A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cow's milk

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed. Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37ºC in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by double CAMP and plasma coagulation tube test. All 14 isolates developed a synergistic haemolysis with Rhodococcus equi (ATCC 6939) and inverse CAMP phenomenon with Staphylococcus aureus and coagulated rabbit plasma. Final diagnosis was confirmed using API Coryne V 2.0 and software program by BioMerieux1, revealing an identity rate of 99.9%, accuracy rate T = 1, test count = 0. Discussion: The first fourteen isolates of Corynebacterium ulcerans have been identified in our country, on the basis of a diagnostic protocol that is proposed in this paper. In our experience double CAMP test, rabbit plasma coagulation, catalase, oxidase tests and selected biochemical parameters, are sufficient as a diagnostic minimum. In the diagnostics of bacterial agents in cow mastitis, the attention of a bacteriologist is mostly limited to most widespread agents of mastitis, the isolation of which is mandatory pursuant to national legislation (Staphylococcus aureus and Streptococcus agalactiae). A more important reason for "missing" Corynebacterium ulcerans in the diagnosis is its colonial morphology that could resemble organisms of the genus Staphylococcus. Complex and expensive diagnostic procedure that is not available to most laboratories is also responsible for the small number of reports of isolation C. ulcerans. Furthermore, in routine work C. ulcerans could be misidentified with Staphylococcus intermedius, because of cultural similarity, positive plasma coagulation tube test and absence of manitol fermentation of both species. This paper is a report on isolation and identification of Corynebacterium ulcerans from milk of cows with mastitis, as well as a suggestion of a diagnostic protocol available for routine work in most veterinary microbiology laboratory. Therefore we suggest as the diagnostic protocol double CAMP test to be used as a complementary method to rabbit plasma coagulation tube test.

A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cows milk / A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cows milk

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed.Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37o C in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by d

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed.Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37o C in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by d

A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cows milk / A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cows milk

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed.Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37o C in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by d

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed.Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37o C in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by d