Bacterial burden and molecular characterization of Coxiella burnetii in shedding pregnant and postpartum ewes from Saint Kitts.

This study aimed to evaluate the bacterial burden and perform molecular characterization of Coxiella burnetii during shedding in pregnant (vaginal, mucus and feces) and postpartum (vaginal mucus, feces and milk) ewes from Saint Kitts. Positive IS1111 DNA (n=250) for C. burnetii samples from pregnant (n=87) and postpartum (n=74) Barbados Blackbelly ewes in a previous investigation were used for this study. Vaginal mucus (n=118), feces (n=100), and milk (n=32) positive IS1111 C. burnetii-DNA were analysed by real time qPCR (icd gene). For molecular characterization of C. burnetii, selected (n=10) IS1111 qPCR positive samples were sequenced for fragments of the IS1111 element and the 16 S rRNA gene. nBLAST, phylogenetic and haplotype analyses were performed. Vaginal mucus, feces and milk had estimated equal amounts of bacterial DNA (icd copies), and super spreaders were detected within the fecal samples. C. burnetii haplotypes had moderate to high diversity, were ubiquitous worldwide and similar to previously described in ruminants and ticks and humans.

Isolation and Identification of Pasteurella multocida and Mannheimia haemolytica from Pneumonic Small Ruminants and Their Antibiotic Susceptibility in Haramaya District, Eastern Ethiopia.

Background: Pasteurella species are frequently encountered as serious diseases in small ruminants. It is the main cause of respiratory pasteurellosis in sheep and goats of all age groups. Methods: The cross-sectional study was conducted from December 2022 to April 2023 in Haramaya district, eastern Ethiopia, to isolate and identify Pasteurella multocida and Mannheimia haemolytica and estimate their prevalence, associated risk factors, and antimicrobial sensitivity of isolates in small ruminants using a purposive sampling method. A total of 384 samples (156 nasal swabs from clinic cases and 228 lung swabs from abattoir cases) were collected. STATA 14 software was used to analyze the data. In addition, multivariable logistic regression analysis was performed to assess an association of risk factors. Results: Out of the 384 samples examined, 164 were positive for pasteurellosis, resulting in a 42.70% prevalence. Similarly, 63 (38.4%) of the 164 positive results were from nasal swabs, while 101 (61.6%) came from lung samples. M. haemolytica accounted for 126 (76.82%) of the isolates, while P. multocida accounted for 38 (23.17%). Of the 63 nasal swab isolates, 33 (37%) were from goats and 30 (42.8%) were from sheep. And 17 (10.89%) and 46 (29.58%), respectively, were P. multocida and M. haemolytica. Of the 46 (40%) of the 101 (44.3%) isolates of the pneumonic lung, samples were from goats, while 55 (48.47%) were from sheep. In this study, the risk factors (species, age, and body condition score) were found to be significant (p < 0.05). Pasteurella isolates evaluated for antibiotic susceptibility were highly resistant to oxacillin (90.90%), followed by gentamycin (72.72%), and penicillin (63.63%). However, the isolates were highly sensitive to chloramphenicol (90.90%), followed by tetracycline (63.63%), and ampicillin (54.54%). Conclusion: This study showed that M. haemolytica and P. multocida are the common causes of mannheimiosis and pasteurellosis in small ruminants, respectively, and isolates were resistant to commonly used antibiotics in the study area. Thus, an integrated vaccination strategy, antimicrobial resistance monitoring, and avoidance of stress-inducing factors are recommended.

Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis.

Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a bfaA gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. Mycobacterium avium subspecies paratuberculosis (Map) growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including Mycobacterium smegmatis. While Map C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type Map strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by bfaB and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the bfaA gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type Map bfaA knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of Map and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for Map.IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the Mycobacterium genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for Mycobacterium tuberculosis. This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, Map strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of Mycobacterium avium subspecies paratuberculosis produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of Map. This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid.

Bacteriological and serological investigation of Clostridium perfringens in lambs.

Clostridium perfringens is one of the most common and important pathogens in livestock due to its ability to produce a diverse arsenal of toxins. Owing to C. perfringens economic importance, this study aimed to determine the types and toxins of C. perfringens in newly born lambs. A total of 200 lambs of less than one-month old were examined, including 100 lambs suffered from diarrhea, 60 freshly dead and 40 apparent healthy. C. perfringens was identified morphologically and biochemically using bacteriological culture in 103 of 200 samples (51.5%). Moreover, serological typing of C. perfringens isolates revealed three serotypes, C. perfringens type A (54.2%), C. perfringens type B (28.8%) and C. perfringens type D (16.9%). The highest prevalence rate for C. perfringens infection was observed in winter (58.25%) in comparison with other seasons. The findings of the present study confirm the presence of enterotoxmia among lambs in localities under study, causing economic losses. The proper vaccination schedule particularly against C. perfringens type A and B is highly recommended.

Risk factors of Shiga toxin-producing Escherichia coli in livestock raised on diversified small-scale farms in California.

The increasing number of diversified small-scale farms (DSSF) that raise outdoor-based livestock in the USA reflects growing consumer demand for sustainably produced food. Diversified farms are small scale and raise a combination of multiple livestock species and numerous produce varieties. This 2015-2016 cross-sectional study aimed to describe the unique characteristics of DSSF in California, estimate the prevalence of Shiga toxin-producing Escherichia coli (STEC) in livestock and evaluate the association between risk factors and the presence of STEC in livestock, using generalised linear mixed models. STEC prevalence was 13.62% (76/558). Significant variables in the mixed-effect logistic regression model included daily maximum temperature (OR 0.95; CI95% 0.91-0.98), livestock sample source (cattle (OR 4.61; CI95% 1.64-12.96) and sheep (OR 5.29; CI95% 1.80-15.51)), multiple species sharing the same barn (OR 6.23; CI95% 1.84-21.15) and livestock having contact with wild areas (OR 3.63; CI95% 1.37-9.62). Identification of STEC serogroups of public health concern (e.g. O157:H7, O26, O103) in this study indicated the need for mitigation strategies to ensure food safety by evaluating risk factors and management practices that contribute to the spread and prevalence of foodborne pathogens in a pre-harvest environment on DSSF.

Fibrolytic rumen bacteria of camel and sheep and their applications in the bioconversion of barley straw to soluble sugars for biofuel production.

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.

Effects of diets with various levels of forage rape (Brassica napus) on growth performance, carcass traits, meat quality and rumen microbiota of Hu lambs.

BACKGROUND: Apart from being an oil crop, forage rape (Brassica napus) can be used to feed ruminants. The objective of this study was to investigate the effects of pelleted total mixed ration (TMR) diets with various levels of forage rape on growth performance, carcass traits, meat quality, meat nutritional value and rumen microbiota of Hu lambs, which was important for the efficient utilization of forage rape and alleviating the shortage of high-quality forage in China. RESULTS: Lambs fed on diets with 200-400 g kg-1 forage rape had greater average daily gain (ADG) and lower feed conversion ratio (FCR) than those fed on diets with 0-100 g kg-1 of forage rape (P < 0.05). As dietary forage rape levels increased, the content of intramuscular α-linolenic acid and a variety of amino acids in the muscle increased linearly (P < 0.05). No difference was found in carcass traits or meat quality among the dietary treatments (P > 0.05). However, the inclusion of forage rape increased the relative abundance of cellulolytic bacteria and short-chain fatty acid producers, including Succiniclasticum, Fibrobacter and members of the Lachnospiraceae. Besides, Succiniclasticum was found to be positively correlated with the final body weight of lambs. CONCLUSION: TMR diets that included 200-400 g kg-1 forage rape could improve the growth performance of lambs, and elevated the content of intramuscular α-linolenic acid and a variety of amino acids in the muscle, accompanied by increased abundance of cellulolytic bacteria in the rumen.

When to suspect contamination rather than colonization - lessons from a putative fetal sheep microbiome.

There is an ongoing controversy around the existence of a prenatal, fetal microbiome in humans, livestock, and other animals. The 'in utero microbial colonization' hypothesis challenges the clinical paradigm of the 'sterile womb' but has been criticized for its reliance on DNA-based evidence to detect microbiomes and the failure to conciliate the routine experimental derivation of germ-free animals from surgically resected embryos with a thriving fetal microbiome. In order to avoid the propagation of misinformation in the scientific literature, a critical assessment and careful review of newly published studies, particularly those that challenge the convincing current clinical dogma of the sterile womb, is of critical importance.We read with interest a recent publication that postulated the presence of a fetal microbiome in sheep, but questioned the plausibility of the reported findings and their meaningfulness to prove "microbial colonisation of the fetal gut […] in utero". We reanalyzed the published metagenomic and metatranscriptomic sequence data from the original publication and identified evidence for different types of contamination that affected all samples alike and could explain the reported findings without requiring the existence of a fetal microbiome.Our reanalysis challenges the reported findings as supportive of a prenatal fetal lamb microbiome. The shortcomings of the original analysis and data interpretation highlight common problems of low-biomass microbiome projects. We propose genomic independence of separate biological samples, i.e. distinctive profiles at the microbial strain level, as a potential new microbiome marker to increase confidence in metagenomics analyses of controversial low-biomass microbiomes.

Sporadic Dissemination of tet(X3) and tet(X6) Mediated by Highly Diverse Plasmidomes among Livestock-Associated Acinetobacter.

The emergence of high-level tigecycline resistance mediated by plasmid-borne tet(X) genes greatly threatens the clinical effectiveness of tigecycline. However, the dissemination pattern of plasmid-borne tet(X) genes remains unclear. We here recovered tet(X)-positive Acinetobacter isolates from 684 fecal and environmental samples collected at six livestock farms. Fifteen tet(X)-positive Acinetobacter isolates were identified, mainly including 9 tet(X3)- and 5 tet(X6)-positive Acinetobacter towneri isolates. A clonal dissemination of tet(X3)-positive A. towneri was detected in a swine farm, while the tet(X6)-positive A. towneri isolates mainly disseminated sporadically in the same farm. A tet(X3)-carrying plasmid (pAT181) was self-transmissible from a tigecycline-susceptible A. towneri strain to Acinetobacter baumannii strain ATCC 17978, causing 64- to 512-fold increases in the MIC values of tetracyclines (including tigecycline). Worrisomely, pAT181 was stably maintained and increased the growth rate of strain ATCC 17978. Further identification of tet(X) genes in 10,680 Acinetobacter genomes retrieved from GenBank revealed that tet(X3) (n = 249), tet(X5)-like (n = 61), and tet(X6) (n = 53) were the prevalent alleles mainly carried by four species, and most of them were livestock associated. Phylogenetic analysis showed that most of the tet(X3)- and tet(X6)-positive isolates disseminated sporadically. The structures of the tet(X3), and tet(X6) plasmidomes were highly diverse, and no epidemic plasmids were detected. However, cross-species and cross-region transmissions of tet(X3) might have been mediated by several plasmids in a small proportion of strains. Our study implies that horizontal plasmid transfer may be insignificant for the current dissemination of tet(X3) and tet(X6) in Acinetobacter strains. Continuous surveillance for tet(X) genes in the context of One Health is necessary to prevent them from transmitting to humans. IMPORTANCE Recently identified plasmid-borne tet(X) genes have greatly challenged the efficiency of tigecycline, a last-resort antibiotic for severe infection, while the dissemination pattern of the plasmid-borne tet(X) genes remains unclear. In this study, we identified a clonal dissemination of tet(X3)-positive A. towneri isolates on a swine farm, while the tet(X6)-positive A. towneri strains mainly disseminated sporadically on the same farm. Of more concern, a tet(X3)-carrying plasmid was found to be self-transmissible, resulting in enhanced tigecycline resistance and growth rate of the recipient. Further exploration of a global data set of tet(X)-positive Acinetobacter genomes retrieved from GenBank revealed that most of the tet(X3)- and tet(X6)-positive isolates shared a highly distant relationship, and the structures of tet(X3) and tet(X6) plasmidomes exhibited high mosaicism. Notably, some of the isolates belong to Acinetobacter species that are opportunistic pathogens and have been identified as sources of nosocomial infections, raising concerns about transmission to humans in the future. Our study evidenced the sporadic dissemination of tet(X3) and tet(X6) in Acinetobacter strains and the necessity of continuous surveillance for tet(X) genes in the context of One Health.

Serological detection of Mycobacterium Tuberculosis complex infection in multiple hosts by One Universal ELISA.

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.

Prevalence and Whole-Genome Sequence-Based Analysis of Shiga Toxin-Producing Escherichia coli Isolates from the Recto-Anal Junction of Slaughter-Age Irish Sheep.

Shiga toxin-producing Escherichia coli (STEC) organisms are a diverse group of pathogenic bacteria capable of causing serious human illness, and serogroups O157 and O26 are frequently implicated in human disease. Ruminant hosts are the primary STEC reservoir, and small ruminants are important contributors to STEC transmission. This study investigated the prevalence, serotypes, and shedding dynamics of STEC, including the supershedding of serogroups O157 and O26, in Irish sheep. Recto-anal mucosal swab samples (n = 840) were collected over 24 months from two ovine slaughtering facilities. Samples were plated on selective agars and were quantitatively and qualitatively assessed via real-time PCR (RT-PCR) for Shiga toxin prevalence and serogroup. A subset of STEC isolates (n = 199) were selected for whole-genome sequencing and analyzed in silico. In total, 704/840 (83.8%) swab samples were Shiga toxin positive following RT-PCR screening, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were shedding STEC O157, and three of these were identified as supershedders. No STEC O26 was isolated. Post hoc statistical analysis showed that younger animals are more likely to harbor STEC and that STEC carriage is most prevalent during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, 15 of which were not yet reported for sheep. Serotype O91:H14 was the most frequently reported. Eight Shiga toxin gene variants were reported, two stx1 and six stx2, and three novel Shiga-toxin subunit combinations were observed. Variant stx1c was the most prevalent, while many strains also harbored stx2b. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) bacteria are foodborne, zoonotic pathogens of significant public health concern. All STEC organisms harbor stx, a critical virulence determinant, but it is not expressed in most serotypes. Sheep shed the pathogen via fecal excretion and are increasingly recognized as important contributors to the dissemination of STEC. In this study, we have found that there is high prevalence of STEC circulating within sheep and that prevalence is related to animal age and seasonality. Further, sheep harbor a variety of non-O157 STEC, whose prevalence and contribution to human disease have been underinvestigated for many years. A variety of Stx variants were also observed, some of which are of high clinical importance.

Serological investigation and genotyping of Mycobacterium avium subsp. paratuberculosis in sheep and goats in Inner Mongolia, China.

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.

Pharmacokinetics of tulathromycin in pregnant ewes (Ovis aries) challenged with Campylobacter jejuni.

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.

Comparative Analysis of the Microbiota Between Rumen and Duodenum of Twin Lambs Based on Diets of Ceratoides or Alfalfa.

In our previous study, diet directly impacted the microbiota of the rumen in twin lambs. The duodenum is the first part of the small intestine, so we seek to determine whether there is a difference in the digesta between the two feed groups HFLP (high fiber, low protein) and LFHP (low fiber, high protein), and its impact on the biodiversity and metabolism of the duodenum. Results showed that the number of Operational Taxonomic Units (OTUs) in the duodenum (2,373 OTUs) was more than those in the rumen (1,230 OTUs), and 143 OTUs were significantly different in the duodenum between the two groups. The two most predominant phyla were Bacteriodetes and Firmicutes, but this ratio was reversed between the rumen and duodenum of lambs fed different feedstuffs. The difference in the digesta that greatly changed the biodiversity of the rumen and duodenum could affect the microbial community in the gastrointestinal tract (GIT). Sixteen metabolites were significantly different in the duodenum between the two groups based on the metabolome analysis. The relationships were built between the microbiome and the metabolome based on the correlation analysis. Some metabolites have a potential role in influencing meat quality, which indicated that the diet could affect the microbiota community and finally change meat quality. This study could explain how the diet affects the rumen and duodenum's microbiota, lay a theoretical basis for controlling feed intake, and determine the relationship between the duodenum's microbiota and metabolism.

Cocultivation of Anaerobic Fungi with Rumen Bacteria Establishes an Antagonistic Relationship.

Anaerobic gut fungi (Neocallimastigomycetes) live in the digestive tract of large herbivores, where they are vastly outnumbered by bacteria. It has been suggested that anaerobic fungi challenge growth of bacteria owing to the wealth of biosynthetic genes in fungal genomes, although this relationship has not been experimentally tested. Here, we cocultivated the rumen bacteria Fibrobacter succinogenes strain UWB7 with the anaerobic gut fungi Anaeromyces robustus or Caecomyces churrovis on a range of carbon substrates and quantified the bacterial and fungal transcriptomic response. Synthetic cocultures were established for at least 24 h, as verified by active fungal and bacterial transcription. A. robustus upregulated components of its secondary metabolism in the presence of Fibrobacter succinogenes strain UWB7, including six nonribosomal peptide synthetases, one polyketide synthase-like enzyme, and five polyketide synthesis O-type methyltransferases. Both A. robustus and C. churrovis cocultures upregulated S-adenosyl-l-methionine (SAM)-dependent methyltransferases, histone methyltransferases, and an acetyltransferase. Fungal histone 3 lysine 27 trimethylation marks were more abundant in coculture, and heterochromatin protein-1 was downregulated. Together, these findings suggest that fungal chromatin remodeling occurs when bacteria are present. F. succinogenes strain UWB7 upregulated four genes in coculture encoding drug efflux pumps, which likely protect the cell against toxins. Furthermore, untargeted nonpolar metabolomics data revealed at least one novel fungal metabolite enriched in coculture, which may be a defense compound. Taken together, these data suggest that A. robustus and C. churrovis produce antimicrobials when exposed to rumen bacteria and, more broadly, that anaerobic gut fungi are a source of novel antibiotics. IMPORTANCE Anaerobic fungi are outnumbered by bacteria by 4 orders of magnitude in the herbivore rumen. Despite their numerical disadvantage, they are resilient members of the rumen microbiome. Previous studies mining the genomes of anaerobic fungi identified genes encoding enzymes to produce natural products, which are small molecules that are often antimicrobials. In this work, we cocultured the anaerobic fungus Anaeromyces robustus or Caecomyes churrovis with rumen bacteria Fibrobacter succinogenes strain UWB7 and sequenced fungal and bacterial active genes via transcriptome sequencing (RNA-seq). Consistent with production of a fungal defense compound, bacteria upregulated genes encoding drug efflux pumps, which often export toxic molecules, and fungi upregulated genes encoding biosynthetic enzymes of natural products. Furthermore, tandem mass spectrometry detected an unknown fungal metabolite enriched in the coculture. Together, these findings point to an antagonistic relationship between anaerobic fungi and rumen bacteria resulting in the production of a fungal compound with potential antimicrobial activity.

Antimicrobial Peptide Modifications against Clinically Isolated Antibiotic-Resistant Salmonella.

Antimicrobial peptides are promising molecules to address the global antibiotic resistance problem, however, optimization to achieve favorable potency and safety is required. Here, a peptide-template modification approach was employed to design physicochemical variants based on net charge, hydrophobicity, enantiomer, and terminal group. All variants of the scorpion venom peptide BmKn-2 with amphipathic α-helical cationic structure exhibited an increased antibacterial potency when evaluated against multidrug-resistant Salmonella isolates at a MIC range of 4-8 µM. They revealed antibiofilm activity in a dose-dependent manner. Sheep red blood cells were used to evaluate hemolytic and cell selectivity properties. Peptide Kn2-5R-NH2, dKn2-5R-NH2, and 2F-Kn2-5R-NH2 (variants with +6 charges carrying amidated C-terminus) showed stronger antibacterial activity than Kn2-5R (a variant with +5 charges bearing free-carboxyl group at C-terminus). Peptide dKn2-5R-NH2 (d-enantiomer) exhibited slightly weaker antibacterial activity with much less hemolytic activity (higher hemolytic concentration 50) than Kn2-5R-NH2 (l-enantiomer). Furthermore, peptide Kn2-5R with the least hydrophobicity had the lowest hemolytic activity and showed the highest specificity to Salmonella (the highest selectivity index). This study also explained the relationship of peptide physicochemical properties and bioactivities that would fulfill and accelerate progress in peptide antibiotic research and development.

Whole genome sequencing reveals possible host species adaptation of Streptococcus dysgalactiae.

Streptococcus dysgalactiae (SD) is an emerging pathogen in human and veterinary medicine, and is associated with several host species, disease phenotypes and virulence mechanisms. SD has traditionally been divided into the subspecies dysgalactiae (SDSD) and subsp. equisimilis (SDSE), but recent molecular studies have indicated that the phylogenetic relationships are more complex. Moreover, the genetic basis for the niche versatility of SD has not been extensively investigated. To expand the knowledge about virulence factors, phylogenetic relationships and host-adaptation strategies of SD, we analyzed 78 SDSD genomes from cows and sheep, and 78 SDSE genomes from other host species. Sixty SDSD and 40 SDSE genomes were newly sequenced in this study. Phylogenetic analysis supported SDSD as a distinct taxonomic entity, presenting a mean value of the average nucleotide identity of 99%. Bovine and ovine associated SDSD isolates clustered separately on pangenome analysis, but no single gene or genetic region was uniquely associated with host species. In contrast, SDSE isolates were more heterogenous and could be delineated in accordance with host. Although phylogenetic clustering suggestive of cross species transmission was observed, we predominantly detected a host restricted distribution of the SD-lineages. Furthermore, lineage specific virulence factors were detected, several of them located in proximity to hotspots for integration of mobile genetic elements. Our study indicates that SD has evolved to adapt to several different host species and infers a potential role of horizontal genetic transfer in niche specialization.

Dental biofilm and its ecological interrelationships in ovine periodontitis.

Introduction. Periodontitis, one of the most common oral disorders in sheep, is caused by a mixed and opportunistic microbiota that severely affects the health and welfare of animals. However, little is known about the ecological processes involved and the composition of the microbiota associated with the development of the disease.Hypothesis/Gap Statement. Using high-throughput sequencing of the 16S ribosomal RNA gene and network analysis it would be possible to discriminate the microbiomes of clinically healthy sheep and those with periodontitis and possibly identify the key microorganisms associated with the disease.Aim. The present study aimed to characterise the composition of dental microbiomes and bacterial co-occurrence networks in clinically healthy sheep and animals with periodontitis.Methodology. Dental biofilm samples were collected from ten sheep with periodontitis and ten clinically healthy animals. Bacteria were identified using high-throughput sequencing of the 16S ribosomal RNA gene.Results. The most prevalent genera in the dental microbiota of sheep with periodontitis were Petrimonas, Acinetobacter, Porphyromonas and Aerococcus. In clinically healthy animals, the most significant genera were unclassified Pasteurellaceae, Pseudomonas, and Neisseria. Fusobacterium was found at high prevalence in the microbiomes of both groups. The dental microbiota of sheep in the two clinical conditions presented different profiles and the diversity and richness of bacteria was greater in the diseased animals. Network analyses showed the presence of a large number of antagonistic interactions between bacteria in the dental microbiota of animals with periodontitis, indicating the occurrence of a dysbiotic community. Through the interrelationships, members of the Prevotella genus are likely to be key pathogens, both in the dental microbiota of healthy animals and those with periodontitis. Porphyromonas stood out among the top three nodes with more centrality and the largest number of hubs in the networks of animals with periodontitis.Conclusion. The dental biofilm microbiota associated with ovine periodontitis is dysbiotic and with significant antagonistic interactions, which discriminates healthy animals from diseased animals and highlights the importance of key bacteria, such as Petrimonas, Porphyromonas, Prevotella and Fusobacterium species.

Analysis of environmental dust in goat and sheep farms to assess Coxiella burnetii infection in a Q fever endemic area: Geographical distribution, relationship with human cases and genotypes.

Real-time PCR analysis of environmental samples (dust and aerosols) is an easy tool to investigate the presence of Coxiella burnetii in the farm environment. The aim of this study was to assess the distribution of C. burnetii DNA in dust collected inside animal premises from 272 small ruminant farms in Bizkaia (northern Spain), a region with recent reports of human Q fever cases and outbreaks. Within each farm, 5 samples of dust were collected from difference surfaces, and data on animal census, management procedures, characteristics of the premises and geographic location were collected. Real-time PCR analysis of the dust samples detected presence of C. burnetii DNA in 98 farms (36.0%), flock-prevalence being higher in sheep (38.9%) or mixed ovine-caprine production systems (36.8%), compared to goats (25.0%). Larger bacterial burdens were observed in mixed farms, compared to sheep (p < .05). Single nucleotide polymorphism (SNP) analysis identified 5 different genotypes, with SNP8 being the predominant genotype (73%), followed by SNP6 (11%), SNP2 (9%), SNP4 (5%) and SNP1 (2%). Proportion of farms where C. burnetii DNA was detected differed among the different agricultural counties, and a higher proportion of C. burnetii DNA positive farms was associated with the occurrence of recent human Q fever outbreaks at several geographical locations. Dust sampling in domestic ruminant farms coupled with real-time PCR to screen for the presence of C. burnetii and estimate bacterial load can be a useful tool to identify herds and regions with high prevalence, define priority actions and monitor the effect of control measures. If combined with molecular genotyping and spatial distribution maps, it can help to identify farm contamination sources and trace the origin of human outbreaks.

A Trifecta of New Insights into Ovine Footrot for Infection Drivers, Immune Response, and Host-Pathogen Interactions.

Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industry's largest welfare problems. The complex etiology of footrot makes in situ or in vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved and how they may interact with the host, ultimately providing a way to identify targets for future hypothesis-driven investigative work. Here, we present the first combined global analysis of bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intratissue and surface bacterial populations and the most abundant bacterial transcriptomes were analyzed, demonstrating that footrot-affected skin has reduced diversity and increased abundances of not only the causative bacterium Dichelobacter nodosus but also other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica. Host transcriptomics reveals the suppression of biological processes related to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot-affected interdigital skin compared to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome, which could lead to the identification of future therapeutic targets.