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A rapid method for determination of somatic cell count and isolation of neutrophil elastase from bovine milk

Zlatina BECHEVA, Tzonka GODJEVARGOVA

Abstract: A rapid method for SCC with YOYO1 was developed. Dying the cells fluorescent is an advantageous technology with accurate and repeatable results. PMN elastase from bovine milk was obtained and proven by a RP-HPLC. The activity of the PMN elastase was measured and compared with porcine pancreatic elastase. Measuring the PMN elastase activity through created method is helpful for determination of the cattle health.

Keywords: SCC; mastitis; PMN elastase; elastase activity; YOYO1; HPLC; milk;

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Immunofluorescence microscope assay of neutrophils and somatic cells in bovine milk

Zlatina BECHEVA, Katya GABROVSKA, Tzonka GODJEVARGOVA

Abstract: Polyclonal antibody specific for bovine neutrophils was prepared. Serum titer was observed at 75th and 105th day of injection period. The first titer was 1250-fold serum dilution and the second – 31,250-fold serum dilution. Antibody was purified with affinity chromatography. Antibody reactivity to neutrophil fragments, Ficoll-separated bovine blood cells – granulocytes (neutrophils, basophils and eosinophils) and mononuclear cells, and bovine milk somatic cells (SC) were studied. The antibody has detection limit 5μg/mL neutrophil fragments and reacts only with neutrophils. The antibody was conjugated to fluorescein isothiocyanate (FITC). The specificity of the fluorescent conjugate was analyzed with Ficoll-separated blood cells and milk SC by fluorescence microscopic analysis. The microscope images prove the conjugate reactivity with blood and milk neutrophils. It was found that when SC count (SCC) was 100,000 cells/mL neutrophils were 30%, but when SCC exceeded 800,000 cells/mL neutrophils were 88%. The proposed antibody–FITC method was compared with commercial Giemsa method.

Keywords: Bovine milk; somatic cell count; neutrophil; FITC; conjugate; ELISA;

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Preparation of Anti-Elastase Antibody Conjugated With Quantum Dots 710 Nm and Fluorescein Isothiocyanate for Immunoassay of Milk Somatic Cells

Zlatina BECHEVA, Tzonka GODJEVARGOVA

Abstract: A novel immunofluorescence microscopic method for a neutrophil and somatic cell count was developed by using anti-elastase antibody. Two different conjugates - anti-bovine neutrophil elastase antibody-fluorescein isothiocyanate conjugate and anti-bovine neutrophil elastase antibody-Quantum Dots 710nm conjugate were prepared. The coupling of Quantum Dots (QDs) and fluorescein isothiocyanate (FITC) to antibody has been demonstrated by comparison of ultraviolet absorption spectra and fluorescence spectra of conjugate and initial components. The activities of conjugates were measured by indirect ELISA at the same conditions. It was found that the sensitivity of the immunoassay with QDs-conjugate was higher than the sensitivity of the immunoassay with FITC-conjugate. The storage stability of anti- bovine neutrophil elastase antibody-QDs710nm conjugate and anti-bovine neutrophil elastase antibody-FITC conjugate was studied. The residual fluorescence intensity of anti-elastase antibody-QDs was 95% at 30th day, while the intensity of anti- elastase antibody-FITC conjugate was reduced to 60% at same day. The obtained conjugates were applied in real test with bovine milk somatic cells. The fluorescence imaging proved that QDs-conjugate has better fluorescence intensity than the FITC- conjugate, and is more appropriate for determination of somatic cells in milk.

Keywords: Quantum Dots; FITC; Antibody; Conjugate; Somatic cells;

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Diurnal variation of milk somatic and differential leukocyte counts of Murrah buffaloes as influenced by different milk fractions, seasons and parities

Karuna Bombade, Aarti Kamboj, Mohanned Naif Alhussien, Ashok Kumar Mohanty & Ajay Kumar Dang

Abstract: Milk cell counts are good indicators of a mammary infection and milk quality. The present study was done to record diurnal rhythmicity in the milk somatic and differential cell counts during different seasons, milk strips and parity in Murrah buffaloes. Milk somatic cell counts (SCC) were measured by SCC counter and milk differential cell counts were measured microscopically after making a milk smear and staining it to identify neutrophils, lymphocytes and macrophages. Maximum milk SCC was observed in the summer season. Milk neutrophils were lowest during thermoneutral (TN), intermediate during the winter season and highest during the summer season. Milk lymphocytes were highest during the winter season, intermediate in the TN and lowest in the summer season. Diurnal rhythm in the milk SCC and neutrophil percentage is noticed in the summer season only. Maximum milk SCC values were observed in the late strip but neutrophils were highest in the early strip. Diurnal rhythm was observed in the late strip for neutrophils and in mid strip for the lymphocytes. Milk SCC and milk neutrophils were found to be the highest in the multiparous buffaloes and diurnal rhythm was observed only in the lymphocytes of primiparous buffaloes. Milk macrophages were higher in the morning samples of primiparous as compared to the multiparous buffaloes. In this pioneer study, diurnal rhythms in the milk cell counts of buffaloes have been studied extensively. This will help in maintaining low milk cell counts in buffalo and thus help in getting more milk per buffalo during stress periods.

Keywords: Milk somatic cells; differential cells; diurnal rhythm; seasons; strip; parity; buffalo;

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Diurnal rhythm in the counts and types of milk somatic cells, neutrophil phagocytosis and plasma cortisol levels in Karan Fries cows during different seasons and parity

Mohanned Naif Alhussien, Ajay Kumar Dang

Abstract: Chances of mammary infections are comparatively higher in high producing cows during harsh environmental conditions and are usually characterised by changes occurring in various somatic cells secreted in the milk and their activities. The present study was conducted to record diurnal rhythmicity in milk somatic cell counts (SCC), neutrophil: macrophage (N: M) ratio, phagocytic activity (PA) of milk neutrophils and plasma cortisol concentrations during different seasons and parity in high producing Karan Fries cows. Values of milk SCC, N: M ratio and plasma cortisol levels were lowest during thermoneutral (TN), intermediate in winter and highest during the summer season. Diurnal rhythm in the milk SCC and N: M ratio was noticed in the summer while plasma cortisol exhibited diurnal rhythm in both winter and summer seasons. Milk SCC, N: M ratio and plasma cortisol increased in multiparous cows, but diurnal variation was noticed only in the N: M ratio and plasma cortisol in cows having more than four parity. Phagocytic activity of milk neutrophils was highest during TN, intermediate in winter and lowest during the summer season. Phagocytic activity was higher and similar in cows up to fourth parity but decreased in subsequent lactation cycles. Diurnal rhythm in the PA was noticed in winter and summer seasons and in cows having more than four parity where morning samples showed higher phagocytosis as compared to the evening samples. These results can be used for immunomodulatory interventions and therapeutic approaches in treating mastitis of crossbred cows reared under tropical conditions.

Keywords: Milk somatic cell; neutrophil: macrophage ratio; season; parity; cow;

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