Evaluation of the McLintock syringe as a cause of non-specific reactions in the intradermal tuberculin test used for the diagnosis of bovine tuberculosis.

The objective of this study was to elucidate whether the use of the McLintock syringe, used to inject tuberculin in cattle in several countries and based on an intradermal inoculation by needle, may, in itself, cause skin reactions that can be interpreted as positive reactions regardless of the real tuberculosis (TB) infection status of the animals. Forty-four cattle from an officially TB-free (OTF) herd were selected for the experiment. Each animal received four inoculations [one with sterile phosphate buffer saline (PBS) with 10% of glycerol and three with bovine purified protein derivative (PPD), as performed during the single intradermal tuberculin (SIT) test], two on each side of the neck (n = 176 inoculations). Three different McLintock syringes (n = 132 inoculations, PBS and bovine PPD) and one Dermojet syringe (n = 44 inoculations, PBS) were used to carry out the inoculations. No positive reactions (increase in skin-fold thickness > 3 mm) in response to the bovine PPD or PBS inoculations were observed regardless of the syringe used. No significant differences (p > 0.05) in the skin fold thickness increase (in mm) were observed between inoculation sites. Significant differences (p < 0.05) in the skin fold thickness were observed when PPD was injected in comparison to the PBS but no differences between McLintock and Dermojet were detected when PBS was injected. The McLintock syringe did not cause reactions per se that could be misunderstood as positive in TB-free cattle demonstrating that it is not a significant factor associated with the previously reported imperfect specificity of the SIT test.

Evaluation of the use of a needle-free injection syringe as a cause of non-specific reactions in the intradermal tuberculin test used for the diagnosis of bovine tuberculosis.

The objective of the study was to elucidate whether the use of the needle-free Dermojet syringe, which is based on a high pressure inoculation and is used to inject tuberculin in cattle in several countries, may, in itself, cause skin reactions that can be interpreted as positive reactions to the intradermal tests that are not, in fact, related to the real infection status of the animals. Forty-four cattle from an officially tuberculosis-free (OTF) herd were selected, and four single intradermal tuberculin (SIT) tests were performed on each animal, two on each side of the neck. Three different Dermojet (D1, D2 and D3) and one McLintock (M4) syringes were used to carry out sterile phosphate buffer saline (PBS) with 10% of glycerol and bovine PPD injections. No positive reactions to the SIT test were observed when using the D1-D3 syringes in the case of either bovine PPD or PBS. With regard to M4 (PBS), all the tests were negative when using a standard interpretation but three were positive in the case of the severe interpretation. Significant differences (p < 0.05) in the skin fold thickness measured were found only between certain Dermojet and McLintock syringes at certain inoculation sites. The results showed that the needle-free Dermojet syringe used for PPD intradermal testing in cattle did not cause significant reactions that could be misunderstood as positives.

Assessing the variability in transmission of bovine tuberculosis within Spanish cattle herds.

In Spain, despite years of efforts to eradicate bovine tuberculosis (bTB), the disease is still endemic, with some areas of high prevalence. In this context, the surveillance and control plans may need to be re-evaluated, and understanding the dynamics of bTB spread within Spanish herds may help to develop new strategies for reducing the time for detection of infected herds and for the elimination of bTB from the herds already infected. Here, we developed a compartmental stochastic model to simulate bTB within-herd transmission, fed it with epidemiological data from 22 herds (obtained from a previous work) and carried out parameter inference using Approximate Bayesian Computing methods We also estimated the "Within-herd transmission potential Number" (Rh), i.e. the average number of secondary cases generated by a single animal infected introduced into a totally susceptible herd, considering different scenarios depending on the frequency of controls. The median global values obtained for the transmission parameters were: for the transmission coefficient (ß), 0.014 newly infected animals per infectious individual per day (i.e. 5.2 per year), for the rate at which infected individuals become infectious (α), 0.01 per day (equivalent to a latent period of 97 days), and for the rate at which infected individuals become reactive to the skin test (α1), 0.08 per day (equivalent to a period of 12 days for an infected animal to become reactive). However, the results also evidenced a great variability in the estimates of those parameters (in particular ß and α) among the 22 herds. Considering a 6-month interval between tests, the mean Rh was 0.23, increasing to 0.82 with an interval of 1 year, and to 2.01 and 3.47 with testing intervals of 2 and 4 years, respectively.

Antibody detection tests improve the sensitivity of tuberculosis diagnosis in cattle.

We evaluated the sensitivity (Se) of the single cervical intradermal tuberculin (SIT) test, two interferon-gamma (IFN-γ) assays and three different antibody detection techniques for bovine tuberculosis (bTB) diagnosis in 131 mixed beef breed cattle. The results of the diagnostic techniques performed over the whole herd, and over the animals confirmed as infected based on the presence of lesions compatible with the disease and/or M. bovis isolation were compared to determine apparent prevalence (AP) and Se. The Se of the SIT test (severe interpretation) was 63.7% (95% CI, 54.54-72.00), while the Se of the IFN-γ assays ranged between 60.2% and 92%. The proportion of infected cattle detected by the different antibody detection techniques ranged from 65.5% to 87.6%. Three of the antibody detection techniques yielded a significant higher (p<0.05) Se than that achieved with the official diagnostic techniques. In addition, the interpretation in parallel of cellular and antibody detection techniques reached the highest Se: 98.2% (95% CI, 93.78-99.51) suggesting that the use of diagnostic techniques detecting both cellular and humoral responses could be considered as an alternative in the control of bTB outbreaks in high prevalence settings.

Molecular characterization and antimicrobial resistance of STEC strains isolated from healthy cattle in 2011 and 2013 in Spain.

Prevention of Shiga toxin-producing Escherichia coli (STEC) foodborne outbreaks is hampered by its complex epidemiology. We assessed the distribution of virulence genes (VGs), main serogroups/serotypes for public health [haemolytic uraemic syndrome (HUS)-related], antimicrobial resistance (AMR) profiles and pulsed-field gel electrophoresis (PFGE) patterns in a collection of STEC isolates obtained from cattle hide (n = 149) and faecal (n = 406) samples collected during a national survey conducted in Spain in 2011 and 2013. Isolates were cultured using McConkey and CT-SMAC agar after enrichment, and confirmed as STEC by PCR. STEC prevalence in hides (15·4%) was higher than in faeces (10·7%) and O157:H7 was more frequent in the former (2·7% vs. 0·99%). Non-O157 HUS-related serogroups were present albeit at low frequencies. The non-O157 isolates were more heterogeneous than O157:H7 in their VG patterns, with 25/64 presenting VGs from both STEC and enterotoxigenic pathotypes (hybrid isolates). Of the STEC isolates, 62·5% were resistant at least to one antimicrobial, and no differences in AMR between O157:H7 and non-O157 were detected. All isolates had different profiles by PFGE and did not form a cluster. Overall, our results demonstrated that STEC in the cattle reservoir is still a matter of concern for human health due to the presence of HUS-related serogroups, the occurrence of certain VGs, AMR and the additional risks that hybrid isolates may pose, and thus warrants further investigation.

Sheep as a Potential Source of Bovine TB: Epidemiology, Pathology and Evaluation of Diagnostic Techniques.

Bovine tuberculosis (TB) infection is infrequently diagnosed in sheep. Most reports are from single individual cases or flock outbreaks. However, in Spain several outbreaks have been reported recently, all of which had epidemiological links with TB-infected cattle herds. A total of 897 sheep suspected of being infected with TB and belonging to 23 flocks cohabiting with TB-infected cattle herds and/or goats were tested between 2009 and 2013 in Galicia (north-western Spain), using pathological, immunological and molecular techniques. Of these, 50.44% were positive by culture, 83.23% by histopathology and 24.92%, 4.86% and 59.42% by single intradermal tuberculin test (SITT), interferon-γ and ELISA, respectively. Results suggest that in circumstances akin to those in our study, sheep may be considered as a potential source of TB. We conclude that under similar conditions, serious consideration should be given to TB testing sheep, as they may represent a potential risk to other susceptible co-habiting species. The SITT and ELISA are recommended as the simplest and most cost-effective initial approaches for the diagnosis of TB in sheep under field conditions. However, when possible, interferon-γ should be applied to increase sensitivity.

Risk factors for bovine tuberculosis persistence in beef herds of Southern and Central Spain.

In order to assess risk factors related to bovine tuberculosis (bTB) persistence, a case-control study, comparing persistent versus transient bTB infected beef farms from Central and Southern Spain, was conducted. Farms were matched by herd size and geographical location (county). A questionnaire administered by personal interview was conducted on 150 herds (80 controls and 70 cases) from Andalucia and Castilla La Mancha regions. The questionnaire included questions related to the personnel involved in routine diagnostics, structure of the farm and of the herd, management, presence of other domestic species and of wildlife reservoirs. According to the results of our study, farms with large pasture areas and bTB infected neighbors had more difficulties in eradicating the disease, and therefore, were more likely to suffer a persistent bTB infection. The odds of bTB persistence were between 1.2 and 5.1 (i.e., 95% confidence interval of the OR) times higher in those herds that had a neighbor infected herd. Farms with large pasture areas had odds between 1.2 and 12.7 (i.e., 95% confidence interval of the OR) times higher of having a persistent bTB episode than farms with small pasture areas.

Comparison of depopulation and S19-RB51 vaccination strategies for control of bovine brucellosis in high prevalence areas.

RB51 vaccination can minimise the diagnostic problems associated with S19 vaccination of adult cattle, but its use for bovine brucellosis (BB) control remains controversial. Here, the evolution of BB prevalence in five high prevalence areas in Spain subjected to different control measures is described: herd depopulation of infected herds (I-III) or mass vaccination with RB51 and S19-RB51 vaccination of replacement heifers (IV-V). Annual data from the eradication campaigns were analysed at the special incidence area (SIA) level and the time to obtain herd prevalence levels of <1 per cent ('controlled status') was obtained at the local veterinary unit (LVU) level and compared using Cox's proportional hazard model. A higher annual rate of decrease in herd prevalence was observed in the SIAs subjected to vaccination (46.9%, 95% CI 43.5% to 50.0%) compared with those managed using stamping out (14.9%, 95% CI 9.6% to 19.9%). No significant differences in the time to achieve controlled status were observed between the stamping-out and vaccination strategies used at the LVU level, with median times of 60 (stamping-out LVUs) and 63 (vaccination LVUs) months. These results suggest that RB51 mass vaccination, in combination with the S19-RB51 vaccination of replacement heifers and strict implementation of other eradication measures, may provide results at least comparable with those resulting from a herd depopulation based strategy.

Analysis of the spatial variation of Bovine tuberculosis disease risk in Spain (2006-2009).

In this study we explored the spatial variation of Bovine tuberculosis (BTB) risk of being positive, new positive or persistently positive, as well as the risk of eliminating BTB in positive herds throughout Spain from 2006 to 2009 by means of hierarchical Bayesian models. The results of the models showed that the risk of infection (positive or new positive herds), persistence and elimination was lower in counties located in north and north-eastern of Spain, and in the Balearic and Canary islands than in the rest of the country. In some counties the risk of positivity was high during the four years of study, whereas there were others where the risk of positivity was high only in some of the years. With regard to the risk of persistence of BTB positive herds, counties located in the central, western and south-western part of the country had a higher risk in the three studied periods. This study has identified some specific areas of increased BTB risk in Spain, information that is useful for disease management.