PREDICTION OF THE SUSCEPTIBILITY OF AN AT RISK PATIENT FOR DEVELOPING OR REDEVELOPING CLOSTRIDIUM DIFFICILE INFECTION
20170242006 · 2017-08-24
Assignee
Inventors
Cpc classification
G01N2469/20
PHYSICS
International classification
Abstract
A method for prediction of the susceptibility of an at risk patient to developing or redeveloping an infection with Clostridium difficile, having the determination by immunoassay, in a stool sample from said patient, of the level of antibody IgA anti-toxin B of Clostridium difficile, and comparing this level with a reference value S determined beforehand using two populations of patients exposed to the bacterium, one population not having developed or redeveloped such an infection and the other population having developed or redeveloped such an infection, —a level lower than said reference value S signifying that the patient is a patient with a heightened risk of developing or redeveloping a Clostridium difficile infection, and —a level higher than said reference value S signifying that the patient is not a patient with a heightened risk of developing or redeveloping a Clostridium difficile infection.
Claims
1. A method for predicting the susceptibility of an at-risk patient for developing or redeveloping Clostridium difficile infection, comprising determining, by immunoassay, in a stool sample from said patient, the level of IgA antibodies against toxin B of Clostridium difficile, and comparing this level against a reference value S determined beforehand with two populations of patients exposed to the bacterium, one not having developed or redeveloped such an infection and the other having developed or redeveloped such an infection, a level below said reference value S signifying that the patient is a patient with increased risk of developing or redeveloping Clostridium difficile infection, and a level above said reference value S signifying that the patient is not a patient with increased risk of developing or redeveloping Clostridium difficile infection.
2. The method as claimed in claim 1, wherein the stool sample used in the immunoassay had been treated beforehand with an acidic sample treatment buffer, preferably at pH 2.5.
3. The method as claimed in claim 2, wherein, in the sample pretreatment step, the sample is brought into contact with the acidic sample pretreatment buffer for at most 30 min, and it then undergoes separation, notably by filtration or sedimentation, the filtrate or supernatant then being recovered, to be used in the immunoassay.
4. The method as claimed in claim 2, wherein the sample pretreatment step does not employ a neutralizing step before the pretreated sample is used in the immunoassay.
5. A method for determining, by immunoassay, the level of at least one IgA antibody directed against a protein that is unaffected in the presence of acid, preferably directed against toxin B of Clostridium difficile, in a patient's biological sample that may contain said at least one IgA antibody, comprising bringing one or more binding partners to said at least one IgA antibody, used for performing the immunoassay, into contact with an acidic reaction mixture comprising said biological sample pretreated with an acidic sample treatment buffer, without neutralization before it is used in the immunoassay.
6. The method as claimed in claim 5, wherein, the sample pretreated with the acidic buffer undergoes separation, notably by filtration or sedimentation, the filtrate or supernatant then being the reaction mixture recovered for use in the immunoassay.
7. The method as claimed in claim 5, wherein the acidic sample pretreatment buffer has pH 2.5.
8. A kit for determining, by immunoassay, the level of at least one IgA antibody directed against a protein that is unaffected in the presence of acid, notably directed against toxin B of Clostridium difficile, in a patient's biological sample that may contain said at least one IgA antibody, comprising (i) one or more binding partners to said at least one IgA antibody for performing the immunoassay, and (ii) an acidic sample treatment buffer, it being understood that said kit does not contain any neutralizing solution.
9. The kit as claimed in claim 8, in which the biological sample consists of stool or rectal enema from the patient.
10. The kit as claimed in claim 8, for immunoassay detection of IgA antibodies against toxin B of Clostridium difficile in a patient's stool sample that may contain said IgAs, also comprising (iii) at least one control sample, which is a sample containing a known amount of IgA antibodies against toxin B of Clostridium difficile.
Description
[0083] The invention will be better understood from the following examples, which are given for purposes of illustration and are nonlimiting, as well as from
EXAMPLES
Example 1
Preparation of Recombinant Toxins A and B of Clostridium difficile
[0084] The tcdA gene coding for toxin A of Clostridium difficile and the tcdB gene coding for toxin B of Clostridium difficile were derived from the reference strain VPI 10463, toxinotvpe 0, ribotype 087-(toxin A: Swissprot accession No. P16154 and toxin B: Swissprot accession No. P18177). For each of the genes, the whole sequence was optimized by Geneart (Invitrogen) for expression in E. coli and 8 histidines were added on the N-term side to allow purification by metal-chelate affinity chromatography. The synthetic genes obtained were cloned into the pET3d vector (Novagen) by means of the NcoI and BamHI restriction sites.
[0085] The expression plasmids thus constructed are introduced into E. coli BL21 bacteria and derivatives (Stratagene, Agilent Technologies). The cultures are carried out in 2× YT medium (Difco), in the presence of 100 μg/mL, ampicillin and 10% glucose, at 24° C. with stirring. Induction of expression of the protein is effected by adding 1 mM of IPTG (isopropyl beta-D-1-thiogalactopyranoside) for 4 h, once it is in the exponential growth phase. At the end of culture, the bacteria are collected by centrifugation at 10000 g, at 4° C., for 30 min. The bacterial deposits are frozen at −80° C.,
[0086] The bacterial deposits are taken up in PBS buffer 2× (phosphate buffered saline) and lysed. The lysates are centrifuged at 3000 g for 30 min at 4° C. The supernatant contains the purified soluble proteins, recombinant toxin A or toxin B depending on the expression plasmid used initially.
[0087] The proteins are purified by one-step metal-chelate affinity chromatography. The supernatant obtained after centrifugation is loaded on an Ni-NTA-Agarose resin (Qiagen). After a washing cycle, the protein (toxin A or toxin B) is eluted in the presence of 250 mM imidazole. The protein is dialyzed in a 50 mM phosphate, 150 mM NaCl buffer,
Example 2
Immunoassay for Detecting Fecal IgA Against Toxin B
[0088] The immunoassays were carried out using the VIDAS® automatic immunoanalyzer (bioMérieux). The disposable cone serves both as solid phase for the reaction and as pipetting system. The cartridge comprises 10 wells (X0 to X9) covered with aluminum foil, sealed and labeled. The first well (X0) comprises a pre-cut part to facilitate sample insertion. The last well (X9) is an optical cuvette in which the fluorescence of the substrate is measured. The various reagents required for the analysis are contained in the intermediate wells (X1 to X8). All the steps of the assay are carried out automatically by the instrument. They consist of a succession of cycles of aspiration/discharge of the reaction mixture.
[0089] a) Sensitization and Passivation of the Cones
[0090] The cones were sensitized with 300 μL of a solution of recombinant or native toxin B diluted to 2 μg/mL in PBS buffer, pH 7.2. The inactivated native toxin B (Cat. No. CDB-TDL) was obtained from The Native Antigen Company (Upper Heyford, United Kingdom). After about 20 h of incubation at +18/25° C. with the sensitizing solution, the cones are emptied. Then 300 μL of a solution of 200 mM Tris containing 5 g/L of bovine albumin is added. Passivation is continued at +18/25° C. overnight. The cones are emptied, dried, and then stored at +4° C. until use, away from moisture.
[0091] b) Sample Pretreatment
[0092] One volume of stool is brought into contact with 3 volumes of a sample treatment buffer (dilution ¼). Improvement of the composition of this buffer is illustrated in example 3. Manual homogenization is carried out, followed by stirring in a Vortex. This step takes between about 30 seconds and 2 minutes, depending on stool consistency. The samples are centrifuged for 5 minutes at 12000 g to recover the soluble proteins. Immunoassay is carried out on this supernatant, which is deposited directly in well X1 of the VIDAS® cartridge.
[0093] c) Immunoassay Procedure
[0094] Well X1 contains 300 μL of sample diluent, with composition identical to the sample treatment buffer. 200 μL of the supernatant obtained in step b) is transferred to well X1 in order to perform additional dilution. As soon as the VIDAS® cone is in contact with the sample, the first step of the immunologic reaction begins. This step allows specific binding of fecal anti-toxin B IgAs, present or not in the stool sample supernatant, to the toxin B adsorbed on the cone. After 4 minutes of incubation at 37° C., the components that are not bound are removed by washing with 200 mM Tris buffer pH 7.8, NaCl 300 mM, Tween® 20 0.25%. In the second step, the cone is incubated with a solution of conjugate containing about 0.5 μg/mL of an anti-human IgA mouse IgG (bioMérieux) coupled to alkaline phosphatase, in a 10 mM phosphate buffer, containing 300 mM of NaCl and 5 g/L of bovine serum albumin. Well X5 contains 400 μL of this solution, which the cone aspirates/discharges for 5 minutes, still at 37° C. The second step results in the formation of a complex between the fecal anti-toxin B IgAs and the anti-IgA conjugate coupled to alkaline phosphatase. This step is followed by 3 successive washing operations to remove the compounds that are not fixed.
[0095] In the final detection step, the substrate 4-methylombelliferyl phosphate is aspirated and then discharged in the cone; the enzyme of the conjugate catalyzes the reaction of hydrolysis of this substrate to 4-methylombelliferone, whose fluorescence emitted is measured at 450 nm. The value of the fluorescence signal (RFV=relative fluorescence value) is proportional to the concentration of fecal anti-toxin B IgAs present in the sample.
[0096] Table I below presents the fluorescence signals (RFV=relative fluorescence value) determined by the VIDAS® automatic instrument using buffer R1 as sample treatment buffer. This buffer forms part of the VIDAS® C. difficile Toxin A&B kit (Cat. No. 30118, bioMérieux) for detecting toxins A and B in the stool. Its pH is 7.2, as described in the instructions with the kit. It is used for extracting the toxins from human stool. The stool samples used were obtained from patients who consulted the department of Dr. Robert Spencer, Health Protection Agency Regional Laboratory, Bristol, United Kingdom with suspected Clostridium difficile infection.
TABLE-US-00001 TABLE 1 Detection of the fecal IgAs in human stool. Anti-toxin B IgAs Anti-toxin B IgAs Sample VIDAS ® signal Interpretation of VIDAS ® VIDAS ® code (RFV) the assay GDH Tox A & B S700 36 Neg Neg ND S706 9 Neg Neg ND S709 114 Pos Neg ND S713 91 Neg Pos Neg S719 831 Pos Neg ND S720 56 Neg Pos Neg Neg = negative; Pos = positive; ND = not determined
[0097] The result of the immunoassay is interpreted by comparing the RFV signal measured for each sample at the limit of detection of the VIDAS® assay, which is defined as follows; mean background noise (RFV signal measured with a buffer) +3 standard deviations. The VIDAS® signals above this limit of detection are regarded as positive: anti-toxin B IgA antibodies are present. The VIDAS® signals below this limit of detection are regarded as negative: no anti-toxin B IgA antibodies are present.
[0098] It is important to note that sample S709 with a signal of 114 RFV is classified as positive whereas sample S713 with a signal of 91 RFV is classified as negative. Two signals that are close in absolute value lead to a different biological interpretation: this situation is not satisfactory. It therefore needs to be improved.
Example 3
Optimization of Extraction of the Fecal IgAs
[0099] Buffer R1 suitable for extraction of toxins A and B, used in example 2, showed that it was possible to improve the analysis of the fecal IgAs. Another sample treatment buffer was therefore tested using the stool samples characterized in example 2, with the same treatment time as before (30 s and 2 min). This buffer is a hydrochloric acid-glycine buffer at pH 2.5 (1M glycine pH 2.5). Table 2 below presents the fluorescence signals (RFV=relative fluorescence value) determined by the VIDAS® automatic instrument. The results presented in this experiment were obtained using cones on which recombinant toxin B was immobilized.
TABLE-US-00002 TABLE 2 Detection of the fecal IgAs extracted with different buffers from human stool. Acid buffer = 1M glycine pH 2.5 and buffer R1 described in example 2. Anti-toxin B IgAs, VIDAS ® signal (RFV) Sample Prescence Buffer R1 code of IgA (state of the art) Acid buffer S700 Neg 36 127 S706 Neg 9 60 S713 Neg 91 137 S720 Neg 56 169 S709 Pos 114 1303 S719 Pos 831 2797 Pos = positive; Neg = negative
[0100] The 1M glycine acidic buffer pH 2.5 makes it possible to increase the RFV signals obtained for the positive samples significantly, from 3 to 7 times more than with buffer R1. The extraction solution at acid pH is selected for the rest of the experiments.
Example 4
Investigation of the Contact Time Between the Sample and the Sample Treatment Buffer
[0101] The duration of contact between the sample and the acidic sample treatment buffer (condition in example 3) was studied using a high positive sample (S719), a low positive sample (S709) and a negative sample (S700). The results are presented in
Example 5
Comparison of Recombinant Toxin B and Native Toxin B for Detecting the Anti-Toxin B IgAs by Immunoassay
[0102] We compared the fluorescence signals obtained when using a recombinant toxin B and a native toxin B for preparing the capture phase (adsorption on the cones). The two types of toxin B were immobilized on the solid phase at the same concentration (2 μg/mL). The results are presented in Table 3 below. Samples SP023 and SP pool were obtained from Professor M. Delmée, Clostridium difficile National Reference Center, Catholic University of Louvain, Brussels, Belgium.
[0103] The procedure described in example 3 above was repeated.
[0104] Apart from sample S709, the VIDAS® signals obtained for all the samples are stronger when the solid phase has the native toxin than the recombinant toxin. For the negative samples, the gain is negligible (+37 to +67 RFV), whereas for 3 of the 4 positive samples the gain is very large (+640 to +1797 RFV). Thus, use of the native toxin B for capture allows better discrimination of the positive and negative samples.
[0105] According to the results presented in Table 3, the positivity threshold when using recombinant toxin B is fixed at 200 RFV whereas the positivity threshold when using native toxin B is fixed at 250 RFV.
TABLE-US-00003 TABLE 3 Detection of the fecal anti-toxin B IgAs by recombinant toxin B or native toxin B. Pretreatment of the stool samples was carried out in the presence of 1M glycine acidic buffer pH 2.5, according to example 3. Anti-toxin B IgAs, VIDAS ® signal (RFV) Sample Presence Recombinant Native ΔSignal code of IgA Toxin B Toxin B Native − Recomb. S700 Neg 127 194 +16 S706 Neg 60 97 +37 S713 Neg 137 193 +56 S720 Neg 169 215 +46 S709 Pos 1303 623 −680 S719 Pos 2797 3437 +640 SP023 Pos 498 1221 +723 SP pool Pos 1637 3434 +1797 Pos = positive; Neg = negative
Example 6
Investigation for Fecal IgAs in Patients with Multiple Reinfection and Determination of the Reference Values S
[0106] The stool samples used in this example were obtained from patients who had been infected repeatedly with Clostridium difficile, called patients with multiple reinfection. These patients consulted the Clinical Microbiology Division, in the Medical Department of the Sir Mortimer B. Davis—Jewish General Hospital, in Montreal, Quebec, Canada. Pretreatment of the stool samples was carried out in the presence of the 1M glycine acidic buffer pH 2.5, according to the conditions in example 3. The different types of toxin were all immobilized on the solid phase at the same concentration (2 μg/mL). The fecal IgAs against toxin B, as well as the fecal IgAs against toxin A, were investigated by immunoassay using the toxins described in example 1 according to the procedure described in example 2. The results are presented in Table 4.
TABLE-US-00004 TABLE 4 Investigation for fecal IgAs against toxin B and against toxin A in the patients' stools with multiple reinfection. VIDAS ® signal (RFV) VIDAS ® signal Anti-toxin B IgAs Anti-toxin A IgAs Native Recombinant Recombinant Sample code Toxin B Toxin B Toxin A MR001 11 5 6 MR002 867 191 156 MR003 97 35 66 MR004 129 37 60 MR005 161 42 60 MR006 20 14 NAv MR007 380 145 138 MR008 337 214 NAv MR009* 1001 609 462 NAv = not available, the test could not be carried out, owing to insufficient sample volume.
[0107] In 3 patients with multiple reinfection, among the 8 tested, it was possible to detect fecal IgAs against toxin B (MR002, MR007, MR008). For all these patients, the signal obtained is above the positivity threshold of the assay, of 250 RFV. Moreover, these results confirm independently that the immunoassay using recombinant toxin B is less sensitive than the method using native toxin B: patient MR007 is not detected as positive and patient MR002 is at the limit of positivity with a signal of 191 RFV, for a threshold at 200 RFV.
[0108] Clinical follow-up of the patients showed that all the patients MR001 to MR008 presented at least one reinfection after their consultation and obtaining of the samples used in this example. Our assays show that some of these patients (MR002, MR007, MR008) have anti-toxin B IgA antibodies, and in some cases anti-toxin A IgA antibodies, or neither of these two types of antibodies. It may be concluded that the titer of the fecal anti-toxin B IgA antibodies that these patients possess is not sufficient to protect against such a reinfection. This group of patients therefore constitutes the group of patients with increased risk of developing or redeveloping Clostridium difficile infection.
[0109] Patient MR009 was initially classified in the group of patients with multiple reinfection according to the clinical picture. Supplementary biological investigations showed that this is in fact a poor classification. This patient was monitored for 3 months after this first assay. Investigation for the bacterium Clostridium difficile by culture always proved negative despite several attempts. No reinfection was observed. It should therefore be considered that patient MR009 is protected against reinfection. This patient has both anti-toxin B IgAs (1001 RFV) and anti-toxin A IgAs (462 RFV). This patient therefore constitutes the group of patients without an increased risk of developing or redeveloping Clostridium difficile infection.
[0110] Based on all of these results obtained for these groups of patients, we are able to define the reference value S. For a given capture antigen, the reference value S is selected in such a way that all the VIDAS® signals obtained for the patients with multiple reinfection MR001 to MR008 are below it and those for patient MR009 are above it.
[0111] For the anti-toxin B antibodies, the reference value S is between 870 and 1000 RFV, or fixed at about 930 RFV, when a native toxin B is used for capture. It is between 200 and 600 RFV, or fixed at about 400 RFV, when recombinant toxin B is used for capture. For the anti-toxin A antibodies, the reference value S is between 160 and 460 RFV, or fixed at about 310 RFV, when a recombinant toxin A is used for capture.
Example 7
Investigation for Fecal IgAs in Toxin A and B Negative Patients with Suspected Clostridium difficile Infection
[0112] The stool samples used in this example were obtained from patients who presented with suspected Clostridium difficile infection, in the department of Dr. Robert Spencer, Health Protection Agency Regional Laboratory, Bristol, United Kingdom. The samples in our possession were characterized using the kits VIDAS® C. difficile GDH (Cat. No. 30125, bioMérieux) and VIDAS® C. difficile Toxins A and B (Cat. No. 30118, bioMérieux). Investigation for fecal IgAs was carried out according to the protocols described in examples 2, 3 and 6 using native toxin B and recombinant toxin A.
[0113] The samples selected for the investigation in example 7 are all toxin A and toxin B negative, as well as GDH negative (Table 5). This selection includes two subgroups of patients. In a first subgroup, these two assays are negative because the patients had never been in contact with the bacterium C. difficile. The symptoms that led to suspicion of infection with C. difficile are due to another cause. The second subgroup corresponds to the patients who had actually been in contact with the bacterium but were able to eliminate it or limit its multiplication and thus recover from infection. We do not have the clinical data that would allow us to distinguish between these two subgroups. In contrast, if we detect anti-toxin B IgAs and/or anti-toxin A IgAs in the patients' stool, they are undoubtedly patients who have had a symptomatic infection or are carriers of Clostridium difficile. Thus, we showed that 6 patients out of the 12 tested (50%) have levels of anti-toxin B antibodies above the reference value S defined in example 6. Of these 6 patients, only 5 also have anti-toxin A antibodies that are present above the reference value S defined in example 6 (Table 5). Thus, if only investigated for anti-toxin A IgAs, patient SP252 would not be detected as having anti-Clostridium difficile secretory antibodies.
[0114] The results obtained for this cohort show that detection of the anti-toxin B IgAs is more sensitive than detection of the anti-toxin A IgAs.
TABLE-US-00005 TABLE 5 Investigation for fecal IgAs against toxin B and toxin A in the stool from patients with suspected Clostridium difficile infection. Interpretation (relative to the VIDAS ® signal (RFV) reference value) Sample VIDAS ® VIDAS ® Anti-toxin Anti-toxin Anti-toxin Anti-toxin code GDH CDAB B IgAs A IgAs B IgAs A IgAs SP29 Neg Neg 2034 390 Pos Pos SP32 Neg Neg 2094 1056 Pos Pos SP37 Neg Neg 84 24 Neg Neg SP38 Neg Neg 108 60 Neg Neg SP40 Neg Neg 9684 4338 Pos Pos SP43 Neg Neg 1044 378 Pos Pos SP228 Neg Neg 24 18 Neg Neg SP232 Neg Neg 12 18 Neg Neg SP234 Neg Neg 42 24 Neg Neg SP236 Neg Neg 2274 636 Pos Pos SP251 Neg Neg 36 24 Neg Neg SP252 Neg Neg 1026 240 Pos Neg Pos = positive; Neg = negative
Example 8
Investigation for Fecal IgAs in Patients with Clostridium Difficile Infection and Prediction of Reinfection
[0115] The stool samples used in this example were obtained from patients with proven Clostridium difficile infection. These patients presented in the Clinical Microbiology Division, in the Medical Department of the Sir Mortimer B. Davis—Jewish General Hospital, Montreal, Quebec, Canada. Wherever possible, two consecutive stool samples, from 10 to 35 days apart, were collected for each patient. The number of days between the first sample and the second is shown in Table 6. For all the patients, PCR investigation for the genetic material of the bacterium Clostridium difficile was carried out on the extracts from the first stool samples. This investigation proved positive for all the patients included in the study presented here, thus proving the clinical suspicion of Clostridium difficile infection.
[0116] The investigation for fecal IgAs was carried out according to the protocols described in examples 2, 3 and 6 using native toxin B. The samples for which we obtain a signal above the reference value S determined in example 6 (930 RFV when native toxin B is used for capture) are regarded as positive and those for which the signal is below this value are negative. The results are presented in Table 6.
TABLE-US-00006 TABLE 6 Investigation for fecal IgAs against toxin B in the stool of patients with Clostridium difficile infection. 1st sample 2nd sample Clinical Anti-toxin B IgAs Anti-toxin B IgAs Risk of Number of Patient RFV VIDAS ® RFV reinfection reinfections code signal Interpretation Day CDAB signal Interpretation (prediction) observed CD001 10451 Pos NA NAv NAv NAv NAv No follow-up CD002 3623 Pos NA NAv NAv NAv NAv 1 CD003 413 Neg +13 NAv 2708 Pos Low 1 CD004 675 Neg +12 Pos 3291 Pos Low 1 CD005 1891 Pos NA NAv NAv NAv NAv No follow-up CD006 6881 Pos +19 Pos 2254 Pos Low 1 CD007 NAv NAv +34 Pos 7528 Pos Low 1 CD008 3480 Pos +25 NAv 922 Neg High 2 CD009 2107 Pos +17 Pos 1475 Pos Low 1 Pos = positive; Neg = negative; NAv = not available, the test could not be carried out, owing to insufficient sample volume; NA = not applicable
[0117] To evaluate the risk of reinfection, it is useful to investigate for fecal IgAs against toxin B in a stool sample obtained 10 to 38 days, preferably 15 to 25 days after the infection with Clostridium difficile regarded as primary infection.
[0118] If the stool sample contains a level of anti-toxin B IgAs above the reference value S, the patient is at LOW risk of reinfection: he will have 0 or 1 reinfection at most after the episode of infection with Clostridium difficile regarded as primary infection.
[0119] If the stool sample contains a level of anti-toxin B IgAs below the reference value S, the patient is at HIGH risk of reinfection: he will have 2 reinfections or more after the episode of infection with Clostridium difficile regarded as primary infection.
[0120] The predictions made by applying this rule are presented in the column “Risk of reinfection” in Table 6, which also shows the number of reinfections actually observed in these same patients. It will be noted that there is perfect agreement between the predictions and the clinical observations.
[0121] For 3 patients (CD001, CD002 and CD005), it was not possible to obtain a second sample 10 to 35 days after the first. However, the analyses show that the first stool samples obtained from these patients contain high levels of fecal anti-toxin B IgAs.
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