Development of an improved selective medium for isolation of leptospires from clinical material

Adler, Ben; Faine, S.; Christopher, W. L.; Chappel, R.J.

doi:10.1016/0378-1135(86)90087-8

Cited by 34 publications

(26 citation statements)

References 6 publications

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“…ADLER et al (1986) and BOLIN et al (1989) recognized the difficulty in isolating leptospires, despite the presence of leptospires in samples. MOREIRA (1994), using 420 urine samples from bovines naturally infected, corresponding to 2100 cultured tubes, obtained two leptospiras isolation.…”

Section: Discussionmentioning

confidence: 99%

“…SCHÖNBERG (1981) states that the contaminant microorganisms make the isolation difficult because they multiply quickly and consequently impede leptospira growth. FAINE (1982) and ADLER et al (1986) stated that undesirable microorganism growth could be inhibited by the addition of antibiotics to the culture medium without modifying the leptospira cell multiplication. The Leptospirosis Laboratory at DMVP-UEL used only four of the seven antibiotics tested by SCHÖNBERG (1981) plus 5-fluorouracil at the concentration recommended by HEER et al (1982).…”

Section: Discussionmentioning

confidence: 99%

“…Isolation of leptospira from tissues and fluids both in animals of economic interest and pets has been simplified with the availability of culture mediums, antibiotics and improvement of sample handling and dilution techniques (SCHÖNBERG, 1981;THIERMANN, 1984;ADLER et al, 1986). ADLER et al (1986) stated that the main problem in culturing leptospires is contamination with other microorganisms, especially when attempting to culture from non-sterile sources such as urine and fetal tissues. The inclusion of antibiotics to inhibit the contamination in the culture medium has been recommended, but inhibitory substances had a detrimental effect on the multiplication phase of the leptospiras (SCHÖNBERG, 1981).…”

Section: Introductionmentioning

confidence: 99%

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Isolation of Leptospira spp from dogs, bovine and swine naturally infected

et al. 2004

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Isolation of Leptospira spp from dogs, bovine and swine naturally infected

et al. 2004

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“…Leptospires are slow growing organisms having a generation time of approximately 24 hours at 30ºC and the requirement for a rich medium at neutral pH predisposes the cultivation of those microorganisms sometimes troublesome, particularly from natural sources ( (1,3,7,8).…”

Section: Introductionmentioning

confidence: 99%

EMJH medium with 5-fluorouracil and nalidixic acid associated with serial dilution technique used to recover Leptospira spp from experimentally contaminated bovine semen

Miraglia¹,

Moraes²,

Melville³

et al. 2009

Braz. J. Microbiol.

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Bovine semen experimentally contaminated with Leptospira santarosai serovar Guaricura was submitted to the modified EMJH medium with 5-fluorouracil (300mg/L) and nalidixic acid (20mg/L), named as "selective medium" and using the serial dilution technique, in order to evaluate the percentage of recovery of the added microorganism. The selective EMJH medium was found with higher percentage of recovery of leptospiras and minor losses of samples due to contamination with opportunistic microorganisms than the non-selective EMJH medium: 151/376 (40.0%) of positive growth; and 38/376 (10.0%) contamination and 58/376 (15%) and 129/376 (34.0%), respectively. These results were statistically significant (p<0. 0001; Fisher). Differences were found when the frequencies of positive leptospires recovery have been compared in the serial dilution technique (10 -1 to 10 -4 ) between the selective and non-selective media at different dilution factors. At 1/10 th dilution the percentages found were (0%, 0/80) and (38%, 30/80), at 1/100 th dilution, (3%, 2/80) and (49%, 39/ 80) and at 1/1,000 th dilution, (25%, 20/80) and (50%, 40/80), respectively. The percentage of recovery of leptospires was found to be directly proportional to the dilution used. The methodology of the serial dilution technique (setting at least three dilutions) and the use of selective EMJH medium have been found to be efficient for the isolation of leptospires from the bovine semen samples.

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“…canis QE13, which had been maintained as frozen glycerol stocks, were cultured in Brucella broth (Becton Dickinson) or Brucella broth containing 1.5% agar [7]. L. interrogans serotype Pomona (ATCC23478) were grown in liquid EMJH medium [1] at 30°C under aerobic conditions to a density of about 10 8 bacteria per ml when counted by the method of Faine [3]. Semen samples were collected from three brucellosis-free dogs, and then used for bacteriological and PCR amplification assays to detect B. canis or L. interrogans.…”

mentioning

confidence: 99%

Detection of Brucella canis and Leptospira interrogans in Canine Semen by Multiplex Nested PCR

Kim

Lee

Suzuki

et al. 2006

The Journal of Veterinary Medical Science

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ABSTRACT. Brucella canis and Leptospira interrogans are pathogenic bacteria that cause brucellosis and leptospirosis in dogs around the world. Both diseases can be diagnosed serologically, but the direct detection of these organisms in canine semen is needed when it is used for artificial reproduction. We have been attempting the artificial reproduction of guide dogs for greater breeding efficiency and for this purpose have developed a multiplex nested PCR technique for the detection of B. canis and L. interrogans in the semen and cryoprotective agent (CPA). Our results demonstrated the high sensitivity and simplicity of this technique in the detection of these organisms in canine semen and that will be useful in routine diagnosis. Since they have been found to stay alive in canine semen and CPA up to 48 hr, canine semen for breeding purposes should be checked for contamination by the PCR assay. KEY WORDS: Brucella canis, detection, Leptospira interrogans, PCR.J. Vet. Med. Sci. 68(6): 615-618, 2006 Canine brucellosis and leptospirosis are widely distributed around the world and they are important diseases due to the economic losses in animal production, and their risks to human health [2]. Reproductive disorders such as abortions and premature births will be the clinical signals of these bacterial diseases in pregnant animals. Though both diseases can be diagnosed from the detection of serum specific antibodies, the direct detection of these organisms in canine semen is needed when the semen is used for artificial reproduction. Bacteriological isolation is usually employed in diagnosis, but this is difficult, time consuming and dangerous [5,8].In Japan, there are 895 guide dogs which provide physical and mental support to the sight impaired. Since the raising of guide dogs is not easy, and the labor and other costs are huge [6], artificial reproduction of guide dogs will be needed to solve these problems. In the present study, we describe a method of detecting Burucella canis and Leptospira interrogans in canine semen for artificial reproduction using multiplex PCR, which is easier to perform and more rapid than other methods.B. canis QE13, which had been maintained as frozen glycerol stocks, were cultured in Brucella broth (Becton Dickinson) or Brucella broth containing 1.5% agar [7]. L. interrogans serotype Pomona (ATCC23478) were grown in liquid EMJH medium [1] at 30°C under aerobic conditions to a density of about 10 8 bacteria per ml when counted by the method of Faine [3]. Semen samples were collected from three brucellosis-free dogs, and then used for bacteriological and PCR amplification assays to detect B. canis or L. interrogans. Specific primer sequences were designed to detect the virB2 gene for Brucella spp. and 16S rRNA gene for Leptospira spp [4,9]. The sequences of oligonucleotide primers for PCR and nested PCR and expected sizes of amplicons are shown in Table 1. The accession number of the chromosome on the NCBI Enterez Genome website was AF226278 for B. abortus and M71241 for L. interrogans...

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Development of an improved selective medium for isolation of leptospires from clinical material

Cited by 34 publications

References 6 publications

Isolation of Leptospira spp from dogs, bovine and swine naturally infected

Isolation of Leptospira spp from dogs, bovine and swine naturally infected

EMJH medium with 5-fluorouracil and nalidixic acid associated with serial dilution technique used to recover Leptospira spp from experimentally contaminated bovine semen

Detection of Brucella canis and Leptospira interrogans in Canine Semen by Multiplex Nested PCR

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