The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated from tissues and faeces

Veterinary Microbiology 95 (2003) 271–282

The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated from tissues and faeces L.A. Reddacliff a,∗ , A. Vadali a , R.J. Whittington b a

Elizabeth Macarthur Agricultural Institute, New South Wales Agriculture, PMB 8, Camden, NSW, Australia b Faculty of Veterinary Science, University of Sydney, Private Bag 3, Camden, NSW 2570, Australia Received 29 November 2002; received in revised form 24 April 2003; accepted 20 May 2003

Abstract The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated in BACTEC cultures from clinical samples was assessed by spiking tissues and faeces at various points during the decontamination procedure. Routine protocols in the laboratory were shown to decrease the number of organisms isolated per sample by about 2.7 log10 and 3.1 log10 for faeces and tissues, respectively. These findings are important for the interpretation of negative culture results and may be useful in epidemiological studies. Addition of a centrifugation step to the tissue protocol increased the recovery by about 1 log, but resulted in increased contamination of BACTEC cultures. These studies may also facilitate future improvements to decontamination procedures. © 2003 Elsevier B.V. All rights reserved. Keywords: Mycobacterium; Paratuberculosis; Decontamination; Sheep; Radiometric culture

1. Introduction For the isolation of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) from clinical samples (e.g. tissues collected at necropsy, or faeces), decontamination protocols are required to prevent contamination of culture media by other gut flora. During these decontamination procedures, numbers of viable organisms may be reduced by exposure to disinfectants or antibiotics, by losses during centrifugation or sedimentation and by ∗

Corresponding author. Tel.: +61-246406314; fax: +61-246406400. E-mail address: [email protected] (L.A. Reddacliff). 0378-1135/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1135(03)00181-0

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removal of aliquots for inoculation of culture media. Various techniques are used by different laboratories and much effort has gone into optimising the balancing act between too little decontamination leading to contaminated cultures and too rigorous decontamination leading to false negative cultures (Whipple et al., 1992; Stabel, 1997; Eamens et al., 2000). Regardless of the method used, all protocols may reduce the numbers of M. a. paratuberculosis isolated from clinical samples. Several authors have measured or estimated this loss to be about 1 log10 –2 log10 , using different culture techniques for cattle (C) strains of M. a. paratuberculosis (Merkal, 1973; Jorgensen, 1982; Whitlock and Rosenberger, 1990), but results are often contradictory. For example, one study (Mokresh et al., 1989) found a reduction by about 2 log10 after exposure to 0.75% hexadecylpyridinium chloride (HPC) for 18 h, although other workers have reported no effect of HPC for up to 5 days (Whipple and Merkal, 1983). There are no published reports of the effects of decontamination procedures on the sheep (S) strains of M. a. paratuberculosis, and it would be unwise to simply extrapolate from C strain findings. S strains are very different to C strains in their cultural requirements and have only recently been reliably cultured in liquid media (Whittington et al., 1999). A knowledge of the effects of decontamination protocols on numbers is important to properly interpret negative culture results, in epidemiological studies (for example when attempting to quantify environmental contamination from the faeces of infected sheep) or in pathogenesis studies (for determination of the levels of infection in different tissues). The main aim of this study was to quantify the total effect of the routine decontamination protocols used in our laboratory for faecal and tissue samples on viable numbers of S strain M. a. paratuberculosis. This was addressed in Experiment 1, along with a secondary aim to identify the steps within the procedures where the major losses occurred. In Experiment 2, a modification of the routine tissue protocol by the addition of a centrifugation step, which was shown in Experiment 1 to have less effect on numbers, was evaluated using naturally infected tissue samples.

2. Methods 2.1. Experiment 1: spiked tissue and faecal samples 2.1.1. Tissue and faecal samples Terminal ileum and faeces were collected at necropsy from several sheep from a flock monitored negative for ovine Johne’s disease and stored at −80 ◦ C until processed. The samples were thawed, pooled to produce one pool of tissues and one pool of faeces, homogenised with a small amount of saline in a blender, dispensed into 2 ml aliquots and stored at −80 ◦ C until required. Prior to the main experiment, several aliquots of tissue and faeces were thawed, processed by the routine decontamination procedures (below) and cultured in BACTEC vials to confirm negative status for M. a. paratuberculosis, and also to assess potential contamination of cultures. 2.1.2. Decontamination of faecal samples for BACTEC culture A double incubation method was used (Whittington et al., 1998). Briefly, an aliquot of faeces was mixed with 12 ml of sterile normal saline in a 15 ml polypropylene tube. After

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mixing, the tube was allowed to stand for 30 min at room temperature. A 5 ml aliquot of the surface fluid was transferred to a 35 ml polystyrene tube containing 25 ml of 0.9% HPC (Sigma Chemical Co., St Louis, MO, USA) in half-strength brain heart infusion broth (BHI) (Oxoid, Basingstoke, England) and allowed to stand at 37 ◦ C for 24 h. The tube was then centrifuged at 900 × g for 30 min. The pellet was resuspended in 1 ml of sterile water with vancomycin (100 ␮g/ml), nalidixic acid (100 ␮g/ml) and amphotericin B (50 ␮g/ml) (VAN) and incubated for 72 h at 37 ◦ C. Sediment was then resuspended by vigorous agitation, and 100 ␮l aliquots were used to inoculate BACTEC vials. 2.1.3. Decontamination of tissue samples for BACTEC culture Two protocols were used: a previously described sedimentation method which is routinely used in our laboratory (Whittington et al., 1998, 1999), and a modification including a centrifugation step. Briefly, an aliquot of tissue was mixed with 25 ml of 0.75% HPC in a 35 ml polystyrene tube, then left undisturbed at room temperature for 72 h. In the sedimentation method, 100 ␮l aliquots were then carefully removed from near the bottom of the tube by aspiration using a 25 gauge needle on a tuberculin syringe, and inoculated into BACTEC vials. In the alternate method, after 72 h in HPC the tube was centrifuged at 900 × g for 30 min. The pellet was resuspended in 1 ml of 0.75% HPC by vigorous agitation and vortexing, and 100 ␮l aliquots were used to inoculate BACTEC vials. 2.1.4. BACTEC culture BACTEC vials were incubated at 37 ◦ C for up to 12 weeks. The modified BACTEC 12B radiometric medium consisted of 4 ml enriched Middlebrook 7H9 medium (BACTEC 12B; Becton Dickinson, Sparks, MD, USA) with 200 ␮l PANTA PLUS (Becton Dickinson), 1 ml egg yolk, 5 ␮g of mycobactin J (Allied Monitor Inc., Fayette, MO, USA) and 0.7 ml of water (Whittington et al., 1999). Growth indices (GI) were measured with an automatic ion chamber (BACTEC 460; Johnston Laboratories, Towson, MD, USA). PCR for IS900 and restriction endonuclease analysis (REA) were performed on material from GI positive vials to confirm that the observed GI were due to M. a. paratuberculosis (Whittington et al., 1998; Cousins et al., 1999). 2.1.5. Preparation and enumeration of stock suspension of M. a. paratuberculosis M. a. paratuberculosis (fifth passage of material originally isolated from faeces of infected sheep) was stored at −80 ◦ C in modified BACTEC medium. This was thawed, reinoculated into modified BACTEC medium, and modified 7H10 slopes (Whittington et al., 1999) were inoculated from the fresh BACTEC vial when the weekly GI reached 999. Colonies were harvested in phosphate-buffered saline with 0.1% v/v Tween-80 (PBST) as previously described (Reddacliff et al., 2003). The resulting suspension was diluted tenfold in PBST, vortexed for 1 min, passed 10× through a 26 g needle, then filtered through an 8 ␮m filter. This filtered suspension was further diluted 1:10 in PBST, dispensed into 1 ml aliquots and stored at −80 ◦ C. The stored material was termed stock suspension. Previous studies had shown no decrease in numbers of S strain M. a. paratuberculosis in PBST suspensions after at least 12 months storage at −80 ◦ C (Reddacliff, 2002). The number of viable M. a. paratuberculosis in the stock suspension was 8.1 log10 per ml. This was determined using a serial dilution (most probable number) technique (Anon, 1991; Reddacliff et al., 2003).

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In the main experiment, aliquots of the stock suspension were thawed at 4 ◦ C within 1 h of use and tenfold dilution series were made in PBST. Hundred microlitre aliquots of undiluted, 10−2 , 10−4 and 10−6 dilutions of the stock suspension were used to spike samples with 7.1, 5.1, 3.1 or 1.1 log10 viable M. a. paratuberculosis, respectively. This range was selected to span the range of number of organisms likely to be encountered in clinical material. 2.1.6. Experimental design Faecal samples were all prepared using the same decontamination protocol (above) and were spiked at four different stages during this procedure. These are shown Fig. 1a. In Group F1, faecal samples were spiked before the start of the procedure, so that the M. a. paratuberculosis organisms were exposed to the whole protocol. The steps in the protocol where loss of numbers could occur included the initial sedimentation to remove heavy faecal solids (all organisms may not remain in suspension), the effect of removing only 5 ml of surface fluid from a 12 ml total volume, 24 h exposure to HPC/BHI, centrifugation (all organisms may not be in the pellet), 72 h exposure to VAN, the effect of removing a 100 ␮l aliquot from a well mixed 1 ml volume (a 1 log10 reduction), and finally any carryover effect of VAN in the inoculum on the ability of the remaining organisms to grow in BACTEC culture. In Group F2, samples were spiked immediately after the 5 ml aliquot was added to the HPC/BHI, thus avoiding the effects of the initial sedimentation and 5 ml aliquot removal. Group F3 samples were spiked immediately after the pellet was resuspended in VAN, so that only the effect of 72 h in VAN and subsequent steps reduced numbers. Group F4 samples were spiked immediately before the final 100 ␮l aliquots were removed from the 1 ml of VAN, so that the number of M. a. paratuberculosis were reduced only by aliquot removal (1 log10 ), and by brief exposure to VAN plus any carryover effect. For the tissue cultures, two different decontamination protocols were assessed, and these are shown in Fig. 1b. In group T1, tissue samples were spiked before the start of the routine (sedimentation) protocol. Thus, numbers of M. a. paratuberculosis organisms were likely to be reduced by exposure to HPC for 72 h, by the effect of removing a 100 ␮l aliquot from the bottom of a 27 ml volume after 72 h of sedimentation, and by any carryover effect of HPC. It was not possible to separate the effects of sedimentation from those of exposure to HPC within this protocol. The centrifugation protocol which was used to prepare tissue samples from groups T2 and T3 was devised so that the effect of HPC alone could be assessed. Group T2 samples were spiked before the start of the procedure so that numbers of M. a. paratuberculosis were reduced by 72 h exposure to HPC, by any losses during centrifugation, by removal of a 100 ␮l aliquot from 1 ml (1 log10 ), and finally by any carryover effect of HPC. Group T3 samples were spiked immediately before the final 100 ␮l aliquots were removed from the 1 ml of HPC, so that numbers of M. a. paratuberculosis were reduced only by aliquot removal (1 log10 ), and by brief exposure to HPC plus any carryover effect. Within each treatment group above, four separate samples were processed, each spiked with 7.1, 5.1, 3.1 or 1.1 log10 viable M. a. paratuberculosis. For each sample, triplicate BACTEC vials were inoculated to determine viable numbers of M. a. paratuberculosis remaining. All samples were processed so that the inoculation into BACTEC vials was done at the same time.

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(a) Decontamination procedure for each faecal sample

2 g faecal sample

Spiking with

Treatment Group

Faeces spiked prior to start of decontamination

F1

HPC/BHI spiked at start of incubation

F2

VAN spiked at start of incubation

F3

VAN spiked immediately prior to aliquot removal

F4

Faeces mixed with 12 mL saline, sedimentation for 30 min

5 mL aliquot removed from near surface

5 mL aliquot added to HPC/BHI, incubated 24 h, then centrifuged

Pellet resuspended in 1 mL VAN

Incubated in VAN 72 h, then remixed

100 µL aliquots removed and inoculated into 3 replicate BACTEC vials

(b) Decontamination procedures for tissue samples

Sedimentation protocol 2 g tissue sample

Spiking with

Treatment Group

Tissues spiked prior to start of decontamination

T1

Tissues spiked 30 min prior to start of decontamination

T2

HPC spiked immediately prior to aliquot removal

T3

Tissue mixed with HPC, incubated and sedimented 72 h

100µL aliquots removed from bottom of tube and inoculated into 3 replicate BACTEC vials

Centrifugation protocol 2 g tissue sample

Tissue mixed with HPC, incubated 72 h, then centrifuged

Pellet resuspended in 1 mL HPC

100 µL aliquots removed and inoculated into 3 replicate BACTEC vials

Fig. 1. Experimental design shows the points in the procedures where samples were spiked with M. a. paratuberculosis: decontamination protocol used for (a) all faecal samples; (b) tissue samples.

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log10 inoculum size

6 5 4 3 2 1 0 15

20

25

30

35

40

Days for CGI to reach 1000

Fig. 2. Regression of inoculum size for the untreated organisms on dCGI1000: 10−1 (䊏),10−3 (䊉) and 10−5 (䉱) dilutions of the stock suspension were used in the regression. Error bars show the range of dCGI1000 across the three replicate BACTEC vials. The 10−7 dilution (䊐), for which only one of the three vials had growth, was not used.

2.1.7. Enumeration of viable M. a. paratuberculosis after decontamination GI in BACTEC cultures were measured weekly until growth was observed, then as often as necessary to prevent their going off-scale, until the cumulative growth index exceed 1000. A linear regression of inoculum size on days taken for the CGI to reach 1000 (dCGI1000) was produced as previously described (Reddacliff et al., 2003), using the results from triplicate BACTEC vials inoculated with 6.1, 4.1, 2.1 or 0.1 log10 viable M. a. paratuberculosis (100 ␮l of 10−1 , 10−3 , 10−5 and 10−7 dilutions of stock suspension). This regression is shown in Fig. 2. The approximate number of viable organisms in inocula for each sample was determined from this regression using the mean dCGI1000 of the replicate vials. 2.1.8. Statistical analysis The results for each sample within each treatment group were expressed as the log10 reduction in organisms: (number log10 used to spike sample) − (number log10 isolated in BACTEC culture). Mean log10 reductions were compared between selected groups using Student’s t-test. 2.2. Experiment 2: comparison of sedimentation and centrifugation methods for naturally infected tissue samples Tissues obtained at necropsy from 8 to 21 month old Merino lambs grazing pasture heavily contaminated with M. a. paratuberculosis were stored at −80 ◦ C for up to 3 months until cultured. A total of 409 tissues were used in this study. Each tissue sample (1–2 g) was homogenised in 2 ml of sterile saline and equal aliquots from the resulting homogenate were prepared in parallel by the sedimentation and centrifugation tissue decontamination

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methods. A single BACTEC vial was inoculated for each method, GI were read weekly for 12 weeks, and M. a. paratuberculosis was identified in GI positive vials by IS900 PCR and REA as described above. The dCGI1000 of the cultures prepared by the centrifugation method was estimated from the weekly data as previously described (Reddacliff et al., 2003), and the approximate number of viable organisms inoculated into the BACTEC vials was determined from a previously described general predictive relationship (Reddacliff et al., 2003). 2.2.1. Statistical analysis Sensitivity and contamination rates for the sedimentation and centrifugation methods were compared using McNemar’s Chi-square test for paired observations (Motulsky, 1995). Student’s t-test was used to compare the mean dCGI1000 of samples which were culturepositive by both methods with that of samples positive by the centrifugation method only.

3. Results 3.1. Experiment 1 Preliminary cultures of the pooled faecal and tissue samples revealed no growth of M. a. paratuberculosis and contamination was not observed. The numbers of viable M. a. paratuberculosis remaining after the various decontamination procedures are shown in Fig. 3. The log10 reduction in numbers was similar across the different levels of inoculation, and the group means using data for levels of 7.1, 5.1 and 3.1 (log10 ) inoculated organisms are shown in Table 1. The faeces decontamination protocol reduced numbers by 2.7 log10 . The reduction in numbers for tissues processed by the sedimentation method (3.1 log10 ) was significantly greater than that for the centrifugation method (2.2 log10 ) (P < 0.05). Table 1 Effect of decontamination procedures on numbers of viable M. a. paratuberculosis isolated in BACTEC culture Treatment group (steps likely to influence numbers)

Log10 reduction Mean

S.D.

Untreated organisms None (used to establish regression)

0.0

0.03

Faecal culture procedures F4 (VAN carryover, final aliquot) F3 (72 h in VAN plus F4 above) F2 (24 h HPC/BHI plus F3 above) F1 (full faeces protocol)

1.4 2.7 2.3 2.7

0.36 0.34 0.21 0.50

Tissue culture procedures T3 (HPC carryover, final aliquot) T2 (full centrifugation protocol) T1 (full sedimentation protocol)

1 2.2 3.1

0.15 0.26 0.06

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(a)

Number isolated in BACTEC culture

7 6 5 4 3 2

*

1 0 7.1

5.1

3.1

1.1

-1 Number of organisms (log10) used to spike sample

(b) Number isolated in BACTEC culture

7 6 5 4 3 2 1 0 7.1

5.1

3.1

1.1

-1 Number of organisms (log10) used to spike sample

Fig. 3. Effect of decontamination procedures on numbers of viable M. a. paratuberculosis inoculated into BACTEC vials. Numbers (log10 ) were estimated by regression on dCGI1000. Values less than 0 indicate no growth in any of the three replicate vials. No growth in one or two replicate vials is indicated as (*). Error bars show the range of estimated numbers across the three replicate vials for each sample. (a) Faecal samples: F4, brief exposure to VAN (䊏); F3, 72 h in VAN ( ); F2, 24 h in HPC/BHI, then 72 h in VAN (䊐); F1, full faeces protocol ( ). (b) Tissue samples: T3, brief exposure to HPC (䊏); T2, full centrifugation protocol ( ); T1, full sedimentation protocol ( ).

3.2. Experiment 2 Results are summarised in Table 2. With the sedimentation method, 47/409 tissue samples (11.5%) were positive for M. a. paratuberculosis. With the centrifugation method 59/409

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Table 2 Comparison of routine (sedimentation) and centrifugation protocols for recovery of M. a. paratuberculosis from ovine tissue samples Sample result Positive by both methods Positive by centrifugation only Positive by sedimentation only Negative by both methods

No. of samples 44 15 3 347

Mean dCGI1000 29.3 (centrifugation), 36 (sedimentation) 43 53

were positive (14.4%). Thus the centrifugation method detected about 25% more infected tissue samples than the sedimentation method. The difference was highly significant (P < 0.01). Only 2/409 samples (0.5%) were contaminated (GI detected but negative for M. a. paratuberculosis by IS900 PCR) using the sedimentation method, whereas 16/409 (3.9%) were contaminated using the centrifugation method. One sample was contaminated in both methods. The difference was very highly significant (P < 0.001). The mean dCGI1000 (centrifugation method) for samples positive by both methods was 29.3 days, and that for samples, only positive in the centrifugation method was 43 days. This difference was highly significant (P < 0.01), indicating that samples positive by the centrifugation method alone contained fewer viable organisms.

4. Discussion The results for samples spiked at the start of the decontamination procedures show the total effects of these procedures for S strain M. a. paratuberculosis. The loss of numbers was 2.7 log10 per faecal sample, 3.1 log10 for tissue samples prepared by the routine (sedimentation) method, and 2.2 log10 for tissue samples prepared using centrifugation. Using these values, and assuming a sample size of 2 g, the detection limit was 2.4 log10 (2.5 × 102 ) organisms per gram for routine faecal culture, 2.8 log10 (6.3 × 102 ) for sedimentation tissue culture and 1.9 log10 (7.9 × 101 ) for centrifugation tissue culture. Obviously, when small numbers of organisms are present in clinical samples false negative cultures will be frequent. This is an important consideration in pathogenesis studies or diagnosis of Johne’s disease, especially in the subclinical stages when levels of infection in tissues and faeces are low. Comparison of the results for samples spiked at various points during the decontamination procedures indicated the steps of those procedures most responsible for losses in numbers of viable organisms. The loss due to the final selection of 100 ␮l aliquots for inoculation of BACTEC cultures included the effect of aliquot removal, and any loss due to carryover effects of the VAN (faecal cultures) or HPC (tissue cultures), and is indicated by the results for Group F4 (1.4 log10 ) and Group T3 (1.0 log10 ), respectively. The loss due to aliquot removal was obviously 1 log10 (a 100 ␮l aliquot was removed from a 1 ml volume), and would be difficult to avoid. One millilitre is the minimum practical volume for resuspension of the pellet resulting from about 2 g of faeces or tissues, and the use of more than 100 ␮l for inoculation

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of BACTEC vials may interfere with the concentration of substrates necessary for growth of the fastidious S strain M. a. paratuberculosis. In this experiment there was no carryover effect of HPC on M. a. paratuberculosis numbers, since the reduction of numbers in Group T3 was exactly that expected due to aliquot removal alone. A small (0.4 log10 ) carryover effect of VAN was seen, but was not statistically significant in this experiment. In the initial processing of faecal samples, there was a sedimentation step in a total volume of 12–14 ml of saline and faeces, with removal after 30 min of a 5 ml aliquot from the surface layer. The effect of this step was revealed by comparison of the results for groups F1 and F2. This step resulted in a small, but statistically insignificant loss (0.4 log10 ), about equal to the expected effect of taking a 5 ml sub-sample from a total volume of about 12 ml. In this experiment, faeces were spiked just 30 min before the start of decontamination, and the M. a. paratuberculosis organisms were apparently well suspended after mixing with saline. In real clinical samples, the association of organisms with faecal solids which settle out may be different, so this result may not reflect the situation for clinical samples. In the faeces protocol there was a significant (P < 0.05) 1.3 log10 reduction in numbers due to the 72 h in VAN (compare group F3 with F4), but there was no further reduction in numbers due to the 24 h in HPC/BHI and subsequent centrifugation (compare group F2 with F3). In fact, there was some suggestion that this step may have minimised the effects on M. a. paratuberculosis of subsequent exposure to VAN. In the samples spiked with 3.1 log10 organisms, no growth was seen in any of the three BACTEC vials following 72 h exposure to VAN alone, whereas M. a. paratuberculosis was recovered from all three vials after treatment with HPC/BHI followed by VAN. For the tissue decontamination protocols, 72 h in 0.75% HPC was associated with a significant (P < 0.01) 1.2 log10 reduction in numbers (compare group T2 with T3). This reduction is intermediate between reported reductions in viable numbers for C strains of M. a. subsp. paratuberculosis exposed to HPC (Whipple and Merkal, 1983; Mokresh et al., 1989). The significantly greater loss (0.9 log10 ) of organisms from tissue samples using the routine protocol compared to the centrifugation modification indicates that the removal of a 100 ␮l aliquot from the bottom of the tube after 72 h sedimentation was less effective than centrifugation in concentrating M. a. paratuberculosis. Whether the centrifugation modification would be useful to increase culture sensitivity from real samples was assessed in Experiment 2. Despite a significant increase in contamination rates, this method detected about 25% more M. a. paratuberculosis-positive tissues than the sedimentation method. The difference was likely to be a result of fewer viable organisms remaining in inocula prepared by the sedimentation method. In samples positive by both methods, the mean dCGI1000 was 29.3 when centrifugation was used, compared to 36 for the sedimentation method. These figures suggest that inocula prepared by the centrifugation method contained about 1 log10 more viable organisms than those prepared by sedimentation (Reddacliff et al., 2003), consistent with the findings for spiked samples in Experiment 1. Moreover, samples positive by both methods contained larger numbers of viable organisms than samples positive only in the centrifugation method. The dCGI1000 from samples positive only by centrifugation (43 days) indicate that very few M. a. paratuberculosis organisms (10 or less) were present in the inocula (Reddacliff et al., 2003). It is then reasonable to expect that the sedimentation method (demonstrated to have a detection limit of about 1 log10 higher than the centrifugation method) would fail to detect M. a. subsp. paratuberculosis in many such samples.

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In conclusion, this study has established that routine decontamination protocols for faecal and tissue samples reduces the number of S strain M. a. paratuberculosis ultimately isolated in BACTEC culture by about 2.7 log10 and 3.1 log10 , respectively. A modification of the routine tissue protocol including a centrifugation step improved recovery by 0.9 log10 , and was shown to improve detection of M. a. paratuberculosis from field samples. This study may also facilitate further refinement of the decontamination protocols to minimise the losses of viable S strain M. a. paratuberculosis during processing. For example, the use of a 100 ␮l aliquot to inoculate BACTEC vials was responsible for almost half the losses in the modified tissue protocol. Whether larger aliquot volumes can be used without compromising BACTEC cultures might be investigated. In the faecal culture technique, exposure to VAN was the biggest cause of reduced numbers—about 1.7 log10 if carryover effects are included. Changes in VAN concentration or length of exposure might be worthy of investigation.

Acknowledgements This work formed part of Ph.D. studies of the senior author, and was funded by Meat and Livestock Australia and New South Wales Agriculture. All procedures were undertaken at the Elizabeth Macarthur Agricultural Institute, New South Wales Agriculture.

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