Development and Application of a Loop‐mediated Isothermal Amplification Assay for Rapid Detection of Borrelia burgdorferi s. l. in Ticks

A loop‐mediated isothermal amplification (LAMP) assay was developed to detect Borrelia burgdorferi s. l. in ticks, which is a pathogen that causes Lyme disease. Cross‐reactions with Chlamydia psittaci, Mycoplasma mycoides subsp. capri and some tick‐borne pathogens were excluded. Analytical sensitivity of LAMP showed its detection limit was from 0.02 to 0.2 pg of DNA in detection of the reference samples at 65°C for 40 min. The performance of LAMP was assessed by testing 110 samples from susceptible tick species and comparing the results with conventional and nested PCR tests previously described. The results demonstrated that LAMP was significantly more sensitive than the conventional PCR (32.7% versus 15.5%, P < 0.05) and slightly more sensitive, although not significantly so, than nested PCR (32.7% versus 26.4%, P > 0.05). The assay was used to analyse a total of 1052 ticks collected from eight provinces in China. The results showed that the infection rates of B. burgdorferi s. l. varied from 12.5% to 88.9% across the different geographical sites. Selected positive samples were subjected to sequencing and sequence analysis for conformation of the accuracy of the assay. Here we report a highly sensitive, specific and easy diagnostic assay based on LAMP technology. These data indicate that LAMP is a useful approach for detecting B. burgdorferi s. l. in field‐collected ticks and has the potential as an alternative tool for the ecological and epidemiological surveillance of Lyme disease.     

Characterization of a Tembusu Virus Isolated from Naturally Infected House Sparrows (Passer domesticus) in Northern China

The house sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Tembusu virus (TMUV) strain, TMUV‐SDHS, was isolated from house sparrows living around the poultry farms in Shandong Province, Northern China. Genetic analysis of E and NS5 genes showed that it had a close relationship with that of the YY5 strain, which can cause severe egg drop in ducks. Pathogenicity studies showed that the virus is highly virulent when experimentally inoculated into the ducks. These findings show that house sparrows carrying the Tembusu virus may play an important role in transmitting the virus among other species.     

Assessment of Preparation of Samples Under the Field Conditions and a Portable Real‐Time RT‐PCR Assay for the Rapid On‐Site Detection of Newcastle Disease Virus

Newcastle disease virus (NDV), also known as virulent forms of avian paramyxovirus serotype 1 (AMPV‐1), is the causative agent of Newcastle disease affecting many species of birds and causing heavy losses to the poultry industry worldwide. Early, rapid and sensitive detection of the viruses or the viral nucleic acids is very important for disease diagnosis and control. This study aimed to evaluate sample preparation under field conditions and the application of a real‐time RT‐PCR method in the portable T‐COR4 platform for the rapid, on‐site detection of NDV on a farm. In the laboratory setting, the portable real‐time RT‐PCR assay had a similar performance compared with that obtained with a larger, stationary Rotor Gene real‐time thermocycler. In the field conditions, viral nucleic acids were manually extracted just outside of animal units with minimal equipment and real‐time RT‐PCR detection was performed with the portable thermocycler T‐COR4 placed in a nearby room. The portable assay at the farm detected viral RNA in 15 samples and reached an agreement of 83% (39/47) when the same RNA preparations were tested in the Rotor Gene thermocycler under the laboratory setting. The results demonstrated the feasibility of performing field detection but also the need to improve and further simplify sample preparation procedures.     

Experimental Infection of Giraffe (Giraffa camelopardalis) With SAT‐1 and SAT‐2 Foot‐and‐Mouth Disease Virus

The potential role of giraffe (Giraffa camelopardalis) in the epidemiology and spread of foot‐and‐mouth disease (FMD) SAT types was investigated by experimental infection and detection of virus in excretions using virus isolation on primary pig kidney cell cultures. In two experiments separated by a period of 24 months, groups of four animals were needle infected with a SAT‐1 or SAT‐2 virus, respectively and two in‐contact controls were kept with each group. Viraemia was detected 3–9 days post‐infection and virus isolated from mouth washes and faeces only occasionally up to day 13. The SAT‐1 virus was transmitted to only one in‐contact control animal, probably via saliva that contained virus from vesicles in the mouth of a needle‐infected animal. None of the animals infected with the SAT‐2 virus had any vesicles in the mouth, and there was no evidence of transmission to the in‐contact controls. No virus was detected in probang samples for the duration of the experiments (60 days post‐infection), indicating that persistent infection probably did not establish with either of these isolates. Giraffe most likely do not play an important role in FMD dissemination. Transmission of infection would possibly occur only during close contact with other animals when mouth vesicles are evident.     

Detection and Quantification of Bovine Papilloma Virus Type 2 (BPV‐2) by Real‐time PCR in Urine and Urinary Bladder Lesions in Enzootic Bovine Haematuria (EBH)‐Affected Cows

This study was conducted with the objectives of detecting bovine papillomavirus type 2 (BPV‐2) in urine samples and urinary bladder lesions in bovines using polymerase chain reaction (PCR) and real‐time PCR‐based molecular diagnostic tests, and quantifying BPV‐2 in urinary bladder lesions especially in enzootic bovine haematuria (EBH)‐affected animals. BPV‐2 viral DNA was detected in urine samples (50%) and urinary bladder tissue (68.6%). Cloning and sequencing results showed a close homology with other Indian BPV‐2 sequences. Quantitative real‐time PCR (SYBR Green assay) showed that the BPV‐2 load was low and similar irrespective of inflammatory or neoplastic lesions in the bladder. It was concluded that BPV‐2 DNA is frequently present in urine and urinary bladder lesions in cows in an EBH endemic region and virus load was low in urinary bladder lesions.     

Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC)‐mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of caspase‐3, mitochondria dysfunction and nitric oxide (NO) in human osteogenic sarcoma U‐2 OS cells

Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), a member of the isothiocyanate family, have been shown to exhibit antineoplastic ability against many human cancer cells. In this study, we found that exposure of human osteogenic sarcoma U‐2 OS cells to BITC and PEITC led to induce morphological changes and to decrease the percentage of viable cells in a time‐ and dose‐dependent manner. BITC and PEITC induced cell cycle arrest at G2/M phase at 48 h treatment and inhibited the levels of cell cycle regulatory proteins such as cyclin A and B1 in U‐2 OS cells but promoted the level of Chk1 and p53 that led to G2/M arrest. BITC and PEITC induced a marked increase in apoptosis (DNA fragmentation) and poly(ADP‐ribose)polymerase (PARP) cleavage, which was associated with mitochondrial dysfunction and the activation of caspase‐9 and ‐3. BITC and PEITC also promoted the ROS production in U‐2 OS cells and the N‐acetylcysteine (NAC, an antoxidant agent) was pretreated and then treated with both compounds which led to decrease the levels of ROS and increase the cell viability. Interestingly, BITC and PEITC promoted the levels of NO production and increased the iNOS enzyme. Confocal laser microscope also demonstrated that BITC and PEITC promoted the release of cytochrome c and AIF, suggesting that both compounds induced apoptosis through ROS, caspase‐3 and mitochondrial, and NO signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC and PEITC‐caused growth inhibition, G2/M arrest, and apoptotic death of U‐2 OS cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1199–1209, 2011     

Molecular Detection of Tick‐Borne Pathogens of the Family Anaplasmataceae in Brazilian Brown Brocket Deer (Mazama gouazoubira,Fischer, 1814) and Marsh Deer (Blastocerus dichotomus,Illiger, 1815)

Deer are important natural reservoir hosts of Anaplasmataceae. The present study used nested PCR and nucleotide sequencing to evaluate the occurrence of Anaplasmataceae species in 23 free‐living and six captive specimens of the cervids Mazama gouazoubira and Blastocerus dichotomus in Minas Gerais State, Brazil. Blood samples were tested for the presence of Ehrlichia and Anaplasma spp. using nPCR assays and sequencing of the msp4, msp1 and 16S rRNA genes. The identity of each sequence was confirmed by comparison with sequences available from GenBank using BLAST software. Of the animals investigated, 93.1% (27/29) were infected with haemoparasites including Anaplasma marginale (79.3%), Ehrlichia chaffeensis (3.4%), Anaplasma bovis (3.4%) and Anaplasma spp. (assigned to A. platys and A. phagocytophilum) (17.2%). Co‐infection occurred in 20% (6/29) of the deer examined. Four (13.8%) were infected with A. marginale and Anaplasma sp., one (3.4%) was infected with A. marginale and E. chaffeensis, and one (3.4%) was infected with A. marginale and A. bovis. The results of the present study suggest that cross‐protection does not occur in these deer. Immunological cross‐reaction occurs when sera are tested diagnostically because these bacteria are closely related taxonomically, reinforcing the importance of molecular diagnosis followed by nucleotide sequencing.     

Attenuation of cartilage pathogenesis in post‐traumatic osteoarthritis (PTOA) in mice by blocking the stromal derived factor 1 receptor (CXCR4) with the specific inhibitor,AMD3100

SDF‐1 was found to infiltrate cartilage, decrease proteoglycan content, and increase MMP‐13 activity after joint trauma. In this study, we tested the hypothesis that interference of the SDF‐1/CXCR4 signaling pathway via AMD3100 can attenuate pathogenesis in a mouse model of PTOA. We also tested the predictive and confirmatory power of fluorescence molecular tomography (FMT) for cartilage assessment. AMD3100 was continuously delivered via mini‐osmotic pumps. The extent of cartilage damage after AMD3100 or PBS treatment was assessed by histological analysis 2 months after PTOA was induced by surgical destabilization of the medial meniscus (DMM). Biochemical markers of PTOA were assessed via immunohistochemistry and in vivo fluorescence molecular tomography (FMT). Regression analysis was used to validate the predictive power of FMT measurements. Safranin‐O staining revealed significant PTOA damage in the DMM/PBS mice, while the DMM/AMD3100 treated mice showed a significantly reduced response with minimal pathology. Immunohistochemistry showed that AMD3100 treatment markedly reduced typical PTOA marker expression in chondrocytes. FMT measurements showed decreased cathepsins and MMP activity in knee joints after treatment. The results demonstrate that AMD3100 treatment attenuates PTOA. AMD3100 may provide a viable and expedient option for PTOA therapy given the drug's FDA approval and well‐known safety profile. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1071–1078, 2015.