J. Comp. Path. 2011, Vol. 145, 1e5
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Brucellosis of the European Brown Hare (Lepus europaeus) M. Gyuranecz*, K. Erd elyi†, L. Makrai*, L. Fodor*, B. Sz epe‡, R an†, L. Dencs} o†, E. Fassang* and L. Szeredi† A. aczn e Mesz aros†, A. D *Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent Istv an University, Central Agriculture Office Veterinary Diagnostic Directorate, Budapest and ‡ Medo kft., Tiszan ana, Hungary
Summary The European brown hare (Lepus europaeus) is an important reservoir of Brucella suis biovar 2 and also of the lifethreatening zoonotic agent Francisella tularensis. Since both bacteria can produce similar gross pathological lesions in this species, laboratory tests are necessary for the final diagnosis. The aim of the present study was to develop an immunohistochemical method for the detection of B. suis infection and to describe the pathological and histological lesions caused by B. suis in European brown hares. Hyperimmune serum for immunohistochemistry (IHC) was produced by subcutaneous infection of mice with 2 109 colony forming units of live B. suis biovar 2, injected four times at 1-week intervals. The antiserum did not react with F. tularensis or Yersinia pseudotuberculosis in IHC and displayed only weak cross-reaction with B. canis. Numerous, yellowewhite necrotic foci (0.1e0.5 cm diameter) were found in the spleen of five B. suis-infected female European brown hares and also in the lung, uterus, kidney or liver of four of these cases. Microscopically, the foci comprised single or coalescing granulomas with a central necrotic area. Both bacterial isolation and IHC gave positive results for B. suis infection in these animals. B. suis antigens were found as granular or amorphous extracellular material in the necrotic centre of several granulomas. IHC appears to be a suitable complementary diagnostic method for the detection of B. suis infection in the European brown hare. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: Brucella suis; European brown hare; immunohistochemistry
Brucellosis is a zoonotic disease. Domestic or wild animal reservoirs are always the source of human infection (Godfroid et al., 2005). The European brown hare (Lepus europaeus) is one of the most significant European game species and is an important reservoir of Brucella suis biovar 2 (Godfroid et al., 2005). Hares can maintain the bacterium and infect domestic animals (grazing pigs and cows) even in the absence of a wild boar population (Godfroid et al., 2005). In addition to brucellosis, the European brown hare plays a significant role in the ecology of tularaemia, the life-threatening zoonotic disease caused by Francisella tularensis (Gyuranecz et al., 2010). Since these two bacteria share common antigens, which can cross-react in serological tests and produce similar gross pathologi-
Correspondence to: M. Gyuranecz (e-mail: [email protected]
). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2010.11.013
cal lesions in infected organs, additional laboratory tests are necessary to distinguish these two zoonotic diseases (Sterba, 1983; Gyuranecz et al., 2010). Several immunohistochemical tests have been developed for the detection of B. abortus antigens in tissues; however, no report has been published describing the detection of B. suis by immunohistochemistry (IHC) (Meador et al., 1986; Perez et al., 1998; Xavier et al., 2009). The aim of the present study was to develop an immunohistochemical test for the detection of B. suis infection and to describe the gross and microscopical lesions caused by B. suis in European brown hares. Five-hundred and ten European brown hares, shot at different locations in Hungary during several hunting events over three winter hunting seasons (2007e2008, 2008e2009 and 2009e2010), were screened by the Rose-Bengal test (Bioveta Inc., Ó 2010 Elsevier Ltd. All rights reserved.
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Ivanovice na Hane, Czech Republic) using stained bacteria and whole blood (Sterba and Halackova, 1987). Seropositive animals were used for laboratory examinations. A F. tularensis-specific slide agglutination test using stained bacteria (Bioveta Inc.) and whole blood was also performed on the Brucella seropositive hares. Carcasses were subject to necropsy examination under appropriate biosafety conditions on the day of the hunting events. The body condition of each animal was estimated using a simplified, categorical (good, moderate or poor) version of the kidney/fat index (Pintur et al., 2006). Tissue samples (heart, lung, liver, spleen, lymph nodes, kidney, small and large intestine, ovary and uterus) were collected for microscopical examination and fixed in 10% neutral buffered formalin. Sections (4 mm) of formalinfixed and paraffin wax-embedded tissue samples were stained with haematoxylin and eosin (HE) and examined by light microscopy. Hyperimmune serum was produced in mice (Naval Medical Research Institute [NMRI] mouse, approximately 20 g) by subcutaneous infection of two animals once weekly for 4 weeks each with 2 109 colony forming units (cfu) of live B. suis biovar 2 isolated from one of the hares in accordance with all applicable institutional and national guidelines. After dewaxing, the sections were incubated in 3% H2O2 solution for 10 min and then in a 2% solution of skimmed milk powder for 20 min. The sections were incubated overnight at 4 C with the mouse hyperimmune serum. Antibody binding was detected by a horseradish peroxidase-labelled polymer (EnVisionÔ + Kit; Dako, Glostrup, Denmark). In order to find the optimal dilution of the hyperimmune serum, tenfold serial dilutions (from 1 in 20 to 200,000) were tested on tissue sections prepared from the organs of a fetal pig from a sow with B. suis biotype 1-induced abortion. The optimal dilution was 1 in 20,000. The pig tissues were used as a positive control and a serial section incubated with phosphate buffered saline (PBS) in place of antiserum was used as a negative
control. The occurrence of F. tularensis was examined on serial tissue sections as described previously (Gyuranecz et al., 2010). The B. suisspecific immunohistochemical method was also performed on tissue sections from animals with known B. canis, F. tularensis and Yersinia pseudotuberculosis infections in order to exclude cross-reaction. The specificity and sensitivity of the IHC assay were determined as described previously (Moore et al., 1988). Using aseptic precautions, foci in spleens were excised and approximately 1 g of each sample was homogenized, inoculated onto blood agar and incubated at 37 C in 5% CO2 for 5 days. Growth, morphological and biochemical characteristics of the isolated strains were examined using standard methods (Barrow and Feltham, 1993). The utilization of 95 different carbon sources by the isolated strains was examined using a commercial test (MicroLogÔ MicroStationÔ System, GN2 Microplates; Biolog Inc., Hayward, California, USA). Single nucleotide polymorphism (SNP) typing was used for the molecular characterization of the strains as described previously (Fretin et al., 2008). F. tularensis isolation was attempted as described earlier (Gyuranecz et al., 2010). All procedures were performed under biosafety level 3 (BSL-3) conditions. A total of five animals were found to be infected with Brucella by serology and submitted for a detailed necropsy examination (Table 1). The most significant gross lesion was the presence of numerous yellowe white necrotic foci in the spleen (Fig. 1). Additionally a variable number of necrotic foci were found in the lung, kidney, uterus (Fig. 2) and liver of a few cases (Table 1). The foci (0.1e0.5 cm diameter) were well demarcated, spherical, often protruded onto the serosal surface and had a yellowewhite, caseous or dry central content. Microscopically, these foci were found to comprise single large or several small coalescing granulomas. Several small granulomas were observed only microscopically (Fig. 1). The
Table 1 Summary of the body condition, gross, microscopical, immunohistochemical and bacteriological examinations in Brucella-seropositive European brown hares Case number
Granulomas on gross examination
Spleen, lung, uterus, kidney Spleen, lung
3 4 5
Moderate Poor Poor
Spleen, lung Spleen Spleen, liver
Granulomas on microscopical examination Spleen, lung, uterus, kidney, lymph nodes Spleen, lung, lymph nodes Spleen, lung Spleen Spleen, liver
Isolation of B. suis
Spleen, lung, uterus, kidney, lymph nodes Spleen, lung
Spleen, lung Spleen Spleen, liver
Positive Positive Positive
Brucellosis of the European Brown Hare
Fig. 1. Spleen; case 2. Numerous yellowewhite necrotic foci deform the shape of spleen, which is significantly enlarged. Inset: Spleen; case 1. A small granuloma showing amorphous eosinophilic necrotic material and basophilic debris with some heterophils in the centre. This area is encircled by a wide zone of epithelioid cells, containing a small number of heterophils, while the outermost zone consists of several epithelioid cells, large numbers of heterophils, moderate numbers of lymphocytes and a few fibrocytes. HE. Bar, 150 mm.
normal tissue architecture was completely replaced by granulomas in the spleen. In case 1, the endometrium was multifocally eroded by granulomas (Fig. 2). Amorphous eosinophilic material and/or ba-
sophilic debris and a few heterophils were observed in the centre of the granulomas, which were encircled by a wide zone of epithelioid cells, containing a small number of heterophils and occasional Langhans-type
Fig. 2. Uterus; case 1. Yellowewhite necrotic foci in the uterine wall, which occasionally protrude onto the serosal surface (arrows). Inset: Uterus; case 1. The endometrium is eroded by a granuloma in the uterine wall (asterisk). The necrotic centre is absent from this section. HE. Bar, 250 mm.
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Fig. 3. Spleen; case 3. Moderate amounts of B. suis antigen, present in the form of amorphous labelling within the necrotic centre of a large granuloma. IHC. Bar, 400 mm. Inset: Spleen; case 4. Langhans-type giant cell (arrow) surrounded by epithelioid cells and few lymphocytes. HE. Bar, 100 mm.
giant cells (Fig. 3). The outermost zone consisted of epithelioid cells, large numbers of heterophils, moderate numbers of lymphocytes and a few fibrocytes (Fig. 1). Immunolabelling for B. suis was observed in all seropositive hares examined. Low to moderate amounts of B. suis antigen were found in the form of granular or amorphous extracellular material within the central necrotic area of the granulomas (Fig. 3). Immunolabelling was absent in several granulomas. Additionally, B. suis antigen was found multifocally in the cytoplasm of a few macrophages located in the spleen, in the myometrium of the uterus and in the lymph nodes of case 1. All five animals were seropositive in the F. tularensis slide agglutination assay, but tissue samples were negative when tested by F. tularensis-specific IHC. The B. suis-specific mouse hyperimmune serum produced strong intra- and extracellular labelling in samples from a case of swine abortion caused by B. suis biotype 1. Very weak labelling was found in sections from a case of B. canis infection and no labelling was evident in PBS-treated sections (negative control) and in F. tularensis- or Y. pseudotuberculosispositive tissues. IHC showed 100% specificity and sensitivity when compared with bacterial culture. Bacteria growing in greyewhite colonies (1 mm diameter) were isolated in pure culture from all five cases after 4 days of incubation. Isolated bacteria were gramnegative, less than 1 mm long, non-motile rods. They stained with the K€oster method and exhibited both
oxidase and catalase activity. The strains were identified as B. suis by the carbon-source utilization test and further characterized as B. suis biovar 2 by SNP typing. F. tularensis was not isolated from any of the hares. Previous reports of the gross and microscopical lesions in B. suis-infected European brown hares suggested that the most prominent changes occurred in the reproductive organs and liver (Vıtovec et al., 1976; Sterba, 1982; Sterba, 1984). These lesions were similar to those described in the present study, but the most severely affected organ in the current study was the spleen. The microscopical lesion in these animals was a granuloma with central necrosis, but as several infections may produce similar lesions in the European brown hare, additional laboratory tests were necessary for the final diagnosis of brucellosis (Sterba, 1983). IHC has been shown to be a useful and sensitive method for the detection of Brucella infection in domestic and wild animals (Meador et al., 1986; Perez et al., 1998; Xavier et al., 2009; Gonzalez-Barrientos et al., 2010). The advantage of IHC is that the infectivity of tissue samples is eliminated by formalin fixation, while it also enables examination of the causative agent in the context of the tissue lesions. There is only a single commercially available Brucella antiserum, which can detect B. abortus in formalinfixed and paraffin wax-embedded tissue sections in indirect IHC (Perez et al., 1998). We have also detected B. suis using this reagent (data not presented);
Brucellosis of the European Brown Hare
however, since this antiserum was produced in rabbits, it could not be used on hare samples because of the strong background staining. To overcome this problem, we produced a B. suis-specific mouse hyperimmune serum. The mouse antiserum did not crossreact with F. tularensis or Y. pseudotuberculosis, which can infect European brown hares (Sterba, 1983; Gyuranecz et al., 2010), but there was weak crossreactivity with B. canis. The further examination of the specificity of the hyperimmune serum by IHC was not possible, because of the lack of tissue samples infected with other Brucella species. This is the first study describing the immunohistochemical detection of B. suis in European brown hares. The sensitivity and specificity of IHC were the same as for bacterial culture. Although only a small number of cases was examined in the present study, IHC appears to be a suitable additional diagnostic method for the detection of B. suis infection in the European brown hare.
Acknowledgments This work was funded by the OTKA 78139 grant.
Conflict of Interest The authors declare no conflicts of interest.
References Barrow GI, Feltham RKA (1993) Cowan and Steel’s Manual for the Identification of Medical Bacteria, 3rd Edit. Cambridge University Press, New York, pp. 105e106. Fretin D, Whatmore AM, Al Dahouk S, Neubauer H, Garin-Bastuji B et al. (2008) Brucella suis identification and biovar typing by real-time PCR. Veterinary Microbiology, 131, 376e385. Godfroid J, Cloeckaert A, Liautard JP, Kohler S, Fretin D et al. (2005) From the discovery of the Malta fever’s agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Veterinary Research, 36, 313e326. Gonzalez-Barrientos R, Morales JA, Hern andez-Mora G, Barquero-Calvo E, Guzm an-Verri C et al. (2010) Pathology of striped dolphins (Stenella coeruleoalba) infected with Brucella ceti. Journal of Comparative Pathology, 142, 347e352.
Gyuranecz M, Szeredi L, Makrai L, Fodor L, Mesz aros RA et al. (2010) Tularemia of European brown hare (Lepus europaeus): a pathological, histopathological and immunohistochemical study. Veterinary Pathology, 47, 958e963. Meador VP, Tabatabai LB, Hagemoser WA, Deyoe BL (1986) Identification of Brucella abortus in formalin-fixed, paraffin-embedded tissues of cows, goats, and mice with an avidinebiotineperoxidase complex immunoenzymatic staining technique. American Journal of Veterinary Research, 47, 2147e2150. Moore FM, Petrak ML, McMillan MC (1988) Diagnosis of chlamydial infection in pet birds: comparison of cloacalswab culture and peroxidaseeantiperoxidase methods. Avian Diseases, 32, 157e162. opez J, Casquet O, Sierra MO et al. Perez J, Quezada M, L (1998) Immunohistochemical detection of Brucella abortus antigens in tissues from aborted bovine fetuses using a commercially available polyclonal antibody. Journal of Veterinary Diagnostic Investigation, 10, 17e21. Pintur K, Popovic N, Alegro A, Severin K, Slavica A et al. (2006) Selected indicators of brown hare (Lepus europaeus Pallas, 1778) population dynamics in northwestern Croatia. Veterinary Archives, 76, 199e209. Sterba F (1982) Pathomorphological changes in brucellosis in hares. Veterin arnı medicına, 27, 437e448. Sterba F (1983) Differential pathomorphologic diagnosis of brucellosis in hares. Veterin arnı medicına, 28, 293e308. Sterba F (1984) Brucellosis in rabbits induced by an experimental infection with Brucella suis germs. Veterin arnı medicına, 29, 231e244. Sterba F, Halackov a M (1987) Use of the Rose-Bengal test in the diagnosis of brucellosis in hares. Veterin arnı medicına, 32, 441e447. ahor Z, Slab y V (1976) MorphologVıtovec J, Vladık P, Z ical study of 70 cases of brucellosis in rabbits caused by Brucella suis. Veterin arnı medicına, 21, 359e368. Xavier MN, Paixao TA, Poester FP, Lage AP, Santos RL (2009) Pathological, immunohistochemical and bacteriological study of tissues and milk of cows and fetuses experimentally infected with Brucella abortus. Journal of Comparative Pathology, 140, 149e157.
June 6th, 2010 ½ Received, Accepted, November 23rd, 2010