Disease Control In Aquaculture Research Articles

Antibacterial activity of betel leaf (Piper betle L.) leaves extract on Vibrio harveyi

The use of herbs as a natural medicine for disease control in aquaculture is safer than using antibiotics. Antibiotic resistance from microorganisms is a problem that can impact public health. The purpose of this study was to determine the antibacterial ability of betel leaf (Piper betle L.) toward the pathogenic bacteria, Vibrio harveyi by in vitro. The research design used a completely randomized design. The number of treatments was 12 concentrations of betel leaf extract with three replications for each treatment. The parameters observed were Minimum Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC) from the ethanol extract of betel leaf. The OD value data of the MIC test results were analyzed by Analysis of Variance (ANOVA) and Duncan’s Multiple Range Test. The results proved that betel leaf extract has the potential to be antibacterial with the ability to inhibit the growth of V. harveyi at a minimum concentration of 0.19% (OD: 2.057) while at a minimum concentration of 0.39% it can kill it.

Application of Outer Membrane Protein-Based Vaccines Against Major Bacterial Fish Pathogens in India.

Aquaculture is one of the fastest-growing food-producing sectors in the world. However, its growth is hampered by various disease problems due to infectious microorganisms, including Gram-negative bacteria in finfish aquaculture. Disease control in aquaculture by use of antibiotics is not recommended as it leads to antibiotic residues in the final product, selection, and spread of antibiotic resistance in the environment. Therefore, focus is on disease prevention by vaccination. All Gram-negative bacteria possess surface-associated outer membrane proteins (OMPs), some of which have long been recognized as potential vaccine candidates. OMPs are essential for maintaining the integrity and selective permeability of the bacterial membrane and play a key role in adaptive responses of bacteria such as solute and ion uptake, iron acquisition, antimicrobial resistance, serum resistance, and bile salt resistance and some adhesins have virulence attributes. Antigenic diversity among bacterial strains even within the same bacterial species has constrained vaccine developments, but OMPs that are conserved across serotypes could be used as potential candidates in vaccine development, and several studies have demonstrated their efficacy and potential as vaccine candidates. In this review, we will look into the application of OMPs for the design of vaccines based on recombinant proteins, subunit vaccines, chimeric proteins, and DNA vaccines as new-generation vaccine candidates for major bacterial pathogens of fish for sustainable aquaculture.

Antibacterial activity of Anadendrum microstachyum and Selaginella plana against two pathogenic bacteria (Edwardsiella ictaluri and Streptococcus agalactiae) causing freshwater fish diseases

Aquatic plants are potential to use as antibacterial to control pathogens that cause disease in fish. The antibacterial activity of extracts of Anadendrum microstachyum and Selaginella plana were tested against Edwardsiella ictaluri and Streptococcus agalactiae using disc diffusion Kirby-Bauer method. Plant extracts of A. microstachyum and S. plana at concentration 0.2 g could inhibit E. ictaluri and S. agalactiae; therefore A. microstachyum and S. plana are potential be developed as antibacterial compounds to substitute the use of antibiotic in fish farming and as candidates new drug to prevent and control of diseases in aquaculture.

Enhancement of shrimp health and immunity with diets supplemented with combined probiotics: application to Vibrio parahaemolyticus infections.

The application of probiotics for disease control in aquaculture is now a convincing approach towards replacement of antibiotics, which can cause adverse effects in aquatic animals and humans. In this study, we combined 2 probiotics, Lactobacillus acidophilus and Saccharomyces cerevisiae, with shrimp feed to create 2 formulas (WU8 and WU9), which were fed for 10 d to juvenile shrimp Penaeus vannamei. The shrimps were then subjected to a challenge infection with Vibrio parahaemolyticus, the causative agent of acute hepatopancreas necrosis disease (AHPND). The protective effects of probiotics against bacterial infection were investigated through histopathology of the hepatopancrease and immunological evaluation of shrimp. Both WU8 and WU9 probiotic mixtures (1:1, at 108 and 109 CFU kg diet-1) increased blasenzellen hepatopancreatic epithelial cells and reduced pathology caused by AHPND. After 10 d of feeding, hemocyte parameters, including the total hemocyte count, percent of granular hemocytes, and phenoloxidase activity, increased significantly and were still increasing at 24 h post infection. Crustin and penaeidin 3 genes were also highly upregulated in hemocytes before and after 24 h of bacterial challenge and significantly upregulated in the hepatopancreas 1 to 5 d post-infection. A significantly higher survival rate was observed in shrimp fed with the probiotic supplemented diet (>90%) in comparison to the control group (60%). In conclusion, probiotic mixtures of L. acidophilus and S. cerevisiae reduced hepatopancreas pathology and protected shrimp from a challenge with AHPND.

Characterization of a novel Yersinia ruckeri serotype O1-specific bacteriophage with virulence-neutralizing activity.

A lytic bacteriophage (φNC10) specific to serotype O1 Yersinia ruckeri has been identified and evaluated as a model to assess the potential use of bacteriophages and their products for disease control in aquaculture. Electron microscopy of purified φNC10 revealed a virion particle with a small (70nm) polyhedral head and short tail. φNC10 infected only serotype O1 strains of Y.ruckeri and failed to bind a defined Y.ruckeri mutant strain lacking O1 lipopolysaccharides (O1-LPS), suggesting that φNC10 uses O1-LPS as its receptor. In addition, spontaneous φNC10-resistant mutants of Y.ruckeri exhibited defects in O1-LPS production and were sensitive to rainbow trout serum. Purified φNC10 displayed a polysaccharide depolymerase activity capable of degrading Y.ruckeri O1-LPS and thereby sensitizing Y.ruckeri to the bactericidal effects of rainbow trout serum. The φNC10-associated polysaccharide depolymerase activity also reduced the ability of Y.ruckeri cells to cause mortality following intraperitoneal injection into rainbow trout. These data demonstrate a potential utility of φNC10 and its associated polysaccharide depolymerase activity for Y.ruckeri disease prevention.

Expression Response Profiles of Toll-Like Receptor Family Members in Cyprinus Carpio after Lipopolysaccharide Stimulation, and Implication for Disease Control in Aquaculture

In this study, response profiles of 17 Toll-like receptors (TLRs) to lipopolysaccharide (LPS) stimulation were studied at mRNA levels in the liver of the common carp Cyprinus carpio. Three types of gene response profiles were observed in TLR family. The first pattern was that the expressions of some TLRs were initially up-regulated and then decreased with time. This finding indicated that the responses of these TLRs were sensitive to LPS stimulation, and it was suggested that these TLRs were involved in responding and coping with LPS, implying that these TLRs were important in immune defense from Gram-negative bacteria. The second pattern was that some TLR expressions showed no obvious change after LPS stimulation, demonstrating that they were not sensitive to LPS and probably did not recognize LPS, implying that these TLRs should not be regarded as the major receptors to be regulated to prevent disease caused by Gram-negative bacteria. The third pattern was that gene expressions of partial TLRs were significantly downregulated after a period of LPS stimulation, indicating that their gene expressions were inhibited by LPS action, and implying that these TLRs did not initiate the signal pathway for inflammation reaction to clear Gramnegative bacteria. This study is based on the different response patterns of TLRs to LPS, that imply that the sensitive response TLR genes to LPS might be selected as target genes regulated by drugs that control inflammation reaction of diseased fish in aquaculture.

El uso de hongos macroscópicos como inmunoestimulantes en peces teleósteos: estado del arte al 2018

Background. Different methods are currently used for the prevention and control of diseases in aquaculture. Prophylaxis with immunostimulants from microscopic fungi provides protection against diseases and increases resistance to parasites. In this context, Basidiomycota fungi could have high potential for use in aquaculture because they contain different compounds, such as fungal proteins, polysaccharides, terpenoids, vitamins, and minerals, which could work as immunostimulants. Goals. Collect and disseminate the information on species of macroscopic fungi that have been used as immunostimulants in the farming of teleost fish and the fungi that have not been tested in fish but have shown positive results in other organisms. Methods. We gather, discuss and compare the experimental, scientific and theoretical literature related to the immunostimulant effect of macroscopic fungi in the cultivation of teleosts, as well as fungi whose stimulating effect has been positive in other organisms. Results. We evaluated more than 50 species of fungi with immunostimulating properties. Nevertheless, in recent years, the effects of only eight species of macromycetes, belonging to the genera Ganoderma, Inonotus, Lentinula, Pleurotus, and Trametes, have been evaluated in 17 published papers regarding the immune response of nine species of fish. Seven of these papers are about isolated glucans, six about crude and alcoholic extracts, two about pulverized basidiomata, and one about a mushroom fermented by-product. Conclusions. Due to the limited information on the immunostimulating effect of macromycetes on fish, more research is needed regarding other fungi species with immunostimulant properties, the correct determination of fungi species, their culture, and in vivo and in vitro tests that confirm their immune system effects on the model organism and target organism.

Biological Approaches for Disease Control in Aquaculture: Advantages, Limitations and Challenges.

Although aquaculture activity has experienced a great development over the past three decades, infectious diseases have become a limiting factor for further intensification. Because the use of antibiotics has led to the widespread emergence of antibiotic resistance, the search for alternative environmentally friendly approaches is urgently needed. This Opinion paper offers an update on the successes and challenges of biological approaches for bacterial disease prevention and control in aquaculture. Although most of these approaches are still in research and development stages, some of them have shown promising results in field trials. Therefore, a better understanding of the mechanisms of action of these approaches will help to maximise their beneficial properties.

Multiple Isoforms of Anti-Lipopolysaccharide Factors and Their Antimicrobial Functions in the Ridgetail Prawn Exopalaemon carinicauda.

As a kind of antimicrobial peptides (AMP) in crustacean, anti-lipopolysaccharide factors (ALFs) have broad spectrum antimicrobial activities. In the present study, we identified four ALF genes, EcALF2-5, from the ridgetail prawn Exopalaemon carinicauda. Tissue distribution analysis showed that EcALF2 and EcALF4 transcripts were mainly located in gill, epidermis, and stomach, while EcALF3 and EcALF5 were mainly in hemocytes. Peptides corresponding to the LPS binding domain (LBD) of EcALFs were synthesized for analyzing their antimicrobial activities. Minimal inhibitory concentration (MIC) analysis showed that the synthetic LBD peptides of EcALF3 and EcALF4 could inhibit the growth of Gram-positive and Gram-negative bacteria, while the synthetic LBD peptides of EcALF2 and EcALF5 showed antibacterial activity against Vibrio. Incubation of white spot syndrome virus (WSSV) with the synthetic LBD peptides of EcALF3, EcALF4, and EcALF5 could reduce the in vivo viral copy number in WSSV-infected prawns. After silencing of EcALFs, Vibrio exhibited a rapid proliferation in the hepatopancreas of the prawn. The present data showed the important function of different EcALFs in modulating the in vivo bacterial and viral propagation in E. carinicauda. This study will provide new clues into the disease control in aquaculture.

Trends in aquaculture sciences: from now to use of nanotechnology for disease control

AbstractAquaculture is increasingly important in global food production. Consequently, the control of diseases in aquaculture is essential, due to the potential environmental impacts, notably in terms of the states of health of freshwater bodies and the oceans. In this study, we review the possible uses of different management systems, highlighting essential oils and novel nanotechnological strategies, for the control of diseases in aquaculture.

Identification and characterization of two arasin-like peptides in red swamp crayfish Procambarus clarkii

Antimicrobial peptides (AMPs) are small effectors in host defense by directly targeting microorganisms or by indirectly modulating immune responses. In the present study, two arasin like AMPs, named as Pc-arasin1 and Pc-arasin2, were identified in red swamp crayfish Procambarus clarkii with sequence similarity to the arasins found in Hyas araneus. Both Pc-arasins consisted of signal peptide, N-terminal proline-rich region and C-terminal region containing four conserved cysteine residues. The similarity of two Pc-arasins was 44.44%, and Pc-arasin2 contained several additional residues in the N-terminus. Multiple alignment of arasin family suggested the conservation of the C-terminus and the variation of the N-terminus of Pc-arasins. Both AMPs were found hemocytes-specific, and the expression could be induced the challenge of bacteria, espeacially by the pathogenic bacterium Aeromonas hydrophila. Knockdown of each Pc-arasin expression by double strand RNA would suppress the host immunity against A. hydrophila, and the commercially synthetic Pc-arasins could rescue the knockdown consequence. Both synthetic peptide showed broad antimicrobial activity towards 3 Gram-positive bacterium and 3 Gram-negative bacterium, and the minimal inhibitory concentrations varied from 6.25 μM to 50 μM. These results presented new data about the sequence, expression and function of arasin family, and emphasized the role of this family in host immune response against bacterial pathogens. The characterization of Pc-arasins also provided potential of therapeutic agent development for disease control in aquaculture based on these two newly identified AMPs.

A single WAP domain-containing protein from Litopenaeus vannamei possesses antiproteinase activity against subtilisin and antimicrobial activity against AHPND-inducing Vibrio parahaemolyticus

The single WAP domain-containing protein (SWD) is a type III crustin antimicrobial peptide whose function is to defense the host animal against the bacterial infection by means of antimicrobial and antiproteinase activities. A study of SWD from Litopenaeus vannamei (LvSWD) is reported herein about its activities and function against bacteria, particularly the AHPND-inducing Vibrio parahaemolyticus (VPAHPND) that causes acute hepatopancreatic necrosis disease (AHPND). The LvSWD is mainly synthesized in hemocytes and up-regulated in response to VPAHPND infection. Over-expressed mature recombinant LvSWD (rLvSWD) and its WAP domain (rLvSWD-WAP) are able to strongly inhibit subtilisin but not trypsin, chymotrypsin and elastase. The rLvSWD inhibits subtilisin with the inhibition constant (Ki) of 14.3 nM. However, only rLvSWD exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Unlike the rLvSWD, the rLvSWD-WAP does not possess antimicrobial activity. Therefore, the killing effect of rLvSWD on VPAHPND and Bacillus megaterium was studied. The MIC of 30 μM against VPAHPND is bactericidal whereas the MIC against B. megaterium is not. With four times the MIC of rLvSWD, the VPAHPND-treated post larval shrimp are able to survive longer with 50% survival rate as long as 78 h as compared to 36 h of the infected shrimp without rLvSWD. The antimicrobial activity of LvSWD against the VPAHPND infection suggests its potential application for disease control in aquaculture.

Evolution of the angiopoietin-like gene family in teleosts and their role in skin regeneration

BackgroundThe skin in vertebrates is a protective barrier and damage is rapidly repaired to re-establish barrier function and maintain internal homeostasis. The angiopoietin-like (ANGPTL) proteins are a family of eight secreted glycoproteins with an important role in skin repair and angiogenesis in humans. In other vertebrates their existence and role in skin remains largely unstudied. The present study characterizes for the first time the homologues of human ANGPTLs in fish and identifies the candidates that share a conserved role in skin repair using a regenerating teleost skin model over a 4-day healing period.ResultsHomologues of human ANGPTL1-7 were identified in fish, although ANGPTL8 was absent and a totally new family member designated angptl9 was identified in fish and other non-mammalian vertebrates. In the teleost fishes a gene family expansion occurred but all the deduced Angptl proteins retained conserved sequence and structure motifs with the human homologues. In sea bream skin angptl1b, angptl2b, angptl4a, angptl4b and angptl7 transcripts were successfully amplified and they were differentially expressed during skin regeneration. In the first 2 days of skin regeneration, re-establishment of the physical barrier and an increase in the number of blood vessels was observed. During the initial stages of skin regeneration angptl1b and angptl2b transcripts were significantly more abundant (p < 0.05) than in intact skin and angptl7 transcripts were down-regulated (p < 0.05) throughout the 4-days of skin regeneration that was studied. No difference in angptl4a and angptl4b transcript abundance was detected during regeneration or between regenerating and intact skin.ConclusionsThe angptl gene family has expanded in teleost genomes. In sea bream, changes in the expression of angptl1b, angptl2b and angptl7 were correlated with the main phases of skin regeneration, indicating the involvement of ANGPTL family members in skin regeneration has been conserved in the vertebrates. Exploration of the fish angptl family in skin sheds new light on the understanding of the molecular basis of skin regeneration an issue of importance for disease control in aquaculture.

Identification and function analysis of an anti-lipopolysaccharide factor from the ridgetail prawn Exopalaemon carinicauda

Anti-lipopolysaccharide factor is a kind of antimicrobial peptide (AMP) with broad-spectrum activities against bacteria, virus, and fungi in crustacean. Different isoforms of ALFs showed different activities to virus or bacteria. Therefore, discovery of more ALFs will provide new insights into drug development and disease control. Here we reported an ALF gene, EcALF1, isolated from the ridgetail prawn Exopalaemon carinicauda. Its transcripts was mainly detected in hemocytes. Silencing of EcALF1 caused a lesion of hepatopancreas and finally led to death of the prawn. In vivo bacteria detection to the hepatopancreas showed that the colonies of thiosulphate citrate bile salts (TCBS) cultured bacteria in EcALF1-silenced prawn increased dramatically. We identified the dominant bacteria cultured in TCBS and found that Vibrio alginolyticus and Vibrio parahaemolyticus were dominant bacteria which were pathogenic species to prawn. We further synthesized the functional domain, LPS binding domain (LBD) of EcALF1, and its antibacterial and antiviral activities were detected. The synthetic EcLBD1 peptide showed very strong antibacterial activity against Micrococcus luteus and V. alginolyticus. Pre-incubation of WSSV with EcLBD1 peptide reduced the pathogenicity of WSSV infection to the prawn. The present data showed the important roles of an ALF gene in modulating the in vivo bacterial proliferation in E. carinicauda and provided new insight in disease control in aquaculture.

The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product.

The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.

Zebrafish as a useful model for immunological research with potential applications in aquaculture

AbstractThe Zebrafish (Danio Rerio) is a Teleost fish, which offers several practical advantages concerning manipulation, maintenance and versatility as well as short life cycle, low cost and physiological similarity with most farmed species. Zebrafish have been used as model for several aquaculture‐relevant infectious agents, since the cost of infections to aquaculture can be great, making failure of immunity a major risk for commercial fish farming. This have allowed scientist to understand pathogens biology as well as fish immune response against infection, and thus have helped to improve disease control in aquaculture. This review summarizes the recent reports and main knowledge concerning the cellular and molecular aspects of the immune response in zebrafish and its potential applications in the fish farming industry. Several research results in zebrafish and other teleosts reveal that mucosal tissues have a strong and well‐structured immune response involving both innate and adaptive components. Together with the advantages of low maintenance cost, easy handling and genetic tools available, zebrafish comes up as a useful and reliable animal model for in vivo study of fish immune response and mechanisms as well as fish‐affecting infectious and inflammatory diseases which are a cause of concern for the aquaculture industry. Also, vaccines development, drugs screening and immunologically focused fish food assessment seem to be promising fields where zebrafish model can be used to generate positive impacts to the aquaculture practice.

Phaeobacter inhibens as probiotic bacteria in non-axenic Artemia and algae cultures

The growing aquaculture industry is in need for non-antibiotic based disease control strategies to reduce risk of bacteria developing and spreading antibiotic resistance. We have previously, in axenic model systems of live larval feed, demonstrated that bacteria from the Roseobacter clade can antagonize fish pathogens such as Vibrio anguillarum and Vibrio harveyi and that they can reduce larval mortality in challenge trials. However, in the aquaculture production, a natural microbiota is present at all stages and may affect the efficacy of the probiotic bacteria. The purpose of the present study was to determine if marine roseobacters in non-axenic systems were capable of antagonizing fish pathogenic vibrios. We added a controlled background microbiota of four bacterial strains to axenic Artemia and algae (Duniella) and these bacteria had a marginal but significant reducing effect on inoculated Vibrio anguillarum that grew to 107 in control samples but to a level 1–2 log lower in samples with background microbiota. The addition of the Roseobacter-clade bacteria, Phaeobacter inhibens, caused a significant reduction in growth of the pathogen that reached levels 3–4 log lower than in the control. In non-axenic natural Artemia and algae (Tetraselmis) received from an aquaculture unit, Vibrio anguillarum grew to 107CFU/ml but only reached 104CFU/ml when P. inhibens was also added. P. inhibens was added at a concentration 106CFU/ml in all systems and remained at this concentration at the end of the study, irrespective of the background microbiota. We therefore conclude that P. inhibens are indeed promising as probiotic bacteria in marine larvi-culture where it in natural live feed can suppress fish larval pathogens. Statement of relevanceWe and others have in several studies demonstrated the potential effect of probiotic bacteria in axenic or gnotobiotic models. Here, we for the first time present data that demonstrate that probiotic roseobacters also are efficient in non-axenic systems; even in commercial algae and Artemia from a commercial fish farmer. This adds further promise to probiotics as disease control in aquaculture.

Characterization of Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss) feed and larvae: safety, DNA fingerprinting, and bacteriocinogenicity.

The use of lactic acid bacteria (LAB) as probiotics constitutes an alternative or complementary strategy to chemotherapy and vaccination for disease control in aquaculture. The objectives of this work were (1) the in vitro safety assessment of 8 Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) feed and larvae; (2) the evaluation of their genetic relatedness; (3) the study of their antimicrobial/bacteriocin activity against fish pathogens; and (4) the biochemical and genetic characterization of the bacteriocin produced by the strain displaying the greatest antimicrobial activity. Concerning the safety assessment, none of the pediococci showed antibiotic resistance nor produced hemolysin or gelatinase, degraded gastric mucin, or deconjugated bile salts. Four strains (50%) produced tyramine or putrescine, but the corresponding genes were not amplified by PCR. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting allowed clustering of the pediococci into 2 well-defined groups (68% similarity). From the 8 pediococci displaying direct antimicrobial activity against at least 3 out of 9 fish pathogens, 6 strains (75%) were identified as bacteriocin producers. The bacteriocin produced by P. acidilactici L-14 was purified, and mass spectrometry and DNA sequencing revealed its identity to pediocin PA-1 (PedPA-1). Altogether, our results allowed the identification of 4 (50%) putatively safe pediococci, including 2 bacteriocinogenic strains. ERIC-PCR fingerprinting was a valuable tool for genetic profiling of P. acidilactici strains. This work reports for the first time the characterization of a PedPA-1-producing P. acidilactici strain isolated from an aquatic environment (rainbow trout larvae), which shows interesting properties related to its potential use as a probiotic in aquaculture.

Enhancing incentives for disease control in aquaculture: a research agenda

Enhancing incentives for disease control in aquaculture: a research agenda

Testing the efficacy of probiotics for disease control in aquaculture

Infectious diseases have been estimated to cost the global aquaculture industry billions of dollars annually1,2. With concerns over emerging resistance and residues of antibiotics in food3 many such chemicals are now being banned and environmentally friendly alternatives are being sought. Probiotics influence the composition of the gut microbiota and confer health benefits to their host4,5 and are one of several alternative approaches gaining significant popularity in aquaculture. Whilst primarily used to manage bacterial disease, there is also some evidence that probiotics can provide protection against parasites4 and viruses6. Probiotics can inhibit the growth of pathogens in the gut through the excretion of antagonistic substances including bacterocins6,7; prevent pathogen adhesion in the gut through competition of space and nutrients3 and by modulating the immune system5. Some probiotics have been reported to improve growth and feed utilisation efficiency5 and others can also improve water quality2,8,9, which confers indirect benefits to host healthsup&amp;gt;6.