Alberto Díez-Guerrier

Strategic use of serology for the diagnosis of bovine tuberculosis after intradermal skin testing.

Diagnostic tests based on cell-mediated immunity are used in programmes for eradication of bovine tuberculosis (Mycobacterium bovis). Serological assays could be applied as ancillary methods to detect infected animals. Our objective was to evaluate two serological techniques: M. bovis Ab Test (IDEXX, USA) and Enferplex™ TB assay (Enfer, Ireland) in animals tested simultaneously with the single and comparative intradermal tests and the interferon-gamma assay. This work was performed at two stages. First, a preliminary panel of samples collected prior to intradermal tests from tuberculosis-free (n=60) and M. bovis-infected herds (n=78) was assayed, obtaining high specificity: 100% (M. bovis Ab Test) and 98.3% (Enferplex TB assay) but low sensitivity (detection of M. bovis infected animals): 23.9% (M. bovis Ab Test) and 32.6% (Enferplex TB assay). Subsequently, the use of serological techniques was further studied in two herds with M. bovis infection (n=77) using samples collected prior to, and 72 h and 15 days after PPD inoculation. The highest level of detection of infected animals for serology was achieved at 15 days post-intradermal tests taking advantage of the anamnestic effect: 70.4% and 85.2% in herd A, and 66.7% and 83.3% in herd B, using M. bovis Ab Test and Enferplex TB assay, respectively. Quantitative results (average values obtained with M. bovis Ab Test ELISA and degree of positivity obtained with Enferplex TB assay) were higher in animals showing lesions compatible with tuberculosis. No significant differences were observed in the number of confirmed infected animals detected with either serological technique.

Evaluation of two cocktails containing ESAT-6, CFP-10 and Rv-3615c in the intradermal test and the interferon-γ assay for diagnosis of bovine tuberculosis

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n=23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p>0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p>0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis.

Staphylococcus aureus Carrying mecC Gene in Animals and Urban Wastewater, Spain

To the Editor: A new methicillin resistance mechanism gene, a divergent mecA homologue named mecC (formerly mecALGA251), was recently described in Staphylococcus aureus (1). Methicillin-resistant S. aureus (MRSA) isolates carrying mecC have been recovered from humans, ruminants, pets, and other animals such as rats, seals, and guinea pigs (1–3). It has been suggested that mecC-carrying MRSA isolates might not be detected by using MRSA selective media (4). For mecC-carrying S. aureus isolates, cefoxitin MICs of 4–64 mg/L have been demonstrated (1–2,4), values that would normally include susceptible isolates, according to the epidemiologic cutoff value established by the European Committee on Antibiotic Susceptibility Testing (EUCAST; www.eucast.org). mecC-carrying S. aureus isolates have been classified as heteroresistant (5), and MICs can be affected by the drug-susceptibility testing method used (1,5). These observations led us to retrospectively investigate the presence of mecC gene in a set of 361 mecA-negative S. aureus isolates collected during 2009–2012 (Table), independently of their susceptibility to cefoxitin. Isolates were recovered from healthy carriers in livestock (n = 39), from wild animals (n = 254), and from wastewater (effluents) from an urban sewage plant (n = 68). Specific amplification of the mecC gene was performed as described (6). The mecC-carrying S. aureus isolates were tested by broth microdilution using Microtiter EUST plates (Trek Diagnostic Systems, East Grinstead, UK) for susceptibility to benzylpenicillin, cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, florfenicol, fusidic acid, gentamicin, kanamycin, linezolid, mupirocin, rifampin, sulfamethoxazole, streptomycin, quinupristin-dalfopristin, tetracycline, thiamulin, trimethoprim, and vancomycin. Additionally, susceptibility to oxacillin was determined by using microScan Gram Positive Combo panel 37 (Siemens, Erlangen, Germany). MICs were interpreted according to EUCAST epidemiologic cutoff values. Table Testing of Staphylococcus aureus isolates for presence of methicillin resistance mechanism gene mecC, Spain* mecC was detected in a total of 4 isolates from wild boar (n = 1), fallow deer (n = 2), and urban wastewater (n = 1); these isolates represent 1% of the 361 tested isolates. The 3 isolates recovered from animals were susceptible to all antimicrobial drugs tested other than β-lactams and to oxacillin (MICs 0.5–1 mg/L) but were resistant to penicillin (MICs 0.5–2 mg/L). Two of the isolates were resistant to cefoxitin (MICs 8 and 16 mg/L) and the third was susceptible (MIC 4 mg/L). The wastewater isolate was resistant to penicillin (MIC 2 mg/L) and erythromycin (MIC 16 mg/L) and susceptible to all other antimicrobial drugs tested, including cefoxitin (MIC 4 mg/L) and oxacillin (MIC ≤0.25 mg/L). Previous studies have described mecC-positive isolates as susceptible to all antimicrobial drugs tested except β-lactams (2,3), although sporadic resistance to fluoroquinolones has been found (4,7). We additionally found erythromycin resistance in 1 mecC-carrying S. aureus isolate. For the 4 mecC-carrying S. aureus isolates we detected, MICs of oxacillin were interpreted as susceptible, and 2 isolates were susceptible to cefoxitin according to EUCAST guidelines, findings that agree with previous reports (1–2,4). Thus, mecC presence is not always linked to resistance phenotypes for cefoxitin or oxacillin; such unclear findings could hinder the detection of mecC-carrying isolates. We further characterized the 4 mecC-carrying S. aureus isolates by spa typing and detection of Panton-Valentin leukocidin (PVL) toxin genes (6,8). Multilocus sequence typing (MLST) was performed according to Enright et al. (9) by using self-designed primers arc (down 5′-CGATTTGTTGTTGATTAGGTTC-3′), tpi (up 5′-CATTAGCAGATTTAGGCGTTA-3′), and yqiL (down 5′-GATTGGYTCACCTTTRCGTTG-3′). All 4 isolates were PVL negative. The 3 animal isolates were assigned to a new spa type (t11212) and to clonal complex (CC) 425 and sequence type (ST) 425 (Table). ST425 has been previously associated with mecC-carrying S. aureus isolates in cattle and humans (1–2); the animals we sampled were from a game estate and may have had contact with cattle and with urban wastewater. The wastewater isolate was assigned to spa type t843 and to a new allelic profile, ST2676, in CC130 (Table). ST2676 represents a single-locus variant of ST130 carrying a different allele for the gene aroE. MRSA isolates of CC130 have been associated with humans and animals (1–4,6). This result indicates that mecC-carrying S. aureus isolates can be found in urban wastewater, which may act as an environmental reservoir, as has been demonstrated for mecA-carrying S. aureus (10). In conclusion, we detected the methicillin resistance mechanism gene mecC in nonclinical S. aureus isolates from animals and urban wastewater in Spain. Although our data indicate that the frequency of this resistance mechanism is low, this gene appears to be expanding to new areas. Prospective studies should be performed to evaluate epidemiologic changes and to analyze the genetic lineages that carry this resistance mechanism.

Detection of mecC-Methicillin-resistant Staphylococcus aureus isolates in river water : a potential role for water in the environmental dissemination

Methicillin-resistant Staphylococcus aureus (MRSA) is a public health concern due to limited treatment options. The recent description of a mecA homologue, mecC in human and cattle, led to studies to detect this new variant in human and other animal species. Detection of mecC in wild boar and fallow deer in a Spanish game estate led us to further investigate the presence of mecC-MRSA at this location. Samples from cattle, wild animals, workers and river water were tested. A further three mecC-MRSA isolates were obtained from river water. Molecular characterization (multilocus sequence typing and spa typing) and antimicrobial susceptibility testing (broth microdilution) showed that isolates were similar to those detected in wild animals. Whole genome sequencing confirmed that the isolates from the river water and wild animals in the same geographic area were all closely related isolates of ST425 mecC-MRSA. The presence of mecC-MRSA in the river water highlights the potential role of water in the dissemination of mecC-MRSA.

Evaluation of the performance of cellular and serological diagnostic tests for the diagnosis of tuberculosis in an alpaca (Vicugna pacos) herd naturally infected with Mycobacterium bovis

Tuberculosis (TB) in llamas and alpacas has gained importance in recent years since they are imported into the European Union mainly for serving as pets and for production of natural fibre. The intradermal tuberculin test has been widely used for diagnosis of TB in these species showing lack of sensitivity (Se) although little information has been previously reported evaluating the effect on its performance of different PPD inoculation sites and time of readings. Moreover, different cost-effective serological assays have been developed in the recent years for TB diagnosis in camelids obtaining a variety of results and, for this reason, new assays still being developed. The main objectives of this study were: (1) to evaluate the performance of the intradermal tuberculin test using different inoculation sites (axillary, prescapular and cervical) and times of reading (72 and 120 h) and (2) to test a novel serological assay based on MPB83 antigen in a Mycobacterium bovis naturally infected alpaca herd in Spain. In regards to skin test, single intradermal tuberculin (SIT) test at the prescapular site and reading at 72 h showed the highest proportion of test-positive-culture positive animals among all culture positive animals (T+/C+), ranging from 53.8% (95% CI, 37.2-69.9) to 80% (95% CI, 44.4-97.5) using a more stringent interpretation than typically prescribed although, in general, low T+/C+ was achieved using both SIT and single comparative intradermal tuberculin (SCIT) tests alone. T+/C+ of the serological assay increased using samples collected 15-30 days after PPD injection [76.9% (95% CI, 60.7-88.9) - 100% (95% CI, 69.2-100)]. The best results of T+/C+ were obtained applying in parallel the most sensitive SIT test and serology using samples collected 15-30 days after PPD inoculation [90% (95% CI, 55.5-99.7)-100% (95% CI, 69.2-100)]. Therefore implementation of serology in parallel with the most sensitive skin test could maximize the detection of infected animals.

Development and evaluation of an IS711-based loop mediated isothermal amplification method (LAMP) for detection of Brucella spp. on clinical samples

DNA-based methods have emerged as an additional tool for Brucella infection-confirmation at a herd level. However, their implementation may require the use of specialized equipment. In this context the recently developed loop-mediated isothermal amplification (LAMP) technique may constitute an additional and cost-effective tool for rapid and specific DNA detection, especially in low income areas. In the present study the usefulness of a newly developed LAMP assay aiming at the multicopy-IS711 sequence was assessed on a variety of clinical samples (n=81 from abortions and ewes; cattle, n=3; swine, n=4) that were analyzed in parallel using real-time PCR and bacteriology. Although overall sensitivities obtained with the three methods were comparable (p>0.05), our results highlighted the complementarity between bacteriology and molecular-based methods for increased sensitivity. Significant differences (p<0.05) were observed with all techniques depending on the nature of the sample. Our results demonstrate the potential of the IS711-LAMP technique for direct Brucella detection.

Effect of the inoculation site of bovine purified protein derivative (PPD) on the skin fold thickness increase in cattle from officially tuberculosis free and tuberculosis-infected herds.

The official technique for diagnosis of bovine tuberculosis (bTB) worldwide is the tuberculin skin test, based on the evaluation of the skin thickness increase after the intradermal inoculation of a purified protein derivative (PPD) in cattle. A number of studies performed on experimentally infected or sensitized cattle have suggested that the relative sensitivity of the cervical test (performed in the neck) may vary depending on the exact location in which the PPD is injected. However, quantitative evidence on the variation of the test accuracy associated to changes in the site of inoculation in naturally infected animals (the population in which performance of the test is most critical for disease eradication) is lacking. Here, the probability of obtaining a positive reaction (>2 or 4 millimeters and/or presence of local clinical signs) after multiple inoculations of bovine PPD in different cervical and scapular locations was assessed in animals from five bTB-infected herds (818 cattle receiving eight inoculations) using a hierarchical Bayesian logistic regression model and adjusting for the potential effect of age and sex. The effect of the inoculation site was also assessed qualitatively in animals from four officially tuberculosis free (OTF) herds (two inoculations in 210 animals and eight inoculations in 38 cattle). Although no differences in the qualitative outcome of the test were observed in cattle from OTF herds, a statistically important association between the test outcome and the inoculation site in animals from infected herds was observed, with higher probabilities of positive results when the test was performed in the neck anterior area. Our results suggest that test sensitivity may be maximized by considering the area of the neck in which the test is applied, although lack of effect of the inoculation site in the specificity of the test should be confirmed in a larger sample.

Interferon-gamma responses in sheep exposed to virulent and attenuated Brucella melitensis strains

Antibody detection is the basis of large-scale sheep brucellosis diagnosis because of its sensitivity and specificity. In contrast, information on the cellular mediated immune (CMI) response triggered after Brucella melitensis infection, a cornerstone in the protection against this pathogen, is more limited, particularly regarding the effect of the virulence of the infecting strain in the induced CMI reaction. Here, the interferon-gamma (IFN-γ) profiles evoked after exposure by different routes to virulent (H38) and attenuated (Rev.1) B. melitensis strains in 14 pregnant sheep and 87 ewe lambs, respectively, were characterized accounting for different host-related factors, and compared with their serological response and with the basal IFN-γ responses observed in 155 animals non exposed to Brucella. No significant differences in the IFN-γ response of Rev.1 vaccinated animals depending on the inoculation route was observed, in contrast with their serological results. Response in H38-challenged followed a similar trend although peaked later, and an effect of the abortion on the IFN-γ response was detected. This information could help to understand the interaction bacteria-host that leads to its intracellular survival and could be useful for the design of new diagnostic approaches.

Evaluation of the immunogenicity and diagnostic interference caused by M. tuberculosis SO2 vaccination against tuberculosis in goats

The immunogenicity and diagnostic interference caused by M. tuberculosis SO2, a prototype vaccine first time tested in goats was evaluated. Tuberculosis-free goats were distributed in four groups: [1], non-vaccinated; [2], subcutaneously (SC) BCG vaccinated; [3], intranasally (IN) SO2 vaccinated and [4], SC SO2 vaccinated. Intradermal tuberculin and IFN-γ tests using PPDs and alternative antigenic cocktails containing mainly ESAT-6 and CFP-10 (E/C) were applied at different times post-vaccination. Results showed a significant (p<0.05) increase in the number of reactors detected using both PPD-based intradermal and IFN-γ tests at different times in all the vaccinated groups. No intradermal reactivity was detected in the vaccinated goats using a cocktail containing E/C, Rv3615c and Rv3020c. A higher overall reactivity was observed in the group [4] in comparison with the other vaccinated groups. Results showed that antigens used to differentiate BCG vaccinated animals could be potentially used to differentiate SO2 vaccinated ones.

Evaluation of the immunogenicity and safety of Brucella melitensis B115 vaccination in pregnant sheep

In spite of its limitations, Rev.1 is currently recognized as the most suitable vaccine against Brucella melitensis (the causative agent of ovine and caprine brucellosis). However, its use is limited to young animals when test-and-slaughter programs are in place because of the occurrence of false positive-reactions due to Rev.1 vaccination. The B. melitensis B115 rough strain has demonstrated its efficacy against B. melitensis virulent strains in the mouse model, but there is a lack of information regarding its potential use in small ruminants for brucellosis control. Here, the safety and immune response elicited by B115 strain inoculation were evaluated in pregnant ewes vaccinated at their midpregnancy. Vaccinated (n=8) and non-vaccinated (n=3) sheep were periodically sampled and analyzed for the 108 days following inoculations using tests designed for the detection of the response elicited by the B115 strain and routine serological tests for brucellosis [Rose Bengal Test (RBT), Complement Fixation Test (CFT) and blocking ELISA (ELISAb)]. Five out of the 8 vaccinated animals aborted, indicating a significant abortifacient effect of B115 inoculation at midpregnancy. In addition, a smooth strain was recovered from one vaccinated animal, suggesting the occurrence of an in vivo reversion phenomenon. Only one animal was positive in both RBT and CFT simultaneously (91 days after vaccination) confirming the lack of induction of cross-reacting antibody responses interfering with routine brucellosis diagnostic tests in most B115-vaccinated animals.