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Dermacentor reticulatus Ticks as Possible Vectors of Trueperella pyogenes Infection in European Bison (Bison bonasus): Preliminary Studies
120
ESVP, ECVP and NSVP Proceedings 2015
J. Comp. Path. 2016, Vol. 154, 58e123
CYTOKINE EXPRESSION IN TONSILS IN RESPONSE TO INFECTION WITH DIFFERENT PRRSV-1 STRAINS S.P. Amarilla *, I.M. Rodrıguez-G omez *, J. G omez-Laguna y, L. Carrasco *, S.P. Graham z,x, J.-P. Frossard z, F. Steinbach z,x and F.J. Salguerox *Department of Anatomy and Comparative Pathology, University of Cordoba, y CICAP e Food Research Centre, Department of Virology, Cordoba, Spain, z Animal and Plant Health Agency, Surrey and xDepartment of Pathology and Infectious Diseases, Guildford, UK Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) strains show differences among and within genotypes. The aim of this study was to determine the expression of pro-inflammatory cytokines in the tonsils of pigs infected experimentally with different PRRSV-1 strains. Materials and Methods: Fifty-seven 5-week-old pigs were inoculated with either (1) Lelystad-prototype virus (LV), (2) 215-06, British field strain, (3) SU1-Bel, a highly virulent strain from Belarus, or (4) a commercial modified live vaccine (DV group, considered as the control group). Animals were killed at 3, 7 and 35 dpi. Histomorphometry analysis and immunohistochemistry studies of tonsils were performed by using specific antibodies against PRRSV, IL-1a, TNF-a and IL-6. Cell types were identified based on their morphological characteristics. Results: All three pro-inflammatory cytokines showed the same kinetics in the tonsils, peaking at 7 dpi and decreasing at the end of the study. These kinetics also coincided with viral antigen distribution. Moreover, the expression of TNF-a and IL-6 was higher in those animals infected with SU1-Bel and LV strains, followed by 215-06 when compared with the DV group. The histomorphometry analysis did not reveal any change with regard to lymphoid follicle (LF) diameter or number of cells within each LF in any of the infected groups. Conclusions: The virulent Eastern European strain of PRRSV induced higher expression of pro-inflammatory cytokines in the tonsil, mainly of TNF-a and IL-6, when compared with the Lelystad strain and the British field isolate (215-06). Moreover, no suggestion of LF activation was observed in the tonsils from these animals.
DISSEMINATED MYCOBACTERIUM MALMOENSE INFECTION IN A CAT ackman y, V. Karkamo *, P. Syrj€ a *, M. Speeti y, H. B€ L. Savolainen z, M. Vaara z and S. Sukura* *University of Helsinki, Faculty of Veterinary Medicine, Department of Basic Veterinary Sciences, yHerttoniemi Veterinary Clinic and zHUSLAB, Clinical Microbiology, Bacteriology Unit, Helsinki, Finland Introduction: Mycobacterial infections in cats are divided into tuberculosis caused by M. tuberculosis, M. bovis and M. microti, the feline leprosy syndrome caused by M. lepraemurium, M. visibile, Mycobacterium spp., and non-tuberculous mycobacteriosis (NTM) caused by facultative pathogenic opportunistic saprophytes, like Mycobacterium malmoensis. Originating from the environment, M. malmoensis has been isolated from lung lesions of immunocompromised people. Previously, it has been found in skin lesions and bite wounds in cats. Materials and Methods: An adult, privately owned, domestic short haired cat from suburban Helsinki was examined due to polydipsia and ill thrift. The CBC, clinical chemistry and FIV and FeLV status were determined. The cat was examined radiographically and by abdominal ultrasound. FNA was done from the liver and stained by MayeGruenwaldeGiemsa and ZiehleNeelsen. The cat was humanely destroyed after the presumptive diagnosis and underwent complete necropsy examination with bacterial culture of internal organs. Results: Clinical examination revealed mild enlargement of the peripheral lymph nodes and a diffusely enlarged liver was noted on ultrasound examination. Liver cytology showed large amounts of mycobacteria-like organisms within a severe pyogranulomatous inflammatory response. The cat tested negative for FIV and FeLV. At necropsy examination, severe, disseminated granulomatous inflammation with intralesional M. malmoensis affected several organs including liver, lung, lymph nodes, eyes, brain, kidney and the adrenals. Conclusions: NTM can cause a disseminated mycobacteriosis in cats. The organism was present in large number in this case and was detected by cytology, emphasizing the value of cytology in diagnosing diffuse liver lesions.
DERMACENTOR RETICULATUS TICKS AS POSSIBLE VECTORS OF TRUEPERELLA PYOGENES INFECTION IN EUROPEAN BISON (BISON BONASUS): PRELIMINARY STUDIES M. Rzewuska *, A. Rodo y and W. Bieleckiy *Department of Preclinical Sciences and yDepartment of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences-SGGW, Poland Introduction: Since 1980, balanoposthitis (chronic disease of the external genital organs) has been observed in the bulls of European bison (Bison bonasus) on the Polish side of the Bia1owie_za Primeval Forest. The disease is characterized by oedema and necrosis of the skin, and the presence of exudate. Recent studies on the aetiological agent have reported the correlation between the presence of the bacterium Trueperella pyogenes and the development of disease. This bacteria may also cause pathological lesions in other organs. Moreover, considering the endemic occurrence of the disease, blood-sucking arthropods have been suspected to play an important role in transmission of balanoposthitis. Materials and Methods: Questing and feeding ticks of two species were collected from 16 European bison (five females and 11 males) with clinical signs of T. pyogenes infection. The presence of T. pyogenes in the ticks was determined by PCR. Results: Sixteen Ixodes ricinus ticks (11 females and five males) and 31 Dermacentor reticulatus ticks (six females and 25 males) were studied for the presence of T. pyogenes. The DNA of T. pyogenes was found only in the D. reticulatus ticks, collected from two European bison. Conclusions: The data obtained in this preliminary study indicate Dermacentor reticulatus as a reservoir of T. pyogenes and a possible vector of this pathogen causing infection in European bison (Bison bonasus) on the Polish side of the Bia1owie_za Primeval Forest.
DETECTION OF BOVINE LEUKAEMIA VIRUS INFECTION IN CATTLE WITH THE USE OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD M. Szczotka *, A. Pluta *, J. Kuzmak *, E. Iwan * and A. Szczotka-Bochniarzy *Department of Biochemistry and yDepartment of Swine Diseases, National Veterinary Research Institute, Pulawy, Poland Introduction: The bovine leukaemia virus (BLV) is a causative agent of bovine leukaemia and is classified in the genus Deltaretrovirus, family Retroviridae. The aim of this study was to apply the loopmediated isothermal amplification (LAMP) method for diagnosis of BLV infection in cattle. The LAMP method is a novel technique for the amplification of nucleic acid under isothermal conditions, with the use of specifically designed set of primers and Bst polymerase. Materials and Methods: Blood, lymphoid tissue samples and neural tissue from serologically-positive cattle were investigated using the LAMP method (Mast Isoplex DNA kit, Germany), nested-PCR and real-time PCR. As a positive control, the FLK-BLV cell line was used. The specificity of the LAMP-amplified product was analyzed by digestion with the HaeIII enzyme. Telomerase activity and telomere length was also determined by real-time PCR. Results: The results of LAMP and real-time PCR showed high agreement in all investigated samples. Non-specific reactions were not observed. The detection limit of LAMP was approximately 2 copies per sample. Very high relative telomerase activities were found in dendritic cells, sera of cattle with lymphocytosis and in the FLKBLV cell line. The relative telomere length (RTL) was much shorter in leukaemic animals. Conclusion: The LAMP assay proved to be significantly more sensitive and faster than conventional PCR for BLV identification, therefore it can be recommended for routine diagnosis of the disease. Measurement of telomere length and telomerase activity might be useful in disease stage monitoring and targetted therapy of the tumours.
ESVP, ECVP and NSVP Proceedings 2015
J. Comp. Path. 2016, Vol. 154, 58e123
CYTOKINE EXPRESSION IN TONSILS IN RESPONSE TO INFECTION WITH DIFFERENT PRRSV-1 STRAINS S.P. Amarilla *, I.M. Rodrıguez-G omez *, J. G omez-Laguna y, L. Carrasco *, S.P. Graham z,x, J.-P. Frossard z, F. Steinbach z,x and F.J. Salguerox *Department of Anatomy and Comparative Pathology, University of Cordoba, y CICAP e Food Research Centre, Department of Virology, Cordoba, Spain, z Animal and Plant Health Agency, Surrey and xDepartment of Pathology and Infectious Diseases, Guildford, UK Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) strains show differences among and within genotypes. The aim of this study was to determine the expression of pro-inflammatory cytokines in the tonsils of pigs infected experimentally with different PRRSV-1 strains. Materials and Methods: Fifty-seven 5-week-old pigs were inoculated with either (1) Lelystad-prototype virus (LV), (2) 215-06, British field strain, (3) SU1-Bel, a highly virulent strain from Belarus, or (4) a commercial modified live vaccine (DV group, considered as the control group). Animals were killed at 3, 7 and 35 dpi. Histomorphometry analysis and immunohistochemistry studies of tonsils were performed by using specific antibodies against PRRSV, IL-1a, TNF-a and IL-6. Cell types were identified based on their morphological characteristics. Results: All three pro-inflammatory cytokines showed the same kinetics in the tonsils, peaking at 7 dpi and decreasing at the end of the study. These kinetics also coincided with viral antigen distribution. Moreover, the expression of TNF-a and IL-6 was higher in those animals infected with SU1-Bel and LV strains, followed by 215-06 when compared with the DV group. The histomorphometry analysis did not reveal any change with regard to lymphoid follicle (LF) diameter or number of cells within each LF in any of the infected groups. Conclusions: The virulent Eastern European strain of PRRSV induced higher expression of pro-inflammatory cytokines in the tonsil, mainly of TNF-a and IL-6, when compared with the Lelystad strain and the British field isolate (215-06). Moreover, no suggestion of LF activation was observed in the tonsils from these animals.
DISSEMINATED MYCOBACTERIUM MALMOENSE INFECTION IN A CAT ackman y, V. Karkamo *, P. Syrj€ a *, M. Speeti y, H. B€ L. Savolainen z, M. Vaara z and S. Sukura* *University of Helsinki, Faculty of Veterinary Medicine, Department of Basic Veterinary Sciences, yHerttoniemi Veterinary Clinic and zHUSLAB, Clinical Microbiology, Bacteriology Unit, Helsinki, Finland Introduction: Mycobacterial infections in cats are divided into tuberculosis caused by M. tuberculosis, M. bovis and M. microti, the feline leprosy syndrome caused by M. lepraemurium, M. visibile, Mycobacterium spp., and non-tuberculous mycobacteriosis (NTM) caused by facultative pathogenic opportunistic saprophytes, like Mycobacterium malmoensis. Originating from the environment, M. malmoensis has been isolated from lung lesions of immunocompromised people. Previously, it has been found in skin lesions and bite wounds in cats. Materials and Methods: An adult, privately owned, domestic short haired cat from suburban Helsinki was examined due to polydipsia and ill thrift. The CBC, clinical chemistry and FIV and FeLV status were determined. The cat was examined radiographically and by abdominal ultrasound. FNA was done from the liver and stained by MayeGruenwaldeGiemsa and ZiehleNeelsen. The cat was humanely destroyed after the presumptive diagnosis and underwent complete necropsy examination with bacterial culture of internal organs. Results: Clinical examination revealed mild enlargement of the peripheral lymph nodes and a diffusely enlarged liver was noted on ultrasound examination. Liver cytology showed large amounts of mycobacteria-like organisms within a severe pyogranulomatous inflammatory response. The cat tested negative for FIV and FeLV. At necropsy examination, severe, disseminated granulomatous inflammation with intralesional M. malmoensis affected several organs including liver, lung, lymph nodes, eyes, brain, kidney and the adrenals. Conclusions: NTM can cause a disseminated mycobacteriosis in cats. The organism was present in large number in this case and was detected by cytology, emphasizing the value of cytology in diagnosing diffuse liver lesions.
DERMACENTOR RETICULATUS TICKS AS POSSIBLE VECTORS OF TRUEPERELLA PYOGENES INFECTION IN EUROPEAN BISON (BISON BONASUS): PRELIMINARY STUDIES M. Rzewuska *, A. Rodo y and W. Bieleckiy *Department of Preclinical Sciences and yDepartment of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences-SGGW, Poland Introduction: Since 1980, balanoposthitis (chronic disease of the external genital organs) has been observed in the bulls of European bison (Bison bonasus) on the Polish side of the Bia1owie_za Primeval Forest. The disease is characterized by oedema and necrosis of the skin, and the presence of exudate. Recent studies on the aetiological agent have reported the correlation between the presence of the bacterium Trueperella pyogenes and the development of disease. This bacteria may also cause pathological lesions in other organs. Moreover, considering the endemic occurrence of the disease, blood-sucking arthropods have been suspected to play an important role in transmission of balanoposthitis. Materials and Methods: Questing and feeding ticks of two species were collected from 16 European bison (five females and 11 males) with clinical signs of T. pyogenes infection. The presence of T. pyogenes in the ticks was determined by PCR. Results: Sixteen Ixodes ricinus ticks (11 females and five males) and 31 Dermacentor reticulatus ticks (six females and 25 males) were studied for the presence of T. pyogenes. The DNA of T. pyogenes was found only in the D. reticulatus ticks, collected from two European bison. Conclusions: The data obtained in this preliminary study indicate Dermacentor reticulatus as a reservoir of T. pyogenes and a possible vector of this pathogen causing infection in European bison (Bison bonasus) on the Polish side of the Bia1owie_za Primeval Forest.
DETECTION OF BOVINE LEUKAEMIA VIRUS INFECTION IN CATTLE WITH THE USE OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD M. Szczotka *, A. Pluta *, J. Kuzmak *, E. Iwan * and A. Szczotka-Bochniarzy *Department of Biochemistry and yDepartment of Swine Diseases, National Veterinary Research Institute, Pulawy, Poland Introduction: The bovine leukaemia virus (BLV) is a causative agent of bovine leukaemia and is classified in the genus Deltaretrovirus, family Retroviridae. The aim of this study was to apply the loopmediated isothermal amplification (LAMP) method for diagnosis of BLV infection in cattle. The LAMP method is a novel technique for the amplification of nucleic acid under isothermal conditions, with the use of specifically designed set of primers and Bst polymerase. Materials and Methods: Blood, lymphoid tissue samples and neural tissue from serologically-positive cattle were investigated using the LAMP method (Mast Isoplex DNA kit, Germany), nested-PCR and real-time PCR. As a positive control, the FLK-BLV cell line was used. The specificity of the LAMP-amplified product was analyzed by digestion with the HaeIII enzyme. Telomerase activity and telomere length was also determined by real-time PCR. Results: The results of LAMP and real-time PCR showed high agreement in all investigated samples. Non-specific reactions were not observed. The detection limit of LAMP was approximately 2 copies per sample. Very high relative telomerase activities were found in dendritic cells, sera of cattle with lymphocytosis and in the FLKBLV cell line. The relative telomere length (RTL) was much shorter in leukaemic animals. Conclusion: The LAMP assay proved to be significantly more sensitive and faster than conventional PCR for BLV identification, therefore it can be recommended for routine diagnosis of the disease. Measurement of telomere length and telomerase activity might be useful in disease stage monitoring and targetted therapy of the tumours.