Development of Immune Response to Experimental Bovine Trichophyton vervucosum Infection

Abstract— Cell mediated and humoral immune responses to experimental Trichophyton verrucosum infection were assessed by sequential cutaneous biopsies, antibody assessments and microscopic monitoring of fungal presence. Histopathologic examination showed the accrual of lymphocytes and other inflammatory cells in the dermis of infected sites. Immunoperoxidase staining of frozen sections with monoclonal antibody preparations revealed an influx of macrophages, BoCD4+ and B0CD8+ lymphocytes and γδ T cells from the 5th day to the 33rd day of infection. A moderate influx of B cells was observed. Protein G-colloidal gold staining revealed the presence of immunoglobulins in the dermis and superficial epidermal layers. Trichophyton specific serum antibodies appeared between days 33 and 55. Microscopic assessment of infected tissues revealed an increase in T, verrucosum elements (mycelium and ectothrix spores) from days 19 to 55. Fungal elements in infected areas did not decrease until after both humoral and cell mediated elements of the immune response were established. These responses imply a combination of cell mediated and humoral events were associated with T. verrucosum immunity and clearance in the calf. Résumé— Le réponse immunitaire humorale et cellulaire a une infection expérimentale àT. verrucosum a été appréciée par des biospsies cutanées successives, des dosages d'anticorps et la recherche microscopique de champignons. L'examen histopathologique a montré un afflux de lymphocytes et d'autres cellules inflammatoires dane le derme des sites infectés. Les marquages en immunopéroxydase par un anticorps monoclonal de coupes congelées a montré un influx de macrophages, lymphocytes BoCD4+ et BoCD8+ et des cellules T γδ, du 5e au 33e jour de l'infection. Un marquage par une protéine G—or colloidal a révélé d'immunogolglobulines dans le derme et les couches supéerficielles de l'épiderme. Les anticorps spécifiques de Trichophyton sont apparus entre 33 et 55 jours. L'examen microscopique des tissus infectés a révélé une augmentation du nombre d'éléments de T. verrucosum (mycelium et spores ectothrix) du 19e au 55e jours. Les éléments fongiques dans les zones infectées n'ont pas diminué avant que les réponses humorales et cellulaires ne solent établies. Ces résponses impliquent qu'une coopération des réponses humorales et cellulaires étaient associées dans l'immunité et la défense contre T. verrucosum. Zusammenfassung— Die zellvermittelten und humoralen Immunantworten auf die experimentelle Infektion mit T. verrucosum wurden durch eine Serie von Hautbiopsien, Antikörperuntersuchungen und mikroskopischer Untersuchung auf das Vorhandensein von Pilzen ausgewertet. Die histopathologische Untersuchung zeigte eine Ansammlung von Lymphozyten und anderen Entzündungszellen in der Dermis der infizierten Stellen. Die Immunperoxidasefärbung der Gefrierschnitte mit monoklonalen Antikörperzubereitungen zeigte einen Influx von Makrophagen, BoCd4-und BoCD8-Lymphozyten und gamma-delta-T-Zellen vom 5. bis zum 33. Tag der Infektion. Es wurde auch ein mäßiger Influx von B-Zellen beobachtet. Die Protein-G-kolloidale Goldfärbung zeigte die Anwesenheit von Immunglobulinen in der Dermis und den oberflächlichen epidermalen Schichten. Trichophyton-spezifische Serumantikörper traten zwischen Tag 33 und 55 auf. Die mikroskopische Untersuchung infizierter Gewebe zeigte eine Zunahme von T. verrucosum-Bestandteilen (Myzel und exktothrixe Sporen) vom Tag 19 bis 55. Pilzteile in infizierten Bereichen verminderten sich weder, nachdem humorale, noch nachdem zellvermittelte Elemente der Immunantwort auftraten. Diese Reaktionen deuten an, daß eine Kombination von zellvermittelten und humoralen Vorgängen im Zusammenhang mit T. verrucosum-Immumtät und Abheilung beim Kalb vorliegt. Resumen Por medio de biposias cutáneas secuenciales, medida de anticuerpos y exámen microscópico de presencia de hongos, se estudió la respuesta inmunitaria de tipo humoral y celular producida por la infección experimental con T. verrucosum. El exámen histopatológico reveló la presencia de agregación de linfocitos y otras células inflamatorias procedentes de la dermis de los puntos infectados. La tintura por medio de inmunoperoxidasa de las secciones congeladas, con preparaciones monoclonales de anticuerpos, demostró un aflujo de macrófagos BoCD4 + y BoCD8 + linfocitos y linfocitos, Tαδ, desde el quinto hasta el día 33 la infección. También se observó un aflugo moderado de linfocitos B. La tintura aúrica de proteina coloidal G reveló la presencia de inmunoglobulinas en al dermis y capas superficiales de la epidermis. Los anticuerpos específicos para la especie Trichophyton aparecieron entre los días 33 y 55. El exámen microscópico de los tejidos afectados demostró un incremento, de los elementos füngicos T. verrucosum (micelio y esporas exótricas) desde los días 19 al 55. Los elementos fúngicos en áreas infectadas no disminuyeron hasta después del establecimiento de ambos tipos de respuesta inmunitaria, humoral y celular. Estas respuestas implican que la combinación de ciertos fenómenos de inmunidad celular y humoral, están relacionados con la desaparición y la inmunidad de la infección producia por T. verrucosum en el ternero.     

Improving the specificity of immunodiagnosis for porcine brucellosis

This report describes the use of cell mediated immunity to improve specificity of current diagnosis for Brucella suis. Diagnosis is problematic due to cross reactions that lead to false positive serological reactions (FPSR) in the standard diagnostic tests. A common cause of this cross reactivity is infection with the organism Yersinia enterocolitica O:9. Gottingen™ mini-pigs were experimentally infected with B. suis biovar I field strain or Y. enterocolitica serotype O:9 biotype 3. Infection was followed for 70 days. During this time whole blood stimulation assays were set up using Brucella specific antigen. IFNγ was measured in the supernatants (SN) from these assays by ELISA. Concurrent standard serological tests were carried out. The results indicate that the IFNγ assay is specifically able to distinguish Y. enterocolitica O:9 infection from a B. suis infection in experimentally infected mini-pigs. These results represent an improvement in diagnostic specificity compared to currently used serological tests. Thus suggesting that in a surveillance setting this test could be applied as a confirmatory test in the face of FPSR. The authors are British Civil Servants and as such their work is subject to British Crown Copyright. This means the exclusive copyright for the article cannot be transferred.     

Molecular evidence for cosmopolitan distribution of platyhelminth parasites of tunas (Thunnus spp.)

Global distribution of platyhelminth parasites and their host specificities are not well known. Our hypothesis was that platyhelminth parasites of large pelagic fishes are common around the world. We analysed molecular variation in three different taxa of platyhelminth parasites infecting four species of tunas: yellowfin tuna (Thunnus albacares, Scombridae) from Western Australia, southern bluefin tuna (Thunnus maccoyii, Scombridae) from South Australia, Pacific bluefin tuna (Thunnus orientalis, Scombridae) from Pacific Mexico and northern bluefin tuna (T. thynnus, Scombridae) from two localities in the Mediterranean (Spain and Croatia). Comparisons of ITS2 and partial 28S rDNA demonstrated two congeneric species of blood flukes (Digenea: Sanguinicolidae) from multiple hosts and localities: Cardicola forsteri from southern bluefin and northern bluefin tunas, and Cardicola sp. from Pacific bluefin and northern bluefin tunas; and a gill fluke, Hexostoma thynni (Polyopisthocotylea: Hexostomatidae), from yellowfin, southern bluefin and northern bluefin tunas. Partial 28S rDNA indicates that a second type of fluke on the gills, Capsala sp. (Monopisthocotylea: Capsalidae), occurs on both southern bluefin and Pacific bluefin tunas. This appears to be the first report of conspecific platyhelminth parasites of teleosts with a wide‐ranging geographical distribution that has been confirmed through molecular approaches. Given the brevity of the free‐living larval stage of both taxa of flukes on the gills (H. thynni and Capsala sp.), we conclude that the only feasible hypothesis for the cosmopolitan distribution of these flatworms is migrations of host tunas. Host migration also seems likely to be responsible for the widespread occurrence of the two species of blood flukes (Cardicola spp.), although it is also possible that these were translocated recently by the spread of infected intermediate hosts.     

Influence of high dietary α-tocopherol intakes on specific immune response, nonspecific resistance factors and disease resistance of healthy and aflatoxin B1-induced immunocompromised Indian major carp, Labeo rohita (Hamilton)

The effect of aflatoxin treatment and/or feeding of a high level of α‐tocopherol on immune response and disease resistance was investigated in Indian major carp, Labeo rohita. Group A served as a healthy control, group B was treated with aflatoxin, group C was fed a high level of α‐tocopherol whereas group D was exposed both to aflatoxin and a high level of dietary α‐tocopherol for 60 days. Aflatoxin B₁ (AFB₁) was injected once intraperitoneally into fish on the first day of the experiment (groups B & D). High levels of DL‐α‐tocopherol (1000 mg kg^–1 feed) were provided to healthy as well as AFB₁‐treated immunocompromised fish for 60 days (groups C & D). At the end of the experiment blood samples were assayed for changes in nonspecific immunity and humoral protein levels. Disease resistance against two common bacterial pathogens viz., Aeromonas hydrophila and Edwardsiella tarda were evaluated in all groups. Significant (P < 0.05) suppression of specific immunity as measured through haemagglutination (HA) titre against sheep red blood cells (SRBCs) as well as bacterial (formalin‐killed E. tarda) agglutination titre; nonspecific resistance factors viz., globulin level, serum bactericidal and lysozyme activities, neutrophil activities, and disease resistance against two bacterial pathogens only in aflatoxin‐treated fish with respect to the control group, clearly indicated the immunosuppressive nature of aflatoxin. Feeding of a high level of α‐tocopherol to AFB₁‐treated immunocompromised fish significantly (P < 0.05) raised specific immunity, nonspecific resistance factors and disease resistance capacity when compared with aflatoxin‐exposed fish. Disease resistance and enhancement of immune status through feeding of high levels of α‐tocopherol to healthy as well as AFB₁‐treated immunocompromised fish confirmed the potential of α‐tocopherol in carp feed for prevention of disease and for combating natural/environmental immunosuppressants.     

Species identification method for <Emphasis Type="Italic">Scombrops boops</Emphasis> and <Emphasis Type="Italic">Scombrops gilberti</Emphasis> based on polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA

ABSTRACT:   Gnomefish Scombrops boops and Scombrops gilberti are commercially important fishes in Japan, but these species are often confused in the markets because of their morphological similarity. To identify these two species, we performed nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis on 16S ribosomal RNA (rRNA) gene and the control region in mitochondrial DNA. Five and 12 nucleotide substitutions were observed between species in the 777-bp 16S rRNA gene and 471-bp control region, respectively. Diagnostic restriction sites for discriminating between S. boops and S. gilberti were found in the 16S rRNA gene, but not in the control region. Polymerase chain reaction (PCR)–RFLP analysis using two enzymes, Eco NI and Mva I, clearly discriminated between S. boops and S. gilberti identified by meristic characters. The PCR–RFLP analysis identified most of the 168 Scombrops young caught in the coastal waters of the Izu and Miura peninsulas as S. boops , suggesting that S. gilberti juveniles are rare in this area.