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Immunohistochemical Study of the Inflammatory Response in the Rumen and Reticulum of Calicophoron daubneyi-infected Cattle
82
ESVP/ECVP Proceedings 2012
148:1, 2013
BALANTIDIUM COLI IN PMWS-SUSPECTED PIGS A. Szczotka and Z. Pejsak National Veterinary Research Institute, Department of Swine Diseases, Partyzantow 57, 24-100 Pulawy, Poland Introduction: Porcine circovirus type 2 (PCV2) is an aetiological agent of post weaning multisystemic wasting syndrome (PMWS) in pigs. Diarrhoea can sometimes be present in diseased animals. The aim of this study was to analyze the presence of PCV2 in tissues from PMWS-suspected pigs. Materials and Methods: Lymph nodes and intestines from three 13- to 15-week-old fatteners from a pig herd with signs of PMWS and persistent diarrhoea were analyzed by in-situ hybridization for the presence of PCV2. Sections were also stained by haematoxyin and eosin. Faeces and scrapings from the intestinal mucosa were subjected to routine bacteriological examination. Results: All of the pigs were PMWS negative. No pathogenic microflora was identified in the faecal samples. However, Balantidium coli was found in the intestines. The protozoons were present not only in the intestinal lumen, but also in the intestinal mucosa. Conclusions: B. coli is an opportunistic parasite of mammals, including man. The infection is usually subclinical, but in immunocompromised individuals may lead to fulminant infection with bloody and mucus-containing diarrhoea. The identification of B. coli in pigs showing symptoms of PMWS and antibiotic non-responsive diarrhoea may indicate that these animals might have already recovered from PMWS, but due to PCV2-related immunosuppression B. coli infection complicated the clinical presentation of the disease.
IMMUNOHISTOCHEMICAL STUDY OF THE INFLAMMATORY RESPONSE IN THE RUMEN AND RETICULUM OF CALICOPHORON DAUBNEYI-INFECTED CATTLE M.C. Ferreras *, V. P erez *, M.C. Gonz alez-Lanza *, J. Benavides *, M. Mezo y, M. Fuertes *, M. Gonz alez-Warleta *, L. Delgado *, A. Martınez-Ibeas * and M.Y. Manga-Gonz alez* *Instituto de Ganaderıa de Monta~na CSIC-ULE, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Leon, Campus de Vegazana s/ n, 24071 Leon and yCentro de Investigaciones Agrarias de Mabegondo, 15318 Abegondo, ACoru~na, Spain Introduction: The importance of changes caused by the rumen and reticulum trematode parasite Calicophoron daubneyi (Digenea: Paramphistomidae) is unclear. Most authors suggest that the adult paramphistomes are relatively harmless. The aim of this work was to characterize the inflammatory cells in the rumen and reticulum from cattle infected naturally with C. daubneyi. Materials and Methods: Samples of rumen and reticulum of 38 calves infected with C. daubneyi flukes were examined by histology and immunohistochemistry using antibodies against macrophages (CD68), T cells (CD3, CD4 and CD8) and B cells (CD79). Results: Numerous inflammatory cells were seen in rumen papillae and reticular folds where the ventral sucker of the parasites was attached. CD4+ T lymphocytes, CD68+ macrophages, eosinophils and globular leucocytes were found in intraepithelial and subepithelial locations. A mixed population of lymphocytes, eosinophils, mast cells and macrophages was present in the reticular and ruminal lamina propria. Most of the cells from lymphoid aggregates and follicles were positive for the B-cell marker. CD3+ T lymphocytes were mostly CD4+, although sporadic CD8+ cells were observed. Conclusions: In natural conditions, adult flukes of C. daubneyi have pathogenic effects and may induce a humoral and cell-mediated local immune response in cattle.
EARLY HEPATIC CHANGES AND IMMUNE RESPONSE IN GOATS VACCINATED WITH A RECOMBINANT GLUTATHIONE TRANSFERASE SIGMA CLASS AND CHALLENGED WITH FASCIOLA HEPATICA R. Zafra *, I.L. Pacheco y, L. Buffoni y, A. Escamilla y, A. Martınez-Moreno y, E.J. LaCourse z, P.M. Brophy z and J. P erezy *University of Las Palmas, UK, yUniversity of Cordoba, Spain and z Aberystwyth University, Aberystwyth, UK Introduction: The aim of the present work was to study early hepatic changes and local immune response in goats immunized with recombinant GST sigma class and experimentally infected with F. hepatica. Materials and Methods: Twenty-seven Malague~ na breed goats were divided into three groups. Group 1 (n 5 7) was unimmunized and uninfected; group 2 (n 5 10) was immunized with adjuvant Quil A and infected; group 3 (n 5 10) was immunized with rFhGST-S1 and infected. Three goats from each group were killed at 7e9 days post infection (dpi) to evaluate early changes and immune response by histopathology and immunohistochemistry. Results: In the rFhGST-S1 group, two out of three goats showed low hepatic damage at early infection stages. This response was associated with a severe infiltrate of eosinophils in peritoneal fluid and hepatic necrotic foci, high iNOS expression in peritoneal cells and abundant infiltration of eosinophils surrounding hepatic migrating flukes. CD2+, CD4+ and CD8+ T lymphocytes were found in the vicinity of the necrotic areas, but they were absent in the vicinity of migrating larvae. Conclusions: Results of the present work suggest that the early response mediated by eosinophils plays a key role in the effective response against F. hepatica in goats.
FLOW CYTOMETRY FROM WHOLE BLOOD OF GOATS INFECTED EXPERIMENTALLY WITH FASCIOLA HEPATICA R. Zafra *, L. Buffoni y, F.J. Martınez-Moreno y, M.J. Bautista y, erez y and A. MartınezA. Escamilla y, I.L. Pacheco y, J. P Morenoy *University of Las Palmas de Gran Canaria and yUniversity of Cordoba, Spain Introduction: Fasciolosis caused by Fasciola hepatica is an important disease causing important economic losses worldwide and affecting ruminants, but also man (zoonosis). The aim of this work was to study lymphocyte subsets (CD4+, CD8+ and WC1+) using flow cytometry from whole blood in order to characterize the immune response in goats infected experimentally with F. hepatica. Materials and Methods: Twenty-four Malague~ na breed goats were divided into four groups of six animals each. Group 1 was immunized with recombinant CL1, group 2 with recombinant GST sigma class, group 3 with the adjuvant Quil A, and group 4 was the uninfected control. Blood samples were collected at: (1) pre-immunization, (2) at the 5th week post infection, and (3) at the 12th week post infection. Flow cytometry was carried out using a COULTER EPICS-XL cytometer. Results: Flow cytometry assay showed that at the 5th week post infection there was a significant decrease (P 5 0.012) in CD4+ lymphocytes in vaccinated groups in comparison with group 4 (uninfected and unimmunized) animals. No significant differences were found among the immunized groups. Conclusions: This is the first report of flow cytometry from whole blood in ruminants infected experimentally with Fasciola hepatica. The decrease in CD4+ cells in the 5th week post infection coincides with the migratory stage of the parasite and could explain the lack of effective immune response during this critical period of the infection.
ESVP/ECVP Proceedings 2012
148:1, 2013
BALANTIDIUM COLI IN PMWS-SUSPECTED PIGS A. Szczotka and Z. Pejsak National Veterinary Research Institute, Department of Swine Diseases, Partyzantow 57, 24-100 Pulawy, Poland Introduction: Porcine circovirus type 2 (PCV2) is an aetiological agent of post weaning multisystemic wasting syndrome (PMWS) in pigs. Diarrhoea can sometimes be present in diseased animals. The aim of this study was to analyze the presence of PCV2 in tissues from PMWS-suspected pigs. Materials and Methods: Lymph nodes and intestines from three 13- to 15-week-old fatteners from a pig herd with signs of PMWS and persistent diarrhoea were analyzed by in-situ hybridization for the presence of PCV2. Sections were also stained by haematoxyin and eosin. Faeces and scrapings from the intestinal mucosa were subjected to routine bacteriological examination. Results: All of the pigs were PMWS negative. No pathogenic microflora was identified in the faecal samples. However, Balantidium coli was found in the intestines. The protozoons were present not only in the intestinal lumen, but also in the intestinal mucosa. Conclusions: B. coli is an opportunistic parasite of mammals, including man. The infection is usually subclinical, but in immunocompromised individuals may lead to fulminant infection with bloody and mucus-containing diarrhoea. The identification of B. coli in pigs showing symptoms of PMWS and antibiotic non-responsive diarrhoea may indicate that these animals might have already recovered from PMWS, but due to PCV2-related immunosuppression B. coli infection complicated the clinical presentation of the disease.
IMMUNOHISTOCHEMICAL STUDY OF THE INFLAMMATORY RESPONSE IN THE RUMEN AND RETICULUM OF CALICOPHORON DAUBNEYI-INFECTED CATTLE M.C. Ferreras *, V. P erez *, M.C. Gonz alez-Lanza *, J. Benavides *, M. Mezo y, M. Fuertes *, M. Gonz alez-Warleta *, L. Delgado *, A. Martınez-Ibeas * and M.Y. Manga-Gonz alez* *Instituto de Ganaderıa de Monta~na CSIC-ULE, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Leon, Campus de Vegazana s/ n, 24071 Leon and yCentro de Investigaciones Agrarias de Mabegondo, 15318 Abegondo, ACoru~na, Spain Introduction: The importance of changes caused by the rumen and reticulum trematode parasite Calicophoron daubneyi (Digenea: Paramphistomidae) is unclear. Most authors suggest that the adult paramphistomes are relatively harmless. The aim of this work was to characterize the inflammatory cells in the rumen and reticulum from cattle infected naturally with C. daubneyi. Materials and Methods: Samples of rumen and reticulum of 38 calves infected with C. daubneyi flukes were examined by histology and immunohistochemistry using antibodies against macrophages (CD68), T cells (CD3, CD4 and CD8) and B cells (CD79). Results: Numerous inflammatory cells were seen in rumen papillae and reticular folds where the ventral sucker of the parasites was attached. CD4+ T lymphocytes, CD68+ macrophages, eosinophils and globular leucocytes were found in intraepithelial and subepithelial locations. A mixed population of lymphocytes, eosinophils, mast cells and macrophages was present in the reticular and ruminal lamina propria. Most of the cells from lymphoid aggregates and follicles were positive for the B-cell marker. CD3+ T lymphocytes were mostly CD4+, although sporadic CD8+ cells were observed. Conclusions: In natural conditions, adult flukes of C. daubneyi have pathogenic effects and may induce a humoral and cell-mediated local immune response in cattle.
EARLY HEPATIC CHANGES AND IMMUNE RESPONSE IN GOATS VACCINATED WITH A RECOMBINANT GLUTATHIONE TRANSFERASE SIGMA CLASS AND CHALLENGED WITH FASCIOLA HEPATICA R. Zafra *, I.L. Pacheco y, L. Buffoni y, A. Escamilla y, A. Martınez-Moreno y, E.J. LaCourse z, P.M. Brophy z and J. P erezy *University of Las Palmas, UK, yUniversity of Cordoba, Spain and z Aberystwyth University, Aberystwyth, UK Introduction: The aim of the present work was to study early hepatic changes and local immune response in goats immunized with recombinant GST sigma class and experimentally infected with F. hepatica. Materials and Methods: Twenty-seven Malague~ na breed goats were divided into three groups. Group 1 (n 5 7) was unimmunized and uninfected; group 2 (n 5 10) was immunized with adjuvant Quil A and infected; group 3 (n 5 10) was immunized with rFhGST-S1 and infected. Three goats from each group were killed at 7e9 days post infection (dpi) to evaluate early changes and immune response by histopathology and immunohistochemistry. Results: In the rFhGST-S1 group, two out of three goats showed low hepatic damage at early infection stages. This response was associated with a severe infiltrate of eosinophils in peritoneal fluid and hepatic necrotic foci, high iNOS expression in peritoneal cells and abundant infiltration of eosinophils surrounding hepatic migrating flukes. CD2+, CD4+ and CD8+ T lymphocytes were found in the vicinity of the necrotic areas, but they were absent in the vicinity of migrating larvae. Conclusions: Results of the present work suggest that the early response mediated by eosinophils plays a key role in the effective response against F. hepatica in goats.
FLOW CYTOMETRY FROM WHOLE BLOOD OF GOATS INFECTED EXPERIMENTALLY WITH FASCIOLA HEPATICA R. Zafra *, L. Buffoni y, F.J. Martınez-Moreno y, M.J. Bautista y, erez y and A. MartınezA. Escamilla y, I.L. Pacheco y, J. P Morenoy *University of Las Palmas de Gran Canaria and yUniversity of Cordoba, Spain Introduction: Fasciolosis caused by Fasciola hepatica is an important disease causing important economic losses worldwide and affecting ruminants, but also man (zoonosis). The aim of this work was to study lymphocyte subsets (CD4+, CD8+ and WC1+) using flow cytometry from whole blood in order to characterize the immune response in goats infected experimentally with F. hepatica. Materials and Methods: Twenty-four Malague~ na breed goats were divided into four groups of six animals each. Group 1 was immunized with recombinant CL1, group 2 with recombinant GST sigma class, group 3 with the adjuvant Quil A, and group 4 was the uninfected control. Blood samples were collected at: (1) pre-immunization, (2) at the 5th week post infection, and (3) at the 12th week post infection. Flow cytometry was carried out using a COULTER EPICS-XL cytometer. Results: Flow cytometry assay showed that at the 5th week post infection there was a significant decrease (P 5 0.012) in CD4+ lymphocytes in vaccinated groups in comparison with group 4 (uninfected and unimmunized) animals. No significant differences were found among the immunized groups. Conclusions: This is the first report of flow cytometry from whole blood in ruminants infected experimentally with Fasciola hepatica. The decrease in CD4+ cells in the 5th week post infection coincides with the migratory stage of the parasite and could explain the lack of effective immune response during this critical period of the infection.