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.     

The Effect of Lead and Zinc on Plant Growth and Chlorophyll Content of Holcus lanatus L.

Root length of Holcus lanatus L. declined rapidly with increasing lead and zinc concentrations in nutrient solution. In all used Pb and Zn concentrations root growth of genotypes coming from a Pb-Zn mine area, was greater than that of the control, suggesting that these genotypes evolved tolerance to Pb and Zn.
Negative correlation was also observed between chlorophyll content and increased heavy metal concentrations. The greater chlorophyll content found in tolerant genotypes, in different Pb and Zn concentrations, in comparison with the control, could be served to distinguish tolerant and non-tolerant genotypes.     

Comparison of Three Solvent Systems for Extraction of Chlorophyll a from Fish Pond Phytoplankton Communities

Chloroform-methanol (2:1 v/v), absolute methanol, and 90% acetone were evaluated for their effectiveness as extractants of chlorophyll a from samples of phytoplankton communities collected from catfish ponds. Chloroform-methanol consistently extracted more chlorophyll a than either 90% acetone or methanol. Precision for the methanol extraction was also unacceptably low, with an average coefficient of variation of 17%. Average coefficients of variation for the chloroform-methanol and 90% acetone extraction procedures were 6 and 5%, respectively. Filtered samples should be steeped in chloroform-methanol for at least 4 h to obtain maximum chlorophyll extraction, and the addition of MgCO₃ to the extractant as a buffer is not necessary.     

Epibulbar melanoma in a cat

In a nine-year-old female cat, the diagnostic procedure, therapy and differential diagnoses of a rare, primary, malignant, epibulbar melanoma are presented. Post-operatively no recidivation or metastases occurred within 18 months.     

Microcirculation in the follicles and corpora lutea of the bovine ovary--a corrosion anatomical study

With the help of a new synthetic material follicles and corpora lutea from the ovaries of 80 cows in different stages of the sexual cycle were examined for modifications of their blood vessel architecture. In the theca externa the main artery which runs towards the follicle, separates into arterioles of which capillaries derive in the theca interna, building a basket-like network around the zona granulosa. After ovulation the follicle wall forms characteristic fields. Capillaries grow into the zona granulosa and thus the folds form the lobular structure of the corpus luteum. Capillaries which rise from the ruptured part of the follicle, spread centripetally and together with lutein cells they build up the not lobulated cupola of the corpus luteum. The regression of the corpus luteum starts around the 16th day of the cycle, beginning at the capillary network of the cupola. A contraction of the vessels and a quick decrease of the volume follow.     

The effect of hypervitaminosis D3 on the bones of the chicken. 2. Electron microscopic studies

In an experiment of 36 days duration 46 one-day-old chicks were divided into 5 groups and fed with different concentrations of vitamin D3. The animals of the group which lacked vitamin D3, showed the typical rachitic lesions. After a 15 days lack of vitamin D3 the chicks of another group were treated with standard food (2000 I.U. vitamin D3/kg food) with the consequence of an approximation of the analyzed parameters to those of the control group within 3 weeks. When fed with 60,000 I.U. of vitamin D3 after a 15 days lack of this vitamin, the animals showed an over-hasty healing process, ending up with signs of intoxication which were even more conspicuous when fed with 120,000 I.U. of vitamin D3. Besides an increasing calcification of osteoblasts and endothelial cell membranes as well as a degeneration of osteoblasts, a clear increase of eosinophilic granulocytes could be noticed. In all groups free erythrocytes within the ground substance were found. There was no evidence of necroses of osteocytes or of bone.     

The Content of β-endorphin-like Immunoreactivity in Porcine Corpus Luteum and the Potential Roles of Progesterone, Oxytocin and Prolactin in the Regulation of β-endorphin Release from Luteal Cells in vitro

The amount of β‐endorphin‐like immunoreactivity (β‐END‐LI) in porcine corpora lutea from several stages of the oestrous cycle and the effects of progesterone, oxytocin, and prolactin on β‐END‐LI secretion in vitro by luteal cells were studied. Porcine corpora lutea obtained on days 1–5, 6–10, 11–13, 14–18, and 19–21 of the cycle were used to prepare extracts for β‐END‐LI determination. Additionally, corpora lutea from days 11–13 and 14–18 were enzymatically dissociated and isolated luteal cells were used for further study of β‐endorphin secretion in vitro. Cells were cultured in serum‐free defined M 199 medium (10⁶ cells/ml) at 37°C under 5% CO₂ in air, for 12 h. The influences of the following factors on β‐END‐LI secretion by luteal cells were tested: progesterone (10^–9, 10^–7 and 10^–5M ), oxytocin (0.01, 0.1, 1 and 10 ng/ml), and prolactin (0.1, 1, 10 and 100 ng/ml). The β‐END‐LI contents in extracts and media were measured by radioimmunoassay. The tissue concentration of β‐END‐LI was lowest on days 1–5 of the cycle (0.35 ± 0.03 ng/g wet tissue). Subsequently, it constantly increased to the highest value on days 14–18 (16.58 ± 0.52 ng/g wet tissue) and on days 19–21 it declined (11.10 ± 0.52 ng/g wet tissue). Progesterone at a low dose (10^–9 M ) resulted in significant (p < 0.05) increases and decreases in β‐END‐LI secretion by luteal cells from days 11–13 and 14–18, respectively. Higher doses of progesterone (10^–7 and 10^–5 M ) had no effect on β‐END‐LI release, compared with the control group. All dose‐levels of oxytocin used decreased β‐END‐LI secretion by luteal cells on days 11–13 and 14–18 of the cycle. Prolactin at doses of 0.1 and 1 ng/ml on days 11–13, and all doses tested on days 14–18 resulted in decreases in β‐END‐LI release from luteal cells. These results document evident changes in β‐END‐LI content in the pig corpus luteum during its development and indicate the potential roles of progesterone, oxytocin, and prolactin in luteal cell secretion of β‐END‐LI.     

Effectiveness of Cabergoline for Termination of Pregnancy in Silver Fox (Vulpes vulpes fulva)

Red foxes ( Vulpes vulpes ) are a major pest species in Europe and Australia. Traditional methods of control such as hunting or poisoning are no longer sufficient or feasible. As with domestic dogs and cats, prolactin (PRL) in the vixen is an essential luteotropin during the second half of gestation. Hence, PRL inhibitors such as cabergoline have been used to induce abortions. Eighteen mated silver fox vixens (three groups of six foxes each) were treated orally with a placebo of paraffin oil (I), or with 15  μ g/kg cabergoline in feed once (II) or twice (III), on day 30 (I and II) or days 30 and 32 (III) post-coitum. Blood samples were taken prior to and after treatments and concentrations of PRL and progesterone (P₄) were determined. Normal parturitions were observed in five of six, five of six and two of six vixens in groups I, II and III, respectively. In group III plasma concentrations of PRL and P₄ decreased significantly but only temporarily. This drop in hormone concentrations was more pronounced in the vixens that did not carry to term. In conclusion, doses in excess of 15  μ g/kg of cabergoline are likely to prevent the development of fetuses to term in pregnant vixens.     

Comparison of partial ribosomal DNA sequences of Babesia gibsoni occurring in Miyazaki prefecture, Japan.

Nucleotide sequences of ribosomal DNA (rDNA) of Babesia (B.) gibsoni occurring in Miyazaki, western Japan, were examined using blood samples obtained from seven dogs suffering from natural canine babesiosis. DNA isolated from these blood samples was subjected to the polymerase chain reaction (PCR). The nucleotide sequences of the PCR products were determined and compared with other rDNA sequences of B. gibsoni isolated from Asia, Europe and U.S.A. Although homology values between our isolates and those isolated from Europe and U.S.A. were both 84.0%, respectively, our isolates were identical to the Asian types. In conclusion, B. gibsoni occurring in Miyazaki was revealed to have the genotype Asia 1 or Asia 2 from a comparison of the partial rDNA sequences.