhinitis, infection with influenza A H7N7, that have not or only rarely been described in ratites extend the spectrum of disorders in these species.

Acknowledgements We thank Mrs. Buck, Mrs. Grünig, Mrs. Herrmann and Mrs. Schütz for the excellent technical assistance. A. Bello received a grant from ―Fundación Gran Mariscal de Ayacucho‖, Venezuela and from ―Deutscher Akademischer Austauschdienst‖ (DAAD), Germany. This study was in part supported by the Niedersachsen-Research Network on Neuroinfectiology of the Ministry of Science and Culture of Lower Saxony.

Declaration of conflicting interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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46. Buckles EL, Eaton KA, Swayne DE. Cases of spirochete-associated necrotizing typhlitis in captive common rheas (Rhea americana). Avian Dis. 1997;41(1):144– 8. doi: 10.2307/1592454. 47. Sagartz JE, Swayne DE, Eaton KA, Hayes JR, Amass KD, Wack R, et al. Necrotizing typhlocolitis associated with a spirochete in rheas (Rhea americana). Avian Dis. 1992;36(2):282–9. doi: 10.2307/1591502. 48. Ponce Gordo F, Herrera S, Castro AT, Garc a Dur n B, Mart n ez D a z A. Parasites from farmed ostriches (Struthio camelus) and rheas (Rhea americana) in Europe. Vet Parasitol. 2002;107(1–2):137–60. doi: 10.1016/S0304- 4017(02)00104-8. 49. Hoberg EP, Lloyd S, Omar H. Libyostrongylus dentatus n. sp. (Nematoda: Trichostrongylidae) from ostriches in North America, with comments on the genera Libyostrongylus and Paralibyostrongylus. J Parasitol. 1995;81(1):85–93. doi: 10.2307/3284011. 50. Schulze C, Grossmann E, Krone O. [Case report: Libyostrongylus douglassii- associated proventriculitis in ostriches in Germany]. Dtsch Tierarztl Wochenschr. 2006;113(6):240-2. PubMed PMID: 16856612. 51. Landman WJ, Bronneberg RG. Libyostrongylus douglassii in ostriches (Struthio camelus ssp.) in the Netherlands: case report and review. Tijdschr Diergeneeskd. 2001;126(14-15):484–7. PubMed PMID: 11510368. 52. Mitchinson MJ, Keymer IF. Aortic rupture in ostriches (Struthio camelus)—A comparative study. J Comp Pathol. 1977;87(1):27–33. doi: 10.1016/0021- 9975(77)90076-7. 53. Ferreras MC, Gonzalez J, Pérez V, Reyes L, Gómez N, Pérez C, et al. Proximal aortic dissection (dissecting aortic aneurysm) in a mature ostrich. Avian Dis. 2001;45(1):251–6. doi: 10.2307/1593037. 54. Baptiste KE, Pyle RL, Robertson JL, Pierson W, Larsen C, Warthen K. Dissecting aortic aneurysm associated with a right ventricular arteriovenous shunt in a mature ostrich (Struthio camelus). J Avian Med Surg. 1997;11(3):194–200. 55. Vanhooser SL, Stair E, Edwards WC, Labor MR, Carter D. Aortic rupture in ostrich associated with copper deficiency. Vet Hum Toxicol. 1994;36(3):226–7.

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56. Graham CLG. Copper levels in livers of turkeys with naturally occurring aortic rupture. Avian Dis. 1977;21(1):113–6. doi: 10.2307/1589369. 57. Schmidt RE, Reavill DR, Phalen DN. Cardiovascular System. In: Schmidt RE, Reavill DR, Phalen DN, editors. Pathology of Pet and Aviary Birds. 2nd ed. Ames, IA: John Wiley & Sons; 2015. p. 1–20. 58. Beaufrère H. Avian Atherosclerosis: Parrots and Beyond. J Exot Pet Med. 2013;22(4):336–47. doi: 10.1053/j.jepm.2013.10.015. 59. Vandevelde M, Higgins RJ, Oevermann A. Degenerative diseases. In: Vandevelde M, Higgins RJ, Oevermann A, editors. Veterinary Neuropathology: essentials of theory and practice. West Sussex, UK: John Wiley & Sons, Inc.; 2012. p. 182–7. 60. Wells GAH, Pohlenz J, Hawkins SAC, Matthews D. Portrait of a spongiform encephalopathy in birds and the transmissibility of mammalian prion diseases to birds. In: Hörnlimann B, Riesner D, Kretzschmar H, editors. Prions in Humans and Animals. Berlin, DE: De Gruyter; 2007. p. 279–83. 61. Cantile C, Youssef S. Nervous system. In: Maxie MG, editor. Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. 1. 6th ed. Philadelphia, PA: Saunders Elsevier; 2016. p. 250-406. 62. Fitzgerald SD, Moisan PG. Mycotic rhinitis in an ostrich. Avian Dis. 1995;39(1):194-6. doi: 10.2307/1592003. 63. Alexander DJ. A review of avian influenza in different bird species. Vet Microbiol. 2000;74(1-2):3–13. doi: 10.1016/S0378-1135(00)00160-7. 64. Capua I, Mutinelli F, Dalla Pozza M, Donatelli I, Puzelli S, Cancellotti FM. The 1999–2000 avian influenza (H7N1) epidemic in Italy: veterinary and human health implications. Acta Trop. 2002;83(1):7–11. doi: doi:10.1016/S0001- 706X(02)00057-8. 65. Pocknell AM, Miller BJ, Neufeld JL, Grahn BH. Conjunctival mycobacteriosis in two emus (Dromaius novaehollandiae). Vet Pathol. 1996;33(3):346-8. doi: 10.1177/030098589603300314.

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66. García A, LeClear CT, Gaskin JM. Mycobacterium avium infection in an ostrich (Struthio camelus). J Zoo Wildl Med. 2001;32(1):96-100. doi: 10.1638/1042- 7260(2001)032[0096:MAIIAO]2.0.CO;2. 67. Kelly P, Jahns H, Power E, Bainbridge J, Kenny K, Corpa JM, et al. Mycobacteriosis in ostriches (Struthio camelus) due to Infection with Mycobacterium bovis and Mycobacterium avium complex. Avian Dis. 2013;57(4):808–11. doi: 10.1637/10581-052313-Case.1. 68. Terzich M, Vanhooser S. Postmortem findings of ostriches submitted to the Oklahoma Animal Disease Diagnostic Laboratory. Avian Dis. 1993;37(4):1136– 41. doi: 10.2307/1591926. 69. Cooper RG, Horbanczuk JO, Fujihara N. Nutrition and feed management in the ostrich (Struthio camelus var. domesticus). Anim Sci J. 2004;75(3):175–81. doi: 10.1111/j.1740-0929.2004.00173.x. 70. Philbey AW, Button C, Gestier AW, Munro BE, Glastonbury JRW, Hindmarsh M, et al. Anasarca and myopathy in ostrich chicks. Aust Vet J. 1991;68(7):237–40. doi: 10.1111/j.1751-0813.1991.tb03215.x. 71. El-Attrache J, Villegas P, O'Connor B, Buhr JR, Rowland GN. Adenovirus pathogenicity in immature ostriches. Avian Dis. 2001;45(2):442–6. doi: 10.2307/1592985. 72. Eisenberg SWF, Van Asten AJAM, Van Ederen AM, Dorrestein GM. Detection of circovirus with a polymerase chain reaction in the ostrich (Struthio camelus) on a farm in The Netherlands. Vet Microbiol. 2003;95(1-2):27–38. doi: 10.1016/S0378- 1135(03)00122-6. 73. Raines AM, Kocan A, Schmidt R. Experimental inoculation of adenovirus in ostrich chicks (Struthio camelus). J Avian Med Surg. 1997;11(4):255–9.

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Figure legends Figure 1-2. Intraosseous fibrosarcoma, left tarso-metatarsal bone, emu, case No. 53. Figure 1. Adjacent to the gray-white neoplastic tissue that occupies partially the marrow cavity (asterisk) the oblique fracture line of the bone is visible (arrow). Figure 2. The fibrosarcoma is composed of spindle-shaped neoplastic cells embedded in abundant amounts of collagen fibers. Hematoxylin and eosin (HE).

Figure 3-6. Proliferative enteritis caused by Lawsonia intracellularis, intestine, rhea, case No. 41. Figure 3. Red-brown discoloration of the intestinal mucosa with fleshy appearance of the duodenal wall. P = pancreas. Figure 4. Marked thickening of the intestinal mucosa (asterisks). Figure 5. Subgross magnification reveals severe elongation of the intestinal crypts. HE. Figure 6. Severe crypt hyperplasia (asterisks) and moderate inflammatory infiltration of the adjacent lamina propria. HE.

Figure 7-8. Fibrino-necrotizing typhlocolitis caused by Brachyspira sp., intestine, rhea, case No. 31. Figure 7. Brown-greenish tinged fibrin attached to the cecal mucosa (arrows). Figure 8. Multifocal fibrino-necrotic mucosal lesions (asterisks). HE. Inset: Immunolabelling of spirochetes using a polyclonal antiserum against B. hyodysenteriae. Immunofluorescence.

Figure 9. Hypertrophy and hyperplasia of the tunica media, cardiac artery, rhea, case No. 22. Marked increase in thickness of the tunica media (asterisks). HE.

Figure 10. Spongy degeneration, brain, ostrich, case No. 19. Oval to spherical vacuoles of variable diameter (arrows) are present in the perikaryon of several neurons of the red nucleus. HE.

Figure 11-12. Spongy degeneration and lipofuscin deposition, brain, ostrich, case No. 34. Figure 11. Oval vacuole (arrowhead) and deposition of a coarse granular golden-brown pigment (arrow) in the perikaryon of some brain stem neurons. HE.

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Figure 12. PAS-positive coarse granular pigment deposition suggestive of lipofuscin (arrow) in a brain stem neuron. PAS-reaction.

Figure 13-15. Avian influenza, larynx and trachea, rhea, case No. 62. Figure 13. Severe necro-suppurative laryngo-tracheitis with diffuse hyperemia and numerous white, necrotic foci. Figure 14. Severe, diffuse, necro-suppurative tracheitis. HE. Figure 15. Intralesional immunolabelling of influenza A virus nucleoprotein (arrows). Immunohistochemistry.

Figure 16-17. Avian mycobacteriosis and aspergillosis, trachea and lung, emu, case No. 63. Figure 16. Multifocal yellow granulomas (arrows) in the trachea caused by M. avium subsp. avium and focal white granuloma caused by Aspergillus sp. (arrowhead). Figure 17. Necro-granulomatous pneumonia. HE.

Figure 18. Fading ostrich syndrome, subcutis, ostrich, case No. 35. Generalized subcutaneous edema (anasarca) and purulent omphalitis (arrow).

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Figures

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Chapter 5: Discussion

In this retrospective study about necropsied ratites kept in northwestern Germany the pathological findings and the results of additional etiological investigations are described. The frequency of lesions is related to different age groups. In many animals more than one morphological diagnosis attributable to different disease processes was found.

5.1 General aspects Ostriches represent with more than three-quarters the predominant ratite species of the retrospectively evaluated necropsy cases indicating that they are probably the most prevalent species in northwestern Germany. More than 80% of the cases originated from zoological collections. Approximately 65% of the reviewed cases died spontaneously and about half of the cases were adult individuals. The body condition was regarded as good in almost half of all investigated animals which might indicate that fatal diseases or disorders requiring euthanasia are of acute courses instead of chronic debilitating illnesses.

5.2 Musculoskeletal system In adult ratites, alterations of leg, neck and trunk bones associated with soft tissue injuries were most likely caused by traumatic injuries and represent the most frequent cause of disease. This high incidence of trauma-related lesions in adult ratites has also been observed in other studies (KÖSTERS et al. 1996). When ostriches and other ratites are chased or frightened they run into fences or obstacles possibly favored by unsuitable housing situations on farms and in zoological institutions (HUCHZERMEYER 1998). Fractures of femur, tibiotarsus or tarsometatarsus may result in severe hemorrhage leading to fatal hypovolemic shock. Surgical setting of leg fractures is extremely difficult and these cases often end up in euthanasia (HUCHZERMEYER 1998). A predisposing lesion for a fracture was found only in one individual, i. e. emu No. 53. It suffered from an intraosseous fibrosarcoma with subsequent pathological fracture. In ratites this tumor variant has generally not been described before. Avian fibrosarcomas are most frequently seen in pet birds (REAVILL 2004) and may occur anywhere on the body,

Chapter 5: Discussion

particularly in the oral cavity, associated with long bones, or in the abdominal cavity (LIGHTFOOT 2006). Systemic muscular degeneration in ratites may be caused by muscular overexertion, nutritional muscular dystrophy related to selenium or vitamin E deficiency, furazolidone and ionophore intoxications (STEWART 1994; BAIRD et al. 1997; MUSHI et al. 1998; WOTTON and HEWITT 1999; MINKA and AYO 2008; MENON et al. 2014; KUMMROW 2015). Pale discoloration of the skeletal musculature is present at gross examination and swelling, vacuolation, fragmentation, interstitial edema and necrosis of muscle fibers are observed histologically (MUSHI et al. 1998). Chronic lesions are characterized by firm white streaks representing dystrophic calcifications. In an 11-month-old ostrich (case No. 60), muscular degeneration and death was suggestive of capture myopathy because the animal was submitted with a history of transportation. Transportation is considered to be one of the most stressful procedures in managing ratites (WOTTON and HEWITT 1999; MINKA and AYO 2008). However, the ostrich No. 60 suffered additionally from a severe multifocal myocardial degeneration. Similar to monensin intoxication (BAIRD et al. 1997), capture myopathy is reported to be restricted to the skeletal muscles (HUCHZERMEYER, 1998). Therefore, in this case a nutritional muscular and cardiac degeneration most likely related to selenium or vitamin E deficiency has alternatively or additionally to be considered, because this disorder involves skeletal musculature, myocardium and the gizzard muscle (HUCHZERMEIER 1998). Furthermore, it cannot be excluded that the acute manifestation is simply based on a subclinical chronic vitamin E and selenium deficiency triggered by transportation because an insufficient supplementation is more commonly observed in young ratites due to higher demands required for growth (STEWART 1994). Vitamin E and selenium values were not determined in that case. Slipped tendon, a lay term used for the lateral displacement of the gastrocnemius tendon, occurred in juvenile ratites. In domestic fowl a similar disorder is termed “perosis”, however, in this disorder the displacement of the tendon is medial (HUCHZERMEIER 1998). Slipped tendon is reported in all ratite species, but ostriches and emus are most frequently affected. Genetic predisposition and malnutrition (manganese, biotin, pantothenic acid, folic acid and choline deficiency associated with deformation of the hock joint) are regarded as possible predisposing factors in relation with a triggering trauma (MACWHIRTER 1994;

Chapter 5: Discussion

SPEER 1996). However, essential nutrient values were not determined in the cases of this study. Inadequate mineralization of bone may be caused by rickets due to a lack of vitamin D supplementation (HUCHZERMEIER 1998) or hypophosphatemia (GRÖNE et al. 1995; MORROW et al. 1997). Animals most likely affected by rickets, like case No. 15, are typically juvenile and display soft bone tissue with fractures. As differential diagnosis fibrous osteodystrophy caused by secondary hyperparathyroidism has to be considered (SCHMIDT 2015).

5.3 Alimentary system In this study, gastro-intestinal disorders were the major cause of disease in young ratites. Perforation of the stomach as in the young rhea No. 24 was presumably caused by ingestion of a sharp foreign body. Ratites consume or try to consume most items they can reach resulting in gastro-intestinal impaction or perforation often resulting in death. Examples of swallowed foreign bodies include large amounts of earth, stones, wood, straw, thorns, sharp metal objects, nails, bullets, cartridge cases, handkerchiefs, gloves, pencils, and wire pieces, in one case up to 2.5 m of barbed wire (DEGEN et al. 1989; STEWART 1994; SPEER 1996; SAMSON 1997; HUCHZERMEIER 1998). Causes of foreign body ingestion are overgrazing of the pasture, insufficient grazing, insufficient energy intake, lack of fiber in the diet, desertion and disorientation stress (HUCHZERMEIER 1998). Gastric endoparasitosis was observed in a juvenile ostrich (case No. 68) associated with severe purulent inflammation of the proventriculus and cachexia. The localization of the parasites is typical for the hematophagous trichostrongylid nematode Libyostrongylus sp. that already has been described in ostriches in Germany (SCHULZE et al. 2006). This wireworm is considered as a primary pathogen in ostriches. The parasite lives in deep mucosal glands of the glandular stomach and under the koilin layer of the muscular stomach. Disruption of the gastric barrier results in secondary bacterial infections and suppurative inflammation (HUCHZERMEYER 1998, 2002). Intestinal intussusception was observed in one poorly nourished juvenile ostrich (case No. 64). Invagination of the intestine occurs occasionally in ostrich chicks (KEFFEN 1984; MUSHI et al. 1998) and has also been described in rheas (FRASCA and KHAN, 1997).

Chapter 5: Discussion

Predisposing causes include enteritis that may be frequently associated with other intestinal disorders such as gastro-intestinal displacement, torsion or volvulus, as well as tumors, parasites and abrupt diet change with higher fiber content (STEWART, 1994). However, in some cases, as in ostrich No. 64, the cause of abnormal peristaltic contractions remains undetermined (KEFFEN 1984; SHANE and TULLY 1996). Bacterial pathogens associated with enteritis in the current study include Lawsonia intracellularis, C. perfringens, C. sordellii, Bacillus lentus and Enterococcus durans (case No. 41), and Brachyspira sp. (case No. 31). Lawsonia intracellularis has been described as cause of proliferative enteritis among many species including ostriches (COOPER et al., 1997) and emus (LEMARCHAND et al. 1997). In this study, a rhea, case No. 41, showed morphological findings similar to those described in the literature for other ratite species. The demonstration of genome fragments of Lawsonia intracellularis in the intestinal content suggests a causal association with the morphological changes which represents the first documented case in this avian species. Infections of various ratite species with this not-host-specific pathogen may have epidemiological implications, particularly in zoological collections with multi-species exhibitions in the same enclosure. Brachyspira sp. is considered as the causative pathogen of a severe fibrino-necrotizing typhlocolitis with intralesional spirochetes in a juvenile rhea (case No. 31). The manifestation in juvenile individuals as well as the morphological findings are similar to those already described in rheas (SAGARTZ et al. 1992; BUCKLES et al. 1997; KUTZER et al. 2007), that generally are termed avian intestinal spirochetosis (MAPPLEY et al. 2014). Spirochete- associated enteritis causes high mortality rates up to 80% in rhea flocks (SAGARTZ et al. 1992). B. hyodysenteriae and B. pilosicoli have been identified in affected rheas (KUTZER et al. 2007; MAPPLEY et al. 2014). However, in the case of this study detailed molecular analysis of the causative agent was not performed. Transmission may occur by contact between pigs and ratites. However, in cases without evidence of contact to pigs or pig manure (KUTZER et al. 2007), feral birds, rodents and domestic animals that may harbour Brachyspira sp. or contaminated water have to be considered as possible source of infection (MAPPLEY et al. 2014).

Chapter 5: Discussion

The aetiology of the lympho-histiocytic and lympho-plasmacytic hepatitis observed in three animals (case Nos. 21, 51, 53) remains undetermined. Infectious and non-infectious causes have to be considered including intoxications, viral or bacterial infections.

5.4 Cardiovascular system A degenerative cardiomyopathy with extensive hyaline degeneration of cardiomyocytes and histiocytic inflammation was found in two ostriches and one rhea (case Nos. 2, 47, 60). Vitamin E and/or selenium deficiency has to be considered as primary cause in cases with skeletal muscle involvement (case No. 60), because ionophore intoxication and capture myopathy in ratites typically lack cardiac degeneration (BAIRD et al. 1997; HUCHZERMEIER 1998). The cause of the degenerative cardiomyopathy in the other two cases remains undetermined. Possible aetiologies include local perfusion disturbances, chronic myocarditis or intoxications, e. g. avocado (Persea americana var. guatemalensis) poisoning (BURGER et al. 1994). Valvular endocarditis with intralesional Gram-positive bacteria was detected in an adult ostrich (case No. 51) indicative for a systemic bacterial infection. However, involvement of other organs and tissues was not recorded and a portal of entry was not evident in this case. An adult ostrich in this study (case No. 56) died due to rupture of a dissecting aortic aneurysm. As predisposing factor degenerative changes of the aortic wall were found in this case. Aortic rupture occurs in growing and adult ostriches and may be associated with overfeeding and lack of exercise (HUCHZERMEIER 1998). Furthermore, copper deficiency has been incriminated to play a role in the pathogenesis (VANHOOSER et al. 1994; BAPTISTE et al. 1997; FERRERAS et al. 2001). Hormonal influences, diet, lathyrism, zinc deficiency, pharmaceuticals, and parasites have to be considered as additional factors (Van VEEN 1999). One ostrich (case No. 57; 80 kg body weight) suffered from a left ventricular hypertrophic cardiomyopathy with an absolute heart weight of 1.3 kg corresponding to a relative heart weight of 1.63%. The reference range for the absolute heart weight in adult ostriches (122.1 ± 3.9 kg body weight) is reported to be 1.054 kg (± 172.34 g) with a relative heart weight of 0.86% (+/- 0.14%) (TADJALLI et al. 2009). Hypertrophic cardiomyopathy

Chapter 5: Discussion

has been associated with degenerative valvular changes described as endocardiosis in adult ostriches (KUBBA and AL-AZAREG 2013). However, semilunar and atrio-ventricular valves in the case of this study displayed no morphological alterations and the cause of the hypertrophic cardiomyopathy remained undetermined. Furthermore, morphological findings indicating decompensation such as atrial hypertrophy and dilatation, chronic pulmonary and/or hepatic congestion or effusions were not recorded. In addition, linear intimal elevations in the aorta consisting of plaque-like fibrous tissue were observed in ostrich no. 57 and are interpreted as plaque-like intima sclerosis or arteriosclerotic plaques (ROBINSON and ROBINSON 2016). Similarly, the pathogenesis of this vascular change remained undetermined. In several animals of this study hypertrophy and hyperplasia of vascular smooth muscle cells with variable extent were found in different organs. Increased blood pressure and platelet- or monocyte/macrophage-derived growth factors are important triggering factors for muscular hypertrophy and proliferation (ROBINSON and ROBINSON 2016). However, due to the lack of other cardiovascular-related lesions these changes were regarded most likely as incidental findings. Disseminated intravascular coagulopathy (DIC) represents a pathological activation of the coagulation system resulting in systemic consumption of clotting factors. Numerous bacteria, helminths, protozoa, viruses, toxins, as well as particular conditions like neoplasms, neoplasia, gastric dilation and volvulus, are known to induce DIC. Morphological findings in animals that die of DIC include hyaline thrombi, granular thrombi consisting of platelets and hyaline globules/”shock bodies” derived from fibrin degradation products (ROBINSON and ROBINSON 2016). In the presented case (case No. 9), E. coli and Salmonella typhimurium were cultured from the liver and lung suggesting septicaemia with concurrent DIC as the possible cause of hypoxia and encephalomalacia observed in this juvenile ostrich. Gram- negative bacterial endotoxins can cause endothelial damage, which exposes subendothelial collagen and leads to activation of platelets and Hageman factor (ROBINSON and ROBINSON 2016).

Chapter 5: Discussion

5.5 Nervous system The principal change encountered in the central nervous system (CNS) was a spongy degeneration in six adult ostriches (case Nos. 7, 8, 17, 19, 20, 34) two adult emus (case Nos. 18, 55) and three juvenile ostriches (case nos. 12, 29, 30). Clinical signs together with the morphological findings in case Nos. 7, 8, 12, 17 to 20 during the years 1986 to 1994 and the simultaneous emergence of bovine spongiform encephalopathy in the United Kingdom with subsequent spread in Europe raised the possibility of a causal link between both diseases. However, so far no causal relationship between bovine transmissible spongiform encephalopathy and the central nervous system lesions in ratites was detected (WELLS et al. 2006). In addition, in this study different PrP-specific antibodies that are routinely used for the diagnostics of ruminant transmissible spongiform encephalopathy did not show an affinity to ratite PrP. Therefore, the aetiology of the spongy degeneration in ratites remains unresolved. As alternate causes for spongy CNS changes toxins, gene defects and infections as well as metabolic processes have to be considered. In two cachectic juvenile ostriches (case Nos. 29, 30) apathy, somnolence and opisthotonus occurred prior to death. Histologically, a spongy degeneration of the brain was observed in the cerebral cortex, brain stem and cerebellum. Whether the clinical signs are related to the CNS changes remains undetermined. In addition, the aetiopathogenesis of the CNS lesions is unclear. A causal relationship to the poor nutritional status cannot be excluded completely. In case Nos. 34 and 55, no nervous signs were reported and the spongy changes were interpreted as possible age-related findings similar to neuronal lipofuscin accumulations. In few other cases (case Nos. 49, 52 and 59) lipofuscin depositions were present without spongy lesions in the CNS. In one adult emu (case Nos. 53) and one adult ostrich (case No. 54), inflammatory lesions of the brain were found associated with multiple haemorrhages on the head and neck. Presumably, acute neurologic disturbance may have led to polytrauma in case No. 54. The cause of the encephalitis remained undetermined in both cases, but an infectious agent, most likely a virus, has to be considered.

Chapter 5: Discussion

5.6 Respiratory system Respiratory diseases were recorded in ostriches and rheas including four cases of rhinitis (case Nos. 30, 57, 61, 68), one case of tracheitis, laryngitis and parabronchitis (case No. 62) due to influenza A virus infection, and three cases of granulomatous pneumonia (case Nos. 1, 34, 59). In two cases (case Nos. 30, 57) mycotic rhinitis characterized by ulcerative, granulomatous and necrotizing inflammations was found. As causative pathogen most likely Aspergillus sp. has to be considered. Infection is usually contracted by inhalation of spores from moulds growing in the environment, especially on rotten feed (HUCHZERMEIER 1998, 1999). In ostrich No. 57, the infection was restricted to the nasal cavity, which is unusual and has been described in ratites only rarely (FITZGERALD and MOISAN 1995). In three adult ostriches (case Nos. 1, 34, 59) and one adult emu (case No. 63) granulomatous pneumonia with intralesional septated fungal hyphae indicative of Aspergillus sp. was recorded. In case Nos. 34 and 63 mycotic pneumonia represents a concurrent infection to a mycobacterial infection. Respiratory mycosis occurs preferentially in young ostriches (COOPER 2005) and other ratites, but can also affect older birds under immunosuppression or suboptimal management conditions (HUCHZERMEYER 1998; KUMMROW 2015). In ostrich No. 68 an ulcerative and purulent rhinitis was associated with intralesional nematodes (not further specified). A nematode that specifically inhabits the nasal cavity of ratites is not known. Therefore, an aberrant localization of a ratite parasite or an infection with a facultative parasite similar to Halicephalobus deletrix in horses and humans (ONDREJKA et al. 2010) has to be considered. In rhea No. 62 an infection with low pathogenic influenza A virus H7N7 had caused a severe laryngo-tracheitis and parabronchitis. Ratites are highly susceptible to avian influenza, and various subtypes have already been isolated including H1N2, H3N2, H4N2, H4N6, H5N2, H5N9, H6N2, H7N1, H7N3, H9N2, H10N4 and H10N7 (ALEXANDER 2000; ABOLNIK et al. 2012), however, the low pathogenic subtype H7N7 has not been reported in ratites so far. Morphological lesions and distribution of viral antigen share similarities with findings reported in ostriches infected with other low pathogenic subtypes like H10N7 (WOOLCOCK et al. 2000). Wild and migratory birds may play an important role in

Chapter 5: Discussion

transmitting the virus by co-mingling with ratite flocks (SHANE and TULLY 1996). In addition, the direct or indirect contact to domestic poultry and/or products may be implicated in the spread of the disease (VERWOERD 2000).

5.7 Urogenital system In the urogenital system, degenerative and inflammatory changes of mainly mild extent were found and interpreted as secondary or incidental findings without great clinical importance.

5.8 Eyes and ocular adnexae Granulomatous conjunctivitis caused by Mycobacterium sp. infection in ostrich No. 34 was part of a disseminated mycobacteriosis. Conjunctival manifestation of systemic avian tuberculosis in ratites is not unusual (POCKNELL et al. 1996; GARCÍA et al. 2001). However, as differential aetiologies of conjunctival granulomas foreign bodies (ŠEVČİKOVÁ et al. 1999), infections with fungi (HUCHZERMEIER 1998), or Morexella phenylpyruvica (GÜREL et al. 2004) and granulomatous dacryoadenitis (SAROGLU et al. 2003) have to be considered. Bilateral retinal detachment was recorded in an adult emu (case No. 55). The cause of this disorder remained undetermined. Retinal detachment may be trauma-associated (WILLIAMS 1994), but it has also been described as idiopathic bilateral ocular disorder in pheasants (RANDALL et al. 1986).

5.9 Systemic diseases Three adult ratites animals (case Nos. 34, 49, 63) showed granulomatous inflammation of multiple organs with intralesional acid-fast positive bacteria indicative for mycobacteriosis, presumably an infection with Mycobacterium avium subsp. avium. However, bacteriological investigation failed to isolate the pathogen. In the emu (case No. 63) a spill-over from or to wallabies housed together with the ratites is likely, because one wallaby was also infected with the same bacterium. Typically, ratites are infected by repeated ingestion of the pathogen, which is present in soil, water and feed. Avian mycobacteriosis may result in an atypical form with diffuse enlargement of affected organs, a lepromatous form with circumscribed

Chapter 5: Discussion

subcutaneous lesions, and a tubercular form characterized by discrete granulomas (DONELEY et al. 1999). The latter type was present in the cases of this study. Fading ostrich syndrome (FOS), also termed “chick fading syndrome”, represented the most common cause of disease and death observed in this study among eggs and neonates. This syndrome occurs exclusively in animals held in captivity and is characterized by generalized oedema (anasarca), depression, weakness, anorexia, reluctance or unability to exercise, and high mortality rates 3 to 5 days after onset of clinical signs in ostriches of less than 3 weeks (PHILBEY et al. 1991; KUMMROW 2015). There are several most likely concurrent factors causing FOS including inadequate management practices and husbandry like improper incubation and rearing conditions, and possibly viral infections such as circovirus infection or immunosuppressive mycotoxins (SPEER 1996; EISENBERG et al. 2003; KUMMROW 2015). In addition, isolation of E. coli and/or Klebsiella pneumoniae from various organs including umbilicus, yolk sac, liver and air sacs, has been regarded as criterion for FOS (TERZICH and VANHOOSER 1993). Subsequently, chicks often develop enteritis, yolk sac retention and infection, gastric stasis and impaction, bacterial hepatitis and enteritis, fungal proventriculitis, pneumonia and septicemia (SPEER 1996; KUMMROW 2015).

5.10 Conclusions In summary, the diseases in captive ratites that originated from zoos and farms in northwestern Germany presented a broad spectrum of various disorders. The high frequencies of musculo-skeletal and alimentary alterations as well as numerous cases of FOS are most likely related to housing and management conditions. However, the retrospective casuistic work-up revealed also disorders that have been not or only rarely described in ratites including mycotic rhinitis, intraosseous fibrosarcoma, Lawsonia intracellularis infection in rheas, spongy CNS degeneration, or infection with subtype H7N7 of avian influenza A virus. Knowledge about ratites diseases is gaining particular importance in future because these flightless birds are expected to be used more and more in commercial farming in Germany.

Chapter 6: S u m m a r y

Casuistic evaluation of necropsied ratites (Struthioniformes) in northwestern Germany - a retrospective study - By: Aimara Bello

Chapter 6: Summary

The spectrum of spontaneous diseases was retrospectively analysed in 71 captive ratites including ostriches, emus, and rheas kept in zoos or farms in northwestern Germany. Reports and histological slides of the animals necropsied between 1968 and 2014 were reviewed, additional histo- and immunohistochemical stainings were performed and findings were interpreted with respect to virological, microbiological and parasitological results. In many animals more than one morphologic diagnosis attributable to different disease processes was found. In adult ratites, the most commonly altered organ systems were the musculoskeletal, the digestive, and the cardiovascular systems affected by traumatic lesions, inflammation and degenerative changes, respectively. The high frequency of traumatic lesions underlines the susceptibility of ratites to mechanical injuries. In one ostrich an intraosseus fibrosarcoma, that has not been described in ratites so far, represented a predisposing lesion for a pathological leg fracture. Lesions observed in the nervous system included spongy degeneration; however, immunohistochemistry and western blotting failed to detect pathologic prion protein. A similar disorder is not known in wild ratites, and hereditary or toxic causes have to be considered. Lawsonia intracellularis was recognized as cause of proliferative enteritis for the first time in a rhea. Severe necrotizing inflammation of the respiratory tract in a rhea was caused by the low pathogenic avian influenza virus A H7N7, a subtype that has not been reported in ratites so far. In juvenile animals, the digestive system and the musculoskeletal system were predominantly affected by inflammatory and traumatic lesions. Bacterial infections of the alimentary tract were an important cause of disease in the present study. Whereas alimentary disorders are very rare in wild ratites, they are often reported in captive birds, even in their natural habitat. In eggs and neonates the major cause of death was the fading ostrich syndrome that is a worldwide disease occurring exclusively in farm husbandry. Among

Chapter 6: S u m m a r y

various factors particularly inadequate management practices such as improper incubation conditions for eggs contribute to this disorder. The retrospective analysis of diseases in various ratite species revealed many disorders that were associated with housing or management conditions in farms and zoos. Some disorders were found that were never or rarely described in these flightless birds.

Chapter 7: Zusammenfassung

Retrospektive Untersuchung der Erkrankungs- und Todesursachen von Ratiten (Struthioniformes) in Nordwest-Deutschland

Von: Aimara Bello

Chapter 7: Zusammenfassung

Das Spektrum der spontanen Erkrankungen von 71 in menschlicher Obhut gehaltenen Laufvögeln (Strauße, Nandus, Emus) aus Zoos und Farmen in Nordwest-Deutschland wurde retrospektiv analysiert. Die Sektionsberichte und histologischen Präparate der zwischen 1968 und 2014 obduzierten Tiere wurden geprüft, zusätzliche histochemische und immunhistologische Färbungen durchgeführt. Alle Befunde wurden unter Berücksichtigung der virologischen, mikrobiologischen und parasitologischen Befunde interpretiert. Bei vielen Tieren waren mehrere, voneinander unabhängige, morphologische Diagnosen zu stellen. Die am häufigsten veränderten Organsysteme adulter Ratiten waren der Bewegungsapparat, der Verdauungstrakt und das Herz-Kreislauf-System, die traumatisch bedingte Verletzungen, Entzündungen und degenerative Veränderungen aufwiesen. Die große Zahl traumatischer Alterationen unterstreicht die Anfälligkeit von Laufvögeln für mechanische Schädigungen. Bei einem Strauß bestand ein intraossäres Fibrosarkom, das bislang bei Laufvögeln noch nicht beschrieben wurde. Zu den Veränderungen im zentralen Nervensystem zählte eine spongiöse Degeneration. Allerdings gelang weder durch Immunohistochemie noch durch Western-Blotting ein spezifischer Nachweis von pathologischem Prion-Protein. Eine vergleichbare Erkrankung ist bei wildlebenden Laufvögeln nicht bekannt. Eine erbliche oder toxische Ursache ist in Erwägung zu ziehen. Lawsonia intracellularis wurde erstmals bei einem Nandu als Ursache einer proliferativen Enteritis festgestellt. Eine ausgeprägte nekrotisierende Entzündung des Respirationstraktes wurde durch das niedrig pathogene aviäre Influenza A-Virus H7N7 verursacht, ein Subtyp, der bislang noch nicht bei Ratiten nachgewiesen wurde. Bei Jungtieren waren vor allem der Verdauungs- und Bewegungsapparat von entzündlichen und traumatischen Veränderungen betroffen. Bakterielle Infektionen des Verdauungstrakts stellten in der vorliegenden Studie eine wichtige Krankheitsursache dar.

Chapter 7: Zusammenfassung

Während diese Erkrankungen bei wild lebenden Laufvögeln sehr selten sind, werden sie bei in Gefangenschaft gehaltenen Laufvögeln häufig beobachtet, auch wenn sie in ihrem natürlichen Lebensraum leben. Bei Eiern und Neugeborenen stellte das „Fading Ostrich Syndrome“ die wesentliche Todesursache dar. Diese weltweit verbreitete Erkrankung tritt ausschließlich bei in menschlicher Obhut gehaltenen Laufvögeln auf. Unter anderem tragen insbesondere unzulängliche Management-Praktiken, beispielsweise ungeeignete Inkubationsbedingungen für die Eier, zur Pathogenese bei. Die retrospektive Analyse von Erkrankungen verschiedener Laufvogelarten ergab, dass zahlreiche Alterationen mit den Haltungsbedingungen in Farmen und Zoos assoziiert sind. Aber es wurden auch einige Erkrankungen festgestellt, die bislang noch nicht oder nur selten bei diesen flugunfähigen Vögeln beschrieben wurden.

Chapter 8: R e f e r e n c e s

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Chapter 9: Acknowledgements

I would like to express my sincere gratitude to my supervisor Prof. Dr. Wolfgang

Baumgärtner, for his constant support and constructive criticism.

I would also like to express my thanks to Dr. Peter Wohlsein, for his helpful discussions, great support and time.

It gives me immense pleasure to thank my Family and Mu, for understanding and supporting me in their own ways, and also to Jessica Freundt, for all the great and not so great moments we shared until now!!! You were always there for me and I think just saying thanks is not enough.

My sincere thanks also goes to Daniel Kupka, meine Liebe!! You changed my live!!

I would also like to thank Felipe and Alejandro, life was boring before you guys.

I also would like to express thanks to Vanessa and Annika, my dear colleagues!! For your friendship and supporting me all the time, to Annika and Dr. M. Peters for providing the great pictures used in this presentation, and to all my colleagues and personnel from Pathology, for your great support, understanding and the wonderful time I had in Hannover.

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