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Ling Wang

Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Dairy Science

First Advisor

Bhushan Jayarao


A study was conducted to study the prevalence of Gram-negative bacteria in bulk tank milk (n = 131) in eastern South Dakota and western Minnesota. A total of 130 bulk tanks (BTs) were examined for coliforms. Coliforms were detected in 62.3% of BTs, and ranged from 0-4.7 log10 cfu/ml, with a mean of 2.6 log10 cfu/ml. A total of 131 BTs were examined for non-coliform counts. The non-coliform counts were observed in 76.3% of BTs, and ranged from 0-6.2 log10 cfu/ml, with a mean of 4.2 log10 cfu/ml. A total of 234 isolates from bulk tank milk (B TM) were identified till species level, of which 201 isolates belonged to 28 species. Coliforms and non-coliforms accounted for 32.9 and 67.1 % of the total isolates, respectively. Bacteria belonging to the genera Agrobacterium, Bordetella, Comamonas, Listonella, Ochrobactrum, and Oligella were isolated from BTM. These organisms have not been reported previously in BTM. Pseudomonas jl.uorescens accounted for 29. 9% of all species and was the most predominant species in BTM. A total of 55 isolates of Ps. fluorescens were examined for phenotypic (biotypes, proteolytic and lipolytic profiles) and genotypic (plasmid and 16S-23S PCR ribotyping) characteristics. The phenotypic (biotypes, proteolytic and lipolytic profiles) and genotypic (16S-23 S PCR ribotyping) characteristics were used to conduct phylogenetic analysis. Ps. fluorescens belonged to 28 API 20 NE biotypes and 14 proteolytic and lipolytic profiles. It was observed that 80, 91, and. 58% of the isolates were proteolytic at 7, 22, and 32°C, respectively. While only 7, 44, and 7% of the isolates were lipolytic at the same 3 temperatures. Ps. fluorescens isolates were more likely to produce proteinases at 7 and 22°C and lipases at 22°C. Ps. fluorescens isolates were more proteolytic than lipolytic at 7°C. Nine of 55 isolates of Ps. fluorescens harbored plasmids. Based on 16S-23S PCR ribotyping, Ps. fluorescens belonged to 14 subtypes. Based on the results of the study, it can be inferred that: 1) The coliform and non-coliform counts were higher than previously reported and could be probably due to severe weather condition at the time of the study, 2) A total of 28 bacterial species were identified, some of them were reported for the first time, 3) The most predominant species isolated from BTM was Ps. fluorescens, and 4) For the given geographical area and the time of sampling, phylogenetic analysis based on phenotypic and genotypic characteristics suggested that Ps. fluorescens belonged to a large heterogeneous group of organisms.

Library of Congress Subject Headings

Milk -- Microbiology
Gram-negative bacteria
Milk tanks
Pseudomonas fluorescens


Includes bibliographical references (pages 37-43)



Number of Pages



South Dakota State University


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