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Brucellosis is a zoonotic disease that can result in symptoms including infertility, abortion, testicular inflammation, and arthritis in affected sheep and cattle. The disease can spread through a range of mechanisms, making outbreaks difficult to control such that affected farms often suffer severe economic losses. In addition, humans can be affected by brucellosis, and the number of cases continues to rise annually. As rates of brucellosis in Xinjiang, China have been increasing substantially in recent years, this study was developed to assess brucellosis seroprevalence among herds of cattle, sheep, and camels in Xinjiang through the use of common diagnostic procedures like the Rose Bengal test (RBT) and PCR. A cross-sectional approach was used to analyze the seroprevalence of brucellosis across 720 total farm animals (320 camels, 250 sheep, and 150 cattle). RBT incidence rate for brucellosis was detected in 60 serum samples, 14 (4.38%) from camels, 45 (18%) from sheep, and 1 (0.67%) from cattle. PCR was performed as a confirmatory approach for these RBT-positive samples, with 55 ultimately being confirmed to be positive 13 (4.06%), 41 (16.4%), and 1 (0.67%) samples from camels, sheep, and cattle, respectively. In this survey, sheep exhibited the highest brucellosis seroprevalence using these two analytical techniques, while cattle exhibited the lowest seroprevalence and camels exhibited an intermediate rate. AMOS-PCR analyses identified Brucella melitensis as the unknown bacterium in some of these samples. These results offer new insight regarding brucellosis seroprevalence among farm animals that can be used to formulate more appropriate prevention and control policies, while also improving awareness of epidemic prevention efforts, the need for routine quarantine and disinfection, the benefits of scientific breeding and management, and approaches to improving breeding efficiency for the camel, cattle, and sheep industries., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Xingxing et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The economic impact of gastrointestinal (GI) nematode infections on livestock production is well documented worldwide. Increasing evidence supports the hypothesis that parasite colonization induces significant changes in the GI tract environment and, therefore, in the landscape where the microbiota and parasites occur. Understanding the interactions between bacterial and parasite populations in the digestive tract of livestock may be useful to design parasite control strategies based on microbiota modification. The aims of this work were to investigate the impact of the oxytetracycline-mediated manipulation of the gut microbial community on the composition of GI nematode populations in naturally infected sheep and to explore changes in the GI microbial communities after nematode population treatment with the anthelmintic compound monepantel. Extensive manipulation of the GI microbiota with a therapeutic dose of the long-acting oxytetracycline formulation did not induce significant changes in the GI nematode burden. The gut microbiota of treated animals returned to control levels 17 days after treatment, suggesting strong resilience of the sheep microbial community to antibiotic-mediated microbiota perturbation. A significant decrease of the bacterial Mycoplasmataceae family (Log2FC = -4, Padj = 0.001) and a marked increase of the Methanobacteriaceae family (Log2FC = 2.9, Padj = 0.018) were observed in the abomasum of sheep receiving the monepantel treatment. While a comprehensive evaluation of the interactions among GI mycoplasma, methanobacteria and nematode populations deserves further assessment, the bacteria-nematode population interactions should be included in future control programs in livestock production. Understanding how bacteria and parasites may influence each other in the GI tract environment may substantially contribute to the knowledge of the role of microbiota composition in nematode parasite establishment and the role of the parasites in the microbiota composition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mate et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Coxiella burnetii is the worldwide zoonotic infectious agent for Q fever in humans and animals. Farm animals are the main reservoirs of C. burnetii infection, which is mainly transmitted via tick bites. In humans, oral, percutaneous, and respiratory routes are the primary sources of infection transmission. The clinical signs vary from flu-like symptoms to endocarditis for humans' acute and chronic Q fever. While it is usually asymptomatic in livestock, abortion, stillbirth, infertility, mastitis, and endometritis are its clinical consequences. Infected farm animals shed C. burnetii in birth products, milk, feces, vaginal mucus, and urine. Milk is an important source of infection among foods of animal origin. This study aimed to determine the prevalence and molecular characterization of C. burnetii in milk samples of dairy animals from two districts in Punjab, Pakistan, as it has not been reported there so far. Using a convenience sampling approach, the current study included 304 individual milk samples from different herds of cattle, buffalo, goats, and sheep present on 39 farms in 11 villages in the districts of Kasur and Lahore. PCR targeting the IS1111 gene sequence was used for its detection. Coxiella burnetii DNA was present in 19 of the 304 (6.3%) samples. The distribution was 7.2% and 5.2% in districts Kasur and Lahore, respectively. The results showed the distribution in ruminants as 3.4% in buffalo, 5.6% in cattle, 6.7% in goats, and 10.6% in sheep. From the univariable analysis, the clinical signs of infection i.e. mastitis and abortion were analyzed for the prevalence of Coxiella burnetii. The obtained sequences were identical to the previously reported sequence of a local strain in district Lahore, Sahiwal and Attock. These findings demonstrated that the prevalence of C. burnetii in raw milk samples deserves more attention from the health care system and veterinary organizations in Kasur and Lahore of Punjab, Pakistan. Future studies should include different districts and human populations, especially professionals working with animals, to estimate the prevalence of C. burnetii., Competing Interests: Competing interests: The authors have declared that no competing interests exists., (Copyright: © 2024 Shujat et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lignocellulosic biomass such as barley straw is a renewable and sustainable alternative to traditional feeds and could be used as bioenergy sources; however, low hydrolysis rate reduces the fermentation efficiency. Understanding the degradation and colonization of barley straw by rumen bacteria is the key step to improve the utilization of barley straw in animal feeding or biofuel production. This study evaluated the hydrolysis of barley straw as a result of the inoculation by rumen fluid of camel and sheep. Ground barley straw was incubated anaerobically with rumen inocula from three fistulated camels (FC) and three fistulated sheep (FR) for a period of 72 h. The source of rumen inoculum did not affect the disappearance of dry matter (DMD), neutral detergent fiber (NDFD). Group FR showed higher production of glucose, xylose, and gas; while higher ethanol production was associated with cellulosic hydrolysates obtained from FC group. The diversity and structure of bacterial communities attached to barley straw was investigated by Illumina Mi-Seq sequencing of V4-V5 region of 16S rRNA genes. The bacterial community was dominated by phylum Firmicutes and Bacteroidetes. The dominant genera were RC9_gut_group, Ruminococcus, Saccharofermentans, Butyrivibrio, Succiniclasticum, Selenomonas, and Streptococcus, indicating the important role of these genera in lignocellulose fermentation in the rumen. Group FR showed higher RC9_gut_group and group FC revealed higher Ruminococcus, Saccharofermentans, and Butyrivibrio. Higher enzymes activities (cellulase and xylanase) were associated with group FC. Thus, bacterial communities in camel and sheep have a great potential to improve the utilization lignocellulosic material in animal feeding and the production of biofuel and enzymes., Competing Interests: The authors have declared that no competing interests exist.

Tuberculosis (TB), a contagious disease mainly caused by Mycobacterium tuberculosis (M. tb), Mycobacterium bovis (M. bovis), and Mycobacterium caprae (M. caprae), poses a major global threat to the health of humans and many species of animals. Developing an ante-mortem detection technique for different species would be of significance in improving the surveillance employing a One Health strategy. To achieve this goal, a universal indirect ELISA was established for serologically detecting Mycobacterium tuberculosis complex infection for multiple live hosts by using a fusion protein of MPB70, MPB83, ESAT6, and CFP10 common in M. tb, M. bovis, and M. caprae as the coating antigen (MMEC) and HRP-labeled fusion protein A and G as a secondary antibody. After testing the known positive and negative sera, the receiver operating characteristic curves were constructed to decide the cut-off values. Then, the diagnostic sensitivity and specificity of MMEC/AG-iELISA were determined as 100.00% (95% CI: 96.90%, 100.00%) and 100.00% (95% CI: 98.44%, 100.00%) for M. bovis infection of cattle, 100.00% (95% CI: 95.00%, 100.00%) and 100.0% (95% CI: 96.80%, 100.00%) for M. bovis infection of sheep, 90.74% (95% CI: 80.09%, 95.98%) and 98.63% (95% CI: 95.14%, 99.76%) for M. bovis infection of cervids, 100.00% (95% CI: 15.81%, 100.00%) and 98.81% (95% CI: 93.54%, 99.97%) for M. bovis infection of monkeys, 100.00% (95% CI: 86.82%, 100.00%) and 94.85% (95% CI: 91.22%, 97.03%) for M. tb infection of humans. Furthermore, this MMEC/AG-iELISA likely detects M. caprae infection in roe deer. Thus this method has a promising application in serological TB surveillance for multiple animal species thereby providing evidence for taking further action in TB control., Competing Interests: The authors have declared that no competing interests exist.

Paratuberculosis a contagious and chronic disease in domestic and wild ruminants, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Typical clinical signs include intractable diarrhea, progressive emaciation, proliferative enteropathy, and mesenteric lymphadenitis. Paratuberculosis is endemic to many parts of the world and responsible for considerable economic losses. In this study, different types of paratuberculosis and MAP in sheep and goats were investigated in Inner Mongolia, a northern province in China contiguous with two countries and eight other provinces. A total of 4434 serum samples were collected from six cities in the western, central, and eastern regions of Inner Mongolia and analyzed using the ELISA test. In addition, tissue samples were collected from seven animals that were suspected to be infected with MAP. Finally, these tissues samples were analyzed by histopathological examination followed by polymerase chain reaction (PCR), IS1311 PCR-restriction enzyme analysis (PCR-REA), and a sequence analysis of five genes. Among all 4434 ruminant serum samples collected from the six cities in the western, central, and eastern regions of Inner Mongolia, 7.60% (337/4434) measured positive for the MAP antibody. The proportions of positive MAP antibody results for serum samples collected in the western, central, and eastern regions were 5.10% (105/2058), 6.63% (85/1282), and 13.44% (147/1094), respectively. For the seven suspected infected animals selected from the herd with the highest rate of positivity, the gross pathology and histopathology of the necropsied animals were found to be consistent with the pathological features of paratuberculosis. The PCR analysis further confirmed the diagnosis of paratuberculosis. The rest of the results demonstrated that herds of sheep and goats in Inner Mongolia were infected with both MAP type II and type III. To the best of our knowledge, this is the first study of the two subtypes of MAP strains in sheep and goats in Inner Mongolia., Competing Interests: The authors have declared that no competing interests exist.

The purpose of this study was to evaluate the pharmacokinetics of tulathromycin in the plasma and maternal and fetal tissues of pregnant ewes when administered within 24 hours of a single, IV Campylobacter jejuni (C. jejuni) challenge. Twelve, pregnant ewes between 72-92 days of gestation were challenged IV with C. jejuni IA3902 and then treated with 1.1 ml/45.36 kg of tulathromycin subcutaneously 18 hours post-challenge. Ewes were bled at predetermined time points and euthanized either at a predetermined time point or following the observation of vaginal bleeding or abortion. Following euthanasia, tissues were collected for bacterial culture, pharmacokinetics and histologic examination. The maximum (geometric) mean tulathromycin plasma concentration was estimated at 0.302 μg/mL, with a peak level observed at around 1.2 hours. The apparent systemic clearance of tulathromycin was estimated at 16.6 L/h (or 0.28 L/kg/h) with an elimination half-life estimated at approximately 22 hours. The mean tissue concentrations were highest in the uterus (2.464 μg/g) and placentome (0.484 μg/g), and were lowest in fetal liver (0.11 μg/g) and fetal lung (0.03 μg/g). Compared to previous reports, results of this study demonstrate that prior IV administration of C. jejuni appeared to substantially alter the pharmacokinetics of tulathromycin, reducing both the peak plasma concentrations and elimination half-life. However, additional controlled trials are required to confirm those observations., Competing Interests: The authors have declared that no competing interests exist.

Ruminants are dependent on their rumen microbiota to obtain energy from plants. The composition of the microbiome was well-known to be associated with health status, and production traits, but published results are difficult to reproduce due to large sources of variation. The objectives of this study were to evaluate the associations of ruminal microbiota and its association with genetic lines selected by somatic cell score (SCS) or milk persistency (PERS), as well as milk production, somatic cell score, fat and protein contents, and fatty acids and proteins of milk, using the principles of compositional data. A large sample of 700 Lacaune dairy ewes from INRAE La Fage feeding the same diet and belonging to two divergent genetic lines selected for SCS or PERS was used. The ruminal bacterial metagenome was sequenced using the 16S rRNA gene, resulting in 2,059 operational taxonomic units affiliated with 112 genera. The abundance data were centred log-transformed after the replacement of zeros with the geometric Bayesian method. Discriminant analysis of the SCS showed differences between SCS+ and SCS- ewes, while for PERS no difference was obtained. Milk traits as fat content, protein content, saturated fatty acids and caseins of milk were negatively associated with Prevotella (R = [-0.08;-0.16]), Suttonella (R = [-0.09;-0.16]) and Ruminococcus (R = [-0.08;-0.16]), and positively associated with Lachnospiraceae (R = [0.09;0.16]) and Christensenellaceae (R = [0.09;0.16]). Our findings provide an understanding of the application of compositional data to microbiome analysis, and the potential association of Prevotella, Suttonella, Ruminococcaceae and Lachnospiraceae with milk production traits such as milk fatty acids and proteins in dairy sheep., Competing Interests: The authors have declared that no competing interests exist.

Background: To monitor the prevalence of antimicrobial resistance (AMR), methods for interpretation of susceptibility phenotypes of bacteria are needed. Reference limits to declare resistance are generally based on or dominated by data from human bacterial isolates and may not reflect clinical relevance or wild type (WT) populations in livestock or other hosts., Methods: We compared the observed prevalence of AMR using standard and bespoke interpretations based on clinical breakpoints or epidemiological cut-offs (ECOFF) using gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria from sheep as exemplars. Isolates were obtained from a cross-sectional study in three lowland sheep flocks in Scotland, and from a longitudinal study in one flock in Norway. S. aureus (n = 101) was predominantly isolated from milk or mammary glands whilst E. coli (n = 103) was mostly isolated from faecal samples. Disc diffusion testing was used to determine inhibition zone diameters, which were interpreted using either clinical breakpoints or ECOFF, which distinguish the bacterial wild type population from bacteria with acquired or mutational resistance to the compound of interest (non-wild type). Standard ECOFF values were considered as well as sheep-specific values calculated from the data using Normalized Resistance Interpretation (NRI) methodology., Results: The prevalence of AMR as measured based on clinical breakpoints was low, e.g. 4.0% for penicillin resistance in S. aureus. Estimation of AMR prevalence based on standard ECOFFs was hampered by lack of relevant reference values. In addition, standard ECOFFS, which are predominantly based on human data, bisected the normal distribution of inhibition zone diameters for several compounds in our analysis of sheep isolates. This contravenes recommendations for ECOFF setting based on NRI methodology and may lead to high apparent AMR prevalence. Using bespoke ECOFF values based on NRI, S. aureus showed non-wild type for less than 4% of isolates across 13 compounds, and ca. 13% non-wild type for amoxicillin and ampicillin, while E. coli showed non-wild type for less than 3% of isolates across 12 compounds, and ca. 13% non-wild type for tetracyclines and sulfamethoxazole-trimethoprim., Conclusion: The apparent prevalence of AMR in bacteria isolated from sheep is highly dependent on interpretation criteria. The sheep industry may want to establish bespoke cut-off values for AMR monitoring to avoid the use of cut-offs developed for other host species. The latter could lead to high apparent prevalence of resistance, including to critically important antimicrobial classes such as 4th generation cephalosporins and carbapenems, suggesting an AMR problem that may not actually exist., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to evaluate the effects of medicinal herbal mixtures rich in phenolic, flavonoid and alkaloid compounds on ruminal fermentation and microbial populations, and fatty acid (FA) concentrations and lipid oxidation in tissues of lambs infected with the gastrointestinal nematode (GIN) parasite (Haemonchus contortus). Parallel in vitro and in vivo studies were performed using two different herbal mixtures (Mix1 and Mix2). The in vitro study was conducted in a 2 (infection status; non-infected versus infected) × 3 (diets; control, Mix1 and Mix2) factorial design. In the in vivo study, 24 lambs were equally divided into four treatments: non-infected lambs fed a control diet, infected lambs fed the control diet, infected lambs fed a diet with Mix1 and infected lambs fed a diet with Mix2. Herbal mixtures (100 g dry matter (DM)/d) were added to the basal diets of meadow hay (ad libitum) and a commercial concentrate (500 g DM/d). The experimental period lasted for 70 days. Ruminal fermentation characteristics and methane production were not affected by infection in vivo or in vitro. Both herbal mixture supplementation increased total volatile fatty acid (VFA) concentrations (P < 0.01) and DM digestibility (P < 0.01) in vitro. Archaea population was slightly diminished by both herbal mixtures (P < 0.05), but they did not lower methane production in vitro or in vivo (P > 0.05). Infection of H. contortus or herbal mixtures modulated FA proportion mainly in the liver, especially the long chain FA proportion. Concentrations of thiobarbituric acid reactive substances (TBARS) in serum were significantly higher after 70 days post-infection in the infected lambs. Herbal Mix1 supplementation reduced TBARS concentrations in meat after seven days of storage. In conclusion, supplementing of herbal mixtures to the diets of GIN parasite infected lambs did not affect the basic ruminal fermentation parameters. Herbal mixtures may improve few FA proportions mainly in liver as well as decrease lipid oxidation in meat., Competing Interests: The authors have declared that no competing interests exist.

Microbial community profiles have been associated with a variety of traits, including methane emissions in livestock. These profiles can be difficult and expensive to obtain for thousands of samples (e.g. for accurate association of microbial profiles with traits), therefore the objective of this work was to develop a low-cost, high-throughput approach to capture the diversity of the rumen microbiome. Restriction enzyme reduced representation sequencing (RE-RRS) using ApeKI or PstI, and two bioinformatic pipelines (reference-based and reference-free) were compared to bacterial 16S rRNA gene sequencing using repeated samples collected two weeks apart from 118 sheep that were phenotypically extreme (60 high and 58 low) for methane emitted per kg dry matter intake (n = 236). DNA was extracted from freeze-dried rumen samples using a phenol chloroform and bead-beating protocol prior to RE-RRS. The resulting sequences were used to investigate the repeatability of the rumen microbial community profiles, the effect of laboratory and analytical method, and the relationship with methane production. The results suggested that the best method was PstI RE-RRS analyzed with the reference-free approach, which accounted for 53.3±5.9% of reads, and had repeatabilities of 0.49±0.07 and 0.50±0.07 for the first two principal components (PC1 and PC2), phenotypic correlations with methane yield of 0.43±0.06 and 0.46±0.06 for PC1 and PC2, and explained 41±8% of the variation in methane yield. These results were significantly better than for bacterial 16S rRNA gene sequencing of the same samples (p<0.05) except for the correlation between PC2 and methane yield. A Sensitivity study suggested approximately 2000 samples could be sequenced in a single lane on an Illumina HiSeq 2500, meaning the current work using 118 samples/lane and future proposed 384 samples/lane are well within that threshold. With minor adaptations, our approach could be used to obtain microbial profiles from other metagenomic samples., Competing Interests: The authors have declared that no competing interests exist.

The nutritional and economic potentials of livestock systems are compromised by the emergence and spread of antimicrobial resistance. A major driver of resistance is the misuse and abuse of antimicrobial drugs. The likelihood of misuse may be elevated in low- and middle-income countries where limited professional veterinary services and inadequately controlled access to drugs are assumed to promote non-prudent practices (e.g., self-administration of drugs). The extent of these practices, as well as the knowledge and attitudes motivating them, are largely unknown within most agricultural communities in low- and middle-income countries. The main objective of this study was to document dimensions of knowledge, attitudes and practices related to antimicrobial use and antimicrobial resistance in livestock systems and identify the livelihood factors associated with these dimensions. A mixed-methods ethnographic approach was used to survey households keeping layers in Ghana (N = 110) and Kenya (N = 76), pastoralists keeping cattle, sheep, and goats in Tanzania (N = 195), and broiler farmers in Zambia (N = 198), and Zimbabwe (N = 298). Across countries, we find that it is individuals who live or work at the farm who draw upon their knowledge and experiences to make decisions regarding antimicrobial use and related practices. Input from animal health professionals is rare and antimicrobials are sourced at local, privately owned agrovet drug shops. We also find that knowledge, attitudes, and particularly practices significantly varied across countries, with poultry farmers holding more knowledge, desirable attitudes, and prudent practices compared to pastoralist households. Multivariate models showed that variation in knowledge, attitudes and practices is related to several factors, including gender, disease dynamics on the farm, and source of animal health information. Study results emphasize that interventions to limit antimicrobial resistance should be founded upon a bottom-up understanding of antimicrobial use at the farm-level given limited input from animal health professionals and under-resourced regulatory capacities within most low- and middle-income countries. Establishing this bottom-up understanding across cultures and production systems will inform the development and implementation of the behavioral change interventions to combat antimicrobial resistance globally., Competing Interests: The authors have declared that no competing interests exist.

Footrot is a worldwide economically important, painful, contagious bacterial foot disease of domestic and wild ungulates caused by Dichelobacter nodosus. Benign and virulent strains have been identified in sheep presenting with mild and severe lesions, respectively. However, in Alpine ibex (Capra ibex ibex), both strains have been associated with severe lesions. Because the disease is widespread throughout sheep flocks in Switzerland, a nationwide footrot control program for sheep focusing on virulent strains shall soon be implemented. The aim of this cross-sectional study was to estimate the nationwide prevalence of both strain groups of D. nodosus in four wild indigenous ruminant species and to identify potential susceptible wildlife maintenance hosts that could be a reinfection source for domestic sheep. During two years (2017-2018), interdigital swabs of 1,821 wild indigenous ruminant species (Alpine ibex, Alpine chamois (Rupicapra rupicapra), roe deer (Capreolus capreolus), red deer (Cervus elaphus)) were analysed by Real-Time PCR. Furthermore, observed interspecies interactions were documented for each sample. Overall, we report a low prevalence of D. nodosus in all four indigenous wild ruminants, for both benign (1.97%, N = 36, of which 31 red deer) and virulent (0.05%, N = 1 ibex) strains. Footrot lesions were documented in one ibex with virulent strains, and in one ibex with benign strains. Interspecific interactions involving domestic livestock occurred mainly with cattle and sheep. In conclusion, the data suggest that wild ungulates are likely irrelevant for the maintenance and spread of D. nodosus. Furthermore, we add evidence that both D. nodosus strain types can be associated with severe disease in Alpine ibex. These data are crucial for the upcoming nationwide control program and reveal that wild ruminants should not be considered as a threat to footrot control in sheep in this context., Competing Interests: The authors have declared that no competing interests exist.

This study investigated the associations between abundance of rumen ciliate protozoa and the proportion of the main bioactive fatty acids related to rumen biohydrogenation, as 18:0, t10-18:1, t11-18:1, c9,t11-18:2, 18:3n-3 and 18:2 n-6, in rumen and meat of growing lambs, using data derived from 3 production experiments. A global correlation analysis and a linear regression analysis considering the effect of the experiment were performed. Ten of the 86 lambs involved in the experiments did not present ciliate cells in rumen liquor and the remaining lambs presented an average of 1.35 × 106ciliates / ml rumen liquor. From the nine genera of ciliates identified, Entodinium was the most abundant, averaging 1.17 × 106 cells / ml of rumen liquor. A large variation among lambs was observed for both rumen concentration and community structure of ciliates. Rumen t11-18:1 (P < 0.001) and meat deposition of t11-18:1 (P < 0.001) and of c9,t11-18:2 (P < 0.001) increased linearly with total ciliates, whereas the t10/t11 ratio in rumen (P = 0.002) and in meat (P = 0.036) decreased linearly. Entodiniomorphids seems to be strongly related with meat deposition oft11-18:1 and c9,t11-18:2 and with the reduction of the trans-10 shifted pathway. Completeness of RBH decreased linearly with Holotrichs (P = 0.029), Entodiniomorphids (P = 0.029), Isotricha (P = 0.011) and Epidinium (P = 0.027) abundances. Rumen 18:0 also decreased linearly with increasing counts of total ciliates (P = 0.015), Holotrichs (P = 0.020), Entodiniomorphids (P = 0.010) and Isotricha (P = 0.014). Rumen protozoa were positively linked with the deposition of healthy bioactive FA and simultaneously negatively associated with the occurrence of trans-10 shift., Competing Interests: The authors have declared that no competing interests exist.

Oregano essential oil (OEO), which has antimicrobial properties, may be used for altering the ruminal pH and microbial populations of sheep, as observed by the altered volatile fatty acid patterns. To further elucidate the effects of OEO on ruminal pH and microbial populations of sheep, 3 German merino sheep × local sheep crossbred rams with permanent ruminal fistulas were randomly assigned to a 3 × 3 Latin square design with 12-d periods. The treatments were as follows: control (CON); OEO4: OEO supplied at 4 g•d-1; and OEO7: OEO supplied at 7 g•d-1. Starting on day 11, rumen fluid was collected at 0 h, and at 4, 8, 12, 24 and 48 h after supplying OEO, and then pH values of rumen fluid were immediately measured. The abundance of microbial populations was determined by using qPCR. The ruminal pH values were similar among the sheep from all treatments. The abundance of ruminal fungi was higher for the sheep supplied OEO7 compared with the sheep supplied CON and OEO4, especially at 4 and 12 h. The abundance of ruminal protozoa decreased with supplied OEO, indicating that OEO could inhibit the protozoa. The abundance of the total ruminal bacteria was similar for the sheep from all treatments, but R. flavefaciens, R. albus and F. succinogenes increased in the sheep supplied OEO4 compared with those in the sheep supplied CON, however, the sheep supplied OEO7 had higher abundances of R. flavefaciens than the sheep supplied CON. These results demonstrated that supplying OEO to sheep did not affect the ruminal pH but could shift the rumen microbial population to one with less protozoa. Supplying OEO can preferentially enhance the growth of certain rumen microbial populations, but the shifts were influenced by the supply rate. Therefore, supplying low amount (i.e. 4 g•d-1) of OEO could have positive effects on ruminal microbial populations, whereas supplying elevated doses of OEO could be detrimental to those same ruminal microbial populations., Competing Interests: The authors have declared that no competing interests exist.

In China, thirteen species of tick-borne rickettsiales bacteria pathogenic to human have been reported in ticks and host animals, and human patients caused by them also has been identified. However, investigation for rickettsiales bacteria circulating in Xi'an wasn't performed although diseases resembling human diseases caused by these organisms have been found. In this study, domestic animals and ticks in Xi'an, China, were tested for the presence of rickettsiales bacteria pathogenic to humans. Besides A. ovis, a high prevalence of A. capra was observed suggesting a high public health risk exists. In addition, two novel Anaplasma species closely related to A. phagocytophilum were identified and formed distinct lineages in the phylogenetic trees, with more than 98.3% identities for rrs gene, while divergences up to 20.2% and 37.0% for groEL and gltA genes, respectively. Both of these two novel Anaplasma species were found to circulate in goats and further assessment of their pathogenicity is needed. Ca. R. jingxinensis, with potential pathogenicity, was also detected in H. longicomis ticks with high prevalence. However, other causative agents were not identified although they were distributed in other areas of China., Competing Interests: The authors have declared that no competing interests exist.

The Qinghai-Tibet plateau is a natural plague focus and is the largest such focus in China. In this area, while Marmota himalayana is the primary host, a total of 18 human plague outbreaks associated with Tibetan sheep (78 cases with 47 deaths) have been reported on the Qinghai-Tibet plateau since 1956. All of the index infectious cases had an exposure history of slaughtering or skinning diseased or dead Tibetan sheep. In this study, we sequenced and compared 38 strains of Yersinia pestis isolated from different hosts, including humans, Tibetan sheep, and M. himalayana. Phylogenetic relationships were reconstructed based on genome-wide single-nucleotide polymorphisms identified from our isolates and reference strains. The phylogenetic relationships illustrated in our study, together with the finding that the Tibetan sheep plague clearly lagged behind the M. himalayana plague, and a previous study that identified the Tibetan sheep as a plague reservoir with high susceptibility and moderate sensitivity, indicated that the human plague was transmitted from Tibetan sheep, while the Tibetan sheep plague originated from marmots. Tibetan sheep may encounter this infection by contact with dead rodents or through being bitten by fleas originating from M. himalayana during local epizootics., Competing Interests: The authors have declared that no competing interests exist.

Culture-independent methods were used to study the microbiota of adult worms, third-stage larvae and eggs, both in faeces and laid in vitro, of Haemonchus contortus, a nematode parasite of the abomasa of ruminants which is a major cause of production losses and ill-health. Bacteria were identified in eggs, the female reproductive tract and the gut of adult and third-stage larvae (L3). PCR amplification of 16S rRNA sequences, denaturing gradient gel electrophoresis (DGGE) and clone libraries were used to compare the composition of the microbial communities of the different life-cycle stages of the parasites, as well as parasites and their natural environments. The microbiomes of adult worms and L3 were different from those in the abomasum or faeces respectively. The H. contortus microbiota was mainly comprised of members of the phyla Proteobacteria, Firmicutes and Bacteroidetes. Bacteria were localised in the gut, inside eggs and within the uterus of adult female worms using the universal FISH Eub338 probe, which targets most bacteria, and were also seen in these tissues by light and transmission electron microscopy. Streptococcus/Lactococcus sp. were identified within the distal uterus with the probe Strc493. Sequences from the genera Weissella and Leuconostoc were found in all life-cycle stages, except eggs collected from faeces, in which most sequences belonged to Clostridium sp. Bacteria affiliated with Weissella/Leuconostoc were identified in both PCR-DGGE short sequences and clone libraries of nearly full length 16S rRNA bacterial sequences in all life-cycle stages and subsequently visualised in eggs by fluorescent in situ hybridisation (FISH) with group-specific probes. This strongly suggests they are vertically transmitted endosymbionts. As this study was carried out on a parasite strain which has been maintained in the laboratory, other field isolates will need to be examined to establish whether these bacteria are more widely dispersed and have potential as targets to control H. contortus infections.

Brucellosis has been an endemic disease of cattle and humans in Costa Rica since the beginning of XX century. However, brucellosis in sheep, goats, pigs, water buffaloes, horses and cetaceans, has not been reported in the country. We have performed a brucellosis survey in these host mammal species, from 1999-2016. In addition, we have documented the number of human brucellosis reported cases, from 2003-2016. The brucellosis seroprevalence in goat and sheep herds was 0.98% and 0.7% respectively, with no Brucella isolation. Antibodies against Brucella were not detected in feral or domestic pigs. Likewise, brucellosis seroprevalence in horse and water buffalo farms was estimated in 6.5% and 21.7%, respectively, with no Brucella isolation. Six cetacean species showed positive reactions against Brucella antigens, and B. ceti was isolated in 70% (n = 29) of striped dolphins (Stenella coeruleoalba). A steady increase in the diagnosis of human brucellosis cases was observed. Taking into account the prevalence of brucellosis in the various host mammals of Costa Rica, different measures are recommended.

This study investigated the pathogenesis of two variant strains (LLG and POS) of Chlamydia abortus, in comparison to a typical wild-type strain (S26/3) which is known to be responsible for late term abortion in small ruminants. Challenge with the three strains at mid-gestation resulted in similar pregnancy outcomes, with abortion occurring in approximately 50-60% of ewes with the mean gestational lengths also being similar. However, differences were observed in the severity of placental pathology, with infection appearing milder for strain LLG, which was reflected in the lower number of organisms shed in vaginal swabs post-partum and less gross pathology and organisms present in placental smears. Results for strain POS were somewhat different than LLG with a more focal restriction of infection observed. Post-abortion antibody responses revealed prominent differences in seropositivity to the major outer membrane protein (MOMP) present in elementary body (EB) preparations under denaturing conditions, most notably with anti-LLG and anti-POS convalescent sera where there was no or reduced detection of MOMP present in EBs derived from the three strains. These results and additional analysis of whole EB and chlamydial outer membrane complex preparations suggest that there are conformational differences in MOMP for the three strains. Overall, the results suggest that gross placental pathology and clinical outcome is not indicative of bacterial colonization and the severity of infection. The results also highlight potential conformational differences in MOMP epitopes that perhaps impact on disease diagnosis and the development of new vaccines.

Background: Q fever is a main zoonotic disease around the world. The aim of this meta-analysis was to estimate the overall seroprevalence of Coxiella burnetii among human and animal population in Iran., Methods: Major national and international databases were searched from 2005 up to August 2016. We extracted the prevalence of Q fever antibodies (IgG) as the main primary outcome. We reported the prevalence of the seropositivity as point and 95% confidence intervals., Results: The overall seroprevalence of IgG phase I and II antibodies of Q fever in human was 19.80% (95% CI: 16.35-23.25%) and 32.86% (95% CI: 23.80-41.92%), respectively. The herd and individual prevalence of C. burnetii antibody in goat were 93.42% (95% CI: 80.23-100.00) and 31.97% (95% CI: 20.96-42.98%), respectively. The herd and individual prevalence of Q fever antibody in sheep's were 96.07% (95% CI: 89.11-100.00%) and 24.66% (95% CI: 19.81-29.51%), respectively. The herd and individual prevalence of C. burnetii antibody in cattle were 41.37% (95% CI: 17.88-64.86%) and 13.30% (95% CI: 2.98-23.62%), respectively. Individual seropositivity of Q fever in camel and dog were 28.26% (95% CI: 21.47-35.05) and 0.55% (0.03-2.68), respectively., Conclusion: Seroprevalence of Q fever among human and domestic animals is considerable. Preventative planning and control of C. burnetii infections in Iran is necessary. Active surveillance and further research studies are recommended, to more clearly define the epidemiology and importance of C. burnetii infections in animals and people in Iran.

Domestic goats are raised for meat, milk and hair production, in herds for rangeland weed control, and as pack animals. Domestic sheep, goats and wild bighorn sheep are all susceptible to a multifactorial pneumonia. We sampled 43 herd goats from 7 herds and 48 pack goats from 11 herds for viral and bacterial serology, parasitology, and Pasteurellaceae microbiology. The goats in this study were in generally good health, although most goats did harbor various pathogens and parasites including several bacteria, specifically Pasteurellaceae, which have been associated with pneumonia in free-ranging bighorn sheep. It is not known if domestic goats can transmit the Pasteurellaceae or other pathogens found in this study readily to wild bighorn sheep. However, due the possibility of transmission, domestic goats in areas in or near bighorn sheep habitat should be managed to minimize the risk of spreading disease agents to bighorn sheep.

Background: Brucellosis is a worldwide zoonosis with significant impact on rural livelihoods and a potentially underestimated contributor to febrile illnesses. The aim of this study was to estimate the seroprevalence of brucellosis in humans and small ruminants in The Gambia., Methods: The study was carried out in rural and urban areas. In 12 rural villages in Kiang West district, sera were collected from humans (n = 599) and small ruminants (n = 623) from the same compounds. From lactating small ruminants, milk samples and vaginal swabs were obtained. At the urban study sites, sera were collected from small ruminants (n = 500) from slaughterhouses and livestock markets. Information on possible risk factors for seropositivity was collected through questionnaires. Sera were screened for antibodies against Brucella spp. with the Rose Bengal Test, ELISA and Micro Agglutination Test (human sera only). PCR was performed on 10 percent of the milk samples and vaginal swabs from small ruminants., Results: One human and 14 sheep sera were positive by the Rose Bengal Test. The rest were negative in all serological tests used. The PCR results were all negative., Conclusions: The results suggest that brucellosis is currently not a generalized problem in humans or small ruminants in The Gambia., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to investigate the presence of β-lactamase producing or fluoroquinolone-resistant members of the family Enterobacteriaceae in European mouflons (Ovis orientalis musimon). The mouflon samples originated from nasal and perineal swabs and/or organ samples in cases of a suspected infection. Only one of the 32 mouflons was tested positive for the presence of Enterobacteriaceae that displayed either an ESBL/AmpC phenotype or were resistant to ciprofloxacin. The positively tested swab originated from a sample of the jejunal mucosa of a four-year old female mouflon. Two different colony morphotypes were identified as Escherichia coli and Klebsiella pneumoniae. These isolates were phenotypically and genotypically characterized in detail by a polyphasic approach. Both isolates were multi-drug resistant. The E. coli isolate belonged to the phylogenetic group B1 and sequence type (ST) 744 and harboured the β-lactamase genes blaCTX-M-15 and blaOXA-1. The K. pneumoniae, identified as ST11, harboured the β-lactamase genes blaSHV-11, blaOXA-1, and blaDHA-1 as well as the plasmid-mediated quinolone resistance (PMQR) gene qnrB55. The present study demonstrates that wild animals can acquire human-derived resistance determinants and such findings may indicate environmental pollution with resistance determinants from other sources.

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

Background: Brucellosis and coxiellosis are known to be endemic in ruminant populations throughout Afghanistan, but information about their prevalence and factors that affect prevalence in householders and livestock under diverse husbandry systems and pastoral settings is sparse., Methods/principal Findings: We conducted a cross-sectional survey to investigate the seroprevalence of brucellosis and Coxiella burnetii in humans and livestock in six secure districts in Herat from 26th December 2012-17th January 2013. A total of 204 households with livestock were surveyed in six Kuchi and five sedentary type villages. Blood samples from 1,017 humans, 1,143 sheep, 876 goats and 344 cattle were tested for brucellosis and Q fever. About one in six households (15.7%) had at least one Brucella seropositive person, about one in eight households (12.3%) had at least one Brucella seropositive animal and about one in four (24.5%) had either seropositive animals or humans. Ninety-seven percent of households had at least one C. burnetii seropositive person and 98.5% of households had one or more C. burnetii seropositive animals. Forty- seven householders had serological evidence of exposure to both C. burnetii and Brucella and eight animals were serologically positive for both diseases. Drinking unpasteurised milk (OR 1.6), treating animals for ticks (OR 1.4), milking sheep (OR 1.4), male gender (OR 1.4) and seropositivity to Brucella (OR 4.3) were identified as risk factors for seropositivity to C. burnetii in householders. Household factors associated with households having either Brucella seropositive animals or humans were Kuchi households (OR 2.5), having ≤ 4 rooms in the house (OR 2.9) and not owning land (OR 2.9)., Conclusions: The results from this study provide baseline information for the planning and monitoring of future interventions against these diseases. The implementation of this study greatly improved collaboration, coordination and capability of veterinary and public health professionals from government, NGOs and donor funded projects.

Background: Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany., Results: Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2β effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all., Conclusion: Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2β components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk assessment in food analyses and clinical diagnostics.

Improvement of knowledge, attitudes and practices among urban livestock farmers could have a significant impact on the reduction of many zoonotic infections in urban farming. This study aimed to describe and evaluate weak areas in knowledge, attitudes and practices with regards to brucellosis among urban and peri-urban small-scale dairy farmers in a low income country to generate information essential for control programmes and public health interventions. The cross-sectional study was conducted during six weeks in 2011. The study subjects were small-scale dairy farmers living in the urban and peri-urban area of the capital Dushanbe in Tajikistan. In total, 441 farmers were interviewed using a questionnaire with questions about demographic characteristics, knowledge, attitudes and practices relating to brucellosis. Descriptive statistics were used and a logistic regression model applied to evaluate potential predictors to knowledge about brucellosis. The majority (85%) of the farmers had never heard of brucellosis. Low educational level was found to be associated with low awareness of brucellosis (P = < 0.001). Respondents who talked about animal health issues with family members or friends were less likely to have heard of brucellosis compared to those who often talked to veterinarians (P = 0.03). Sixty three per cent of the participants wanted more information about brucellosis. Seventeen per cent sold unpasteurized dairy products on a regular basis direct to consumers. Almost 30% of the households consumed unpasteurized dairy products on regular basis. A majority of the respondents did not use any protection when handling cows having an abortion or when dealing with aborted materials. Poor knowledge, high-risk behaviours and a willingness to learn more strengthens the logic for including health education as part of control programmes.

Despite control/eradication programs, brucellosis, major worldwide zoonosis due to the Brucella genus, is endemic in Northern Africa and remains a major public health problem in the Maghreb region (Algeria/Morocco/Tunisia). Brucella melitensis biovar 3 is mostly involved in human infections and infects mainly small ruminants. Human and animal brucellosis occurrence in the Maghreb seems still underestimated and its epidemiological situation remains hazy. This study summarizes official data, regarding Brucella melitensis infections in Algeria, from 1989 to 2012, with the purpose to provide appropriate insights concerning the epidemiological situation of human and small ruminant brucellosis in Maghreb. Algeria and Europe are closely linked for historical and economical reasons. These historical connections raise the question of their possible impact on the genetic variability of Brucella strains circulating in the Maghreb. Other purpose of this study was to assess the genetic diversity among Maghreb B. melitensis biovar 3 strains, and to investigate their possible epidemiological relationship with European strains, especially with French strains. A total of 90 B. melitensis biovar 3 Maghreb strains isolated over a 25 year-period (1989-2014), mainly from humans, were analysed by MLVA-16. The obtained results were compared with genotypes of European B. melitensis biovar 3 strains. Molecular assays showed that Algerian strains were mainly distributed into two distinct clusters, one Algerian cluster related to European sub-cluster. These results led to suggest the existence of a lineage resulting from socio-historical connections between Algeria and Europe that might have evolved distinctly from the Maghreb autochthonous group. This study provides insights regarding the epidemiological situation of human brucellosis in the Maghreb and is the first molecular investigation regarding B. melitensis biovar 3 strains circulating in the Maghreb.

The manufacturing processes of royal demolition explosive (RDX), or hexahydro-1,3,5-trinitro-1,3,5-triazine, have resulted in serious water contamination. As a potential carcinogen, RDX can cause a broad range of harmful effects to humans and animals. The ovine rumen is capable of rapid degradation of nitroaromatic compounds, including RDX. While ruminal RDX-degrading bacteria have been identified, the genes and pathways responsible for RDX degradation in the rumen have yet to be characterized. In this study, we characterized the metabolic potential of the ovine rumen using metagenomic approaches. Sequences homologous to at least five RDX-degrading genes cloned from environmental samples (diaA, xenA, xenB, xplA, and xplB) were present in the ovine rumen microbiome. Among them, diaA was the most abundant, likely reflective of the predominance of the genus Clostridium in the ovine rumen. At least ten genera known to harbor RDX-degrading microorganisms were detectable. Metagenomic sequences were also annotated using public databases, such as Pfam, COG, and KEGG. Five of the six Pfam protein families known to be responsible for RDX degradation in environmental samples were identified in the ovine rumen. However, increased substrate availability did not appear to enhance the proliferation of RDX-degrading bacteria and alter the microbial composition of the ovine rumen. This implies that the RDX-degrading capacity of the ovine rumen microbiome is likely regulated at the transcription level. Our results provide metagenomic insights into the RDX-degrading potential of the ovine rumen, and they will facilitate the development of novel and economic bioremediation strategies.

The analysis of the early macrophage responses, including bacterial growth within macrophages, represents a powerful tool to characterize the virulence of clinical isolates of Mycobcaterium avium susbp. paratuberculosis (Map). The present study represents the first assessment of the intracellular behaviour in ovine monocyte-derived macrophages (MDMs) of Map isolates representing distinct genotypes (C, S and B), and isolated from cattle, sheep, goat, fallow deer, deer, and wild boar. Intracellular growth and survival of the selected isolates in ovine MDMs was assessed by quantification of CFUs inside of the host cells at 2 h p.i. (day 0) and 7 d p. i. using an automatic liquid culture system (Bactec MGIT 960). Variations in bacterial counts over 7 days from the baseline were small, in a range between 1.63 to 1.05-fold. After 7 d of infection, variations in the estimated log10 CFUs between all the tested isolates were not statistically significant. In addition, ovine MDMs exhibited enhanced anti-inflammatory, antiapoptotic and antidestructive responses when infected with two ovine isolates of distinct genotype (C and S) or with two C-type isolates from distinct hosts (cattle and sheep); which correlated with the successful survival of these isolates within ovine MDMs. A second objective was to study, based on an in vitro granuloma model, latter stages of the infection by investigating the capacity of two Map isolates from cattle and sheep to trigger formation of microgranulomas. Upon 10 d p.i., both Map isolates were able to induce the formation of granulomas comparable to the granulomas observed in clinical specimens with respect to the cellular components involved. In summary, our results demonstrated that Map isolates from cattle, sheep, goats, deer, fallow-deer and wild boar were able not only to initiate but also to establish a successful infection in ovine macrophages regardless of genotype.

Q fever is a widespread zoonosis that is caused by Coxiella burnetii (C. burnetii), and ruminants are identified as the main sources of human infections. Some human cases have been described, but very limited information was available about Q fever in ruminants on Reunion Island, a tropical island in the Indian Ocean. A cross-sectional study was undertaken from March 2011 to August 2012 to assess the Q fever prevalence and to identify the major risk factors of C. burnetii infection in ruminants. A total of 516 ruminants (245 cattle, 137 sheep and 134 goats) belonging to 71 farms and localized in different ecosystems of the island were randomly selected. Samples of blood, vaginal mucus and milk were concomitantly collected from females, and a questionnaire was submitted to the farmers. Ticks from positively detected farms were also collected. The overall seropositivity was 11.8% in cattle, 1.4% in sheep and 13.4% in goats. C. burnetii DNA was detected by PCR in 0.81%, 4.4% and 20.1% in cow, sheep and goat vaginal swabs, respectively. C. burnetii shedding in milk was observed in 1% of cows, 0% in sheep and 4.7% in goats. None of the ticks were detected to be positive for C. burnetii. C. burnetii infection increased when the farm was exposed to prevailing winds and when there were no specific precautions for a visitor before entering the farm, and they decreased when a proper quarantine was set up for any introduction of a new ruminant and when the animals returned to the farm at night. MLVA genotyping confirmed the role of these risk factors in infection.

Background: Waddlia chondrophila (W. chondrophila) is an emerging abortifacient organism which has been identified in the placentae of humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial abortifacients, such as Chlamydia abortus (C. abortus). This study investigates the growth of the organism and its effects upon pro-inflammatory cytokine expression in a ruminant placental cell line which we have previously utilised in a model of C. abortus pathogenicity., Methodology/principal Findings: Using qPCR, fluorescent immunocytochemistry and electron microscopy, we characterised the infection and growth of W. chondrophila within the ovine trophoblast AH-1 cell line. Inclusions were visible from 6 h post-infection (p.i.) and exponential growth of the organism could be observed over a 60 h time-course, with significant levels of host cell lysis being observed only after 36 h p.i. Expression of CXCL8, TNF-α, IL-1α and IL-1β were determined 24 h p.i. A statistically significant response in the expression of CXCL8, TNF-α and IL-1β could be observed following active infection with W. chondrophila. However a significant increase in IL-1β expression was also observed following the exposure of cells to UV-killed organisms, indicating the stimulation of multiple innate recognition pathways., Conclusions/significance: W. chondrophila infects and grows in the ruminant trophoblast AH-1 cell line exhibiting a complete chlamydial replicative cycle. Infection of the trophoblasts resulted in the expression of pro-inflammatory cytokines in a dose-dependent manner similar to that observed with C. abortus in previous studies, suggesting similarities in the pathogenesis of infection between the two organisms.

Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.

Ureaplasma species are the microorganisms most frequently associated with adverse pregnancy outcomes. The multiple banded antigen (MBA), a surface-exposed lipoprotein, is a key virulence factor of ureaplasmas. The MBA demonstrates size variation, which we have shown previously to be correlated with the severity of chorioamnion inflammation. We aimed to investigate U. parvum serovar 3 pathogenesis in vivo, using a sheep model, by investigating: MBA variation after long term (chronic) and short term (acute) durations of in utero ureaplasma infections, and the severity of chorioamnionitis and inflammation in other fetal tissues. Inocula of 2 × 10(7) colony-forming-units (CFU) of U. parvum serovar 3 (Up) or media controls (C) were injected intra-amniotically into pregnant ewes at one of three time points: day 55 (69d Up, n = 8; C69, n = 4); day 117 (7d Up, n = 8; C7, n = 2); and day 121 (3d Up, n = 8; C3, n = 2) of gestation (term = 145-150d). At day 124, preterm fetuses were delivered surgically. Samples of chorioamnion, fetal lung, and umbilical cord were: (i) snap frozen for subsequent ureaplasma culture, and (ii) fixed, embedded, sectioned and stained by haematoxylin and eosin stain for histological analysis. Selected fetal lung clinical ureaplasma isolates were cloned and filtered to obtain cultures from a single CFU. Passage 1 and clone 2 ureaplasma cultures were tested by western blot to demonstrate MBA variation. In acute durations of ureaplasma infection no MBA variants (3d Up) or very few MBA variants (7d Up) were present when compared to the original inoculum. However, numerous MBA size variants were generated in vivo (alike within contiguous tissues, amniotic fluid and fetal lung, but different variants were present within chorioamnion), during chronic, 69d exposure to ureaplasma infection. For the first time we have shown that the degree of ureaplasma MBA variation in vivo increased with the duration of gestation.

In this study the enzymatic activity of Mycoplasma agalactiae MAG&#95;5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG&#95;5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG&#95;5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG&#95;5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG&#95;5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG&#95;5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG&#95;5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG&#95;5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG&#95;5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.

The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×10⁴ CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32-170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.

Current post-epidemic sero-surveillance uses random selection of animal holdings. A better strategy may be to estimate the benefits gained by sampling each farm and use this to target selection. In this study we estimate the probability of undiscovered infection for sheep farms in Devon after the 2001 foot-and-mouth disease outbreak using the combination of a previously published model of daily infection risk and a simple model of probability of discovery of infection during the outbreak. This allows comparison of the system sensitivity (ability to detect infection in the area) of arbitrary, random sampling compared to risk-targeted selection across a full range of sampling budgets. We show that it is possible to achieve 95% system sensitivity by sampling, on average, 945 farms with random sampling and 184 farms with risk-targeted sampling. We also examine the effect of ordering samples by risk to expedite return to a disease-free status. Risk ordering the sampling process results in detection of positive farms, if present, 15.6 days sooner than with randomly ordered sampling, assuming 50 farms are tested per day.

Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis.