Quantitative measurement of reproductive output in the American oyster, Crassostrea virginica (Gmelin), using an enzyme-linked immunosorbent assay (ELISA)

Abstract. A quantitative gonadal index was developed for oysters, Crassostrea virginica (Gmelin. 1791), using polyclonal antibodies from eggs and sperm. Percoll used in the purification of oyster eggs and sperm greatly improved the purity of antigens compared to filtering the egg or sperm through a fine mesh only. The antigen-antibody reaction was tested with indirect sandwich ELISA using alkaline phosphatase-conjugated goat anti-rabbit igG as a secondary antibody. Rabbit anti-oyster egg IgG and anti-oyster sperm IgG initially exhibited a weak cross-reactivity over somatic tissue. Absoring with acetone-dried oyster tissue powder removed this cross-reactivity. Both antisera exhibited strong specific immunological reactions to oyster eggs or sperm respectively. The quantity of eggs or sperm was measured using ELISA and a quantitative gonadosomatic index (dry wt of egg or sperm/dry wt oyster) (GSI) calculated. GSI from ELISA correlated with gonadal stage measured histologically. Monthly mean GSI of female oysters was highest during late spring to early summer (0·157–0·201) and lowest during early winter to early spring (0·002-0·000). Maximum GSI observed during the study was 0·422 for female oysters and 0·446 for male oysters. Female oysters produce 3·7–65·4 million eggs, with an average of 21·1 million during each spawning. A positive correlation was observed between the number of eggs produced and oyster size; the number of eggs in the gonad increased as oyster size (i.e. total dry wt) increased (r= 0·67); however, the relationship was non-linear. Large oysters contained proportionally fewer eggs. Prevalence of Perkinsus marinus parasitism was high, 90–100%, during the study, as was weighted incidence, 1·33 to 2·67, No statistically significant correlation was observed between infection intensity and the per cent weight of oyster eggs or egg number.     

Antibody responses after vaccination against equine influenza in the Republic of Korea in 2013

In this study, antibody responses after equine influenza vaccination were investigated among 1,098 horses in Korea using the hemagglutination inhibition (HI) assay. The equine influenza viruses, A/equine/South Africa/4/03 (H3N8) and A/equine/Wildeshausen/1/08 (H3N8), were used as antigens in the HI assay. The mean seropositive rates were 91.7% (geometric mean antibody levels (GMT), 56.8) and 93.6% (GMT, 105.2) for A/equine/South Africa/4/03 and A/equine/Wildeshausen/1/08, respectively. Yearlings and two-year-olds in training exhibited lower positive rates (68.1% (GMT, 14) and 61.7% (GMT, 11.9), respectively, with different antigens) than average. Horses two years old or younger may require more attention in vaccination against equine influenza according to the vaccination regime, because they could be a target of the equine influenza virus.     

Immunological study of an attenuated Salmonella Typhimurium expressing ApxIA,ApxIIA, ApxIIIA and OmpA of Actinobacillus pleuropneumoniae in a mouse model

Salmonella Typhimurium strain expressing the Actinobacillus pleuropneumoniae antigens, ApxIA, ApxIIA, ApxIIIA and OmpA, was previously constructed as a vaccine candidate for porcine pleuropneumonia. This strain was a live attenuated (∆lon∆cpxR∆asd)Salmonella as a delivery host and contained a vector containing asd. An immunological study of lymphocyte proliferation, T-lymphocyte subsets and cytokines in the splenocytes of a mouse model was carried out after stimulation with the candidate Salmonella Typhimurium by intranasal inoculation. The splenic lymphocyte proliferation and the levels of IL-4, IL-6 and IL-12 of the inoculated mice were significantly increased, and the T- and B-cell populations were also elevated. Collectively, the candidate may efficiently induce the Th1- and Th2-type immune responses.     

Optimal placement of the region of interest for bolus tracking on brain computed tomography angiography in Beagle dogs

This study aimed to determine the optimal placement of the region of interest (ROI) among four anatomical sites—pulmonary artery (PA), pulmonary vein (PV), aortic arch (AA), and carotid artery (CA)—in computed tomography (CT) brain angiography with automatic bolus tracking in healthy beagle dogs. Six beagles were included, and CT brain angiography was performed four times for each dog, to cover each ROI. The scan parameters, amount, and injection rate of the contrast medium were the same. The major intracranial arteries were selected for quantitative and qualitative evaluation: caudal cerebellar artery (CcA), basilar artery (BA), rostral cerebellar artery (RcA), caudal cerebral artery (CCA), middle cerebral artery (MCA), and rostral cerebral artery (RCA). Quantitative evaluation showed significantly higher CT attenuation values for the RcA, CCA, and MCA in the PA group and RcA and MCA in the PV group than in the CA group. Qualitative analysis revealed significantly higher scores for the BA, CCA, and MCA in the PA and PV groups than in the CA group. Venous contamination did not differ significantly among the ROIs, but the mean scores of the AA and CA groups were higher than those of the PA and PV groups. CT brain angiography using bolus tracking in the beagle dogs showed that the ROI should be placed at the PA or PV rather than at the CA for optimal images with strong contrast enhancement of the BA, RcA, CCA, and MCA and minimal venous contamination.     

A rapid molecular method for diagnosing epidemic dermatophytosis in a racehorse facility

Reasons for performing study: Identification of the species and strain of dermatophyte can play an effective role in control of disease outbreaks by establishing the source of infection. Current methods of identification are based on cultural and microscopic methods, often involving weeks before a positive identification are made. A rapid molecular diagnostic method would therefore be an important laboratory technique, but requires confirmation in equine clinical practice. Objectives: To test the sensitivity and specificity of molecular diagnostic methods applied to a racehorse herd from the Korean Racehorse Authority (KRA). Methods: A total of 57 DNA samples were collected from hairs and crusts of skin lesions in KRA racehorses with histories and clinical signs suggestive of dermatophytosis, which was confirmed by dermatophyte‐specific PCR amplification analysis using the primer pair for the chitin synthase 1 (CHS1) gene. Results: Thirty‐eight racehorses were definitively diagnosed with dermatophytosis using molecular and traditional diagnostic methods. PCR fingerprinting profiles using simple repetitive (GACA)₄ primers showed that all diagnosed horses had the same pattern profile. Oligonucleotide sequencing of CHS1 gene PCR products confirmed Trichophyton mentagrophytes as the infectious agent. Conclusions: This study demonstrates that the PCR‐based molecular diagnostic method is sensitive and specific and offers fast precise diagnosis of dermatophytosis in horses.     

Comparing recombinant MPB70/SahH and native 20-kDa protein for detecting bovine tuberculosis using ELISA

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. Test-and-cull protocols and gross pathological examinations of abattoir animals as well as milk pasteurization have been implemented to prevent the spread of tuberculosis from animals to humans worldwide. Despite the importance of precise and rapid diagnostic tests, conventional methods including intradermal skin tests and γ-interferon assays are limited by the high rate of false-negative results for cattle in the late infectious stage and due to laborious and time-consuming procedures. Therefore, antibody detection methods such as enzyme-linked immunosorbent assay (ELISA) are urgently needed to supplement the established approaches and expand the diagnostic window. This study was conducted to develop a bTB ELISA by evaluating recombinant and native proteins and various assay parameters. We produced recombinant MPB70 and SahH (M70S) and a native 20-kDa protein (20K) and optimized the ELISA protocol. The 20K ELISA showed 94.4% sensitivity and 98.2% specificity with an optimal sample-to-positive ratio cut-off of 0.531. The sensitivity and specificity of M70S ELISA were 94.4% and 97.3%, respectively, with an optimal sample-to-negative ratio cut-off of 1.696. Both assays showed acceptable diagnostic efficiency and could be used for bTB diagnosis in combination with established methods for herd screening and to expand the diagnostic window.     

Ultraviolet B radiation-mediated stress ethylene emission from rice plants is regulated by 1-aminocyclopropane-1-carboxylate deaminase-producing bacteria

Dear Editor,Industrial emissions have played a major role in damaging the ozone layer,leading to increased atmospheric penetration of ultraviolet B (UV-B)(290–320 nm) radiation (Jordan,2002).The recovery rate of the damaged ozone layer in the mid-latitudes and tropics has been insignificant since 1979(Chipperfield et al.,2017),even after the adoption of the Montreal Protocol (Protocol,1987),and the recovery process is expected to take many decades.In the meantime,UV-B radiation continues to penetrate the atmosphere (McKenzie et al.,2011;Bais et al.,2019).The characteristic short length and high energy of this radiation wavelength can directly affect major molecules,such as DNA,proteins,and lipids,which are important to living beings (Hideg et al.,2013).Rice is one of the major global crops,which is generally cultivated at lower latitudes and can potentially be exposed to a higher flux of UV radiation due to greater solar angles and longer duration of sunlight (Kataria and Guruprasad,2012).