title: Evaluation of confirmatory stains used for direct microscopic somatic cell counting of sheep milk.
abstract: Current FDA regulatory screening and confirmatory methods, electronic cell counting and the direct microscopic somatic cell count (DMSCC), differ for the detection of abnormal milk in sheep and goats. The DNA-specific electronic SCC screening methods such as Fossomatic (Foss, Hillerød, Denmark) can be used for both sheep and goat milk; however, the nonspecific methylene blue-based stains used for DMSCC in sheep cannot be used for goats as they nonspecifically stain cytoplasmic particles naturally present in goat milk. The DNA-specific stain pyronin Y-methyl green (PMG) is currently used for DMSCC in goats. Sheep also shed cytoplasmic particles during the milk secretory process, but in fewer numbers than goats. The objective of this study was to determine whether the nonspecific, methylene blue-based Levowitz-Weber (L-W) stain is the appropriate regulatory stain to use for DMSCC in sheep milk. Composite milk samples from 42 commercial dairy ewes were collected every 4 wk for the duration of each ewe's lactation for a total of 10 sample days. Milk samples were subjected to 3 methods of SCC determination: automated Fossomatic counting, DMSCC with L-W stain, and DMSCC with PMG stain conducted according to FDA regulatory procedures (2400 series forms). The DMSCC from milk smears stained with L-W were greater than those from smears stained with PMG and those from the Fossomatic analysis on 6 of the 10 sampling days. Milk smears stained with PMG did not differ from Fossomatic analysis at any sampling. The average milk SCC for L-W, PMG, and Fossomatic were (mean±SE) 275±36×10(3), 174±24×10(3), and 164±24×10(3) cells/mL, respectively. The DMSCC for milk stained with L-W was 58% greater than that with PMG and 68% greater than that obtained with the Fossomatic analysis. In conclusion, DMSCC of sheep milk stained with the nonspecific, methylene blue L-W stain resulted in a marked increase in SCC over that of the DNA-specific stain PMG and Fossomatic SCC analysis. The findings of this study support the continued use of Fossomatic SCC but recommend the replacement of the methylene blue-based stains with DNA-specific PMG for determination of DMSCC in sheep milk.
authors.affiliation: Department of Fisheries, Animal and Veterinary Science, University of Rhode Island, Kingston, RI 02881, USA. kpetersson@uri.edu
authors.forename: K H
authors.initials: KH
authors.lastname: Petersson
authors.forename: L A
authors.initials: LA
authors.lastname: Connor
authors.forename: C S
authors.initials: CS
authors.lastname: Petersson-Wolfe
authors.forename: K A
authors.initials: KA
authors.lastname: Rego
mesh_terms: D000818:Animals; D002452:Cell Count / Q000379:methods / Q000662:veterinary; D005243:Feces / Q000382:microbiology; D005260:Female; D008739:Methyl Green; D008751:Methylene Blue; D008853:Microscopy / Q000662:veterinary; D008892:Milk / Q000166:cytology*; D011754:Pyronine; D012756:Sheep; D013045:Species Specificity; D013194:Staining and Labeling / Q000379:methods / Q000662:veterinary*
chemical_list: D008739:Methyl Green; D008751:Methylene Blue; D011754:Pyronine