Veterinary Microbiology 116 (2006) 325–328 www.elsevier.com/locate/vetmic
Short communication
Mycobacterium bovis cultured from commercially pasteurized cows’ milk: Laboratory cross-contamination Darling Me´ndez a, Francisco Gime´nez a, Arnelly Escalona b, Omaira Da Mata b, Alexandra Gonza´lez b, Howard Takiff c, Jacobus H. de Waard b,* a
Laboratorio de Zoonosis A´ngel Mele´ndez, Decanato Veterinario Universidad Centrooccidente Lisandro Alvarez, Barquisimeto, Venezuela b Laboratorio de Tuberculosis, Instituto de Biomedicina, Caracas, Venezuela c Laboratorio de Gene´tica Molecular, Centro de Microbiologı´a y Biologı´a Celular, Instituto Venezolana de Investigacio´n Cientı´fica, Altos de Pipe, Venezuela Received 16 February 2006; received in revised form 2 April 2006; accepted 3 April 2006
Abstract The ability of Mycobacterium paratuberculosis to survive the commercial pasteurization process of raw milk remains controversial. In a study undertaken in Venezuela to culture M. paratuberculosis from commercially pasteurized cows’ milk, 83– 200 ml containers of milk were processed and cultured on Herrold’s egg yolk slants. No M. paratuberculosis was cultured but a total of six colonies of Mycobacterium bovis were isolated from one container each from two different milk providers. Because laboratory cross-contamination was suspected, the laboratory records were reviewed and spoligotyping was carried out on the isolated individual colonies. On the day before these milk specimens were processed, the biological safety cabinet had been used for the isolation of M. bovis from lymph nodes from infected cattle. Spoligotyping showed that that the colonies isolated from the milk all had the same pattern as the strains isolated from the lymph nodes that were processed the previous day. As far as we know, this is the first report of cross-contamination in a veterinary mycobacterial laboratory. False-positive cultures in the mycobacterial laboratory are not rare. In this setting M. bovis was isolated because it is the most common manipulated organism in this laboratory. We believe that reports on the isolation of M. paratuberculosis from commercially pasteurized milk should exclude cross-contamination before reporting, especially when this organism is routinely isolated from animal material in the same lab. # 2006 Elsevier B.V. All rights reserved. Keywords: Mycobacterium bovis; Mycobacterium paratuberculosis; Laboratory cross-contamination; Spoligotyping; Pasteurized milk
1. Introduction * Corresponding author at: Laboratorio de Tuberculosis, Instituto de Biomedicina, al lado del Hospital Vargas, San Jose´, Caracas, Venezuela. Tel.: +58 212 8607095x1007; fax: +58 212 8611258. E-mail address:
[email protected] (J.H. de Waard).
Paratuberculosis is a chronic enteric disease of ruminant animals (Cocito et al., 1994) and is endemic in Venezuela. Although complete information is not
0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.04.021
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available for the country, a seroprevalence ranging from 20 to 50% was found in six tested cattle herds (authors’ unpublished results) Mycobacterium paratuberculosis is the causative agent of paratuberculosis and it has been suggested that this organism is associated with Crohn’s disease in humans (Chacon et al., 2004). Milk is a potential source of human exposure to this organism. At least four studies have demonstrated that M. paratuberculosis is not completely inactivated by pasteurization, and that humans could possibly be exposed to this pathogen in milk (Millar et al., 1996; Grant et al., 2002; Ellingson et al., 2005; Ayele et al., 2005). To examine this possibility, a study was undertaken to demonstrate the presence of viable mycobacteria in commercially pasteurized cow’s milk in Venezuela.
2. Materials and methods Between November 2002 and April 2003, 83 cartons, 200 ml each, of commercially pasteurized cow’s milk were bought at random from retail outlets throughout the town of Barquisimeto, and brought to the Veterinary Research Institute for processing. Before processing, special care was taken to avoid contamination with environmental mycobacteria: the outsides of the cartons were decontaminated with 0.5% sodium hypochlorite. The samples were processed in a biological safety cabinet class II which was cleaned with 0.5% sodium hypochlorite and 70% alcohol followed by UV light for 30 min with the air circulating. Only heat sterilized centrifuge tubes with screw tap were used. The milk samples (200 ml) were decontaminated with 0.75% hexadecylpyridinium
chloride (HPC) for 5 h and centrifuged at 3000 g for 20 min (Dundee et al., 2001). The sediment was inoculated onto Herrold’s egg yolk slants, and incubated for 36 weeks at 37 8C.
3. Results and discussion Of all 83 specimens cultured, colonies grew only from two cartons of milk, one from each of two different retailers. A total of six colonies of acid-fast bacilli were cultured after 4–6 weeks of incubation. These two cartons were processed on the same day, and the strains isolated were nitrate reductase negative and identified by IS6110 PCR and PRA (Telenti et al., 1993) as Mycobacterium bovis. No viable organisms of M. paratuberculosis or other mycobacteria were recovered. Because laboratory cross-contamination was suspected, the laboratory records were reviewed. It appeared that the day before the culture positive milk samples were processed, 43 lymph nodes from tuberculin positive cattle from a single cattle farm were processed in the same biological safety cabinet. Twelve isolates of M. bovis were obtained from these lymph nodes, and all these isolates were typed using spoligotyping (Kamerbeek et al., 1997), a molecular technique commonly used for the genotyping of M. bovis strains. This technique revealed that only one genotype is circulating on this farm, a unique genotype rather different from other M. bovis strains isolated in our laboratory. Spoligotyping of the six colonies obtained from the two milk samples confirmed the previous identification of M. bovis (absence of the spacers 39–43) and showed that the six colonies all
Fig. 1. Spoligotypes patterns of amplified mycobacterial DNA of M. tuberculosis strain 14323, the six M. bovis strains isolated from the milk samples (LTC24S, LCT21S, LCT21H, LCT23H, LCT24H and LCT2H) and two clinical isolates of M. tuberculosis from Venezuelan patients (2C6 and 9G3). The second lane is the negative control.
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had the same spoligotype pattern, which was identical to that of the strain circulating on the cattle farm (Fig. 1). The spoligotyping thus confirmed that the colonies that grew from the milk samples were most probably the result of cross-contamination in the laboratory with the M. bovis strain that originated from the lymph nodes processed the day before processing of the milk samples. The possible source of the crosscontamination stays unclear. It is unlikely that the contamination was airborne and occurred in the biological safety cabinet that had been thoroughly cleaned and sterilized. More likely the false-positive cultures arose from contamination of one of the reagents or contaminated equipment used in processing the milk samples and the lymph nodes. This is the first report of laboratory crosscontamination in a veterinary mycobacterial laboratory. Numerous reports have found that crosscontamination, causing cultures that are false-positives for M. tuberculosis, is a relatively common problem in human clinical tuberculosis laboratories (reviewed in Burman and Reves, 2000). False-positive cultures were found in 13 of the 14 (93%) studies that evaluated >100 patients, with a median false-positive rate of 3.1%, and an interquartile range between 2.2 and 10.5%. As stated by these authors, false-positive cultures for M. tuberculosis are not rare but are infrequently recognized by laboratory personnel. With our experience in mind, we believe that the possibility of cross-contamination must be excluded when reporting on the isolation of M. paratuberculosis from pasteurized milk samples, especially when the reporting laboratory routinely isolates M. paratuberculosis from clinical samples with a high mycobacterial load. In most articles reporting the isolation of M. paratuberculosis from pasteurized milk this is not done, nor are the number of colonies isolated indicated. The mycobacterial load in pasteurized milk is probably very low. It has been documented that cows with clinical disease caused by M. paratuberculosis or asymptomatic cows with heavy fecal shedding may excrete less than 1 CFU/ml of milk (Sweeney et al., 1992). In another publication only 4–20 CFU of M. paratuberculosis were isolated from 50 ml of raw milk collected aseptically from the udder (Ayele et al., 2005). Further, a significant dilution of contaminated milk with milk from healthy cows will occur at the processing plant where milk from many farms may be
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mixed together, and in this diluted milk the pasteurization process will kill a substantial part of the if not all of the M. paratuberculosis present. The decontamination procedure, necessary to overcome overgrowth of the culture medium with other bacteria present in the milk samples, will kill another 75– 99.9% of the survivors, depending the applied decontamination method (Dundee et al., 2001). These factors make detection of the viable microorganism fairly difficult if not impossible, and if isolated, it is unlikely that more than only a few colonies will grow. In the human mycobacterial laboratory the isolation of only a few colonies from a sample should always raise the possibility of cross-contamination (Burman and Reves, 2000) because the inoculum of material that contaminates another specimen is generally minimal (e.g., a droplet) and contains few bacilli. We therefore recommend that in future publications reporting the growth of individual colonies from milk specimens, the isolates must be studied by a genotyping method to confirm that their genotypes are distinct from those of laboratory and clinical strains recently isolated or in regular use within the testing laboratory. However, strain differentiation techniques for M. paratuberculosis have been shown to have only moderate discriminative power and therefore have limited applicability in these studies.
Acknowledgement This investigation was financed by the FONACIT project S1-2001000860.
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