Varroa destructor has emphasized the significance of viruses as pathogens of honey bee (Apis mellifera) populations. In particular, the association of deformed wing virus (DWV) with V. destructor and its devastating effect on honey bee colonies has led to that virus now becoming one of the most well-studied insect viruses. However, there has been no opportunity to examine the effects of Varroa mites without the influence of DWV. In Papua New Guinea (PNG), the sister species, V. jacobsoni, has emerged through a host-shift to reproduce on the local A. mellifera population. After initial colony losses, beekeepers have maintained colonies without chemicals for more than a decade, suggesting that this bee population has an unknown mite tolerance mechanism. Using high throughput sequencing (HTS) and target PCR detection, we investigated whether the viral landscape of the PNG honey bee population is the underlying factor responsible for mite tolerance. We found A. mellifera and A. cerana from PNG and nearby Solomon Islands were predominantly infected by sacbrood virus (SBV), black queen cell virus (BQCV) and Lake Sinai viruses (LSV), with no evidence for any DWV strains. V. jacobsoni was infected by several viral homologs to recently discovered V. destructor viruses, but Varroa jacobsoni rhabdovirus-1 (ARV-1 homolog) was the only virus detected in both mites and honey bees. We conclude from these findings that A. mellifera in PNG may tolerate V. jacobsoni because the damage from parasitism is significantly reduced without DWV. This study also provides further evidence that DWV does not exist as a covert infection in all honey bee populations, and remaining free of this serious viral pathogen can have important implications for bee health outcomes in the face of Varroa.The global spread of the parasitic mite  Apis mellifera) populations, with none more so than the parasitic mite Varroa destructor and several associated viruses [A. cerana) host last century, V. destructor has spread globally, causing significant harm to A. mellifera populations by feeding on developing pupae and adult bees. Importantly, this host shift also led to the emergence of virulent iflavirus strains of deformed wing virus (DWV), of which there are three known master variants  that are effectively vectored by V. destructor [V. destructor, and from this it has been generalised that DWV exists latently in all A. mellifera populations [A. mellifera population, which has yet to be invaded by parasitic bee mites, and the absence of DWV from New Zealand until the arrival of V. destructor [V. destructor and DWV have left few opportunities to examine their individual impacts [A. mellifera population that does not have V. destructor but has recently experienced a host shift from the sister mite species V. jacobsoni [Pathogens have a major impact on honey bee  are not native to PNG. The two species currently found there, A. mellifera and A. cerana, were introduced in the last 80 years through human activities. European honey bees (A. mellifera) were first introduced in the late 1940s from Australia [A. mellifera in PNG are descendants of bees originally imported from Australia and New Zealand. Apis cerana were first introduced into Indonesian Papua  from Indonesian Java during the 1970s with human transmigration [A. cerana also brought the parasitic mite, V. jacobsoni, of which it is the native host [A. cerana for ca. three decades, invasively spreading with A. cerana to nearby Solomon Islands in 2003 [A. mellifera in PNG by 2008. After initial colony losses, PNG beekeepers were reportedly managing to withstand V. jacobsoni infestations without any chemical or cultural control (pers. comm J. Buka IHBA).Honey bees  the miticide Bayvarol was used for the first time in PNG in efforts to eradicate T. mercedesae [V. destructor, PNG beekeepers have continued to persist without any targeted mite management [A. mellifera population to better resist the impact of parasitic mites.Soon after the emergence of pathogenic ndonesia ,18. DWV ndonesia ,23. The rcedesae . This wanagement . This suV. jacobsoni is related to a different viral landscape compared with V. destructor-affected honey bee populations. There is very little knowledge of the viruses infecting honey bees in PNG, although previous serological testing of A. mellifera colonies identified the common bee viruses, sacbrood virus (SBV), black queen cell virus (BQCV), Kashmir bee virus (KBV) and chronic bee paralysis virus (CBPV) [A. mellifera in PNG were originally imported from Australia, which is free of DWV, and New Zealand before DWV was present there, perhaps this population is also not infected by strains of DWV, and hence their absence may confer a level of tolerance toward V. jacobsoni. To assess this, we used a combination of high-throughput sequencing (HTS) and reverse-transcription PCR to identify RNA viruses infecting the bees and mites in PNG and bees in nearby Solomon Islands.In this study, we investigated if the mechanism conferring tolerance to s (CBPV) . We hypoV. jacobsoni that were reproducing on A. mellifera were collected during their initial discovery in 2008 from brood cells of two different A. mellifera colonies in Goroka, PNG. Mites were collected in RNAlater  and stored at −20 °C.Female A. mellifera and A. cerana worker bees were collected from colonies across the PNG Highlands in 2014, 2015 and 2018, and from Solomon Islands in 2014  extraction protocol, except for precipitated RNA, which was left in 70% ethanol. The samples were then purified using the Purelink RNA mini kit  and on-column Purelink DNase treatment protocol  before being eluted in 50 µL of RNase-free water. Illumina mRNA libraries were prepared from the extracted RNA and 100 bp paired-end sequences were generated on an Illumina HiSeq 2000 .A. mellifera samples collected in May 2014 from PNG and September 2014 from Solomon Islands were used for HTS with viral enrichment steps. First, 50 worker bees per apiary location were macerated in 5 mL of 0.05 M potassium phosphate buffer in an extraction bag . Two pooled samples were created using 1 mL of each apiary sample  into a screw-cap centrifuge tube and adjusted with 0.05 M potassium phosphate buffer for a total volume of 20 mL. 3 mL diethyl ether and 3 mL chloroform were added, shaken vigorously and centrifuged at 6000 rpm for 30 min . Supernatants were transferred to Ultraclear SW28 tubes  and centrifuged at 21,500 rpm for 3.5 h at 4 °C . Pelleted samples were dissolved in 1 mL of 0.05 M potassium phosphate buffer, then passed through a 0.22 µm filter to remove bacterial contamination. A total of 340 µL of each filtered sample was mixed with 10 µL of RNase, 10 µL DNase and 40 µL of DNase I buffer and incubated at 37 °C for 2 h. RNA was extracted from the treated samples using the Purelink viral RNA extraction kit . Illumina mRNA libraries were prepared and 100 bp paired-end sequences were generated on an Illumina HiSeq 2500 .A. mellifera (11 samples) and A. cerana (six samples) samples collected in November 2015 across the PNG Highlands were tested by RT-PCR for DWV. As above, 30 worker bees per apiary location were macerated in 5 mL of 0.05 M potassium phosphate buffer in an extraction bag . RNA was extracted from 200 µL of sample using the Purelink viral RNA/DNA extraction kit  and tested by real-time RT-PCR for DWV-A and DWV-B using the primers developed by Kevill et al. [® No-ROX One-Step mix  alongside synthetic positive controls for the target region of DWV-A and DWV-B produced by GeneArt™ . RNA quality of each sample was also confirmed by amplification of the honey bee gene vitellogenin [l et al. . One-stellogenin .A. mellifera (13 samples) and A. cerana (three samples) samples collected in May and October 2018 from PNG were tested by RT-PCR for DWV and also used for HTS with only limited viral enrichment. As above, 30 worker bees per apiary location were macerated in 5 mL of 0.05 M potassium phosphate buffer in an extraction bag  and 200 µL used for RNA extraction for real-time RT-PCR. In addition, 1 mL of each sample was centrifuged at 17,000 g for 3 min and the supernatant passed through a 0.45 µm filter as suggested by Conceição-Neto et al. [A. mellifera and A. cerana. Total RNA libraries were made and 50 bp paired end sequences were generated on the NovaSeq 6000 . Library preparation and sequencing was carried out at the Biomedical Research Facility .o et al.  for viraV. jacobsoni libraries and between 53% and 71% for honey bee libraries receiving viral particle enrichment. Unmapped reads were collected and then mapped to the virus reference genome database retrieved from NCBI using a length fraction of 0.5 and similarity fraction of 0.8. Consensus sequences were manually inspected for genome coverage and similarity to mapped reference genomes using BLASTn. Unmapped reads were also de novo assembled in CLC Genomics workbench using default parameters and a minimum contig length of 1000 nucleotides. Contigs were compared to the NCBI virus reference genome database using BLASTn to identify known viruses and compared to the NCBI non-redundant protein database using BLASTx to identify putative novel virus genomes. Sequence alignments, annotation and phylogenetic analysis of viral genomes were done in Geneious v2020.0.5 . Raw data and virus sequences have been submitted to GenBank (MT482464-MT482499).Sequence analysis was carried out with CLC Genomics Workbench v11  with raw data first quality trimmed and adapter sequences removed before trimmed reads were mapped to reference host genomes from the NCBI GenBank database. Unmapped reads ranged from 27% to 37% for V. jacobsoni in PNG, was to undertake HTS of two pooled samples of pathogenic V. jacobsoni collected in 2008. Sequence reads were first mapped to Varroa reference genomes and the unmapped reads collected for viral analysis by mapping to the NCBI virus reference database and de novo assembly with contigs BLAST searched for similarity to known viruses. These two approaches revealed that homologous isolates to several known Varroa-associated viruses were present in these samples (V. destructor isolates. We have designated these novel variants as Varroa jacobsoni rhabdovirus 1 and 2 (VJRV-1 and VJRV-2). Several contigs in both samples were most similar to the novel iflavirus VDV-2 with partial genomes for two major variants emerging. Consensus sequences for these variants shared ca 96% amino acid identity across the polyprotein and were between 88% and 92% similar to the VDV-2 ISR and UK isolates. These viruses were dominant in both mite samples and are named here as Varroa jacobsoni virus-2 (VJV-2), PNG isolate 1 and 2. Both samples also had genome length contigs with 84% amino acid identity to VDV-4, a novel unclassified +ssRNA virus detected in V. destructor on A. cerana from Thailand [Our first opportunity to explore the viruses associated with the new pathogenic  samples . Two rhaThailand . Lastly,A. mellifera samples collected across PNG in May 2014 and Solomon Islands in September 2014. Read mapping to the honey bee genome and de novo assembly of the unmapped reads revealed the known honey bee viruses, SBV, BQCV, IAPV and LSVs in both samples  and only low detection of other bee viruses. The A. mellifera sample had a similar viral profile to the 2014 sample but also had low presence of VJV-1, which would have been excluded from the 2014 samples due to the viral enrichment method used affecting enveloped viruses. Few novel virus sequences were detected from these samples compared with the 2014 samples, which we suspect is also due to the different enrichment approaches and shorter (50 bp) library sequencing. Two partial RNA virus genomes were found in A. mellifera and one partial virus genome was found in A. cerana. Interestingly, one of the virus sequences in A. mellifera was identical to the PNG bee virus 9 detected in the 2014 sample, suggesting this virus is a persistent infection. The other virus sequence in A. mellifera was identical to the virus sequence recovered from A. cerana. This putative novel virus (PNG bee virus 14) appears to be an Alphanodavirus of the Nodaviridae, which have a bipartite genome. A Noda-like virus was also reported from A. mellifera in New Zealand, although no sequence information is available for comparison [Lastly, we used HTS to analyse two pooled honey bee samples of variants . The A. mparison .A. mellifera and 14 A. cerana samples collected between 2014 and 2018 from the PNG Highlands and Solomon Islands. In all cases, there was no evidence of either DWV strains infecting any A. mellifera or A. cerana sample.In addition to HTS, we also screened for DWV-A and DWV-B by real-time RT-PCR in a total of 48 V. jacobsoni shifted hosts to become pathogenic to A. mellifera in PNG. Using HTS and real-time RT-PCR viral analysis, we demonstrated that the viral profiles of V. jacobsoni, A. mellifera and A. cerana in PNG and A. mellifera in Solomon Islands have several common honey bee viruses but not any strain of DWV. This is very different from most other honey bee populations around the world, were DWV has become ubiquitous alongside V. destructor [Varroa parasitism on colony health. Our findings suggest that the absence of DWV in PNG is a key underlying factor in the apparent tolerance of this A. mellifera population to V. jacobsoni. Multiple resistance traits have evolved in different A. mellifera populations around the world that suppress mite levels through honey bee behaviours [V. destructor) populations in the absence of DWV [V. destructor, suggesting that additional tolerance mechanisms may be involved. This may also be the case for V. jacobsoni in PNG, with potentially lower virulence to A. mellifera than its sister species V. destructor. Further investigations are still needed in PNG to better understand the impacts of V. jacobsoni and T. mercedesae on colony health and identify potential tolerance traits that have evolved.Our study brings together viral data from over a decade since structor . The unistructor ,33,34,35haviours ,37 or cohaviours ,39. It ihaviours . Indeed,e of DWV . A simile of DWV . HoweverA. mellifera populations as a covert infection until V. destructor disrupts this balance and allows virulent strains in the bee to proliferate [A. mellifera population recently shown to be free of DWV strains [Varroa mites have not reached have also not detected DWV . In facdetected  but was  Zealand . This prnd stock , it appeA. mellifera in PNG and Solomon Islands that showed low similarity to DWV strains. The virus sequence found in Solomon Islands was closely related (94%) to the recently identified Bundaberg bee virus 6, whereas the PNG virus sequence represents a third DWV-like virus detected from this region—with Bundaberg bee virus 6 and Darwin bee virus 3 being the other DWV-like viruses detected from Australian A. mellifera [V. jacobsoni [V. jacobsoni, there were several viruses with relatively high abundance compared with the known honey bee viruses in PNG which may also warrant further investigation.Interestingly, partial genomes for two putative novel viruses were recovered from ellifera . The relacobsoni ,35,50. TV. jacobsoni and honey bees in PNG or Solomon Islands. Aside from DWV, other honey bee viruses have been commonly detected in V. destructor [V. jacobsoni, despite the high abundance of SBV in PNG A. mellifera and A. cerana. The only shared virus was VJRV-1, which is homologous to ARV-1, and found in low abundance in the PNG A. mellifera 2018 sample. ARV-1 has been shown to replicate in both A. mellifera and V. destructor and can spill over into bumblebees [V. jacobsoni and possibly do not replicate in A. mellifera. VJV-1 and VJV-2 are quite divergent from ARV-1 and ARV-2, with less than 73% amino acid identity, and could feasibly have a different ecology. Interestingly, ARV-2 was found to be common in Thai A. cerana, whereas no Varroa-associated virus was detected in A. cerana in PNG. We also detected a V. jacobsoni homolog (VJV-4) of the virga-like virus recently discovered infecting V. destructor in Thailand [There was almost no overlap in the viral profiles of structor ,52. In omblebees ,53. HoweThailand , suggestThailand ,55, indiV. jacobsoni, which is consistent with the findings of Herrero et al. [V. destructor from multiple geographic populations. Herrero et al. [A. mellifera pupae, but suggest this was contamination from feeding mites and not active infections. In our study, we did not detect VJV-2 in adult honey bees nor did Levin [A. mellifera. Interestingly, we found two VJV-2 strains co-occurring in V. jacobsoni at similarly high levels. In contrast, only single strains of VDV-2 (Israel and UK strains) have been reported infecting V. destructor. Potentially, these two strains represent master variants similar to that described for the honey bee iflaviruses DWV, SBV and slow bee paralysis virus (SBPV), and perhaps the presence of two master variants is a common feature of invertebrate iflaviruses. This mirroring of viral profiles between two Varroa species is also interesting and suggests a long association that may extend back to a common ancestor. Determining the prevalence and pathology of these V. jacobsoni viruses will help to understand their importance and provide useful comparative insights with V. destructor.The two iflavirus VJV-2 isolates (VDV-2 homologs) were the most abundant viruses in o et al.  and Levio et al. , who fouo et al.  also fouid Levin  when theA. mellifera, where the absence of DWV appears to have limited the impact of V. jacobsoni on colony health. However, this also suggests that there is potential for a virulent viral pathogen to emerge in this population, whether from the introduction of DWV or the emergence of a novel virus that can exploit this niche. Therefore, the impact of V. jacobsoni could change over time and should be monitored, alongside any changes to the viral landscape. Determining whether other Varroa tolerance mechanisms have also evolved or if there are any biological differences that reduce the virulence of V. jacobsoni compared with V. destructor also have valuable implications for understanding this novel host–pathogen relationship that has emerged in PNG.This study has highlighted a unique mechanism for Varroa tolerance in PNG


An individual’s cardiovascular state is a crucial aspect of a healthy life. However, it is not routinely assessed outside the clinical setting. Smart wearables use photoplethysmography (PPG) to monitor the arterial pulse wave (PW) and estimate heart rate. The PPG PW is strongly influenced by the ejection of blood from the heart, providing an opportunity to monitor cardiac parameters using smart wearables. The aim of this study was to investigate the feasibility of monitoring left ventricular ejection time (LVET) and left ventricular contractility (LVC) from the PPG PW at the wrist. PPG PWs were simulated under a range of cardiovascular conditions using a numerical model of PW propagation. LVET and LVC were estimated from the first and second derivatives of the PPG PWs and compared to reference values extracted from the blood pressure PW at the aortic root. There was strong agreement between the estimated and reference values of LVET, indicating that it may be feasible to assess LVET from PPG signals, including those acquired by smart watches. The correlations between the estimated and reference values of LVC were less strong, indicating that further work is required to assess contractility robustly using smart wearables. This study demonstrated the feasibility of assessing LVET using smart wearables that could allow individuals to monitor their cardiovascular state on a daily basis. FitnessPhotoplethysmography is the optical measurement of arterial blood volume changes in a tissue bed. Smart wearables usually obtain the PPG at the wrist by illuminating the skin with an LED and measuring the level of reflected light. A pulsatile signal consisting of PWs is produced as the arterial blood volume increases and decreases with each heart beat, as shown in LVET is the time period of blood flowing through the aortic valve. LVET is influenced by several cardiac factors, including heart rate, stroke volume, preload, afterload and inotropic drugs . Consequin vivo, it can be quantified as the maximal rate of intraventricular pressure rise during systole [Left ventricular contractility (LVC) is the strength of the contractile force generated by the heart . Whilst  systole . Drugs c systole . HoweverThe aim of this pilot study was to investigate whether LVET and LVC could be accurately estimated from peripheral PPG signals. Simultaneous BP and PPG PWs were simulated at the aortic root, wrist and carotid artery using a numerical model of pulse wave propagation. LVET and LVC values were estimated from PPG and BP PWs at the wrist and carotid artery, and compared to reference values obtained from the aortic root.22.1The dataset used in this study was adapted from the database of simulated PWs presented in , which i2.2LVET and LVC were estimated from each of the radial BP and PPG waves using similar pulse wave analysis techniques to those reported in Reference . The tec2.32, the square of Pearson’s correlation coefficient. Results were compared between PPG and BP PWs to determine whether it could be beneficial to include BP PW monitoring more widely in smart wearables.A fixed reference value for LVET of 310 ms was used, as this was not varied in the original database . Errors 33.13.2The results of estimating LVET at the wrist from the PPG and BP PWs are shown in 3.32 = 0.03). This indicates that further work is required to obtain useful assessments of LVC at the wrist. However, the strength of the correlation was higher at arterial sites closer to the heart . In particular, the correlation of R2 = 0.41 at the carotid artery may be strong enough to provide useful assessments of LVC. Such assessments could potentially be performed using an imaging PPG signal acquired using a smartphone at the neck.The results of estimating LVC from the PPG at the wrist are shown in 3.4The main limitation of this pilot study was that it used simulated signals. This had the benefits of ensuring that reference parameter values were known precisely, without measurement error, and allowing the performance of techniques for estimating LVET and LVC to be assessed across a wide range of cardiovascular conditions. A second limitation was the use of a fixed value for LVET. The study indicated that further work is required to develop techniques to estimate LVC. In addition, the PPG signals routinely acquired by wearable sensors may be sufficient to assess LVET, without the need to acquire BP signals, which are more difficult to acquire from wearables.in vivo. Initial validation should be performed in controlled laboratory settings, where it is easier to acquire reference measurements. The technique could then be assessed in daily living, where less-precise reference measurements could be acquired to validate the technique. If validated, then this approach would allow LVET to be continuously monitored in daily life, improving health monitoring. It would also allow for large-scale research studies to be conducted into the utility of LVET for clinical decision making and diagnosis in various types of cardiovascular diseases, including aortic valve disease, left ventricular failure, stenosis and aortic regurgitation [Future studies should assess the performance of the technique for estimating LVET from smart wearables gitation .3.5The inclusion of PPG sensors in smart wearables provides opportunity to monitor cardiovascular states unobtrusively in daily life. Photoplethysmography was first reported in the 1930s , and beg4The main finding of this pilot study was that LVET could be estimated from PPG signals at the wrist with sufficient precision for use in some settings. Clinical studies are warranted to determine whether this approach could be used to estimate LVET precisely from wrist-worn smart wearables. LVC could not be estimated precisely from the wrist using the trialed technique. Further development of pulse wave analysis techniques may improve the performance of LVC estimates, although this study indicates that they are likely to perform better when obtained from signals acquired at the neck, such as imaging PPG signals.


Tetraselmis chuii microalgal biomass, a sustainable source of protein and bioactive compounds. The impact of different levels of T. chuii  on the GF doughs and breads’ structure was studied. Microdough-Lab mixing tests and oscillatory rheology were conducted to evaluate the dough´s structure. Physical properties of the loaves, total phenolic content  and antioxidant capacity (DPPH and FRAP) of the bread extracts were assessed. For the low additions of T. chuii (1% and 2%), a destabilising effect is noticed, expressed by lower dough viscoelastic functions (G’ and G’’) and poor baking results. At the higher level (4%) of microalgal addition, there was a structure recovery with bread volume increase and a decrease in crumb firmness. Moreover, 4% T. chuii bread presented higher total phenolic content and antioxidant capacity when compared to control. Bread with 4% T. chuii seems particularly interesting since a significant increase in the bioactivity and an innovative green appearance was achieved, with a low impact on technological performance, but with lower sensory scores.The objective of this work is to increase the nutritional quality of gluten-free (GF) bread by addition of  This study is part of Algae2Future project, that intends to explore the microalgae potential to be a low-carbon/nitrogen-footprints healthy food ingredient. These photosynthetic unicellular organisms have a huge importance in terms of the carbon dioxide mitigation  and nitrTetraselmis chuii is a green microalga approved by EFSA (Regulation UE 2017/2470) which has a high protein content, that is an important requirement to be used in bread with a specific nutritional profile. In the last few years, several works have been developed about the incorporation of microalgae into food. Many innovative food systems enriched with algae have been proposed, namely pasta 1/2. The measurements were replicated at least six times under artificial fluorescent light using a white standard .The bread colour was measured using a Minolta CR-400 (Japan) colorimeter with standard illuminant D65 and a visual angle of 2°. The results use the CIELAB system: L*—lightness (0 to 100), a*—greenness to redness (−60 to 60), and b*—blueness to yellowness (−60 to 60). The total colour difference between breads containing the microalgal biomass and the Control sample was calculated using the equation ΔE* = [(ΔL*)To evaluate the bioactivity of breads, aqueous extracts were prepared by freeze-drying the bread samples and then milling into homogeneous fine powders using an electric blender. Then, 500 mg of each sample was stirred in 50 mL of distilled water using a magnetic bar for 30 min at room temperature, and then filtered by Whatman n° 4 paper. This procedure was repeated in duplicate for each bread. For all the bioactivity analysis, three replicates were performed for each extract, correspondent to six measurements for each bread sample.Folin–Ciocalteu reagent and mixed with 1200 μL of sodium carbonate (7.5%) after 10 min. The mixtures were incubated in the dark at room temperature for 2 h, and then the absorbance was measured at 760 nm in a UNICAM UV4 UV/Vis Spectrometer. For blank testing, the extract was replaced by the same volume of water. The TPC was reported as milligrams of gallic acid equivalents per gram of extract (db).The total phenolic content (TPC) of bread extract was evaluated using the method reported by Oktya et al. , with soThe scavenging effect of bread extracts was determined using the DPPH  methodology . ExtractHedonic sensory evaluation was performed for breads with 1 and 4% of T. chuii, as well as for control sample, using an untrained panel of 32 consumers, randomly chosen among the staff and students from the Instituto Superior de Agronomia Food Science Department, 14 males and 18 females, with ages between 16 and 65. The three breads were analysed in terms of colour, smell, taste, texture and general acceptance using a 5-point hedonic scale from “very unpleasant” (1) to “very pleasant” (5). The assays were conducted in a standardized sensory analysis room, according to the standard EN ISO 8589: 2007.p < 0.05).The analysis of variance (one-way ANOVA) of the experimental data was performed using Origin Pro 8.0 software , followed by Tukey’s test. Correlation analysis was performed by using STATISTICA  and the function Bivariate Scatterplot. The significance level was set to 95% (The mixing curves obtained from the MicrodoughLab are represented in w/w) and 69% of water absorption, similar peak values were obtained, without being necessary to adjust the water content of microalgal-containing formulations. From this empirical rheology test, it was concluded that T. chuii addition had no significant (p < 0.05) impact on the rheology parameters extracted from de mixing curves: optimum water absorption, peak development time, stability and softening.Our proposed methodology is to assess optimum water absorption values  for the formulations containing microalgal biomass in order to achieve a peak of 56 nN.m ± 5 mN.m. This peak value corresponds to a control-dough  with good bread baking characteristics, previously optimised by testing different water hydrations (data not presented). The technological parameters obtained from the mixing curves are presented in The GF mix was not evaluated using Micro-doughLab since water hydration followed the recipe indicated on the label of this commercial product.T. chuii biomass addition on dough structure, after fermentation  are significantly (p < 0.05) higher than for 2% T. chuii, and are similar to the commercial mix, control-sample and 1% T. chuii. These values correspond to a structure recovery at 4% level of microalgae addition.Frequency sweeps were performed to evaluate the impact of entation . For eacThe frequency dependence of G’ (elastic modulus) and G’’ (viscous modulus) could be described by the power law Equations (1) and (2):Values of a and b are determined by performing a linear regression on log G’ and G’’ versus log frequency, where a’ and a’’ are the y-intercepts and b’ and b’’ are the slopes of the resulting line . AccordiChlorella vulgaris has a negative impact on the dynamic viscoelastic properties of the wheat dough.Flours from grains without gluten, such as rice, and from pseudocereals, such as buckwheat, have been used as ingredients to produce GF breads. Addition of pseudocereals considerably improves dough viscosity and the texture, volume and nutritional quality of GF products ,36 and cT. chuii, a destabilisation of this structure can be noticed , the proteins should take the lead in the structure network, playing an important role by replacing the former structure, contributing to a new matrix, dominated by protein interactions, where the starch granules and HPMC will be embedded with some compatible reinforcement of the amylose released from starch granules. This structure is different from the previous one, more compact, as it can be seen by the smaller size of the crumb alveoli.The structure of complex GF dough systems is mainly accounted by starch and HPMC which could entrap the air. When small amounts of protein are added, coming from  noticed , mainly T. chuii incorporation in the GF bread shape and colour can be accessed through The impact of T. chuii (p < 0.05) reduction of bread crumb firmness comparing to 2%, not differing from 1% T. chuii bread. Nevertheless, this formulation with 4% of microalgal biomass presented a significant (p < 0.05) lower cohesiveness when compared to all the others, including the control-bread and the bread prepared with a commercial mix.To study the relation between overall bread quality parameters, raw materials and dough properties, the texture and the volume of bread loaves were evaluated. For bread crumb differing in composition, crumb firmness increased with microalgal addition until 2% T. chuii  and therT. chuii . HoweverT. chuii incorporation, microalgal protein induces a destabilisation of the network formed by starch and HPMC. This is revelled by a significant reduction of bread volume (the bread becomes more compact), as well as a significant increase of firmness. However, for the highest protein content of 4%, microalgal protein plays an important role on the GF dough structure, increasing the capacity to retain the gas bubbles.Starch gelatinization plays an important role in GF formulations, due to the ability of starch to form a matrix in which gas bubbles are entrapped . Incorpop < 0.05) decrease from control (19.2) to 1% T. chuii bread (16.2), increasing with 2% of algae (27.1) and decreasing again for 4% T. chuii (22.2). These results are related to the pigments of this Chlorophyceae green algae, namely chlorophylls, carotenoids (such as fucoxanthin and β-carotene) and α-tocopherol (Vitamin E) [T. chuii crumbs was higher than 64, despite the small differences between 2% and 4% T. chuii bread colours. Several authors consider that the human eye can differentiate colours when the total colour difference ΔE* > 5 [T. chuii is contributing to a darkening effect, as it is evident by visual observation  . The tot ΔE* > 5 , therefoervation  and throT. chuii incorporation were obtained. In 6.283 rad/s), bread volume vs elastic modulus (G’6.283 rad/s), and bread volume vs bread firmness. High G’ values are related with greater elastic contribution and this is negatively correlated with the bread firmness (softening effect), followed by a bread volume increase. As expected, bread volume presented a negative correlation with bread firmness.Using correlation analysis, the relationships between dough rheology and breadmaking performance of breads with different amounts of Similar results were described by Martínez & Gómez , showingT. chuii breads, considering the previous results and the objective of achieving high levels of microalgae incorporation.Total phenolic content and antioxidant capacity (DPPH and FRAP) were evaluated only for the control and 4% w/w) resulted in a significant (p < 0.05) increase in the total phenolic content (TPC)  , which wcroalgae  and may croalgae ,45.−1 and 0.33 mg·g−1 ascorbic acid equivalents obtained by DPPH and FRAP, respectively), the incorporation of microalgal biomass led to a significant (p < 0.05) increase in the antioxidant capacity of the breads . Even upon baking, the antioxidant activity of these breads is interesting. Some other authors reported similar results, Rózylo et al. [Chlorella vulgaris.The antioxidant capacity was tested using the DPPH and FRAP methods. Compared with the control-bread , the microalgal proteins with starch and HPMC build up another type of structure, characterised by higher values of the viscoelastic functions (G‘and G’’) producing higher bread volume and a softening effect. This study shows that the structure of 4% T. chuii bread is competitive with the control-bread with the advantage of having an improved bioactivity (phenolics and antioxidants) with possible positive impacts on health. The use of T. chuii at 4% level is interesting, but low sensory scores postpone its utilization.


Mosquito-borne viruses will continue to emerge and generate a significant public health burden around the globe. Here, we provide a longitudinal perspective on how the emergence of mosquito-borne viruses in the Americas has triggered reactionary funding by sponsored agencies, stimulating a number of publications, innovative development of traps, and augmented capacity. We discuss the return on investment (ROI) from the oscillation in federal funding that influences demand for surveillance and control traps and leads to innovation and research productivity. While outbreaks of infectious diseases are devastating to human and animal populations, surges in private and public funding for outbreak pathogens have positively influenced research and innovation. Contemporary outbreak response efforts now incorporate digital media; electronic surveillance tools; mathematical modelling; sequencing; and teams of experts that include anthropologists, social scientists, and other diverse disciplinarians . OutbreaThe introduction and spread of numerous mosquito-borne viruses around the globe has also shepherded a wave of innovation to improve upon industry-standard sampling techniques that more efficiently capture the target vector species, physiological cohort, or virus-infected population of vectors . RamirezMany arboviruses cause a significant disease burden to the public each year. West Nile virus (WNV) (introduced in 1999), St. Louis encephalitis virus (SLEV), Jamestown Canyon virus (JCV), La Crosse encephalitis virus (LACV), Eastern equine encephalitis virus (EEEV), and Powassan  are tracked through the CDC database, ArboNET . In the Aedes-borne viruses in the Americas coupled with the expanding range of Ae. aegypti mosquitoes presents a continual threat of these viruses gaining a foothold in the United States as well [In 2013, chikungunya (CHIKV) was first reported in the Americas, followed by Zika virus (ZIKV) two years later ,23. Thes as well ,29,30. T as well ,32.When a mosquito-borne virus rapidly spreads around the world and becomes a public health emergency of international concern, federal agencies allocate substantial funds to fight the virus. In the U.S., Congress approves these funding packages, such as the $1.1 billion for Zika virus in September, 2016 . This stAedes (Stegomyia) mosquitoes. Once again, along with the NIH who spent over $400 million in 2016 on ZIKV-related projects, the CDC administered $97 million in supplementary Zika ELC stimulus funds to states to help programs re-bound and shift from a Culex-centric focus to Aedes [Aedes mosquitoes, although the fading of Zika virus in the national news and the reduction of these ELC funds [In response to WNV and ZIKV, the CDC supports detection and response capacity to local agencies by providing states Epidemiology and Laboratory Capacity (ELC) cooperative agreements with all 50 states and US territories. For the first five years following the introduction of WNV into the U.S., the CDC issued ELC funds which were reduced in 2004. A survey by the Council of State and Territorial Epidemiologists in 2005 showed several enhancements to surveillance, laboratory, and control capacity that were made possible as the result of ELC funding . Howeverto Aedes . This puLC funds  has resuAlong with the pulse in funding and attention by media and the public, surges in peer-reviewed publications occur following the emergences of arboviruses. To quantify these publications, we performed a “Basic Search” of the Web of Science Core Collection for the key words of “West Nile virus”, “chikungunya virus”, and “Zika virus”. As with the NIH RePORTER search, we included “Eastern equine encephalitis virus”, “St Louis encephalitis virus”, and “La Crosse encephalitis virus”. The search for St. Louis encephalitis virus returned more publications with “St” than with “Saint”. The year range for the search was 1985 to 2020, and the search was conducted on May 16, 2020. The results yielded 11,868 records for WNV; 5088 for CHIKV; 8074 for ZIKV; 634 for EEEV; 604 for SLEV; and 238 for LACV . The yeaCulex and Aedes mosquitoes has followed suit, being driven by the emergence of these viruses, the resulting resources generated by federal agencies, and consumer demand. To visualize trends in trap usage over time, we evaluated BioQuip Products sales data in each main trap category between 2003 and 2019  as attractants. Between 2003 and 2010, trap sales were dominated by suction traps using light and CO2 as attractants, and gravid traps, largely for Culex vectors of WNV and SLEV. Due to the sustained threat of WNV year after year [Culex vectors are expected to remain a mainstay in the industry. The CDC light trap has become an industry standard for collection of mosquitoes for the purposes of arbovirus surveillance. The similar EVS  trap  is also ter year , traps t2-baited trap that was not reliant on a power source and could be used to sample arbovirus vectors in more remote areas [Several specific innovations have either improved the functionality of the standard light trap or targeted different physiological cohorts of mosquitoes. In 2006, BioQuip released the first commercially available light-emitting-diode (LED) CDC-style light trap with customizable colors . Bayonette areas . A collate areas . A photote areas . PassiveAe. aegypti mosquitoes requires very different tools and strategies than for those used for Culex-borne viruses. These urbanized, day-biting, container-breeding mosquitoes are generally not attracted to the suction and gravid traps that are so efficient at catching Culex mosquitoes [Aedes mosquitoes led to the innovation of the BG Sentinel Mosquito Trap in 2006  [Aedes traps (GAT) [Surveillance for viruses transmitted by squitoes . The groGermany) , among ops (GAT) ,46 were ps (GAT) , these tps (GAT)  are lighThe emergence of mosquito-borne viruses has occurred repeatedly in recent decades, and studies predict that these emergences will continue. Much of our response to these events is reactionary, triggered by the increase in attention, funding, publication, innovation, and preventive measures for public health. The long-term impact or return on investment of outbreak spending is evidenced by scientific advancements (publications) and innovation, but we advocate for a more sustainable, economical, and effective approach by minimizing the oscillations of boom and bust in funding and capacity for mosquito-borne viruses. Our goal should be to optimize the cost-effectiveness of budgetary spending by adopting resource allocation for biosecurity threats that maximize benefits while minimizing the total cost given anticipated expenditures incurred in the event of mosquito-borne viral outbreaks ,50.


In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV).A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASFV.  The virus infects domestic pigs and their relatives and ticks2. ASF outbreaks had been recorded in Africa and Europe, but in recent years the disease has spread to China, Vietnam, Cambodia, Mongolia, Hong Kong, and Korea, becoming a threat to the swine industry worldwide3. ASFV is a member of nucleocytoplasmic large DNA viruses (NCLDVs) with an average diameter of 200 nm. Although some related viruses, such as faustovirus4, kaumoebavirus5, and pacmanvirus6, have been reported, ASFV is the only member of the Asfarviridae family7. In the present paper, we describe a virus likely to be the closest ASFV relative found to date; this novel virus was isolated as the presumptive causative agent of abalone amyotrophia.African swine fever virus (ASFV) is the causative agent of African swine fever (ASF). The virus causes a hemorrhagic fever with high mortality, with rates approaching 100% in domestic pigs8. Diseased abalone show reduced ability to adhere to the substrate, and some diseased abalone exhibit incisions on the front margin of the shell and brown pigmentation inside of the shell9. Histopathological evaluation has revealed the presence of abnormal cell masses that are produced extensively, primarily in the ganglion and peripheral nerve of the foot muscle9. Cumulative mortality can reach 50% and higher10. Abalone herpesvirus (AbHV)12 and abalone shriveling syndrome-associated virus (AbSV)13 also cause mortality accompanied by amyotrophia, but the latent period differs between these two viral infections and abalone amyotrophia. Abalone infected with AbHV14 and AbSV13 show signs of disease onset or mortality several days after artificial infection compared with approximately 2 months after challenge in abalone amyotrophia15. The causative agent is thought to be a virus, based on the demonstrated infectivity of a 0.22-μm-filtered homogenate derived from affected juvenile abalone16. Although radiation of the water supply with ultraviolet light successfully prevents the occurrence of this disease17, the disease still occurs occasionally in facilities that do not employ UV sterilizers.Mass mortalities of abalone have been reported since the early 1980s, during seed production in Japan. The disease was designated abalone amyotrophia because diseased abalone develop muscle atrophy in the mantle and foot26. Pathogen identification for shellfish diseases, especially for viral diseases, is difficult because of the lack of cell lines available for virus isolation and serological methods are hampered by the absence of antibodies and pathogen-specific immune mechanisms in mollusks27. For these reasons, searches for pathogenic viruses have relied primarily on electron microscopic observation25. Indeed, tissues from diseased abalone have been screened for pathogenic viruses by electron microscopy, but those studies were not successful.Shellfish are an important food source for people in many countries, but resources are sometimes decreased due to infectious diseaseset al.28 succeeded in isolating and culturing spherical viruses with a diameter of about 120 nm from affected abalone using primary cultured hemolymph cells. The resemble virus also was cultured from affected abalone obtained from different areas, but these virions did not show pathogenicity in infection tests28. Virus-like particles with a diameter of 100 nm were observed in cells near the nerve trunk of affected abalone, but the pathogenicity of these particles was not demonstrated29. Virus-like particles with a diameter of 50–60 nm were observed in secondary lysosomes in the cytoplasm of cells of affected abalone9. These authors described that these particles were not likely to be the etiological agent because the size of the causative agent was estimated to be between 100–220 nm by filtration experiments16.Subsequently, Nakatsugawa 33 and unculturable bacteria34 that infect shellfish have been found using next-generation sequencing (NGS) technologies. Therefore, we applied NGS in the search for the causative agent of abalone amyotrophia. Samples of diseased abalone were collected from hatcheries where mass mortalities occurred. We analyzed the pathogenic semi-purified fraction prepared from diseased abalone homogenate by NGS. Sequences specific to diseased abalone were selected by PCR-based epidemiological survey. As we report here, the presumptive causative agent of abalone amyotrophia was identified as a virus with genomic similarity to ASFV; we propose provisionally designating the new virus as abalone asfa-like virus (AbALV).Recently, new virusesHaliotis discus discus) to confirm the presence of an etiological agent in a semi-purified fraction that had been prepared from a homogenate of diseased black abalone. In the positive control group , the mortality began on the 56th day post-injection (dpi), with cumulative mortality reaching 80% at 136 dpi . Among the surviving abalone, abnormal cell masses, which are characteristic of the disease, were observed only in animals of the positive control group and semi-purified fraction-injected groups . Of the total scaffolds and singletons, 2,160 scaffolds consisted only of reads obtained from the semi-purified fraction of diseased abalone. Among these 2,160 scaffolds, 70 scaffolds with lengths exceeding 500 bp were selected for further analysis. Among those 70 scaffolds, 15 showed homology to the abalone sequence when subjected to BLASTN analysis; sequences of these 15 scaffolds were not studied further. The remaining 55 scaffolds  were used for a PCR-based epidemiological survey intended to identify scaffolds corresponding to the causative agent.In the genome sequence of the semi-purified fraction prepared from the diseased abalone, 6.51 × 107 reads consisting of 2.8 × 109 bases of nucleotide sequence; assembly of these sequences generated 1,198 contigs. Among these contigs, 4 with a length exceeding 500 bp were selected for an RT-PCR-based epidemiological survey intended to identify sequences corresponding to the causative agent.Pyrosequencing runs on an RNA library from the semi-purified fraction yielded 2.35 × 10Fifty-five scaffolds from DNA sequencing and 4 contigs from RNA sequencing were screened by PCR and RT-PCR, respectively, using nucleotides extracted from diseased and healthy abalone as samples. In the first screening using pooled DNA samples, 13 primer sets  generated amplicons of the expected sizes from the two pools of samples from diseased abalone, and not from the two pools of samples from healthy abalone  individual DNA preparations from each abalone. With two exceptions, all the tested primer sets generated amplicons with the expected product sizes from the 12 diseased abalone, but not from the healthy animals  searches of the virus and non-redundant (nr) sequence databases with each of the 159 predicted proteins are summarized in Table Among the 12 scaffolds that were specifically and universally detected in diseased abalone, 10 scaffolds  were linked into one sequence (Accession No. LC506465) by gap-closing PCR. A draft genome sequence consisting of 1,551,181 bp with a GC content of 31.7% was obtained by the assembly of the NGS sequence and Sanger sequencing of the 9 gap regions. The RAST programTwo scaffolds (Scaffolds 4 and 38) were not linked by gap-closing PCR. Thirty-eight ORFs were predicted from Scaffold 4, and one ORF encoded a protein with sequence similarity to a protein encoded by a Planctomycetes bacterium (as assessed by BLASTP against the nr database). Notably, none of these 38 ORFs yielded hits against the virus database. Scaffold 38 was predicted to harbor 4 ORFs, but none of the resulting predicted proteins exhibited similarities by BLASTP against either the nr or virus database.The predicted DNA polymerase B Fig. , topoiso36 revealed large syntenic regions and overall conservation of gene order, albeit with inversion and translocation  were generated based on the longer major capsid-encoding gene (ORF68). The two probes yielded similar positive signals only in infected abalone. The results of Probe 1 yielded a slightly stronger signal  encoding homologs of known viral proteins. However, proof of this hypothesis will require isolation and purification of the putative causative agent.Approximately 30 years have passed since abalone amyotrophia was first reportedThe gap-closed sequence consisted of 1,551,181 bp with a GC content of 31.7% and 159 predicted ORFs. When BLASTP was used to search for sequences with similarity to the 159 predicted proteins encoded by this extended sequence, the majority of the top hits were obtained with ASFV. The phylogeny of five proteins encoded by the gap-closed sequence yielded similar results. With the exception of the short MCP, all the predicted proteins were closest to respective proteins encoded by ASFV. Furthermore, conserved synteny was observed between the partial genome sequence of the abalone virus and ASFV. Therefore, the pathogen is assumed to be a virus that evolved from an ancestor shared with that of ASFV. The virus found from amyotrophic abalone is most closely related to ASFV; we therefore propose provisionally designating this virus as abalone asfa-like virus (AbALV).37. Because the AbALV genome remains incomplete, comparison of the entire genome to those of other viruses cannot be performed. Nonetheless, the sequenced region of AbALV spans 155 kb, contains 159 ORFs, and exhibits a GC content of 31.7%; all three of these parameters are similar to those of ASFV. Although the overall structures of the ASFV and AbALV genomes are conserved, separate inversion and translocation events were observed in the AbALV genome relative to the ASFV genome, as shown in Fig. 5 and faustoviruses38. Thus, the splitting of MCP-encoding sequences into 2 genes in AbALV resembles the case in kaumoebavirus and faustoviruses. The virological or evolutionary implications of these AbALV genome characteristics are unknown.The ASFV genome consists of a single-molecule double-stranded DNA with a linear genome length of about 170–193 kb containing between 150 and 167 ORFs, depending on the isolate; the GC content of the ASFV genome is approximately 39%37. The ASFV capsid was reported to have side-to-side dimensions of 172–191 nm39. The diameter of the amyotrophia-causing virus was estimated to be 100–220 nm by filtration experiments16; therefore, the inferred size of AbALV virions would be consistent with the characteristics of ASFV. ASFV is a member of the NCLDV superfamily and replicates primarily in the cytoplasm of macrophages4. ISH in the present study yielded positive signals throughout the entire cytoplasm of infected cells, another property that is consistent with that of ASFV. Among viruses reported by electron microscopic (EM) observation of amyotrophic abalone29, that observed by Otsu29 was the most similar to ASFV. Notably, Otsu observed virus-like particles with a diameter of approximately 100 nm in the cytoplasm of cells near the nerve trunk of diseased black abalone. However, no information is available that links the virus observed by Otsu to AbALV. While we also performed transmission EM (TEM) evaluation of diseased black abalone, we did not observe virus-like particles (data not shown). Difficulty in observing the causative virus by EM likely reflects dispersion of infected cells throughout the connective tissue, as shown by ISH. Additionally, we suspect that there are large individual differences in the amount of virus in the tissue. Specifically, the titer of the AbALV genome (a parameter that was measured to select samples for ISH) in the muscle of 20 diseased animals ranged from 2.3 × 101 to 1.9 × 105 copies/mg wet tissue weight as judged by AbALV-targeted quantitative PCR. Negative staining and TEM observation of the semi-purified fraction used for NGS also was attempted, but spheroids of various sizes were found in the fractions prepared from both healthy and diseased abalone, precluding identification of the virus by EM  as host cells, but no cytopathic effect (CPE) was observed, nor was virus proliferation detected by quantitative PCR (data not shown). Failure to isolate the causative virus using a primary culture of abalone hemocytes from the amyotrophic abalone had been reported previously28. Based on our ISH results, we infer that AbALV likely infects a cell type other than hemocytes. Identification of viral target cells also will be necessary for isolation of the virus using host cells, as well as to understand the pathological nature of AbALV.ASFV primarily infects monocytes and macrophagesPhacochoerus africanus)42, bushpig (Potamochoerus spp.)43, and soft tick (Ornithodoros moubata)2, ASFV has low pathogenicity while causing persistent infections. In its natural state, ASFV is maintained in the sylvatic cycle in the warthog, bushpig, and soft tick without inducing clinical signs of disease4. It is not clear which organisms are the evolutionally original host of ASFV, but the present work indicates that a virus similar to ASFV was detected in abalone, which belongs to the protostome clade similar to ticks. This observation supports the idea that ASFV originally was a virus that used an arthropod  as a host, but not mammals , before evolution to gain the ability to infect and propagate in the warthog, a tick host. Analysis of the virological properties of AbALV is expected to facilitate protecting abalone from disease while also bringing new insight into ASFV pathology and evolution.ASFV causes an acute, high-mortality disease in domestic pigs. However, in its natural hosts the warthog (H. discus discus) and giant abalone (H. madaka) with high mortality were obtained respectively from the city of Omaezaki, Shizuoka Prefecture, on June 30, 2015, and from the town of Ikata, Ehime Prefecture, on June 9, 2016. The diseased black abalone exhibited incisions on the front margin of the shells and brown pigmentations on the inner surface of the shell .Abalone was preserved in Davidson’s solutiong for 10 min at 4 °C, and the supernatants were passed through a 0.22-μm syringe filter (Millipore). Aliquots of the filtrates were stored at −80 °C for use as the infectious sources for the positive (diseased) and negative  control groups. The balance of the filtrate from diseased abalone was diluted with autoclaved seawater to 25 mL and recombinant DNase I and RNase A (TAKARA Bio) were added to final concentrations of 100 U/mL and 200 µg/mL, respectively. Immediately following the addition of the nucleases, samples were centrifuged at 150,000 × g for 1 h at 25 °C to sediment the particles; digestion of free nucleic acids proceeded during the ultracentrifugation. The resulting supernatant was decanted and 12 mL of autoclaved seawater was added to each tube; the tubes then were incubated for 2 h on ice to resuspend the pellet. Again, DNase I (100 U/mL) and RNase A (200 µg/mL) were added to each tube, and the samples were subjected to another round of ultracentrifugation . The resulting supernatant was decanted and 12 mL of autoclaved seawater was added to each tube; the tubes were incubated for 12 h on ice to resuspend the pellet before being subjected to another round of ultracentrifugation to remove remaining nuclease. The resulting supernatant was decanted and 1 mL of autoclaved seawater was added to each tube; the tubes were incubated for 2 h on ice to resuspend the pellet. The samples then were centrifuged at 15,000 × g for 10 min at 4 °C, and the resulting supernatant (designated as the semi-purified sample) was aliquoted at 200 μL/tube and stored at −80 °C until analysis.Approximately 0.5 g of soft-body tissues, excluding the midgut, were recovered from each of 6 healthy black abalone from Owase in 2015 and from each of 6 diseased black abalone from Omaezaki at the indicated times. Tissues were minced with a razor, combined with 10 volumes of autoclaved seawater, and crushed with a Potter-type glass homogenizer. The crushed samples were centrifuged at 15,000 × 4 and 7.3 × 102 copy/animal in inocula for positive control and semi-purified fraction-infected group respectively, based on absolute quantification using quantitative PCR (methods are described below). For the semi-purified fraction-infected group (N = 40), abalone were divided into 2 groups (N = 20 each) and reared in separate 56-L tanks. The two control groups were reared in separate tanks. All groups were reared in running seawater maintained at 19 ± 2 °C and fed with a commercial pelleted diet once per week. Three surviving animals from each group were recovered for histological observation at the end of the infection test.Healthy black abalone  were used as the recipient of the infection test. The semi-purified sample was diluted 2-fold with autoclaved seawater and injected into the foot muscles of 40 healthy black abalone  with a 30-gauge needle. Filtrates prepared as described above  from diseased and healthy abalone were used as inocula for the positive and negative control groups, respectively, and injected into 20 healthy abalone/group using the same procedure as for the semi-purified sample. The amount of virus was estimated to be 7.5 × 10RNA and DNA were isolated from the semi-purified samples using NucleoSpin RNA NX and NucleoSpin tissue XS (TAKARA Bio), respectively. DNA also was purified from the muscle of a healthy abalone. RNA-seq libraries were prepared from RNA sample using the ScriptSeq v2 RNA-Seq Library Preparation kit (Illumina), and sequenced on the NextSeq 500 sequencer (Illumina) with single-end reads of 151 bp according to the manufacturer’s protocol. DNA from the semi-purified fraction was amplified using Illustra Single Cell GenomiPhi DNA Amplification Kits . The libraries were constructed from amplified semi-purified fraction DNA and healthy abalone DNA using TruSeq Nano DNA LT Sample Prep Kits and sequenced on the NextSeq 500 sequencer with paired-end reads of 2 × 151 bp according to the manufacturer’s protocol.45 v. 0.3.6 . Trimmed reads obtained from the semi-purified fraction library and healthy abalone library were mixed and de novo assembled with Platanus 1.2.4. (http://platanus.bio.titech.ac.jp) under default parameters to generate scaffolds. Scaffolds that formed only from reads obtained from the semi-purified fraction library were extracted. Scaffolds longer than 500 bp were selected and homology-searched against the NCBI nr database; sequences classified as abalone sequences were removed. Residual scaffolds were subjected to further analysis.RNA sequencing data were assembled de novo with the CLC Genomics Workbench 7.0.3 (Qiagen) under default parameters, and contigs longer than 500 bp were selected for further analysis. Processing of the DNA sequencing data was conducted as follows. The sequencing data were filtered and trimmed using Trimmomatichttp://bioinfo.ut.ee/primer3-0.4.0/), primers were designed to target the 55 diseased abalone-specific scaffolds that were obtained from DNA sequencing, and 4 contigs longer than 500 bp that were obtained from RNA sequencing  were performed with each of the predicted proteins.The RAST program46.Maximum-likelihood phylogenetic trees were constructed using protein sequences predicted from the genes encoding DNA polymerase B, topoisomerase, MCPs, and RNA polymerases 1 and 2. The protein sequences included those encoded by the type species for each NCLDV, related species, and the respective sequence obtained in the present study. Trees were created using 1,000 bootstrap replicates of the sequence alignments in MEGA 736 and visualized for syntenic comparison using GenomeMatcher47.The genome sequence of the putative causative agent was compared with the reference genome of ASFV Georgia 2007/1For quantitative PCR (qPCR), specific primers were designed  that generate 127-bp amplicons within the MCP gene (GenBank Accession No. BBO54023) of AbALV. The complete MCP gene of AbALV was PCR amplified from diseased abalone sample from Omaezaki using specific primer sets  and inserted into the pCR2.1 plasmid vector to generate standards for quantification.qPCR assays were performed using the Stratagene Mx3000p qPCR system and companion software, MxPro (Stratagene). KOD SYBR qPCR Mix (Toyobo) was used for the reactions according to the manufacturer’s protocol. Briefly, qPCR was performed in 20-μL reactions containing 10 μL of kit-supplied buffer (2×), 50 pmol of primers, 0.1 μL of ROX, and 1 μL of sample DNA. qPCR assays were performed under the following cycling conditions: activation of Hot-Start DNA polymerase at 95 °C for 10 min, followed by 35 amplification cycles of 95 °C for 10 s, 60 °C for 10 s, and 68 °C for 30 s. Specificity was evaluated based on melting curve analyses conducted after amplification, as follows: 95 °C for 1 min, 55 °C for 1 min, followed by an increase in temperature of 0.5 °C every 10 s from 55 °C to 95 °C. The melting curve data were plotted according to the manufacturer’s instructions (Stratagene). Standard curves were constructed using a plasmid vector containing the MCP fragment. Plasmid vector copy number was determined based on molecular weight. Gene copy number was calculated per microliter for inoculum or per milligram of tissue.An infection test was conducted to generate diseased black abalone for ISH. Twenty diseased abalone were injected (as described above) with diseased black abalone homogenate; at 28 dpi, tissue samples from each animal were screened for titer of the putative AbALV agent using qPCR. Two animals with high levels of AbALV genomic DNA infection titers were selected and used as sources of tissue samples for ISH. ISH was performed under contract with Genostaff Co., Ltd. The longer MCP-encoding gene (ORF68) of AbALV was cloned into the pGEM-T Easy vector (Promega) and used for generation of digoxigenin (DIG)-labeled probes via the DIG Probe Synthesis Kit (Roche Diagnostics) according to the manufacturer’s instructions. The resulting probes corresponded to nucleotide positions 385–942  and 1440–1553  of ORF68. Tissue samples from four abalone total  were fixed with G-Fix (Genostaff), embedded in paraffin, and sectioned at 6-µm thicknesses. ISH was performed with the ISH Reagent Kit (Genostaff) according to the manufacturer’s instructions. Hybridization was performed separately with either of the two probes at concentrations of 150 ng/mL in G-Hybo-L (Genostaff) for 16 h at 40 °C. Following hybridization, the sections were washed with 50% formamide in 2xG-Wash (Genostaff) and then with Tris-buffered saline containing 0.1% Tween 20 (TBST). The washed sections were incubated with alkaline phosphatase-conjugated anti-DIG antibody (Roche Diagnostics) diluted 1:2000 with 50x G-Block (Genostaff) in TBST for 1 h at room temperature. Color development was performed with NBT/BCIP solution (Sigma-Aldrich), and sections then were counterstained with Kernechtrot stain solution.p value of <0.05 was considered significant.Where applicable, data are presented as mean ± standard deviation (SD). Cumulative mortality was analyzed statistically with Fisher’s exact tests. A Supplementary informationSupplementary information2


Thoracoscopic lobectomy has become the preferred approach for surgical management of early stage lung cancer, but the potential higher operative costs limit its widespread use. Theoretically, higher direct costs may be significantly counterbalanced by lower indirect costs, resulting in lower overall costs for thoracoscopic than for open lobectomy. To support this hypothesis, we reviewed the literature until May 2020, analyzing all papers comparing the cost of thoracoscopic versus open lobectomy.A total of 20 studies provided the most applicable evidence to evaluate this issue. In all the studies apart from one, thoracoscopic lobectomy was associated with higher operative costs due to the increased use of disposable instruments, and prolonged operative time. By contrast, in 17 studies the increased operative costs were significantly offset by indirect costs which were lower in thoracoscopic than in open lobectomy due to fewer postoperative complications, faster recovery, and lower readmission rates. It translated into lower overall costs for thoracoscopic than for open lobectomy in 10 studies, similar costs in seven, and higher in three, despite the lower hospitalization costs. The low bed fees and high prices of disposable instruments in these three studies may explain the discordance. The careful use of disposable instruments, and the minimizing hospitalization costs can reduce the total costs of thoracoscopic lobectomy to levels similar or to below those of open lobectomy. The worry that video‐assisted thoracoscopic surgery lobectomy (VATSL) might be associated with an increased overal cost is thus not warranted, and should not be used as an excuse against the use of VATS in surgery for early stage lung cancers. Is video‐assisted thoracoscopic lobectomy associated with higher overall costs compared with open surgery? A review of the literature until May 2020 was performed, and 20 studies provided the most applicable evidence to evaluate this issue.Thoracoscopic lobectomy was associated with higher operative costs than open lobectomy, but lower indirect costs. It translated into lower overall costs for thoracoscopic than for open lobectomy in 10 studies, similar in seven, and higher in three studies.The worry that thoracoscopic lobectomy might be associated with increased total cost is thus not warranted, and should not be used as an excuse against the use of thoracoscopy in surgery for early stage lung cancers. You plan to start a VATS lobectomy (VATSL) program, but the manager of your hospital has some concerns about the cost. He underlines that VATSL may be associated with an increase in hospital costs due to longer operative time, and greater consumption of disposable instruments than open lobectomy (OPENL). You reply that the analysis of VATSL costs should include not only the direct costs related to surgery, but also the other indirect costs related to the length of hospital stay (LOHS), pharmacy and manpower consumption for increased complications, output clinical visits, and readmission. Since VATSL is associated with shorter LOHS and lower postoperative morbidity and mortality, it leads to less health care use after discharge, resulting in lower costs over a longer time horizon than OPENL. You undertake a literature search to support your hypothesis.Surgery is the only curative treatment for early stage non‐small cell lung cancer (NSCLC), and lobectomy is still the most effective resection.The higher operative costs for VATSL than for OPENL is one of the main concerns for widespread adoption of VATSL. Although this issue has been evaluated in several studies,The study design was structured according to the PRISMA protocol.Additional papers, abstracts, chapter of books, letters and editorials were retrieved from bibliographies by manual research. The Science Citation Index was used to cross reference for further studies that met the criteria of the study.Papers were included in the review if they fulfilled the following criteria: (i) papers published in English; (ii) a study population including patients who had undergone VATSL and OPENL; (iii) results comparing costs between OPENL and VATSL. We excluded (i) studies published in languages other than English; (ii) reviews, meta‐analyses, abstracts, case reports and case series; (iii) papers from the same groups. In these cases, only the most recent publication was reported to avoid duplication; (iv) papers comparing only the outcomes, but not the costs between the two procedures.First, the titles of papers were inspected to decide whether they were appropriate for the purpose of the study. Second, the abstracts of the selected papers were evaluated, and those that were not appropriate were excluded. Third, the remaining articles were thoroughly inspected to decide whether they should be included. Any disagreements were judged by the three senior reviewers  after referring to the original articles.n = 235), and the additional manual (n = 15) searches from the references of the selected articles. A total of 135 papers were excluded as being duplicates. Among the 115 papers screened, 88 were excluded based on the titles and abstracts. Of the remaining 27 studies, seven studies were further excluded. Thus, 20 papers were included in the analysis. The authors, year of publication, country, study design, level of evidence based on the criteria of Centre for Evidence Based Medicine,The flow chart of the study is listed in Figure nual n = 5 searcheet al.n = 294) and OPENL (n = 588). They found no difference in the hospital stay costs (P = 0.5) and hospital care costs (P = 0.1) between the two procedures. Compared to OPENL, however, VATSL was associated with lower three‐year lung cancer‐related costs (P = 0.02) due to lower outpatient physician care (P = 0.01), and drug prescriptions (P = 0.06). OPENL included a higher number of patients who underwent adjuvant therapy, which could explain the increased lung cancer related costs in that group.Marijic et al.n = 103) and OPENL (n = 103). The total costs for VATSL was lower than for OPENL (P < 0.001). VATSL was associated with a longer operative time (P < 0.001), but shorter LOHS (P < 0.001). The post‐discharge costs were lower after VATSL, and this difference was primarily associated with lower costs of readmissions (P < 0.001), and outpatient clinics (P = 0.012).Bendixen et al.n = 4608) versus OPENL (n = 8501). VATSL compared to OPENL was associated with lower rates of postoperative morbidity (P < 0.001) and mortality (P < 0.001), shorter LOHS (P < 0.001), and fewer 90‐day readmissions (P < 0.001), translating into lower index hospitalization cost (P < 0.001), and 90‐day cost (P < 0.001). After adjusting for patient age, gender, income, comorbidities, and hospital teaching status, VATSL was still less expensive than OPENL.Subramanian et al.n = 240) versus VATSL (n = 161). Procedural cost in the operating room was lower for OPENL, as it was associated with the shortest operating room times (P = 0.05) and the least expensive equipment. However, VATSL compared to OPENL was associated with shorter LOHS (P < 0.001) and fewer costly events, and, as a result, in similar overall hospital cost (P = 0.6). These results were confirmed even after carefully adjusting for patient selection.Kneuertz et al.n = 38) versus VATSL (n = 32). VATSL was associated with higher operative costs (P = 0.01) mainly driven by staplers, but lower LHOS (P = 0.000008). The lower hospitalization costs and the high cost of staplers might explain the higher total hospital costs for VATSL than for OPENL (P = 0.05).Lipinska et al.n = 2.200) or OPENL (n = 3.166). VATSL compared to OPENL was associated with higher operative costs (P = 0.0001), but this difference was not significantly balanced by lower costs related to anesthesia (P = 0.007), ordinary ward (P < 0.0001), ICU (P < 0.0001), nursing (P < 0.0001), and pharmacy (P < 0.0001), and hospitalization, resulting into higher total costs for VATSL than for OPENL (P = 0.02). By contrast, 30‐day post discharge costs were lower for VATSL than for OPENL (P = 0.0001).Wang et al.P < 0.0001), and higher payment to hospitals (P = 0.009), and physicians (P = 0.01). OPENL had 1.28‐times and 1.14‐times more health care utilization days within 90‐day (P = 0.0002), and 365 day (P = 0.03), respectively, after the operation compared with VATSL, translating into increased expenditures of $3260 at 90 days, and $822 at 365 days for OPEN procedures. No significant differences in utilization were noted between OPEN and VATS wedge resections, except for fewer outpatient visits within 90 days in the OPEN group.Watson et al.n = 69) versus VATSL (n = 58) for early stage lung cancer. Complication rates (P = 0.94) and LOHS (P = 0.11) were similar between the two groups. Operative costs related to operative time (P = 0.02) and disposable instrument costs (P < 0.001) were higher for VATSL than for OPENL, but this difference was offset by lower ICU costs (P = 0.002) and lower laboratory costs (P < 0.001), resulting in similar overall costs between the two procedures (P = 0.2).Deen et al.n = 6893) or VATSL (n = 3069). VATSL compared to OPENL was associated with significantly lower total unadjusted 90‐day (P = 0.001), index hospitalization (P = 0.002), and outpatient use (P = 0.04) costs. After adjusting costs for age, sex, comorbidity index, health plan, and use of epidural anesthesia, 90‐day costs were $3476 lower for VATS lobectomy than for OPENL (P < 0.001). VATSL was associated with a lower rate of patients with prolonged LOHS (>14 days) than OPENL (P < 0.0001), explaining the difference in total costs between the two groups. In fact, adding prolonged LOHS as a covariate to the regression model reduced the differential cost by 63% (−$1276), and the difference between VATSL versus OPENL was no longer significant. In the fully adjusted model, PLOS was associated with the highest cost differential .Farajad et al.n = 32) and OPENL (n = 49). LOHS in the VATSL group was significantly shorter than for OPENL (P < 0.05), but VATSL was associated with higher costs of disposable surgical instruments (P < 0.05). More expensive disposable surgical instruments and cheaper hospital stay charges lead to higher overall costs in VATSL than in OPENL group (P = 0.002).Alpay et al.n = 108) or OPENL (n = 104). VATSL was associated with shorter LOHS (P < 0.0012), lower complication rate (P < 0.0006) and ICU admission rate (P < 0.0027), but higher theater costs (P = 0.0001) due to increased utilization of staplers (P = 0.0001). The significantly higher hospital costs and ICU costs after OPENL did not compensate for the higher theater costs of VATSL, translating into higher total hospital costs for VATSL than for OPENL (P = 0.004).Piwkowski et al.n = 98) versus OPENL (n = 189). VATSL compared to OPENL was associated with increased intraoperative costs (P < 0.0001) due to increased use of disposable surgical instruments and staplers (P < 0.001), longer operative time (P < 0.001). In VATSL, upper‐right lobectomy and segmentectomy were more expensive than other types of resection. However, the increased surgical costs of VATSL were offset by the lower hospital stay (P < 0.001), laboratory (P < 0.001), and radiology (P = 0.01) costs, resulting in lower overall cost for VATSL than for OPENL (P < 0.0001).Ramos et al.n = 1054) versus OPENL (n = 2907). VATSL was associated with longer operative time (P < 0.000), but lower LOHS (P < 0.000) and lower risk of adverse events (P < 0.019), resulting in lower total hospital costs than OPENL (P = 0.02). These differences persisted even after adjusting for patient and hospital characteristics. Only for VATSL the economic impact was magnified as the surgeon's experience increased (P < 0.05).Swanson et al.n = 86) versus OPENL (n = 97) for lung cancer. VATSL compared to OPENL was associated with lower postoperative morbidity (P < 0.000); lower chest tube duration (P = 0.000); and LOHS (P = 0.000), but higher surgical material costs (P < 0.000). Cost comparisons were then adjusted for postoperative complications, type of lobectomy, and surgeon's experience. No significant difference was found between VATSL and OPENL among all patients (P = 0.09), and among only noncomplicated patients (P = 0.8). The overall cost for the VATSL was lower than for the OPENL in cases of right lower lobectomy, left upper lobectomy, and left lower lobectomy, while only the cost of anesthesia was affected by surgeon's experience, being higher for the early than for the experienced period (P = 0.009).Cho et al.n = 12 860) or VATSL (n = 759). VATSL was associated with a higher risk of intraoperative complications than OPENL (P = 0.04), but no differences were found regarding postoperative mortality (P = 0.6), complications (P = 0.5), and LOHS (P = 0.8), resulting in similar overall hospitalization costs (P = 0.1). A higher percentage of patients with an annual income >$59 000 underwent VATS lobectomy than patients with an income <$59 000 (P < 0.0001). Theoretically, patients of higher socioeconomic status choose VATS because it implicates smaller incisions, and allows the patient to return to work earlier.Gopalds et al.n = 37) versus VATSL (n = 76). Total costs were significantly greater for OPENL than for VATSL (P = 0.0012), with overall savings of approximately $2000 per patient. The costs were less for TL at all phases of patient care, and the most dramatic savings were in the preoperative phase where OPENL was almost twice as expensive as VATSL. One explanation could be that fewer preoperative tests of patient fitness were performed due to the perceived minimally surgical trauma related to VATS. There was also more surgical staging performed in the prelobectomy setting within the OPENL, perhaps reflecting the surgeon's wishes to confirm the absence of mediastinal involvement before performing a thoracotomy. Even after adjusting for lung cancer stage, total costs for OPENL were still higher than for VATSL (P = 0.005).Burfeind et al.n = 93) versus OPENL (n = 253) for stage I or II lung cancer. Total costs for VATSL were lower than for OPENL (P = 0.00002). Despite theater room costs being twice as high for VATSL over OPENL (P = 0.00001), this difference was significantly offset by reduced costs related to HDU (P = 0.00001) and ward‐bed stays (P = 0.00001). The operating costs varied according to the type of resection. Among VATS resections, upper bilobectomy was associated with the highest theater cost, €1400 more than left lower lobectomy, which was the least expensive. In this case, the reduced postoperative costs were not able to offset the intraoperative costs. Upper lobectomies and right lower lobectomies were associated with the highest intraoperative cost differences between VATSL versus OPENL, ranging between €2000 and €2500.Casali et al.n = 269) versus VATSL (n = 82). OPENL was associated with higher LOHS (P < 0.001) and complication rate (P < 0.001) compared to VATSL, resulting in $5098 of additional cost.Park et al.P = 0.0064), anesthesia (P > 0.05), disposable devices (P < 0.0001), and LOHS (P = 0.0015). Thus, the total hospital costs for VATS surgery were lower than for OPEN resection (P = 0.0012). However, 64 of 66 OPEN patients underwent lobectomy, whereas only eight of 36 VATS patients had lobectomy. This difference undoubtedly favored the VATS group and explained the decreased costs and hospital stay in that group.Nakajima et al.n = 10) versus OPENL (n = 20). VATSL compared to OPENL was associated with higher overall costs (P > 0.05) due to higher disposable costs (P < 0.05), and longer operative time (P < 0.05) and similar LOHS (P > 0.05).Sugi The present analysis included 19 retrospective cohort studies,In all studiesIn 17 studiesIn this analysis, the assessment of the comparative costs for VATSL and OPENL was limited by several issues. All papersIn conclusion, the current evidence showed that VATSL was associated with higher operative costs than OPENL, but lower indirect costs during and after discharge. This translated into lower or similar overall costs for VATSL than for OPENL in most of the studies. Thus, the careful use of disposable instruments, and minimizing the health costs during and after discharge can reduce the overall costs of VATSL to levels similar or below those of OPENL. The worry that VATSL might be associated with increased total cost is thus not warranted, and should not be used as an excuse against the use of VATS in surgery for early stage lung cancers.Alfonso Fiorelli, Stefano Forte, Francesco Paolo Caronia, Francesco Ferrigno, Mario Santini and Wentao Fang disclose no conflict of interest and no funding for the present paper. René Horsleben Petersen is speaker fee from Medtronic.


Bullfighting is booming in South Thailand, attracting tourists, and stimulating local economies. The bulls are well raised and practiced, but in many cases, the owners lack knowledge and understanding of the prevention of animal diseases, including parasitic infections. This study aimed to determine the occurrence of gastrointestinal (GI) parasite infection in fighting bulls.A total of 1501 fecal samples were collected from bulls aged 2-5 years visiting the animal hospital of Prince of Songkla University during 2016-2019. The formalin ethylether concentration method was used to detect GI parasites in feces.Eurytrema spp. (2.83%), Fasciola spp. (2.47%), Trichuris spp. (0.35%), and Moniezia spp. (0.14%). Two protozoan genera were identified, Balantidium coli (6.64%) and Eimeria spp. (3.53%). Coinfection was observed in 33.99%. The five most common coinfections were rumen fluke with strongyles (20.85%), B. coli (4.66%), Eimeria spp. (1.55%), Eurytrema spp. (1.34%), and Fasciola spp. (1.06%).The overall rate of GI parasite infection was 94.27%. Rumen fluke eggs were detected in 97.17% of all infected animals, followed in prevalence by strongyles (26.29%), In addition to high GI parasite infection rates, zoonotic parasites were observed. Therefore, it is recommended that farmers should follow good sanitation and prevention practices to control parasitic infections in bulls, and proper hygienic precautions should be taken by the owners. Implementation of deworming programs using appropriateanthelmintic drugs as well as rotation of anthelmintic drug that have different chemical agent to prevent further drug resistance should be considered. The promotion of bull health management is highly recommended to protect humans from zoonotic diseases. The raising of bulls for bullfighting is very popular in South Thailand. Fighting bulls are bred from indigenous cattle by searching for animals with distinctive properties. Bulls with the desired characteristics are raised on specialized ranches equipped with mosquito nets. The main feed includes all types of grass. The trainer walks the animals every morning at dawn for a distance of 5-10 km. After walking and exercising, the trainer takes the bull for showering and sunbathing to improve patience and strength. Although the raising of fighting bulls is more specialized than that of other types of cattle, in most cases, the owners lack the knowledge and understanding to look after and prevent several types of animal diseases. However, to keep the bulls competitive, the owner must pay attention to the bulls’ health. The owners, therefore, bring their bulls for initial physical check-ups at the animal hospital. However, local veterinarians may not have sufficient basic knowledge of the treatment and prophylaxis of infections recommended for the raising of fighting bulls.Strongyles spp., Fasciola spp., and Moniezia spp. [Trichuris spp., Moniezia spp., Eurytrema spp., Fasciola spp., and Eimeria spp. [In South Thailand, the number of bulls is approximately 10,000 . The repzia spp. . In dairria spp. . The repria spp. .With regard to the prevalence of GI parasitic infections in cattle according to region, the highest prevalence has been found in the southern region of Thailand. The impact of GI parasite infection in cattle varies from mild to severe clinical symptoms. Mild or subclinical symptoms include losses in animal productivity such as reduced milk production and weight gain, altered carcass composition, and decreased conception rate. Moderate-to-severe symptoms include hair coat roughness, anemia, edema, and diarrhea. Even subclinical effects can be of major economic importance to the bull farmers.Therefore, this study aimed to identify the prevalence of GI parasite infection in fighting bulls in South Thailand, thereby providing data that can inform future deworming programs.This study was conducted on 1501 animals at the animal hospital of Prince of Songkla University under Animal Ethics of Thailand .Fecal samples were collected from 1501 fighting bulls aged 2 to 5 years and presented for physical health examination at the Prince of Songkla University Animal Hospital from November 2016 to October 2019. The IDs and breeding areas of the bulls were also recorded.This study used a standard protocol for the formalin ether concentration technique to detect parasite eggs . Five grEggs of helminths and (oo)cysts of protozoa were identified by microscopy based on the morphological identification keys described by William .Data were analyzed as percentages using Microsoft Excel  and further by descriptive analysis.Eurytrema spp. (2.83%), Fasciola spp. (2.47%), Trichuris spp. (0.35%), and Moniezia spp. (0.14%)  and Eimeria spp. (3.53%) (B. coli (4.66%), Eimeria spp. (1.55%), Eurytrema spp. (1.34%), and Fasciola spp. (1.06%)  were positive for at least one enteric parasitic genus; coinfection with up to seven genera was seen. The most commonly observed GI parasite eggs were those of rumen fluke (97.17%), followed in frequency by strongyles (26.29%),  (0.14%) . In addi (3.53%) . A total (1.06%) .As there are no data available on GI parasite infection in fighting bulls, this study aimed to identify the prevalence of GI parasite infections in such animals with the aim of supplying data that can be used to inform choices of anthelmintic drugs for bulls. Moreover, the data serve to identify the extent of the reservoir of zoonotic parasitic infections represented by fighting bulls. The data indicated a high prevalence of GI parasite infections in bulls 94.27%); however, the results differed from those in beef cattle in the Nan Province (61%) . Our fin4.27%; hoB. coli, Eimeria spp., Eurytrema spp., and Fasciola spp., some of which are zoonotic. These results imply that parasite infections in bulls may be a reservoir of human GI parasitic infections. The prevalence of strongyle nematode infections (26.29%) in this study was higher than that observed in a previous study in cattle (10.76%) in Udon Thani, Thailand [Coinfection with GI parasite mostly involved rumen fluke with parasites such as strongyles, Thailand ; meanwhiThailand .Eurytrema spp. (2.83%) and Fasciola spp. (2.47%). Eurytrema spp. are pancreatic flukes found in the pancreatic ducts and occasionally the bile ducts of sheep, pigs, and cattle [Fasciola spp. are associated with diseases in cattle, sheep, and goats [Fasciola infections, fasciolosis has been identified by the WHO as a reemerging neglected tropical disease associated with endemic and epidemic outbreaks of diseases in human populations.There were two species of trematodes identified in the bull samples, d cattle -15. Infed cattle . Fasciolnd goats . AlthougB. coli and Eimeria spp. B. coli is a common opportunistic protozoan of man and animals and causes gastroenteritis, also known as balantidiasis, which mostly arises by ingesting infective cysts from food and water contaminated by feces from pigs or cattle [B. coli in HIV patients [Eimeria spp. are parasitic protozoa that cause human coccidiosis, found mostly in tropical and subtropical regions. This affects people of all ages and has an important impact on public health. The infections may be symptomatic, depending on the population. Symptoms may include diarrhea, cramps, bloating, nausea, and vomiting and may be prolonged [Eimeria spp. is a common problem in HIV infection which the pooled prevalence of Isospora was 2.5%  [The protozoa found in coinfections with rumen fluke in the bulls were r cattle . Human ir cattle . There apatients ,21. Eimerolonged . Disease.1-2.9%) .Several risk factors affect the level of GI parasitic infection in cattle, such as the season, pasture area, age, breed, and parasitic drugs used as well as the presence of snails on the farmland ,24,25. TSome of the identified GI parasites have zoonotic potential. Therefore, the information should be distributed to the bulls’ owners and their family members so that they understand the dangers of those parasites. Good hygiene should be practiced by individuals who have close contact with the bulls to avoid infection by zoonotic parasites.This study is the first report of GI parasitic infections in fighting bulls in South Thailand. The high prevalence of GI infections reflects the potential risk of substantial economic losses in livestock production and determines the reservoir of zoonotic parasite infections in humans represented by these animals. Future studies should focus on the relationship between types and levels of parasitism with fighting endurance and animal production, and evaluate the parasitic dynamics throughout the year and the potential paths of spread to humans.RN planned and designed the experiment, data analysis, drafted, and revised the manuscript. DK collected the samples, conducted the experiment, and interpreted the results. RW collected the samples and conducted the experiment. SI conducted the experiment and interpreted the results. All the authors have read and approved the final manuscript.The authors declare that they have no competing interests.Veterinary World remains neutral with regard to jurisdictional claims in published institutional affiliation.