Whole genome sequencing of uropathogenic E. coli from Ireland reveals diverse resistance mechanisms and strong correlation with phenotypic (EUCAST) susceptibility testing.

Urinary tract infections (UTI) caused by uropathogenic Escherichia coli (UPEC) pose a global health concern. Resistance mechanisms, including genetic mutations in antimicrobial target genes, efflux pumps, and drug deactivating enzymes, hinder clinical treatment. These resistance factors often spread through mobile genetic elements. Molecular techniques like whole genome sequencing (WGS), multilocus sequence typing (MLST), and phylotyping help decode bacterial genomes and categorise resistance genes. In this study, we analysed 57 UPEC isolates from different UTI patients following EUCAST guidelines. A selection of 17 representative strains underwent WGS, phylotyping, MLST, and comparative analysis to connect laboratory susceptibility data with predictive genomics based on key resistance genes and chromosomal mutations in antimicrobial targets. Trimethoprim resistance consistently correlated with dfr genes, with six different alleles detected among the isolates. These dfr genes often coexisted with class 1 integrons, with the most common gene cassette combining dfr and aadA. Furthermore, 52.9% of isolates harboured the blaTem-1 gene, rendering resistance to ampicillin and amoxicillin. Ciprofloxacin-resistant strains exhibited mutations in GyrA, GyrB and ParC, plasmid-mediated quinolone resistance genes (qnrb10), and aac(6')-Ib-cr5. Nitrofurantoin resistance in one isolate stemmed from a four amino acid deletion in NfsB. These findings illustrate the varied strategies employed by UPEC to resist antibiotics and the correlation between clinical susceptibility testing and molecular determinants. As molecular testing gains prominence in clinical applications, understanding key resistance determinants becomes crucial for accurate susceptibility testing and guiding effective antimicrobial therapy.

Infectious mononucleosis: new concepts in clinical presentation, epidemiology, and host response.

PURPOSE OF REVIEW: Infectious mononucleosis (IM) is an infectious disease that presents clinically in only a small percentage of individuals despite almost universal infection with the causative agent. Here, we review the latest concepts in the clinical presentation, epidemiology, and host response of this disease. RECENT FINDINGS: Several recently published papers/reviews describe IM as a condition caused by one of several etiologic agents including, cytomegalovirus (HHV-5), Roseola virus (HHV-6) and Toxoplasmosis amongst others; this review focuses on IM as solely caused by the human herpes virus 4 (HHV-4). Since the initial discovery of the virus in the 1960s and its subsequent discovery as the primary etiologic agent for IM it has been associated with several human cancers and autoimmune disorders. Recent published findings show a correlation between HHV-4 and the autoimmune disorder, multiple sclerosis (MS), suggesting earlier IM could possibly act as a causative factor. Considering the important links being made with IM to so many cancers and autoimmune disorders it is surprising that a standard investigative procedure has yet to be determined for this disease. A standard approach to the investigation of IM would ensure more cases are diagnosed, particularly atypical cases, this would benefit epidemiological studies, and more immediately help practitioners distinguish viral from bacterial throat infections, enabling them to treat accordingly. SUMMARY: The understanding of the latest concepts in clinical presentation, epidemiology and host response to IM would benefit greatly from the introduction of a standard procedure for its investigation and diagnosis.

Uropathogenic Escherichia coli (UPEC)-Associated Urinary Tract Infections: The Molecular Basis for Challenges to Effective Treatment.

Urinary tract infections (UTIs) are among the most common bacterial infections, especially among women and older adults, leading to a significant global healthcare cost burden. Uropathogenic Escherichia coli (UPEC) are the most common cause and accounts for the majority of community-acquired UTIs. Infection by UPEC can cause discomfort, polyuria, and fever. More serious clinical consequences can result in urosepsis, kidney damage, and death. UPEC is a highly adaptive pathogen which presents significant treatment challenges rooted in a complex interplay of molecular factors that allow UPEC to evade host defences, persist within the urinary tract, and resist antibiotic therapy. This review discusses these factors, which include the key genes responsible for adhesion, toxin production, and iron acquisition. Additionally, it addresses antibiotic resistance mechanisms, including chromosomal gene mutations, antibiotic deactivating enzymes, drug efflux, and the role of mobile genetic elements in their dissemination. Furthermore, we provide a forward-looking analysis of emerging alternative therapies, such as phage therapy, nano-formulations, and interventions based on nanomaterials, as well as vaccines and strategies for immunomodulation. This review underscores the continued need for research into the molecular basis of pathogenesis and antimicrobial resistance in the treatment of UPEC, as well as the need for clinically guided treatment of UTIs, particularly in light of the rapid spread of multidrug resistance.

Novel metallomic profiling and non-carcinogenic risk assessment of botanical ingredients for use in herbal, phytopharmaceutical and dietary products using HR-ICP-SFMS.

Knowledge of element concentrations in botanical extracts is relevant to assure consumer protection given the increased interest in plant-based ingredients. This study demonstrates successful multi-element investigations in order to address the lack of comprehensive profiling data for botanical extracts, while reporting for the first time the metallomic profile(s) of arnica, bush vetch, sweet cicely, yellow rattle, bogbean, rock-tea and tufted catchfly. Key element compositions were quantified using a validated HR-ICP-SFMS method (µg kg-1) and were found highly variable between the different plants: Lithium (18-3964); Beryllium (3-121); Molybdenum (75-4505); Cadmium (5-325); Tin (6-165); Barium (747-4646); Platinum (2-33); Mercury (5-30); Thallium (3-91); Lead (12-4248); Bismuth (2-30); Titanium (131-5827); Vanadium (15-1758); Chromium (100-4534); Cobalt (21-652); Nickel (230-6060) and Copper (1910-6340). Compendial permissible limits were not exceeded. Overall, no evidence of a health risk to consumers could be determined from consumption of the investigated plants at reasonable intake rates. Mathematical risk modelling (EDI, CDI, HQ, HI) estimated levels above safe oral thresholds only for Cd (16%) and Pb (8%) from higher intakes of the respective plant-derived material. Following high consumption of certain plants, 42% of the samples were categorised as potentially unsafe due to cumulative exposure to Cu, Cd, Hg and Pb. PCA suggested a potential influence of post-harvest processing on Cr, Ti and V levels in commercially-acquired plant material compared to wild-collected and farm-grown plants. Moreover, a strong correlation was observed between Pb-Bi, Be-V, Bi-Sn, and Tl-Mo occurrence. This study may support future research by providing both robust methodology and accompanying reference profile(s) suitable for the quality evaluation of essential elements and/or metal contaminants in botanical ingredients.

An Investigation of the Effect of Water Additives on Broiler Growth and the Caecal Microbiota at Harvest.

Campylobacter is the most common foodborne pathogen in developed countries and most cases are associated with poultry. This study investigated the effect of three anti-Campylobacter water additives on broiler growth and on the caecal microbiota at harvest using 16S rRNA amplicon sequencing. Mixtures of organic acids (OA) and essential oils (EO) were administered to broilers for the entirety of the production cycle (35 d) and medium-chain fatty acids (MCFA) for 5 d immediately before harvest, under commercial conditions. Bird weight gain was significantly (p < 0.001) reduced in broilers receiving the OA and EO treatments. While this was most likely due to reduced water intake and corresponding lower feed consumption, changes to the caecal microbiota may also have contributed. Firmicutes made up over 75% of the bacteria regardless of sample type, while the minor phyla included Bacteroidetes, Actinobacteria, Melainabacteria, and Proteobacteria. There were no significant (p > 0.05) differences in the alpha diversity as measured using ACE, Chao1, and Shannon indices, except for control (water) versus MCFA and OA versus MCFA, using the Wilcox test. In contrast, there was a significant (p < 0.05) difference in beta diversity when the treated were compared to the untreated control and main flock samples, while linear discriminant analysis effect size (LeFSe) identified three OTUs that were present in the control but absent in the treated birds. It was concluded that the water additives tested adversely affected broiler performance, which may, at least in part, be due to changes in the caecal microbiota, assuming that the altered microbiota at day 35 is indicative of a change throughout the production cycle.

Genomic diversity, virulence and source of Campylobacter jejuni contamination in Irish poultry slaughterhouses by whole genome sequencing.

AIMS: The aim was to exploit whole genome sequencing (WGS) to assess genomic diversity, identify virulence genes and deduce the proportion of Campylobacter colonized broilers that directly contaminate their carcasses. METHODS AND RESULTS: Campylobacter jejuni isolates (107) from caeca and carcass neck skin samples (50 pairs from the same batch plus 7 individual caeca) sampled at three poultry slaughterhouses over a one-year period were selected for sequencing (MiSeq; Illumina). FastQ files were submitted to BioNumerics for analysis using the wgMLST scheme for allele calling. Campylobacter cgMLST and hierarchical clustering was performed by applying the single linkage algorithm. Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into clonal complexes (CCs) using the Campylobacter PubMLST.org database. Virulence genes were determined by downloading core sequences from the virulence factor database (VFDB) and the National Center for Biotechnology Information (NCBI). A high degree of diversity was observed with 23 different STs identified. ST257 and CC-21 were the most common STs and CCs, respectively. cgMLST analysis suggested that 56% of carcass contamination was a direct result of contamination from caeca from the same batch. Virulence genes known to play a role in human C. jejuni infection were identified such as the wlaN gene and the genes associated with lipooligosaccharide synthesis, which were identified in 30% of isolates. CONCLUSIONS: Caecal colonization was the more plausible occurring source of C. jejuni contamination of broiler carcasses, compared with cross-contamination from another batch or the environment. The high rate of genetic diversity observed amongst caecal isolates is consistent with a wide variety of Campylobacter strains circulating in poultry flocks in Ireland. SIGNIFICANCE AND IMPACT OF STUDY: The results will further inform broiler processors and regulators about the influence and importance of on-farm colonization versus slaughterhouse cross-contamination and the relationship between C. jejuni in caeca and carcasses during processing.

Effect of Doxycycline Use in the Early Broiler Production Cycle on the Microbiome.

16S rRNA amplicon sequencing was used to investigate changes in the broiler gastrointestinal tract (GIT) microbiota throughout the rearing period and in combination with antibiotic treatment. Thirty birds (from a commercial flock) were removed at multiple points throughout the rearing period on days 13, 27, and 33, euthanised, and their GIT aseptically removed and divided into upper (the crop, proventriculus, and the gizzard), middle (the duodenum, jejunum, and ileum) and lower (the large intestine, the caeca, and the cloaca) sections. In a separate commercial flock, on the same farm with similar husbandry practices and feed, doxycycline (100 mg/ml per kg body weight) was administered in drinking water between day 8 and 12 (inclusive) of the production cycle. Birds were removed on days, 13, 27, and 33 and GIT samples prepared as above. The contents of three merged samples from each GIT section were pooled (n = 60), the DNA extracted and analysed by 16S rRNA amplicon metagenomic sequencing and analysed. Major changes in the broiler microbiota were observed as the birds aged particularly with the Firmicutes/Bacteroidetes ratio (F:B) of the lower GIT. Moreover, Chao1, ACE, and Shannon indices showed the antibiotic treatment significantly altered the microbiota, and this change persisted throughout the rearing period. Further research is required to investigate the effect of these changes on bird performance, susceptibility to infections and Campylobacter carriage.

Prevalence and levels of Campylobacter in broiler chicken batches and carcasses in Ireland in 2017-2018.

In 2008, an EU wide baseline survey of broilers revealed a high Campylobacter prevalence. To assist with industry-wide controls, updated data were required. The primary objective of this study was to establish up-to-date data on Campylobacter carriage and carcass contamination in Irish broilers. Monthly samples were collected from the three largest broiler processing plants in Ireland over a twelve-month period. Samples were taken from both first and final thin birds (partial and full depopulation) from 358 batches of broilers. From each batch, a composite sample of 10 caecal contents (n = 358) and 5 neck skins (n = 1790) were collected and numbers of Campylobacter in each sample were determined. Of the 1790 neck skin samples tested, 53% were Campylobacter positive. Campylobacter was detected in the caecal contents of 66% of all batches tested. Depopulation and/or age had a significant effect on Campylobacter prevalence with 67% of final thin broilers yielding Campylobacter-positive neck skin samples in contrast to 38% of first thin broilers that yielded positive neck skin samples (P ≤ 0.002). A significant seasonal variation was observed in the rate of Campylobacter-positive caecal samples with higher prevalence seen in July (85%) than the colder months of November (61%), December (50%), January (61%) March (57%) and April (59%). Neck skin samples were 7 times more likely to be Campylobacter positive if the caecal contents from the same batch were positive (odds ratio = 7.1; P ≤ 0.0001). The decrease in Campylobacter prevalence observed in neck skin and caecal contents demonstrates the improvements and progress made in reducing prevalences of this important enteropathogen in the Irish poultry industry since the 2008 EU baseline survey. It also provides further supporting data on the impact of thinning, the processing environment and season on Campylobacter prevalence.

The Effects of Freeze-Thaw and UVC Radiation on Microbial Survivability in a Selected Mars-like Environment.

The purpose of this study was to determine survivability of Escherichia coli, Deinococcus radiodurans and Paraburkholderia fungorum under Mars-simulated conditions for freeze-thawing (-80 °C to +30 °C) and UV exposure alone and in combination. E. coli ATCC 25922, D. radiodurans and P. fungorum remained viable following 20 successive freeze-thaw cycles, exhibiting viabilities of 2.3%, 96% and 72.6%, respectively. E. coli ATCC 9079 was non-recoverable by cycle 9. When exposed to UV irradiation, cells withstood doses of 870 J/m2 (E. coli ATCC 25922), 200 J/m2 (E. coli ATCC 9079), 50,760 J/m2 (D. radiodurans) and 44,415 J/m2 (P. fungorum). Data suggests P. fungorum is highly UV-resistant. Combined freeze-thawing with UV irradiation showed freezing increased UV resistance in E. coli ATCC 25922, E. coli DSM 9079 and D. radiodurans by 6-fold, 30-fold and 1.2-fold, respectively. Conversely, freezing caused P. fungorum to exhibit a 1.75-fold increase in UV susceptibility. Strain-dependent experimentation demonstrated that freezing increases UV resistance and prolongs survival. These findings suggest that exposure to short wavelength UV rays (254 nm) and temperature cycles resembling the daily fluctuating conditions on Mars do not significantly affect survival of D. radiodurans, P. fungorum and E. coli ATCC 25922 following 20 days of exposure.

From Monographs to Chromatograms: The Antimicrobial Potential of Inula helenium L. (Elecampane) Naturalised in Ireland.

With antimicrobial resistance rising globally, the exploration of alternative sources of candidate molecules is critical to safeguard effective chemotherapeutics worldwide. Plant natural products are accessible, structurally diverse compounds with antimicrobial potential. The pharmacological applications of plants in medicine can be guided by the attestation of traditional use, as demonstrated in this study. In Irish ethnomedical literature, Inula helenium L. (elecampane) is often indicated for respiratory and dermal ailments. This is the first assessment of antimicrobial sesquiterpene lactones from the roots of elecampane, naturalised in Ireland. Traditional hydro-ethanolic extracts were prepared from multi-origin elecampane roots. A novel clean-up strategy facilitated the bioactivity-guided fractionation of a subset of anti-staphylococcal fractions (the compositions of which were investigated using HPLC-DAD, supported by 1H NMR). The natural products attributing to the antimicrobial activity, observed in vitro, were identified as alantolactone (1), isoalantolactone (2), igalan (3), and an unseparated mixture of dugesialactone (4) and alloalantolactone (5), as major compounds. The findings suggest that the geographical origin of the plant does not influence the anti-bacterial potency nor the chemical composition of traditional elecampane root. Considering the prevalence of staphylococci-associated infections and associated broad spectrum resistance in Irish hospitals, currently, further research is warranted into the usage of the identified compounds as potential candidates in the control of staphylococcal carriage and infection.

Phenotypic and genetic analyses of two Campylobacter fetus isolates from a patient with relapsed prosthetic valve endocarditis.

Campylobacter fetus can cause intestinal and systemic disease in humans and are well-established veterinary and economic pathogens. We report the complete genomic sequences of two C. fetus subsp. fetus (Cff) isolates recovered in 2017 (CITCf01) and 2018 (CITCf02) from a case of recurrent prosthetic valve endocarditis. Both were capable of growth aerobically. Their genomes were found to be highly conserved and syntenic with 99.97% average nucleotide identity (ANI) while differences in their respective sap loci defined the temporal separation of their genomes. Based on core genome phylogeny and ANI of 83 Cff genomes belonging to the previously described human-associated Cff lineage, CITCf01 and CITCf02 grouped in a clade of 11 sequence type (ST)3 Cff (including the Cff type strain NCTC 10842T). CITCf01 and CITCf02 were marked for their lack of unique genomic features when compared to isolates within the subspecies and the type strain in particular. We identified point mutations in oxidative stress response genes, among others, that may contribute to aerobiosis. We report a case of Cff causing relapsed prosthetic valve endocarditis and we highlight the sap island as a polymorphic site within the genetically stable ST3 lineage, central to pathogenicity.

The efficacy of organic acid, medium chain fatty acid and essential oil based broiler treatments; in vitro anti-Campylobacter jejuni activity and the effect of these chemical-based treatments on broiler performance.

AIMS: This research tested the anti-Campylobacter properties of organic acids (OA), medium chain fatty acids (MCFA) and essential oils (EO) in vitro and commenced in vivo suitability testing focused on broiler performance. METHODS AND RESULTS: Nine active compounds were tested at different concentrations and times against Campylobacter jejuni in sterile distilled water, Mueller Hinton broth and grower feed digestate (GFD). Sodium caprate (1.5%, v/v), thymol (0.25% and 2.5%, v/v), carvacrol (1.25%, v/v) and potassium sorbate (1.5%, v/v) each achieved C. jejuni reductions of ≥4.5 log10  CFU per ml in GFD, the matrix most representative of the broiler gut, after 60 s. Similar reductions were achieved after 60 min with lactic acid (1.25%, v/v), formic acid (3.1%, v/v), sodium caprylate (1.5%, v/v) and carvacrol (1.25%, v/v). However, in vivo these compounds adversely affected broiler performance, resulting in dimished water intake and reduced weight. CONCLUSIONS: OA, MFCA and EO based compounds are effective anti-Campylobacter treatments in laboratory model studies but cannot be applied in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates that OAs, MCFAs and EOs can achieve significant reductions in Campylobacter in vitro but identifies a major issue, inhibition of broiler performance, preventing their use in practice.

Campylobacter majalis sp. nov. and Campylobacter suis sp. nov., novel Campylobacter species isolated from porcine gastrointestinal mucosa.

Strains LMG 7974T and LMG 8286T represent single, novel Campylobacter lineages with Campylobacter pinnipediorum and Campylobacter mucosalis as nearest phylogenomic neighbours, respectively. The results of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analyses of LMG 7974T, LMG 8286T and type strains of species of the genus Campylobacter confirmed that these strains represent novel species of the genus Campylobacter. The 16S rRNA gene sequences of both strains showed highest identity towards C. mucosalis (97.84 and 98.74 %, respectively). Strains LMG 7974T and LMG 8286T shared 72.5 and 73.7% ANI, respectively, with their nearest phylogenomic neighbours and less than 21 % dDDH. The draft genome sizes of LMG 7974T and LMG 8286T are 1 945429 bp and 1 708214 bp in length with percentage DNA G+C contents of 33.8 and 37.2 %, respectively. Anomalous biochemical characteristics and low MALDI-TOF mass spectrometry log scores supported their designation as representing novel species of the genus Campylobacte. We therefore propose to classify strain LMG 7974T (=CCUG 20705T) as the type strain of the novel species Campylobacter majalis sp. nov. and strain LMG 8286T (=CCUG 24193T, NCTC 11879T) as the type strain of the novel species Campylobacter suis sp. nov.

Effect of Sub-Inhibitory Concentrations of Nitrofurantoin, Ciprofloxacin, and Trimethoprim on In Vitro Biofilm Formation in Uropathogenic Escherichia coli (UPEC).

The purpose of this study was to determine the effect of sublethal concentrations of nitrofurantoin, ciprofloxacin, and trimethoprim on biofilm formation in 57 uropathogenic Escherichia coli strains (UPEC). The minimum inhibitory concentration of nitrofurantoin, ciprofloxacin, and trimethoprim was determined and the biofilm formation for each isolate with and without sub-lethal concentrations of each antibiotic was then quantified. The statistical significance of changes in biofilm formation was ascertained by way of a Dunnett's test. A total of 22.8% of strains were induced to form stronger biofilms by nitrofurantoin, 12% by ciprofloxacin, and 19% by trimethoprim; conversely 36.8% of strains had inhibited biofilm formation with nitrofurantoin, 52.6% with ciprofloxacin, and 38.5% with trimethoprim. A key finding was that even in cases where the isolate was resistant to an antibiotic as defined by EUCAST, many were induced to form a stronger biofilm when grown with sub-MIC concentrations of antibiotics, especially trimethoprim, where six of the 22 trimethoprim resistant strains were induced to form stronger biofilms. These findings suggest that the use of empirical treatment with trimethoprim without first establishing susceptibility may in fact potentiate infection in cases where a patient who is suffering from a urinary tract infection (UTI) caused by trimethoprim resistant UPEC is administered trimethoprim. This emphasizes the need for laboratory-guided treatment of UTI.

Cryptosporidium spp. diagnosis and research in the 21st century.

The protozoan parasite Cryptosporidium has emerged as a leading cause of diarrhoeal illness worldwide, posing a significant threat to young children and immunocompromised patients. While endemic in the vast majority of developing countries, Cryptosporidium also has the potential to cause waterborne epidemics and large scale outbreaks in both developing and developed nations. Anthroponontic and zoonotic transmission routes are well defined, with the ingestion of faecally contaminated food and water supplies a common source of infection. Microscopy, the current diagnostic mainstay, is considered by many to be suboptimal. This has prompted a shift towards alternative diagnostic techniques in the advent of the molecular era. Molecular methods, particularly PCR, are gaining traction in a diagnostic capacity over microscopy in the diagnosis of cryptosporidiosis, given the laborious and often tedious nature of the latter. Until now, developments in the field of Cryptosporidium detection and research have been somewhat hampered by the intractable nature of this parasite. However, recent advances in the field have taken the tentative first steps towards bringing Cryptosporidium research into the 21st century. Herein, we provide a review of these advances.

Misdiagnosis of SARS-CoV-2: A Critical Review of the Influence of Sampling and Clinical Detection Methods.

SARS-CoV-2 infection has generated the biggest pandemic since the influenza outbreak of 1918-1919. One clear difference between these pandemics has been the ability to test for the presence of the virus or for evidence of infection. This review examined the performance characteristics of sample types via PCR detection of the virus, of antibody testing, of rapid viral antigen detection kits and computerised tomography (CT) scanning. It was found that combined detection approaches, such as the incorporation of CT scans, may reduce the levels of false negatives obtained by PCR detection in both symptomatic and asymptomatic patients, while sputum and oral throat washing sample types should take precedence over swabbing when available. Rt-PCR assays for detection of the virus remain the gold-standard method for SARS-CoV-2 diagnosis and can be used effectively on pooled samples for widespread screening. The novel Oxford antibody assay was found to have the highest sensitivity and specificity of four currently available commercial antibody kits but should only be used during a specific timeframe post-symptom onset. Further research into transmission modes between symptomatic and asymptomatic patients is needed. Analysis of the performance characteristics of different sampling and detection methods for SARS-CoV-2 showed that timing of sampling and testing methods used can greatly influence the rate of false-positive and false-negative test results, thereby influencing viral spread.

Development of a novel, high resolution melting analysis based genotyping method for Cryptosporidium parvum.

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes. The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.

A novel genotyping method for Cryptosporidium hominis.

Cryptosporidiosis remains the leading protozoan induced cause of diarrhoea-associated mortality worldwide. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in many countries. This study applied high resolution melting analysis, a post-real-time PCR application, to the differentiation of six globally prevalent C. hominisgp60-subtypes. This novel method targeted three microsatellite, tandem repeat containing genetic markers, gp60, the genetic marker upon which current Cryptosporidium subtype nomenclature is based, MSB, and MSE, by which to differentiate between C. hominis isolates. This multi-locus approach successfully differentiated between all six C. hominisgp60-subtypes studied, some of which, such as IbA10G2, are known to exhibit global ubiquity. Thus, this method has the potential to be universally employed as a sensitive, cost effective and highly reproducible means to rapidly differentiate between C. hominisgp60-subtypes. Such a method would be of particular utility in epidemiological studies and outbreak scenarios, providing cost effective, clinically accessible alternative to DNA sequencing. The success of this preliminary study also supports further analysis of an expanded C. hominisgp60-subtype range and the potential expansion of the multi-locus panel in order to improve the discriminatory power of this approach.

Maximising Productivity and Eliminating Campylobacter in Broilers by Manipulating Stocking Density and Population Structure Using 'Biosecurity Cubes'.

This study investigates the effect of stocking density and population dynamics on broiler growth rates and productivity, while further validating the ability of the biosecurity cubes (BC) to protect birds from Campylobacter. In our methodology, six BC were constructed in a commercial broiler house containing approximately 28,500 birds. During three trials, the BC were stocked at densities of 12, 14, 16, 18, 20 and 22 birds/m2, with the main flock (20 birds/m2) considered the control. Periodically, 10 birds per density were weighed and examined. The Campylobacter status of the birds was monitored via faecal samples using the ISO 10272: 2017. The stocking density for maximum calculated yield was 20 (trials 1 and 2) or 22 birds/m2 (trial 3), followed by 18, 16, 14 and 12. At the stocking rate of 20 birds/m2, the birds in the pen grew faster than those at the same density in the main flock achieving 2 Kg 3-6 days faster. Birds in the BC were observed to be generally healthier, and in some cases, remained Campylobacter negative, even after the main flock was infected. Our results conclude that dividing the flock into sub-flocks of approximately 20 birds/m2 using BC could increase productivity up to 20%, while preventing Campylobacter.