Diagnostic pathology in microbial diseases of sheep or goats

G Model VETMIC 7031 No. of Pages 12

Veterinary Microbiology xxx (2015) xxx–xxx

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Diagnostic pathology in microbial diseases of sheep or goats J. Benavidesa , L. Gonzálezb , M. Dagleishc, V. Péreza,* a Departamento de Sanidad Animal, Instituto de Ganadería de Montaña (CSIC-ULE), Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain b Animal Health and Veterinary Laboratories Agency (AHVLA), Pentlands Science Park, Bush Loan, Midlothian EH26 0PZ, Scotland, UK c Moredun Research Institute, Pentlands Science Park, Bush Loan, Midlothian EH26 0PZ, Scotland, UK

A R T I C L E I N F O

Keywords: Ancillary methods Diagnosis Organs Pathology Tissue

A B S T R A C T

Post-mortem examination is a key step in the diagnostic process of infectious diseases in sheep and goats. Diagnostic pathology deals with identification and study of lesions, at the same time providing also significant clues regarding pathogenesis of the diseases. This article reviews the salient pathological findings associated with the most significant infectious diseases of sheep and goats present in countries where small ruminants are a relevant agricultural industry. Lesions are reviewed according to the different organ systems where they occur. Emphasis has been given in the description of the salient lesional patterns than can be identified in each organ and which can be of help in the differential diagnosis of the lesions caused by bacteria, viruses, fungi or prions. Finally, a review of the usefulness of ancillary tests that may be used on various tissue samples for performing an aetiological diagnosis, is included; the application of various techniques, from immunohistochemistry to molecular biology-based tests, is described. ã 2015 Elsevier B.V. All rights reserved.

1. Introduction Diagnostic pathology deals with study and diagnosis of disease through examination of organs, tissues or cells from affected animals. Pathology is a key tool in the diagnosis, understanding and control of infectious diseases and essential when studying their pathogenesis (Caswell and Callanan, 2014). A thorough post-mortem examination is probably the most useful tool for achieving a correct pathological diagnosis and the only way to identify the majority of lesions (King et al., 1999). Tissue or other (e.g., gastrointestinal content, fluid) samples are collected for further analysis and to establish a diagnosis of the disease. In small ruminants, due to their size and economic value, post-mortem examination is an affordable and frequently used method of diagnosis of infectious diseases or other conditions affecting the animal. In order to avoid overlooking gross changes, it is recommended that the necropsy should be performed methodically always following the same routine (King et al., 1999). On the basis of the authors’ experience in the field of diagnostic pathology in small ruminants, this article reviews the relevant literature on pathological findings associated with the most relevant infectious diseases of sheep and goats. The review does

* Corresponding author. E-mail address: [email protected] (V. Pérez).

not contain an exhaustive list of lesions, but aims to provide some guidelines that permit the identification of pathological changes that appear in the different organ systems, paying special attention to the differential diagnosis. Finally, some information is provided regarding ancillary techniques, based on pathological methods, which can be used to arrive to a diagnosis from examining the affected tissues. 2. Alimentary system 2.1. Oral cavity Superficial stomatitis (i.e., with lesions limited to the mucosa) is mainly caused by viruses leading to the formation of vesicles, erosions-ulcers or papules. Foot-and-mouth disease is caused by a virus of the Picornaviridae family that results in a systemic disease primarily of cloven-hoofed animals. Small ruminants are, in general, less susceptible than pigs or cattle and lesions in the mouth appear initially as blanched areas that subsequently develop into fluid-filled vesicles, which rapidly progress to shallow erosions (Alexandersen et al., 2003). The dental pad and the tongue are the most common locations for lesions and erosions usually heal after a few days with little exudate or scarring. Pestedes-petits-ruminants virus of the Paramyxoviridae family causes a disease currently recognised in several parts of Africa and Asia. The virus has an affinity for the alimentary epithelium where it causes

http://dx.doi.org/10.1016/j.vetmic.2015.07.012 0378-1135/ ã 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Benavides, J., et al., Diagnostic pathology in microbial diseases of sheep or goats. Vet. Microbiol. (2015), http:// dx.doi.org/10.1016/j.vetmic.2015.07.012

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necrosis. Subsequent abrasions in the affected area are responsible for the sloughing of necrotic tissue resulting in erosions and ulcers, primarily in the caudal oral cavity (Truong et al., 2014). Goats and sheep are also susceptible to bluetongue, a viral disease caused by a virus of the Reoviridae family, which induces vascular damage leading to oedema, haemorrhage and thrombosis in several organs (MacLachlan et al., 2008), including the oral mucosa. A relevant detailed review of the lesions has been published recently (MacLachlan et al., 2009). During the acute phase, lesions are characterised by catarrhal stomatitis, punctuate haemorrhages in the lips and gums and oedema and congestion of the tongue resulting in cyanosis, giving the disease its name. Subsequently, small erosions and ulcers are present especially in the hard palate and dental pad. Contagious ecthyma, or for contagious pustular dermatitis, is probably the most common viral disease affecting the oral cavity in small ruminants. It is caused by a parapoxvirus that affects primarily young lambs and kids (Reid and Rodger, 2007). Outbreaks can also occur in adult sheep. Initially, lesions appear as raised reddened or greyish areas, surrounded by hyperaemia, that rapidly evolve into papules (Supplementary material 1), which become necrotic and slough, originating therefore erosions and ulcers (McElroy and Basset, 2007). These lesions are most frequent in the gums, associated with erupting teeth, as well as in the hard and soft palate and in the lateral and dorsal zones of the tongue (McElroy and Basset, 2007). In long-standing clinical cases, we have observed that lesions become chronic and progress to proliferative stomatitis, with pseudo-papillomatous growths in the affected areas (Supplementary material 2), comprised of epithelial hyperplasia and granuloma formation that can persist for several months (Reid and Rodger, 2007). This may result in deformation of the tongue. Microscopically, there is epithelial hyperplasia with ballooning degeneration and eosinophilic cytoplasmic inclusions (McKeever et al., 1988). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. A common consequence of mucosal lesions causing disruption of the oral epithelium, especially in orf-affected lambs, is secondary bacterial infection. Fusobacterium necrophorum, the aetiologic agent of oral necrobacillosis, is probably the most common cause of deep, often necrotising, stomatitis (i.e., lesions extend to deeper layers of the oral wall) in small ruminants (Nagaraja et al., 2005). The early lesions are characterised by welldemarcated white–grey–yellowish foci of necrosis surrounded by an hyperaemic ring and are commonly seen on the lateral or dorsal aspects of the tongue (Brown et al., 2007). Lesions can extend to form a locally extensive or even diffuse fibrino-necrotic stomatitis affecting the gums, palate, lingual aspects of the cheeks and the pharynx (Supplementary material 3). Microscopically, the necrotic tissue shows loss of histological architecture and peripheral vascular hyperaemia, leucocytes and, very chronic cases, granulation tissue (MacLeod and Watt, 1967). Bacteria within the lesions can be identified as Gram-negative filamentous rods around the periphery of the necrotic areas. Actinobacillosis,caused by Actinobacillus ligneriesii, can affect sheep causing chronic multifocal pyogranulomatous glossitis and stomatitis (Rycroft and Garside, 2000). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 2.2. Forestomachs and abomasum The only lesion caused by an infectious agent of some significance in the forestomachs is necrotising inflammation caused by F. necrophorum or fungi, usually zygomycetes (Chiyaya et al., 1988; Nagaraja et al., 2005). In both cases, acidosis is a significant predisposing factor. Visible lesions include necrotic foci

surrounded by a hyperaemic halos (Supplementary material 4) that frequently sloughs resulting in ulcers. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Haemorrhagic abomasitis is the most common lesion observed in the abomasum caused by infectious agents and are usually associated with clostridial infections. Exotoxins produced by Clostridium septicum infection are the cause of ‘bradshot’ or ‘braxy’, in sudden deaths in lambs in cold climates. At post-mortem examination (Ellis et al., 1983), besides other signs of toxaemia, the abomasum is severely and diffusely congested together with oedema of the folds, in which haemorrhages, ulcers or emphysema may be present. Infection by Cl. sordelli in lambs can also result in haemorrhagic abomasitis (Lewis, 2007). In our experience, multifocal punctuate mucosal haemorrhages together with abomasal milk overload can be seen in young sucking lambs dying from colibacillosis (Supplementary material 5). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 2.3. Intestine 2.3.1. Enteritis in young lambs or kids Enteritis in young animals is usually of multifactorial aetiology, with influences from management procedures, climatic factors, infectious agents, etc. Among these, Escherichia coli is an important cause of enteritis in lambs or kids during the first weeks of life (Muñoz et al., 1996). Newborn lambs are affected in their first hours of life by ‘watery mouth disease’, an endotoxic colibacillosis the predisposing factor of which is the inadequate intake of colostrum (Gilmour et al., 1985). At post-mortem examination, there is absence of colostrum in the abomasum, which is filled with the mucus seen typically in the foetuses, the presence of meconium in the large intestine and, occasionally, dilation of areas of the jejunum. During the first weeks of life, E. coli is also an important cause of diarrhoea that can also cause septicaemia. The most typical lesion is sero-mucous (catarrhal) enteritis, characterised by diffuse or segmental dilation of the jejunal loops and ileum due to abundant yellowish sero-mucous content (Supplementary material 6) (Tzipori et al., 1981). In our experience, the abomasum frequently contains coagulated milk with dark red spots due to small haemorrhages (Supplementary material 5). When septicaemia is present, many petechiae and larger haemorrhages can be observed on serosal surfaces. A sero-mucous enteritis is also a characteristic of cryptosporidiosis or rotavirus infections. The latter, although reported in lambs and kids, are not considered to be a significant cause of enteritis (Muñoz et al., 1996; Chatzopoulos et al., 2012). The main pathological distinction is made microscopically where, in contrast to colibacillosis, a marked villous atrophy is seen in the small intestine. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. The role of the several types of Cl. perfringens causing enteritis in small ruminants has been reviewed in detail by Uzal and Songer (2008). Cl. perfringens type B is the aetiologic agent of ‘lamb dysentery’, characterised by haemorrhagic and necrotising enteritis (Supplementary material 7), predominantly in the ileum, with ulcer formation and presence of blood and fibrin strands in the intestinal content, in the small intestines of lambs younger than 14 days. Cl. perfringens type C can also cause haemorrhagic enteritis in neonatal lambs. Occasionally, Salmonella species, mainly S. arizonae, or Yersinia enterocolitica have been incriminated in cases of diarrhoea in young lambs or goat kids and are characterised by a fibrinous enteritis (Richards et al., 1993). A condition termed ‘terminal ileitis ‘ or ‘regional ileitis’, for which the aetiology has not been established, has been described in

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lambs up to 6-month-old and is characterised by a proliferative enteritis in distal small intestine (Green et al., 1992). Microscopically, a marked hyperplasia of the mucosa with associated mononuclear inflammation is seen. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 2.3.2. Enteritis in adult animals Toxins produced by Cl. perfringens type D is the most common form of clostridial enterotoxaemia in sheep and goats (Uzal and Songer, 2008). It occurs in lambs, especially when they are introduced to feedlots, and in adult sheep. Typical lesions occur in several organs (kidney, pericardial cavity, lung, brain) including the intestine. Although not always present, a marked hyperaemia of the mucosa together with slight to abundant reddish contents is seen in the small intestine. In goats, a fibrino-haemorrhagic colitis and also ileitis is described. Infection by Cl. perfringens type C in adult animals result in ‘struck’, a disease that causes rapid death due to severe haemorrhagic and necrotising enteritis. Salmonella spp. or Y. enterocolitica can affect adult animals in a sporadic manner (Slee and Button, 1990; Richards et al., 1993). The most typical lesions in subacute to chronic forms is the presence of multifocally distributed abscesses or ‘buttons’ showing a necrotic center with fibrin deposition (Supplementary material 8), seen mainly in the caecum, colon and ileum (Slee and Button, 1990). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Probably the most important enteric infectious disease in adult ruminants is paratuberculosis or Johne's disease. Infection by Mycobacterium avium subsp. paratuberculosis causes a granulomatous enteritis affecting mainly the ileum, the jejunum and the upper large intestine (Pérez et al., 1996; Corpa et al., 2000). In the most advanced cases, the mesentery shows serous atrophy of the fat, the affected intestinal wall appears markedly enlarged and the lymphatic vessels of the serosa appear as whitish thickened cords. Upon opening, the intestinal mucosa is thickened and shows marked corrugated folds (Supplementary material 9). Although rarely, and only in sheep, a yellowish colour can be seen in the inflamed mucosa due to the existence of pigmented ovine strains of the organism (Supplementary material 9). In small ruminant paratuberculosis, serosal lymphatic dilation (Supplementary material 10) can be the earliest recognised gross change and the absence of notable gross thickening of the intestinal mucosa, in spite of severe wasting and muscular atrophy, is a common finding (Pérez et al., 1996). In goats, it has been reported that the jejunal mucosa is usually more thickened than the ileum (Corpa et al., 1996). Microscopically (Pérez et al., 1996; Corpa et al., 2000), there is a diffuse infiltrate of macrophages together with lymphocytes. The majority of the cases show the so-called lepromatous forms, with large numbers of macrophages containing high amounts of the organism. However, in some animals lymphocytes are the most predominant cell with groups of macrophages and Langhans giant cells scattered among them, with none or very few bacilli (paucibacillary forms). Microscopic examination of areas of intestine, where lymphoid tissue is still present in adult animals, particularly the ileocaecal junction, has great importance, since focal granulomas seen in the initial or latent forms of paratuberculosis can be detected in these areas in subclinical infected animals. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 2.4. Liver Specific hepatic lesions associated with infectious diseases are bacterial hepatitis. In hepatic necrobacillosis occurring in

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young animals, F. necrophorum reaches the liver following omphalophlebitis (Nagaraja et al., 2005; Orfanou et al., 2012). Multifocal white-yellow, slightly elevated, coagulative necrotic foci with peripheral hyperaemia are the most typical lesions (Supplementary material 11). They are composed of a core of coagulative necrosis surrounded by necrotic phagocytic cells (Scanlan and Edwards, 1990). A similar lesion can be seen in adult sheep due to toxins produced by Cl. novyi type B infection (‘blackdisease’) associated with migrating larvae of Fasciola hepatica, which carry the bacterial spores from the soil (Lewis, 2007). A similar portal of entry is employed by Cl. haemolyticum, the aetiologic agent of bacillary haemoglobinuria. The pathogen also causes necrosis in the liver, but in that infection the necrotic foci are usually singular and larger (Lewis, 2007). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Hepatic abscesses, caused mainly by Trueperella (formerly Arcanobacter) pyogenes or F. necrophorum, are the result of omphalitis or rumenitis secondary to acidosis. These bacteria, present in the ruminal contents, gain access to the liver via the portal vein, after invasion of the compromised ruminal wall (Scanlan and Edwards, 1996; Nagaraja et al., 2005). Initially, they cause necrotic foci similar to those already described for hepatic necrobacillosis that evolve to an abscess with soft, green, purulent material in the center and surrounded by a thick fibrous capsule. 3. Respiratory system 3.1. Upper respiratory tract Acute rhinitis, pharyngitis, laryngitis and tracheitis, pathologically characterised by hyperaemia/petechial haemorrhages and swelling/oedema of the respective mucosal linings, can be caused by a variety of agents. When of infectious aetiology, these are often part of generalised acute respiratory diseases (e.g., caused by Mannheimia haemolytica or respiratory viruses) or of systemic diseases (e.g., caused by Bibersteinia trehalosi or morbilliviruses). Amongst the conditions with clinical appearance of chronic rhinitis, enzootic nasal adenocarcinoma (enzootic nasal tumour) deserves special mention. This betaretrovirus-induced neoplasm of the nasal mucosa secretory cells affects both sheep and goats with a nearly world-wide distribution. The ovine and caprine infections are caused by two related but distinct viruses: ENTV-1 affecting sheep (Cousens et al., 1999) and ENTV-2 affecting goats (Ortín et al., 2003), but the ensuing pathology is the same in both species. The tumour grows in the ethmoid turbinate, either uni- or bilaterally and appears as soft, greyish, cauliflower-like masses covered in mucus that progressively involve the conchae, fill the nasal cavity and extend into the sinuses (Supplementary material 12) (De las Heras et al., 2003b). Microscopically, the tumour consists of proliferating secretory, non-ciliated cells adopting acinar, papillary, tubular or cystic arrangements. The low degree of anisokaryosis, low mitotic rate and apparent absence of metastases define the tumour as a ‘low grade adenocarcinoma’. Tumour cells express the envelope protein of the virus(es), which can be detected by immunohistochemistry (Walsh et al., 2013) and retrovirus-like particles can be observed in the tumours by electron microscopy (De las Heras et al., 2003b). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Other infections of the nasal cavity must be considered in the differential diagnosis of enzootic nasal adenocarcinoma. Chronic proliferative rhinitis caused by S. arizonae (Meehan et al., 1992) or S. enterica subsp. diarizonae (Lacasta et al., 2012) has been described in sheep and is characterised by bilateral nodular to polypoid proliferations affecting the ventral concha

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(Supplemantary material 13). Chronic rhinitis of fungal (Conidiobolus sp.) or fungus-like (Pythium sp.) aetiology has also been described (Ubiali et al., 2013). The former appears as white or yellow firm granulomas located in the ethmoid turbinate and nasopharynx, while rhinitis caused by Pythium sp. consists of brown or red friable necrotic masses located in the anterior part of the nasal cavity. Nasal abscesses caused by Staphylococcus spp. and caseous rhinitis by Arcanobacterium sp. have also been described (Ferrer et al., 2002). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 3.2. Lungs 3.2.1. Generalised lung involvement M. haemolytica infection of young lambs and kids often results in peracute septicaemia as do infections by other bacteria, such as B. trehalosi (Donachie, 2007). In those cases, generalised pulmonary hyperaemia/cyanosis, disseminated subpleural haemorrhages and oedema, with accumulation of foamy fluid in bronchi and trachea, may be the only changes evident. Maedi/visna of sheep and caprine arthritis/encephalitis are two lentiviral infections capable of causing multi-systemic disease. Ovine progressive pneumonia (Maedi) is a common manifestation of the sheep infection, while lung involvement in caprine arthritis/encephalitis is occurring less frequently than lesions in the joints or brain. The pulmonary pathology and other aspects of these infections have been extensively reviewed (Bulgin, 1990; Campbell and Robinson, 1998). Affected lungs fail to collapse when the thoracic cavity is opened and appear large, heavy and pale yellow to greyish, with occasional scattered foci of grey consolidation (Supplemantary material 14). On closer inspection, a fine grey mottling is seen under the pleura, which histologically corresponds to a lymphofollicular hyperplasia of the bronchialassociated lymphoid tissue. Other microscopic changes include infiltration of the alveolar septa by mononuclear cells, hyperplasia of smooth muscle and mild fibrosis. In areas of most severe interstitial pneumonia, which correspond to the scattered areas of consolidation, there is some hyperplasia of type II pneumocytes leading to pseudoepithelialisation. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 3.2.2. Focal and multifocal lung lesions Two pathological forms, classical and atypical, of ovine pulmonary adenocarcinoma (pulmonary adenomatosis) have been described. Both are associated with infection by a widely prevalent betaretrovirus, Jaagsiekte sheep retrovirus (García-Goti et al., 2000; De las Heras et al., 2003a; Griffiths et al., 2010). Although mainly a disease of sheep, ithas also been reported in goats (De las Heras et al., 2003a). In atypical pulmonary adenocarcinoma, single or multiple, white, hard, dry, non-coalescing nodules can be found anywhere in the lung and fluid is absent from the air passages (Supplemantary material 15). The histopathological appearance of the tumour nodules is similar to that of classical ovine pulmonary adenocarcinoma (see below). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Contagious caprine pleuropneumonia is a disease of goats and wild ungulates caused by Mycoplasma capricolum subsp. capripneumoniae (Caswell and Williams, 2007; Nicholas et al., 2012). Unilateral pleural and pulmonary involvement rather than bilateral is the norm and lesions can be either focal, multifocal or extensive (Wesonga et al., 2004; Nicholas and Churchward, 2012). Pleurisy (and pericarditis) can be fibrinous or fibrous and serous fluid is often present in the pleural cavity (Supplemantary

material 16). Lung lesions are those of granular consolidation and focal necrosis, sometimes evolving to sequestra, but no thickening of interlobular septa (as in the bovine homologue). Histologically, acute lesions consist of a fibrino-purulent to necrotic bronchopneumonia with vasculitis and thrombosis and peribronchiolar lymphoid cuffs; in advanced cases, pulmonary fibrosis and granulation tissue in alveoli are observed (Wesonga et al., 2004). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Mycobacterium bovis and M. caprae, both members of the M. tuberculosis complex, are capable of inducing lesions of tuberculosis in sheep and goats. Gross pulmonary lesions are characterised by the presence of granulomas of variable size, sometimes confluent, in various stages of development and, preferentially, located in the diaphragmatic lobes (Daniel et al., 2009; Pérez de val et al., 2013). Some nodules have a solid appearance, while others show the characteristic central caseous necrosis and encapsulation (Supplemantary material 17); at later stages, the necrotic areas may undergo mineralisation and fibrosis or liquefaction to cavity formation (open tuberculosis). Histologically, the caseous necrotic centre, with or without calcified areas, is surrounded by a halo of inflammatory cells (epithelioid macrophages, multinucleated giant cells and lymphocytes) and an outer layer of connective tissue (Buendía et al., 2013). Mycobacterium spp. microorganisms can be visualised inside multinucleated cells, in the necrotic areas and are often numerous at the luminal side of the cavities (Sánchez et al., 2011). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Multifocal, randomly distributed foci of coagulative or caseous necrosis, suppurative or necrotic pneumonia and pulmonary abscesses are often the result of thromboembolic dissemination of different bacteria, e.g., F. necrophorum, Corynebacterium pseudotuberculosis and Tr. pyogenes from the vascular system. Multifocal lung lesions can be induced also by air-borne microorganisms, e.g., A. fumigatus, which may cause a granulomatous pneumonia, or by saprophytic bacteria that are brought in or contaminate wounds and caused by aspirated material resulting in aspiration/necrotic pneumonia (Ferrer et al., 2002). 3.2.3. Diffuse cranioventral involvement of the lungs Experimental inoculation with Parainfluenza type 3 virus or one of the various ovine adenoviruses can result in appearance of patchy areas of reddish consolidation in apical and cardiac lobes, histologically characterised by hyperplasia of the bronchiolar epithelium, mononuclear interstitial inflammation and nonsuppurative bronchiolitis (Sharp and Nettleton, 2007). In natural disease, however, these viruses, as well as other ones, e.g., various ovine respiratory syncytial viruses (Bryson et al., 1988), various reoviruses and Bovine herpesvirus 4 (Sharp and Nettleton, 2007) are considered to play only a predisposing or exacerbating role for acute bacterial pneumonias. Peste-des-petits-ruminants, caused by a morbilllivirus, can cause outbreaks of pneumonia. Lesions are not confined to the lungs, but extend to the upper respiratory tract. In the lungs, multifocal to coalescing areas of consolidation involve the cranioventral parts of the lung, sometimes accompanied by fibrinous pleuritis. The affected areas show histological changes characteristic of bronchointerstitial pneumonia with necrosis of the epithelium of bronchi and bronchioles, alveolar type II proliferation and syncytial formation (Caswell and Williams, 2007). Intranuclear and intracytoplasmic eosinophilic inclusions are often present in all cell types, where virus-specific antigens can be detected (Brown et al., 1991). In classical pulmonary adenocarcinoma, multiple, whitish to greyish, raised nodules coalesce to occupy large portions of the

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apical and cardiac lung lobes (Supplementary material 18). The cut surface of such areas is moist, with a frothy fluid pouring from the airways; fibrous, adhesive pleurisy is commonly seen. As with atypical pulmonary adenocarcinoma, the histological appearance is that of a bronchioloalveolar carcinoma, in which neoplastic cells (type II pneumocytes and Clara cells) proliferate to form papillary and acinar structures. Characteristically, the alveoli adjacent to the neoplastic nodules are filled with enlarged, foamy macrophages. In addition, nodular, spiral-shaped proliferations of myxomatous tissue have been reported. The main differences between classical and atypical ovine pulmonary adenocarcinoma is that in the latter, para-adenomatous macrophages are much less abundant while infiltration of the tumour stroma by inflammatory cells and connective tissue is more prominent (García-Goti et al., 2000; De las Heras et al., 2003a). Jaagsiekte sheep retrovirus capsid and envelope proteins can be detected in the neoplastic cells by immunohistochemistry, not only in the affected lung (Griffiths et al., 2010) but also in intra- and extra-thoracic metastases (Minguijón et al., 2013). Jaagsiekte sheep retrovirus particles can be visualised in ovine pulmonary adenocarcinoma affected lungs (De las Heras et al., 2003a). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. A chronic, non-progressive pneumonia of lambs and goat kids, also known as atypical pneumonia, is the result of a combined infection by Mycoplasma ovipneumoniae and M. haemolytica (Gilmour et al., 1979). Gross lesions consist of more or less extensive, clearly demarcated, areas of red/brown/grey, firm consolidation affecting the cranioventral parts of the lungs (Supplementary material 19); on closer inspection, the affected areas show widespread grey rosettes, which correspond to peribronchiolar proliferation of lymphoid tissue. In addition to those lymphoid cuffs, histological changes include hyperplasia of the bronchiolar epithelium, exudative pneumonia made up of macrophages and neutrophils, pseudoepithelialisation and presence of hyaline scars next to the bronchiolar wall (Jones and Gilmour, 1991). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. In acute and subacute M. haemolytica infection, gross lesions consist of areas of dark red consolidation involving mostly the cranial and cardiac lobes, covered by fibrinous pleurisy (Supplementary material 20). Sometimes, beneath the pleura, but more often on cut surface, the parenchyma shows irregular, sharply demarcated areas of necrosis. Microscopic lesions are those of a fibrino-purulent bronchopneumonia, in which neutrophils and characteristic ‘oat cells’ fill the alveoli. The necrotic lesions affect all parenchymal structures and appear demarcated by macrophages and oat cells (Donachie, 2007). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 4. Central nervous system 4.1. Transplacental infection of the developing foetus Several viruses, collectively known as teratogenic viruses, when infecting pregnant ewes or does can reach the central nervous system (CNS) of the foetus and cause developmental defects. Such viruses belong to three main genera: Pestivirus (Border disease virus), Orbivirus (Bluetongue virus) or Orthobunyavirus (Akabane virus, Cache Valley virus, Schmallenberg virus and others). Viruses of a fourth genus, Flavivirus (Wesselsbron disease virus), although with primary tropism for liver, can also replicate in foetal brain with teratogenic results (Coetzer, 1980; cited by Maxie and Youssef, 2007).

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Gross lesions in aborted foetuses, stillbirths and neonates are not confined to the central nervous system, but are often evident, particularly in Orthobunyavirus infections, as skeletal defects such as brachygnathia, kyphosis, torticollis and arthrogryposis. These are most likely due to muscular atrophy and decreased foetal motility secondary to tissue damage. Gross lesions in the central nervous system are similar in all those infections and variations are more related to time of embryonic life at infection and intra-genera strain variation than to inter-genera differences. They include cerebellar dysgenesis/hypoplasia (Supplementary material 21), particularly prominent in Border disease and Schmallenberg disease, internal hydrocephalus and/or hydranencephaly, which are less frequent in Border disease, and leucomalacia/porencephaly Infrequent occurrence of micrencephaly and lissencephaly has been described in experimental Border disease. Microscopic changes include lymphohistiocytic or necrotising meningoencephalitis and/or myelitis, with perivascular cuffing and multifocal glial-histiocytic inflammation, neuroparenchymal necrosis with gitter cells and mineralisation and neuronal chromatolysis and necrosis (Sawyer, 1992; MacLachlan et al., 2009; Toplu et al., 2011; van der Sluijs et al., 2011; Herder et al., 2012). Particularly prominent in Border disease are lesions of hypomyelination, cerebellar cortex dysplasia and necrosis/loss of Purkinje cells (Løken and Bjerkås, 1991; Sawyer, 1992; Toplu et al., 2011). Viral proteins can be detected by immunohistochemistry in neurons of Schmallenberg virus infected foetuses (Herder et al., 2013) and in a variety of cell types in cases of Border disease (Thür et al., 1997). However, detection of orthobunyaviruses or orbiviruses by immunohistochemistry (or PCR) may be hampered by their quick clearance from the brain (Wouda et al., 2009). In these cases, detection of specific antibodies in foetal fluids, in paired serum samples from the dams or in pre-colostral serum samples from newborns may be more informative. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 4.2. Post-natal infections by neurotropic viruses Neurotropic viral infections of small ruminants include those by flaviviruses (louping ill), lyssaviruses (rabies), herpesviruses (Aujeszky’s disease or pseudorabies) and bornaviruses (Borna disease). These viral infections rarely result in detectable gross pathology and, therefore, a systematic approach to collecting samples from the central nervous system for histopathological examination is necessary (Supplementary material 22) (Vandevelde et al., 2012). In this way, the chance of missing histological changes is minimised and a presumptive diagnosis can be achieved based on lesion type(s) and distribution. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. In sheep and goats, cases of tick-borne encephalitis include primarily louping-ill, which, in endemic areas (British Isles and some mountain ranges of continental Europe), is mostly a disease of young animals. After replication in lymphoid tissues, Louping ill virus reaches the brain from the blood. Other viruses initially infect peripheral nerve endings, either after bite injuries (rabies) or after oro-nasal exposure to aerosols (Borna disease, Aujeszky’s disease), reaching the brain by retrograde axonal transport. Although infrequent, rabies and Aujeszky’s disease can also affect small ruminants, while Borna disease is limited in sheep in central Europe. Lesions in the central nervous systemare those of a non-suppurative polioencephalitis or poliomyelitis, in some cases accompanied of mononuclear meningitis (Borna disease and louping ill; Supplementary material 23) or ganglioneuritis (rabies and pseudorabies), although lesions can be very subtle

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or even absent in clinical cases of rabies and pseudorabies. Characteristic lesions in all four diseases include lymphoplasmacytic perivascular cuffs and foci of gliosis (Supplementary material 23); these may be inconspicuous in Borna disease. Phenomena of chromatolysis and neuron necrosis are observed in pseudorabies and louping ill, but only in the case of louping ill induce neuronophagia; apoptosis in rabies and T cell-mediated pathology in Borna disease have been described. Viral inclusion bodies are observed in the cytoplasm of neurons in cases of rabies (Negri bodies) and in neuronal nuclei in cases of Aujeszky’s disease (Cowdry type-A) and Borna disease (Joest-Degen bodies). In all four diseases, viral proteins can be detected by immunohistochemistry in the target areas, mostly in neurons. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. In louping ill, lesions are mainly found in the brainstem, cerebellum and spinal cord. In rabies and pseudorabies, the spinal cord and brainstem, and in rabies also the cerebellum, basal ganglia and hippocampus are most often targeted. In Borna disease, lesions mainly involve the frontal and olfactory cortex, basal ganglia and hippocampus. Descriptions of the histopathology and other aspects of these diseases can be found in Maxie and Youssef (2007) and in Vandevelde et al. (2012), as well as specifically for louping ill in (Doherty and Reid, 1971; González et al., 1987; Sheahan et al., 2002), for rabies in Perl and Good (1991) and Suja et al. (2011), for Aujeszky’s disease in Dow and McFerran (1964, 1966; Schmidt et al., 1992) and Henderson et al. (1995) and for Borna disease in Richt et al. (1997), Stitz et al. (1995) and Wahlenkamp et al. (2002). 4.3. Non-neurotropic lentiviruses Lentiviral infections of small ruminants, namely maedi/visna in sheep and caprine arthritis-encephalitis in goats can induce neurological disease. While visna is almost only seen in adult sheep, the encephalic form of caprine arthritis/encephalitis mostly affects kids; lesions in the central nervous system are, in general, similar in the two diseases. Lentiviruses arrive to the brain from blood or cerebrospinal fluid. On rare occasions, gross lesions can be seen as softening of the periventricular or funicular white matter (leucomalacia) or as a granular appearance of the choroids plexus (Benavides et al., 2009; Vandevelde et al., 2012). Histological changes include a lymphoproliferative choroiditis (Supplementary material 24) and a non-suppurative, granulomatous to necrotic leucoencephalomyelitis with secondary leucomalacia (Supplementary material 24) or primary demyelination; in some cases, mononuclear meningitis can be observed (Bulgin, 1990; Campbell and Robinson, 1998; Benavides et al., 2009). Lymphohistiocytic perivascular cuffs and adjacent, more or less extensive areas of macrophage infiltration of the neuroparenchyma leading to its destruction have a typical periventricular and periaqueductal distribution (funicular in the spinal cord), although they can progress to a more disseminated distribution. Viral antigens can be demonstrated by immunohistochemistry in macrophages and glial cells (Benavides et al., 2009). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 4.4. Bacterial meningoencephalitis Listerial encephalitis is the most prevalent and relevant bacterial meningoencephalitis in small ruminants. In addition to abortion, septicaemia and enteritis, infection by Listeria monocytogenes can result in encephalitis after entry of bacteria through

wounds in the oral/gingival mucosa and retrograde transport through branches of facial nerves. Lesions, almost always only microscopically evident, are mainly located in the brain stem: pons and rostral medulla initially, extending rostrally and caudally in severe or advanced cases (Oevermann et al., 2008, 2010). Lesions can be defined as non-suppurative meningoencephalitis (myelitis also in some cases) with microabscess formation (Supplementary material 25), since the meningeal infiltrates (with some exceptions) and the thick perivascular cuffs in the neuroparenchyma are comprised of lymphocytes and macrophages, while neutrophils are present in clusters in the adjacent parenchyma. These microabscesses constitute the response to bacterial colonisation and, often, result in non-selective destruction of neurons and neuropils; in subacute or long-standing cases, macrophages progressively replace neutrophils in those inflammatory foci. Immunohistochemistry shows good performance in confirming presence of bacterial antigens within microabscesses (Oevermann et al., 2010). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Suppurative meningitis, sometimes accompanied with choroiditis and purulent inflammation of the ependyma, is a frequent consequence of bacterial septicaemia in young small ruminants. Bacterial genera often involved include Escherichia, Salmonella, Pasteurella and Staphylococcus, with lesions visualised as clouding of the meninges or even purulent depositions (Supplementary material 26), which microscopically correspond to leptomeningeal infiltration by neutrophils and presence of bacteria. Epidural abscesses in the spinal cord may be observed after tail docking of newborns, in cases of bacterial invasion of the wound. Abscesses in the brain may be caused by bacterial contamination of skull injuries, but are more often the result of thromboembolic sepsis; in the first case, they are usually single, while in the second scenario they tend to be multiple and, regardless of their aetiology being bacterial (e.g., Histophilus spp.) or fungal (e.g., Aspergillus spp.), have the histological appearance of multifocal haemorrhagic, purulent or necrotic inflammation (Vandevelde et al., 2012). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 4.5. Transmissible spongiform encephalopathies (TSEs) Classical scrapie of sheep and goats is the archetypal transmissible spongiform encephalopathy or prion disease, which behaves as infectious and contagious under natural circumstances. The oral route is the most relevant in natural infections and two possible pathways, neural or haematogenous, have been proposed for the agent to reach the brain (Sisó et al., 2010). Lesions in the central nervous system are exclusively microscopic and consist of vacuolation of neuronal processes (neuropil) and cell bodies (Supplementary material 27). Vacuoles vary in size and can be single or multiple; they initially appear in specific nuclei of the medulla oblongata and progressively extend to other neuroanatomical areas and become more severe to give the characteristic appearance of symmetrical spongiform change. Other lesions include gliosis and, in some cases, neuronal loss and/or amyloid plaque formation (Jeffrey and González, 2004, 2007). Vacuolar changes are preceded by the accumulation of an abnormally misfolded, disease-associated form of the prion protein (PrPd) that can be visualised by immunohistochemistry (Supplementary material 27) (Jeffrey and González, 2004, 2007). Immunohistochemistry also indicates that the morphological and cell-associated types of PrPd in the brain (the ‘PrPd profile’) vary depending on the interaction between infecting strain and host genotype (González et al., 2012) and is a useful tool to

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discriminate between scrapie and experimental ovine and caprine infection with the bovine spongiform encephalopathy agent (Jeffrey and González, 2007). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. A newer form of transmissible spongiform encephalopathies in sheep and goats, Nor98 or ‘atypical scrapie’, has been observed worldwide as a result of intensive active surveillance for the disease. Unlike classical scrapie, the disease does not appear to be contagious, although sheep to sheep transmission has been experimentally achieved. Classical and atypical scrapie also differ in the morphological appearance of PrPd deposits and their distribution (Benestad et al., 2003), which in atypical scrapie are fine particulate (grey matter) or globular (white matter), always extracellular and mostly affecting the cerebral or cerebellar cortices (Supplementary material 27). 5. Genital system and mammary gland 5.1. Placenta A common lesion observed in ewes and does is intercotyledonary placentitis. This lesion is characterised by thickening and opacity of the intercotyledonary membranes, which acquire a leathery texture and show a yellow-brown exudate on the surface (Supplementary material 28). This type of lesion is usually indicative of a bacterial infection in the placenta (Moeller, 2012). Many organisms causing septicaemia, in pregnant animals may invade the placenta and result in sporadic abortion (Tr. pyogenes, Bacillus spp., E. coli or Staphylococcus spp.). However, there are some bacteria that specifically target the placenta, e.g., Chlamydophila abortus, Coxiella burnetti or Brucella spp., and can cause a high number of abortions in a flock in the same season. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Ovine enzootic abortion, caused by Ch. abortus, is a serious reproductive disorder of small ruminants, causing abortion at late pregnancy or delivery of stillborn or weak premature liveborn lambs. Sheep and goats of any age are susceptible to infection and would abort, especially when infection has not occurred in the flock before. This is also a clinical feature of other relevant bacterial reproductive diseases such as Q fever (C. burnetti infection) and brucellosis (Br. melitensis infection). Affected placentas in all these diseases show multifocal to coalescent thickened areas of intercotyledonary placentitis, with grey to yellow exudate and necrotic, reddish cotyledons (Schlafer, 2007). In brucellosis, aborted placentas also show necrotic cotyledons, covered by a red to brown/yellow exudate (Anderson et al., 1986). Histologically, lesions found in these three diseases are very similar: there is a marked inflammatory infiltrate, usually mixed to purulent, and necrosis of the chorioallantoic membranes are particularly severe at the hilial zone of the placentome (Foster, 2012). Vasculitis of the large and medium vessels, a prominent feature of Ch. abortusplacentitis (Sammin et al., 2009), although it can be also found, with less relevance, in the other diseases (Schlafer and Miller, 2007). The presence of intracellular aggregates of bacteria can be demonstrated, generally inside trophoblast cells. (Moeller, 2012). Protozoal infections also cause necrotic placentitis, but only limited in the cotyledons. Detailed descriptions of these conditions, which fall outside the scope of the current review, can be found elsewhere (Buxton, 1998).

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organs. The latter are seen as white/yellow miliary foci on the liver, which microscopically correspond to multifocal areas of necrosis. Although necrotic hepatitis could be caused by several infectious agents, it is especially common in bacterial infections with L. monocitogenes (Supplementary material 29), Y. pseudotuberculosis or Salmonella spp. In addition to the hepatic lesions, histological lesions associated with bacterial abortions include also suppurative or mixed inflammatory cell foetal pneumonia and mild perivascular inflammation in the epicardium (Moeller, 2012). Similar lesions are present in the liver and lung in foetuses aborted due to Caprine herpesvirus type 1infections in goats. Microscopically, these lesions are characterised by areas of necrosis with occasional neutrophilic infiltrates. Intranuclear eosinophilic inclusion bodies can be found in cells adjacent to the areas of necrosis (Chenier et al., 2004). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Infections by Campylobacter fetus subsp. fetus and C. jejuni,are a relevant disease in small ruminants. Aborted foetuses frequently show as more specific lesions large foci of necrosis in the liver, up to 2 to 4 cm of diametre, with a red depressed center (Supplementary material 30). When campylobacter infection causes septicaemia in the foetus, there can be fibrinous peritonitis and suppurative bronchopneumonia, with neutrophils in the airways and alveolar ducts. Lesions in the placenta, when present, are characterised by necrotic cotyledons, which appear enlarged and covered with a red/yellowish exudate. Colonies of Gram-negative bacteria are present inside the trophoblast cells but also, as emboli, inside the capillaries of the placenta (Hedstrom et al., 1987). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 5.3. Mammary gland 5.3.1. Acute clinical mastitis Bacteria are the primary cause of acute mastitis, mainly Staphylococus spp., but other pathogens (e.g., Streptococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, M. haemolytica) are also involved (Anderson et al., 2002). Besides, isolated and severe cases of acute mastitis have been rarely caused by other pathogens, e.g., A. fumigatus or Burkholdelia cepacia, have also been reported (Perez et al., 1998; Contreras et al., 2007). Mammary glands with bacterial mastitis are, usually, red, hot and swollen. Usually, only one gland is affected and its secretion becomes thickened, serous or sanguineous and contains flakes, purulent discharge or fibrin (Supplementary material 31). Microscopically, there is a massive infiltration of leucocytes, mainly neutrophils, into the alveoli and ducts. Necrosis and loss of the alveolar epithelium is frequently found in these lesions (Watkins and Jones, 2007). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 5.3.2. Severe acute clinical mastitis This type of mastitis is caused mainly by St. aureus infection. Affected glands are oedematous and enlarged, udder skin is red or purple (Supplementary material 32) and, after the initial phase, becomes cold to the touch. Histologically, there is extensive necrosis of the parenchyma, oedema and thrombosis. If the dam survives the acute episode, the affected gland may slough off (Winter, 2001). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012.

5.2. Foetus Lesions caused by infectious agents in the foetus are mainly malformations (Section 4) and multifocal necrotic foci in several

5.3.3. Subclinical mastitis This type of mastitis is not accompanied by clinical signs or macroscopic lesions. Its diagnosis is based on demonstration of

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increased numbers of inflammatory or desquamated cells in milk samples and isolation of the causative bacteria from these (Contreras et al., 1996; Fragkou et al., 2014). It is caused by a variety of bacteria, similar to those which cause acute mastitis, although the most frequent agents are coagulase-negative staphylococci. Nevertheless, histological changes do occur, the predominant ones being neutrophilic infiltration at the acute stage, followed by lysis of neutrophils, destruction of alveoli and, in late stages of the infection, infiltration of lymphocytes and fibrous tissue proliferation (Fthenakis and Jones, 1990). 5.3.4. Long-standing mastitis Bacterial infections can result in long-standing mastitis, which usually manifests as hardness of the affected mammary gland, with presence of abscesses or milk cysts (Watkins and Jones, 2007). In the para-Mediterranean countries, a common cause of long-standing mastitis is Mycoplasma spp. infection, especially by M. agalactiae (contagious agalactia). In the initial phase, the mammary form of the disease has those characteristics of acute mammary infection. As chronicity ensues, there is progressive atrophy and fibrosis of the gland (Supplementary material 33), histologically is characterised by a lymphoplasmocytic infiltrate, finally becoming organised in subepithelial lymphoid follicles (Castro-Alonso et al., 2010). Long-standing mastitis is the most frequent form of contagious agalactia, which also causes arthritis, keratoconjuntivitis, pneumonia and abortion. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Long-standing mastitis can be also caused by lentiviral infection. Affected mammary glands show bilateral increased firmness (induration) without no macroscopically evident changes in milk. Microscopically, there is interstitial diffuse infiltration of lymphocytes with formation of prominent lymphoid follicles around ducts, but also in relation to the acini (van der Molen and Houwers, 1987). 5.4. Testes and epididymides Infectious epididymitis and orchitis, mostly bacterial, are more common in sheep (ovine contagious epididymitis) than goats. A great variety of bacteria (e.g., Tr. pyogenes, Corynebacterium ovis, Histophilus spp., Salmonella enterica subsp. diarizoniae) (Gouletsou and Fthenakis, 2006; Ferreras et al., 2007) have been recovered from sporadic cases, where they caused acute, purulent orchitis/ epididymitis or pyogranulomatous inflammation (Gouletsou et al., 2004; Ferreras et al., 2007; Smith and Sherman, 2009b). However, the most relevant cause for infectious infertility in rams, especially mature animals, is Br. ovis infection. In that disease, there is an initial swelling of the epididymis with development of single or multiple abscesses. Histologically, there is, initially, diffuse infiltration of lymphocytes in the epididymis, which develops into a mixed or suppurative abundant infiltrate that, together with fibrosis and ductal hyperplasia, occlude the lumen of the epididymal ducts. This obstruction causes a buildup of spermatozoa, the development of sperm granulomas, and multifocal to coalescent foci of pressure necrosis in the testicles (Mobini et al., 2002). 6. Haemolymphatic system 6.1. Lymph nodes Most lesions observed in the lymph nodes are not specific for the cause. Acute or peracute bacterial infections (i.e., acute pneumonia, acute clostridial enteritis, acute mastitis etc) are usually accompanied by acute lymphadenitis of the regional lymph

nodes, which become enlarged, soft, markedly reddened, due to hyperaemia, and oedematous. In chronic infections, the lymph nodes related to the affected organ show lymphoid hyperplasia or chronic lymphadenitis, characterised by their marked enlargement and appearing firm and white-grey in color, usually with absence of circulatory changes. Caseous lymphadenitisis a disease caused by Corynebacterium pseudotuberculosis that has a primary location in the lymph nodes of sheep and goats. The organism gains access through wounds or abrasions of the skin and reaches the lymph nodes, where it causes a diffuse lymphadenitis with a center of soft, white to green caseous necrotic material surrounded by thin fibrous capsule (Baird and Fontaine, 2007) (Supplementary material 34). The consistency of the necrotic material varies from soft to dry and can be laminated showing concentric rings. The most affected lymph nodes are those located in the head and neck. Spread from peripheral lymph nodes can produce lesions in other locations (Laven et al., 1997), e.g., in the thoracic lymph nodes, the liver, the spleen, the kidneys, the mesenteric lymph nodes. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. In lambs and goat kids up to 5–6 months a condition termed ‘abscess disease’ (De la Fuente and Suárez, 1985), caused by S. aureus subsp. anerobius, is characterised by presence of purulent lymphadenitis in the superficial lymph nodes (Supplementary material 35). These become markedly enlarged and contain abundant, green, soft, dense, purulent content, which occasionally can fistulate. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Tuberculosis caused by Mycobacterium caprae or M. bovis is an important disease in goats and more rarely is seen in sheep. Besides lesions in the lungs, respiratory lymph nodes are always affected (Daniel, 2009; Buendía et al., 2013). Initially (primary complex) tracheal and mediastinal lymph nodes show small white-yellow, dry and usually calcified necrotic foci (Supplementary material 36) that can coalesce and cause an irregular increase of the node size. When pulmonary lesions are generalised, regional lymph nodes are markedly enlarged and show extensive whiteyellowish areas of caseous necrosis that microscopically usually shows abundant mycobacteria (Sánchez et al., 2001). When tuberculosis has spread to other organs (e.g., spleen, liver, intestine), regional lymph nodes can show similar lesions. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 6.2. Spleen Lymphoid cells present in the spleen are in contact with antigens or infectious agents circulating in the blood. As in the lymph nodes, non-specific lesions are commonly seen in the spleen in relation to infections. Hyper-immune states, which can be the consequence of chronic infections, cause lymphoid hyperplasiaS characterised by an increase in the number of cells seen in the white pulp of the spleen. In this lesion, the spleen is not significantly enlarged but when a section is made, multiple white nodules representing the follicular structure of the spleen are seen (Supplementary material 37). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Sheep affected by haemolytic diseases, such as Cl. perfringens type A infection, can show a spleen markedly enlarged that, when sectioned, shows a reddish, abundant pulpy parenchyma (Uzal and Sanger, 2008). Infections by Bacillus anthracis cause anthrax and, although animals are not usually necropsied to avoid spore contamination, a very dark, uniformely enlarged spleen containing unclotted blood is a characteristic hallmark (Okoh, 1981).

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7. Cardiovascular system Specific infectious diseases of the cardiovascular system of small ruminants are rare. The finding of pericardial inflammation, either fibrinous or fibrous, is frequently related to the extension of pleuropneumonic inflammations as an incidental finding. Pericardial effusion, or hydropericardium, can be characteristically found in Ehrlichia ruminantium infection (heartwater disease) (Prozesky, 1987), present in tropical and subtropical areas, and also in enterotoxaemia caused by Cl. perfringens type D, where it is frequent to find fibrin floccules floating within the exudate (Uzal and Songer, 2008) (Supplementary material 38). Foot and mouth virus can also cause an acute lympho-histiocytic myocarditis (Aleksandersen et al., 2003) in lambs or kids, which die suddenly showing, as gross lesions, multiple white spots or strips (tiger heart) in the myocardium. Another viral disease that can affect the heart is bluetongue, which can cause haemorrhages in the endocardium and at the base of the pulmonary artery (Worwa et al., 2010), although this last lesion could be seen in other septicaemic conditions, such as pasteurellosis (Lujan et al., 2005). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 8. Urinary system There are very few infectious diseases specifically targeting the urinary system of small ruminants, e.g., caprine herpesvirus vulvovaginitis and balanoposthitis, caused by Caprine herpesvirus 1, or enzootic balanoposthitis of rams, both disease causing epithelial erosions in the vulval, vaginal or penile mucosa (Belknam and Pugh, 2002). Most of the lesions in this system, especially in the kidneys, are a consequence of systemic diseases or thromboembolic dissemination of bacteria. Enteretoxaemia, caused by the e- toxinof Cl. perfringens type D, is also known as ‘pulpy kidney disease’, due to the frequent finding of softer than normal renal consistency in animals dying as a result of this disease (Supplementary material 39). It is thought that this reflects accelerated autolysis of the carcass (Uzal and Songer, 2008). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Abscesses in the kidney are caused by haematogenous spread of bacteria like Staphylococcus, Salmonella, Chlamydia or Streptococcus species Belknam and Pugh (2002), and can, in extreme cases of C. pseudotuberculosis infection, completely destroy the renal parenchyma (Supplementary material 40). Pyelonephritis, denoted by presence of a purulent exudate in the pelvis, is caused by ascending infection, usually of E. coli or Corynebacterium renale (Belknam and Pugh, 2002). Bilateral enlargement and pallor of the renal cortex, which histologically corresponds to diffuse nephrosis, can be found in lambs younger than one-month (Supplementary material 41). The cause of this condition is unknown (Angus et al., 1989), but, in our experience, it is commonly observed in lambs with gastrointestinal lesions consistent with colibacillosis. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 9. Musculoskeletal System 9.1. Joints Septic polyarthritis is caused by bacteria and mainly occurs in neonates, usually after an episode of omphalophlebitis and subsequent septicaemia, although older lambs, under six months, can be also affected. This syndrome may be consequence of the infection by a great variety of bacteria, usually E. coli, Tr. pyogenes, Streptococcus spp. or Staphylococcus spp. Commonly, several joints

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can be affected, the carpal and tarsal joints being the most frequent locations for lesions, although any joint could be involved (Angus, 1991). Post-mortem examination reveals abundant purulent or fibrinous exudate, synovitis and erosions in the cartilage (Supplementary material 42). As a consequence of the septicaemia, thromboembolic inflammation can be found in other organs, e.g., in lungs, meninges or liver. There is usually abscesses formation in the navel. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Mycoplasma spp. is also a cause of arthritis. In contagious agalactia, adult animals may show arthritis characterised by mononuclear infiltration of periarticular and synovial tissues. In the acute phase, the joints, mainly the carpal and tarsal joints, are swollen, hot and painful and contain fibrinopurulent exudate. In chronic lesions, the joint may develop ankylosis. In newborns, particularly kids, several species of mycoplasma can cause arthritis after the instauration of septicaemia. Affected joints show fibrinous exudate, the cartilage may be eroded and the periarticular tissue may be thickened. In addition to arthritis, various other lesions may be present elsewhere, e.g., interstitial pneumonia, fibrinous pericarditis or peritonitis (DaMassa et al., 1992). Maedi visna and caprine arthritis can show an arthritic form in adult animals, which is a chronic proliferative arthritis characterised by thickening of the articular capsule, with foci of mineralisation and cartilage erosion and histological evidence of lymphoproliferative arthritis with synovial hyperplasia, infiltration of lymphocytes, plasma cells and macrophages and formation of lymphoid follicles (Narayan and Cork, 1985). 9.2. Muscles Clostridial myonecrosis is due to Cl. chauvoei, the aetiological agent of ‘blackleg’, and Cl. septicum, Cl. sordelli and Cl. novyi, the causes of ‘malignant oedema’ (Lewis, 2007). These diseases appear when vegetative organisms, present in contaminated wounds (malignant oedema) or in the muscle after internal dissemination from the gastrointestinal tract (blackleg), germinate under anaerobic conditions and begin to proliferate and liberate necrotic exotoxins. Clinical signs of blackleg are often unnoticed, as it is rapidly fatal, while animals suffering from malignant oedema show hot, swollen and painful areas around the wound. Affected muscles are dark, swollen and may show gas bubbles (Supplementary material 43), which may be also found in other organs such as liver (Supplementary material 44) or kidney. When external wounds are involved, subcutaneous oedema is also present (Lewis, 2007). Microscopically, there is extensive necrosis of the muscle, gas bubbles, incipient infiltration of neutrophils and clostridial organisms. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. 10. Skin and appendages Most of the viral diseases affecting the oral cavity cause lesions in the skin. Animals suffering from foot-and-mouth disease can develop vesicles in the muzzle, teats, mammary gland, prepuce and most consistently around the mouth and on the feet. Lesions in the feet, that are not a constant finding in sheep, include vesicles in the coronary band and the shedding of claws (Alexandersen et al., 2003). Bluetongue (MacLachlan et al., 2009) initiates with hyperaemic areas in the muzzle and skin, including axillae and inguinal folds, which progress to crusts and excoriations in several areas. Congestion and haemorrhages in the hoof are also characteristic. Lambs infected with orf virus show initially papulo-erosive areas that become pustular and necrotic, principally in the muzzle, the

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lips, skin around the mouth and the nostrils (Reid and Rodger, 2007) (Supplementary material 45). In lactating females, the teats and skin of the mammary gland can be infected by the lambs at the time of suckling. Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.07.012. Pox (Bhanuprakash et al., 2006), caused by a pox virus, is an infectious disease characterised by presence of papules all over the body that progress to scabs and scars. Lesions can also occur in the mucous membranes of the nose or mouth, can be distinguished from those of orf (contagious ecthyma), where lesions in the skin are rarely occur. Staphylococcal dermatitis and infections by dermatophytes (ringworm) are more commonly seen in goats than sheep (Smith and Sherman, 2009a). Multiple small pustules that can coalesce and discharge purulent exudate are frequent on the teats and udder, perineum or the tail and are the hallmarks of staphylococcal dermatitis. Ringworm is characterised by alopecic areas, scaling and crusts most commonly seen on the face, the neck and the external ears. Foot rot is a contagious severe disease of sheep and, more rarely, goats. It is a multifactorial condition closely linked to predisposing factors such as wet and warm weather and pasture conditions favouring the maceration of the interdigital skin. Once devitalised, the skin can be colonised by F. necroforum and Dichelobacter nodosus, the aetiologic agents of the disease (Bennet et al., 2009). Lesions vary from mild dermatitis in the interdigital skin to severe, necrotic dermatitis and separation of the hoof (Whittington and Nicholls,1995). Dermatophilus congolensis infection causes a disease called ‘strawberry foot-rot’, characterised by dry crusts on the coronary band (Norris et al., 2008) that can also be seen in other skin areas.

compared to immunohistochemistry, due to the heating steps in the protocol of the former, whereas immunohistochemistry is limited by the availability and specificity of suitable antibodies. Another molecular method involves extraction of nucleic acids from thick wax embedded tissue sections (2  10 mm) and inclusion into DNA/RNA free containers for detection of pathogen specific nucleic acid sequences by the polymerase chain reaction (PCR) (McPherson and Møller, 2000). Despite this technique being exquisitely sensitive, the pathogen cannot be localised visually to the histological lesion and can only be used for making a definitive diagnosis in diseases caused by obligate pathogens with no possibility of carrier or latent states. A refinement of this technique to address this deficit is the use of laser-capture microdissection where specific cells can be removed from tissue section prior to being subjected to PCR analysis (Espina et al., 2006). 12. Concluding remarks The examination of altered tissues, grossly or histologically, can provide valuable and useful information for diagnosis of infectious diseases in sheep and goats. Although only in few occasions diagnostic pathology provides a definitive diagnosis of a disease, lesions can orientate or suggest a tentative diagnosis based on the lesions encountered and greatly narrow the necessity of further analyses to achieve an aetiological diagnosis. In this process, it is fundamental to know how the various pathological phenomena (e.g., inflammation, neoplasia) are manifested in the several organs or tissues of the animal body. Besides, an accurate aetiological diagnosis can be accomplished by using different ancillary techniques on the same tissue samples that perfectly complement the examination of the lesions. Conflict of interest statement

11. Ancillary diagnostic techniques in pathological examinations Several techniques are available for use on paraffin-wax embedded fixed tissues, which can be used to determine the definitive diagnosis or reduce the number of possible aetiologies in a differential diagnosis. A myriad of histochemical staining techniques are available (Bancroft and Gamble, 2007), including the Gram-stain which differentiates between Gram-positive and Gram-negative bacteria, the Ziehl-Nielsen stain which highlights Mycobacteria and Nocardia bacteria, the Warthin-Starry stain for spirochetes and the Grocott Gomori methenamine silver stain for fungal hyphae. Additionally, indirect associations can be made, e.g., Martius-Scarlet Blue to detect perivascular serum leakage in the brain associated with the presence of e-toxin from Cl. perfringens type D infections. Immunohistochemistry is the use of an antibody, which has been raised to recognise an antigen (protein or carbohydrate) specific to a pathogen, family of pathogens (e.g., pan-Pestivirus) or proteinaceous agent (e.g., prion). The antibody is applied to unstained tissue sections and various detection systems are used to localise where the antibody and antigen have bound (Dagleish et al., 2010). A similar method, in-situ hybridisation (Stoler, 1990), uses synthesised probes instead of antibodies to detects sequences of DNA or RNA, which code for proteins specific to chosen pathogens and specificity. Both methods allow localisation of the antigen and subjective quantitation within histological lesions, which is usually required for suggesting causality. This is essential in many diseases, especially those caused by facultative pathogens, as these may be part of the commensal microflora in healthy animals and only become pathogenic under certain circumstances, e.g., Pasteurella multocida. However, morphology of tissue sections is significantly compromised with in situ hybridisation (ISH)

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Please cite this article in press as: Benavides, J., et al., Diagnostic pathology in microbial diseases of sheep or goats. Vet. Microbiol. (2015), http:// dx.doi.org/10.1016/j.vetmic.2015.07.012