Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep.

Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health.

Complete Genome Sequence of a New Zealand Mycobacterium tuberculosis Strain Responsible for Ongoing Transmission over the Past 30 Years.

We report here the complete genome sequence of Mycobacterium tuberculosis strain Colonial S-type 1 (CS1), which has been responsible for ongoing outbreaks of tuberculosis in New Zealand over the past 30 years. CS1 appears to be highly transmissible, with greater rates of progression to active disease, compared to other circulating M. tuberculosis strains; therefore, comparison of its genomic content is of interest.

Globetrotting strangles: the unbridled national and international transmission of Streptococcus equi between horses.

The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi, establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such 'carrier' animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing national and international transmission events that drive this endemic disease in horse populations throughout the world. Our data argue for the recognition of the international importance of strangles by the Office International des Épizooties to highlight the health, welfare and economic cost of this disease. The Pathogenwatch cgMLST web bioresource described herein is available for tailored genomic analysis of populations of S. equi and its close relative S. equi subspecies zooepidemicus that are recovered from horses and other animals, including humans, throughout the world. This article contains data hosted by Microreact.

Dispersal of Mycobacterium tuberculosis Driven by Historical European Trade in the South Pacific.

Mycobacterium tuberculosis (Mtb) is a globally distributed bacterial pathogen whose population structure has largely been shaped by the activities of its obligate human host. Oceania was the last major global region to be reached by Europeans and is the last region for which the dispersal and evolution of Mtb remains largely unexplored. Here, we investigated the evolutionary history of the Euro-American L4.4 sublineage and its dispersal to the South Pacific. Using a phylodynamics approach and a dataset of 236 global Mtb L4.4 genomes we have traced the origins and dispersal of L4.4 strains to New Zealand. These strains are predominantly found in indigenous Maori and Pacific people and we identify a clade of European, likely French, origin that is prevalent in indigenous populations in both New Zealand and Canada. Molecular dating suggests the expansion of European trade networks in the early 19th century drove the dispersal of this clade to the South Pacific. We also identify historical and social factors within the region that have contributed to the local spread and expansion of these strains, including recent Pacific migrations to New Zealand and the rapid urbanization of Maori in the 20th century. Our results offer new insight into the expansion and dispersal of Mtb in the South Pacific and provide a striking example of the role of historical European migrations in the global dispersal of Mtb.

The Interaction of Selenium with Chemotherapy and Radiation on Normal and Malignant Human Mononuclear Blood Cells.

Selenium, a trace element with anticancer properties, can reduce harmful toxicities of chemotherapy and radiotherapy without compromising efficacy. However, the dose-response relationship in normal versus malignant human cells is unclear. We evaluated how methylseleninic acid (MSA) modulates the toxicity and efficacy of chemotherapy and radiation on malignant and non-malignant human mononuclear blood cells in vitro. We specifically investigated its effects on endoplasmic reticulum stress induction, intracellular glutathione concentration, DNA damage and viability of peripheral blood mononuclear cells and THP1 monocytic leukaemia cells in response to radiation, cytosine arabinoside or doxorubicin chemotherapy. MSA, at lower concentrations, induced protective responses in normal cells but cytotoxic effects in malignant cells, alone and in conjunction with chemotherapy or radiation. However, in normal cells higher concentrations of MSA were directly toxic and increased the cytotoxicity of radiation but not chemotherapy. In malignant cells higher MSA concentrations were generally more effective in combination with cancer treatments. Thus, optimal MSA concentrations differed between normal and malignant cells and treatments. This work supports clinical reports that selenium can significantly reduce dose-limiting toxicities of anticancer therapies and potentially improve efficacy of anticancer treatments. The optimal selenium compound and dose is not yet determined.

Vitamin D3 supplementation in adults with bronchiectasis: A pilot study.

Vitamin D supplementation prevents acute respiratory infections and, through modulating innate and adaptive immunity, could have a potential role in bronchiectasis management. The primary aims of this pilot study were to assess serum 25-hydroxyvitamin D (25(OH)D) levels in New Zealand adults with bronchiectasis, and their 25(OH)D levels after vitamin D3 supplementation. Adults with bronchiectasis received an initial 2.5 mg vitamin D3 oral loading dose and 0.625 mg vitamin D3 weekly for 24 weeks. The primary outcome was serum 25(OH)D levels before and after vitamin D3 supplementation. Secondary outcomes (time to first infective exacerbation, exacerbation frequency, spirometry, health-related quality of life measures, sputum bacteriology and cell counts and chronic rhinosinusitis) were also assessed. This study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN 12612001222831). The initial, average 25(OH)D level was 71 nmol/L (95% confidence interval (CI): [58, 84]), rising to 218 nmol/L (95% CI: [199, 237]) at 12 weeks and 205 nmol/L (95% CI: [186, 224]) at 24 weeks. The initial serum cathelicidin level was 25 nmol/L (95% CI: [17, 33]), rising to 102 nmol/L (95% CI: [48, 156]) at 12 weeks and 151 nmol/L (95% CI: [97, 205]) at 24 weeks. Over the 24-week study period, we observed statistically significant changes of 1.11 (95% CI: [0.08, 2.14]) in the Leicester Cough Questionnaire and -1.97 (95% CI: [-3.71, -0.23]) in the Dartmouth COOP charts score. No significant adverse effects were recorded. Many New Zealand adults with bronchiectasis have adequate 25(OH)D levels. Weekly vitamin D3 supplementation significantly improved 25(OH)D levels.

Rapid molecular diagnosis of the Mycobacterium tuberculosis Rangipo strain responsible for the largest recurring TB cluster in New Zealand.

Despite New Zealand being a low-tuberculosis (TB) burden country, there are disproportionately high rates of TB in particular populations. Here, we report a rapid molecular diagnosis of the Mycobacterium tuberculosis Rangipo strain responsible for the largest recurring TB cluster in New Zealand.

Development of a qPCR Method to Measure Mitochondrial and Genomic DNA Damage with Application to Chemotherapy-Induced DNA Damage and Cryopreserved Cells.

DNA damage quantitation assays such as the comet assay have focused on the measurement of total nuclear damage per cell. The adoption of PCR-based techniques to quantify DNA damage has enabled sequence- and organelle-specific assessment of DNA lesions. Here we report on an adaptation of a qPCR technique to assess DNA damage in nuclear and mitochondrial targets relative to control. Novel aspects of this assay include application of the assay to the Rotor-Gene platform with optimized DNA polymerase/fluorophore/primer set combination in a touchdown PCR protocol. Assay validation was performed using ultraviolet C radiation in A549 and THP1 cancer cell lines. A comparison was made to the comet assay applied to peripheral blood mononuclear cells, and an estimation of the effects of cryopreservation on ultraviolet C-induced DNA damage was carried out. Finally, dose responses for DNA damage were measured in peripheral blood mononuclear cells following exposure to the cytotoxic agents bleomycin and cisplatin. We show reproducible experimental outputs across the tested conditions and concordance with published findings with respect to mitochondrial and nuclear genotoxic susceptibilities. The application of this DNA damage assay to a wide range of clinical and laboratory-derived samples is both feasible and resource-efficient.

Keratin and S100 calcium-binding proteins are major constituents of the bovine teat canal lining.

The bovine teat canal provides the first-line of defence against pathogenic bacteria infecting the mammary gland, yet the protein composition and host-defence functionality of the teat canal lining (TCL) are not well characterised. In this study, TCL collected from six healthy lactating dairy cows was subjected to two-dimensional electrophoresis (2-DE) and mass spectrometry. The abundance and location of selected identified proteins were determined by western blotting and fluorescence immunohistochemistry. The variability of abundance among individual cows was also investigated. Two dominant clusters of proteins were detected in the TCL, comprising members of the keratin and S100 families of proteins. The S100 proteins were localised to the teat canal keratinocytes and were particularly predominant in the cornified outermost layer of the teat canal epithelium. Significant between-animal variation in the abundance of the S100 proteins in the TCL was demonstrated. Four of the six identified S100 proteins have been reported to have antimicrobial activity, suggesting that the TCL has additional functionality beyond being a physical barrier to invading microorganisms. These findings provide new insights into understanding host-defence of the teat canal and resistance of cows to mastitis.

Strangles in horses can be caused by vaccination with Pinnacle I. N.

The differentiation of live attenuated vaccine strains from their progenitor and wild-type counterparts is important for ongoing surveillance of product safety and improved guidelines on their use. We utilised a genome sequencing approach to confirm that two cases of strangles in previously healthy horses that had received the Pinnacle I. N. vaccine (Zoetis) were caused by the vaccine strain. Our data shed new light on the safety of this vaccine and suggest that factors beyond the maturity of the animal's immune system influence the development of adverse reactions.

Whole genome sequencing of Mycobacterium tuberculosis reveals slow growth and low mutation rates during latent infections in humans.

Very little is known about the growth and mutation rates of Mycobacterium tuberculosis during latent infection in humans. However, studies in rhesus macaques have suggested that latent infections have mutation rates that are higher than that observed during active tuberculosis disease. Elevated mutation rates are presumed risk factors for the development of drug resistance. Therefore, the investigation of mutation rates during human latency is of high importance. We performed whole genome mutation analysis of M. tuberculosis isolates from a multi-decade tuberculosis outbreak of the New Zealand Rangipo strain. We used epidemiological and phylogenetic analysis to identify four cases of tuberculosis acquired from the same index case. Two of the tuberculosis cases occurred within two years of exposure and were classified as recently transmitted tuberculosis. Two other cases occurred more than 20 years after exposure and were classified as reactivation of latent M. tuberculosis infections. Mutation rates were compared between the two recently transmitted pairs versus the two latent pairs. Mean mutation rates assuming 20 hour generation times were 5.5 X 10(-10) mutations/bp/generation for recently transmitted tuberculosis and 7.3 X 10(-11) mutations/bp/generation for latent tuberculosis. Generation time versus mutation rate curves were also significantly higher for recently transmitted tuberculosis across all replication rates (p = 0.006). Assuming identical replication and mutation rates among all isolates in the final two years before disease reactivation, the u 20 hr mutation rate attributable to the remaining latent period was 1.6 × 10(-11) mutations/bp/generation, or approximately 30 fold less than that calculated during the two years immediately before disease. Mutations attributable to oxidative stress as might be caused by bacterial exposure to the host immune system were not increased in latent infections. In conclusion, we did not find any evidence to suggest elevated mutation rates during tuberculosis latency in humans, unlike the situation in rhesus macaques.

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry.

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequence-specificity of four VapC proteins from two different organisms (VapC(PAE0151) and VapC(PAE2754) from Pyrobaculum aerophilum, and VapC(Rv0065) and VapC(Rv0617) from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.

The effects of hypertonic fluid administration on the gene expression of inflammatory mediators in circulating leucocytes in patients with septic shock: a preliminary study.

OBJECTIVE: This study was designed to investigate the effect of hypertonic fluid administration on inflammatory mediator gene expression in patients with septic shock. DESIGN AND SETTING: Prospective, randomized, controlled, double-blind clinical study in a 15-bed mixed intensive care unit in a tertiary referral teaching hospital. INTERVENTIONS: Twenty-four patients, who met standard criteria for septic shock, were randomized to receive a bolus of hypertonic fluid (HT, 250 ml 6% HES/7.2% NaCl) or isotonic fluid (IT, 500 ml 6% HES/0.9% NaCl) administered over 15 minutes. Randomization and study fluid administration was within 24 hours of ICU admission for all patients. This trial is registered with ANZCTR.org.au as ACTRN12607000259448. RESULTS: Blood samples were taken immediately before and 4, 8, 12, and 24 hours after fluid administration. Real-time reverse transcriptase polymerase chain reaction (RT rtPCR) was used to quantify mRNA expression of different inflammatory mediators in peripheral leukocytes. In the HT group, compared with the IT group, levels of gene expression of MMP9 and L-selectin were significantly suppressed (p = 0.0002 and p = 0.007, respectively), and CD11b gene expression tended to be elevated (p = NS). No differences were found in the other mediators examined. CONCLUSIONS: In septic shock patients, hypertonic fluid administration compared with isotonic fluid may modulate expression of genes that are implicated in leukocyte-endothelial interaction and capillary leakage.The study was performed at the Intensive Care Department, Waikato Hospital, and at the Molecular Genetics Laboratory, University of Waikato, Hamilton, New Zealand. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN12607000259448.

Vitamin D, innate immunity and outcomes in community acquired pneumonia.

BACKGROUND AND OBJECTIVE: Vitamin D regulates the production of the antimicrobial peptides cathelicidin and beta-defensin-2, which play an important role in the innate immune response to infection. We hypothesized that vitamin D deficiency would be associated with lower levels of these peptides and worse outcomes in patients admitted to hospital with community acquired pneumonia. METHODS: Associations between mortality and serum levels of 25-hydroxyvitamin D, cathelicidin and beta-defensin-2 were investigated in a prospective cohort of 112 patients admitted with community acquired pneumonia during winter. RESULTS: Severe 25-hydroxyvitamin D deficiency (<30nmol/L) was common in this population (15%) and was associated with a higher 30-day mortality compared with patients with sufficient 25-hydroxyvitamin D (>50nmol/L) (odds ratio 12.7, 95% confidence interval: 2.2-73.3, P=0.004). These associations were not explained by differences in age, comorbidities, or the severity of the acute illness. Neither cathelicidin nor beta-defensin-2 levels predicted mortality, although there was a trend towards increased mortality with lower cathelicidin (P=0.053). Neither cathelicidin nor beta-defensin-2 levels correlated with 25-hydroxyvitamin D. CONCLUSIONS: 25-hydroxyvitamin D deficiency is associated with increased mortality in patients admitted to hospital with community acquired pneumonia during winter. Contrary to our hypothesis, 25-hydroxyvitamin D levels were not associated with levels of cathelicidin or beta-defensin-2.

Connexin36 knockout mice display increased sensitivity to pentylenetetrazol-induced seizure-like behaviors.

OBJECTIVE: Large-scale synchronous firing of neurons during seizures is modulated by electrotonic coupling between neurons via gap junctions. To explore roles for connexin36 (Cx36) gap junctions in seizures, we examined the seizure threshold of connexin36 knockout (Cx36KO) mice using a pentylenetetrazol (PTZ) model. METHODS: Mice (2-3months old) with Cx36 wildtype (WT) or Cx36KO genotype were treated with vehicle or 10-40mg/kg of the convulsant PTZ by intraperitoneal injection. Seizure and seizure-like behaviors were scored by examination of video collected for 20min. Quantitative real-time PCR (QPCR) was performed to measure potential compensatory neuronal connexin (Cx30.2, Cx37, Cx43 and Cx45), pannexin (PANX1 and PANX2) and gamma-aminobutyric acid type A (GABA(A)) receptor α1 subunit gene expression. RESULTS: Cx36KO animals exhibited considerably more severe seizures; 40mg/kg of PTZ caused severe generalized (≥grade III) seizures in 78% of KO, but just 5% of WT mice. A lower dose of PTZ (20mg/kg) induced grade II seizure-like behaviors in 40% KO vs. 0% of WT animals. There was no significant difference in either connexin, pannexin or GABA(A) α1 gene expression between WT and KO animals. CONCLUSION: Increased sensitivity of Cx36KO animals to PTZ-induced seizure suggests that Cx36 gap junctional communication functions as a physiological anti-convulsant mechanism, and identifies the Cx36 gap junction as a potential therapeutic target in epilepsy.

The vapBC operon from Mycobacterium smegmatis is an autoregulated toxin-antitoxin module that controls growth via inhibition of translation.

The largest family of bacterial toxin-antitoxin (TA) modules is formed by the vapBC operons, and these are grouped together by virtue of their toxin components belonging to the PilT N-terminal domain family of proteins that are thought to function as ribonucleases. We have identified a single vapBC operon in the genome of Mycobacterium smegmatis and herein report the molecular and biochemical characterisation of this TA module. In M. smegmatis, the vapBC genes are transcribed as a leaderless mRNA that is constitutively synthesised throughout the growth cycle. The vapBC operon is autoregulated by the VapBC protein complex as demonstrated by a threefold increase in vapBC expression (promoter-vapB-lacZ) in a DeltavapBC mutant. Electrophoretic mobility shift assays using purified VapBC protein complex show that the complex binds to inverted repeat DNA sequences in the vapBC promoter region that overlap the -35 and -10 promoter elements, thus explaining the autoregulation and the low-level constitutive expression of this operon in M. smegmatis. Neither a DeltavapBC nor a DeltavapB mutant strain exhibited any phenotypic deviation to that of the isogenic wild-type parent strain under normal laboratory growth conditions, but conditional overexpression of VapC in M. smegmatis inhibited growth by a bacteriostatic mechanism and this phenotype is exacerbated in a DeltavapBC mutant. This effect is mediated through VapC-dependent inhibition of translation, not inhibition of DNA replication or transcription. The growth inhibitory effect of VapC was neutralised when co-expressed with its cognate antitoxin VapB. Western blot analysis revealed the overproduction of VapC under inducing conditions and that the VapC protein is not produced in the DeltavapB mutant despite the presence of mRNA transcript. Taken together, these data demonstrate that VapBC from M. smegmatis has all the hallmarks of a TA module with the capacity to cause growth inhibition by regulating translation.

Heifer teats sprayed in the dry period with an iodine teat sanitizer have reduced Streptococcus uberis teat-end contamination and less Streptococcus uberis intra-mammary infections at calving.

Heifers managed under pastoral conditions are at risk from Streptococcus uberis mastitis infections at calving. A total of 397 heifers from six farms around New Zealand were enrolled in a study to identify and enumerate S. uberis on teat-ends of heifers in the peri-partum period, and to understand the effect of teat-spraying in the pre-calving period on the prevalence and incidence of S. uberis mastitis post-calving. Heifers were randomly assigned to Control or Sprayed groups. Sprayed heifers were teat-sprayed once, three times a week (Monday, Wednesday and Friday) with a commercial iodine-based teat sanitizer, starting at 3 weeks prior to calving and ending at day of calving. Across three farms, all glands of cows in both groups were sampled at calving to determine S. uberis intra-mammary infection (IMI) prevalence. For all farms, clinical mastitis (CM) cases detected during the week after calving were sampled and submitted for bacteriological analysis. Swabbing of teat-ends of 54 heifers from one farm showed that heifers had a pre-existing S. uberis contamination averaging 610 colony-forming units per swab (cfu/swab), at 3 weeks prior to calving. At calving, teat-end contamination was 560 cfu/swab for Sprayed heifers and 1775 cfu/swab for Control heifers. Two weeks after calving, teat-end contamination was similar between both groups, at 30 cfu/swab. The prevalence of S. uberis IMI was significantly lower in the Sprayed (3.5% glands) vs. the Control (7.4%) heifers in the first week after calving. There was a trend for Sprayed heifers (3.6% heifers) to have a lower incidence of S. uberis CM compared with Control heifers (7.4% heifers). It is concluded that teat-spraying in the dry period is a management option that could contribute to controlling heifer S. uberis mastitis in the transition period.

Expression of innate resistance factors in mammary secretion from periparturient dairy heifers and their association with subsequent infection status.

Mastitis in dairy heifers in the peripartum period is a common and costly problem for producers, and mammary innate resistance is of key importance in defense of the gland from bacterial invasion. A prospective observational study was undertaken in 97 dairy heifers to measure associations between expression of eight innate resistance factors in mammary secretions collected from the same animals within 14 days prior to calving and at calving, and intramammary infection (IMI) status at calving, and to describe changes in expression of these factors over time. Relative expression (RE) of eight candidate resistance mediator genes from cells from intramammary secretion was measured by quantitative real time polymerase chain reaction. Glands which were IMI-free pre-calving and did not develop a new IMI had significantly higher RE of molecule possessing ankyrin-repeat (MAIL) and beta defensin (Bdef) genes compared to glands which subsequently did develop a new IMI. Also, Bdef RE increased up to the day of calving in glands that did not develop a new IMI, but was unresponsive in glands that did develop a new IMI. Relative expression of complement 5 alpha receptor, interleukin 1beta and interleukin 8 increased in glands that did develop a new IMI. Serum amyloid A3 and toll-like receptor 2 RE increased in all glands up to the day of calving. Transforming growth factor beta RE was not associated with new infection status or time relative to calving. These findings support further investigation of function and gene polymorphisms of MAIL and Bdef as potential markers of mastitis resistance in dairy heifers.

Application of Streptococcus uberis multilocus sequence typing: analysis of the population structure detected among environmental and bovine isolates from New Zealand and the United Kingdom.

We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.