Insect identification and preservation of voucher specimens is integral to pest diagnostic and surveillance activities; yet bulk-trapped insects are a diagnostic challenge due to high catch numbers ...and the susceptibility of samples to environmental damage. Many insect trap catches rely on examination of morphological characters for species identifications, which is a time consuming and highly skilled task, hence there is a need for more efficient molecular approaches. Many bulk DNA extraction methods require destructive sampling of specimens, resulting in damaged, or fully destroyed, voucher specimens. We developed an inexpensive, rapid, bulk DNA isolation method that preserves specimens as pinned vouchers to a standard that allows for post-extraction morphological examination and inclusion in insect reference collections. Our protocol was validated using a group of insects that are time-consuming to identify when trapped in large numbers-the dacine fruit flies (Diptera: Tephritidae: Dacinae). In developing our method, we evaluated existing protocols against the following criteria: effect on morphology; suitability for large trap catches; cost; ease of handling; and application to downstream molecular diagnostic analyses such as real-time PCR and metabarcoding. We found that the optimum method for rapid isolation of DNA extraction was immersing flies in a NaOH:TE buffer at 75°C for 10 minutes, without the need for proteinase K or detergents. This HotSOAK method produced sufficient high-quality DNA whilst preserving morphological characters suitable for species-level identification with up to 20,000 flies in a sample. The lysates performed well in down-stream analyses such as loop-mediated isothermal amplification (LAMP) and real-time PCR applications, while for metabarcoding PCR the lysate required an additional column purification step. Development of this method is a key step required for upscaling our capacity to accurately detect insects captured in bulk traps, whether for biodiversity, biosecurity, or pest management objectives.
Abstract
The cue-lure-responding New Guinea fruit fly,
Bactrocera
trivialis
, poses a biosecurity risk to neighbouring countries, e.g., Australia. In trapping programs, lure caught flies are usually ...morphologically discriminated from non-target species; however, DNA barcoding can be used to confirm similar species where morphology is inconclusive, e.g.,
Bactrocera
breviaculeus
and
B.
rufofuscula
. This can take days—and a laboratory—to resolve. A quicker, simpler, molecular diagnostic assay would facilitate a more rapid detection and potential incursion response. We developed LAMP assays targeting cytochrome
c
oxidase subunit I (COI) and Eukaryotic Translation Initiation Factor 3 Subunit L (EIF3L); both assays detected
B.
trivialis
within 25 min. The BtrivCOI and BtrivEIF3L assay anneal derivatives were 82.7 ± 0.8 °C and 83.3 ± 1.3 °C, respectively, detecting down to 1 × 10
1
copies/µL and 1 × 10
3
copies/µL, respectively. Each assay amplified some non-targets from our test panel; however notably, BtrivCOI eliminated all morphologically similar non-targets, and combined, the assays eliminated all non-targets. Double-stranded DNA gBlocks were developed as positive controls; anneal derivatives for the COI and EIF3L gBlocks were 84.1 ± 0.7 °C and 85.8 ± 0.2 °C, respectively. We recommend the BtrivCOI assay for confirmation of suspect cue-lure-trapped
B.
trivialis
, with BtrivEIF3L used for secondary confirmation when required.
Abstract
Bactrocera tryoni
and
Bactrocera neohumeralis
are morphologically similar sibling pest fruit fly species that possess different biological attributes, geographic distributions, and host ...ranges. The need to differentiate between the two species is critical for accurate pest status assessment, management, biosecurity, and maintenance of reference colonies. While morphologically similar, adults may be separated based on subtle characters; however, some characters exhibit intraspecific variability, creating overlap between the two species. Additionally, there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between
B. neohumeralis
and
B. tryoni
; therefore, ambiguous samples remain undiagnosed. Here we report the first molecular marker that can consistently distinguish between
B. tryoni
and
B. neohumeralis
. Our diagnostic region consists of two adjacent single nucleotide polymorphisms (SNPs) within the
pangolin
(
pan
) gene region. We confirmed the genotypes of each species are consistent across their distributional range, then developed a tetra‐primer amplification refractory mutation system (ARMS) PCR assay for rapid diagnosis of the species. The assay utilizes four primers in multiplex, with two outer universal primers, and two internal primers: one designed to target two adjacent SNPs (AA) present in
B. tryoni
and the other targeting adjacent SNPs present in
B. neohumeralis
(GG). The assay accurately discriminates between the two species, but their SNP genotypes are shared with other nontarget tephritid fruit fly species. Therefore, this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches; maintaining pure colony lines; and in Sterile Insect Technique management responses.
Objective. Clinical interest in C-reactive protein (CRP) - a component of the innate immune system - has focused mainly on its worth as an indicator of disease activity. There has been a resurgence ...of interest in CRP in the Crohn's disease (CD) literature because several trials of new treatments for active CD have been characterized by both a large proportion of patients with low CRP (<10 mg/l) at entry to the trials and by a negative therapeutic outcome. It is therefore of interest to study the clinical characteristics of patients who are thought to have at the same time both active CD and a low CRP. Material and methods. Patients were prospectively recruited as part of the Brisbane IBD clinical and research programme. Subjects were included in the low CRP group only if there were complete datasets for CRP on all occasions of active CD, and CRP was < 10 mg/l. Active disease was defined as CD activity index (CDAI) > 200. The low CRP group was compared with patients in the raised CRP group for a range of clinical variables as well as the major NOD2 variants. Results. There were data sets for 223 CD patients, with a mean disease duration of 12 years. Of these, 22 patients fulfilled the criteria for low CRP. The low CRP group (group 1) showed significant differences for disease site (p<0.01) and for BMI (p=0.006) compared to the raised CRP group (group 2). Specifically, group 1 had a predominance of pure ileal disease (95% versus 53%) and lack of pure colonic disease (0% versus 24%) compared to group 2, and their BMI was significantly lower (20.3 kg/m2 versus 25.0 kg/m2). Groups 1 and 2 did not differ with respect to Vienna behaviour at diagnosis, smoking, appendicectomy, extra-intestinal manifestations of CD, or NOD2 SNP variants. There was a trend for low CRP patients with previous ileal resection to evolve to a stricturing phenotype. Fat wrapping was noted in 11/13 (85%) of low CRP patients undergoing ileal resections. Conclusions. Patients with CD and a persistently low CRP in the face of active disease were characterized by an almost exclusive ileal disease distribution and a low BMI, compared to those with a raised CRP. These patients had a similar frequency and distribution of NOD2/CARD15 variants. Stricturing (v inflammatory or penetrating) behaviour may explain some low CRP. Despite the abnormally low BMI, fat wrapping was noted in the majority of low CRP patients undergoing ileal resection.
Crohn's disease (CD) has been associated with CARD15/NOD2 mutations in Caucasians. The R702W, G908R, and 1007fs mutations represent 82% of the mutated chromosomes. The relative risk of developing CD ...in homozygous or compound heterozygous people has been estimated as between 10 and 40 times that of the general population. This high risk may support the opinion that CARD15/NOD2 variants are strong CD risk factors at the individual and population levels.
The allele and genotype frequencies were calculated for the R702W, G908R, and 1007fs mutations in 3,575 Caucasian healthy controls recruited by 15 groups distributed on three continents. Geographic homogeneity was tested and the observed proportion of double mutants was compared with the expected value using chi2 tests.
The allele frequencies of the R702W, G908R, and 1007fs mutations were 4.3% (3.6-4.9), 1.2% (0.8-1.6), and 2.3% (1.8-2.8), respectively, with large geographic fluctuations of the G908R, 1007fs, and wild-type alleles (P<0.0001). At the population level, no simple relationship was observed between mutation frequencies and the disease incidences in the studied populations. At the individual level, no significant deficit of double-dose mutation carriers among healthy controls was found, providing strong evidence that the penetrances of the most at-risk genotypes are low.
Altogether, these data confirm that CARD15/NOD2 acts in interaction with other unknown risk cofactors.
Bovine viral diarrhoea virus 1 (BVDV-1) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. To date many ...subgenotypes have been reported for BVDV-1, currently ranging from subgenotype 1a to subgenotype 1u. While BVDV-1 has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. As an example, BVDV-1 subgenotypes 1a and 1b are frequently detected in North America and Europe, while the subgenotype 1c is rarely detected. In contrast, BVDV-1 subgenotype 1c is by far the most commonly reported in Australia. Despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. The aim of this study was to characterise the in vivo properties of five strains of BVDV-1 subgenotype 1c in cattle infection studies. No overt respiratory signs were reported in any of the infected cattle regardless of strain. Consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. The quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. Further studies are required to fully understand the variability and importance of the BVDV-1 subgenotype 1c.
Angiotensin II (AII) is a powerful splanchnic vasoconstrictor with pro-inflammatory and pro-fibrotic properties. Angiotensin converting enzyme (ACE) inhibitors and AII Receptor Antagonists (ARBs) are ...therapeutic in animal models of colitis. The aim of this case-control study is to determine the expression of angiotensinogen and related genes in human ileal Crohn's disease.
Using quantitative real-time polymerase chain reaction (RT-PCR), we measured mRNA expression levels of angiotensinogen (AGT), hypoxia inducible factor (HIF)1α and melanoma cell adhesion molecule (MCAM; CD146) in 101 human samples (69 biopsy, 12 resection) from affected ileum (inflamed CD cases, n=36) and unaffected ileum (non-inflamed CD cases, n=45 and controls, n=20). Immunohistochemistry for angiotensinogen was also performed. The study was of case-control design in a tertiary care setting.
Ileal expression of AGT was lower in CD cases compared to controls (p<0.0001), in inflamed CD samples (p=0.017) and current smokers (p=0.02). HIF1α expression was lower in non-inflamed CD biopsy samples than controls (p=0.006). The presence of disease-associated NOD2 variants was associated with increased expression of markers of angiogenesis (HIF1α p=0.009; MCAM p=0.007) in inflamed CD samples. Angiotensinogen immunohistochemical staining supported the quantitative RT-PCR expression findings.
Angiotensinogen expression is down regulated in human ileal CD, particularly in the presence of inflammation and current cigarette smoking, implicating the mesenteric vasculature and mucosal hypoxia as co-factors in ileal CD pathogenesis. A novel reduction in HIF1α expression in non-inflamed ileal mucosa in CD patients was also demonstrated.
Crohn's disease (CD) and ulcerative colitis (UC) are the two most common forms of inflammatory bowel disease (IBD), representing a significant health-care burden. A variant in the autophagy gene ...ATG16L1 (T300A) has been newly identified as a CD susceptibility locus by genome-wide association. Our aim was to assess the contribution of T300A in determining disease susceptibility and phenotype in two independent Australian IBD cohorts and explore the relationship between T300A and known CD risk factors (NOD2nucleotide-binding oligomerization domain containing 2 status and smoking).
In total, 669 CD and 543 UC cases, and 1,244 controls (study 1), 154 CD cases and 420 controls (study 2), and 702 unaffected parents from both groups were genotyped. We conducted case-control and family association analyses, and investigated relationships between T300A and disease subgroups and between NOD2 status and cigarette smoking (CD only).
The strong association between CD and T300A was confirmed (P < 0.001), with a two-fold increase in disease risk associated with the GG genotype (odds ratio OR 1.96, 95% confidence interval CI 1.49-2.58), while ileal CD risk was almost three-fold (OR 2.73, CI 1.87-4.0). ATG16L1 and NOD2 were found to contribute independently to CD risk. A greater than seven-fold increased CD risk was observed for current smokers with a GG genotype (vs nonsmoking AA genotype; P < 0.001, OR 7.65, CI 4.21-13.91). A significant inverse association was found between T300A and UC (P= 0.002). This was strongest for patients with extensive, severe disease.
We confirm the strong association between T300A and CD, specifically ileal subphenotype, and also report the first strong association of this variant with UC.
Meleagrid herpesvirus 1 (MeHV-1) infectious bacterial artificial chromosomes (iBACs) are ideal vectors for the development of recombinant vaccines for the poultry industry. However, the full ...potential of iBACS as vectors can only be realised after thorough genetic characterisation, including identification of those genetic locations that are non-essential for virus replication. Generally, transposition has proven to be a highly effective strategy for rapid and efficient mutagenesis of iBAC clones. The current study describes the characterisation of 34 MeHV-1 mutants containing transposon insertions within the pMeHV1-C18 iBAC genome.
Tn5 and MuA transposition methods were used to generate a library of 76 MeHV-1 insertion mutants. The capacity of each mutant to facilitate the recovery of infectious MeHV-1 was determined by the transfection of clone DNA into chicken embryo fibroblasts.
Attempts to recover infectious virus from the modified clones identified 14 genetic locations that were essential for MeHV-1 replication in cell culture. Infectious MeHV-1 was recovered from the remaining 14 intragenic insertion mutants and six intergenic insertion mutants, suggesting that the respective insertion locations are non-essential for MeHV-1 replication in cell culture.
The essential and non-essential designations for those MeHV-1 genes characterised in this study were generally in agreement with previous reports for other herpesviruses homologues. However, the requirement for the mardivirus-specific genes LORF4A and LORF5 are reported for the first time. These findings will help direct future work on the development of recombinant poultry vaccines using MeHV-1 as a vector by identifying potential transgene insertion sites within the viral genome.
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