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Bacterial isolates from the urine of cattle affected by urothelial tumors of the urinary bladder
Research in Veterinary Science 93 (2012) 1361–1366
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Bacterial isolates from the urine of cattle affected by urothelial tumors of the urinary bladder Sante Roperto a, Giovanni Di Guardo b,⇑, Leonardo Leonardi c, Ugo Pagnini a, Emmanuele Manco a, Orlando Paciello d, Iolanda Esposito d, Giuseppe Borzacchiello d, Valeria Russo d, Paola Maiolino d, Franco Roperto d a
Department of Pathology and Animal Health, Division of Infectious Diseases, Faculty of Veterinary Medicine, University of Naples Federico II, Via Delpino, 1 - 80137 Naples, Italy Department of Comparative Biomedical Sciences, Faculty of Veterinary Medicine, University of Teramo, Piazza Aldo Moro, 45 - 64100 Teramo, Italy Department of Biopathological Sciences and Hygiene of Animal and Alimentary Productions, Faculty of Veterinary Medicine, University of Perugia, Via S. Costanzo, 4 - 06126 Perugia, Italy d Department of Pathology and Animal Health, Division of Pathology, Faculty of Veterinary Medicine, University of Naples Federico II, Via Delpino, 1 - 80137 Naples, Italy b c
a r t i c l e
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Article history: Received 10 March 2012 Accepted 26 June 2012
Keywords: Bacteria Cattle Urothelial tumors BPV-2 Urinary bladder
a b s t r a c t Microbiological investigations were performed on urine samples from 108 cows affected by urothelial tumors of the urinary bladder. Bacteria, frequently of mixed population, were isolated from 100 animals. Gram-positive bacteria prevailed, with Staphylococcus spp. and Bacillus spp. being the most common. Escherichia coli and Acinetobacter spp. were the most frequently recovered Gram-negative bacteria. E5 oncoprotein was detected in 86 of the 108 urothelial tumors under study. In the majority of cases, bacterial agents and BPV-2 E5 were simultaneously detected. A marked down-regulation of Tamm–Horsfall protein was also observed in the examined cases. In addition, the p65 subunit of the nuclear factor-jB (NF-jB) transcription factor appeared to be overexpressed. In all cases, a mild to severe chronic inflammation was evident in the stroma of urinary bladder tumors. Bacterial components may play a role in the activation of the NF-jB and might cause chronic inflammation resulting in an impaired ability to clear BPV-2 infection, thus cooperating with the virus in cancer development. As in man, therefore, bacteria could play both a direct and an indirect role in bovine bladder carcinogenesis. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In some geographic areas rich in bracken fern (Pteridium spp.), urinary bladder tumors are known to occur at a frequency as high as 90% in cattle (Pamucku et al., 1976; Özkul and Aydin, 1996; Roperto et al., 2010a). Outside these areas, bladder cancers are very rare, accounting for 0.01% of all bovine malignancies (Meuten, 2010). In more than 90% of cases, tumors of the urinary bladder are responsible for a severe clinical syndrome known as chronic enzootic hematuria (CEH) (Maxie and Newman, 2010). Some chemical and infectious agents appear to play an important synergistic role in bladder carcinogenesis of cattle. Ptaquiloside, a norsesquiterpene glycoside of bracken fern, is known to be a powerful carcinogen and is responsible for H-ras activation via initial adenine alkylation, which is believed to be the first step
⇑ Corresponding author. Tel.: +39 0 861 266933; fax: +39 0 861 266865. E-mail address: [email protected] (G. Di Guardo). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.06.009
in the initiation of chemical carcinogenesis (Prakash et al., 1996; Gil da Costa et al., 2010). Bovine papillomavirus type 2 (BPV-2) is the most important infectious agent involved in bladder cancer of cattle, since E5 protein, the major BPV-2 oncoprotein, has been detected in almost 80% of bovine bladder tumors (Campo et al., 1992; Borzacchiello et al., 2003b; Wosiaki et al., 2005; Balcos et al., 2008; Borzacchiello and Roperto, 2008; Roperto et al., 2008, 2010b, 2011). Although it is often very difficult to identify infectious agents as causes of tumors, it has been suggested that viruses are responsible for 12.1% of neoplasms linked to infectious agents, with bacteria also implicated in carcinogenesis and being responsible for approximately 5.6% of global cancer incidence caused by infectious pathogens (zur Hausen, 2009; IARC, 2012). There is a convincing body of evidence that bacteria play an important role in cell transformation through different and complex mechanisms which need to be further investigated (Mager, 2006; Vogelmann and Amieva, 2006; Khan and Shrivastava, 2010; Samaras et al., 2010). Clinical and epidemiological studies have indicated a strong association between bacterial agents and
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chronic infections leading to cell transformation and/or to tumor progression (Monack et al., 2004; Karin et al., 2006; Chen et al., 2007; Nath et al., 2010; Ullman and Itzkowitz, 2011). Overwhelming evidence exists that inflammatory disease conditions increase the risk of developing many types of tumors, including bladder, gastric, intestinal, ovarian, esophageal, prostate and thyroid neoplasms (Vakkila and Lotze, 2004; Michaud, 2007; Mantovani et al., 2008). Although the relationship between inflammation and cancer appears to be unquestionable, the cellular and molecular pathways involved in inflammation-related neoplasia still remain largely unexplained (Porta et al., 2009). The aim of the present paper is to report the spectrum of bacterial agents isolated from the urine of cattle suffering from urinary bladder tumors, in an attempt to better understand their potential role in bladder carcinogenesis. To the best of our knowledge, similar investigations have not previously been reported in veterinary and comparative oncology. 2. Materials and methods 2.1. Microbiological investigations on bovine urine samples Urine samples were aseptically collected, between 2004 and 2011, from 108, 4–13-year-old hematuric cows slaughtered at public slaughterhouses. The animals had been reared in mountain cattle households located in southern Italy, as reported elsewhere (Roperto et al., 2010a), and were known to have grazed on pastures rich in bracken fern. Urine samples were also aseptically collected in the same abattoirs and by the same operators from an additional five regularly slaughtered and healthy cows from the same area. After removal from the pelvic cavity, the neck of each bladder was securely aseptically tied before isolating the organ, in order to minimize the risk of contamination. The external bladder wall was immediately washed with sterile sponges soaked in sterile distilled water and then cleansed with sterile gauzes soaked in sterile, antiseptic soap. Urine was collected by aspiration with a sterile syringe via penetration of the bladder wall. Each urine sample was subsequently cultured on the following solid (agar-based) media, in order to characterize its bacterial and/or fungal microflora: Trypcase Soy Agar, supplemented with 5% sheep blood (bioMèrieux, Marcy L’Etoile, France), for 24–48 h at 37 °C; Brilliant Green Agar (Oxoid, Basingstoke, UK) for 24 h at 37 °C; Baird-Parker Agar (Oxoid) for 24–48 h at 37 °C; MacConkey Agar (Oxoid) for 24 h at 37 °C; and Sabouraud Agar (Oxoid) for 3–5 days at both 25 °C and 30 °C. In addition, a pre-enrichment culture of each urine sample, obtained by inoculating 1 ml into 20 ml of either Nutrient Broth (Oxoid) or Biotone Broth (Biolife Italiana, Milano, Italy) and incubating at 37 °C for 24 h, was also assayed on the aforementioned media, in order to improve the chances of microbial isolation. Microbial isolates were subsequently identified using commercial kits, namely BBL Crystal for Gram negative bacteria, BBL Crystal for Gram positive bacteria (Becton, Dickinson & Co., Franklin Lakes, NJ, USA) and Slidex Staph Plus (bioMèrieux). Antibiotic resistance profiles were determined by means of ATB Vet (bioMèrieux), with doubtful and negative responses being ascertained using Antimicrobial Susceptibility Test Discs (Becton, Dickinson & Co.). 2.2. Morphological investigations Bladder tissue samples from both healthy and urothelial bladder tumor-affected cows were also collected and subsequently processed for histological, ultrastructural and biomolecular studies. Neoplastic bladder lesions were detected in all slaughtered ani-
mals under investigation during the mandatory post-mortem examination. Histological classification of the above tumor lesions was performed according to the morphological criteria recently proposed for bovine urinary bladder neoplasms (Roperto et al., 2010a). 2.3. Immunoprecipitation of E5 oncoprotein For biomolecular studies, samples of tumors and normal urinary bladder mucosa were immediately frozen in liquid nitrogen. The frozen samples were then homogenized in RIPA lysis buffer containing 50 mM Hepes pH 7.5, 150 mM sodium chloride (NaCl), 0.25% sodium deoxycholate, 1% Triton and 1 mM ethylenediaminetetraacetic acid (EDTA). Prior to homogenization, 20 mM sodium pyrophosphate (Na4P2O7), 0.1 mg/ml aprotinin, 2 mM phenylmethylsulphonyl fluoride (PMSF), 10 mM sodium orthovanadate (Na2VO3) and 50 mM sodium fluoride (NaF) (Sigma–Aldrich, Milan, Italy) were added. Lysates were clarified by centrifugation at 11,000g for 30 min. Supernatants were collected and protein concentration measured using the Bradford assay (Bio-Rad Laboratories, Milan, Italy). Proteins (1000 lg) were immunoprecipitated by using 2 lg of anti-E5 antibody (a kind gift by Dr. M.S. Campo, University of Glasgow, Scotland) and 30 ll of A-G protein plus agarose (Santa Cruz Biotechnology, CA, USA). The immunoprecipitates were then resuspended in 1X premixed Laemmli Sample Buffer (Bio-Rad Laboratories, Milan, Italy) and analyzed by SDS polyacrylamide gel electrophoresis (PAGE) (15% polyacrylamide) under reducing conditions. After electrophoresis, proteins were transferred onto nitrocellulose filter membranes (GE Healthcare Life Sciences, Chalfont St Giles, UK) for 1 h at 10 V in 192 mM glycine/ 25 mM Tris–HCl (pH 7.5) and 10% methanol using a Trans-Blot SD Semy Dry cell (Bio-Rad Laboratories, Milan, Italy) according to the manufacturer’s instructions. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS, pH 7.5) for 1 h at room temperature and washed with TBS-0.1% Tween. Thereafter, they were probed with the anti-E5 antibody for an overnight incubation at 4 °C. After three washes in Tris-buffered saline, membranes were incubated with horseradish peroxidase-conjugated rabbit anti-sheep IgG (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Finally, antibody binding was visualized by an enhanced chemiluminescence system (Western Blotting Luminol Reagent, Santa Cruz Biotechnology, CA, USA). 2.4. Western blot analysis of NF-jB p65 and Tamm–Horsfall protein (THP) Bladder tissue samples from all the 108 hematuric and from the five healthy cows under investigation were solubilized at 4 °C in lysis buffer containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl and 1% Triton X-100. Immediately prior to use, the following reagents were added: 1 mM DTT, 2 mM PMSF, 1.7 mg/ml Aprotinin, 25 mM NaF and 1 mM Na3VO4 (Sigma–Aldrich, Milan, Italy). Lysates were clarified at 11,000g for 30 min. Protein concentrations were measured by means of the Bradford assay (Bio-Rad Laboratories, Milan, Italy). For Western blotting, 50 lg of each protein lysate were heated at 100 °C in 4X premixed Laemmli Sample Buffer. Proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (7.5% polyacrylamide) under reducing conditions. Following electrophoresis, proteins were transferred onto nitrocellulose filter membranes (GE Healthcare Life Sciences, Chalfont St Giles, UK) for 1 h at 350 mA in 192 mM glycine/25 mM Tris– HCl (pH 7.5) and 10% methanol. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS, pH 7.5) for 1 h at room temperature and subsequently washed with TBS0.1% Tween. Thereafter, they were probed with both anti-NF-jB
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p65 and anti-THP antibodies (Santa Cruz Biotechnology, CA, USA) for an overnight incubation at 4 °C. After three washes in Tris-buffered saline, membranes were respectively incubated with either horseradish peroxidase-conjugated anti-rabbit IgG (Bio-Rad Laboratories, Milan, Italy) or anti-mouse IgG (Bio-Rad Laboratories, Milan, Italy) for 1 h at room temperature. After appropriate washing steps, antibody binding was visualized by an enhanced chemiluminescence system (Western Blotting Luminol Reagent, Santa Cruz Biotechnology, CA, USA).
3. Results In total, 184 bacterial isolates, 139 Gram-positive and 45 Gramnegative, were obtained from 100 urine cultures, with a substantial proportion consisting of a polymicrobial flora. Eight samples did not yield bacteria. Gram-positive genera isolated were Staphylococcus spp. (45/139), Bacillus spp. (36/139), Corynebacterium spp. (19/ 139) and Enterococcus spp. (15/139). Aerococcus spp. (13/139), Streptococcus spp. (5/139), Micrococcus spp. (4/139) and Gemella spp. (1/139) were detected at lower frequencies (Fig. 1). Staphylococcus saprophyticus (11/45) – a true urinary tract pathogen – and Staphylococcus warneri (7/45) were the predominant staphylococcal, coagulase-negative bacteria. Four pathogenic, coagulase-positive isolates of Staphylococcus aureus (4/45) were also recovered. Bacillus licheniformis (12/36), Bacillus cereus (10/36), Corynebacterium renale group (8/19) and Enterococcus faecalis (7/15) were the other most prevalent Gram-positive isolates (Fig. 1). Gram-negative bacteria consisted of the following genera and species: Acinetobacter spp. (8/45), Escherichia coli (7/45), Klebsiella spp. (6/45) and Pseudomonas spp. (5/45). Furthermore, Agrobacterium spp. (1/45), Citrobacter spp. (1/45), Enterobacter spp. (2/45), Flavobacterium spp. (1/45), Shigella spp. (2/45), Sphingomonas spp. (1/45), Stenotrophomonas spp. (3/45) and Weeksella spp. (1/45) were occasionally recovered (Fig. 2). E. coli (7/45) and Acinetobacter lwoffy (7/ 45) were the most frequently isolated Gram-negative bacteria (Fig. 2). Finally, no bacterial isolates were recovered from the urines of clinically healthy cows, with the exception of one animal from which E. coli was obtained. Biomolecular investigations yielded a statistically significant (P < 0.05) overexpression of p65, a subunit of NF-jB (Fig. 3), along
Fig. 2. Distribution of Gram-negative bacterial isolates recovered from the urine samples under study.
A
B
Fig. 3. (A) Western blot analysis showing overexpression of the NF-jB p65 subunit. (B) Quantitative densitometric analyses of the filter membranes were performed with the Image Lab Software Program (Chemidoc Bio-Rad Labor., CA) and the data analyzed by the Student’s t-test (P<0.05). ⁄The p-value is <0.05.
Fig. 1. Distribution of Gram-positive bacterial isolates recovered from the urine samples under study.
with a significant (P < 0.004) down-regulation of Tamm–Horsfall protein (THP) (Fig. 4). In all bladder tissue samples collected from the 108 tumor-affected cattle, the pattern of the microscopic lesions was consistent with a morphological diagnosis of urothelial neoplasm, 86 (80%) of which were also found to be associated – following detection of the E5 oncoprotein (Fig. 5) – with the presence of BPV-2. Furthermore, histologic changes typical of chronic inflammation were simultaneously found in all the neoplastic tissue specimens under investigation, with inflammatory lesions being mostly located in the lamina propria mucosae of the urinary bladder.
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A
B
Fig. 4. (A) Western blot analysis showing down-regulation of Tamm–Horsfall protein (THP). (B) Quantitative densitometric analyses of the filter membranes were performed with the Image Lab Software Program (Chemidoc Bio-Rad Labor., CA) and the data analyzed by the Student’s t-test (P < 0.004). ⁄⁄The p-value is <0.004.
Fig. 5. Biomolecular evidence of BPV-2 E5 oncoprotein in bladder tumor samples, with BPV-2 antigen being detected by immunoprecipitation (IP).
4. Discussion In the present study, bacterial agents were isolated at a very high frequency (93%) from the urine of cattle affected by tumors of the urinary bladder. In this respect, it is also of interest that microscopic lesions of chronic inflammation were concurrently observed in all the neoplastic bladder tissue specimens under study. These findings are surprisingly unusual in cattle as cystitis is known to occur sporadically in farm animals (Radostits et al., 2000). Recent investigations performed on calves and dairy cattle showed an incidence of urinary tract infections (UTIs) up to 1.6% and 4%, respectively (Yeruham et al., 2004, 2006). Several general and local factors may have facilitated such a high bacterial colonization rate of the urinary bladder in examined cattle. It is known that toxic substances contained in bracken fern are responsible for a severe impairment of the cattle immune system (Borzacchiello and Roperto, 2008). Additionally, Tamm–Horsfall protein (THP), a broad host defense factor against bacterial cystitis (Raffi et al., 2005), was found to be down-regulated. THP plays a significant role in immune dysfunction of the urinary bladder, thereby making it difficult to clear host’s urine and the bladder environment from microbial pathogens. Unfortunately, since many of the isolated bacteria were part of a polymicrobial flora, their independent contribution in causing inflammation of the urinary bladder is difficult to assess, with no clear distinction being possible between their effective role of associated versus truly responsible agents. Interestingly, in all the cases investigated herein, a mild to severe chronic cystitis was histologically apparent, with inflammatory lesions being mostly located in the lamina propria mucosae of the urinary bladder. It is also of substantial concern that, in 86
(80%) of the 108 bladder tumor-affected cows under study, BPV-2 E5 oncoprotein was detected by immunoprecipitation. UTIs are currently regarded as a risk factor for developing bladder cancer (Parker et al., 2004; Dobrovolskaia and Kozlov, 2005; Mantovani et al., 2008; Scrivo et al., 2011). It is believed that approximately 1% of chronic bacterial cystitis leads to bladder cancer (Vakkila and Lotze, 2004). It has been also suggested that understanding the role of inflammation in urothelial carcinogenesis may provide important insights on how to prevent bladder cancer (Michaud, 2007). In this respect, it is worth noting that it has been recently shown, for the first time, that Schistosoma haematobium antigens are able to cause a chronic bladder inflammation leading to urothelial dysplasia (Botelho et al., 2011). Still recently, high-risk human papillomaviruses (HPVs) have been shown to infect urothelial cells and are now believed to be the causative agents of some bladder tumors (Li et al., 2011; Shigehara, 2011; Shigehara et al., 2011). Biologic agents including viruses, helminth parasites and bacteria, are emerging as major causes of human neoplasia (zur Hausen, 2006). Indeed, it has been suggested that up to 20% of all tumors are attributable to infectious agents (Pagano et al., 2004; IARC, 2012). Therefore, the relationship between bacterial pathogens and cancer development is becoming a very intriguing field of investigation. Although studies in animal models convincingly support a causative role for several bacterial pathogens (Vogelmann and Amieva, 2006; Nath et al., 2010; Ullman and Itzkowitz, 2011), their oncogenic role still remains far from being clearly elucidated (Herrera et al., 2005; Mager, 2006). Several pathways for the oncogenic potential of bacteria have been proposed (Collins et al., 2011). Bacteria are usually believed to be indirect carcinogens that act via chronic inflammation and/or immune suppression (IARC, 2012). Furthermore, they are able to elaborate mutagenic toxins (Karin et al., 2006; Nath et al., 2010); very recently, mechanisms involving horizontal DNA transfer have also been proposed (Khan and Shrivastava, 2010). In the present study, we did not investigate the complex interplay between host and bacteria resulting in bladder tumor development. It is reasonable to believe, however, that bacteria may be involved and may have contributed to bovine bladder carcinogenesis through different mechanisms. In most cases, we simultaneously detected bacteria and BPV-2, a virus which is able to infect the urinary mucosa of cattle and remain latent in normal tissue until some biological or chemical factor triggers viral gene expression (Campo et al., 1992; Borzacchiello et al., 2003b). Bacterial pathogens, at their turn, may cause a chronic inflammation resulting in an impaired ability to clear BPV-2 infection, thus permitting a persistent papillomavirus (BPV-2) infection ending up with bladder cancer development. A similar etiopathogenetic mechanism has been also suggested for Chlamydia trachomatis-induced inflammation, coupled with a persistent papillomavirus infection of the female genital tract, in the development of human cervical neoplasia (Silins et al., 2005). Furthermore, the molecular pathways by which chronic infection and inflammation may actually promote tumor growth and progression are still far from being completely understood (Balkwill and Coussens, 2004; Balkwill et al., 2005; Mantovani, 2005; Karin, 2006a, 2006b; Ben-Neriah and Karin, 2011; Ullman and Itzkowitz, 2011; Xiao and Fu, 2011). In this respect, the transcription factor nuclear factor-jB (NF-jB) is believed to be the crucial molecular link between inflammation and cancer (Greten et al., 2004; Pikarsky et al., 2004; Karin and Greten, 2005; Lu et al., 2006; Ben-Neriah and Karin, 2011). Furthermore, it has been suggested that NF-jB plays a key role in this process, from bacteria-related inflammation to subsequent cancer development (Karin, 2006a, 2006b; Huang et al., 2007; Ben-Neriah and Karin, 2011).
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In the present study, we detected an overexpression of the p65 subunit of NF-jB. Furthermore, we have previously reported an overexpression of COX-2 protein in bovine urothelial tumors (Borzacchiello et al., 2003a). Interestingly, in 35 bladder neoplasms included in our study we have also detected an up-regulation of STAT3 protein (S. Roperto, unpublished data). Taken together, these last results allow us to speculate that bacteria, acting either alone or synergistically with other biological (i.e. viruses) and non-biological factors (i.e. necrotic cell products), may be responsible for NF-jB activation which would result, in turn, in STAT3 and COX-2 activation. STAT3 is currently believed to be an oncogene playing a very significant role in tumorigenesis (Aggarwal et al., 2011), with COX-2 being also known to enhance the malignant potential of urothelial carcinoma cells (Wadhwa et al., 2005; de Visser et al., 2006; Shimada et al., 2011). In conclusion, further investigations are needed in order to determine the nature of the relationships between bacteria and bladder cancer in cattle, as well as to better define whether bacteria may be involved as carcinogens or co-carcinogens in initiating and/or promoting the development of urinary bladder neoplasia. Conflict of interest The authors declare they have no financial nor personal relationships with other people or organizations that could inappropriately influence (bias) their work. Acknowledgements The authors wish to thank Dr G. Salvatore from Regione Basilicata and Drs R. La Rizza and F. Luposella from Regione Calabria for their useful technical help. This work was sponsored by grants from Ministero Italiano dell’Università e della Ricerca Scientifica (MIUR), from Ministero Italiano delle Politiche Agricole, Alimentari e Forestali (MIPAAF) and from Assessorato alla Sanità della Regione Basilicata e della Regione Campania. References Aggarwal, B.B., Kunnumakkara, A.B., Harikumar, K.B., Gupta, S.R., Tharakan, S.T., Koca, C., Dey, S., Sung, B., 2011. Signal transducer and activator of transcription3, inflammation, and cancer. Annals of the New York Academy of Sciences 1171, 59–76. Balcos, L.G., Borzacchiello, G., Russo, V., Popescu, O., Roperto, S., Roperto, F., 2008. Association of bovine papillomavirus type-2 and urinary bladder tumours in cattle from Romania. Research in Veterinary Science 85, 145–148. Balkwill, F., Coussens, L.M., 2004. Cancer: an inflammatory link. Nature 431, 405– 406. Balkwill, F., Charles, K.A., Mantovani, A., 2005. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7, 211–217. Ben-Neriah, Y., Karin, M., 2011. Inflammation meets cancer, with NF-kB as the matchmaker. Nature Immunology 12, 715–723. Borzacchiello, G., Ambrosio, V., Galati, P., Perillo, A., Roperto, F., 2003a. Cyclooxygenase-1 and -2 expression in urothelial carcinomas of the urinary bladder in cows. Veterinary Pathology 40, 455–459. Borzacchiello, G., Iovane, G., Marcante, M.L., Poggiali, F., Roperto, F., Roperto, S., Venuti, A., 2003b. Presence of bovine papillomavirus type 2 and expression of the viral oncoprotein E5 in naturally occurring urinary bladder tumours in cows. Journal of General Virology 84, 2921–2926. Borzacchiello, G., Roperto, F., 2008. Bovine papillomaviruses, papillomas and cancer in cattle. Veterinary Research 39, 45–63. Botelho, M.C., Oliveira, P.A., Lopes, C., Correia da Costa, J.M., Machado, J.C., 2011. Urothelial dysplasia and inflammation induced by Schistosoma haematobium total antigen instillation in mice normal urothelium. Urologic Oncology 29, 809–814. Campo, M.S., Jarrett, W.F., Barron, R., O’Neil, B.W., Smith, K.T., 1992. Association of bovine papillomavirus type 2 and bracken fern with bladder cancer in cattle. Cancer Research 52, 6898–6904. Chen, R., Alvero, A.B., Silasi, D.A., Mor, G., 2007. Inflammation, cancer and chemoresistance: tacking advantage of the toll-like receptor signaling pathway. American Journal of Reproductive Immunology 57, 93–107. Collins, D., Hogan, A.M., Winter, D.C., 2011. Microbial and viral pathogens in colorectal cancer. Lancet Oncology 12, 504–512.
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Silins, I., Ryd, W., Strand, A., Wadell, G., Törnberg, S., Hansson, B.G., Wang, X., Arnheim, L., Dahl, V., Bremell, D., Persson, K., Dillner, J., Rylander, E., 2005. Chlamydia trachomatis infection and persistence of human papillomavirus. International Journal of Cancer 116, 110–115. Ullman, T.A., Itzkowitz, S.H., 2011. Intestinal inflammation and cancer. Gastroenterology 140, 1807–1816. Vakkila, J., Lotze, M.T., 2004. Inflammation and necrosis promote tumour growth. Nature Reviews Immunology 4, 641–648. Vogelmann, R., Amieva, M.R., 2006. The role of bacterial pathogens in cancer. Current Opinion in Microbiology 10, 1–6. Wadhwa, P., Goswami, A.K., Joshi, K., Sharma, S.K., 2005. Cyclooxygenase-2 expression increases with the stage and grade in transitional cell carcinoma of the urinary bladder. International Urology and Nephrology 37, 47–53. Wosiaki, S.R., Barreiros, M.A.B., Alfieri, A.F., Alfieri, A.A., 2005. Semi-nested PCR for detection and typing of bovine papillomavirus type 2 in urinary bladder and whole blood from cattle with enzootic haematuria. Journal of Virological Methods 126, 215–219. Xiao, G., Fu, J., 2011. NF-kB and cancer: a paradigm of Yin-Yang. American Journal of Cancer Research 1, 192–221. Yeruham, I., Elad, D., Avidar, Y., Goshen, T., Asis, E., 2004. Four-year survey of urinary tract infections in calves in Israel. Veterinary Record 154, 204–206. Yeruham, I., Elad, D., Avidar, Y., Goshen, T., 2006. A herd level analysis of urinary tract infection in dairy cattle. Veterinary Journal 171, 172–176. zur Hausen, H., 2006. Historical review. In: Zur Hausen, H. (Ed.), Infections Causing Human Cancer. Wiley-VCH Verlag GmbH & Co., Weinheim, Germany, pp. 1–16. zur Hausen, H., 2009. The search for infectious causes of human cancers: Where and why. Virology 392, 1–10.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Bacterial isolates from the urine of cattle affected by urothelial tumors of the urinary bladder Sante Roperto a, Giovanni Di Guardo b,⇑, Leonardo Leonardi c, Ugo Pagnini a, Emmanuele Manco a, Orlando Paciello d, Iolanda Esposito d, Giuseppe Borzacchiello d, Valeria Russo d, Paola Maiolino d, Franco Roperto d a
Department of Pathology and Animal Health, Division of Infectious Diseases, Faculty of Veterinary Medicine, University of Naples Federico II, Via Delpino, 1 - 80137 Naples, Italy Department of Comparative Biomedical Sciences, Faculty of Veterinary Medicine, University of Teramo, Piazza Aldo Moro, 45 - 64100 Teramo, Italy Department of Biopathological Sciences and Hygiene of Animal and Alimentary Productions, Faculty of Veterinary Medicine, University of Perugia, Via S. Costanzo, 4 - 06126 Perugia, Italy d Department of Pathology and Animal Health, Division of Pathology, Faculty of Veterinary Medicine, University of Naples Federico II, Via Delpino, 1 - 80137 Naples, Italy b c
a r t i c l e
i n f o
Article history: Received 10 March 2012 Accepted 26 June 2012
Keywords: Bacteria Cattle Urothelial tumors BPV-2 Urinary bladder
a b s t r a c t Microbiological investigations were performed on urine samples from 108 cows affected by urothelial tumors of the urinary bladder. Bacteria, frequently of mixed population, were isolated from 100 animals. Gram-positive bacteria prevailed, with Staphylococcus spp. and Bacillus spp. being the most common. Escherichia coli and Acinetobacter spp. were the most frequently recovered Gram-negative bacteria. E5 oncoprotein was detected in 86 of the 108 urothelial tumors under study. In the majority of cases, bacterial agents and BPV-2 E5 were simultaneously detected. A marked down-regulation of Tamm–Horsfall protein was also observed in the examined cases. In addition, the p65 subunit of the nuclear factor-jB (NF-jB) transcription factor appeared to be overexpressed. In all cases, a mild to severe chronic inflammation was evident in the stroma of urinary bladder tumors. Bacterial components may play a role in the activation of the NF-jB and might cause chronic inflammation resulting in an impaired ability to clear BPV-2 infection, thus cooperating with the virus in cancer development. As in man, therefore, bacteria could play both a direct and an indirect role in bovine bladder carcinogenesis. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In some geographic areas rich in bracken fern (Pteridium spp.), urinary bladder tumors are known to occur at a frequency as high as 90% in cattle (Pamucku et al., 1976; Özkul and Aydin, 1996; Roperto et al., 2010a). Outside these areas, bladder cancers are very rare, accounting for 0.01% of all bovine malignancies (Meuten, 2010). In more than 90% of cases, tumors of the urinary bladder are responsible for a severe clinical syndrome known as chronic enzootic hematuria (CEH) (Maxie and Newman, 2010). Some chemical and infectious agents appear to play an important synergistic role in bladder carcinogenesis of cattle. Ptaquiloside, a norsesquiterpene glycoside of bracken fern, is known to be a powerful carcinogen and is responsible for H-ras activation via initial adenine alkylation, which is believed to be the first step
⇑ Corresponding author. Tel.: +39 0 861 266933; fax: +39 0 861 266865. E-mail address: [email protected] (G. Di Guardo). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.06.009
in the initiation of chemical carcinogenesis (Prakash et al., 1996; Gil da Costa et al., 2010). Bovine papillomavirus type 2 (BPV-2) is the most important infectious agent involved in bladder cancer of cattle, since E5 protein, the major BPV-2 oncoprotein, has been detected in almost 80% of bovine bladder tumors (Campo et al., 1992; Borzacchiello et al., 2003b; Wosiaki et al., 2005; Balcos et al., 2008; Borzacchiello and Roperto, 2008; Roperto et al., 2008, 2010b, 2011). Although it is often very difficult to identify infectious agents as causes of tumors, it has been suggested that viruses are responsible for 12.1% of neoplasms linked to infectious agents, with bacteria also implicated in carcinogenesis and being responsible for approximately 5.6% of global cancer incidence caused by infectious pathogens (zur Hausen, 2009; IARC, 2012). There is a convincing body of evidence that bacteria play an important role in cell transformation through different and complex mechanisms which need to be further investigated (Mager, 2006; Vogelmann and Amieva, 2006; Khan and Shrivastava, 2010; Samaras et al., 2010). Clinical and epidemiological studies have indicated a strong association between bacterial agents and
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chronic infections leading to cell transformation and/or to tumor progression (Monack et al., 2004; Karin et al., 2006; Chen et al., 2007; Nath et al., 2010; Ullman and Itzkowitz, 2011). Overwhelming evidence exists that inflammatory disease conditions increase the risk of developing many types of tumors, including bladder, gastric, intestinal, ovarian, esophageal, prostate and thyroid neoplasms (Vakkila and Lotze, 2004; Michaud, 2007; Mantovani et al., 2008). Although the relationship between inflammation and cancer appears to be unquestionable, the cellular and molecular pathways involved in inflammation-related neoplasia still remain largely unexplained (Porta et al., 2009). The aim of the present paper is to report the spectrum of bacterial agents isolated from the urine of cattle suffering from urinary bladder tumors, in an attempt to better understand their potential role in bladder carcinogenesis. To the best of our knowledge, similar investigations have not previously been reported in veterinary and comparative oncology. 2. Materials and methods 2.1. Microbiological investigations on bovine urine samples Urine samples were aseptically collected, between 2004 and 2011, from 108, 4–13-year-old hematuric cows slaughtered at public slaughterhouses. The animals had been reared in mountain cattle households located in southern Italy, as reported elsewhere (Roperto et al., 2010a), and were known to have grazed on pastures rich in bracken fern. Urine samples were also aseptically collected in the same abattoirs and by the same operators from an additional five regularly slaughtered and healthy cows from the same area. After removal from the pelvic cavity, the neck of each bladder was securely aseptically tied before isolating the organ, in order to minimize the risk of contamination. The external bladder wall was immediately washed with sterile sponges soaked in sterile distilled water and then cleansed with sterile gauzes soaked in sterile, antiseptic soap. Urine was collected by aspiration with a sterile syringe via penetration of the bladder wall. Each urine sample was subsequently cultured on the following solid (agar-based) media, in order to characterize its bacterial and/or fungal microflora: Trypcase Soy Agar, supplemented with 5% sheep blood (bioMèrieux, Marcy L’Etoile, France), for 24–48 h at 37 °C; Brilliant Green Agar (Oxoid, Basingstoke, UK) for 24 h at 37 °C; Baird-Parker Agar (Oxoid) for 24–48 h at 37 °C; MacConkey Agar (Oxoid) for 24 h at 37 °C; and Sabouraud Agar (Oxoid) for 3–5 days at both 25 °C and 30 °C. In addition, a pre-enrichment culture of each urine sample, obtained by inoculating 1 ml into 20 ml of either Nutrient Broth (Oxoid) or Biotone Broth (Biolife Italiana, Milano, Italy) and incubating at 37 °C for 24 h, was also assayed on the aforementioned media, in order to improve the chances of microbial isolation. Microbial isolates were subsequently identified using commercial kits, namely BBL Crystal for Gram negative bacteria, BBL Crystal for Gram positive bacteria (Becton, Dickinson & Co., Franklin Lakes, NJ, USA) and Slidex Staph Plus (bioMèrieux). Antibiotic resistance profiles were determined by means of ATB Vet (bioMèrieux), with doubtful and negative responses being ascertained using Antimicrobial Susceptibility Test Discs (Becton, Dickinson & Co.). 2.2. Morphological investigations Bladder tissue samples from both healthy and urothelial bladder tumor-affected cows were also collected and subsequently processed for histological, ultrastructural and biomolecular studies. Neoplastic bladder lesions were detected in all slaughtered ani-
mals under investigation during the mandatory post-mortem examination. Histological classification of the above tumor lesions was performed according to the morphological criteria recently proposed for bovine urinary bladder neoplasms (Roperto et al., 2010a). 2.3. Immunoprecipitation of E5 oncoprotein For biomolecular studies, samples of tumors and normal urinary bladder mucosa were immediately frozen in liquid nitrogen. The frozen samples were then homogenized in RIPA lysis buffer containing 50 mM Hepes pH 7.5, 150 mM sodium chloride (NaCl), 0.25% sodium deoxycholate, 1% Triton and 1 mM ethylenediaminetetraacetic acid (EDTA). Prior to homogenization, 20 mM sodium pyrophosphate (Na4P2O7), 0.1 mg/ml aprotinin, 2 mM phenylmethylsulphonyl fluoride (PMSF), 10 mM sodium orthovanadate (Na2VO3) and 50 mM sodium fluoride (NaF) (Sigma–Aldrich, Milan, Italy) were added. Lysates were clarified by centrifugation at 11,000g for 30 min. Supernatants were collected and protein concentration measured using the Bradford assay (Bio-Rad Laboratories, Milan, Italy). Proteins (1000 lg) were immunoprecipitated by using 2 lg of anti-E5 antibody (a kind gift by Dr. M.S. Campo, University of Glasgow, Scotland) and 30 ll of A-G protein plus agarose (Santa Cruz Biotechnology, CA, USA). The immunoprecipitates were then resuspended in 1X premixed Laemmli Sample Buffer (Bio-Rad Laboratories, Milan, Italy) and analyzed by SDS polyacrylamide gel electrophoresis (PAGE) (15% polyacrylamide) under reducing conditions. After electrophoresis, proteins were transferred onto nitrocellulose filter membranes (GE Healthcare Life Sciences, Chalfont St Giles, UK) for 1 h at 10 V in 192 mM glycine/ 25 mM Tris–HCl (pH 7.5) and 10% methanol using a Trans-Blot SD Semy Dry cell (Bio-Rad Laboratories, Milan, Italy) according to the manufacturer’s instructions. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS, pH 7.5) for 1 h at room temperature and washed with TBS-0.1% Tween. Thereafter, they were probed with the anti-E5 antibody for an overnight incubation at 4 °C. After three washes in Tris-buffered saline, membranes were incubated with horseradish peroxidase-conjugated rabbit anti-sheep IgG (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Finally, antibody binding was visualized by an enhanced chemiluminescence system (Western Blotting Luminol Reagent, Santa Cruz Biotechnology, CA, USA). 2.4. Western blot analysis of NF-jB p65 and Tamm–Horsfall protein (THP) Bladder tissue samples from all the 108 hematuric and from the five healthy cows under investigation were solubilized at 4 °C in lysis buffer containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl and 1% Triton X-100. Immediately prior to use, the following reagents were added: 1 mM DTT, 2 mM PMSF, 1.7 mg/ml Aprotinin, 25 mM NaF and 1 mM Na3VO4 (Sigma–Aldrich, Milan, Italy). Lysates were clarified at 11,000g for 30 min. Protein concentrations were measured by means of the Bradford assay (Bio-Rad Laboratories, Milan, Italy). For Western blotting, 50 lg of each protein lysate were heated at 100 °C in 4X premixed Laemmli Sample Buffer. Proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (7.5% polyacrylamide) under reducing conditions. Following electrophoresis, proteins were transferred onto nitrocellulose filter membranes (GE Healthcare Life Sciences, Chalfont St Giles, UK) for 1 h at 350 mA in 192 mM glycine/25 mM Tris– HCl (pH 7.5) and 10% methanol. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS, pH 7.5) for 1 h at room temperature and subsequently washed with TBS0.1% Tween. Thereafter, they were probed with both anti-NF-jB
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p65 and anti-THP antibodies (Santa Cruz Biotechnology, CA, USA) for an overnight incubation at 4 °C. After three washes in Tris-buffered saline, membranes were respectively incubated with either horseradish peroxidase-conjugated anti-rabbit IgG (Bio-Rad Laboratories, Milan, Italy) or anti-mouse IgG (Bio-Rad Laboratories, Milan, Italy) for 1 h at room temperature. After appropriate washing steps, antibody binding was visualized by an enhanced chemiluminescence system (Western Blotting Luminol Reagent, Santa Cruz Biotechnology, CA, USA).
3. Results In total, 184 bacterial isolates, 139 Gram-positive and 45 Gramnegative, were obtained from 100 urine cultures, with a substantial proportion consisting of a polymicrobial flora. Eight samples did not yield bacteria. Gram-positive genera isolated were Staphylococcus spp. (45/139), Bacillus spp. (36/139), Corynebacterium spp. (19/ 139) and Enterococcus spp. (15/139). Aerococcus spp. (13/139), Streptococcus spp. (5/139), Micrococcus spp. (4/139) and Gemella spp. (1/139) were detected at lower frequencies (Fig. 1). Staphylococcus saprophyticus (11/45) – a true urinary tract pathogen – and Staphylococcus warneri (7/45) were the predominant staphylococcal, coagulase-negative bacteria. Four pathogenic, coagulase-positive isolates of Staphylococcus aureus (4/45) were also recovered. Bacillus licheniformis (12/36), Bacillus cereus (10/36), Corynebacterium renale group (8/19) and Enterococcus faecalis (7/15) were the other most prevalent Gram-positive isolates (Fig. 1). Gram-negative bacteria consisted of the following genera and species: Acinetobacter spp. (8/45), Escherichia coli (7/45), Klebsiella spp. (6/45) and Pseudomonas spp. (5/45). Furthermore, Agrobacterium spp. (1/45), Citrobacter spp. (1/45), Enterobacter spp. (2/45), Flavobacterium spp. (1/45), Shigella spp. (2/45), Sphingomonas spp. (1/45), Stenotrophomonas spp. (3/45) and Weeksella spp. (1/45) were occasionally recovered (Fig. 2). E. coli (7/45) and Acinetobacter lwoffy (7/ 45) were the most frequently isolated Gram-negative bacteria (Fig. 2). Finally, no bacterial isolates were recovered from the urines of clinically healthy cows, with the exception of one animal from which E. coli was obtained. Biomolecular investigations yielded a statistically significant (P < 0.05) overexpression of p65, a subunit of NF-jB (Fig. 3), along
Fig. 2. Distribution of Gram-negative bacterial isolates recovered from the urine samples under study.
A
B
Fig. 3. (A) Western blot analysis showing overexpression of the NF-jB p65 subunit. (B) Quantitative densitometric analyses of the filter membranes were performed with the Image Lab Software Program (Chemidoc Bio-Rad Labor., CA) and the data analyzed by the Student’s t-test (P<0.05). ⁄The p-value is <0.05.
Fig. 1. Distribution of Gram-positive bacterial isolates recovered from the urine samples under study.
with a significant (P < 0.004) down-regulation of Tamm–Horsfall protein (THP) (Fig. 4). In all bladder tissue samples collected from the 108 tumor-affected cattle, the pattern of the microscopic lesions was consistent with a morphological diagnosis of urothelial neoplasm, 86 (80%) of which were also found to be associated – following detection of the E5 oncoprotein (Fig. 5) – with the presence of BPV-2. Furthermore, histologic changes typical of chronic inflammation were simultaneously found in all the neoplastic tissue specimens under investigation, with inflammatory lesions being mostly located in the lamina propria mucosae of the urinary bladder.
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A
B
Fig. 4. (A) Western blot analysis showing down-regulation of Tamm–Horsfall protein (THP). (B) Quantitative densitometric analyses of the filter membranes were performed with the Image Lab Software Program (Chemidoc Bio-Rad Labor., CA) and the data analyzed by the Student’s t-test (P < 0.004). ⁄⁄The p-value is <0.004.
Fig. 5. Biomolecular evidence of BPV-2 E5 oncoprotein in bladder tumor samples, with BPV-2 antigen being detected by immunoprecipitation (IP).
4. Discussion In the present study, bacterial agents were isolated at a very high frequency (93%) from the urine of cattle affected by tumors of the urinary bladder. In this respect, it is also of interest that microscopic lesions of chronic inflammation were concurrently observed in all the neoplastic bladder tissue specimens under study. These findings are surprisingly unusual in cattle as cystitis is known to occur sporadically in farm animals (Radostits et al., 2000). Recent investigations performed on calves and dairy cattle showed an incidence of urinary tract infections (UTIs) up to 1.6% and 4%, respectively (Yeruham et al., 2004, 2006). Several general and local factors may have facilitated such a high bacterial colonization rate of the urinary bladder in examined cattle. It is known that toxic substances contained in bracken fern are responsible for a severe impairment of the cattle immune system (Borzacchiello and Roperto, 2008). Additionally, Tamm–Horsfall protein (THP), a broad host defense factor against bacterial cystitis (Raffi et al., 2005), was found to be down-regulated. THP plays a significant role in immune dysfunction of the urinary bladder, thereby making it difficult to clear host’s urine and the bladder environment from microbial pathogens. Unfortunately, since many of the isolated bacteria were part of a polymicrobial flora, their independent contribution in causing inflammation of the urinary bladder is difficult to assess, with no clear distinction being possible between their effective role of associated versus truly responsible agents. Interestingly, in all the cases investigated herein, a mild to severe chronic cystitis was histologically apparent, with inflammatory lesions being mostly located in the lamina propria mucosae of the urinary bladder. It is also of substantial concern that, in 86
(80%) of the 108 bladder tumor-affected cows under study, BPV-2 E5 oncoprotein was detected by immunoprecipitation. UTIs are currently regarded as a risk factor for developing bladder cancer (Parker et al., 2004; Dobrovolskaia and Kozlov, 2005; Mantovani et al., 2008; Scrivo et al., 2011). It is believed that approximately 1% of chronic bacterial cystitis leads to bladder cancer (Vakkila and Lotze, 2004). It has been also suggested that understanding the role of inflammation in urothelial carcinogenesis may provide important insights on how to prevent bladder cancer (Michaud, 2007). In this respect, it is worth noting that it has been recently shown, for the first time, that Schistosoma haematobium antigens are able to cause a chronic bladder inflammation leading to urothelial dysplasia (Botelho et al., 2011). Still recently, high-risk human papillomaviruses (HPVs) have been shown to infect urothelial cells and are now believed to be the causative agents of some bladder tumors (Li et al., 2011; Shigehara, 2011; Shigehara et al., 2011). Biologic agents including viruses, helminth parasites and bacteria, are emerging as major causes of human neoplasia (zur Hausen, 2006). Indeed, it has been suggested that up to 20% of all tumors are attributable to infectious agents (Pagano et al., 2004; IARC, 2012). Therefore, the relationship between bacterial pathogens and cancer development is becoming a very intriguing field of investigation. Although studies in animal models convincingly support a causative role for several bacterial pathogens (Vogelmann and Amieva, 2006; Nath et al., 2010; Ullman and Itzkowitz, 2011), their oncogenic role still remains far from being clearly elucidated (Herrera et al., 2005; Mager, 2006). Several pathways for the oncogenic potential of bacteria have been proposed (Collins et al., 2011). Bacteria are usually believed to be indirect carcinogens that act via chronic inflammation and/or immune suppression (IARC, 2012). Furthermore, they are able to elaborate mutagenic toxins (Karin et al., 2006; Nath et al., 2010); very recently, mechanisms involving horizontal DNA transfer have also been proposed (Khan and Shrivastava, 2010). In the present study, we did not investigate the complex interplay between host and bacteria resulting in bladder tumor development. It is reasonable to believe, however, that bacteria may be involved and may have contributed to bovine bladder carcinogenesis through different mechanisms. In most cases, we simultaneously detected bacteria and BPV-2, a virus which is able to infect the urinary mucosa of cattle and remain latent in normal tissue until some biological or chemical factor triggers viral gene expression (Campo et al., 1992; Borzacchiello et al., 2003b). Bacterial pathogens, at their turn, may cause a chronic inflammation resulting in an impaired ability to clear BPV-2 infection, thus permitting a persistent papillomavirus (BPV-2) infection ending up with bladder cancer development. A similar etiopathogenetic mechanism has been also suggested for Chlamydia trachomatis-induced inflammation, coupled with a persistent papillomavirus infection of the female genital tract, in the development of human cervical neoplasia (Silins et al., 2005). Furthermore, the molecular pathways by which chronic infection and inflammation may actually promote tumor growth and progression are still far from being completely understood (Balkwill and Coussens, 2004; Balkwill et al., 2005; Mantovani, 2005; Karin, 2006a, 2006b; Ben-Neriah and Karin, 2011; Ullman and Itzkowitz, 2011; Xiao and Fu, 2011). In this respect, the transcription factor nuclear factor-jB (NF-jB) is believed to be the crucial molecular link between inflammation and cancer (Greten et al., 2004; Pikarsky et al., 2004; Karin and Greten, 2005; Lu et al., 2006; Ben-Neriah and Karin, 2011). Furthermore, it has been suggested that NF-jB plays a key role in this process, from bacteria-related inflammation to subsequent cancer development (Karin, 2006a, 2006b; Huang et al., 2007; Ben-Neriah and Karin, 2011).
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In the present study, we detected an overexpression of the p65 subunit of NF-jB. Furthermore, we have previously reported an overexpression of COX-2 protein in bovine urothelial tumors (Borzacchiello et al., 2003a). Interestingly, in 35 bladder neoplasms included in our study we have also detected an up-regulation of STAT3 protein (S. Roperto, unpublished data). Taken together, these last results allow us to speculate that bacteria, acting either alone or synergistically with other biological (i.e. viruses) and non-biological factors (i.e. necrotic cell products), may be responsible for NF-jB activation which would result, in turn, in STAT3 and COX-2 activation. STAT3 is currently believed to be an oncogene playing a very significant role in tumorigenesis (Aggarwal et al., 2011), with COX-2 being also known to enhance the malignant potential of urothelial carcinoma cells (Wadhwa et al., 2005; de Visser et al., 2006; Shimada et al., 2011). In conclusion, further investigations are needed in order to determine the nature of the relationships between bacteria and bladder cancer in cattle, as well as to better define whether bacteria may be involved as carcinogens or co-carcinogens in initiating and/or promoting the development of urinary bladder neoplasia. Conflict of interest The authors declare they have no financial nor personal relationships with other people or organizations that could inappropriately influence (bias) their work. Acknowledgements The authors wish to thank Dr G. Salvatore from Regione Basilicata and Drs R. La Rizza and F. Luposella from Regione Calabria for their useful technical help. This work was sponsored by grants from Ministero Italiano dell’Università e della Ricerca Scientifica (MIUR), from Ministero Italiano delle Politiche Agricole, Alimentari e Forestali (MIPAAF) and from Assessorato alla Sanità della Regione Basilicata e della Regione Campania. References Aggarwal, B.B., Kunnumakkara, A.B., Harikumar, K.B., Gupta, S.R., Tharakan, S.T., Koca, C., Dey, S., Sung, B., 2011. Signal transducer and activator of transcription3, inflammation, and cancer. Annals of the New York Academy of Sciences 1171, 59–76. Balcos, L.G., Borzacchiello, G., Russo, V., Popescu, O., Roperto, S., Roperto, F., 2008. Association of bovine papillomavirus type-2 and urinary bladder tumours in cattle from Romania. Research in Veterinary Science 85, 145–148. Balkwill, F., Coussens, L.M., 2004. Cancer: an inflammatory link. Nature 431, 405– 406. Balkwill, F., Charles, K.A., Mantovani, A., 2005. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 7, 211–217. Ben-Neriah, Y., Karin, M., 2011. Inflammation meets cancer, with NF-kB as the matchmaker. Nature Immunology 12, 715–723. Borzacchiello, G., Ambrosio, V., Galati, P., Perillo, A., Roperto, F., 2003a. Cyclooxygenase-1 and -2 expression in urothelial carcinomas of the urinary bladder in cows. Veterinary Pathology 40, 455–459. Borzacchiello, G., Iovane, G., Marcante, M.L., Poggiali, F., Roperto, F., Roperto, S., Venuti, A., 2003b. Presence of bovine papillomavirus type 2 and expression of the viral oncoprotein E5 in naturally occurring urinary bladder tumours in cows. Journal of General Virology 84, 2921–2926. Borzacchiello, G., Roperto, F., 2008. Bovine papillomaviruses, papillomas and cancer in cattle. Veterinary Research 39, 45–63. Botelho, M.C., Oliveira, P.A., Lopes, C., Correia da Costa, J.M., Machado, J.C., 2011. Urothelial dysplasia and inflammation induced by Schistosoma haematobium total antigen instillation in mice normal urothelium. Urologic Oncology 29, 809–814. Campo, M.S., Jarrett, W.F., Barron, R., O’Neil, B.W., Smith, K.T., 1992. Association of bovine papillomavirus type 2 and bracken fern with bladder cancer in cattle. Cancer Research 52, 6898–6904. Chen, R., Alvero, A.B., Silasi, D.A., Mor, G., 2007. Inflammation, cancer and chemoresistance: tacking advantage of the toll-like receptor signaling pathway. American Journal of Reproductive Immunology 57, 93–107. Collins, D., Hogan, A.M., Winter, D.C., 2011. Microbial and viral pathogens in colorectal cancer. Lancet Oncology 12, 504–512.
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