This article was downloaded by: [University of Otago] On: 05 January 2015, At: 03:04 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
The detection of leptospirae in cattle urine a
D.R. Ris & K.L. Hamel
a
a
Wallaceville Animal Research Centre, Research Division , Ministry of Agriculture and Fisheries , Private Bag, Upper Hutt Published online: 23 Feb 2011.
To cite this article: D.R. Ris & K.L. Hamel (1978) The detection of leptospirae in cattle urine, New Zealand Veterinary Journal, 26:10, 246-256, DOI: 10.1080/00480169.1978.34556 To link to this article: http://dx.doi.org/10.1080/00480169.1978.34556
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
246
NEW ZEALAND VETERINARY JOURNAL
VOL 26
The detection of leptospirae in cattle urine D. R. Ris* and K. L. Hamel* N.z. Vel. J. 26: 246. 255-6
Downloaded by [University of Otago] at 03:04 05 January 2015
ABSTRACT
Hamster inoculation, medium inoculation and microscopical methods were used for the detection of Leptospira interrogans serovar pomona organisms in bovine urine. Urine samples were collected from both naturally and experimentally infected animals during the period when leptospiruria could be expected. Inoculation into hamsters of 0.5 ml of urine from each of 14 samples resulted in 5 positives. The inoculation of 0.025 mlof the same sample into semi-solid EMJH medium gave 10 positives. From a cumulative total of 46 urine samples, 28 were positive using dark-ground microscopy of centrifuged urine deposits and. 35 were positive using media inoculation. A cumulative total of 126 urine samples were examined, after centrifugation, under dark-ground microscopy and leptospirae were detected in 60. Fluorouracil (100 mg/ml) proved to be benefiicial for successful isolations in media, whereas neomycin (5 mg/ml) did not.
INTRODUCTION
In the absence of clinical disease, the diagnosis ofleptospiral infection in cattle is often made using serological tests only(6). However, it is sometimes necessary to determine whether or not individual animals are infectious. Methods to detect leptospirae in urine have been described before(5)(13). Doherty(2) compared two of these methods: direct microscopy and guinea-pig isolation techniques. No other reports that compare similar methods are known to the authors. The present study compares results obtained from hamster inoculation, media inoculation and microscopy of centrifuged-urine deposits.
MATERIALS AND METHODS
Cattle Female dairy cattle, either naturally or experimentally infected with Leptospira interrogans serovar pomona, were used. They consisted of calves less than 4 weeks old, 2-year-old maiden heifers and mixed-age dairy cows. Urine samples were collected at a time when leptospiruria could reasonably be expected.
Urine samples Mid-stream urine samples were obtained after manual stimulation, and suitable aliquots were used immediately for inoculation Qfmedia and hamsters. The remainder of the urine samples was preserved by addition of analytical grade formalin to give a final concentration of 1%.
Hamsters Suitability of hamsters for serovar pomona isolation was determined at the end of the experiment, by inoculating groups of *
Wallaceville Animal Research Centre, Research Division, Ministry of Agriculture and Fisheries, Private Bag, Upper Hutt.
4 hamsters intra peritoneally with 0.5 ml of lO-fold dilutions of a culture of I of the serovar pomona strains isolated. The total count of the culture was determined with a Petroff-Hausser counting chamber. This showed that a single organism of the pomona strain isolated could cause infection and death in hamsters. Four hamsters, between 4 and 5 weeks old, were each inoculated intraperitoneally with 0.5 ml of urine. The hamsters were observed for approximately 3 weeks. Kidney samples of dead hamsters were inoculated into media. After 3 weeks, surviving hamsters were bled by heart puncture, killed, and kidney samples inoculated into medium. Media Isolation attempts, both from urine and from the kidneys of inoculated hamsters, were made in semi-solid (1.5 g Difco agar/litre) EMJH medium(7), supplemented with 100 mg/mlof sodium pyruvate(9) and 100 mg/ml of 5-fluorouracil (FU)(8) but without additional copper sulphate. Copper sulphate was omitted because flameless atomic-absorption analysis showed that the medium (without copper sulphate) contained 0.34 mg/ml of copper, whereas Difco medium (with copper sulphate) contained 0.27 mg/ml of copper. The need for FU was established in a preliminary trial, by inoculating 10 urine samples each into media with and without FU. Five pomona strains were isolated, four only in media plus FU and one in both types of medium. Contamination forced the discarding of seven lots of medium without FU and of two with FU. FU was therefore incorporated in all media used for the isolation of pomona from bovine urine. The suitability of the medium for supporting growth of serovar pomona had been determined by inoculating replicate bottles with one drop of culture, diluted lO-fold to extinction. The total count of the culture was determined with a PetroffHaussercountingchamber. This showed that growth occurred in all bottles inoculated with 10 or more organisms, and in 2 out of 3 bottles inoculated with I organism. Hamster kidney cultures were made in the presence of added neomycint (5 mg/ml final concentration)( I) and urine cultures were made in media with and without neomycin. Cultural Techniques One drop (ca. 0.025 ml) of mid-stream urine was inoculated into each of 3 bottles containing 2.5 ml of semi-solid medium, 2 bottles of which had neomycin added. Kidneys were removed aseptically from dead hamsters and from each kidney a "plug" of tissue approximately 3 mm long by I mm diameter, was inoculated in medium containing neomycin. Media were incubated at 29-30°C and were examined microscopically at least weekly for up to 8 weeks. Microscopy of urine samples . F ormalinised urine samples (approx. 12 ml) were centrifuged in plastic conical centrifuge tubes for at least 40 min at 3000 g in a swing-out head. Then, the supernatant fluid was carefully
t
Neomycin sulphate: Sigma Chemical Company (Continued on page 255)
(Continued from page 246) removed until only a large drop remained, in whfch the deposit was re-suspended. This resulted in more than lOO-fold concentration of leptospirae. Wet smears were examined under dark-ground illumination at a magnification of 525 X. Smears were also thoroughly airdried, fixed with analytical grade methanol for at least 10 min and then stained overnight at 30°C according to the method of G. Fowler (unpublished). A stock solution of Giemsa was prepared by dissolving 3.8 g of the solid stain"'''' in 125 ml of analytical grade glycerol at 60°C, and 375 ml of analytical grade methanol was then added. This solution was k
New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
The detection of leptospirae in cattle urine a
D.R. Ris & K.L. Hamel
a
a
Wallaceville Animal Research Centre, Research Division , Ministry of Agriculture and Fisheries , Private Bag, Upper Hutt Published online: 23 Feb 2011.
To cite this article: D.R. Ris & K.L. Hamel (1978) The detection of leptospirae in cattle urine, New Zealand Veterinary Journal, 26:10, 246-256, DOI: 10.1080/00480169.1978.34556 To link to this article: http://dx.doi.org/10.1080/00480169.1978.34556
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
246
NEW ZEALAND VETERINARY JOURNAL
VOL 26
The detection of leptospirae in cattle urine D. R. Ris* and K. L. Hamel* N.z. Vel. J. 26: 246. 255-6
Downloaded by [University of Otago] at 03:04 05 January 2015
ABSTRACT
Hamster inoculation, medium inoculation and microscopical methods were used for the detection of Leptospira interrogans serovar pomona organisms in bovine urine. Urine samples were collected from both naturally and experimentally infected animals during the period when leptospiruria could be expected. Inoculation into hamsters of 0.5 ml of urine from each of 14 samples resulted in 5 positives. The inoculation of 0.025 mlof the same sample into semi-solid EMJH medium gave 10 positives. From a cumulative total of 46 urine samples, 28 were positive using dark-ground microscopy of centrifuged urine deposits and. 35 were positive using media inoculation. A cumulative total of 126 urine samples were examined, after centrifugation, under dark-ground microscopy and leptospirae were detected in 60. Fluorouracil (100 mg/ml) proved to be benefiicial for successful isolations in media, whereas neomycin (5 mg/ml) did not.
INTRODUCTION
In the absence of clinical disease, the diagnosis ofleptospiral infection in cattle is often made using serological tests only(6). However, it is sometimes necessary to determine whether or not individual animals are infectious. Methods to detect leptospirae in urine have been described before(5)(13). Doherty(2) compared two of these methods: direct microscopy and guinea-pig isolation techniques. No other reports that compare similar methods are known to the authors. The present study compares results obtained from hamster inoculation, media inoculation and microscopy of centrifuged-urine deposits.
MATERIALS AND METHODS
Cattle Female dairy cattle, either naturally or experimentally infected with Leptospira interrogans serovar pomona, were used. They consisted of calves less than 4 weeks old, 2-year-old maiden heifers and mixed-age dairy cows. Urine samples were collected at a time when leptospiruria could reasonably be expected.
Urine samples Mid-stream urine samples were obtained after manual stimulation, and suitable aliquots were used immediately for inoculation Qfmedia and hamsters. The remainder of the urine samples was preserved by addition of analytical grade formalin to give a final concentration of 1%.
Hamsters Suitability of hamsters for serovar pomona isolation was determined at the end of the experiment, by inoculating groups of *
Wallaceville Animal Research Centre, Research Division, Ministry of Agriculture and Fisheries, Private Bag, Upper Hutt.
4 hamsters intra peritoneally with 0.5 ml of lO-fold dilutions of a culture of I of the serovar pomona strains isolated. The total count of the culture was determined with a Petroff-Hausser counting chamber. This showed that a single organism of the pomona strain isolated could cause infection and death in hamsters. Four hamsters, between 4 and 5 weeks old, were each inoculated intraperitoneally with 0.5 ml of urine. The hamsters were observed for approximately 3 weeks. Kidney samples of dead hamsters were inoculated into media. After 3 weeks, surviving hamsters were bled by heart puncture, killed, and kidney samples inoculated into medium. Media Isolation attempts, both from urine and from the kidneys of inoculated hamsters, were made in semi-solid (1.5 g Difco agar/litre) EMJH medium(7), supplemented with 100 mg/mlof sodium pyruvate(9) and 100 mg/ml of 5-fluorouracil (FU)(8) but without additional copper sulphate. Copper sulphate was omitted because flameless atomic-absorption analysis showed that the medium (without copper sulphate) contained 0.34 mg/ml of copper, whereas Difco medium (with copper sulphate) contained 0.27 mg/ml of copper. The need for FU was established in a preliminary trial, by inoculating 10 urine samples each into media with and without FU. Five pomona strains were isolated, four only in media plus FU and one in both types of medium. Contamination forced the discarding of seven lots of medium without FU and of two with FU. FU was therefore incorporated in all media used for the isolation of pomona from bovine urine. The suitability of the medium for supporting growth of serovar pomona had been determined by inoculating replicate bottles with one drop of culture, diluted lO-fold to extinction. The total count of the culture was determined with a PetroffHaussercountingchamber. This showed that growth occurred in all bottles inoculated with 10 or more organisms, and in 2 out of 3 bottles inoculated with I organism. Hamster kidney cultures were made in the presence of added neomycint (5 mg/ml final concentration)( I) and urine cultures were made in media with and without neomycin. Cultural Techniques One drop (ca. 0.025 ml) of mid-stream urine was inoculated into each of 3 bottles containing 2.5 ml of semi-solid medium, 2 bottles of which had neomycin added. Kidneys were removed aseptically from dead hamsters and from each kidney a "plug" of tissue approximately 3 mm long by I mm diameter, was inoculated in medium containing neomycin. Media were incubated at 29-30°C and were examined microscopically at least weekly for up to 8 weeks. Microscopy of urine samples . F ormalinised urine samples (approx. 12 ml) were centrifuged in plastic conical centrifuge tubes for at least 40 min at 3000 g in a swing-out head. Then, the supernatant fluid was carefully
t
Neomycin sulphate: Sigma Chemical Company (Continued on page 255)
(Continued from page 246) removed until only a large drop remained, in whfch the deposit was re-suspended. This resulted in more than lOO-fold concentration of leptospirae. Wet smears were examined under dark-ground illumination at a magnification of 525 X. Smears were also thoroughly airdried, fixed with analytical grade methanol for at least 10 min and then stained overnight at 30°C according to the method of G. Fowler (unpublished). A stock solution of Giemsa was prepared by dissolving 3.8 g of the solid stain"'''' in 125 ml of analytical grade glycerol at 60°C, and 375 ml of analytical grade methanol was then added. This solution was k
