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Intestinal and pulmonary mycotic lymphadenitis in cattle
j. Comp. Path 1990Vol. 102
I n t e s t i n a l a n d P u l m o n a r y M y c o t i c L y m p h a d e n i t i s in Cattle H~E. Jensen*]l, H. Schonheyder~++ and J. B. Jorgensen* *National Dlerinary Laboratory, Biilowsvej 27, P.O. Box 373, DK-1503 Copenhagen V and ~Institute of Medical Microbiology, University of Aarhus, DK-8000 Aarhus C, Denmark
Summary Among 4877 slaughtered cattle with tuberculosis-like lesions in lymph nodes, 94 cases (1'9 per cent) revealed fungal hyphae on histopathological examination. The survey period was 12 years and most of the affected animals (> 77 per cent) were beef cattle. Affected nodes were mesenteric in 84 cases (89'4 per cent), mediastinal and/or bronchial in seven cases (7"4 per cent), and in three cases (3"2 per cent), both mesenteric and mediastinal nodes were affected. The incidence of mycotic lymphadenitis was unrelated to year of study or season. Eighty-two of the cases were re-examined histologically and immunohistochemically. All lesions were granulomatous in nature and, in 26 cases, eosinophilic asteroid bodies (rosette formation) around hyphae were found. In 75 cases, immunofluorescence staining identified the agent as a zygomycete, probably Absidia cor~rnbifera and, in one case, there was a concurrent infection with a Candida species. In seven cases that did not react with the antibodies employed, a diagnosis ofzygomycosis was suggested on the basis of hyphal morphology. Hyphae of Aspergillus spp. were not found in any of the lesions. A concurrent fungaI and mycobacterial infection (M. avium) was diagnosed in one case. Thus, zygomycetes are the main cause of macroscopically apparent mycotic lymphadenitis, a sporadic disease most probably caused by feeding with mouldy food stuffs.
Introduction I n Danish cattle, lesions resembling tuberculosis, when [bund d u r i n g any kind o f p o s t - m o r t e m e x a m i n a t i o n but n o r m a l l y at meat inspection, are r e q u i r e d to be submitted to the National Veterinary L a b o r a t o r y for c o n f i r m a t o r y diagnosis. As a result of this practice, a n u m b e r of mycotic lesions are r e c o r d e d every year. Cases of mycotic lymphadenitis in both cattle and pigs are often found on suspicion of" tuberculosis although, especially in pigs, m y c o t i c lymphadenitis m a y be mistaken tbr l y m p h o s a r c o m a (Dion, B u n d z a a n d Dukes, 1987). T h e aetiological diagnosis of mycoses is usually p e r f o r m e d on the basis o f h y p h a l m o r p h o l o g y in tissue sections, which is considered to be difficult (Pallaske, 1967; Cordes a n d Shortridge, 1968; Ossent, 1987; J e n s e n a n d Present addresses: HDepartmentof Veterinary Pathology,Royal Veterinal3"and Agricuhural University, B/ilowsvej 13, DK-1870 FrederlksbergC, Denmark and ;Department of Clinical Microbiology,Aalborg Hospital, DK-9100Aalborg, Denmark. 0021-9975/90/030345+ 11 $03.00/0
© 1990 AcademicPress Limited
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H.E. Jensen et al.
Schonheyder, 1989a). Isolation of fungi from animal tissue is difficult because of post-mortem changes (Dion et al., 1987). Moreover, since mammalian tissues generally are contaminated by fungi (Dion et al., 1987) and since fungi are c o m m o n laboratory contaminants (Cordes and Shortridge, 1968), it is difficult to j u d g e whether an isolate is also the pathogen (Cordes and Shortridge, 1968; Dion et al., 1987). Since the first report of mycotic lymphadenitis in cattle (Gleiser, 1953), one major study (Dion et al., 1987) and a number of case reports (Davis, Anderson a n d McCrory, 1955; Pallaske, 1967; Werffeli and Biihlmann, 1967) have been published. A comprehensive survey including all specimens obtained during a period of years is not accessible. This paper presents 94 cases of mycotic lymphadenitis diagnosed in cattle which had initially been suspected of having tuberculosis, during a period of 12 .,years (1977-1988). T h e aetiological diagnosis was, in most cases, accomplished by immunofluorescence staining of h y p h a e in the tissue sections.
M a t e r i a l s and M e t h o d s
Spe£ill'zens From 1977 to 1988, lymph nodes or parts of lymph nodes from 4877 cattle were submitted to the National Veterinary Laboratory on suspicion of tuberculosis.
Laboratory Examinations The ~bllowing laboratory examinations were performed on lymph nodes with lesions resembling tuberculosis: (1) microscopy of Ziehl-Neelsen-stained smears; (2) inoculation onto several modifications of L6wenstein-Jensen medium for culture of especially Mycobaclerium boris, M. avium complex, and M. paraluberculosis; (3) histopathology of lesions negative for acid-last bacteria/rods by microscopy of smears; (4) mycological culture on Sabouraud's glucose agar, pH 5.7 (incubated at 37°C for 7 days) in three selected cases. Thc diagnosis of mycotic lymphadenitis was made exclusively on the basis of histological demonstration of hyphae in tissue sections. From cases with paraffin wax-embedded tissue blocks stored at the laboratory (n=82), tissue sections (5-gm thick) were cut and re-examined. The tissue sections were stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS), Grocott's methenamine silver (GMS), Van Kossa's stain for calcium and Luna's stain for eosinophils (Luna, 1968). Imrnunojtuorescence Staining The aetiological diagnosis was accomplished by immunofluorescence staining of hyphae in tissue sections with hyperimmune rabbit antisera absorbed with heterologous fungal antigens before use, as described in detail elsewhere (Jensen and Schonheyder, 1989a). In the present study, the panel of antisera was extended by the addition ofanti-Candida albicans antiserum (B 143, Dakopatts, Denmark), diluted 1 in 256 (the plateau end-point) and used without prior absorption. Mycotic lesions in cattle were examined in parallel with sections of routine tissues experimentally infected with Absidia corymbi/'era, AspeNillus spp. and Candida albicans as described elsewhere (Jensen and Schonheyder, 1989a). The immunofluorescence staining system discriminated between zygomycetes (probably Absidia corymblfera), AspergiUus spp. and Candida spp.
347
Mycotic L y m p h a d e n i t i s o f C a t t l e Statistics
I n f o r m a t i o n on n u m b e r s of s l a u g h t e r e d c a t t l e i n D e n m a r k w a s o b t a i n e d f r o m t h e D a n i s h V e t e r i n a r y S e r v i c e a n d the D a n i s h L i v e S t o c k a n d M e a t B o a r d . I n cases w h e r e a farmer had deIivercd more than one animal with mycotic lymphadenitis, he was i n t e r v i e w e d to e l u c i d a t e t h e f e e d i n g r e g i m e o f his cattle.
Results Statistics Lymph nodes from 94 (1.9 per cent) of 4877 cattle examined were found to contain hyphae (Tables 1 and 2). Four cattle had lesions in two nodes. There was a notable increase in the rate of mycotic lymphadenitis among clinical cases studied, but the rate of mycotic lymphadenitis among slaughtered cattle did not change signifcantly over the years. Likewise, no consistent pattern with respect to the month of diagnosis was observed (Table 2). L y m p h nodes with mycotic lesions were predominantly mesenteric, the anatomical distribution being given in Table 3. Most of the affected cattle (n= 73) were less than 2 years old, some were mature (n= 5) and, for some, no age was stated (n = 16). The sex was stated for only 23 animals (five females and 18 males). However, from the agedistribution it appears that most of the cattle were bull calves which normally are slaughtered for beef production at 1 to 1.5 years old. O f the 94 cattle, nine had been bred by one farmer, while three farmers had
Table 1 Yearly distribution of cattle slaughtered in Denmark, number of cases e x a m i n e d on suspicion of tuberculosis, and cases of mycotic lymphadenitis during a 12-year period (1977-1988) Mycotic lymphadenitis
Year
Cattle slaughtered in .Denmark ( x 100 000)
Total number of cases examined
1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988
11.18 10"53 11'13 10.70 10'44 10'07 10.41 10-79 10,19 10'00 9"49 8'73
Total
123.66
*Mean.
No.
Per cent of cases examined
Per 100 000 cattle slaughtered
844 927 745 572 460 354 278 236 203 122 71 65
fi 10 12 9 5 6 5 6 11 6 8 10
0'7 1'1 1.6 1'6 I' 1 1'7 1"8 2'5 5'4 4-9 11 '3 15.4
0'54 0'95 1"08 0,84 0'48 0'60 0.48 0'56 1'08 0"60 0'84 1.15
4877
94
4.1 *
0,77*
348
H . E . J e n s e n e t al.
Table 2 C u m u l a t i v e m o n t h l y d i s t r i b u t i o n of cattle s l a u g h t e r e d in Denmark, n u m b e r of cases e x a m i n e d on s u s p i c i o n of tuberculosis and cases of mycotic l y m p h a d e n i t i s d u r i n g a 12-year period (1977-1988) Mycotw lymphadenitis
Month
Cattle slaughtered in Denmark ( x 100 000)
Total number of cases examined
11.43 9.76 10.55 9.59 10,46 10.70 7"73 10'69 10"74 1 l'62 11'37 10"02 123'66
1 2 3 4 5 6 7 8 9 I0 11 12 Total
No.
Per cent qf cafes eaammed
Per 100 000 cattle daughto ed
399 368 417 438 477 499 332 397 404 429 359 358
5 7 7 6 10 11 6 3 8 10 1f 10
1.3 1.9 1.7 1.4 2' 1 2'2 1'8 0"8 2'0 2'3 3" 1 2'8
0-44 0"72 0"66 0-63 0.96 1"03 0"78 0"28 0-74 0'86 0-97 1' 11
4877
94
2'0*
0.77*
*Mean.
Table 3 Anatomical distribution of mycotic l y m p h a d e n i t i s in 94 cattle examined during a period of 12 years (1977-1988)
Lymph nodes
Number
Pe~ cent
Mesenteric Mesenteric and mediastinal Mediastinal Bronchial Mediastinal and bronchial Lung*
84 3 3 2 I 1
89'4 3'2
Total
94
~ ~ [ ]
7"4
100.0
Lymph nodes evaluated by immunofiuore.~cence ~taming (No.) 74 3 3 0 1 1 82
*The exact position not stated.
raised two cattle each. The farmer with nine affected beef cattle frequently tkd mouldy waste bread from a bakery as a supplementary feed. The three other farmers reported that their beef cattle occasionally had been fed with hay and silage contaminated by moulds. Gross Pathology
The macroscopic appearance of lymph nodes conformed in most cases with the descriptions by Davis et al. (1955) and Dion et al. 11987), enlarged, firm
Mycotic Lymphadenitis
of Cattle
349
greyish-white or yellow, granular, caseocalcareous, with a variable degree of scarring. T h e granulomas occupied the entire lymph node in most cases (Fig. 1), while occasionally the processes appeared as encapsulated lesions inside the affected lymph nodes (Fig. 2). Hislopalhology All lesions were fbund to be granulomatous, and the picture was generally similar to that described by Davis et al. (1955). In most of the nodes, lesions consisted of masses of necrotic tissue containing scattered loci of calcification. T h e necrotic masses were intermingled with areas containing inflammatory cells, including lymphocytes, plasma cells, epithelioid cells and giant cells (Fig. 3). Between the more or less well-marked granulomas, a variable amount of fibrous tissue was seen. In some of the granulomas, small satellite foci ofhyphal g r o w t h were noted in the periphery underneath the fibrous capsule (Fig. 4). In these loci, the cellular infiltrate contained both neutrophils and eosinophils. W h e n granulomas only occupied a part of the lymph nodes, they were mainly restricted to the cortical zone and were separated fi'om the normal lymphatic tissue by a thick zone of fibrous tissue. Eosinophils were found in almost all lesions but, in 29 cases (28 per cent), they were present in large numbers. The eosinophils were located in all parts of the granulomas, but predominantly centrally. H y p h a e were regularly seen in the necrotic areas accompanied by giant cells, but they were, in some cases, situated also in the connective tissue between the granulomas.
Fig. 1. A granulomatous mycotic lesion occupying an entire mesenteric lymph node. x 1-5,
350
H . E . J e n s e n e t al.
Fig. 2.
A calcified, granulomatous, mycotic lesion appears to be encapsulated within a mesenteric lymph node. x 1.5.
Fig. 3.
Mycotic lymphadenitis. Hyphae in giant cells (large arrows) arid intermingling with the granulomatous tissue (small arrows). HE x 130.
Fig, 4.
Mycotic lymphadenitis, A satellite focus (inset) with hyphal growth and infiltration of granulocytes is located at the periphery of a granuloma (arrows) close to the fibrous capsule (C). Normal lymphatic tissue (N). PAS × 53; inset: x 130.
M y c o t i c L y m p h a d e n i t i s of Cattle
351
Fig. 5. Mycotic lymphadenitis. Asteroid bodies are covering the hyphae (arrows) in a mycotic granuloma. HE x 130,
The morphology of fungal elements in all cases suggested the presence of a zygomycete but, in six cases, the presence of an Aspergillus species was also suspected. In one case, a zygomycete, an AspergiIlussp. plus a Candida sp., were suspected (Fig. 6). In 26 cases, eosinophilic asteroid bodies (rosette formation) covered most of the hyphae (Fig. 5). In two cases, asteroid bodies were observed both in the mediastinal and in the mesenteric nodes.
Microbiology In one case, mycological culture yielded growth of A. corymbifera from a mesenteric lymph node whereas, in two other cases, mycological cultures were unsuccessful. Only in one lymph node containing hyphae was there also evidence of mycobacterial infection (M. aviura).
Irnmunofluorescence Staining An aetiologicai diagnosis was obtained by immunofluorescencc staining of hyphae in 75 cases (Table 4, Fig. 6). Staining with an antibody raised to A. corymbifera produced a strong staining in all 75 cases, whereas no reactivity with antiserum raised against hyphal antigens of Aspergillus spp. was obtained. Thus, the presence of an Aspergillus sp. in addition to a zygomycete in seven cases could not be confirmed immunohistochemically. The suggestion of a Candida sp. in one case was confirmed by the uniform immunofluorescence of fungal elements when stained with anti-C, albicans antiserum (Fig. 6). A diagnosis of zygomycosis was suggested on the basis of hyphal morphology in seven cases, but did not react with any of the antibodies employed.
Discussion The present study confirms that fungi of the zygomycetes often cause mycotic lymphadenitis in cattle (Table 4), predominantly affecting the mesenteric and mediastinal lymph nodes (Table 3). The case with concurrent infection of a
352
H . E . J e n s e n e t at.
Fig. 6.
Mycotic lymphadenitis, a, b, c and d represent the same area in a mesenteric lymph node. (a) By Grocott's methenamlne silver (GMS) staitdng the morphology of hyphae suggested the presence of a zygomyeete (small arrows), an Aspergillus sp. (large arrow) and a Candida sp. (arrow heads). (GMS, x 200). (b) The use ofheterologously absorbed anti-Aspergillusspp, antiserum failed to stain any of the fungal elements. ( x 200). (c) By staining with heterologously absorbed anti-A, corymbifera antiserum hypbae resembling both Aspergilhu spp. and zygomycetes were stained uniformly. ( x 200). (d) Staining with anti-C, albi~ans antiserum confirmed the presence of a Candida sp.
( x 200).
Table 4 I m m u n o l o g i c a l identification of fungi in 82 cases of mycotic lyrnphadenitis in cattle
Lymph nodes
Zygomycetes
Candida sp.
Aspergillus sp.
Inconclusive
Mesenteric Mesenteric and mediastinal Mediastinal Mediastlnal and bronchial Lung*
68 2 3 1 1
1** 0 0 0 0
0 0 0 0 0
6*** 1"** 0 0 0
Total
75
1
0
7
*The exact position not stated. **Dual infection with a Candida sp. and a zygornycete. * * * O n the basis of morpbological criteria a diagnosis of zygomyeosis was suggested.
mesenteric lymph node with a zygomycete and a Candida sp. is the first to link Candida spp. to lymphadenitis in cattle. Mycotic lymphadenitis caused by Aspergillus spp. has been described in cattle (Gilmour and Angus, 1969; Angus, Gilmour and Dawson, 1973), but is notably absent in our study. Mycotic lymphadenitis caused by zygomycetes seems to produce gross macroscopic
Mycotic Lymphadenitis of Cattle
35
alterations, whereas those related to Aspergillus spp. have only been fbund by histological examination. Mycotic abortion in cattle has been found to predominate in the winter season (Krogh, 1985) and to be related to the number of rain-days in June, with an increase during the subsequent 12 months (Hugh-Jones and Austwick, 1967; Krogh, 1985). In the present survey, a correlation could not be demonstrated between the occurrence of mycotic lymphadenitis and either year of study (Table 1) or the season (Table 2). The decrease in total numbers of specimens submitted reflects a steady decline of M. avium infections in cattle probably due to the separation of cattle and poultry breeding in modern farming (J. B. Jorgensen, unpublished observation). It is reasonable to assume that the mycotic infections result from feeding mouldy foodstuffs. Most cattle with mycotic lymphadenitis (> 77 per cent) were beef cattle, and it is common practice to reserve the best feed for breeding heifers and cows, of which only five were represented in the present material. Specifically, the four farmers delivering two or more cattle had given their beef herd mouldy feed. In lymph nodes with only focal mycotic lymphadenitis, the lesions were mainly restricted to the cortical zones, which suggests that fungi reach the mesenteric lymph nodes by the afferent lymph vessels. Furthermore, from the finding of apparently fresh hyphal growth at the periphery of some granulomas, it is likely that mycotic lymphadenitis is a locally progressive disease. As no evidence of dissemination was obtained in any of the 94 cattle, it appears that lymph nodes are capable of containing zygomycosis. Whether it is the conidia or the hyphae which penetrate the intestine to reach the lymph nodes is not known. In most of the reported cases of mycotic lymphadenitis in cattle (Gleiser, 1953; Davis et al., 1955; Werffeli and Biihlmann, 1967; Dion el al., 1987), as in the present survey, intestinal tissue was not available for examination. Gilmour and Angus (1969) and Angus et al. (1979) regarded Peyer's patches to be the site of penetration in at least some of the cases. In one of the cases reported by Pallaske (1967), hyphae were found within the necrotic intestinal mucosa. We have recently found the stomach compartments of cattle to be the target organs for mycotic infections (Jensen and Schonheyder, 1989a; Jensen, Basse and Aalbaek, in press), but examination of several lymph nodes draining affected stomach compartments failed to reveal fungi, whereas hyphae were found regularly in thrombosed vessels. This led us to suggest a haematogenous spread in systemic mycoses in cattle where hyphae are present in damaged stomach tissue. Moreover, it was found that A. fumigalus, the predominant cause of mycotic abortion in cows (Krogh, 1985), had a propensity for infecting the omasum (Jensen and Schonheyder, 1989b; Jensen el al., in press). Fenestrations of the omasal leaves, most likely of mycotic origin, were found in up to 16 per cent of apparently healthy cattle and to predominate in the winter season (Brownlee and Elliot, 1960; Jensen, 1987), coinciding with the peak incidence of mycotic abortion (Krogh, 1985). Our present observations indicate that the intestines of cattle are a less important portal of entry than the lung (Austwick, 1962) and the stomach compartments (Jensen el al., in press)
354
H. E, Jensen e t al.
for fungi that disseminate, and this is particularly true for A. fumigatus infections. I n conclusion, zygomycetes were found to be the main cause ofmacroscopically apparent mycotic lymphadenitis in cattle. The disease seems to be effectively contained locally and to affect mainly beef cattle fed with mouldy foodstuffs. Acknowledgment
This study was supported by the Novo Foundation. References
Angus, K.W., Gilmour, N.J.L. and Dawson, C.O. (1973). Alimentary mycotic lesions in cattle: A histological and cultural study. Journal of Medical Microbiology, 6, 207-213. Austwick, P. K. C. (1962). The presence of Aspergillusfumigatus in the lungs &dairy cows. Laboratory Investigation, 11, 1065-1072. Brownlee, A. and Elliot, J. (1960). Studies on the normal and abnormal structure and function of the omasum of domestic cattle. British VeterinaryJournal, 116, 467-473. Cordes, D. O. and Shortridge, E. H. (1968). Systemic phycomycosis and aspergillosis of cattle. New Zealand Veterinary Journal, 16, 65-80. Davis, C. L., Anderson, W.A. and McCrory, B.R. (1955). Mucormycosis in foodproducing animals. A report of twelve cases. Journal of the American Veterinary Medical Association, 126, 261-267. Dion, W. M., Bundza, A. and Dukes, T. W. (1987). Mycotic lymphadenitis in cattle and swine. Canadian Veterinary Journal, 28, 57-60. Gilmour, N.J.L. and Angus, K.W. (1969). Aspergillusfumigatus in the mesenteric lymph nodes and intestinal mucosa of cattle. British Velerinary Journal, 125, xiiixiv,
Gleiser, C. A. (1953). Mucormycosis in animals. A report of three cases. Journal of the American Veterinary Medical Assaciation, 123~ 441-445. Hugh-Jones, M. E. and Austwick, P. K. C. (1967). Epidemiological studies in bovine mycotic abortion. Veterinary Record, 81, 273-276. Jensen, H. E. (1987). Pathological findings in the omasum of cattle. Summariesfrorn the Scandinavian Association for Veterinary Pathology. Annual meeting and symposium, 1987, Helsingfors, Finland. Suomen Ela~nlgiikiirilehti, 93, 305-310. Jensen, H. E. and Schonheyder, H. (1989a). Immunofluorescence staining of hyphae in the histopathological diagnosis of mycoses in cattle. Journal of Medical and Veterinary Mycology, 27~ 33-44. Jensen, H.E. and Sch~nheyder, H. (1989b). Immunodiagnosis of bovine mycoses. Proceedings of the Vth International Symposium, World Association of Veterinary LaboratoO, Diagnosticians, June 25-30, 1989, Guelph, Ontario, Canada. Jensen, H. E., Basse, A. and Aalb~ek, B. (1989). Mycosis in the stomach compartments of cattle. Acta Veterinaria Scandinavica (in press). Krogh, I--I.V. (1985). Bovine mycotic abortion in Denmark. Nordisk Veterimer Medicin, 37~ 27-33. Luna, L. G. (1968). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd edit. McGraw-Hill, New York. Ossent, P. (1987). Systemic aspergillosis and mucormycosis in 23 eats. VeterinaO, Record, 120~ 330-333. Pallaske, G. (1967). Mykosen beim Rind im Bereiche des Verdauungsapparates (zugleieh ein Beitrag zur Differentialdiagnose der Tuberkulose). Deutsche Tierii~ztfiche Wochenschrift, 74, 27-33.
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Werffeli, F. and B/ihlmann, X. (1967). Uber Tuberkulose~ihnliche Ver~inderungen der Lymphknoten beim Rind, verursacht durch pathogene Schimmelpilze. Schweizer Archivfiir Tierheilkunde, 109, 475--480.
[Receivedfor publication, June 8th, 1989]
I n t e s t i n a l a n d P u l m o n a r y M y c o t i c L y m p h a d e n i t i s in Cattle H~E. Jensen*]l, H. Schonheyder~++ and J. B. Jorgensen* *National Dlerinary Laboratory, Biilowsvej 27, P.O. Box 373, DK-1503 Copenhagen V and ~Institute of Medical Microbiology, University of Aarhus, DK-8000 Aarhus C, Denmark
Summary Among 4877 slaughtered cattle with tuberculosis-like lesions in lymph nodes, 94 cases (1'9 per cent) revealed fungal hyphae on histopathological examination. The survey period was 12 years and most of the affected animals (> 77 per cent) were beef cattle. Affected nodes were mesenteric in 84 cases (89'4 per cent), mediastinal and/or bronchial in seven cases (7"4 per cent), and in three cases (3"2 per cent), both mesenteric and mediastinal nodes were affected. The incidence of mycotic lymphadenitis was unrelated to year of study or season. Eighty-two of the cases were re-examined histologically and immunohistochemically. All lesions were granulomatous in nature and, in 26 cases, eosinophilic asteroid bodies (rosette formation) around hyphae were found. In 75 cases, immunofluorescence staining identified the agent as a zygomycete, probably Absidia cor~rnbifera and, in one case, there was a concurrent infection with a Candida species. In seven cases that did not react with the antibodies employed, a diagnosis ofzygomycosis was suggested on the basis of hyphal morphology. Hyphae of Aspergillus spp. were not found in any of the lesions. A concurrent fungaI and mycobacterial infection (M. avium) was diagnosed in one case. Thus, zygomycetes are the main cause of macroscopically apparent mycotic lymphadenitis, a sporadic disease most probably caused by feeding with mouldy food stuffs.
Introduction I n Danish cattle, lesions resembling tuberculosis, when [bund d u r i n g any kind o f p o s t - m o r t e m e x a m i n a t i o n but n o r m a l l y at meat inspection, are r e q u i r e d to be submitted to the National Veterinary L a b o r a t o r y for c o n f i r m a t o r y diagnosis. As a result of this practice, a n u m b e r of mycotic lesions are r e c o r d e d every year. Cases of mycotic lymphadenitis in both cattle and pigs are often found on suspicion of" tuberculosis although, especially in pigs, m y c o t i c lymphadenitis m a y be mistaken tbr l y m p h o s a r c o m a (Dion, B u n d z a a n d Dukes, 1987). T h e aetiological diagnosis of mycoses is usually p e r f o r m e d on the basis o f h y p h a l m o r p h o l o g y in tissue sections, which is considered to be difficult (Pallaske, 1967; Cordes a n d Shortridge, 1968; Ossent, 1987; J e n s e n a n d Present addresses: HDepartmentof Veterinary Pathology,Royal Veterinal3"and Agricuhural University, B/ilowsvej 13, DK-1870 FrederlksbergC, Denmark and ;Department of Clinical Microbiology,Aalborg Hospital, DK-9100Aalborg, Denmark. 0021-9975/90/030345+ 11 $03.00/0
© 1990 AcademicPress Limited
346
H.E. Jensen et al.
Schonheyder, 1989a). Isolation of fungi from animal tissue is difficult because of post-mortem changes (Dion et al., 1987). Moreover, since mammalian tissues generally are contaminated by fungi (Dion et al., 1987) and since fungi are c o m m o n laboratory contaminants (Cordes and Shortridge, 1968), it is difficult to j u d g e whether an isolate is also the pathogen (Cordes and Shortridge, 1968; Dion et al., 1987). Since the first report of mycotic lymphadenitis in cattle (Gleiser, 1953), one major study (Dion et al., 1987) and a number of case reports (Davis, Anderson a n d McCrory, 1955; Pallaske, 1967; Werffeli and Biihlmann, 1967) have been published. A comprehensive survey including all specimens obtained during a period of years is not accessible. This paper presents 94 cases of mycotic lymphadenitis diagnosed in cattle which had initially been suspected of having tuberculosis, during a period of 12 .,years (1977-1988). T h e aetiological diagnosis was, in most cases, accomplished by immunofluorescence staining of h y p h a e in the tissue sections.
M a t e r i a l s and M e t h o d s
Spe£ill'zens From 1977 to 1988, lymph nodes or parts of lymph nodes from 4877 cattle were submitted to the National Veterinary Laboratory on suspicion of tuberculosis.
Laboratory Examinations The ~bllowing laboratory examinations were performed on lymph nodes with lesions resembling tuberculosis: (1) microscopy of Ziehl-Neelsen-stained smears; (2) inoculation onto several modifications of L6wenstein-Jensen medium for culture of especially Mycobaclerium boris, M. avium complex, and M. paraluberculosis; (3) histopathology of lesions negative for acid-last bacteria/rods by microscopy of smears; (4) mycological culture on Sabouraud's glucose agar, pH 5.7 (incubated at 37°C for 7 days) in three selected cases. Thc diagnosis of mycotic lymphadenitis was made exclusively on the basis of histological demonstration of hyphae in tissue sections. From cases with paraffin wax-embedded tissue blocks stored at the laboratory (n=82), tissue sections (5-gm thick) were cut and re-examined. The tissue sections were stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS), Grocott's methenamine silver (GMS), Van Kossa's stain for calcium and Luna's stain for eosinophils (Luna, 1968). Imrnunojtuorescence Staining The aetiological diagnosis was accomplished by immunofluorescence staining of hyphae in tissue sections with hyperimmune rabbit antisera absorbed with heterologous fungal antigens before use, as described in detail elsewhere (Jensen and Schonheyder, 1989a). In the present study, the panel of antisera was extended by the addition ofanti-Candida albicans antiserum (B 143, Dakopatts, Denmark), diluted 1 in 256 (the plateau end-point) and used without prior absorption. Mycotic lesions in cattle were examined in parallel with sections of routine tissues experimentally infected with Absidia corymbi/'era, AspeNillus spp. and Candida albicans as described elsewhere (Jensen and Schonheyder, 1989a). The immunofluorescence staining system discriminated between zygomycetes (probably Absidia corymblfera), AspergiUus spp. and Candida spp.
347
Mycotic L y m p h a d e n i t i s o f C a t t l e Statistics
I n f o r m a t i o n on n u m b e r s of s l a u g h t e r e d c a t t l e i n D e n m a r k w a s o b t a i n e d f r o m t h e D a n i s h V e t e r i n a r y S e r v i c e a n d the D a n i s h L i v e S t o c k a n d M e a t B o a r d . I n cases w h e r e a farmer had deIivercd more than one animal with mycotic lymphadenitis, he was i n t e r v i e w e d to e l u c i d a t e t h e f e e d i n g r e g i m e o f his cattle.
Results Statistics Lymph nodes from 94 (1.9 per cent) of 4877 cattle examined were found to contain hyphae (Tables 1 and 2). Four cattle had lesions in two nodes. There was a notable increase in the rate of mycotic lymphadenitis among clinical cases studied, but the rate of mycotic lymphadenitis among slaughtered cattle did not change signifcantly over the years. Likewise, no consistent pattern with respect to the month of diagnosis was observed (Table 2). L y m p h nodes with mycotic lesions were predominantly mesenteric, the anatomical distribution being given in Table 3. Most of the affected cattle (n= 73) were less than 2 years old, some were mature (n= 5) and, for some, no age was stated (n = 16). The sex was stated for only 23 animals (five females and 18 males). However, from the agedistribution it appears that most of the cattle were bull calves which normally are slaughtered for beef production at 1 to 1.5 years old. O f the 94 cattle, nine had been bred by one farmer, while three farmers had
Table 1 Yearly distribution of cattle slaughtered in Denmark, number of cases e x a m i n e d on suspicion of tuberculosis, and cases of mycotic lymphadenitis during a 12-year period (1977-1988) Mycotic lymphadenitis
Year
Cattle slaughtered in .Denmark ( x 100 000)
Total number of cases examined
1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988
11.18 10"53 11'13 10.70 10'44 10'07 10.41 10-79 10,19 10'00 9"49 8'73
Total
123.66
*Mean.
No.
Per cent of cases examined
Per 100 000 cattle slaughtered
844 927 745 572 460 354 278 236 203 122 71 65
fi 10 12 9 5 6 5 6 11 6 8 10
0'7 1'1 1.6 1'6 I' 1 1'7 1"8 2'5 5'4 4-9 11 '3 15.4
0'54 0'95 1"08 0,84 0'48 0'60 0.48 0'56 1'08 0"60 0'84 1.15
4877
94
4.1 *
0,77*
348
H . E . J e n s e n e t al.
Table 2 C u m u l a t i v e m o n t h l y d i s t r i b u t i o n of cattle s l a u g h t e r e d in Denmark, n u m b e r of cases e x a m i n e d on s u s p i c i o n of tuberculosis and cases of mycotic l y m p h a d e n i t i s d u r i n g a 12-year period (1977-1988) Mycotw lymphadenitis
Month
Cattle slaughtered in Denmark ( x 100 000)
Total number of cases examined
11.43 9.76 10.55 9.59 10,46 10.70 7"73 10'69 10"74 1 l'62 11'37 10"02 123'66
1 2 3 4 5 6 7 8 9 I0 11 12 Total
No.
Per cent qf cafes eaammed
Per 100 000 cattle daughto ed
399 368 417 438 477 499 332 397 404 429 359 358
5 7 7 6 10 11 6 3 8 10 1f 10
1.3 1.9 1.7 1.4 2' 1 2'2 1'8 0"8 2'0 2'3 3" 1 2'8
0-44 0"72 0"66 0-63 0.96 1"03 0"78 0"28 0-74 0'86 0-97 1' 11
4877
94
2'0*
0.77*
*Mean.
Table 3 Anatomical distribution of mycotic l y m p h a d e n i t i s in 94 cattle examined during a period of 12 years (1977-1988)
Lymph nodes
Number
Pe~ cent
Mesenteric Mesenteric and mediastinal Mediastinal Bronchial Mediastinal and bronchial Lung*
84 3 3 2 I 1
89'4 3'2
Total
94
~ ~ [ ]
7"4
100.0
Lymph nodes evaluated by immunofiuore.~cence ~taming (No.) 74 3 3 0 1 1 82
*The exact position not stated.
raised two cattle each. The farmer with nine affected beef cattle frequently tkd mouldy waste bread from a bakery as a supplementary feed. The three other farmers reported that their beef cattle occasionally had been fed with hay and silage contaminated by moulds. Gross Pathology
The macroscopic appearance of lymph nodes conformed in most cases with the descriptions by Davis et al. (1955) and Dion et al. 11987), enlarged, firm
Mycotic Lymphadenitis
of Cattle
349
greyish-white or yellow, granular, caseocalcareous, with a variable degree of scarring. T h e granulomas occupied the entire lymph node in most cases (Fig. 1), while occasionally the processes appeared as encapsulated lesions inside the affected lymph nodes (Fig. 2). Hislopalhology All lesions were fbund to be granulomatous, and the picture was generally similar to that described by Davis et al. (1955). In most of the nodes, lesions consisted of masses of necrotic tissue containing scattered loci of calcification. T h e necrotic masses were intermingled with areas containing inflammatory cells, including lymphocytes, plasma cells, epithelioid cells and giant cells (Fig. 3). Between the more or less well-marked granulomas, a variable amount of fibrous tissue was seen. In some of the granulomas, small satellite foci ofhyphal g r o w t h were noted in the periphery underneath the fibrous capsule (Fig. 4). In these loci, the cellular infiltrate contained both neutrophils and eosinophils. W h e n granulomas only occupied a part of the lymph nodes, they were mainly restricted to the cortical zone and were separated fi'om the normal lymphatic tissue by a thick zone of fibrous tissue. Eosinophils were found in almost all lesions but, in 29 cases (28 per cent), they were present in large numbers. The eosinophils were located in all parts of the granulomas, but predominantly centrally. H y p h a e were regularly seen in the necrotic areas accompanied by giant cells, but they were, in some cases, situated also in the connective tissue between the granulomas.
Fig. 1. A granulomatous mycotic lesion occupying an entire mesenteric lymph node. x 1-5,
350
H . E . J e n s e n e t al.
Fig. 2.
A calcified, granulomatous, mycotic lesion appears to be encapsulated within a mesenteric lymph node. x 1.5.
Fig. 3.
Mycotic lymphadenitis. Hyphae in giant cells (large arrows) arid intermingling with the granulomatous tissue (small arrows). HE x 130.
Fig, 4.
Mycotic lymphadenitis, A satellite focus (inset) with hyphal growth and infiltration of granulocytes is located at the periphery of a granuloma (arrows) close to the fibrous capsule (C). Normal lymphatic tissue (N). PAS × 53; inset: x 130.
M y c o t i c L y m p h a d e n i t i s of Cattle
351
Fig. 5. Mycotic lymphadenitis. Asteroid bodies are covering the hyphae (arrows) in a mycotic granuloma. HE x 130,
The morphology of fungal elements in all cases suggested the presence of a zygomycete but, in six cases, the presence of an Aspergillus species was also suspected. In one case, a zygomycete, an AspergiIlussp. plus a Candida sp., were suspected (Fig. 6). In 26 cases, eosinophilic asteroid bodies (rosette formation) covered most of the hyphae (Fig. 5). In two cases, asteroid bodies were observed both in the mediastinal and in the mesenteric nodes.
Microbiology In one case, mycological culture yielded growth of A. corymbifera from a mesenteric lymph node whereas, in two other cases, mycological cultures were unsuccessful. Only in one lymph node containing hyphae was there also evidence of mycobacterial infection (M. aviura).
Irnmunofluorescence Staining An aetiologicai diagnosis was obtained by immunofluorescencc staining of hyphae in 75 cases (Table 4, Fig. 6). Staining with an antibody raised to A. corymbifera produced a strong staining in all 75 cases, whereas no reactivity with antiserum raised against hyphal antigens of Aspergillus spp. was obtained. Thus, the presence of an Aspergillus sp. in addition to a zygomycete in seven cases could not be confirmed immunohistochemically. The suggestion of a Candida sp. in one case was confirmed by the uniform immunofluorescence of fungal elements when stained with anti-C, albicans antiserum (Fig. 6). A diagnosis of zygomycosis was suggested on the basis of hyphal morphology in seven cases, but did not react with any of the antibodies employed.
Discussion The present study confirms that fungi of the zygomycetes often cause mycotic lymphadenitis in cattle (Table 4), predominantly affecting the mesenteric and mediastinal lymph nodes (Table 3). The case with concurrent infection of a
352
H . E . J e n s e n e t at.
Fig. 6.
Mycotic lymphadenitis, a, b, c and d represent the same area in a mesenteric lymph node. (a) By Grocott's methenamlne silver (GMS) staitdng the morphology of hyphae suggested the presence of a zygomyeete (small arrows), an Aspergillus sp. (large arrow) and a Candida sp. (arrow heads). (GMS, x 200). (b) The use ofheterologously absorbed anti-Aspergillusspp, antiserum failed to stain any of the fungal elements. ( x 200). (c) By staining with heterologously absorbed anti-A, corymbifera antiserum hypbae resembling both Aspergilhu spp. and zygomycetes were stained uniformly. ( x 200). (d) Staining with anti-C, albi~ans antiserum confirmed the presence of a Candida sp.
( x 200).
Table 4 I m m u n o l o g i c a l identification of fungi in 82 cases of mycotic lyrnphadenitis in cattle
Lymph nodes
Zygomycetes
Candida sp.
Aspergillus sp.
Inconclusive
Mesenteric Mesenteric and mediastinal Mediastinal Mediastlnal and bronchial Lung*
68 2 3 1 1
1** 0 0 0 0
0 0 0 0 0
6*** 1"** 0 0 0
Total
75
1
0
7
*The exact position not stated. **Dual infection with a Candida sp. and a zygornycete. * * * O n the basis of morpbological criteria a diagnosis of zygomyeosis was suggested.
mesenteric lymph node with a zygomycete and a Candida sp. is the first to link Candida spp. to lymphadenitis in cattle. Mycotic lymphadenitis caused by Aspergillus spp. has been described in cattle (Gilmour and Angus, 1969; Angus, Gilmour and Dawson, 1973), but is notably absent in our study. Mycotic lymphadenitis caused by zygomycetes seems to produce gross macroscopic
Mycotic Lymphadenitis of Cattle
35
alterations, whereas those related to Aspergillus spp. have only been fbund by histological examination. Mycotic abortion in cattle has been found to predominate in the winter season (Krogh, 1985) and to be related to the number of rain-days in June, with an increase during the subsequent 12 months (Hugh-Jones and Austwick, 1967; Krogh, 1985). In the present survey, a correlation could not be demonstrated between the occurrence of mycotic lymphadenitis and either year of study (Table 1) or the season (Table 2). The decrease in total numbers of specimens submitted reflects a steady decline of M. avium infections in cattle probably due to the separation of cattle and poultry breeding in modern farming (J. B. Jorgensen, unpublished observation). It is reasonable to assume that the mycotic infections result from feeding mouldy foodstuffs. Most cattle with mycotic lymphadenitis (> 77 per cent) were beef cattle, and it is common practice to reserve the best feed for breeding heifers and cows, of which only five were represented in the present material. Specifically, the four farmers delivering two or more cattle had given their beef herd mouldy feed. In lymph nodes with only focal mycotic lymphadenitis, the lesions were mainly restricted to the cortical zones, which suggests that fungi reach the mesenteric lymph nodes by the afferent lymph vessels. Furthermore, from the finding of apparently fresh hyphal growth at the periphery of some granulomas, it is likely that mycotic lymphadenitis is a locally progressive disease. As no evidence of dissemination was obtained in any of the 94 cattle, it appears that lymph nodes are capable of containing zygomycosis. Whether it is the conidia or the hyphae which penetrate the intestine to reach the lymph nodes is not known. In most of the reported cases of mycotic lymphadenitis in cattle (Gleiser, 1953; Davis et al., 1955; Werffeli and Biihlmann, 1967; Dion el al., 1987), as in the present survey, intestinal tissue was not available for examination. Gilmour and Angus (1969) and Angus et al. (1979) regarded Peyer's patches to be the site of penetration in at least some of the cases. In one of the cases reported by Pallaske (1967), hyphae were found within the necrotic intestinal mucosa. We have recently found the stomach compartments of cattle to be the target organs for mycotic infections (Jensen and Schonheyder, 1989a; Jensen, Basse and Aalbaek, in press), but examination of several lymph nodes draining affected stomach compartments failed to reveal fungi, whereas hyphae were found regularly in thrombosed vessels. This led us to suggest a haematogenous spread in systemic mycoses in cattle where hyphae are present in damaged stomach tissue. Moreover, it was found that A. fumigalus, the predominant cause of mycotic abortion in cows (Krogh, 1985), had a propensity for infecting the omasum (Jensen and Schonheyder, 1989b; Jensen el al., in press). Fenestrations of the omasal leaves, most likely of mycotic origin, were found in up to 16 per cent of apparently healthy cattle and to predominate in the winter season (Brownlee and Elliot, 1960; Jensen, 1987), coinciding with the peak incidence of mycotic abortion (Krogh, 1985). Our present observations indicate that the intestines of cattle are a less important portal of entry than the lung (Austwick, 1962) and the stomach compartments (Jensen el al., in press)
354
H. E, Jensen e t al.
for fungi that disseminate, and this is particularly true for A. fumigatus infections. I n conclusion, zygomycetes were found to be the main cause ofmacroscopically apparent mycotic lymphadenitis in cattle. The disease seems to be effectively contained locally and to affect mainly beef cattle fed with mouldy foodstuffs. Acknowledgment
This study was supported by the Novo Foundation. References
Angus, K.W., Gilmour, N.J.L. and Dawson, C.O. (1973). Alimentary mycotic lesions in cattle: A histological and cultural study. Journal of Medical Microbiology, 6, 207-213. Austwick, P. K. C. (1962). The presence of Aspergillusfumigatus in the lungs &dairy cows. Laboratory Investigation, 11, 1065-1072. Brownlee, A. and Elliot, J. (1960). Studies on the normal and abnormal structure and function of the omasum of domestic cattle. British VeterinaryJournal, 116, 467-473. Cordes, D. O. and Shortridge, E. H. (1968). Systemic phycomycosis and aspergillosis of cattle. New Zealand Veterinary Journal, 16, 65-80. Davis, C. L., Anderson, W.A. and McCrory, B.R. (1955). Mucormycosis in foodproducing animals. A report of twelve cases. Journal of the American Veterinary Medical Association, 126, 261-267. Dion, W. M., Bundza, A. and Dukes, T. W. (1987). Mycotic lymphadenitis in cattle and swine. Canadian Veterinary Journal, 28, 57-60. Gilmour, N.J.L. and Angus, K.W. (1969). Aspergillusfumigatus in the mesenteric lymph nodes and intestinal mucosa of cattle. British Velerinary Journal, 125, xiiixiv,
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Werffeli, F. and B/ihlmann, X. (1967). Uber Tuberkulose~ihnliche Ver~inderungen der Lymphknoten beim Rind, verursacht durch pathogene Schimmelpilze. Schweizer Archivfiir Tierheilkunde, 109, 475--480.
[Receivedfor publication, June 8th, 1989]