Diagnostic Pathology Immunohistochemical localization of mdm-2, p27 Kip1 and bcl-2 in Warthin's tumor of the parotid gland

Ehab S Abd-Elhamid
Oral Pathology Department
Faculty of Dentistry
Ain Shams University
CairoEgypt

Marwa M Elshafei
Oral Pathology Department
Faculty of Dentistry
Misr International University
CairoEgypt

Diagnostic Pathology Immunohistochemical localization of mdm-2, p27 Kip1 and bcl-2 in Warthin's tumor of the parotid gland
10.1186/1746-1596-4-14BioMed Central Page 1 of 7 (page number not for citation purposes) Research * Corresponding author
Background: Warthin's tumor is a benign monomorphic adenoma with unclear origin that almost occur exclusively in the parotid gland. Etiology of Warthin's tumor as well as its malignant potential are still unclear. Therefore immunohistochemical assessment of Warthin's tumor may be useful to detect its origin or its malignant transformation potential.Aims and objectives:The present study aims to investigate the immunohistochemical expression of murine double minute-2 (mdm-2), p27 Kip1 and B cell lymphoma-2 (bcl-2) in Warthin's tumor of parotid gland and also to clarify the role of these proteins in the behavior of that tumor.Methods: Twenty paraffin blocks of cases previously diagnosed as Warthin's tumor were collected for immunohistochemical staining with primary antibodies against mdm-2, p27 Kip1 and bcl-2 using streptavidin-biotin immunoperoxidase staining system.

Background

Warthin's tumor is classified as a benign monomorphic adenoma of salivary gland that occurs exclusively in the parotid gland. Its etiology is still controversial; some authors suggested a bi-layered excretory ductal structure of the neoplastic epithelium in Warthin's tumor, as a result of hyperplastic process of the glandular epithelium that interacts with the excessive lymphoid tissue of the stroma [1]. Some authors consider it as a tumor like condition, not a monomorphic adenoma as it resulted from the proliferation of salivary gland ductal cells that were entrapped in parotid lymph nodes during embryonal life [2]. Although, other investigators detected, rare, histological change from Warthin's tumor to mucoepidermoid carcinoma that may be implicated in squamous or goblet cell metaplasia of epithelial cells. It is considered that the neoplastic cells of Warthin's tumor acquire malignant genotypes simultaneously with this dual differentiation leading to the formation of mucoepidermoid carcinoma, mostly low grade [3,4]. Therefore immunohistochemical assessment of Warthin's tumor may be useful to detect its origin or its malignant transformation potential.

Mdm2 is identified as an evolutionary conserved gene and as a dominant transforming oncogene on the long arm of the chromosome twelve. It encodes a number of alternatively spliced mRNAs that give rise to proteins ranging in size from 40 kilo Dalton (KDa) to 90 KDa: p90, p57-58, p74, p76 and p85 [5].

Mdm2 performs several functions; some of them are related to p53. By binding to p53, mdm2 has at least two functions: first it can target p53 for ubiquitin-degradation. The process of ubiquitination is subjected to a feedback loop as mdm2 protein binds to p53 to be degraded. This lowers the concentration of p53 and reduces transcription of mdm2 gene, closing the feedback loop and allowing p53 levels to rise again [6].

Second, mdm2 can also bind to the acidic activation domain of p53, concealing it from the transcriptional machinery i.e. blocking the ability of p53 to be recruited into active transcriptional complexes [7]. P90, the major product of mdm2, is now considered to form a tight complex with both the wild-type and mutant p53 tumor-suppressor gene product and inactivate wildtype p53 function by masking the N-terminal acidic transactivating domain of p53 protein, indicating that abnormalities of mdm2 gene may be closely associated with tumorigenesis and/or tumor development [8].

The smaller mdm2 protein, p76, increases the level of p53 by blocking the function of p90. Therefore, although, p90 and p57 bind to p53 and inhibit its nuclear accumulation, p74/76 can stabilize p53 by inhibiting the ability of p90 to stimulate its degradation without affecting the p90/p53 interaction [8].

Mdm2 may have a role in preventing apoptosis, either through inactivation of p53 or through some other mechanism independent of p53 [6]. On the other hand, it may down-regulate the anti-apoptotic protein Bcl-2 [9].

Inactivation of mdm2-induced G0/G1 arrest may contribute to tumor development [10], hence, mdm2 appears to play dual roles, as a tumor suppressor and as an oncogene, depending on the levels of mdm2 being expressed in the cell [11]. Mantesso et al [12] and Schlott et al [13] reported that mdm2 gene seems to be an early event related to the development of benign salivary gland tumors and play a part in tumor progression in salivary gland neoplasms. P27 Kip1 , a member of kinase inhibitory protein (KIP) family was identified as an inhibitory protein induced by tansforming growth factor-β (TGF-β) that played an important role in differentiation, senescence and cellular responses to negative growth-regulatory cytokines and to DNA damage and hence plays an essential role in the proliferative arrest that accompanies differentiation as well as is integrally involved in ensuring an orderly progression through the cell cycle [14].

Levels of p27 Kip1 are high in normal quiescent cells (G0/ G1 cells) but decrease in cycling cells, rapidly after mitogenic stimulation, when it gets sequestered into excess cyclin-cyclin-dependent kinase complexes. It remains low thereafter in actively proliferating cells [15].

Different mechanisms appear to regulate apoptosis in resting or cycling cells; deregulated activation of cyclindependent kinases results in apoptosis in resting cells, whereas cyclin-dependent kinase inhibition induces apoptosis in cycling cells. Therefore, apoptosis may result from activation of pathways that promote apoptosis upon inappropriate expression of cell cycle regulatory molecules. P27 Kip1 functions at a critical switch point where growth arrest is followed either by differentiation or apoptosis [16].

Apoptosis is modulated by several proteins or antigens such as the bcl-2 family and p53. Regulation of apoptosis is dependent on interactions between effector and suppressor molecules. Deregulation of the genes controlling apoptosis may contribute to the process of tumourigenesis by reducing the rate of cell death and allowing the accumulation of other genetic defects [17].

Bcl-2 emerged as a new type of proto-oncogene that suppresses cell death rather than stimulating proliferation. However, it does not inhibit apoptosis occurring in all circumstances as it failed to block apoptosis induced by cytotoxic T-lymphocytes [18].

The involvement of bcl-2 in p53-induced apoptosis was first suggested by the observation that p53-induced cell death can be prevented by bcl-2 expression. Wild-type p53-dependent apoptosis occurs at least partly through its suppressive effect on bcl-2 expression or its function. Support for this has come from in vitro studies of lymphoid cells where ectopic p53 was shown to down-regulate endogenous bcl-2 expression while simultaneously increasing the expression of bax [19].

The aim of the present study is to investigate the immunohistochemical expression of mdm-2, p27 Kip1 and bcl-2 in Warthin's tumor of parotid gland and also to clarify the role of these proteins in the behavior of that tumor.

Method

Tissue samples

Twenty paraffin blocks of cases previously diagnosed as Warthin's tumor were collected from the archives of the Oral Pathology Department, Faculty of Dentistry, Ain Shams University. Haematoxylin and Eosin stained sections were prepared for each specimen to confirm the diagnosis. From each of the selected paraffin blocks three sections of 4 μm thick were cut and mounted on positively charged slides (Biogenex Corp, California, USA) for immunohistochemical staining.

Reagents

Monoclonal antibodies against mdm2, p27 Kip1 and bcl-2 were used. These antibodies recognized their corresponding proteins in paraffin-embedded sections. Monoclonal IgG-Kappa antibody against mdm2 was purchased from (Zymed Laboratories Inc., USA). It recognizes epitope located within the amino acid 26-169 of the human mdm2 protein. While monoclonal antibody against p27 Kip1 was purchased from (DAKO Corporation, USA) and monoclonal antibody against bcl-2 was purchased from (Zymed Laboratories Inc., USA).

A streptavidin-biotin immunoperoxidase staining system was used for immuno-detection. It included the following reagents: The detection systems were purchased from DAKO Corporation, USA and Zymed Laboratories Inc., USA.
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The retrieval solution was purchased from BioGenex, USA.

Immunohistochemistry

Sections were deparaffinized in hot xylene. Rehydration of slides was performed by immersing slides in descending alcohol concentrations and then washed in distilled water. The sections were rinsed in two washes of phosphate-buffered saline for 5 minutes each (PBS; pH 7.4). For staining with mdm2 antibody, the sections were microwave antigen retrieved with citrate buffer solution (pH 4.8 for 15 minutes). P27 Kip-1 and bcl-2 antibodies required antigen retrieval by microwave processing in 0.2 M glycine buffer (Glyca, pH 6.2 ± 0.2, for 15 min.). Sections were again rinsed in two washes of PBS. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in water. The primary antibodies anti-mdm2, anti-p27 Kip-1 , and anti-bcl-2 were applied and the slides were incubated in a humid chamber overnight at room temperature. The sections were then washed in PBS. The secondary (Link) antibody (Biotinylated anti-mouse IgG, dilution 1:25) was then applied and the slides were incubated for 20 minutes at room temperature. After washing with PBS, the slides were incubated with peroxidase-labelled streptavidin complex for 20 minutes at room temperature, and then washed with PBS. The slides were then incubated with a solution of 3% diamino-benzidine (DAB) as the chromogen for 20 minutes. Finally, the tissues were lightly counter stained with Mayer's Haematoxylin and mounted with DPX permanent mounting medium.

Assessment

In this study, the immunostaining of each antibody was assessed in 5 serial sections from each specimen. For mdm2, tumor cells were considered immunopositive when they displayed a brownish nuclear and/or cytoplasmic immunoreactivity, p27 Kip-1 immunostaining was considered positive when tumor cells showed a nuclear immunoreactivity while bcl-2 was considered positive if the tumor cells showed cytoplasmic or perinuclear localization of immunoreactivity.

Results and discussion

Mdm2 Immunohistolochemical Results

All specimens were positively stained with mdm2. Most of the neoplastic epithelial cells forming papillae showed cytoplasmic immunopositivity, both the cuboidal basal cells and the columnar luminal cells ( fig. 1), while goblet cells were immunonegative ( fig. 2).

P27 Kip-1 Immunohistolochemical Results

All specimens showed positive immunoreactivity. Both layers of the neoplastic epithelium (basal and columnar cells) revealed positive nuclear immunostaining, surrounded by a negatively stained stroma ( fig. 3). Basal epithelial cells showed a faint immunppositive reaction. However, a combination of cytoplasmic and nuclear staining was observed ( fig. 4). Myoepithelial cells were negatively stained ( fig. 4).

Bcl-2 Immunohistolochemical Results

Eighteen out of 20 cases (90%) were positively stained. Both layers of the neoplastic epithelium (basal and luminal cells) showed positive immunoreactivity, with cytoplasmic localization, surrounded by a negatively stained stroma ( fig. 5). Few basal cells were faintly positive while others were immunonegative. Myoepithelial and goblet cells were also negative ( fig. 6). Areas of budding in epithelial cells showed immunopositive reaction ( fig. 6).

Discussion

This study was aimed to investigate the immunoexpression of mdm2 oncoprotein, p27 Kip1 tumor suppressor protein as well as bcl-2 anti-apoptotic protein in Warthin's tumor as a trial to clarify the possible role of these families in the pathogenesis of this unique tumor of salivary glands.

immunopositive epithelial cells forming papillary growth Figure 1 immunopositive epithelial cells forming papillary growth. The C.T. stroma is negative (anti-mdm2 ×100).

Higher magnification of figure 1 showing cytoplasmic immu-nopositivity in both basal and luminal cells Earlier studies focused on the analysis of p53 gene in tumors of the salivary glands; however the connection between salivary gland carcinogenesis and the mdm2 oncoprotein was still elusive [8]. Some authors demonstrated that not only p53 gene is a potential target in neoplastic transformation, but also genes involved in the regulation of its function since mdm2 can potently regulate p53, and functions as an oncogene in the process of cell transformation [20]. Some authors considered both nuclear and cytoplasmic staining or exclusively cytoplasmic staining as positive results [21], and this was chosen for assessment of immunopositive reaction of mdm2 in this study, because a strong evidence is now available that mdm2 is an RNA binding protein that can shuttle between the nucleus and the cytoplasm [22], and both mdm2 and p53 proteins contain a nuclear-import and nuclear-export signals (NES) that enable them to be directed into the nucleus and out again towards the cytoplasm [7]. This NES of mdm2 is essential for p53 degradation by interacting with cytoplasmic proteasomes, where p53 is specifically degraded [23].

Overexpression of mdm2 protein in glandular epithelium leads to abnormal development, altered histological structure and eventually to overt epithelial neoplasia [21]. Mdm2 is considered an oncoprotein through its p53dependent and p53-independent functions, therefore the expression of mdm2 in both epithelial cell layers in Warthin's tumor might indicate a high proliferation rate of this tumor.

But since Warthin's tumor is reported to be a benign, nonaggressive tumor [24], therefore the increase amount of mdm2 in these lesions may be attributed to the expression of a smaller protein of the mdm2 molecule called p74/76 that binds to p53 and doesn't inhibit its action or even preserve its presence rather than degrade it. This unique role of p74/76 is mediated through binding with p90 leading to inhibition of its function.

Brown et al [25] stated that the human oncoprotein mdm2 arrests the cell cycle and elimination of its cell cycle inhibitory function induces tumorigenesis. They attributed this finding to the dual role of mdm2, which suggests that full-length mdm2 can induce growth arrest in some cell lines, including some normal cells. Only elimination or inactivation of the mdm2-induced G0/G1 arrest may contribute to one of the steps of tumorigenesis. Some tumor-derived cells are partially insensitive to the mdm2mediated cell cycle inhibition and mdm2 can be stably overexpressed in these tumor cells. This dual function of mdm2, suggested by other investigators [26], might explain the high level of expression of mdm2 in Warthin's tumor.

The dual role of mdm2 could depend on the level of mdm2 expressed in the individual cell and the balance of some positive and negative regulators of the cell cycle which is critical for control of cell proliferation and small differences in the levels of any of these proteins may mean large differences in cell activities [26].

Immunohistochemical results of the present study also revealed that Warthin's tumour expressed a high level of p27 Kip1 and bcl-2, with p27 Kip1 immunopositivity detected in all 20 cases, while 18/20 cases were bcl-2 immunopositive. P27 Kip1 protein was detected in both layers of the neoplastic epithelium (basal and luminal) indicating the low proliferation rate of these cells. The more faintly stained basal cells indicated their higher rate of proliferation [27].

On the other hand, bcl-2 immunopositivity was also detected in both layers of the neoplastic epithelium. Areas of budding were also immunopositive indicating the attempt of this protein to maintain the survival of these more proliferative cells and that these epithelial cells might play a role as stem cells [28].

The mutual presence of p27 Kip1 and bcl-2 proteins in these same cells indicated the protection of these slowly proliferating cells from apoptosis to maintain their survival. This conclusion may put forward that mdm2 in this lesion is not acting as oncogene, but rather act as a tumor suppressor gene or a smaller protein is expressed. High levels of p27 Kip1 favor apoptosis, which can be counteracted by other regulatory factors as bcl-2. It was observed that elevated bcl-2 expression did confer significant protection against p27 Kip1 -mediated apoptosis [27]. Bcl-2 protein promotes cell survival in slowly growing tumors and this might decreased the rate of acquiring further defects. Therefore, bcl-2 expression appears to be associated with less aggressive tumor behavior [28].

Bcl-2-Immunonegativity noted in only 2 out of 20 cases of the present study might indicated failure of bcl-2 to block apoptosis in certain circumstances such as apoptosis induced by cytotoxic T-lymphocytes [18], a finding that might indicated a reactive nature of the lymphoid tissue in Warthin's tumor [29].

Some lesions showed proliferating bud at a part of the neoplastic epithelium, that might suggest an aggressive clinical behavior of this lesion or that this lesion may turn into malignancy, however the result of this study attesting the positivity of the neoplastic epithelium for the three antibody used, it is suggested that the proliferating bud is only a part of the non-aggressive lesion as p27 Kip1 and mdm2, in this lesion, act as tumor suppressor proteins while the bcl-2 would in part protect these slowly proliferating cells from apoptosis. Therefore, it could be estimated that Warthin's tumor is slowly but continuously proliferating tumor.

Conclusion

• Mdm-2 could play a tumor-suppressor role that might be implicated with the benign behavior of Warthin's tumor.

• Mutual expression of both p27 Kip1 and bcl-2 suggested a protective role of these slowly proliferating cells from apoptosis to maintain their survival but not to increase their malignant potentiality.

Figure 2
2Higher magnification of figure 1 showing cytoplasmic immunopositivity in both basal and luminal cells. Goblet cells are immunonegative (anti-mdm2 ×200). immunopositive neoplastic epithelial cells forming papillary growth Figure 3 immunopositive neoplastic epithelial cells forming papillary growth. The C.T. stroma is negative (anti-p27 ×200). Higher magnification of figure 3 showing intense immunore-activity in both layers of neoplastic epithelial cells Figure 4 Higher magnification of figure 3 showing intense immunoreactivity in both layers of neoplastic epithelial cells. Some cells are showing both nuclear and cytoplasmic immunopositivity (anti-p27 ×400).

Positive cytoplasmic immunostaining of luminal cells and few basal epithelial cellsFigure 5 Positive cytoplasmic immunostaining of luminal cells and few basal epithelial cells. Myoepithelial cells are immunonegative. The surrounding stroma is also negative (anti-bcl2 ×200). Higher magnification of figure 5 showing intense positive cytoplasmic immunostaining of luminal cells while few basal epithelial cells are faintly positive Figure 6 Higher magnification of figure 5 showing intense positive cytoplasmic immunostaining of luminal cells while few basal epithelial cells are faintly positive. (anti-bcl2 ×400).

Monoclonal Linking Reagent: biotinylated antimouse immunoglobulin • Streptavidin Enzyme Label: horseradish peroxidaseconjugated streptavidin • Buffered Substrate: Hydrochloric acid (HCl) buffer, pH 7.5 • Enzyme Substrate: 0.6% hydrogen peroxide • DAB Chromogen Concentrate: 3.3' diaminobenzidine in chromogen solution • Buffered Wash Solution: phosphate buffered saline (PBS), 0.1 mol/l, pH 7.4 ± 2
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• Elevated bcl-2 expression in Warthin's tumor suggests a possible protection against p27 Kip1 -mediated apoptosis.
AcknowledgementsThe authors acknowledge Dr/Adel Mohamed Abd-Elazim, the chairman of oral pathology department, Faculty of Dentistry, Ain Shams University for his kind permission of collecting paraffin tissue sections from the archives of oral pathology lab and also his kind permission for utilizing the research microscope and video camera for capturing the photomicrographs included in the results of this study.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsEAE participated in the study design, collection of the background references, photomicrography of the immunohistochemical results, displaying the results of the study, writing the discussion of the results, carried out the sequence alignment and drafted the manuscript. MME participated in the study design, collection of the background references, carried out the immunohistochemical technique, participated in displaying the results of the study, writing the discussion of the results and alignment of the references.
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Epidemiological aspects of bovine trypanosomosis in an endemic focus of eastern Zambia: The role of trypanosome strain variability in disease pattern

Justin Masumu firstname.lastname@example.com
Southern African Centre for Infectious Disease Surveillance
Chuo Kiikuu
MorogoroTanzania

Laboratoire Vétérinaire Central
KinshasaDemocratic Republic of the Congo

Institut National de Recherche Biomédicale
Kinshasa I
Avenue de la DemocratieRDC

G Tshilenge
Southern African Centre for Infectious Disease Surveillance
Chuo Kiikuu
MorogoroTanzania

Laboratoire Vétérinaire Central
KinshasaDemocratic Republic of the Congo

V Mbao
Centre for Ticks and Tick-borne Diseases
LilongweMalawi

Chuo KiikuuPO Box 3297MorogoroTanzania

Epidemiological aspects of bovine trypanosomosis in an endemic focus of eastern Zambia: The role of trypanosome strain variability in disease pattern
FB016A66CF4660DCDE2648A32B0B25C810.4102/ojvr
Bovine trypanosomosis displays various epidemiological aspects in various areas.In some instances the disease has a high prevalence in animals with high impact on production whereas in other cases the disease has a low impact on production despite a high level of infection in animals.In addition epidemiological changes are frequently observed in various areas and are related to many factors including the vectors, the host, the parasites, the environment as well as the livestock management.However the implication of these factors in these changes is not fully elucidated.In eastern Zambia, factors predicting the establishment of severe infection in cattle are all present.However trypanosomosis occurring in cattle in this area has a low impact on livestock production.Several studies on the characterisation of trypanosome strains circulating in domestic and wild animals have been conducted in order to clarify the epidemiology of this disease in this area.These studies aimed at evaluating genetic and biological characteristics of these strains including their virulence profiles, their transmissibility by tsetse flies, their resistance to drugs and interference between different strains.In this review these findings are analysed in order to elucidate the implication of trypanosome strain variability in the distribution and the expression of this disease in the study area.The evolutionary trends of the situation occurring in this study area are also explained.Use of these findings is the context of disease control in the study area is further discussed.

Bovine trypanosomosis in eastern Zambia: Background

Bovine trypanosomosis is endemic in eastern province of Zambia.Previous epidemiological studies using parasitological, serological or molecular techniques showed that the prevalence of the disease varies in different districts (Machila, Sinyangwe & Mubanga 2001;Sinyangwe, Delespaux & Brandt 2004;Simukoko, Marcotty & Phiri 2007).The same variation is observed within a particular district where a high prevalence of the disease is observed in some villages compared to other.In some cases no infection has been detected in cattle kept in some villages.In 1996, trypanosome infections were found in cattle in only 74% of sampling sites with a prevalence of infection varying between villages from 0% to 64% (Sinyangwe et al. 2004).Such variations are related to many factors including the habitat fragmentation of tsetse flies that affects tsetse density in various areas (Ducheyne, Mweempwa & De Pus 2009).For example the decrease prevalence of the infection in cattle from 1998 to 2003 in Petauke where tsetse habitat has widely been fragmented supports this observation.

The temporal trends of the infection in cattle revealed no (Sinyangwe et al. 2004) or little (Simukoko et al. 2007) variations between seasons although higher prevalence seems to be observed during the rainy season (Simukoko et al. 2007) when tsetse flies are more abundant (Van den Bossche & De Deken 2002).Such variations influence drug management and, although farmers treat their animals throughout the year, most drugs are selectively administered during this period (Van den Bossche, Doran & Connor 2000).

In this area, the same selection approach is used by farmers in administrating trypanocides drugs in animals.In most cases oxen and cows are more treated (Van den Bossche et al. 2000) since these animals categories, especially oxen are more infected with trypanosomes (Simukoko et al. 2007).Although this selective treatment resulted in the reduction (1.5 treatments per year) of drugs administered to animals (Van den Bossche et al. 2000) it could not limit the development and spread of drug resistance in the area.From 1996 (Sinyangwe et al. 2004) to 2003 (Delespaux, Dinka & Masumu 2008), drug resistance to diminazene aceturate increased fivefold although drug use didn't change during this period of time (Van den Bossche et al. 2000).Despite a high proportion of resistant strains in the area farmers continue using trypanocides which effect needs to be evaluated.

Trypanosoma congolense remains the most prevalent trypanosome species affecting cattle in this area with more than 95% of the infections (Sinyangwe et al. 2004;Simukoko et al. 2007).In eastern province only the Savannah type has been previously characterised in livestock (Masumu, Marcotty & Geysen 2006a).This type was previously shown to be highly virulent in cattle (Bengaly, Sidibe & Ganaba 2002).Furthermore it is efficiently transmitted by Glossina morsitans morsitans, the main vector of trypanosomes in this area.Bloodmeal analyses revealed that cattle provide 75% of meals to G. m. morsitans in this area (Van den Bossche & Staak 1997) and thus are highly exposed to infections with these highly virulent T. congolense Savannah strains circulating in this area.However the cattle breed (Angoni) reared on the plateau of eastern province are not trypanotolerant.Taken together, these factors, including a high level of resistance to trypanocides in trypanosome strains circulating in cattle and the restricted number of treatment per year, favour for the development of an epidemic situation in cattle on the plateau of eastern province.However economical surveys revealed that the disease has a low impact on livestock production (Doran 2000).

The isolation and characterisation of trypanosome isolates from cattle in this endemic area revealed sound features that explain the epidemiology of bovine trypanosomosis in this particular area.In this review trypanosome characteristics observed in this area will be analysed for their implication in the epidemiology of bovine trypanosomosis on the plateau of eastern province in Zambia.

Biological characteristics of trypanosome strains circulating in eastern province

Trypanosomes can only influence the distribution and expression of the disease in various areas and different hosts only if they are genetically and phenotypically different.In order to test this, T. congolense circulating in the domestic and wild animals in eastern province were characterised at genetic level (Masumu, Geysen & Vansnick 2006b;Masumu, Geysen & Van den Bossche 2009a) as well as for biological characters including the virulence profile (Masumu et al. 2006a;Van den Bossche, Chitanga & Masumu 2011), the transmissibility by tsetse flies (Masumu, Marcotty & Ndeledje 2006c;Masumu, Akoda & Van den Bossche 2010) and the possible existence of cross-protection between different strains (Masumu et al., 2009).It appears that many strains of T. congolense circulate in the eastern province (Masumu et al. 2006b;2009a).Very few isolates co-circulate in different areas indicating a more restricted distribution.In trypanosomes circulating in domestic animals most of the strains were of low virulence (Masumu et al. 2006a).Only 20% of them were highly virulent.On the other hand most of the trypanosomes circulating in wild animals were highly virulent (Van den Bossche et al. 2011) highlighting the trypanotolerant trait of these animals.The presence of high prevalence of virulent strains in wild animals is also a clear indication of these animals being reservoirs of these strains in eastern province.In domestic cycle where trypanosomes circulate only in domestic animals, geographical distribution of these strains indicated that high virulent trypanosome strains are present in limited villages (4 out of 11) and different virulent profiles were observed in various areas suggesting an uneven distribution of those virulent trypanosome strains (Masumu et al. 2006a).Whether the circulation of low virulent trypanosome strains in domestic animals could protect them from the adverse affect of the high virulent strains was also assessed (Masumu et al. 2009b).Indeed there is a clear indication of the existence of cross-protection between trypanosomes of high and low virulence but the degree of protection varies in different combinations of strains.This finding suggests that the effect of virulent trypanosome strains may vary depending on the combination of the high and low virulent strains in a particular area.Only in areas where most virulent strains do not cross-react with the majority of low virulent strains will the disease have high impact on animal health and thus livestock production.In areas where high level of cross-protection is observed between strains of high and low virulence the effects of such virulent strains will be minimal.The epidemiological importance of the virulent strains was further stress by the fact they were efficiently transmitted by tsetse flies compared to the strains of low virulence (Masumu et al. 2006c).

Involvement of the trypanosome strain variability in the geographic distribution of bovine trypanosomosis in eastern province

The geographical distribution of trypanosomosis depend on multiple factors including the abundance and infection rate of tsetse flies, the livestock management, the level of drug resistance and drug use in a particular area and the degree of livestock to resist to tsetse fly bites as well as to trypanosome strains circulating in the area.In the context of eastern province, significant variations are observed in the distribution of tsetse in various areas.In most cases high prevalence of the disease is observed in areas highly infested by tsetse flies.Within the same geographic area such variations are related to the abundance of tsetse flies throughout the year.In eastern province it appears that tsetse flies are more abundant during the rainy season (Van den Bossche & De Deken, 2002) explaining the high prevalence of the disease previously observed during this period (Simukoko et al. 2007).Such variations may also be explained by the variations observed in the strains of T. congolense circulating in these areas with a high prevalence of the disease likely being observed in areas where most trypanosome strains are efficiently transmitted by tsetse flies.Although highly virulent strains are efficiently transmitted that low virulent ones, further analyses revealed that during the chronic phase of infection the transmission of low virulent strains increases (Masumu et al. 2010).This indicates that high and low virulent strains are all transmitted by tsetse flies in this area since most low virulent strains induced chronic infections in cattle.

The distribution of the disease can also be influenced by the level of drug resistance to trypanocides in strains circulating in various areas.It can be assumed that high prevalence of infection will be observed in areas where the level of drug resistance is also high.This can partially be explained by the persistence of infection after treatment with trypanocides in animals infected with resistant strains.In addition although there is not evident correlation between resistance and transmission to tsetse flies (personal observation) the level of drug resistance in individual trypanosome strains may influence the infection rate in individual tsetse flies in case of persistence of drugs in tsetse after feeding on treated animals prior infection through an infected bloodmeal.In eastern province high prevalence was observed in areas where the level of drug resistance was also high (Sinyangwe et al. 2004).

Involvement of the trypanosome strain variability in the expression of bovine trypanosomosis in eastern province

The expression of trypanosomosis in various areas varies considerably (Van den Bossche 2001).In most cases severe infections are observed when animals are kept near game areas whereas a rather mild infection occurs in areas where wild animals are rather absent.In eastern province bovine trypanosomosis is rather mild on the plateau but when livestock are kept near the Luangwa valley where wild animals are abundant they suffer from severe infections.This was previously suggested to result from the presence of heterologous challenge in wild areas as a consequence of the circulation of diverse strains in these areas (Van den Bossche 2001).Our findings revealed that even in areas where wild animals are absent several trypanosome strains circulate in domestic animals suggesting that the number of trypanosome strains is not important for the expression of the disease in a given area (Masumu et al. 2009a).Instead from the findings on virulence and cross-protection obtained in eastern province it appears that the expression of the disease in various areas is largely dependent on the virulence of trypanosome strains circulating in each particular area with severe disease being observed in areas where virulent strains are more prevalent.Our findings on high prevalence of virulent trypanosome strains in the wild environment compared to the plateau where wild animals are absent and where most strains are of low virulence support this observation (Van den Bossche 2001).However as indicated previously high level of interference between high and low virulent strains may attenuate the effects of the disease in animals previously infected with low virulent strains even after challenge with virulent strains (Masumu, Marcotty & Geerts 2009b).The low impact the disease has on the plateau of eastern province may be explained by the circulation in animals of a high prevalence of low virulent trypanosome strains and the existence of a high level of cross-protection between the few virulent strains and the high prevalent low virulent strains.On the other hand the high prevalence of virulent strains in the wild environment explains the severity of the disease observed in these areas.

Trypanosome strain variability and the development of drug resistance in eastern province

The rapid development of drug resistance in eastern province arise many questions.On the plateau of eastern province a fivefold increase of drug resistance was observed from 1996 (Sinyangwe et al. 2004) to 2003 (Delespaux et al. 2008).In the absence of factors underlying the development of drug resistance in the area (Van den Bossche et al. 2000), such increase may be explained by the circulation of resistant strains between different areas.However our findings on genetic characterisation on the plateau indicate that the circulation of trypanosome strains is very limited (Masumu et al. 2009a).Even in geographically close villages similar strains were rarely observed.Indeed the circulation of trypanosome strains in various areas can be facilitated by the movement of the infected host or infected vectors.In this area livestock movement is very restricted.In addition tsetse flies do not move freely in various areas due to the fragmentation of their habitats.Variations in the distribution of tsetse flies are more linked to the distribution of cattle (Van den Bossche & Staak 1997).These two factors are in favour of a more restriction of trypanosome strains circulating in various areas.Such restriction cannot facilitate the circulation of resistant trypanosome strains.Instead the development of drug resistance seems to be induced locally.Although genetic exchange was only reported in Trypanosoma brucei (Jenni, Marti & Schweizer 1986) not T. congolense, it may be assumed that the spread of drug resistance in eastern province is related to genetic exchange (Delespaux, Dinka & Masumu 2008) between resistant and susceptible trypanosome strains.Indeed resistant and susceptible trypanosome strains do circulate in animals belonging to the same village (Masumu et al. 2006a).Since two different strains can infect an individual animal, and tsetse flies can be infected more than once, the presence of two different strains in the same fly will favour the exchange of genetic material.Further studies are needed to clarify the role of genetic exchange in the spread of drug resistance.

From the past to the future

The high prevalence of low virulent trypanosome strains and the presence of high level of interference between strains of high and low virulence in eastern province, an area where tsetse flies take their blood meal principally from a susceptible animal i.e. cattle (Van den Bossche & Staak 1997), is a perfect illustration of an endemic situation where the vector (tsetse flies), the parasite (trypanosomes) and the host (cattle) live in harmony.The current situation results from the fragmentation of tsetse habitat and the elimination of wild animals.Indeed many decades from now wild animals were prevalent in this area.Consequently the virulence profile of trypanosome strains was similar to that occurring in Luangwa valley where the trypanotolerant trait of wild animals selects against trypanosomes of low virulence.However the elimination of wild animals on this plateau led to a situation where tsetse depends on livestock for their survival (Van den Bossche & Staak 1997) and trypanosome circulate only in susceptible animals.Since the presence of virulent strains induces severe infections in these susceptible animals, the later are either treated or die from their infection.This results in the elimination of these virulent strains and the subsequently high prevalence of strains that affect animal health to a lesser degree.Only virulent strains that can interfere with strains of low virulence can persist in the susceptible host animal thus explaining the persistence of 20% of the virulent strains in this area (Masumu et al. 2006a).

The situation prevailing on the plateau of eastern province indicates clearly that bovine trypanosomosis is endemic in this area.Although the prevalence of the disease varies largely in different areas (Machila et al. 2001;Sinyangwe et al. 2004;Simukoko et al. 2007), further changes are still to occur in the future in various areas.For example, Petauke district that had a high prevalence of the disease and a high prevalence of drug resistance in 1990s (Sinyangwe et al. 2004) is no longer the major focus of bovine trypanosomosis in eastern province.The high level of fragmentation resulting from the increased surface of cultivation of crops induced a spectacular reduction of tsetse fly abundance and subsequently a low prevalence in animals (Simukoko et al. 2007).On the other hand Katete district remains an area of concern since the level of infection in animals is still high (Simukoko et al. 2007) and more important the increase of drug resistance is alarming (Delespaux et al. 2008).

It's obvious that this situation is far from being stable in the near future.Indeed the presence of a wild environment in Luangwa valley where about 14% of wild animals are infected with trypanosomes (Anderson, Mubanga & Fevre 2011) constitutes a serious threat to livestock production in eastern province.Although habitat fragmentation renders difficult to tsetse flies to move towards districts like Katete, Petauke, other district like Msoro that is located near the game is constantly at high risk of trypanosomosis.In addition this district may constitute a buffer zone between the high risk area of the valley and the plateau of eastern province.In this district the high challenge of tsetse flies and the tranfer of highly virulent trypanosome strains from the wild environment will result in a high infection rate in livestock including pigs and small ruminants that are rather resistant to trypanosomes.This was demonstrated in a survey where all livestock species (cattle, pigs, sheep and goats) kept in Msoro district were highly infected (> 10%) with trypanosomes contrarily to the situation occurring on the plateau where most infections are restricted to cattle (Simukoko et al. 2007).Indeed in the absence of wild animals, the presence of such infections in livestock other than cattle is epidemiologically very important.First, in such areas of high risk of trypanosomosis farmers prefer exploiting small ruminants and pigs for their relative tolerance to T. congolense.Their relative number will exceed that of cattle.Further since these animals are trypanotolerant and rarely treated with trypanocides they will maintain trypanosomial infection for a long period of time and thus increase the risk of tsetse flies being infected with trypanosomes even virulent to cattle.Such risk will increase the severity of the disease in cattle in those particular areas explaining the habitual low production observed in cattle in such areas.Second this encroachment of people and livestock to the game area constitutes a new epidemiological situation where virulent trypanosome strains circulating in wild animals ( Van den Bossche et al. 2011) will gradually be introduced into the livestock management area and finally reach the plateau of eastern Zambia.Whilst such movement of virulent trypanosome strains from the wild to the interface will be favoured by tsetse flies movement, the transfer of these strains from the interface to the plateau of eastern province will probably be occasioned by livestock movement.Since cattle movement is very restricted small ruminants and pigs will be the major factor for the circulation of trypanosomes between areas and the introduction of virulent trypanosome strains in various areas including the plateau where the situation is currently more endemic and rather stable.This will be facilitated for example by the commercial and social exchanges between families in the area that increases the movement of animals from one area to another.When infected with trypanosomes, pigs and small ruminants will favour the circulation of these virulent strains even in areas further away from the game areas.It would be advisable to conduct a follow up of trypanosome strains circulating on the plateau e.i. in Katete district where trypanosomes are more resistant but rather low virulent and at the interface e.i. in Msoro district where a high prevalence of virulent strains may be present whilst being less or no resistant to trypanocides.

Trypanosome-related factors and disease control strategies

Several control strategies have been used in eastern province for bovine trypanosomosis.The extent of drug resistance to either diminazene aceturate or isometamidium chloride indicates clearly that caution should be made in using these drugs to control trypansomosis in cattle.It is not well known how cattle withstand the infection in this particular area where drug use is minimal (Van den Bossche et al. 2000) and a high proportion of trypanosome strains are resistant to either or both drugs (Delespaux et al. 2008;Sinyangwe et al. 2004).Amongst the reasons is the circulation of a high prevalence of low virulent strains in livestock.Indeed infections induced by these strains have minimal effect on livestock production.In most cases low virulent strain can affect reproductive parameters but do not necessarily cause animal death.Like in theileriosis the presence of such infection can be beneficial in protecting animals against adverse effects of virulent strains.

In this area, farmers use production-oriented strategy where only sick animals are treated.Although this strategy could not reduce the development of drug resistance it reduces animal mortality (Van den Bossche et al. 2000).Our findings and the presence of high prevalence of resistant strains are in favour of such control strategy.Not only treating sick animals also is economically acceptable by resource-limited farmers, it favours the establishment of premonition state in animals that are subsequently protected against adverse effects of virulent strains.It's possible that this strategy will remain the strategic option used by farmers in the future although it does not boost the reproduction of infected animals (Van den Bossche et al. 2000).Such control strategy cannot be applied in areas displaying a different epidemiological situation like in Msoro or the game areas of Luangwa valley where the trypanosome profiles are rather threatening for livestock.

Conclusion

Many factors can influence the epidemiology of bovine trypanosomosis in various areas.Very few studies have so far been deeply conducted for the understanding of the involvement of the parasite in the epidemiology of this disease.Indeed this can only be possible if sound information is yielded at genetic and biological levels.This implies the development of appropriate tools for genotypic and phenotypic characterisation.In the situation prevailing on the plateau of eastern province of Zambia where wild animals are not present and tsetse flies depends largely on a susceptible host, the outcome of the disease on livestock health and production was found to be correlated with the profiles of trypanosome strains circulating in animals.Disease control through parasite management leading to the establishment of premonition state in animals is thus encouraged and further supports the control strategy so far adopted by farmers.Similar features can likely be observed in areas displaying a similar epidemiological situation although cautious is needed to generalise these findings.Finally the approach adopted here to clarify the epidemiology of a particular disease in a specific epidemiological condition can be applied to any disease provided appropriate tools are available for such studies.
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Acknowledgements Competing InterestThe authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper.
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