Aj Guidry

Differentiation and enumeration of somatic cells in goat milk

Non-leukocytic cell-like particles commonly observed in goat milk were examined ultrastructurally and cytochemically. Transmission electron microscopy indicated that these particles were generally membrane-bound and anucleate. They contained granular material in the dilated cisternae of the endoplasmic reticulum and homogeneous electron translucent inclusions that resembled lipid. Histochemical and fluorescent staining indicated that the particles contained large amounts of protein, some lipid, but no deoxyribonucleic acid. Several methods routinely used for estimating somatic cell counts in cow milk were compared to determine which one would give accurate estimates of somatic cell counts in goat milk. No significant difference was found (P> .05) among methods which specifically measure deoxyribonucleic acid. These included Membrane Filter-DNA, direct microscopic somatic cell counts using Pyron in Y-methyl green stain, and Fossomatic cell counts. Results of the Wisconsin Mastitis Test did not differ significantly from Fossomatic cell counts. Because Coulter electronic counts and direct microscopic somatic cell counts using Levowitz-Weber stain could not differentiate between the cell-like particles and the actual leukocytes, these methods resulted in elevated cell counts that were highly variable. Results indicate that only those counting methods that are specific for deoxyribonucleic acid can distinguish cell-like particles from somatic cells, and thereby give reliable estimates of somatic cell numbers in goat milk.

Effect of Fat and Casein on Intracellular Killing of Staphylococcus aureus by Milk Leukocytes

Summary Suspensions of PMN isolated from milk and S. aureus were incubated together at different ratios and with different types and concentrations of mammary fluids. Results showed that greater numbers of S. aureus were left unphagocytosed and alive within PMN at the higher S. au-reus: PMN ratios (2:1) than at the lower ratios. This effect was more noticeable in whole milk than in skimmed milk. Phagocytosis and destruction of S. aureus by PMN (ratio 1:10) were reduced (p < 0.01) to a greater extent in whole milk or in skimmed milk containing 10% cream than in either skimmed milk or whey. There was no difference between skimmed milk or whey. Increasing the concentration of either whole milk or skimmed milk in the incubation mixture from 10 to 40% resulted in fewer (p < 0.01) S. aureus phagocytosed but had no affect on intracellular kill. The data show that the milk fat globule is a major deterrent to the phagocytosis and destruction of S. aureus by PMN. Casein inhibited phagocytosis, but only when the concentration of skimmed milk in the incubation medium was increased to 40%. Casein did not affect intracellular kill. We thank Jane Hawes for technical assistance.

Encapsulation of Streptococcus uberis: Influence of storage and cultural conditions

Streptococcus uberis (n = 100) isolated from bovine mammary secretions were assessed by India ink for expression of capsule. Organisms were evaluated under four conditions; (1) after primary culture on blood agar, (2) following 5 passages on blood agar, (3) after 5 passages in Trypticase Soy Broth (TSB), and (4) after storage in 10% skim milk. Strains from primary culture (44 of 100) were positive for an unstained halo (capsule) by the India ink method. Number of strains expressing capsule decreased greatly after passage and following storage. Freeze-etching followed by electron microscopy confirmed results of India ink preparations. Strains were also cultured in various media to determine influence of medium components on capsule expression. Todd-Hewitt medium supplemented with either serum or egg yolk enhanced the size of capsule expressed. Results of this study may aid researchers investigating the pathogenicity of S. uberis.

Monoclonal antibodies to subpopulations of bovine neutrophils

Monoclonal antibodies were developed to neutrophil surface antigens in an effort to identify neutrophil subpopulations. Eleven monoclonals were positive for neutrophils using the ELISA and flow cytometry. Four monoclonals recognised neutrophils and monocytes, but more importantly each recognised subpopulations of neutrophils. Percentage binding of the same population of neutrophils by each monoclonal was 19H7, 23%; 36H10, 34%; 46B3, 16%; 9D13, 37%. All four showed additive percentage binding to neutrophils when reacted with each other. Cow differences in percentage binding of monoclonals were also evident, i.e., 46B3 — cow no. 8437, 8%; cow no. 2820, 22%. Monocytes showed the most variation, both by cow and by clone within cow, i.e., cow no. 8813, 46B3 = 98% and 9D13 = 6%; clone 19H7, cow no. 8813 = 88% and cow no. 8679 = 2%. Percentage binding to eosinophils was low for all clones except 46B3 which averaged 46%, 5%, 72% and 76% for four cows tested. Percentage binding to lymphocytes tended to be high for all clones across all cows, averaging 62%. Clone 36H10 tended to be the lowest of the clones, averaging 38%. The ability of these clones to identify subpopulations of neutrophils and the significant among cow variation make them useful tools for studying the relationship of different cell-surface antigens to cell function within cow and among cows.