Phenotypic and Genotypic Antimicrobial Resistance Profiles of Campylobacter jejuni Isolated from Cattle, Sheep, and Free-Range Poultry Faeces

Minimum inhibitory concentrations (MIC) of 13 antimicrobial agents were determined by broth microdilution for 72 Campylobacter jejuni strains from livestock. Twenty-three (31.9%) isolates were fully susceptible; all isolates were susceptible to erythromycin, chloramphenicol, streptomycin, gentamicin, sulfamethoxazole, and meropenem, and all but one to kanamycin. Resistance to quinolones was highest (52.8%), reaching similar values among poultry, dairy cattle, and sheep, but lower in beef cattle. Resistance to tetracyclines (48.6%) was mainly associated to dairy cattle and β-lactams (26.4%) to poultry. Multidrug resistance was mainly detected in dairy cattle (28.6%) and poultry (21.0%), whereas beef cattle had the highest percentage of fully susceptible isolates. Two real-time PCR assays to detect point mutations associated to quinolone (C257T in the gyrA gene) and macrolide (A2075G in the 23S rRNA genes) resistance were developed and validated on these strains. The analysis of a further set of 88 isolates by real-time PCR confirmed the absence of macrolide resistance and demonstrated the reproducibility and processability of the assay

Selection of ovine housekeeping genes for normalisation by real-time RT-PCR; analysis of PrP gene expression and genetic susceptibility to scrapie

BACKGROUND: Cellular prion protein expression is essential for the development of transmissible spongiform encephalopathies (TSEs), and in sheep, genetic susceptibility to scrapie has been associated to PrP gene polymorphisms. To test the hypothetical linkage between PrP gene expression and genetic susceptibility, PrP mRNA levels were measured by real-time RT-PCR in six ovine tissues of animals with different genotypes. RESULTS: Previous to the PrP gene expression analysis the stability of several housekeeping (HK) genes was assessed in order to select the best ones for relative quantification. The normalisation of gene expression was carried out using a minimum of three HK genes in order to detect small expression differences more accurately than using a single control gene. The expression stability analysis of six HK genes showed a large tissue-associated variation reflecting the existence of tissue-specific factors. Thereby, a specific set of HK genes was required for an accurate normalisation of the PrP gene expression within each tissue. Statistical differences in the normalised PrP mRNA levels were found among the tissues, obtaining the highest expression level in obex, followed by ileum, lymph node, spleen, cerebellum and cerebrum. A tendency towards increased PrP mRNA levels and genetic susceptibility was observed in central nervous system. However, the results did not support the hypothesis that PrP mRNA levels vary between genotypes. CONCLUSION: The results on PrP gene expression presented here provide valuable baseline data for future studies on scrapie pathogenesis. On the other hand, the results on stability data of several HK genes reported in this study could prove very useful in other gene expression studies carried out in these relevant ovine tissues

Diet induced changes in the microbiota and cell composition of rabbit gut associated lymphoid tissue (GALT)

[EN] The gut associated lymphoid tissue (GALT) is the largest immune organ of the body. Although the gut transient and mucosa-associated microbiota have been largely studied, the microbiota that colonizes the GALT has received less attention. The gut microbiome plays an important role in competitive exclusion of pathogens and in development and maturation of immunity. Diet is a key factor affecting the microbiota composition in the digestive tract. To investigate the relation between diet, microbiota and GALT, microbial and cell composition of vermiform appendix (VA) and sacculus rotundus (SR) were studied in two groups of New Zealand white rabbits on different diets. Diet shifted the lymphoid tissue microbiota affecting the presence and/or absence of certain taxa and their abundances. Immunohistochemistry revealed that a higher fibre content diet resulted in M cell hyperplasia and an increase of recently recruited macrophages, whereas T-cell levels remained unaltered in animals on both high fibre and standard diets. These findings indicate that diet has an impact on the microbiota and cell composition of the GALT, which could act as an important microbial recognition site where interactions with beneficial bacteria can take place favouring microbiota replacement after digestive dysregulationsSIAuthors thank Félix Blanco, Sergio Ayuso and Fidel Goiri for animal care and handling. The research was funded by grant (AGL2012-39818-C02-02) from the Spanish Ministry of Economy and Competiveness (MINECO), and by the Department of Economy and Infrastructures (DEI) of the Basque Government. RA held a pre-doctoral fellowship (BFI-2012-237) and a visiting fellowship (EP_2015_1_53) from the Department of Education, Universities and Research of the Basque Governmen

Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays

BACKGROUND: Piroplasmosis in cattle is caused by tick-borne haemoprotozoan parasites of the genera Theileria and Babesia. Molecular detection techniques offer higher sensitivity and specificity than microscopy examination methods and serological tests. A reverse line blot (RLB) macroarray that included generic and species-specific probes for Theileria annulata, Theileria buffeli, Babesia bovis, Babesia bigemina, Babesia divergens and Babesia major was used to study the presence and identity of the piroplasm species infecting 263 bovine blood samples from 79 farms, most of them in Northern Spain. Microscopy examination of blood smears and haematology were also performed whenever possible to identify animals with parasitaemia. RESULTS: RLB hybridisation identified infection in 54.0% of the samples, whereas only 28.8% were positive by microscopy examination. The most frequently found species was T. buffeli, present in 42.6% of the samples. T. annulata was found in 22 samples (8.4%) from 12 farms, including 9 farms (14 samples) located in Northern Spain where presence of the vector is not very common. Babesia infections were less frequently detected: B. major was found in 3.0% of the samples, B. bigemina in 2.7%, B. bovis in 2.3% and B. divergens in 1.1%. Mixed infections were detected in 14 samples, accounting for six different combinations of species. CONCLUSION: This is the first report in which B. major and B. divergens have been detected in Spain using molecular identification techniques and the first time that B. bovis has been detected in Northern Spain. The detection of T. annulata in Northern Spain suggests that the distribution of Mediterranean theileriosis might be changing. Samples with positive RLB hybridisation but negative microscopy had haematology values within the normal ranges suggesting that they corresponded to chronic carriers that may serve as reservoirs of the infection. In this sense, sensitive and specific laboratorial tests like RLB that clearly identify the parasite and can detect subclinical infections are essential to establish good control measures

Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in european wild boar

<p>Abstract</p> <p>Background</p> <p>Bovine tuberculosis (bTB) remains a significant problem in some parts of Spain largely because of contacts between cattle and wildlife reservoirs in extensive grazing systems. European Wild boar (<it>Sus scrofa</it>) is one of the species involved in the transmission of the disease to other species. Fast and simple detection methods would be critical for assessing infection prevalence, study the mechanisms of pathogen transmission and monitoring the effects of TB control measures.</p> <p>Results</p> <p>An enzyme-linked immunosorbent assay (ELISA) to detect antibodies against <it>Mycobacterium bovis </it>in wild boar serum was developed and validated on 185 sera from TB positive and negative wild boar. Based on antigen inoculation of captive animals as well as tuberculosis compatible lesions, culture results and molecular analysis of hunted individuals, animals were allocated into two groups: tuberculosis positive group and tuberculosis negative group. After optimization of the positive to negative ratio using different combinations of serum dilutions and conjugate concentrations, the test yielded a sensitivity of 72.60% and a specificity of 96.43% for the best cut-off.</p> <p>Conclusion</p> <p>Although some negative group animals showed an ELISA positive reaction (< 3%), this assay showed a high potential for accurate diagnosis of TB in wild boar, as its large dynamic range supported a good discriminatory power and a satisfactory balance between sensitivity and specificity.</p

Assessment of exposure to piroplasms in sheep grazing in communal mountain pastures by using a multiplex DNA bead-based suspension array

BACKGROUND: Piroplasms are tick-borne hemoprotozoans with a major impact on extensive management systems. Detection of sub-clinical low-level carriers, which can act as source of infection for vector ticks, is key to protect livestock trade and facilitate preventive control programs. The purpose of this study was to develop a method for the detection of ovine piroplasms and to use it in a field study aimed at investigating piroplasms infection in semi-extensive production systems in the Basque Country (northern Spain). METHODS: A DNA bead-based suspension array using the Luminex® xMAP technology that included a generic Theileria-Babesia control probe, 6 species-specific probes, and an internal control probe was developed to detect and identify piroplasms that infect sheep. To monitor piroplasm infection in clinically healthy sheep from 4 flocks that share communal mountain pastures, blood samples were collected during 2 grazing seasons. RESULTS: Piroplasms were detected in 48% (214/446) of blood samples, nearly half of them (49.1%, 105/214) as mixed infections. Five different piroplasms were identified: Theileria sp. OT3 in 34.8% of the samples, Theileria ovis in 20.9%, and at lower prevalences Babesia motasi (12.3%), Theileria luwenshuni/OT1 (10.5%) and Babesia ovis (6.3%). Despite differences among flocks associated to differences in management, an increasing trend in the incidence of piroplasm infection with increasing age of animals after increased tick exposure was observed. This increment could be attributed to continued re-infection associated with re-exposure to ticks at grazing. Ticks were collected from animals (4 species) and vegetation (8 species), and associations between tick abundance seasonality and risk of infection with the different piroplasms were established. CONCLUSION: The multiplex Luminex® xMAP procedure is a rapid and high throughput technique that provided highly specific and sensitive identification of single and mixed piroplasm infections in blood of sheep carriers. This study confirmed a situation of endemic stability for piroplasm infection in the region, where infection is present in the absence of clinical signs, and mountain grazing allows for sufficient inoculation rates to maintain such situation

Comparative analysis of Mycobacterium avium subsp. paratuberculosis isolates from cattle, sheep and goats by short sequence repeat and pulsed-field gel electrophoresis typing

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(Map) causes the chronic enteritis called paratuberculosis mainly in cattle, sheep and goats. Evidences that point out an association between Map and Crohn's Disease in humans are increasing. Strain differentiation among Map isolates has proved to be difficult and has limited the study of the molecular epidemiology of paratuberculosis. In order to asses the usefulness of the PCR based short sequence repeat (SSR) analysis of locus 1 and locus 8 in the epidemiological tracing of paratuberculosis strains we here compare for the first time the results of SSR and <it>Sna</it>BI-<it>Spe</it>I pulsed-field gel electrophoresis (PFGE) typing methods in a set of 268 Map isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar).</p> <p>Results</p> <p>A total of nineteen different multi-locus SSR (SSR1_SSR8) types were identified amongst the 268 isolates compared to the 37 multiplex profiles differentiated by the <it>Sna</it>BI-<it>Spe</it>I PFGE. SSR type 7_4 was the predominant genotype (51.2% of all isolates and 54.3% of cattle isolates), but combined with PFGE results the abundance of the most prevalent genotype (7_4&{2-1}) dropped down to 37.7%. SSR types 7_3 and 14_3 were significantly spread amongst isolates recovered from small ruminants. The comparison of SSR1_SSR8 and <it>Sna</it>BI-<it>Spe</it>I PFGE typing of these isolates has shown that both methods perform at similar discriminatory level. These were 0.691 and 0.693, respectively for SSR and PFGE as indicated Simpson's Index of Diversity, and 0.82 when calculated for combined SSR and PFGE genotypes. Overall, SSR1_SSR8 analysis seemed to detect higher levels of within-farm strain diversity and seemed to give higher year-related information. Combination of both typing methods revealed 20 multi-type farms out of the 33 bovine farms studied with more than one isolate.</p> <p>Conclusion</p> <p>The particular SSR and PFGE typing approaches described here are in general agreement but they showed some discrepancies that might reflect differing evolutionary processes of Map strains. Both methods are able to reciprocally complement their results and neither should be replaced with the other if sufficient material and time is available. Overall, the results of our comparative analyses suggest that, based on current methodologies available, a combined approach that includes SSR and PFGE seems to provide the highest level of discrimination for Map strain typing with meaningful epidemiological information.</p

Seroepidemiological study of Q fever in domestic ruminants in semi-extensive grazing systems

<p>Abstract</p> <p>Background</p> <p>Q fever, a worldwide zoonotic disease caused by <it>Coxiella burnetii</it>, is endemic in northern Spain where it has been reported as responsible for large series of human pneumonia cases and domestic ruminants' reproductive disorders. To investigate pathogen exposure among domestic ruminants in semi-extensive grazing systems in northern Spain, a serosurvey was carried out in 1,379 sheep (42 flocks), 626 beef cattle (46 herds) and 115 goats (11 herds). Serum antibodies were analysed by ELISA and positive samples were retested by Complement Fixation test (CFT) to detect recent infections.</p> <p>Results</p> <p>ELISA anti-<it>C. burnetii </it>antibody prevalence was slightly higher in sheep (11.8 ± 2.0%) than in goats (8.7 ± 5.9%) and beef cattle (6.7 ± 2.0%). Herd prevalence was 74% for ovine, 45% for goat and 43% for bovine. Twenty-one percent of sheep flocks, 27% of goat and 14% of cattle herds had a <it>C. burnetii </it>seroprevalence ≥ 20%. Only 15 out of 214 ELISA-positive animals reacted positive by CFT. Age-associated seroprevalence differed between ruminant species with a general increasing pattern with age. No evidence of correlation between abortion history and seroprevalence rates was observed despite the known abortifacient nature of <it>C. burnetii </it>in domestic ruminants.</p> <p>Conclusions</p> <p>Results reported herein showed that sheep had the highest contact rate with <it>C. burnetii </it>in the region but also that cattle and goats should not be neglected as part of the domestic cycle of <it>C. burnetii</it>. This work reports basic epidemiologic patterns of <it>C. burnetii </it>in semi-extensive grazed domestic ruminants which, together with the relevant role of <it>C. burnetii </it>as a zoonotic and abortifacient agent, makes these results to concern both Public and Animal Health Authorities.</p

Bovine Intelectin 2 Expression as a Biomarker of Paratuberculosis Disease Progression

[EN]Paratuberculosis (PTB), a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), is responsible for important economic losses in the dairy indus-try. Our previous RNA-sequencing (RNA-Seq) analysis showed that bovine intelectin 2 (ITLN2) precursor gene was overexpressed in ileocecal valve (ICV) samples of animals with focal (log2 fold-change = 10.6) and diffuse (log2 fold-change = 6.8) PTB-associated lesions compared to animals without lesions. This study analyzes the potential use of ITLN2, a protein that has been described as fundamental in the innate immune response to infections, as a biomarker of MAP infection. The presence of ITLN2 was investigated by quantitative immunohistochemical analysis of ICV samples of 20 Holstein Friesian cows showing focal (n = 5), multifocal (n = 5), diffuse (n = 5) and no histological lesions (n = 5). Significant differences were observed in the mean number of ITLN2 immunostained goblet and Paneth cells between the three histopathological types and the control. The number of immunolabelled cells was higher in the focal histopathological type (116.9 ± 113.9) followed by the multifocal (108.7 ± 140.5), diffuse (76.5 ± 97.8) and control types (41.0 ± 81.3). These results validate ITLN2 as a post-mortem biomarker of disease progression.SIThis work has been funded by the National Institute for Agricultural Research (INIA RTA- 2014-00009-C02), the Ministerio de Ciencia, Innovación y Universidades (MCIU) and the Agencia Estatal de Investigación (AEI) reference project RTI2018-094192-R-C22 (FEDER co-funded). Cristina Blanco Vázquez and Maria Canive were supported by a grant from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Spain)

Heat inactivated mycobacteria, alpha-Gal and zebrafish: Insights gained from experiences with two promising trained immunity inductors and a validated animal model

Trained immunity (TRAIM) may be defined as a form of memory where innate immune cells such as monocytes, macrophages, dendritic and natural killer (NK) cells undergo an epigenetic reprogramming that enhances their primary defensive capabilities. Cross-pathogen protective TRAIM can be triggered in different hosts by exposure to live microbes or microbe-derived products such as heat-inactivated Mycobacterium bovis or with the glycan α-Gal to elicit protective responses against several pathogens. We review the TRAIM paradigm using two models representing distinct scales of immune sensitization: the whole bacterial cell and one of its building blocks, the polysaccharides or glycans. Observations point out to macrophage lytic capabilities and cytokine regulation as two key components in non-specific innate immune responses against infections. The study of the TRAIM response deserves attention to better characterize the evolution of host-pathogen cooperation both for identifying the aetiology of some diseases and for finding new therapeutic strategies. In this field, the zebrafish provides a convenient and complete biological system that could help to deepen in the knowledge of TRAIM-mediated mechanisms in pathogen-host interactions.This study was funded by Junta de Comunidades de Castilla-La Mancha, Spain, and EU-FEDER, projects MYCOTRAINING SBPLY/19/180501/000174 and CCM17-PIC-036 (SBPLY/17/180501/000185), Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación MCIN/AEI/10.13039/501100011033, Spain and EU-FEDER (Grant BIOGAL PID2020-116761GB-I00) and by Principado de Asturias, PCTI 2021–2023 (GRUPIN: IDI2021-000102) and FEDER. EFC holds a PhD contract from the UCLM cosupported by the European Social Fund (2020/3836).S