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Caseous Lymphadenitis Caused by Corynebacterium pseudotuberculosis in Alpine Chamois (Rupicapra r. rupicapra): a Review of 98 Cases
J. Comp. Path. 2018, Vol. 161, 11e19
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INFECTIOUS DISEASE
Caseous Lymphadenitis Caused by Corynebacterium pseudotuberculosis in Alpine Chamois (Rupicapra r. rupicapra): a Review of 98 Cases L. Domenis, R. Spedicato, E. Pepe, R. Orusa and S. Robetto Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta, Valle d’Aosta Department, National Reference Centre for Wildlife Diseases, Regione Amerique 7G, Quart, Italy
Summary Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA) in domestic and wild ruminants. Here we describe CLA in alpine chamois (Rupicapra r. rupicapra) based on a series of 98 cases of C. pseudotuberculosis infection confirmed by bacteriology and gene sequence analysis. The population included 53 males and 45 females distributed within three age groups: up to 18 months (n ¼ 14), 18 months to 4 years (n ¼ 11) and over 4 years (n ¼ 73). Four different gross lesion distribution patterns, observed individually or variably combined in the same animal, were defined: (1) cutaneous/external (i.e. subcutaneous lymph nodes with or without muscle involvement, n ¼ 34); (2) abdominal visceral (i.e. only abdominal organs involved: liver and/or spleen and/or kidney and/or lymph nodes, n ¼ 35); (3) thoracic visceral (i.e. only thoracic organs involved: lung and/or heart and/or lymph nodes, n ¼ 26); and (4) generalized visceral (i.e. abdominal and thoracic organs involved, n ¼ 26). In six particularly severe cases, mammary gland, testis, vertebral bone and the central nervous system were also affected. Macroscopically, most abscesses were characterized by fluid pus, confirmed by microscopy that showed the absence of distinct concentric layers and coagulative necrosis, which are typically seen in sheep and goats raised in areas where the infection is endemic. In three cases amyloid deposits were observed in the liver and kidney. The C. pseudotuberculosis strains isolated were highly homologous to the reference strain ATCC 19410, except for some variability in their ability to ferment maltose and mannitol. Based on the production of nitrate reductase, 95 strains were attributed to the ovis biovar (nitrate reduction negative) and three to the equi biovar (nitrate reduction positive). All strains were sensitive to antibiotics, except to ampicillin (62.3% resistant strains) and gentamicin (83.7% resistant strains). Using an indirect enzyme-linked immunosorbent assay designed for CLA in sheep and goats, seven (58.3%) of 12 serum samples tested positive for antibodies. Ó 2018 Elsevier Ltd. All rights reserved. Keywords: caseous lymphadenitis; chamois; Corynebacterium pseudotuberculosis; Rupicapra r. rupicapra
Introduction Caseous lymphadenitis (CLA), also termed pseudotuberculosis owing to its similarity with tuberculosis lesions, is an infectious disease affecting domestic and wild ruminants worldwide. Considered a minor Correspondence to: L. Domenis (e-mail: [email protected]). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2018.04.003
zoonosis in people exposed occupationally to infected animals (Romero-Perez et al., 2004; Join-Lambert et al., 2006; Heggelund et al., 2015), CLA causes considerable economic losses to the sheep meat industry due to condemnation and downgrading of carcasses, decreased meat yield and reduced wool production (Paton et al., 1988). The aetiological agent, isolated for the first time in 1888 by the French Ó 2018 Elsevier Ltd. All rights reserved.
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bacteriologist Nocard from a case of bovine lymphangitis and some 3 years later by the Bulgarian bacteriologist von Pre€ısz from a ewe (Pre€ısz and Guinard, 1891; Nocard, 1896), is Corynebacterium pseudotuberculosis, a gram-positive and catalase-positive coccobacillus of the order Actinomycetales, which also contains the genera Mycobacterium, Rhodococcus and Nocardia. Two subtypes are distinguished according to nitrate reductase production: equi biovar (isolated from cattle and horses; nitrate reduction positive) and ovis biovar (isolated from goats and sheep; nitrate reduction negative) (Biberstein et al., 1971; Songer et al., 1988). The main virulence factor of C. pseudotuberculosis is phospholipase D, an enzyme that catalyzes the breakdown of proteins and lipids in the cell membrane. In small domestic ruminants, it causes the formation of purulent encapsulated abscesses with a caseous, lamellated (‘onion ring’) appearance, which can develop in peripheral lymph nodes (cutaneous or external form) or internal organs and lymph nodes (visceral or internal form). In wildlife species (free-ranging or in captivity), CLA has been reported in alpine ibex (Capra ibex) (Silinski and Walzer, 2004), Spanish ibex (Capra pyrenaica) (Cadena-Colom et al., 2014), alpine chamois (Rupicapra r. rupicapra) (Bassano et al., 1993), fallow deer (Dama dama) (Perez et al., 1996), red deer (Cervus elaphus) (Matos et al., 2015), elk (Cervus canadensis nelsoni) (Jane Kelly et al., 2012), white-tailed deer (Odocoilecus virginianus) (Stauber et al., 1973), pronghorn antelope (Antilocapra americana) (Clark et al., 1972), huemul (Hippocamelus bisulcus) (Morales et al., 2017), white-tailed gnu (Connochaetes gnou) (M€ uller et al., 2011), Arabian oryx (Oryx leucorix) (Tarello and Theneyan, 2008), cheetah (Acinonyx jubatus) (Boomker and Henton, 1980) and the aardvark (Orycteropus afer) (Roth and Vickers, 1966). Sporadic case reports of CLA in chamois are found in the literature (Bassano et al., 1993). Herein we describe CLA in alpine chamois based on a series of 98 confirmed cases of C. pseudotuberculosis infection, with a focus on gross and microscopical lesions, phenotypic and biochemical features, and antibiotic sensitivity of the strains isolated, as well as the serological response to C. pseudotuberculosis in some of the infected animals.
Materials and Methods Sampling
We reviewed 98 cases of CLA in alpine chamois, collected over the past 16 years according to the availability of anamnestic data (i.e. sex, age and origin), description of gross lesions (i.e. type and distribution)
and bacteriological test results (i.e. strain isolation and characterization). The animals came mainly from the Aosta Valley, a mountainous region of northwestern Italy, and to a lesser extent from bordering Piedmont (altitude 1,000e2,500 m). The provenance of the chamois included: (1) hunted with carcass fit for consumption (n ¼ 45); (2) hunted, but with carcass condemnation due to widespread lesions and/or poor condition (n ¼ 32); or (3) found dead or culled for health reasons (n ¼ 21), generally due to starvation (often associated with hyporeactivity). Before necropsy examination, biometric data were recorded for age estimated from horn growth (range <1 year to 16 years) (Lovari, 1985). Three age groups were defined according to male reproductive physiology: group I, aged <18 months (age at which the male reaches maturity); group II, aged between 18 months and 4 years (age range when the sexually mature male generally does not reproduce due to competitiveness inside the herd); and group III, aged over 4 years (age when the male can mate and breed). Necropsy Examination
The suspected CLA lesions (typically the presence of yellowegreen pus in lymph nodes and/or organs) were recorded according to the following groups: (1) cutaneous/external form (i.e. subcutaneous lymph nodes with or without muscle involvement); (2) abdominal visceral form (i.e. only abdominal organs involved: liver and/or spleen and/or kidney and/or lymph nodes); (3) thoracic visceral form (i.e. only thoracic organs involved: lung and/or heart and/or lymph nodes); and (4) generalized visceral form (i.e. abdominal and thoracic organs involved). More severe cases with bone and/or cerebral involvement or suppurative lesions in mammary gland and testis were also recorded. Bacteriology and Antimicrobial Susceptibility
The gross lesions of all animals were subjected to bacteriological examination for confirmation of macroscopical suspicion of CLA. Bacterial culture was performed using blood agar and MacConkey agar, with aerobic incubation at 37 C for up to 72 h. Typical colonies were confirmed initially by Gram staining and the catalase test, then subsequently identified with the API CoryneÒ system (Biomerieux, La Balme les Grottes, France). Once identified, the C. pseudotuberculosis strains were subjected to a KirbyeBauer antibiogram based on measurement of the zone of inhibition of bacterial growth around the antimicrobial strips at known concentrations (according to CLSI guideline M45, 3rd
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Caseous Lymphadenitis in Alpine Chamois
Edit., 2016). The following antimicrobials were tested: amoxicillin, amoxicillineclavulanic acid, ampicillin, cephalothin, cefoperazone, cloxacillin, enrofloxacin, erythromycin, neomycin, penicillin, streptomycin, sulfamethoxazoleetrimethoprim, tetracycline, thiamphenicol, bacitracin, oxytetracycline, gentamicin and lincomycin.
a good quality serum could be obtained, which is not always feasible in hunted wild animals or those found dead) were subjected to indirect enzyme-linked immunosorbent assay (ELISA; ID Screen CLAÒ Indirect, IDvet, Montpellier, France), originally designed for antibody detection in ovine and caprine serum and plasma. All animals, except one, were over 4 years old.
Gene Sequence Analysis
In 19 cases, when biochemical identification of C. pseudotuberculosis was doubtful, the strains were further typed by 16S rRNA gene sequence analysis. Genetic identification of the organism was carried out by sequencing the 16S rRNA gene with a Micro-Seq 500 system (ABI MicroSEQ 500Ò 16S rDNA Bacterial Identification kit, Applied Biosystems, Foster City, California, USA). Bacterial DNA was extracted from isolated colonies and a fragment of approximately 500 base pairs (bp) of the 16S rRNA gene was amplified using the specific primers provided with the kit. The polymerase chain reaction (PCR) fragment was sequenced with an automatic sequencer (ABI3130, Applied Biosystems) and the obtained sequence compared with those of the Microseq 500 system data bank and the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA) Blast data bank. Histopathology
In some animals (n ¼ 34), where the condition of tissue was excellent, routine histological examination was performed by fixation in 10% neutral buffered formalin, routine processing and embedding in paraffin wax. Sections (3e4 mm) were stained with haematoxylin and eosin (HE) to study the microscopical features of abscesses or for differential diagnosis with other diseases that cause similar lesions (e.g. tuberculosis due to Mycobacterium spp., pyogenic bacterial abscesses caused by Staphylococcus aureus or Trueperella pyogenes). In other cases (n ¼ 3), to confirm the presence and type of amyloid, Congo red staining was performed using a commercial kit (Congo Red StainÒ Kit, Highman, Molekula S.r.L., Rimini, Italy) according to the Highman protocol (Congo red solution for 5 min, differentiation in 0.2% potassium hydroxide solution for 3e10 sec, staining with Mayer’s haemalum solution for 5 min), with and without pretreatment of slices using a solution of potassium permanganate and sulphuric acid, followed by observation under polarized light. Serology
To evaluate antibody response to C. pseudotuberculosis infection, 12 of the 98 chamois (i.e. those from which
Results The distribution of gross lesions (i.e. typical abscesses) is reported in Table 1 and portrayed in Fig. 1. The table presents the different forms (i.e. cutaneous/ external, abdominal visceral, thoracic visceral and generalized visceral) that appeared alone or overlapping in the same animal in relation to the three age classes and sex. The external form (i.e. lymph nodes involvement with or without abscesses in the muscle) was observed in 34 animals. The thoracic visceral form was observed more often than the abdominal visceral form (35 versus 26 animals), the latter having the same occurrence as the generalized visceral forms (26 animals) involving the abdominal and thoracic organs. Among the abdominal organs, abscesses were found mostly in the liver (Fig. 1F), often with adhesion to the diaphragm, and seldom in the spleen (Fig. 1E) or kidney. Among the thoracic organs, the lung (Fig. 1A) and mediastinal lymph nodes (Fig. 1B) were often affected with large, cavernous collections of pus; the heart (Fig. 1C) was involved rarely, with generally small, myocardial pyogranulomas. In a few cases, CLA lesions were observed in the mammary glands, testis (Fig. 1G), bone of the thoracic and/or lumbar vertebrae (Fig. 1D) and the brain (Fig. 1H). Encapsulated abscesses ranged in dimension from miliary up to 15e20 cm in diameter. In general, the pus in all organs was yellowegreen in colour (Figs. 1AeH), with creamy to caseous consistency and signs of liquefaction, the latter especially evident in suppurative osteomyelitis (Fig. 1D) and brain abscesses (Fig. 1H); very rarely (e.g. mediastinal lymph nodes shown in Fig. 1B), the abscesses contained caseous and solid pus with initial classic ‘onion ring’ appearance. Abscesses in the subcutaneous lymph nodes and internal organs were often surrounded by variable amounts of fibrous tissue. Histologically (Fig. 2), the abscesses typically showed necrotic neutrophils inside the core, with few eosinophils at the border, surrounded by macrophages (without giant and epithelioid cells), plasma cells mixed with lymphocytes, and fibroblasts with collagen fibre bundles in the outer layer. Focal areas of calcification were observed rarely in the internal necrotic core, which almost
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L. Domenis et al. Table 1 Gross lesion distribution by sex and age of 98 chamois with caseous lymphadenitis
Gross lesion distribution
CM TV AV TV + CM AV + CM* GV GV + CM† TV + B TV + CM + B GV + CNS GV + CM + CNS Total
Number (%)
11 (11.2) 30 (30.6) 15 (15.3) 2 (2.1) 11 (11.2) 16 (16.3) 8 (8.2) 2 (2.1) 1 (1) 1 (1) 1 (1) 98 (100)
Sex Number (%)
Age group Number (%)
Male
Female
<18 months
18 months to 4 years
>4 years
8 (72.7) 16 (53.3) 6 (40) 2 (100) 4 (36.4) 8 (50) 6 (75) 2 (100) 0 (0.0) 0 (0.0) 1 (100) 53 (54.1)
3 (27.3) 14 (46.7) 9 (60) 0 (0.0) 7 (63.6) 8 (50) 2 (25) 0 (0.0) 1 (100) 1 (100) 0 (0.0) 45 (45.9)
3 (27.3) 5 (16.6) 2 (13.3) 0 (0.0) 2 (18.2) 0 (0.0) 1 (12.5) 0 (0.0) 0 (0.0) 1 (100) 0 (0.0) 14 (14.3)
3 (27.3) 2 (6.6) 3 (20) 0 (0.0) 0 (0.0) 1 (6.2) 0 (0.0) 1 (50) 0 (0.0) 0 (0.0) 1 (100) 11 (10.2)
5 (45.4) 23 (76.7) 10 (66.7) 2 (100) 9 (81.8) 15 (93.8) 7 (87.5) 1 (50) 1 (100) 0 (0.0) 0 (0.0) 73 (74.5)
CM, external form (subcutaneous lymph node involvement with or without muscles); AV, abdominal visceral form (only abdominal organ involvement: liver and/or spleen and/or kidney and/or lymph nodes); TV, thoracic visceral form (only thoracic organ involvement: lung and/ or heart and/or lymph nodes); GV, generalized visceral form (both abdominal and thoracic organ involvement); B, bone involvement; CNS, brain involvement. * 1 female with mammary gland involvement. † 1 male with testis involvement.
never tended to stratify in layers of coagulative necrosis (as observed in goats and sheep). In three animals with liver abscesses, there was extensive amyloid deposition in the portal spaces and Glisson’s capsule, between the rows of hepatocytes and in the vessel walls (Fig. 3A), often accompanied by similar deposits in the kidney, especially in the glomeruli and the intertubular spaces (Fig. 4A). In all cases, the amyloid was classified as secondary type based on the loss of typical green fluorescence after treatment with potassium permanganate solution and staining with Congo red (Figs. 3B, 4B). Nematode infestation (parasitic bronchopneumonia) was frequently associated with lung and/or mediastinal lymph node abscessation; fibrinous bronchopneumonia caused by Pasteurellaceae (i.e. Mannheimia haemolytica and Pasteurella multocida) was occasionally observed in the apical lobes. Blood agar culture yielded pinpoint colonies of C. pseudotuberculosis after 24 h of incubation, with growth to 0.5e1 mm in diameter after 48 h. The colonies were chalky in texture, movable on the surface of the medium and defined by a total haemolytic halo (observed at 24 or 48 or 72 h of incubation in 62, 31 and five strains, respectively). All 98 C. pseudotuberculosis strains showed the phenotypic shape of coccobacilli with individual or palisaded elements, were positive on Gram staining and catalase testing, and were sensitive to all antibiotics tested except for ampicillin (62.3% resistant strains) and gentamicin (83.7% resistant strains). Doubtful biochemical pro-
files (n ¼ 19) were all subsequently attributed to C. pseudotuberculosis by 16 S rRNA gene sequencing. The strains were highly homologous to the reference strain ATCC 19410, except for two and 48 isolates with a positive reaction for mannitol and a negative reaction for maltose, respectively. Based on nitrate reductase production, 95 strains were attributed to the ovis biovar (nitrate reduction negative) and three to the equi biovar (nitrate reduction positive) (Table 2). A positive ELISA result was obtained with serum samples from seven (58.3%) of 12 animals analyzed, and was considered effective (i.e. capable of signalling the presence of disease, based on both positive and doubtful results) (Table 3).
Discussion The observation that CLA was most often detected in adult animals over 4 years of age is consistent with previous reports of the disease in sheep and goats (Batey, 1986; Zavoshti et al., 2012; Abebe and Sisay Tessema, 2015). This reflects the chronic, progressive course of infection, in which generalized lesions (i.e. cutaneous and visceral forms) are more likely to be found in older animals. The classic transmission of infection in sheep and goats is through contamination of superficial wounds during common procedures such as shearing, castration and ear tagging or injuries due to other events (Dorella et al., 2006). After entry into the skin, C.
Caseous Lymphadenitis in Alpine Chamois
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Fig. 1. Abscesses from organs of chamois infected with Corynebacterium pseudotuberculosis. (A) Lung, (B) mediastinal lymph nodes, (C) heart, (D) thoracic vertebrae, (E) spleen, (F) liver, (G) testicle and (H) brain. Note the typical yellowegreen coloured, creamy to caseous pus tending to liquefaction, without a laminated ‘onion ring’ appearance (only partially observed in [B]).
pseudotuberculosis migrates within phagocytes to local draining lymph nodes and then disseminates via the blood or lymphatic system to other loci in the host (Fontaine and Baird, 2008). In chamois, in which the cutaneous route of infection is the most reliable, subcutaneous lymph node lesions were un-
even, probably because sometimes, as occurs in bovine tuberculosis, the smallest abscesses can escape detection at necropsy examination. As shown in Table 1, most of the cases studied fell into the third age group (i.e. over 4 years of age), the period of life when the chamois acquires the ability to reproduce.
Fig. 2. (A, B) Transverse histological sections showing a typical purulent abscess produced by Corynebacterium pseudotuberculosis in chamois. (a) Peripheral connective tissue with fibroblasts and collagen fibres; (b) lymphoplasmacytic cell layer; (c) macrophage layer; (d) central core with necrotic neutrophils and eosinophils. Note the absence of giant cells and calcified foci. HE. Bar A, 200 mm; Bar B, 50 mm.
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Fig. 3. Liver with amyloid deposits between hepatic lobules (*). (A) HE. Bar, 200 mm. (B) Congo red stain. Bar, 50 mm.
The higher number of cases in males than females (53 versus 45), albeit not significant, can be explained by the fact that males are more susceptible to injury sustained during battles during the breeding season, which may promote the entry of C. pseudotuberculosis into the lymphatic system from the environment or the ruptured skin abscesses of infected animals. This hypothesis could also partly explain the observation that males had more thoracic than abdominal lesions (20 versus 15). Similarly, the higher number of abdominal lesions in the females compared with the males (16 versus 10) could have been due to increased exposure of the posterior trunk to wounds related to parturition and suckling. In many cases we noted the presence of thoracic lesions without concomitant subcutaneous lymph node involvement. Based on similar observations by others in infected sheep (Stoops et al., 1984; Pepin et al., 1994; Baird and Fontaine, 2007) and in pulmonary infection after intratracheal administration of bacteria in sheep and goats (Brown and Olander, 1987), we hypothesized that CLA could probably also be transmitted via the airborne route in chamois. Finally, two cases of genital pseudotuberculosis were observed: one in a male characterized by testicular abscessation (associated with thoracic, abdominal and subcutaneous lymph node involvement) and one in a female charac-
terized by apostematous mastitis (associated with abdominal organ and subcutaneous lymph node involvement). The rarity of genital organ infection by C. pseudotuberculosis is consistent with the low frequency reported in goats (Valli and Parry, 1993; Fontaine and Baird, 2008). Microscopically, the abscesses generally showed liquefactive necrosis with a negligible tendency to form distinct concentric layers with calcification, which is considered a containment reaction typically found in domestic animals resident in areas where the infection is endemic (Fontaine and Baird, 2008), as well as in the sheep and goat herds of mountainous regions where the chamois of our study lived. In alpine wildlife such as the chamois, which do not receive pharmacological treatment and need to survive environmental stressors (e.g. extreme temperatures, shortage of pasture or pressure by predators), C. pseudotuberculosis appears to take an aggressive course with diffuse spread of infection throughout the body. The most serious and generalized forms (less common in small domestic ruminants) were observed in cases where the pathogen caused vertebral bone lysis and central nervous system (CNS) abscessation. These severe pathological patterns may also be related to a particular virulence linked to the synthesis of phospholipase D by the genotypes involved, as well as to
Fig. 4. Kidney with amyloid deposits in glomeruli (*) and intertubular space (:). (A) HE. Bar, 200 mm; (B) Congo red stain. Bar, 50 mm.
Caseous Lymphadenitis in Alpine Chamois Table 2 Biochemical characteristics of 98 isolates of C. pseudotuberculosis Test
Nitrate reduction Pyrazinamidase Pyrrolidonyl arylamidase Alkaline phosphatase b-glucuronidase b-galactosidase a-glucosidase N-acetyl-b-glucosaminidase Esculin Urea hydrolysis Gelatin hydrolysis Glucose Ribose Xylose Mannitol Maltose Lactose Saccharose Glycogen Catalase
Positive reaction Number (%)
Negative reaction Number (%)
3 (3.1) 0 (0.0) 1 (1.1) 70 (71.4) 0 (0.0) 0 (0.0) 1 (1.1) 0 (0.0) 2 (2.1) 97 (98.9) 3 (3.1) 98 (100) 97 (98.9) 0 (0.0) 2 (2.1) 50 (51.1) 2 (2.1) 2 (2.1) 1 (1.1) 98 (100)
95 (96.9) 98 (100) 97 (98.9) 28 (28.6) 98 (100) 98 (100) 97 (98.9) 98 (100) 96 (97.9) 1 (1.1) 95 (96.9) 0 (0.0) 1 (1.1) 98 (100) 96 (97.9) 48 (48.9) 96 (97.9) 96 (97.9) 97 (98.9) 0 (0.0)
Number and percentage of positive strains are shown.
flare-up events that could promote the spread of corynebacteria in some individuals depending on their immunocompetence. Co-infection with pulmonary nematodes and or Pasteurellaceae (i.e. P. multocida and M. haemolytica) are to be considered a probable debilitating or complicating factor. As seen with other chronic diseases such as tuberculosis, some individuals responded to C. pseudotuberculosis infection by overproducing acute phase proteins Table 3 Serological testing with sera from 12 chamois with C. pseudotuberculosis infection Animal 1 2 3 4 5 6 7 8 9 10 11 12
Gross lesion distribution
ELISA result
TV (tracheal lymph node) GV (tracheal lymph node and liver) TV (mediastinal lymph node) TV (lung) TV (lung) TV (mediastinal lymph node) TV (lung and mediastinal lymph nodes) TV (mediastinal lymph node) TV (mediastinal lymph node) GV (lung, liver, axillary and retropharyngeal lymph nodes) TV (mediastinal lymph node) GV (lung and liver)
N (13.98) D (61.45) P (89.64) P (97.52) N (19.71) N (4.86) P (87.65) N (51.73) N (0.14) P (110.78) P (73.08) P (122.38)
N, negative; D, doubtful; P, positive. The numbers in brackets are the ratio of the optical density of the sample:optical density of the positive control. GV, generalized visceral form; TV, thoracic visceral form.
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and developing serious forms of secondary amyloidosis in the liver and kidney as a consequence. This aspect, which we first report for CLA in chamois, has been observed in domestic animals, in goats with C. pseudotuberculosis (Eckersall et al., 2007) and in wild animals such as ibex, but in connection with other causative agents of chronic infection such as Sarcoptes scabiei (Raez Bravo et al., 2015) and Erysipelothrix rhusiopathiae (Domenis et al., 2017). Regarding phenotypic characteristics, the isolated strains exhibited typical features of C. pseudotuberculosis colonies. The propensity to rapidly develop haemolysis on blood agar (24 h) was not related to greater pathogenic activity in the host animal, as often happens with other bacteria. The biochemical profiles of the isolates were homologous with the reference strain ATCC 19410 (Literak et al., 1999; Chirino-Zarraga et al., 2006; Dorella et al., 2006), except for some variability in the ability to ferment maltose and mannitol. Furthermore, 95 strains belonged to the ovis biovar (negative nitrate reduction), as normally occurs in isolates cultured from sheep and goats (members of the Caprinae family, like the chamois). However, three strains, positive for nitrate reduction, were included in the equi biovar, suggesting environmental transmission (or, less likely, direct contact) from cattle or horses that shared pastures with the affected individuals. The strains causing the most severe forms of infection (bone lysis or CNS involvement) exhibited biochemical characteristics that differed from the reference strain ATCC 19410: case 1 with CM + VT + B lesions (nitrate reduction positive); case 2 with GV + CM + CNS lesions (esculine positive, lactose positive); case 3 with VG + CNS lesions (gelatin positive, mannitol positive); case 4 with VT + B lesions (pyrrolidonyl arylamidase positive) (see Tables 1 and 2). Previous studies have not shown a significant relationship between phenotypic characteristics and lesion extension caused by the equi biovar of C. pseudotuberculosis in horses (Britz et al., 2014). To verify this possible connection in chamois, further research is needed to characterize the genetic profile and virulence factors of the isolated strains. As concerns antibiotic sensitivity, the strains showed good response to penicillins, except for a consistent number of isolates resistant to ampicillin and gentamicin, which might have been selected by antibiotic pressure during veterinary therapies in domestic animals. In general, resistance to beta-lactams is linked to two different mechanisms (Olender, 2012), the production of beta-lactamase and the modification of penicillin protein binding proteins, while the resistance to gentamicin could be considered normal for C. pseudotuberculosis.
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Serological testing was positive in 58.3% of infected animals tested, all with visceral forms of infection. The highest S/P percentages were observed for the generalized visceral form (i.e. the most severe pseudotuberculosis involving thoracic and abdominal organs). As with other Actinomycetales infections (e.g. by Mycobacterium paratuberculosis and Mycobacterium bovis) and with CLA in sheep, the chamois may show a low sensitivity of the ELISA during certain stages of infection where the cell-mediated immune response may dominate (Binns et al., 2007). It should be noted that the ELISA kit uses a conjugated antibody specific for sheep and goat and not for chamois. These aspects, together with possible cross-reactions not investigated in this study, must be taken into account when using a serological kit for monitoring the disease in chamois, as well as the fact that early forms of the disease, with initial lesions localized to individual lymph nodes, may not be detected by the test. In conclusion, CLA in chamois displays close pathological and histological analogies with the disease described in sheep and goats; however, as evidenced here, some differences are probably related to the living conditions of chamois and the characteristics of the C. pseudotuberculosis strains involved. As pseudotuberculosis is classified among the minor zoonoses, people exposed occupationally to small, wild alpine ruminants such as chamois and ibex (e.g. forest rangers, hunters or veterinarians) should comply with safety and prevention guidelines when handling infected animals.
Acknowledgments We thank the forest rangers and hunters of Piedmont and the Aosta Valley for providing the chamois carcasses used in the present study.
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Biberstein EL, Knight HD, Jang S (1971) Two biotypes of Corynebacterium pseudotuberculosis. Veterinary Record, 89, 691e692. Binns SH, Green LE, Bailey M (2007) Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera. Veterinary Microbiology, 123, 169e179. Boomker J, Henton MM (1980) Pseudotuberculosis in a cheetah (Acinonyx jubatus). South African Journal of Wildlife Research, 10, 63e66. Britz E, Spier SJ, Kass PH, Edman JM, Foley JE (2014) The relationship between Corynebacterium pseudotuberculosis biovar equi phenotype with location and extent of lesions in horses. The Veterinary Journal, 200, 282e286. Brown CC, Olander HJ (1987) Caseous lymphadenitis of goats and sheep: a review. Veterinary Bulletin, 57, 1e12. Cadena-Colom A, Velarde R, Salinas J, Borge C, Garcı´ aBocanegra I et al. (2014) Management of a caseous lymphadenitis outbreak in a new Iberian ibex (Capra pyrenaica) stock reservoir. Acta Veterinaria Scandinavica, 56, 1e11. Chirino-Z arraga C, Scaramelli A, Rey-Valeir on C (2006) A bacteriological characterization of Corynebacterium pseudotuberculosis in Venezuelan goat flocks. Small Ruminant Research, 65, 170e175. Clark KA, Robinson RM, Weishuhn LL, Litton GW, Marburger RG (1972) Caseous lymphadenitis in pronghorns (Antilocapra americana). Journal of Wildlife Diseases, 8, 67e71. CLSI guideline M45 (2016) Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria, 3rd Edit.. Domenis L, Spedicato R, Orusa R, Robetto S (2017) Erysipelothrix rhusiopathiae infection in an alpine ibex (Capra ibex). Journal of Comparative Pathology, 156, 101. Dorella FA, Carvalho Pacheco LG, Costa Oliveira S, Miyoshi A, Azevedo V (2006) Corynebacterium pseudotuberculosis: microbiology, biochemical properties, pathogenesis and molecular studies of virulence. Veterinary Research, 37, 201e218. Eckersall PD, Lawson FP, Bence L, Waterston MM, Lang TL et al. (2007) Acute phase protein response in an experimental model of ovine caseous lymphadenitis. BMC Veterinary Research, 3, 35. Fontaine MC, Baird GJ (2008) Caseous lymphadenitis. Small Ruminant Research, 76, 42e48. Heggelund L, Gaustad P, H avelsrud OE, Blom J, Borgen L et al. (2015) Corynebacterium pseudotuberculosis pneumonia in a veterinary student infected during laboratory work. Open Forum Infectious Diseases, 2, 1e6. Jane Kelly E, Rood KA, Skirpstunas R (2012) Abscesses in captive elk associated with Corynebacterium pseudotuberculosis, Utah, USA. Journal of Wildlife Diseases, 48, 803e805. Join-Lambert OF, Ouache M, Canioni D, Beretti JL, Blanche S et al. (2006) Corynebacterium pseudotuberculosis necrotizing lymphadenitis in a twelve-year-old patient. The Pediatric Infectious Disease Journal, 25, 848e851.
Caseous Lymphadenitis in Alpine Chamois
Literak I, Horvathova A, Jahnova M, Rychlık I, Skalka B (1999) Phenotype and genotype characteristics of the Slovak and Czech Corynebacterium pseudotuberculosis strains isolated from sheep and goats. Small Ruminant Research, 32, 107e111. Lovari S (1985) Correct age determination in chamois. In: The Biology and Management of Mountain Ungulates, W Schr€oder, IV von Elsner-Schack, Eds., Croom Helm, London, pp. 67e70. Matos AC, Dias AP, Morais M, Figueira L, Martins MH et al. (2015) Granuloma co-infection with Mycobacterium bovis, Mycobacterium avium subsp. paratuberculosis, and Corynebacterium pseudotuberculosis in five hunted red deer (Cervus elaphus) in Portugal. Journal of Wildlife Diseases, 51, 793e794. Morales N, Aldridge D, Bahamonde A, Cerda J, Araya C et al. (2017) Corynebacterium pseudotuberculosis infection in Patagonian huemus (Hippocamelus bisulcus). Journal of Wildlife Diseases, 53, 621e624. M€ uller B, de Klerk-Lorist LM, Henton MM, Lane E, Parsons S et al. (2011) Mixed infections of Corynebacterium pseudotuberculosis and non-tuberculous mycobacteria in South African antelopes presenting with tuberculosislike lesions. Veterinary Microbiology, 147, 340e345. Nocard E (1896) Sur une lymphangite ulcereuse simulant le farcin morveux chez le cheval. Annales de l’Institut Pasteur, 10, 609e629. Olender A (2012) Mechanisms of antibiotic resistance in Corynebacterium spp. causing infections. In: Antibiotic Resistant Bacteria: A Continuous Challenge in the New Millennium, M Pana, Ed., IntechOpen Publisher, pp. 387e401. http://cdn.intechopen.com/pdfs-wm/34699.pdf. Paton MW, Mercy AR, Sutherland SS (1988) The influence of shearing and age on the incidence of caseous lymphadenitis in Australian sheep flocks. Proceedings of the 5th International Symposium on Veterinary Epidemiology and Economics, Acta Veterinaria Scandinavia, 29(Suppl. 84), 101e103. Pepin M, Paton M, Hodgson AL (1994) Pathogenesis and epidemiology of Corynebacterium pseudotuberculosis infection in sheep. Current Topics in Veterinary Research, 1, 63e82. € propos Perez L, Leon L, Cubero P, Ganz alez M (1996) A de deux cases de pseudotuberculosis chez le daime (Dama dama). In: 14eme Reunion du Groupe d’Etudes sur l’Ecopathologie de la faune Sauvage de Montagne. Cazorla, Espagne. Pre€ısz H, Guinard L (1891) Pseudo-tuberculose chez le mouton. Journal Medicine Veterinaire, 16, 563e572.
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R aez Bravo A, Granados JE, Cer on JJ, Cano-Manuel FJ, Fandos P et al. (2015) Acute phase proteins increase with sarcoptic mange status and severity in Iberian ibex (Capra pyrenaica, Schinz 1838). Parasitology Research, 114, 4005e4010. Romero-Perez JC, Su~ ner-Machado M, Batista-Dı´ az N (2004) Corynebacterium pseudotuberculosis lymphadenitis in a young patient. Revista Clı´nica Espa~nola, 204, 388e389. Roth HH, Vickers DB (1966) Pseudotuberculosis in an antbear (Orycteropus afer) in Rhodesia. British Veterinary Journal, 122, 296e300. Silinski S, Walzer C (2004) Caseous lymphadenitis in a captive group of alpine ibex (Capra ibex) e management and implications. In: Proceeding of the 5th Scientific Meeting of the European Association of Zoo and Wildlife Veterinarians, Ebeltoft, Denmark, p. 321. Songer JG, Beckenbach K, Marshall MM, Olson GB, Kelley L (1988) Biochemical and genetic characterization of Corynebacterium pseudotuberculosis. American Journal of Veterinary Research, 49, 221e226. Stauber E, Armstrong P, Chamberlain K, Gorgen B (1973) Caseous lymphadenitis in a white-tailed deer. Journal of Wildlife Diseases, 9, 56e57. Stoops SG, Renshaw HW, Thilsted JP (1984) Ovine caseous lymphadenitis: disease prevalence, lesion distribution, and thoracic manifestations in a population of mature culled sheep from western United States. American Journal of Veterinary Research, 45(3), 557e561. Tarello W, Theneyan M (2008) Corynebacterium pesudotuberculosis and Corynebacterium renale isolated from two Arabian oryx (Oryx leucoryx). Veterinary Record, 162, 862e863. Valli VEO, Parry BW (1993) Caseous lymphadenitis. In: Pathology of Domestic Animals, 4th Edit., Vol. 3, KVF Jubb, PC Kennedy, N Palmer, Eds., Academic Press, San Diego, pp. 238e240. Zavoshti FR, Khoojine ABS, Helan JA, Hassanzadeh B, Heydari AA (2012) Frequency of caseous lymphadenitis (CLA) in sheep slaughtered in an abattoir in Tabriz: comparison of bacterial culture and pathological study. Comparative Clinical Pathology, 21, 667e671.
February 13th, 2018 ½ Received, Accepted, April 17th, 2018
Available online at www.sciencedirect.com
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INFECTIOUS DISEASE
Caseous Lymphadenitis Caused by Corynebacterium pseudotuberculosis in Alpine Chamois (Rupicapra r. rupicapra): a Review of 98 Cases L. Domenis, R. Spedicato, E. Pepe, R. Orusa and S. Robetto Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta, Valle d’Aosta Department, National Reference Centre for Wildlife Diseases, Regione Amerique 7G, Quart, Italy
Summary Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA) in domestic and wild ruminants. Here we describe CLA in alpine chamois (Rupicapra r. rupicapra) based on a series of 98 cases of C. pseudotuberculosis infection confirmed by bacteriology and gene sequence analysis. The population included 53 males and 45 females distributed within three age groups: up to 18 months (n ¼ 14), 18 months to 4 years (n ¼ 11) and over 4 years (n ¼ 73). Four different gross lesion distribution patterns, observed individually or variably combined in the same animal, were defined: (1) cutaneous/external (i.e. subcutaneous lymph nodes with or without muscle involvement, n ¼ 34); (2) abdominal visceral (i.e. only abdominal organs involved: liver and/or spleen and/or kidney and/or lymph nodes, n ¼ 35); (3) thoracic visceral (i.e. only thoracic organs involved: lung and/or heart and/or lymph nodes, n ¼ 26); and (4) generalized visceral (i.e. abdominal and thoracic organs involved, n ¼ 26). In six particularly severe cases, mammary gland, testis, vertebral bone and the central nervous system were also affected. Macroscopically, most abscesses were characterized by fluid pus, confirmed by microscopy that showed the absence of distinct concentric layers and coagulative necrosis, which are typically seen in sheep and goats raised in areas where the infection is endemic. In three cases amyloid deposits were observed in the liver and kidney. The C. pseudotuberculosis strains isolated were highly homologous to the reference strain ATCC 19410, except for some variability in their ability to ferment maltose and mannitol. Based on the production of nitrate reductase, 95 strains were attributed to the ovis biovar (nitrate reduction negative) and three to the equi biovar (nitrate reduction positive). All strains were sensitive to antibiotics, except to ampicillin (62.3% resistant strains) and gentamicin (83.7% resistant strains). Using an indirect enzyme-linked immunosorbent assay designed for CLA in sheep and goats, seven (58.3%) of 12 serum samples tested positive for antibodies. Ó 2018 Elsevier Ltd. All rights reserved. Keywords: caseous lymphadenitis; chamois; Corynebacterium pseudotuberculosis; Rupicapra r. rupicapra
Introduction Caseous lymphadenitis (CLA), also termed pseudotuberculosis owing to its similarity with tuberculosis lesions, is an infectious disease affecting domestic and wild ruminants worldwide. Considered a minor Correspondence to: L. Domenis (e-mail: [email protected]). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2018.04.003
zoonosis in people exposed occupationally to infected animals (Romero-Perez et al., 2004; Join-Lambert et al., 2006; Heggelund et al., 2015), CLA causes considerable economic losses to the sheep meat industry due to condemnation and downgrading of carcasses, decreased meat yield and reduced wool production (Paton et al., 1988). The aetiological agent, isolated for the first time in 1888 by the French Ó 2018 Elsevier Ltd. All rights reserved.
12
L. Domenis et al.
bacteriologist Nocard from a case of bovine lymphangitis and some 3 years later by the Bulgarian bacteriologist von Pre€ısz from a ewe (Pre€ısz and Guinard, 1891; Nocard, 1896), is Corynebacterium pseudotuberculosis, a gram-positive and catalase-positive coccobacillus of the order Actinomycetales, which also contains the genera Mycobacterium, Rhodococcus and Nocardia. Two subtypes are distinguished according to nitrate reductase production: equi biovar (isolated from cattle and horses; nitrate reduction positive) and ovis biovar (isolated from goats and sheep; nitrate reduction negative) (Biberstein et al., 1971; Songer et al., 1988). The main virulence factor of C. pseudotuberculosis is phospholipase D, an enzyme that catalyzes the breakdown of proteins and lipids in the cell membrane. In small domestic ruminants, it causes the formation of purulent encapsulated abscesses with a caseous, lamellated (‘onion ring’) appearance, which can develop in peripheral lymph nodes (cutaneous or external form) or internal organs and lymph nodes (visceral or internal form). In wildlife species (free-ranging or in captivity), CLA has been reported in alpine ibex (Capra ibex) (Silinski and Walzer, 2004), Spanish ibex (Capra pyrenaica) (Cadena-Colom et al., 2014), alpine chamois (Rupicapra r. rupicapra) (Bassano et al., 1993), fallow deer (Dama dama) (Perez et al., 1996), red deer (Cervus elaphus) (Matos et al., 2015), elk (Cervus canadensis nelsoni) (Jane Kelly et al., 2012), white-tailed deer (Odocoilecus virginianus) (Stauber et al., 1973), pronghorn antelope (Antilocapra americana) (Clark et al., 1972), huemul (Hippocamelus bisulcus) (Morales et al., 2017), white-tailed gnu (Connochaetes gnou) (M€ uller et al., 2011), Arabian oryx (Oryx leucorix) (Tarello and Theneyan, 2008), cheetah (Acinonyx jubatus) (Boomker and Henton, 1980) and the aardvark (Orycteropus afer) (Roth and Vickers, 1966). Sporadic case reports of CLA in chamois are found in the literature (Bassano et al., 1993). Herein we describe CLA in alpine chamois based on a series of 98 confirmed cases of C. pseudotuberculosis infection, with a focus on gross and microscopical lesions, phenotypic and biochemical features, and antibiotic sensitivity of the strains isolated, as well as the serological response to C. pseudotuberculosis in some of the infected animals.
Materials and Methods Sampling
We reviewed 98 cases of CLA in alpine chamois, collected over the past 16 years according to the availability of anamnestic data (i.e. sex, age and origin), description of gross lesions (i.e. type and distribution)
and bacteriological test results (i.e. strain isolation and characterization). The animals came mainly from the Aosta Valley, a mountainous region of northwestern Italy, and to a lesser extent from bordering Piedmont (altitude 1,000e2,500 m). The provenance of the chamois included: (1) hunted with carcass fit for consumption (n ¼ 45); (2) hunted, but with carcass condemnation due to widespread lesions and/or poor condition (n ¼ 32); or (3) found dead or culled for health reasons (n ¼ 21), generally due to starvation (often associated with hyporeactivity). Before necropsy examination, biometric data were recorded for age estimated from horn growth (range <1 year to 16 years) (Lovari, 1985). Three age groups were defined according to male reproductive physiology: group I, aged <18 months (age at which the male reaches maturity); group II, aged between 18 months and 4 years (age range when the sexually mature male generally does not reproduce due to competitiveness inside the herd); and group III, aged over 4 years (age when the male can mate and breed). Necropsy Examination
The suspected CLA lesions (typically the presence of yellowegreen pus in lymph nodes and/or organs) were recorded according to the following groups: (1) cutaneous/external form (i.e. subcutaneous lymph nodes with or without muscle involvement); (2) abdominal visceral form (i.e. only abdominal organs involved: liver and/or spleen and/or kidney and/or lymph nodes); (3) thoracic visceral form (i.e. only thoracic organs involved: lung and/or heart and/or lymph nodes); and (4) generalized visceral form (i.e. abdominal and thoracic organs involved). More severe cases with bone and/or cerebral involvement or suppurative lesions in mammary gland and testis were also recorded. Bacteriology and Antimicrobial Susceptibility
The gross lesions of all animals were subjected to bacteriological examination for confirmation of macroscopical suspicion of CLA. Bacterial culture was performed using blood agar and MacConkey agar, with aerobic incubation at 37 C for up to 72 h. Typical colonies were confirmed initially by Gram staining and the catalase test, then subsequently identified with the API CoryneÒ system (Biomerieux, La Balme les Grottes, France). Once identified, the C. pseudotuberculosis strains were subjected to a KirbyeBauer antibiogram based on measurement of the zone of inhibition of bacterial growth around the antimicrobial strips at known concentrations (according to CLSI guideline M45, 3rd
13
Caseous Lymphadenitis in Alpine Chamois
Edit., 2016). The following antimicrobials were tested: amoxicillin, amoxicillineclavulanic acid, ampicillin, cephalothin, cefoperazone, cloxacillin, enrofloxacin, erythromycin, neomycin, penicillin, streptomycin, sulfamethoxazoleetrimethoprim, tetracycline, thiamphenicol, bacitracin, oxytetracycline, gentamicin and lincomycin.
a good quality serum could be obtained, which is not always feasible in hunted wild animals or those found dead) were subjected to indirect enzyme-linked immunosorbent assay (ELISA; ID Screen CLAÒ Indirect, IDvet, Montpellier, France), originally designed for antibody detection in ovine and caprine serum and plasma. All animals, except one, were over 4 years old.
Gene Sequence Analysis
In 19 cases, when biochemical identification of C. pseudotuberculosis was doubtful, the strains were further typed by 16S rRNA gene sequence analysis. Genetic identification of the organism was carried out by sequencing the 16S rRNA gene with a Micro-Seq 500 system (ABI MicroSEQ 500Ò 16S rDNA Bacterial Identification kit, Applied Biosystems, Foster City, California, USA). Bacterial DNA was extracted from isolated colonies and a fragment of approximately 500 base pairs (bp) of the 16S rRNA gene was amplified using the specific primers provided with the kit. The polymerase chain reaction (PCR) fragment was sequenced with an automatic sequencer (ABI3130, Applied Biosystems) and the obtained sequence compared with those of the Microseq 500 system data bank and the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA) Blast data bank. Histopathology
In some animals (n ¼ 34), where the condition of tissue was excellent, routine histological examination was performed by fixation in 10% neutral buffered formalin, routine processing and embedding in paraffin wax. Sections (3e4 mm) were stained with haematoxylin and eosin (HE) to study the microscopical features of abscesses or for differential diagnosis with other diseases that cause similar lesions (e.g. tuberculosis due to Mycobacterium spp., pyogenic bacterial abscesses caused by Staphylococcus aureus or Trueperella pyogenes). In other cases (n ¼ 3), to confirm the presence and type of amyloid, Congo red staining was performed using a commercial kit (Congo Red StainÒ Kit, Highman, Molekula S.r.L., Rimini, Italy) according to the Highman protocol (Congo red solution for 5 min, differentiation in 0.2% potassium hydroxide solution for 3e10 sec, staining with Mayer’s haemalum solution for 5 min), with and without pretreatment of slices using a solution of potassium permanganate and sulphuric acid, followed by observation under polarized light. Serology
To evaluate antibody response to C. pseudotuberculosis infection, 12 of the 98 chamois (i.e. those from which
Results The distribution of gross lesions (i.e. typical abscesses) is reported in Table 1 and portrayed in Fig. 1. The table presents the different forms (i.e. cutaneous/ external, abdominal visceral, thoracic visceral and generalized visceral) that appeared alone or overlapping in the same animal in relation to the three age classes and sex. The external form (i.e. lymph nodes involvement with or without abscesses in the muscle) was observed in 34 animals. The thoracic visceral form was observed more often than the abdominal visceral form (35 versus 26 animals), the latter having the same occurrence as the generalized visceral forms (26 animals) involving the abdominal and thoracic organs. Among the abdominal organs, abscesses were found mostly in the liver (Fig. 1F), often with adhesion to the diaphragm, and seldom in the spleen (Fig. 1E) or kidney. Among the thoracic organs, the lung (Fig. 1A) and mediastinal lymph nodes (Fig. 1B) were often affected with large, cavernous collections of pus; the heart (Fig. 1C) was involved rarely, with generally small, myocardial pyogranulomas. In a few cases, CLA lesions were observed in the mammary glands, testis (Fig. 1G), bone of the thoracic and/or lumbar vertebrae (Fig. 1D) and the brain (Fig. 1H). Encapsulated abscesses ranged in dimension from miliary up to 15e20 cm in diameter. In general, the pus in all organs was yellowegreen in colour (Figs. 1AeH), with creamy to caseous consistency and signs of liquefaction, the latter especially evident in suppurative osteomyelitis (Fig. 1D) and brain abscesses (Fig. 1H); very rarely (e.g. mediastinal lymph nodes shown in Fig. 1B), the abscesses contained caseous and solid pus with initial classic ‘onion ring’ appearance. Abscesses in the subcutaneous lymph nodes and internal organs were often surrounded by variable amounts of fibrous tissue. Histologically (Fig. 2), the abscesses typically showed necrotic neutrophils inside the core, with few eosinophils at the border, surrounded by macrophages (without giant and epithelioid cells), plasma cells mixed with lymphocytes, and fibroblasts with collagen fibre bundles in the outer layer. Focal areas of calcification were observed rarely in the internal necrotic core, which almost
14
L. Domenis et al. Table 1 Gross lesion distribution by sex and age of 98 chamois with caseous lymphadenitis
Gross lesion distribution
CM TV AV TV + CM AV + CM* GV GV + CM† TV + B TV + CM + B GV + CNS GV + CM + CNS Total
Number (%)
11 (11.2) 30 (30.6) 15 (15.3) 2 (2.1) 11 (11.2) 16 (16.3) 8 (8.2) 2 (2.1) 1 (1) 1 (1) 1 (1) 98 (100)
Sex Number (%)
Age group Number (%)
Male
Female
<18 months
18 months to 4 years
>4 years
8 (72.7) 16 (53.3) 6 (40) 2 (100) 4 (36.4) 8 (50) 6 (75) 2 (100) 0 (0.0) 0 (0.0) 1 (100) 53 (54.1)
3 (27.3) 14 (46.7) 9 (60) 0 (0.0) 7 (63.6) 8 (50) 2 (25) 0 (0.0) 1 (100) 1 (100) 0 (0.0) 45 (45.9)
3 (27.3) 5 (16.6) 2 (13.3) 0 (0.0) 2 (18.2) 0 (0.0) 1 (12.5) 0 (0.0) 0 (0.0) 1 (100) 0 (0.0) 14 (14.3)
3 (27.3) 2 (6.6) 3 (20) 0 (0.0) 0 (0.0) 1 (6.2) 0 (0.0) 1 (50) 0 (0.0) 0 (0.0) 1 (100) 11 (10.2)
5 (45.4) 23 (76.7) 10 (66.7) 2 (100) 9 (81.8) 15 (93.8) 7 (87.5) 1 (50) 1 (100) 0 (0.0) 0 (0.0) 73 (74.5)
CM, external form (subcutaneous lymph node involvement with or without muscles); AV, abdominal visceral form (only abdominal organ involvement: liver and/or spleen and/or kidney and/or lymph nodes); TV, thoracic visceral form (only thoracic organ involvement: lung and/ or heart and/or lymph nodes); GV, generalized visceral form (both abdominal and thoracic organ involvement); B, bone involvement; CNS, brain involvement. * 1 female with mammary gland involvement. † 1 male with testis involvement.
never tended to stratify in layers of coagulative necrosis (as observed in goats and sheep). In three animals with liver abscesses, there was extensive amyloid deposition in the portal spaces and Glisson’s capsule, between the rows of hepatocytes and in the vessel walls (Fig. 3A), often accompanied by similar deposits in the kidney, especially in the glomeruli and the intertubular spaces (Fig. 4A). In all cases, the amyloid was classified as secondary type based on the loss of typical green fluorescence after treatment with potassium permanganate solution and staining with Congo red (Figs. 3B, 4B). Nematode infestation (parasitic bronchopneumonia) was frequently associated with lung and/or mediastinal lymph node abscessation; fibrinous bronchopneumonia caused by Pasteurellaceae (i.e. Mannheimia haemolytica and Pasteurella multocida) was occasionally observed in the apical lobes. Blood agar culture yielded pinpoint colonies of C. pseudotuberculosis after 24 h of incubation, with growth to 0.5e1 mm in diameter after 48 h. The colonies were chalky in texture, movable on the surface of the medium and defined by a total haemolytic halo (observed at 24 or 48 or 72 h of incubation in 62, 31 and five strains, respectively). All 98 C. pseudotuberculosis strains showed the phenotypic shape of coccobacilli with individual or palisaded elements, were positive on Gram staining and catalase testing, and were sensitive to all antibiotics tested except for ampicillin (62.3% resistant strains) and gentamicin (83.7% resistant strains). Doubtful biochemical pro-
files (n ¼ 19) were all subsequently attributed to C. pseudotuberculosis by 16 S rRNA gene sequencing. The strains were highly homologous to the reference strain ATCC 19410, except for two and 48 isolates with a positive reaction for mannitol and a negative reaction for maltose, respectively. Based on nitrate reductase production, 95 strains were attributed to the ovis biovar (nitrate reduction negative) and three to the equi biovar (nitrate reduction positive) (Table 2). A positive ELISA result was obtained with serum samples from seven (58.3%) of 12 animals analyzed, and was considered effective (i.e. capable of signalling the presence of disease, based on both positive and doubtful results) (Table 3).
Discussion The observation that CLA was most often detected in adult animals over 4 years of age is consistent with previous reports of the disease in sheep and goats (Batey, 1986; Zavoshti et al., 2012; Abebe and Sisay Tessema, 2015). This reflects the chronic, progressive course of infection, in which generalized lesions (i.e. cutaneous and visceral forms) are more likely to be found in older animals. The classic transmission of infection in sheep and goats is through contamination of superficial wounds during common procedures such as shearing, castration and ear tagging or injuries due to other events (Dorella et al., 2006). After entry into the skin, C.
Caseous Lymphadenitis in Alpine Chamois
15
Fig. 1. Abscesses from organs of chamois infected with Corynebacterium pseudotuberculosis. (A) Lung, (B) mediastinal lymph nodes, (C) heart, (D) thoracic vertebrae, (E) spleen, (F) liver, (G) testicle and (H) brain. Note the typical yellowegreen coloured, creamy to caseous pus tending to liquefaction, without a laminated ‘onion ring’ appearance (only partially observed in [B]).
pseudotuberculosis migrates within phagocytes to local draining lymph nodes and then disseminates via the blood or lymphatic system to other loci in the host (Fontaine and Baird, 2008). In chamois, in which the cutaneous route of infection is the most reliable, subcutaneous lymph node lesions were un-
even, probably because sometimes, as occurs in bovine tuberculosis, the smallest abscesses can escape detection at necropsy examination. As shown in Table 1, most of the cases studied fell into the third age group (i.e. over 4 years of age), the period of life when the chamois acquires the ability to reproduce.
Fig. 2. (A, B) Transverse histological sections showing a typical purulent abscess produced by Corynebacterium pseudotuberculosis in chamois. (a) Peripheral connective tissue with fibroblasts and collagen fibres; (b) lymphoplasmacytic cell layer; (c) macrophage layer; (d) central core with necrotic neutrophils and eosinophils. Note the absence of giant cells and calcified foci. HE. Bar A, 200 mm; Bar B, 50 mm.
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L. Domenis et al.
Fig. 3. Liver with amyloid deposits between hepatic lobules (*). (A) HE. Bar, 200 mm. (B) Congo red stain. Bar, 50 mm.
The higher number of cases in males than females (53 versus 45), albeit not significant, can be explained by the fact that males are more susceptible to injury sustained during battles during the breeding season, which may promote the entry of C. pseudotuberculosis into the lymphatic system from the environment or the ruptured skin abscesses of infected animals. This hypothesis could also partly explain the observation that males had more thoracic than abdominal lesions (20 versus 15). Similarly, the higher number of abdominal lesions in the females compared with the males (16 versus 10) could have been due to increased exposure of the posterior trunk to wounds related to parturition and suckling. In many cases we noted the presence of thoracic lesions without concomitant subcutaneous lymph node involvement. Based on similar observations by others in infected sheep (Stoops et al., 1984; Pepin et al., 1994; Baird and Fontaine, 2007) and in pulmonary infection after intratracheal administration of bacteria in sheep and goats (Brown and Olander, 1987), we hypothesized that CLA could probably also be transmitted via the airborne route in chamois. Finally, two cases of genital pseudotuberculosis were observed: one in a male characterized by testicular abscessation (associated with thoracic, abdominal and subcutaneous lymph node involvement) and one in a female charac-
terized by apostematous mastitis (associated with abdominal organ and subcutaneous lymph node involvement). The rarity of genital organ infection by C. pseudotuberculosis is consistent with the low frequency reported in goats (Valli and Parry, 1993; Fontaine and Baird, 2008). Microscopically, the abscesses generally showed liquefactive necrosis with a negligible tendency to form distinct concentric layers with calcification, which is considered a containment reaction typically found in domestic animals resident in areas where the infection is endemic (Fontaine and Baird, 2008), as well as in the sheep and goat herds of mountainous regions where the chamois of our study lived. In alpine wildlife such as the chamois, which do not receive pharmacological treatment and need to survive environmental stressors (e.g. extreme temperatures, shortage of pasture or pressure by predators), C. pseudotuberculosis appears to take an aggressive course with diffuse spread of infection throughout the body. The most serious and generalized forms (less common in small domestic ruminants) were observed in cases where the pathogen caused vertebral bone lysis and central nervous system (CNS) abscessation. These severe pathological patterns may also be related to a particular virulence linked to the synthesis of phospholipase D by the genotypes involved, as well as to
Fig. 4. Kidney with amyloid deposits in glomeruli (*) and intertubular space (:). (A) HE. Bar, 200 mm; (B) Congo red stain. Bar, 50 mm.
Caseous Lymphadenitis in Alpine Chamois Table 2 Biochemical characteristics of 98 isolates of C. pseudotuberculosis Test
Nitrate reduction Pyrazinamidase Pyrrolidonyl arylamidase Alkaline phosphatase b-glucuronidase b-galactosidase a-glucosidase N-acetyl-b-glucosaminidase Esculin Urea hydrolysis Gelatin hydrolysis Glucose Ribose Xylose Mannitol Maltose Lactose Saccharose Glycogen Catalase
Positive reaction Number (%)
Negative reaction Number (%)
3 (3.1) 0 (0.0) 1 (1.1) 70 (71.4) 0 (0.0) 0 (0.0) 1 (1.1) 0 (0.0) 2 (2.1) 97 (98.9) 3 (3.1) 98 (100) 97 (98.9) 0 (0.0) 2 (2.1) 50 (51.1) 2 (2.1) 2 (2.1) 1 (1.1) 98 (100)
95 (96.9) 98 (100) 97 (98.9) 28 (28.6) 98 (100) 98 (100) 97 (98.9) 98 (100) 96 (97.9) 1 (1.1) 95 (96.9) 0 (0.0) 1 (1.1) 98 (100) 96 (97.9) 48 (48.9) 96 (97.9) 96 (97.9) 97 (98.9) 0 (0.0)
Number and percentage of positive strains are shown.
flare-up events that could promote the spread of corynebacteria in some individuals depending on their immunocompetence. Co-infection with pulmonary nematodes and or Pasteurellaceae (i.e. P. multocida and M. haemolytica) are to be considered a probable debilitating or complicating factor. As seen with other chronic diseases such as tuberculosis, some individuals responded to C. pseudotuberculosis infection by overproducing acute phase proteins Table 3 Serological testing with sera from 12 chamois with C. pseudotuberculosis infection Animal 1 2 3 4 5 6 7 8 9 10 11 12
Gross lesion distribution
ELISA result
TV (tracheal lymph node) GV (tracheal lymph node and liver) TV (mediastinal lymph node) TV (lung) TV (lung) TV (mediastinal lymph node) TV (lung and mediastinal lymph nodes) TV (mediastinal lymph node) TV (mediastinal lymph node) GV (lung, liver, axillary and retropharyngeal lymph nodes) TV (mediastinal lymph node) GV (lung and liver)
N (13.98) D (61.45) P (89.64) P (97.52) N (19.71) N (4.86) P (87.65) N (51.73) N (0.14) P (110.78) P (73.08) P (122.38)
N, negative; D, doubtful; P, positive. The numbers in brackets are the ratio of the optical density of the sample:optical density of the positive control. GV, generalized visceral form; TV, thoracic visceral form.
17
and developing serious forms of secondary amyloidosis in the liver and kidney as a consequence. This aspect, which we first report for CLA in chamois, has been observed in domestic animals, in goats with C. pseudotuberculosis (Eckersall et al., 2007) and in wild animals such as ibex, but in connection with other causative agents of chronic infection such as Sarcoptes scabiei (Raez Bravo et al., 2015) and Erysipelothrix rhusiopathiae (Domenis et al., 2017). Regarding phenotypic characteristics, the isolated strains exhibited typical features of C. pseudotuberculosis colonies. The propensity to rapidly develop haemolysis on blood agar (24 h) was not related to greater pathogenic activity in the host animal, as often happens with other bacteria. The biochemical profiles of the isolates were homologous with the reference strain ATCC 19410 (Literak et al., 1999; Chirino-Zarraga et al., 2006; Dorella et al., 2006), except for some variability in the ability to ferment maltose and mannitol. Furthermore, 95 strains belonged to the ovis biovar (negative nitrate reduction), as normally occurs in isolates cultured from sheep and goats (members of the Caprinae family, like the chamois). However, three strains, positive for nitrate reduction, were included in the equi biovar, suggesting environmental transmission (or, less likely, direct contact) from cattle or horses that shared pastures with the affected individuals. The strains causing the most severe forms of infection (bone lysis or CNS involvement) exhibited biochemical characteristics that differed from the reference strain ATCC 19410: case 1 with CM + VT + B lesions (nitrate reduction positive); case 2 with GV + CM + CNS lesions (esculine positive, lactose positive); case 3 with VG + CNS lesions (gelatin positive, mannitol positive); case 4 with VT + B lesions (pyrrolidonyl arylamidase positive) (see Tables 1 and 2). Previous studies have not shown a significant relationship between phenotypic characteristics and lesion extension caused by the equi biovar of C. pseudotuberculosis in horses (Britz et al., 2014). To verify this possible connection in chamois, further research is needed to characterize the genetic profile and virulence factors of the isolated strains. As concerns antibiotic sensitivity, the strains showed good response to penicillins, except for a consistent number of isolates resistant to ampicillin and gentamicin, which might have been selected by antibiotic pressure during veterinary therapies in domestic animals. In general, resistance to beta-lactams is linked to two different mechanisms (Olender, 2012), the production of beta-lactamase and the modification of penicillin protein binding proteins, while the resistance to gentamicin could be considered normal for C. pseudotuberculosis.
18
L. Domenis et al.
Serological testing was positive in 58.3% of infected animals tested, all with visceral forms of infection. The highest S/P percentages were observed for the generalized visceral form (i.e. the most severe pseudotuberculosis involving thoracic and abdominal organs). As with other Actinomycetales infections (e.g. by Mycobacterium paratuberculosis and Mycobacterium bovis) and with CLA in sheep, the chamois may show a low sensitivity of the ELISA during certain stages of infection where the cell-mediated immune response may dominate (Binns et al., 2007). It should be noted that the ELISA kit uses a conjugated antibody specific for sheep and goat and not for chamois. These aspects, together with possible cross-reactions not investigated in this study, must be taken into account when using a serological kit for monitoring the disease in chamois, as well as the fact that early forms of the disease, with initial lesions localized to individual lymph nodes, may not be detected by the test. In conclusion, CLA in chamois displays close pathological and histological analogies with the disease described in sheep and goats; however, as evidenced here, some differences are probably related to the living conditions of chamois and the characteristics of the C. pseudotuberculosis strains involved. As pseudotuberculosis is classified among the minor zoonoses, people exposed occupationally to small, wild alpine ruminants such as chamois and ibex (e.g. forest rangers, hunters or veterinarians) should comply with safety and prevention guidelines when handling infected animals.
Acknowledgments We thank the forest rangers and hunters of Piedmont and the Aosta Valley for providing the chamois carcasses used in the present study.
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February 13th, 2018 ½ Received, Accepted, April 17th, 2018