Development and evaluation of a point-of-care test with a combination of EZ-Fast DNA extraction and real-time PCR and LAMP detection: evaluation using blood samples containing the bovine leukaemia DNA.

Along with progress in globalization of society, the spread of infectious diseases has accelerated worldwide. The deployment of highly sensitive genetic tests is essential for early diagnosis and early containment of potential outbreaks and epidemics, as well as routine surveillance, although tedious and expensive nucleic acid extraction steps represent a major drawback. Here we developed a simple and rapid DNA extraction method, named as an EZ-Fast kit, applicable to the field setting. The kit does not require advanced laboratory equipment or expensive DNA extraction kits and achieves crude DNA extraction within 10 min at extremely low cost and can easily be performed in field settings. When combined with real-time PCR and LAMP analyses, the performance of the POCT, using 183 bovine blood samples, was similar to that of the existing DNA extraction method: 92·5% (135/146) (real-time PCR) and 93·7% (133/142) (LAMP) diagnostic sensitivities, and 100% diagnostic specificities. The developed POCT provides a powerful tool to facilitate on-site diagnosis in a field setting.

Development of a LAMP assay for rapid and sensitive detection and differentiation of Mycobacterium avium subsp. avium and subsp. hominissuis.

Mycobacterium avium causes atypical mycobacterial infection in humans and animals worldwide. M. avium comprises the subspecies avium (MAA), hominissuis (MAH), silvaticum (MAS) and paratuberculosis (MAP). The M. avium complex (MAC), comprising M. avium and M. intracellulare, causes opportunistic infections of humans. M. avium subsp. avium (MAA) mainly causes avian tuberculosis while subsp. hominissuis (MAH) mainly infects pig. Distinguishing between these two subspecies is essential to the effective control of these atypical mycobacterial infections and minimization of the resulting economic loss. For this purpose, we developed a loop-mediated isothermal amplification (LAMP) assay that rapidly and sensitively detects and differentiates MAA and MAH. This MAA-LAMP assay targeting IS901 correctly detected four MAA isolates but did not detect 27 MAH and 19 non-MAA/non-MAH mycobacterial isolates. The MAAH-LAMP assay targeting IS1245 detected four MAA and 27 MAH isolates but not the other 19 mycobacterial isolates. We believe that implementation of this LAMP assay will significantly improve public health and safety. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycobacterium avium, which is pathogenic for humans and animals, represents a continuing threat to public health and safety and to food production. Therefore, improved methods are urgently required to readily and efficiently identify M. avium subspecies. Compared with conventional PCR methods, the LAMP assay herein developed more rapidly detects and better distinguishes between two major M. avium subspecies that cause disease of pig. Importantly, this highly accurate and sensitive LAMP assay detects mycobacterial DNAs using real-time fluorescence or the unaided eye with a colour-change dye, making it ideal for translation to the clinic and slaughterhouse.

Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform.

Effective control and monitoring of foot-and-mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype-specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD-endemic countries have motivated the development of simple diagnostic platforms to support local decision-making. Using a portable thermocycler, the T-COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan-serotype-specific real-time RT-PCR (rRT-PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa-specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan-serotype-specific lyophilized assay were comparable to that of an OIE-recommended laboratory-based rRT-PCR (determined using a panel of 57 FMDV-positive samples and six non-FMDV vesicular disease samples for differential diagnosis). The FMDV-typing assay was able to correctly identify the serotype of 33/36 FMDV-positive samples (no cross-reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal-pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre-clinical, clinical and clinically recovered cattle. These data support the use of field-ready rRT-PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.

Campylobacter and Salmonella are prevalent in broiler farms in Kyushu, Japan: results of a 2-year distribution and circulation dynamics audit.

AIM: To elucidate the distribution and circulation dynamics of Campylobacter and Salmonella in Japanese chicken broiler flocks. METHODS AND RESULTS: A 2-year investigation of the distribution of Campylobacter and Salmonella was conducted in 25 broiler flocks at nine farms in Japan from 2013 to 2014. Campylobacter and Salmonella tested positive in 11 (44·0%) and 24 (96·0%) broiler flocks respectively. One hundred and ninety-five Campylobacter and 184 Salmonella isolates were characterized into 12 Campylobacter (including two novel genotypes) and three Salmonella MLST genotypes. Only Salmonella isolation between caecal and environmental samples were significantly correlated. Further, one litter sample tested positive for Salmonella before new chicks were introduced. The Campylobacter strains rapidly lost culturability within 2-18 days; in contrast, the Salmonella strains survived from 64-211 days in artificially inoculated water samples. CONCLUSION: No persistent circulation-mediated Campylobacter contamination was observed. In contrast, circulation of Salmonella in broiler houses was seen, apparently due to the litter excreted from broiler flocks, as well as Salmonella-contaminated water and feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the distribution, genotypic data and circulation dynamics of Campylobacter and Salmonella as recently observed in Japanese chicken broiler farms.

Development of a loop-mediated isothermal amplification assay for rapid and simple detection of Erysipelothrix rhusiopathiae.

UNLABELLED: Erysipelothrix rhusiopathiae is a causative agent of swine erysipelas. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of E. rhusiopathiae. The LAMP assay correctly detected 39 E. rhusiopathiae strains. No LAMP products were detected from 14 non-rhusiopathiae Erysipelothrix and 16 non-Erysipelothrix strains, including E. tonsillarum serovar 10 strains, which are difficult to be discriminated from E. rhusiopathiae strains. These results were consistent with those obtained by a conventional E. rhusiopathiae-specific PCR assay. Starting with DNA extraction from a single colony, the gel-based PCR assay took 4 h to provide a result, but the LAMP assay was faster, requiring only 37-80 min. The conventional culture test required more than 3-4 days to isolate and identify E. rhusiopathiae in the enrichment cultures. In contrast, the LAMP assay required less than 22 h from the beginning of the enrichment culture to final determination. These results suggest that the LAMP assay is useful as an adjunct to facilitate early diagnosis of swine erysipelas. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a loop-mediated isothermal amplification (LAMP) assay for simple and cost-effective detection of E. rhusiopathiae from swine samples. The LAMP assay provided more rapid detection of the bacterium than conventional PCR and biochemical-based assays, and it may potentially facilitate surveillance and early diagnosis of swine erysipelas in the field.

Imprinted gene expression in in vivo- and in vitro-produced bovine embryos and chorio-allantoic membranes.

Cloning by nuclear transfer is often associated with poor results due to abnormal nuclear reprogramming of somatic donor cells and altered gene expression patterns. We investigated the expression patterns of imprinted genes IGF2 and IGF2R in 33- to 36-day bovine embryos and chorio-allantoic membranes derived from in vivo- and in vitro-produced embryos by somatic cell nuclear transfer (SCNT), parthenogenetic activation, and in vitro fertilization (IVF). There was a lower IGF2 expression rate in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to in vivo and IVF embryos (2.01; P < 0.05). In the chorio-allantoic membranes, IGF2 showed a baseline expression pattern (P < 0.05) in parthenotes (0.001) when compared to in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was less expressed (P < 0.05) in SCNT chorio-allantoic membranes (0.25) when compared to the in vivo group. The low expression of IGF2 in parthenogenetic embryos and chorio-allantoic membranes confirms its imprinted status in cattle. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in SCNT-derived bovine embryos and chorio-allantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes of the low efficiency of SCNT procedures in this species.

Parthenogenetic activation of bovine oocytes using single and combined strontium, ionomycin and 6-dimethylaminopurine treatments.

In vitro-matured (IVM) bovine oocytes were activated with single and combined treatments of strontium (S), ionomycin (I) and 6-DMAP (D). Using oocytes IVM for 26 h, we observed that activation altered cell cycle kinetics (faster progression, MIII arrest, or direct transition from MII to pronuclear stage) when compared to in vitro fertilization. The effect of oocyte age on early parthenogenesis was assessed in oocytes IVM for 22, 26 and 30 h. Better results in pronuclear development were obtained in treatments ISD (81.7%) at 22 h; D (66.7%), IS (63.3%), ID (73.3%) and ISD (76.7%) at 26 h; and D (86.7%), IS (85.0%) and ID (78.3%) at 30 h. Higher cleavage occurred on ISD (80.0%) at 22 h; ID (83.3%) and ISD (91.7%) at 26 h; and I (86.7%), IS (90.0%), ID (85.0%) and ISD (95.0%) at 30 h. More blastocysts were achieved in ID (25.0%) and ISD (18.3%) at 22 h; and in ID at 26 h (45.0%) and 30 h (50.0%). We also observed that IS allowed higher haploid (77.4%) embryonic development, whilst ID was better for diploid (89.1%) development. It was concluded that association of S and D without I was not effective for blastocyst development; treatments using S were less influenced by oocyte age, but when S was associated with D there was a detrimental effect on aged oocytes; treatment ISD promoted higher activation and cleavage rates in young oocytes and ID protocol was the best for producing blastocysts.