Teresa García-Seco

Assessment of genetic diversity of zoonotic Brucella spp. Recovered from livestock in Egypt using multiple locus VNTR analysis

Brucellosis is endemic in most parts of Egypt, where it is caused mainly by Brucella melitensis biovar 3, and affects cattle and small ruminants in spite of ongoing efforts devoted to its control. Knowledge of the predominant Brucella species/strains circulating in a region is a prerequisite of a brucellosis control strategy. For this reason a study aiming at the evaluation of the phenotypic and genetic heterogeneity of a panel of 17 Brucella spp. isolates recovered from domestic ruminants (cattle, buffalo, sheep, and goat) from four governorates during a period of five years (2002–2007) was carried out using microbiological tests and molecular biology techniques (PCR, MLVA-15, and sequencing). Thirteen strains were identified as B. melitensis biovar 3 while all phenotypic and genetic techniques classified the remaining isolates as B. abortus (n = 2) and B. suis biovar 1 (n = 2). MLVA-15 yielded a high discriminatory power (h = 0.801), indicating a high genetic diversity among the B. melitensis strains circulating among domestic ruminants in Egypt. This is the first report of the isolation of B. suis from cattle in Egypt which, coupled with the finding of B. abortus, suggests a potential role of livestock as reservoirs of several zoonotic Brucella species in the region.

Evidence of Leishmania infantum Infection in Rabbits (Oryctolagus cuniculus) in a Natural Area in Madrid, Spain

Leishmaniasis is one of the most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs of Leishmania infantum in Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus) from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT) and direct (PCR, culture) techniques. Fifty-seven (82.6%) of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples. L. infantum was isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir of L. infantum and demonstrate that the prevalence of infection is high in the analyzed area.

Epidemiological factors associated with the exposure of cattle to Coxiella burnetii in the Madrid region of Spain

Domestic ruminants are considered to be the major source of Coxiella burnetii, the causative agent of Q fever. Even though Q fever is considered to be present worldwide, its distribution in many areas and countries remains unknown. Here, a serological assay was used to estimate the seroprevalence of C. burnetii in cattle in the Madrid region of Spain, to assess its spatial distribution, and to identify risk factors associated with positive results. Ten animals from each of 110 herds (n=1100) were randomly selected and analyzed using an ELISA test. In addition, epidemiological information, at both the herd and individual level, was collected. Variables for which an association with test results was detected in a bivariate analysis were included as predictors (main effects) in a multivariable logistic regression model. Herd and individual seroprevalences were 30% (95% CI=22.2-39.1) and 6.76% (95% CI=5.42-8.41), respectively, and a strong spatial dependence was identified at the first neighbour level using the Cuzick-Edwards test. Production type (dairy >beef >bullfighting) and age of animals (old vs. young) were the only variables significantly associated (P<0.05) with positive serological results at the herd and individual levels, respectively. These results indicate that cattle are exposed to C. burnetii in the Madrid region The high herd seroprevalence found in dairy herds (75%) indicates a higher risk of infection (probably for management reasons) whereas no C. burnetii positive bullfighting herds were identified.

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.

Assessment of MALDI-TOF MS as alternative tool for Streptococcus suis identification

The accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying Streptococcus suis isolates obtained from pigs, wild animals, and humans was evaluated using a PCR-based identification assay as the gold standard. In addition, MALDI-TOF MS was compared with the commercial multi-tests Rapid ID 32 STREP system. From the 129 S. suis isolates included in the study and identified by the molecular method, only 31 isolates (24.03%) had score values ≥2.300 and 79 isolates (61.24%) gave score values between 2.299 and 2.000. After updating the currently available S. suis MALDI Biotyper database with the spectra of three additional clinical isolates of serotypes 2, 7, and 9, most isolates had statistically significant higher score values (mean score: 2.65) than those obtained using the original database (mean score: 2.182). Considering the results of the present study, we suggest using a less restrictive threshold score of ≥2.000 for reliable species identification of S. suis. According to this cut-off value, a total of 125 S. suis isolates (96.9%) were correctly identified using the updated database. These data indicate an excellent performance of MALDI-TOF MS for the identification of S. suis.

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.

Usefulness of MALDI-TOF MS as a Diagnostic Tool for the Identification of Streptococcus Species Recovered from Clinical Specimens of Pigs.

The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical specimens of diseased pigs was evaluated. For this proposal, the MALDI BDAL Database (Bruker Daltoniks, Germany) was supplemented with the main spectrum profiles (MSP) of the reference strains of S. porci, S. porcorum and S. plurextorum associated with pneumonia and septicemia. Although these three species showed similar MALDI profiles, several peaks were recognized that can be useful for their differentiation: S. porci (4113, 6133, 7975 and 8228 m/z Da), S. plurextorum (3979, 4078, 4665, 6164, 6491, 6812, 7959 and 9330 m/z Da) and S. porcorum (3385, 3954, 4190, 6772, 7908, and 8381 m/z Da). After adding these MSPs, an evaluation was conducted to determine the accuracy of MALDI-TOF MS for the identification of streptococci from diseased pigs using 74 field isolates. Isolates were identified as S. suis, S. porcinus, S. dysgalactiae, S. hyovaginalis, S. porcorum, S. alactolyticus, S. hyointestinalis and S. orisratti. This is the first time that the latter three species have been reported from clinical specimens of pigs. Overall, there was good concordance (95.9%) between the results obtained from MALDI-TOF MS identification (best hint) and those from genotyping. Our results demonstrate the good performance of MALDI-TOF MS (100% sensitivity and specificity) for identifying most of the species of streptococci that can frequently be isolated from diseased pigs. However, conflicting results were observed in the correct identification of some isolates of S. dysgalactiae and S. alactolyticus.

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.

Control of Animal Brucellosis — The Most Effective Tool to Prevent Human Brucellosis

The World Health Organization classifies brucellosis as one of the seven neglected en‐ demic zoonosis which contribute to the perpetuation of poverty in developing coun‐ tries. Although most of the developed countries are free from this important zoonosis, brucellosis has still a widespread distribution in the Mediterranean region, the Middle East, Central Asia, and parts of Latin America, making it a global problem. Nearly half a million of new cases of human brucellosis are reported each year around the world, in which animals (or products of animal origin) are the most likely source of infection. Brucella melitensis, the main etiologic agent of small ruminant brucellosis, is the most prevalent specie involved in cases of human disease in most parts of the world. Additionally, Brucella abortus (main responsible of bovine brucellosis) and Bru‐ cella suis (the most common etiological agent of porcine brucellosis) are often associat‐ ed with human brucellosis. In animal production, brucellosis has a strong economic impact due not only to its direct consequences (e.g., reproductive failures) but also to indirect loses (e.g., trade restrictions). The problem of brucellosis could be considered a clear example of the need for a “One World, One Health” strategy, given that the only approach to achieve its control and subsequent eradication is the cooperation be‐ tween public and animal health authorities. The prevention of human brucellosis can‐ not be achieved without the control of the disease in the animals, as exemplified by the impact that the early measures adopted in the beginning of the 20th century for‐ bidding the consumption of goat milk had on the prevalence of the disease in the Brit‐ ish soldiers in Malta. When the prevalence of the disease in the animal population is high or when eradication cannot be achieved due to other factors (e.g., lack of eco‐ nomic resources), its control in livestock must be the first objective. When deciding the optimal approach to tackle the disease, the prevalence of animal brucellosis is not the only parameter to consider by the decision makers since other epidemiological and economic aspects should be considered in order to implement the most adequate control strategy in each region. Cooperation between all stakeholders involved is a cornerstone in the success of any control strategy. Strict biosafety and management measures, vaccination, and test-and-slaughter strategy are recognized as the most ef‐

effect of Preventive Chlamydia abortus Vaccination in Offspring Development in sheep challenged experimentally

Ovine enzootic abortion, caused by Chlamydia abortus, leads to important economic losses worldwide. In addition to reproductive failures, infection may impact lamb growth during the first weeks after birth, yet this effect has not been well characterized. Vaccination can help to control the disease but variable efficacy values have been described, possibly related with factors associated with the host, the vaccine, the parameter used for efficacy determination, and the challenge conditions. In this context, we evaluated the efficacy of an inactivated standard commercial vaccine and a 1/2 diluted dose in pregnant sheep challenged with C. abortus by examining multiple indicators of vaccine effect (including incidence of reproductive failures, bacterial excretion, and evolution of weight gain of viable lambs during the first month of life). Three groups of ewes [control non-vaccinated, C (n = 18); vaccinated with standard dose, SV (n = 16); and vaccinated with 1/2 dose, DV (n = 17)], were challenged approximately 90 days post-mating and tested using direct PCR (tissue samples and vaginal swabs) and ELISA (serum) until 31 days post-reproductive outcome. There were not significant differences in the proportions of reproductive failures or bacterial shedding after birth/abortion regardless the vaccination protocol. However, a beneficial effect of vaccination on offspring growth was detected in both vaccinated groups compared with the controls, with a mean increase in weight measured at 30 days of life of 1.5 and 2.5 kg (p = 0.056) and an increase in the geometric mean of the daily gain of 8.4 and 9.7% in lambs born from DV and SV ewes compared with controls, respectively. Our results demonstrate the effect of an inactivated vaccine in the development of the offspring of C. abortus-infected ewes at a standard and a diluted dose, an interesting finding given the difficulty in achieving sufficient antigen concentration in the production of enzootic abortion of ewes-commercial vaccines.