Route of infections in bovine aspergillosis

Journalof Medical& VeterinaryMycology 1996, 34, 379-383 Accepted I I May 1996 Route of infections in bovine aspergillosis J. S A R F A T I , H. E. J E N S E N * & J. P. LATGI~ Laboratoire des Aspergillus, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France; and *Laboratory of Veterinary Pathology, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Copenhagen, Denmark Keywords As'pergillosis, Aspergillus Jumigatus, molecular epidemiology, zetrotransposon Introduction Placentitis and abortion are common features associated with bovine aspergillosis and can account for up to 20% of bovine abortions. Because of the important loss in the production of cattle, efforts have recently been undertaken to obtain an efficient diagnosis of this disease [1-3] as well as to understand the portal of entry of this fungus [4]. In cows, the gastrointestinal tract and almost exclusively the omasum is the primary site of mycotic lesions caused by A. fumigatus, which is the main causative agent of bovine aspergillosis [1]. Experimental infections in pregnant cows and mice as well as histopathological examination of tissue from spontaneously infected cattle suggest that placentitis and pneumonia are secondary infections resulting from haematogenous spread of the fungus from primary gastrointestinal lesions [5 7]. However, a final demonstration of the Aspergillus portal of entry in the cow still remains to be made. Moreover, the ease with which A. Jumigatus can be frequently and repetitively isolated from various organs of healthy cattle [4] makes the search for the source of the infectious inoculum difficult. Tracing the route(s) of infection in bovine aspergillosis requires the use of a typing system which can fingerprint accurately the strains of A. fumigatus isolated from the mycotic lesions of the different infected organs. This paper presents an attempt to identify the portal of entry of A. fumigatus in cows, using a typing system which has Correspondence: Dr J. P. Latg6. (c) 1996 ISHAM shown its efficacy in the epidemiological study of various human aspergillosis syndromes [8-10]. Material and methods The origin of the isolates studied is presented in Table 1. All strains were isolated from cattle examined at the Laboratory of Veterinary Pathology (Copenhagen, Denmark). All aspergillosis cases were spontaneous. Due to necrotizing mastitis, cow 373 had been treated intensively for 6 days with broad-spectrum antibiotics (tetracycline and sulfadiazine with trimethoprim (5:1)) and the anti-inflammatory drug flunixin meglumine. Other cows with aspergillosis lesions, except those with placentitis, had also received an intensive antibacterial treatment because of concomitant bacterial infections. In all animals with aspergillosis (Table 1), the diagnosis was confirmed histopathologically by the demonstration of hyphae in association with an acute or subacute inflammatory response [7]. Lesions within the gastrointestinal tract and mediastinal lymph node of the cow with systemic disseminated aspergillosis (no. 373) were typically subacute, i.e. infiltrating cells were mainly of the mononuclear type. The remaining lesions in animals with aspergillosis, including the disseminated pulmonary lesions of cow no. 373, were typically acute, i.e. dominated by necrosis, thrombosis, vasculitis, and infiltrated with neutrophils. Within lesions, hyphae of Aspergillus sp. were identified immunologically by the application of specific monoclonal antibodies in immunohistochemical assays [1]. Repeated DNA sequences were used to fingerprint strains of Aspergillus fumigatus isolated from a cow with disseminated systemic aspergillosis, cows with single aspergillosis lesions, calves aborted due to bovine aspergillosis, mothers of those calves, and cattle without aspergillosis. The analysis of the Southern blot hybridization patterns obtained suggested that: (i) the portal of entry of aspergillosis in cattle is the gastrointestinal tract, and (ii) infection of aborted calves is due to maternally derived strains. Cattle from the same farm slaughtered on the same day harbour the same strain, suggesting a contamination from feed material. Sarfati et al. Table I Origin and genotypes of strains of A, fumigatus isolated from cattle with or without Aspergillus infection Animal Organ Year of Strain culture number With aspergillosis 373 Abomasum 1989 Peyer's patches Omasum Mediastinal lymphnode Lung Rumen Genotype 373-89-1 373-89-2 373-89-3 373-89-4 373-89-5 373-89-6 A B C D A E 1990 260-90-I 260-90-2 F F 434 Placenta Abomasum of the calf 1990 434-90-I 434-90-2 G H 8075 Lung 1992 8075-92 I 7622 Liver 1992 7622-92 J 185 Placenta 1983 185-83 K 890 Placenta 1984 890-89 L 28 Placenta 1985 28-85 M 113 Placenta 1986 113-86 N 1989 1344-89-1 1344-89-2 O O 1349 Lung Peyer's patches 1989 1349-89-1 1349-89-2 P P 1351 Lung Peyer's patches 1989 1351-89-1 1351-89-2 O O 1353 Lung Peyer's patches 1989 1353-89-1 1353-89-2 O O 1355 Lung Peyer's patches 1989 1355-89-1 1355-89-2 O O Results and discussion Without aspergillosis 1344 Lung Peyer's patches Isolation of A. fumigatus from organs of animals with aspergillosis was carried out as described recently [4]. The A. fumigatus strains obtained from tissues of cattle without aspergillosis (Table 1) were grown from organs of normal slaughterhouse cattle. The absence of lesions in these animals was confirmed histopathologically and immunohistochemically [1]. Culture of isolate, DNA extraction and EcoRI digestion, were carried out as previously described [8-10]. Electrophoresis and Southern blots were performed using Preliminary experiments have confirmed previous studies [9,10] showing that single conidium isolates from every original slant gave identical Southern blot patterns (data not shown). With the exception of some specific situations discussed later, isolates from different cattle displayed different Southern blot patterns without any possible clustering (Figs 1 3). The only cow with systemic aspergillosis yielded six isolates, data from which are presented in Fig. 1. The strain isolated from the lung was similar to that isolated from the abomasum. This result strongly suggests that the lung infections developed secondarily following haematogenous spread of A. jumigatus from the gastrointestinal tract. This is also in agreement with the differences in types of inflammation in the abomasum and the lungs. The disseminated localization of the acute necrohaemorrhagic lesions (a result of haematogenous spread) in the lungs and the histopathological demonstration of hyphal outgrowth from pulmonary vessels are also factors strongly supporting a haematogenous route of spread (data not shown). In addition, in an examination of over 100 cows at the veterinary clinic, primary lung infection has not been observed. Disseminated lung infections are only observed after experimental intravenous inoc (...truncated)