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Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.

The COVID-19 pandemic caused much illness, many deaths, and profound disruption to society. The production of ‘safe and effective’ vaccines was a key public health target. Sadly, unprecedented high rates of adverse events have overshadowed the benefits. This two-part narrative review presents evidence for the widespread harms of novel product COVID-19 mRNA and adenovectorDNA vaccines and is novel in attempting to provide a thorough overview of harms arising from the new technology in vaccines that relied on human cells producing a foreign antigen that has evidence of pathogenicity. This first paper explores peer-reviewed data counter to the ‘safe and effective’ narrative attached to these new technologies. Spike protein pathogenicity, termed ‘spikeopathy’, whether from the SARS-CoV-2 virus or produced by vaccine gene codes, akin to a ‘synthetic virus’, is increasingly understood in terms of molecular biology and pathophysiology. Pharmacokinetic transfection through body tissues distant from the injection site by lipid-nanoparticles or viral-vector carriers means that ‘spikeopathy’ can affect many organs. The inflammatory properties of the nanoparticles used to ferry mRNA; N1-methylpseudouridine employed to prolong synthetic mRNA function; the widespread biodistribution of the mRNA and DNA codes and translated spike proteins, and autoimmunity via human production of foreign proteins, contribute to harmful effects. This paper reviews autoimmune, cardiovascular, neurological, potential oncological effects, and autopsy evidence for spikeopathy. With many gene-based therapeutic technologies planned, a re-evaluation is necessary and timely.

Control of buffalo flies ( , BFs) relies mainly on chemical methods; however, resistance to insecticides is widespread in BF populations. Breeding for resistance to BFs represents a possible alternative, but direct phenotyping of animals is laborious and often inaccurate. The availability of reliable diagnostic biomarker(s) to identify low BF carrier cattle would facilitate rapid and accurate selection for genetic improvement. However, limited information is available regarding differences amongst cattle in host responses to BF infestation. This study investigated the variation in Brangus cattle serum proteomic profiles before (naïve) and after peak BF exposure, in low (LF) and high BF burden (HF) cattle. Cattle were phenotyped for susceptibility based on BF counts on multiple dates using visual and photographic techniques. The relative abundance of serum proteins in cattle before and after exposure to BFs was analysed using sequential window acquisition of all theoretical fragment ion mass spectrometry (SWATH-MS). Exposure to BFs elicited similar responses in HF and LF cattle, with 79 and 70 proteins, respectively, showing significantly different abundances post exposure as compared to their relevant naïve groups. The comparison of serum samples from naïve HF and LF cattle identified 44 significantly differentially abundant (DA) proteins, while 37 significantly DA proteins were identified from the comparison between HF and LF cattle post-exposure to BFs. Proteins with higher abundance in naïve LF cattle were enriched in blood coagulation mechanisms that were sustained after exposure to BFs. Strong immune response mechanisms were also identified in naïve LF cattle, whereas these responses developed in HF cattle only after exposure to BF. High BF cattle also showed active anticoagulation mechanisms in response to BF exposure, including downregulation of coagulation factor IX and upregulation of antithrombin-III, which might facilitate BF feeding. Underlying differences in the abundance of proteins related to blood coagulation and immune response pathways could potentially provide indirect indicators of susceptibility to BF infestation and biomarkers for selecting more BF-resistant cattle.

Cattle enterprises are one of the major livestock production systems globally and are forecasted to have stable growth in the next decade. To facilitate sustainable live weight production, optimal reproductive performance is essential. Microbial colonisation in the reproductive tract has been demonstrated as one of the factors contributing to bovine reproductive performance. Studies also implied that reproductive metagenomes are different at each stage of the estrous cycle. This study applied Oxford Nanopore Technologies’ adaptive long-read sequencing to profile the bovine reproductive microbiome collected from tropical cattle in northern Queensland, Australia. The microbiome samples were collected from cattle of different sexes, reproductive status and locations to provide a comprehensive view of the bovine reproductive microbiome in northern Australian cattle. Ascomycota, Firmicutes and Proteobacteria were abundant phyla identified in the bovine reproductive metagenomes of Australian cattle regardless of sexes, reproductive status and location. The species level taxonomical investigation suggested that gastrointestinal metagenome and the surrounding environment were potentially the origins of the bovine reproductive metagenome. Functional profiles further affirmed this implication, revealing that the reproductive metagenomes of the prepubertal and postpartum animals were dominated by microorganisms that catabolise dietary polysaccharides as an energy substrate while that of the pregnant animals had the function of harvesting energy from aromatic compounds. Bovine reproductive metagenome investigations can be employed to trace the origins of abnormal metagenomes, which is beneficial for disease prevention and control. Additionally, our results demonstrated different reproductive metagenome diversities between cattle from two different locations. The variation in diversity within one location can serve as the indicator of abnormal reproductive metagenome, but between locations inferences cannot be made. We suggest establishing localised metagenomic indices that can be used to infer abnormal reproductive metagenomes which contribute to abortion or sub-fertility.

Fowl cholera, caused by Pasteurella multocida, remains a major problem of poultry worldwide. In the current report, we describe an outbreak in free range organic broilers. In addition to culturing samples from dead broilers, we attempted to isolate P. multocida from feral cats trapped on the farm. The isolates were identified by PCR as P. multocida and then serotyped using the Heddleston scheme and genotyped using both a multilocus sequence typing (MLST) method and an enterobacterial repetitive intergenic consensus (ERIC)-PCR method. A total of 123 isolates of P. multocida were recovered from 12 broilers. All 123 isolates were examined by ERIC-PCR, and only one pattern was identified. A subset of seven broiler isolates were examined by MLST and all were typed as sequence type (ST) 20. A total of 28 isolates of P. multocida were recovered from 17 cats, and five ERIC-PCR genotypes were identified, with one genotype (E-1, shared by 19 isolates) being the same as the ERIC-PCR pattern associated with the broilers. One representative cat strain for each ERIC-PCR pattern was subjected to MLST. The cat isolate with the same ERIC-PCR genotype as the broiler isolates was confirmed as having the same MLST result, ST 20. The other five cat ERIC-PCR patterns were allocated to four STs: E-2 and E-5 to ST 265, E-3 to ST 30, E-4 to ST 20, and E-6 to ST 264. Both genotyping methods confirmed that isolates of P. multocida were common between the feral cats and the chickens. It was not clear whether the strain was transmitted from the cats to the chicken or whether the cats obtained the strain preying on chicken. The study has shown that cats can harbor P. multocida strains with the same genotype found in chickens affected with fowl cholera. Epidemiología del cólera aviar en pollos orgánicos—Estudio de un caso. El cólera aviar, causada por Pasteurella multocida, sigue siendo un problema importante en la avicultura a nivel mundial. En el presente reporte, se describe un brote en pollos de engorde de tipo orgánico. Además de cultivar muestras de pollos muertos, se intentó aislar P. multocida de gatos salvajes atrapados en la granja. Los aislamientos fueron identificados por PCR como P. multocida y luego fueron serotipificados utilizando el esquema de Heddleston y genotipificados utilizando tanto el método de tipificación por secuencia multilocus (MLST) y mediante el método del consenso intergénico repetitivo de enterobacerias (ERIC)-PCR. Se recuperaron un total de 123 aislamientos de P. multocida de 12 pollos de engorde. Todos los 123 aislamientos fueron examinados por ERIC-PCR, y se identificó un solo patrón. Un subconjunto de siete aislamientos de pollos de engorde fueron examinados por el método MLST y todos se tipificaron como secuencia tipo (ST) 20. Un total de 28 aislamientos de P. multocida se recuperaron a partir de 17 gatos, y se identificaron cinco genotipos por ERIC-PCR, con un genotipo (E-1, compartida por 19 aislamientos) siendo el mismo que el patrón de ERIC-PCR asociado con los pollos de engorde. Una cepa representativa de gato de cada patrón de ERIC-PCR fue sometida a MLST. El aislamiento de gato con el mismo genotipo ERIC-PCR del aislado de pollo de engorde se confirmó que mostraba el mismo resultado por MLST, ST 20. Los otros cinco patrones de ERIC-PCR de aislamientos de gatos fueron asignados a cuatro serotipos: E-2 y E-5 a ST 265, E-3 a ST 30, E-4 a ST 20, y E-6 a ST 264. Ambos métodos de genotipificación confirmaron que los aislamientos de P. multocida eran comunes entre los gatos silvestres y los pollos. No estuvo claro si las cepas se transmitían de los gatos al pollo, o si los gatos la obtenían mediante la predación de los pollos. El estudio ha demostrado que los gatos pueden albergar cepas de P. multocida con el mismo genotipo encontrado en los pollos afectados con el cólera aviar.

Undesirable microbial infiltration into the female bovine reproductive tracts, for example during calving or mating, is likely to disturb the commensal microflora. Persistent establishment and overgrowth of certain pathogens induce reproductive diseases, render the female bovine reproductive tract unfavourable for pregnancy or can result in transmission to the foetus, leading to death and abortion or birth abnormalities. This review of culture-independent metagenomics studies revealed that normal microflora in the female bovine reproductive tract is reasonably consistently dominated by bacteria from the phyla Bacteroidetes, Firmicutes, Proteobacteria, following by Actinobacteria, Fusobacteria and Tenericutes. Reproductive disease development in the female bovine reproductive tract was demonstrated across multiple studies to be associated with high relative abundances of bacteria from the phyla Bacteroidetes and Fusobacteria. Reduced bacterial diversity in the reproductive tract microbiome in some studies of cows diagnosed with reproductive diseases also indicated an association between dysbiosis and bovine reproductive health. Nonetheless, the bovine genital tract microbiome remains underexplored, and this is especially true for the male genital tract. Future research should focus on the functional aspects of the bovine reproductive tract microbiomes, for example their contributions to cattle fertility and susceptibility towards reproductive diseases.

Pasteurella multocida is a major animal pathogen that causes a range of diseases including fowl cholera. P. multocida infections result in considerable losses to layer and breeder flocks in poultry industries worldwide. Both killed whole-cell and live-attenuated vaccines are available; these vaccines vary in their protective efficacy, particularly against heterologous strains. Moreover, until recently there was no knowledge of P. multocida LPS genetics and structure to determine precisely how LPS structure affects the protective capacity of these vaccines. In this study we show that defined lipopolysaccharide (LPS) mutants presented as killed whole-cell vaccines elicited solid protective immunity only against P. multocida challenge strains expressing highly similar or identical LPS structures. This finding indicates that vaccination of commercial flocks with P. multocida killed cell formulations will not protect against strains producing an LPS structure different to that produced by strains included in the vaccine formulation. Conversely, protective immunity conferred by vaccination with live P. multocida strains was found to be largely independent of LPS structure. Birds vaccinated with a range of live mutants belonging to the L1 and L3 LPS genotypes, each expressing a specific truncated LPS structure, were protected against challenge with the parent strain. Moreover, birds vaccinated with any of the five LPS mutants belonging to the L1 LPS genotype were also protected against challenge with an unrelated strain and two of the five groups vaccinated with live LPS mutants belonging to the L3 genotype were protected against challenge with an unrelated strain. In summary, vaccination with live P. multocida aroA mutants producing full-length L1 or L3 LPS or vaccination with live strains producing shortened L1 LPS elicited strong protective immunity against both homologous and heterologous challenge.

[This corrects the article DOI: 10.1371/journal.pbio.2003916.].

This study investigated the role of cattle immune responses in the pathogenesis of buffalo fly (Haematobia irritans exigua) (BF) lesions. Brangus steers phenotyped for lesion development were divided into three groups: high lesion susceptibility (HL), low lesion susceptibility (LL) and no lesions (NL), based on lesion severity scores. Each steer was injected intradermally with different concentrations of BF, Onchocerca gibsoni (Og), and Musca domestica (Md) antigens. At 1 h post-injection, wheal areas at BF injection sites were found to be significantly larger in HL than NL cattle, but there were no significant differences (p < 0.05) found between either the HL or NL cattle and LL cattle. At 24, 48, and 72 h post-injection, the skinfold thickness response to both BF and Md antigens was significantly greater in the HL group than the NL group. However, skin thickness was significantly greater for the BF antigens than the Md antigens (p < 0.05). There were no significant differences found between the LL and NL animals in response to the BF antigens at any time, and no significant differences were determined between any of the lesion groups in response to the Og antigens. Histological examination of skin sections taken from the BF antigen injection sites in HL cattle at 72 h post-injection revealed necrosis of the epidermis and superficial dermis, along with severe eosinophilic inflammation. This study suggests that differences in the hypersensitivity to BF antigens underlie differences amongst the cattle in their susceptibility to the development of BF lesions, and breeding for immune-related biomarkers may assist in selecting more BF lesion-resistant cattle.

Bovine respiratory disease (BRD) causes high morbidity and mortality in beef cattle worldwide. Antimicrobial resistance (AMR) monitoring of BRD pathogens is critical to promote appropriate antimicrobial stewardship in veterinary medicine for optimal treatment and control. Here, the susceptibility of Mannheimia haemolytica and Pasteurella multicoda isolates obtained from BRD clinical cases (deep lung swabs at post-mortem) among feedlots in four Australian states (2014–2019) was determined for 19 antimicrobial agents. The M. haemolytica isolates were pan-susceptible to all tested agents apart from a single macrolide-resistant isolate (1/88; 1.1%) from New South Wales (NSW). Much higher frequencies of P. multocida isolates were resistant to tetracycline (18/140; 12.9%), tilmicosin (19/140; 13.6%), tulathromycin/gamithromycin (17/140; 12.1%), and ampicillin/penicillin (6/140; 4.6%). Five P. multocida isolates (3.6%), all obtained from NSW in 2019, exhibited dual resistance to macrolides and tetracycline, and a further two Queensland isolates from 2019 (1.4%) exhibited a multidrug-resistant phenotype to ampicillin/penicillin, tetracycline, and tilmicosin. Random-amplified polymorphic DNA (RAPD) typing identified a high degree of genetic homogeneity among the M. haemolytica isolates, whereas P. multocida isolates were more heterogeneous. Illumina whole genome sequencing identified the genes msr(E) and mph(E)encoding macrolide resistance, tet(R)-tet(H) or tet(Y) encoding tetracycline resistance, and blaROB-1 encoding ampicillin/penicillin resistance in all isolates exhibiting a corresponding resistant phenotype. The exception was the tilmicosin-resistant, tulathromycin/gamithromycin-susceptible phenotype identified in two Queensland isolates, the genetic basis of which could not be determined. These results confirm the first emergence of AMR in M. haemolytica and P. multocida from BRD cases in Australia, which should be closely monitored.