The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

Adenosine triphosphate bioluminescence for hygiene testing of rubber liners and tubes on dairy farms.

Prevention of biofilm formation in milking equipment is important to ensure good hygiene quality of raw milk. Key factors to achieving good results are a successful cleaning procedure and a method to check the cleanliness of milking equipment surfaces. Adenosine triphosphate bioluminescence is a fast and easy method for investigating bacterial contamination of surfaces. However, previous studies on the potential of ATP bioluminescence to assess the hygiene status of milking equipment have been hampered by lack of a validated test procedure. The aim of this work was therefore to establish a test procedure for assessing the cleanliness of milking equipment using ATP bioluminescence, and apply the method on-farm to study the hygiene status of aging rubber material in milking equipment. In developing the test procedure, the effects of sampling location in tubes and liners, sampling of dry versus wet barrels, milking point in the parlor, and acid or alkali detergent on ATP values were investigated. The results showed that, to obtain reproducible results, replicate sampling from the same milking points in the parlor is important. For milk tubes, samples should preferably be taken from the milk meter side, for liners on the inside of the barrel. For best results, sampling should be performed after use of alkali detergent. No beneficial effect was observed of sampling dry liner barrels, so sampling in the standardized test procedure is performed directly after cleaning. The standardized test procedure was used on 3 different commercial farms and sampling was initiated after replacement of old rubber parts. On one of the farms, additional sampling was performed to evaluate total bacteria count and determine the association with ATP level. The results suggest that, provided an efficient cleaning procedure is used, the hygiene quality of milking equipment can be maintained during the recommended lifetime of the rubberware. However, due to occasional variation in cleaning efficiency between milking points and liner barrels, random sampling on single occasions can lead to incorrect conclusions. Replicate sampling over time is therefore important for correct interpretation of ATP bioluminescence data. If ATP levels are very high, complementary sampling for total bacteria count should be used to verify that the level is due to bacterial contamination, and not other organic ATP-contributing material (e.g., milk residues).

Validation of commercial luminometry swabs for total bacteria and coliform counts in colostrum-feeding equipment.

A sufficient quantity and quality of colostrum must be fed quickly to the newborn calf while minimizing bacterial contamination. Adenosine triphosphate bioluminescence swabs offer a potential rapid on-farm alternative to assess bacterial contamination of colostrum. The objective of this study was to validate the Hygiena (Camarillo, CA) AquaSnap Total (AS), SuperSnap (SS), PRO-Clean (PC), and MicroSnap Coliform (MS) swabs as well as visual hygiene assessment for detection of elevated bacterial counts in or on colostrum-feeding equipment. From April to October 2016, 18 esophageal tube feeders, 49 nipple bottles, and 6 pails from 52 dairy farms in Ontario were evaluated for cleanliness. Following visual hygiene assessment, sterile physiological saline (15 mL) was poured into each piece of equipment, mixed for 2 min to ensure total surface coverage, and poured into a sterile collection container through the feeding end. The fluid was split into equal aliquots, with one being evaluated by conventional culture and the other evaluated using the luminometry swabs. Nonparametric receiver operator curves were used to compare the test performance of the luminescence reading (relative light units; RLU) from each type of swab to conventional bacterial culture. The area under the curve comparing the AS swab to total bacterial count (cut point >100,000 cfu/mL) was 0.89, and using a cut point of 631 RLU correctly classified 84% of samples with a sensitivity of 88% and a specificity of 77%. The area under the curve comparing the MS swab to total coliform count (cut point >10,000 cfu/mL) was 0.85, and using a cut point of 44 RLU correctly classified 89% of samples with a sensitivity of 83% and a specificity of 90%. Visual hygiene assessment, PC and SS swabs were not reliable indicators for feeding equipment cleanliness. The results suggest that the AS and MS swabs can be used as an alternative to traditional laboratory bacterial counts to evaluate cleanliness of colostrum-feeding equipment.

Investigation of Medium Chain Fatty Acid Feed Supplementation for Reducing Salmonella Typhimurium Colonization in Turkey Poults.

Studies indicate that persistent Salmonella colonization occurs in poultry that are infected early in life, leading to both food safety and public health concerns. Development of improved preharvest Salmonella management strategies is needed to reduce poultry product contamination. The objective of this study was to evaluate the efficacy of a product containing medium chain fatty acids (MCFA) for reducing early Salmonella colonization in turkey poults. Day-of-hatch turkeys were provided a standard starter diet supplemented with MCFA at 0 (negative and positive controls), 1.5, 3, 4.5, or 6 lbs/ton of feed. Positive control and MCFA treated birds were also crop-gavaged with 108 colony forming units (CFU) of bioluminescent Salmonella Typhimurium. Gastrointestinal tissue samples were collected at 3 days postinoculation for bioluminescence imaging (Meckel's diverticulum to the cloaca) and selective enumeration (cecal contents). Quantification of bioluminescence indicated that the 4.5 and 6 lbs/ton MCFA groups had significantly less colonization than the positive control group (p = 0.0412 and p < 0.0001, respectively). Similarly, significantly lower numbers (1-log10 CFU/g reduction) of Salmonella were observed in the ceca of the 6 lbs/ton MCFA group compared to the positive control group (p = 0.0153). These findings indicate that incorporation of MCFA in turkey diets can significantly reduce early Salmonella colonization. In addition, this study highlights the utility of bioluminescence imaging as a screening methodology for assessing the efficacy of treatments that may reduce Salmonella in poultry.

Effect of tricainemethanesulfonate (MS222), clove oil and electro-anaesthesia on respiratory burst activity in whole blood and serum alternative complement response in rainbow trout (Oncorhynchus mykiss), during the narcosis stage.

There is a little available information on the suppressive effect of anaesthesia on immune response in fish, especially electro-anaesthesia. In the present study, two anaesthetics, MS222 (50 ppm), clove oil (25 ppm), and electro-anaesthesia were tested in rainbow trout (Oncorhynchus mykiss) during the narcosis stage in order to observe their effects on the innate immune system. The results showed that electro-anaesthesia reduces light emission in chemiluminescence assay both 1 and 24 h post anaesthesia. Clove oil and MS222 decreased light emission 24 h post anaesthesia. In addition, clove oil, MS222 and electro-anaesthesia had no effect on alternative complement (ACH50) response. From the perspective of aquaculture practice, these data show that the type of anaesthesia should be taken into account to avoid possible immunosuppression in rainbow trout.

NOD/SCID mouse model of canine T-cell lymphoma with humoral hypercalcaemia of malignancy: cytokine gene expression profiling and in vivo bioluminescent imaging.

Lymphoma is a malignant neoplasm arising from B or T lymphocytes. In dogs, one-third of lymphomas are highly aggressive multicentric T-cell lymphomas that are often associated with humoral hypercalcaemia of malignancy (HHM). There are no cell lines or animal models to investigate the pathogenesis of T-cell lymphoma and HHM in dogs. We developed the first xenograft model by injecting lymphoma cells from an Irish Wolfhound intraperitoneally into NOD/SCID mice. The mice developed multicentric lymphoma along with HHM and increased parathyroid hormone-related protein (PTHrP) as occurs in dogs with T-cell lymphoma. Using cytokine complementary DNA arrays, we identified genes that have potential implications in the pathogenesis of T-cell lymphoma. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of T-cell lymphoma samples from hypercalcaemic canine patients showed that PTHrP likely plays a central role in the pathogenesis of HHM and that hypercalcaemia is the result of a combinatorial effect of different hypercalcaemic factors. Finally, we monitored in vivo tumour progression and metastases in the mouse model by transducing the lymphoma cells with a lentiviral vector that encodes a luciferase-yellow fluorescent protein reporter and showed that in vivo trafficking patterns in this model were similar to those seen in dogs. This unique mouse model will be useful for translational research in lymphoma and for investigating the pathogenesis of T-cell lymphoma and HHM in the dog.

Production of reactive oxygen species (ROS) by devil stinger (Inimicus japonicus) during embryogenesis.

In aqua-cultural industry, the seed production of devil stinger, a valuable fish in Japan, has not succeeded yet due to the cryptogenic mass mortality. We found that survival rate of the larvae of devil stinger increased by the addition of green tea extract rich in catechin into rearing tank. Generation of reactive oxygen species (ROS) was detected in the embryo of devil stinger by chemiluminescence analysis under the normal growth conditions without addition of specific stimulants. Even in the unfertilized egg, certain level of ROS was detected. ROS were continuously detected during the development from fertilized egg to larva and tended to increase gradually. Observation of embryos and post-hatching larvae with hypersensitive photon-counting microscopy indicated that ROS were produced on the surface of embryo and the head region of larva especially peripheries of eyes. When the embryo proteins were analyzed by immunoblotting using antibody against the human neutrophil cytochrome b558 large subunit (gp91 phox), a main band of approximately 91 kDa was detected, suggesting the presence of NADPH oxidase-like ROS generating system in the embryo of devil stinger. After treatment with streptomycin and penicillin G for 1 day, the level of ROS production in larvae decreased with increase in the survival rate of larvae. Our results suggest that devil stinger has ROS generation system that is already activated at fairly early stage of development before the maturation of usual immune system.