Assessment of fecal bacterial viability and diversity in fresh and frozen fecal microbiota transplant (FMT) product in horses.

BACKGROUND: Currently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region. RESULTS: The largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities. CONCLUSIONS: Frozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome.

The equine hindgut as a reservoir of mobile genetic elements and antimicrobial resistance genes.

Antibiotic resistance in bacterial pathogens is a growing problem for both human and veterinary medicine. Mobile genetic elements (MGEs) such as plasmids, transposons, and integrons enable the spread of antibiotic resistance genes (ARGs) among bacteria, and the overuse of antibiotics drives this process by providing the selection pressure for resistance genes to establish and persist in bacterial populations. Because bacteria, MGEs, and resistance genes can readily spread between different ecological compartments (e.g. soil, plants, animals, humans, wastewater), a "One Health" approach is needed to combat this problem. The equine hindgut is an understudied but potentially significant reservoir of ARGs and MGEs, since horses have close contact with humans, their manure is used in agriculture, they have a dense microbiome of both bacteria and fungi, and many antimicrobials used for equine treatment are also used in human medicine. Here, we collate information to date about resistance genes, plasmids, and class 1 integrons from equine-derived bacteria, we discuss why the equine hindgut deserves increased attention as a potential reservoir of ARGs, and we suggest ways to minimize the selection for ARGs in horses, in order to prevent their spread to the wider community.

Devosia equisanguinis sp. nov., isolated from horse blood.

A Gram-stain-negative, aerobic, non-endospore-forming organism isolated from horse blood was studied for its taxonomic allocation. On the basis of 16S rRNA gene sequence similarity comparisons, strain M6-77T grouped within the genus Devosia and was most closely related to Devosia elaeis (97.6 %) and Devosia indica (97.55 %). The 16S rRNA gene sequence similarity to type strains of other Devosia species was below 97.5 %. The average nucleotide identity and digital DNA-DNA hybridization values between the M6-77T genome assembly and those of the closest relative Devosia type strains were <85 and <25 %, respectively. Strain M6-77T grew optimally at 25-37 °C (range: 10-36 °C), at a pH range of pH 6.5-10.5 and in the presence of up to 3 % (w/v) NaCl. The fatty acid profile from whole-cell hydrolysates supported the allocation of the strain to the genus Devosia. Major fatty acids were C18 : 1 ω7c, 11-methyl C18 : 1 ω7c and C16 : 0. The quinone system consisted exclusively of ubiquinone Q-10. The polar lipid profile was composed of the major lipids diphosphatidylglycerol, phosphatidylglycerol and three unidentified glycolipids. In the polyamine pattern, putrescine was predominant and spermidine was detected in moderate amounts. The diamino acid of the peptidoglycan was meso-diaminopimelic acid. In addition, the results of physiological and biochemical tests also allowed phenotypic differentiation of strain M6-77T from the closely related species. Hence, M6-77T represents a new species of the genus Devosia, for which we propose the name Devosia equisanguinis sp. nov., with M6-77T (=CIP 111628T=LMG 30659T=CCM 8868T) as the type strain.

Effects of steam-flaked grains on foals' growth and faecal microbiota.

BACKGROUND: There is little objective information concerning the effect of steam-flaked grains on foal's growth performance and faecal microbiota. To determine the effects of steam-flaked grains on foal's growth performance and faecal microbiota, faecal samples were collection from 18 foals which had been fed either corn, oat or barley diets over the 60 days of the experiment. Body weight and conformation measurements were collected. Next-generation sequencing of the V3 + V4 region of the 16 S rRNA gene was used to assess the microbial composition of faeces. Alpha diversity, Venn graph, Relative abundance and beta diversity are presented. RESULTS: There was a significantly higher larger increase in the body weight of those foals fed barley compared to either corn or oats. There were also significant changes in the Alpha diversity of the gut microbiota. The Shannon and Simpson indices were significantly higher in the barley fed group than those fed corn or oats. The Chao1 index was significantly higher in the oat fed group than the corn or barley fed groups. There were significant changes in the relative abundance of bacteria in the microbiota in terms of phylum, family and genus. The histogram of LDA value distribution showed that the 12 statistically different biomarkers of the bacteria were present. Tax4Fun function annotation clustering heat map showed that functional information was detected from 26 species of bacteria in faecal samples from the foals. CONCLUSIONS: Differences by starch sources were found in overall growth of the foals and in the faecal microbiota if either supplementary corn, oat or barley was fed. Further studies are required to determine the potential impact of the changes in the microbiota on the health and development of foals fed cereal starch of different sources.

Assessing the role of livestock and sympatric wild ruminants in spreading antimicrobial resistant Campylobacter and Salmonella in alpine ecosystems.

BACKGROUND: Livestock play an important role as reservoir of enteric pathogens and antimicrobial resistance (AMR), a health and economic concern worldwide. However, little is known regarding the transmission and maintenance of these pathogens at the wildlife-livestock interface. In this study, we assessed the occurrence, genetic diversity and AMR of Campylobacter spp. and Salmonella spp. shed by sympatric free-ranging livestock and a wild herbivore in an alpine ecosystem. RESULTS: Campylobacter spp. was isolated from 23.3 % of cattle and 7.7 % of sheep but was not isolated from horses nor Pyrenean chamois (Rupicapra pyrenaica). Campylobacter jejuni was the most frequent species. A high genetic diversity and certain host specificity of C. jejuni isolates was observed. The main AMR detected in Campylobacter isolates was to nalidixic acid (88.2 %), ciprofloxacin (82.4 %) and tetracycline (82.4 %); only 11.7 % of the isolates were pan-susceptible and 17.6 % were multi-resistant. Salmonella ser. Newport was isolated only from one Pyrenean chamois and was pan-susceptible. CONCLUSIONS: Results show that free-ranging cattle and sheep are spreaders of Campylobacter as well as their AMR strains in the alpine environment. Therefore, contaminated alpine pastures or streams may constitute a source for the dissemination of AMR enteropathogens. However, apparently, alpine wild ungulates such as Pyrenean chamois play a negligible role in the epidemiology of zoonotic enteropathogens and AMR, and are not potential bioindicators of the burden of alpine environments.

Molecular characterization and antimicrobial resistance of potentially human-pathogenic Escherichia coli strains isolated from riding horses.

BACKGROUND: Transmission of antimicrobial resistant and virulent Escherichia coli (E. coli) from animal to human has been considered as a public health concern. This study aimed to determine the phylogenetic background and prevalence of diarrheagenic E. coli and antimicrobial resistance in healthy riding-horses in Iran. In this research, the genes related to six main pathotypes of E. coli were screened. Also, genotypic and phenotypic antimicrobial resistance against commonly used antibiotics were studied, then phylo-grouping was performed on all the isolates. RESULTS: Out of 65 analyzed isolates, 29.23 % (n = 19) were determined as STEC and 6.15 % (n = 4) as potential EPEC. The most prevalent antimicrobial resistance phenotypes were against amoxicillin/clavulanic acid (46.2 %) and ceftriaxone (38.5 %). blaTEM was the most detected resistance gene (98.4 %) among the isolates and 26.15 % of the E. coli isolates were determined as multi-drug resistant (MDR). Three phylo-types including B1 (76.92 %), A (13.85 %) and D (3.08 %) were detected among the isolates. CONCLUSIONS: Due to the close interaction of horses and humans, these findings would place emphasis on the pathogenic and zoonotic potential of the equine strains and may help to design antimicrobial resistance stewardship programs to control the dissemination of virulent and multi-drug resistant E. coli strains in the community.

Molecular detection and identification of spotted fever group rickettsiae in ticks collected from horses in Cuba.

Spotted fever group (SFG) rickettsiae are obligatory intracellular bacteria that cause disease in humans and other animals. Ixodid ticks are the principal vectors of SFG rickettsiae. The present study aimed to determine the prevalence and species identity of SFG rickettsiae in ticks and horses from urban and rural areas of western Cuba using PCR assays. Tick samples, collected from 79 horses, consisted of 14 Amblyomma mixtum adults, 111 Dermacentor nitens adults and 19 pools of D. nitens nymphs (2-5 individuals/pool). The PCR results revealed the presence of Rickettsia spp. in 64% of the A. mixtum adults, 16% of the D. nitens adults, and 11% of the pooled samples of D. nitens nymphs. In contrast, Rickettsia spp. was not detected in any of the 200 horse blood samples included in this study. DNA sequence data of the rickettsial 17 kDa antigen gene showed that Rickettsia amblyommatis was present in A. mixtum; and Rickettsia felis in D. nitens. This is the first report of R. felis in D. nitens in Cuba. The present study extends our knowledge of the potential vector spectrum and distribution of SFG rickettsiae pathogens in western Cuba.

Diversity and genotypic analysis of tick-borne pathogens carried by ticks infesting horses in Korea.

Ticks account for an extensive range of health and welfare issues in horses. In addition, tick-borne pathogens (TBPs) limit global animal trading and equine sporting events. Here, we assess the prevalence, co-infectivity and risk factors of TBPs in horse ticks in Korea. A total of 245 hard ticks, including 103 male and 142 female adults, were obtained from horses on Jeju Island during the spring to autumn seasons of 2013-2019. All collected ticks were identified as adult Haemaphysalis longicornis. We screened and analyzed each tick for the presence of several TBPs by polymerase chain reaction (PCR) analysis. Among the 245 ticks, we detected genes for three TBPs, Candidatus Rickettsia longicornii (22.9%), Ehrlichia canis (0.4%) and Theileria luwenshuni (0.4%), while Anaplasma spp. was not detected. TBPs were most prevalent in ticks harvested during the autumn season, and more abundant in the female than male adults. This is the first report of the genera Ehrlichia, Rickettsia and Theileria in horse ticks in Korea. TBPs in horse ticks are likely a reservoir for zoonotic transmission to other animals, including humans. Our findings demonstrate the need for further understanding of the prevalence and epidemiology of TBPs in wild and domestic animals.

Characterization of a new bacteriocin-like inhibitory peptide produced by Lactobacillus sakei B-RKM 0559.

The biopreservation strategy allows extending the shelf life and food safety through the use of indigenous or controlled microbiota and their antimicrobial compounds. The aim of this work was to characterize an inhibitory substance with bacteriocin-like activity (Sak-59) produced by the potentially probiotic L. sakei strain from artisanal traditional Kazakh horse meat product Kazy. The maximum production of Sak-59 occurred at the stationary phase of the L. sakei growth. Sak-59 showed inhibitory activity against gram-positive meat spoilage bacteria strains of Listeria monocytogenes, Staphylococcus aureus, and pathogenic gram-negative bacteria strains of Serratia marcescens and Escherichia coli, but not against the tested Lactobacilli strains. Sak-59 activity, as measured by diffusion assay in agar wells, was completely suppressed after treatment with proteolytic enzymes and remained stable after treatment with α-amylase and lipase, indicating that Sak-59 is a peptide and most likely not glycosylated or lipidated. It was concluded that Sak-59 is a potential new bacteriocin with a characteristic activity spectrum, which can be useful in the food and feed industries.

Identification of a mecA/mecC-positive MRSA ST1-t127 isolate from a racehorse in Japan.

OBJECTIVES: MRSA is a known pathogen that affects horses. We investigated an equine MRSA isolate for potential antimicrobial resistance genes, classified the staphylococcal cassette chromosome mec (SCCmec) and identified the strain-specific dissemination in the horse community based on WGS. METHODS: WGS, using short-read sequencing, and subsequent long-read sequencing by hybrid assembly, was conducted to obtain a complete genome sequence. Pairwise sequence alignment of relative SCCmec sequences and core-genome phylogenetic analysis were performed to highlight transmission routes of the SCCmec and MRSA strain-specific lineages. RESULTS: In 2018, we isolated the MRSA JRA307 strain from the pus of a wound on a racehorse and the complete genome sequence suggests that it is a clinically relevant pvl-negative ST1-t127 MRSA that harbours both mecA and mecC on SCCmec-307. SCCmec-307 exhibited marked sequence identity to the previously reported SCCmec-mecC in the Staphylococcus sciuri GVGS2 strain isolated from cattle. The JRA307 mecC gene was classified as a mecC allotype of S. sciuri rather than that of Staphylococcus aureus. CONCLUSIONS: We demonstrated the complete genome sequence of equine isolate JRA307, which is a clinically relevant MRSA harbouring mecA and mecC on SCCmec-307. The finding of mecC MRSA suggests a possible SCCmec transmission between distinct staphylococcal species. To the best of our knowledge, this is the first report of mecC detection in Japan.

Serological evidence of leptospirosis in Iran; A systematic review and meta-analysis.

Leptospirosis is a neglected infectious zoonotic disease that affects domestic animals and wildlife as well as humans. Although leptospirosis is known as an endemic disease in Iran, there is no accurate information on the overall prevalence of this disease in humans and animals. The aim of this systematic review and meta-analysis was to estimate the seroprevalence of leptospirosis among human and domestic and wild animals in Iran. A systematic review of English and Persian articles (since 1998 to December 2017) was conducted using Google Scholar, Medline/PubMed, Science Direct, Scopus, Web of science and Iranian databases Iranmedex, Scientific Information Database (SID), Magiran, and IRANDOC. Search terms include leptospirosis, Leptospira, serology, seroprevalence, seroepidemiology, serological, Iran, cow, goat, sheep, camel, dog, cat, equine, donkey, horse, mule and rodent. In Eventually 66 articles were selected to analyze based on inclusion criteria. Seroprevalence of leptospirosis in human was 27.84% (95% CI: 13.22-22.47) and 19.71% (95% CI: 6.78-32.65%) based on ELISA and MAT, respectively. The pooled prevalence of leptospirosis in cow, sheep, goat and camel was 26.62% (95% CI: 18.76-34.48), 17.38% (95% CI: 13.32-21.43), 12.18% (95% CI: 9.96-14.41) and 22.68% (95% CI: 18.97-26.40), respectively. The prevalence of leptospirosis in horse, donkey, and mule was 19.99% (95% CI: 13.32-26.68), 40.59% (95% CI: 33.20-47.97) and 9.10% (95% CI: 2.90-15.30), respectively. The prevalence in dog and cat were estimated 14.63% (95% CI: 3.49-25.77) and 14.44% (95% CI: 3.25-25.65), respectively. The prevalence of seropositivity in rodents was estimated 20.96% (95% CI: 10.62-31.30). This study is a very comprehensive report on the status of leptospirosis in Iran. Based on our results, leptospirosis has considerable seroprevalence among human and animals in Iran. This high seroprevalence of leptospirosis showed should be given more attention for this disease in Iran and thus health measures must be taken to diagnosis, control and prevent it.

Acholeplasma equirhinis sp. nov. isolated from respiratory tract of horse (Equus caballus) and Mycoplasma procyoni sp. nov. isolated from oral cavity of raccoon (Procyon lotor).

We describe two novel species of Acholeplasma sp. strain N93 and Mycoplasma sp. strain LR5794 which were isolated from the nasopharynx of a horse from the United Kingdom and from the oral cavity of a North American raccoon from Canada, respectively. These strains were phenotypically and genetically characterized and compared to other established Mycoplasma and Acholeplasma species. Both strains are facultative anaerobes, resistant to penicillin, and produce acid from glucose but do not hydrolyze arginine and urea. Both strains grew well in microaerophilic and anaerobic atmospheric conditions at 35-37 °C using PPLO (pleuropneumonia-like organisms) medium. Acholeplasma sp. N93 does not require serum for growth. Colonies of both strains showed a typical fried-egg appearance and transmission electron microscopy of bacterial cells revealed a typical mycoplasma cellular morphology. Molecular characterization included assessment of several genetic loci. The genetic analysis indicated that Acholeplasma sp. N93 and Mycoplasma sp. LR5794 were most closely related to A. hippikon and A. equifetale, and M. molare and M. lagogenitalium, respectively. However, both novel strains were genetically unique in comparison to other well-known Mycoplasma and Acholeplasma species. Based on the isolation source history, phenotypic, genotypic, and phylogenetic characteristics of these novel strains, we propose the name Acholeplasma equirhinis sp. nov. for Acholeplasma sp. isolated from the nasopharynx of a horse [the type strain is N93T (= DSM 106692T = ATCC TSD-139T = NCTC 14351T)], and the name Mycoplasma procyoni sp. nov. for the Mycoplasma sp. isolated from the oral cavity of a North American raccoon [the type strain is LR5794T (= DSM 106703T = ATCC TSD-141T = NCTC 14309T)].

The equine gastrointestinal microbiome: impacts of weight-loss.

BACKGROUND: Obesity is an important equine welfare issue. Whilst dietary restriction is the most effective weight-loss tool, individual animals range in their weight-loss propensity. Gastrointestinal-derived bacteria play a fundamental role in host-health and have been associated with obesity and weight-loss in other species. This study evaluated the faecal microbiome (next-generation sequencing of 16S rRNA genes) of 15 obese Welsh Mountain pony mares, in the same 11-week period across 2 years (n = 8 Year 1; n = 7 Year 2). Following a 4-week acclimation period (pre-diet phase) during which time individuals were fed the same hay to maintenance (2% body mass (BM) as daily dry matter (DM) intake), animals underwent a 7-week period of dietary restriction (1% BM hay as daily DM intake). Faeces were sampled on the final 3 days of the pre-diet phase and the final 3 days of the dietary restriction phase. Bacterial communities were determined using Next Generation Sequencing of amplified V1-V2 hypervariable regions of bacterial 16S rRNA. RESULTS: Losses in body mass ranged from 7.11 to 11.59%. Changes in the faecal microbiome composition following weight-loss included a reduction in the relative abundance of Firmicutes and Tenericutes and a reduction in indices of bacterial diversity. Pre-diet diversity was negatively associated with weight-loss. Pre-diet faecal acetate concentration was a strong predictor of subsequent weight-loss and negatively associated with Sphaerochaeta (Spirochaetes phylum) abundance. When animals were divided into 3 groups (high, mid, low) based overall weight loss, pre-diet bacterial community structure was found to have the greatest divergence between the high and low weight-loss groups (R = 0.67, p <  0.01), following PERMANOVA and ANOSIM analysis. CONCLUSIONS: Weight-loss in this group of ponies was associated with lower pre-diet faecal bacterial diversity and greater pre-diet acetate concentration. Overall, these data support a role for the faecal microbiome in weight-loss propensity in ponies and provide a baseline for research evaluating elements of the faecal microbiome in predicting weight-loss success in larger cohorts.

Field assessment of horse-associated genetic markers HoF597 and mtCytb for detecting the source of contamination in surface waters.

We investigated the specificity and sensitivity of two horse-associated markers, HoF597 and Horse mtCytb, and 12 mitochondrial and bacterial markers of six animal species (human, cow, pig, bird, dog, chicken) in the faecal samples of 50 individual horses. Both horse markers were detected in 48 (96%) faecal samples. Cross-reactivity with dog (BacCan545) and pig (P23-2) occurred in 88% and 72% of horse faecal samples, respectively. Several other bacterial and mitochondrial markers of non-target hosts were also detected; however, their specificities were >80%. Analyses of samples from surface waters (n = 11) on or adjacent to properties from which horse faecal samples had been collected showed only the presence of HoF597 but not horse mitochondrial marker. Our data suggest that while bacterial and (or) mitochondrial markers of other animal species may be present in horse faeces, dog and pig markers may predominantly be present in horse faecal samples, which points to their nonspecificity as markers for microbial source tracking. Although HoF597 and Horse mtCytb are highly sensitive and specific for the detection of horse faecal pollution, because of their low numbers, mitochondrial (mtDNA) markers may not be robust for screening surface waters.

Recent history of psittacosis in Australia: expanding our understanding of the epidemiology of this important globally distributed zoonotic disease.

Psittacosis is a human systemic disease caused by infection with Chlamydia psittaci. Shortly after reports emerged of a global pandemic associated with contact with imported parrots, Australian researchers including Macfarlane Burnet and others demonstrated that C. psittaci was widespread in Australian parrots. Australian cases over the last two decades have revealed that environmental exposure and contact with infected horses are also risk factors in an increasingly complicated epidemiological picture for this zoonotic disease.

Characterization of the normal equine conjunctival bacterial community using culture-independent methods.

BACKGROUND: The equine conjunctival microbiota has often been reported to be dominated by Gram-positive species such as Staphylococcus sp., Bacillus sp., and Corynebacterium sp. However, traditional culture-based methods can only recover a fraction of the bacterial species present in the sample. OBJECTIVES: This pilot study aimed at exploring the diversity of the equine conjunctival microbiota using culture-independent methods. STUDY DESIGN: Eight horses were included in this study, and only eyes with normal ophthalmic examination (n = 15 eyes) were sampled. METHODS: Conjunctival biopsies (culture-independent) were collected, and DNA was extracted from the tissues. Bacterial communities in conjunctival biopsies were characterized by next-generation sequencing of the 16S rRNA genes. Individual reads were ascribed to operational taxonomic units (OTUs) using BLASTn and Greengenes databases. Species richness, evenness, and Good's coverage were determined for each conjunctiva-associated microbial community. RESULTS: Culture-independent samples produced a total of 329 bacterial OTUs. The main OTUs identified in the study belonged to the Gram-negative species Ralstonia mannitolilytica (88.0%), Nicoletella semolina (3.3%), and Pseudomonas tolaasii (1.5%). CONCLUSIONS: Contrary to previously published data based on culture-dependent methods, the horse eye microbial community was dominated by Gram-negative bacteria of the phylum Proteobacteria.

Inter-breed diversity and temporal dynamics of the faecal microbiota in healthy horses.

Understanding gut microbiota similarities and differences across breeds in horses has the potential to advance approaches aimed at personalized microbial modifications, particularly those involved in improving sport athletic performance. Here, we explore whether faecal microbiota composition based on faecal 16S ribosomal RNA gene sequencing varies across six different sport breeds at two time points 8 months apart within a cohort of 189 healthy horses cared for under similar conditions. Lusitano horses presented the smallest and Hanoverians the greatest bacterial diversity. We found subtle but significant differences in ß-diversity between Lusitano, Anglo Arabian and the central European breeds, and we reproduced these results across the two time points. Repeat sampling of subjects showed community to be temporally more stable in Lusitano and Anglo Arabian breeds. Additionally, we found that 27 genera significantly varied in abundance across breeds. Overall, 33% of these taxa overlapped with previously identified taxa that were associated with genetic variation in humans or other species. However, a non-significant correlation was observed between microbial composition and the host pedigree-based kinship. Despite a notable variation in the diversity and composition of the faecal microbiota, breed exerted limited effects on the equine faecal microbiota.

Equine pythiosis in Egypt: clinicopathological findings, detection, identification and genotyping of Pythium insidiosum.

BACKGROUND: Equine pythiosis is an emerging, devastating disease that is hard to treat. The tumour-like nodular skin masses grow rapidly and the outcome is generally fatal, and thus early diagnosis and intervention are important. OBJECTIVES: (i) To highlight the clinical, histological and haematological findings in pythiosis, and (ii) to evaluate the efficacy of direct sample multiplex-PCR targeting the single nucleotide polymorphisms within the ribosomal DNA region for detection and genotyping of Pythium insidiosum. ANIMALS: Two hundred and twenty horses including 204 Arabian and 16 draft horses were surveyed. METHODS: Case series study diagnosis was based on clinical, pathological and haematological findings typical of P. insidiosum infection, culture identification, immunohistochemical investigation and direct sample PCR. RESULTS: The affected horses (24 of 220, 10.91%) presented with unifocal or multiple lesions on the abdomen, limbs, chest, face and mammary gland. Cases commonly had a history of access to stagnant water, ponds and intentionally flooded rice fields. Most were pregnant mares (58.33%). Histopathology revealed granulomatous reaction, blood vessel endotheliosis, heavy infiltration of eosinophils in the dermal layer, multifocal necrosis and Splendore-Hoeppli phenomenon. Unlike direct microscopy (50%) and culture (91.6%), multiplex-PCR assay identified P. insidiosum (Clade II) in all tested samples. To the best of the authors knowledge, this is the first study determining a clade of P. insidiosum causing equine pythiosis in Egypt. CONCLUSIONS AND CLINICAL IMPORTANCE: Direct sample multiplex-PCR assay is a potential tool for the early and rapid diagnosis of equine pythiosis. It overcomes limitations associated with morphological identification and provides a definitive diagnosis.

The Antistaphylococcal Lysin, CF-301, Activates Key Host Factors in Human Blood To Potentiate Methicillin-Resistant Staphylococcus aureus Bacteriolysis.

Bacteriophage-derived lysins are cell-wall-hydrolytic enzymes that represent a potential new class of antibacterial therapeutics in development to address burgeoning antimicrobial resistance. CF-301, the lead compound in this class, is in clinical development as an adjunctive treatment to potentially improve clinical cure rates of Staphylococcus aureus bacteremia and infective endocarditis (IE) when used in addition to antibiotics. In order to profile the activity of CF-301 in a clinically relevant milieu, we assessed its in vitro activity in human blood versus in a conventional testing medium (cation-adjusted Mueller-Hinton broth [caMHB]). CF-301 exhibited substantially greater potency (32 to ≥100-fold) in human blood versus caMHB in three standard microbiologic testing formats (e.g., broth dilution MICs, checkerboard synergy, and time-kill assays). We demonstrated that CF-301 acted synergistically with two key human blood factors, human serum lysozyme (HuLYZ) and human serum albumin (HSA), which normally have no nascent antistaphylococcal activity, against a prototypic methicillin-resistant S. aureus (MRSA) strain (MW2). Similar in vitro enhancement of CF-301 activity was also observed in rabbit, horse, and dog (but not rat or mouse) blood. Two well-established MRSA IE models in rabbit and rat were used to validate these findings in vivo by demonstrating comparable synergistic efficacy with standard-of-care anti-MRSA antibiotics at >100-fold lower lysin doses in the rabbit than in the rat model. The unique properties of CF-301 that enable bactericidal potentiation of antimicrobial activity via activation of "latent" host factors in human blood may have important therapeutic implications for durable improvements in clinical outcomes of serious antibiotic-resistant staphylococcal infections.

Sharpea azabuensis: a ruminal bacterium that produces trans-11 intermediates from linoleic and linolenic acid.

To investigate the metabolism of 18:2n-6 and 18:3n-3 by pure cultures of Sharpea azabuensis, two different strains (RL 1 and ST18) were each incubated in the presence of 40 µg ml-1 18:2n-6 or 18:3n-3. Pure cultures of Butyrivibriofibrisolvens D1 and Butyrivibrio proteoclasticus P18 were included as control treatments. Similar to the metabolism of B. fibrisolvens, both S. azabuensis strains converted 18:2n-6 or 18:3n-3 to cis-9, trans-11 CLA or cis-9, trans-11, cis-15 CLnA, after which it was further reduced to trans-11 18:1 or trans-11, cis-15 18:2, respectively. B. proteoclasticus additionally reduced trans-11 18:1 to 18:0. Trans-11, cis-15 18:2 was also further metabolized by B. proteoclasticus, although trans-11 18:1 did not accumulate, and only minor amounts of 18:0 were formed. The time frame of 18:2n-6 and 18:3n-3 biohydrogenation by S. azabuensis was comparable with B. fibrisolvens, indicating that S. azabuensis and B. fibrisolvens might be alternative biohydrogenators of 18:2n-6 and 18:3n-3 in the rumen.