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Many marine invertebrates, particularly sessile or slow-moving organisms, are a rich source of valuable bioactive metabolites. Among marine invertebrates, sea cucumbers, or holothurians, have been utilized as food and folk medicines by Asia and Middle East communities. Asian people, especially Chinese believe that consuming holothurians may treat a variety of impediments and illnesses such as weakness, impotence, debility of the aged, constipation due to intestinal dryness, and frequent urination. As a consequence, these reported beneficial effects lead to the high demand for holothurians in Chinese markets.
Among holothurians, Stichopus vastus and Holothuria leucospilota were reported to have medicinal application. S. vastus is well-known for its wound healing activities which were proven by pre-clinical test in rats. In addition, the integument tissue is rich in collagen and can be used as a functional ingredient in nutraceuticals, cosmetics and food products. Furthermore, S. vastus contains novel bioactive peptides which inhibit angiotensin I converting enzyme (ACE) and also possesses radical scavenging activities. H. leucospilota is widespread throughout the Red Sea, Persian Gulf and the entire Indo-Pacific. Its main habitat are shallow areas, such as reef flats, shallow costal lagoons, and seagrass beds. In some areas such as the Federated States of Micronesia (FSM), Marshall Islands, Kiribati, Samoa, Tonga, Cook Islands, Papua New Guinea (PNG), Solomon Islands and Fiji, people consume holothurian´s gonad as food delicacies and as additional protein diets. H. leucospilota has shown antioxidant effects as well as anticancer activities against HeLa, human lung carcinoma (A549) and skin melanoma (B16F10) cells. Several bioactive compounds have been isolated from it such as leucospilotaside A to C, echinoside B, holothurin A, holothurin B, and holothurin B2. Organic extracts of body wall, gonad and intestine of H. leucospilota exhibited bacteriostatic rather than bactericidal activity against Gram-positive Bacillus subtilis and Staphylococcus aureus. This bacteriostatic effect from the organic extracts was confirmed against the Gram-negative bacteria like Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa and against the Gram-positive S. aureus and the filamentous fungi Aspergillus niger, A. fumigatus, A. flavus and A. brasilensis.
Numerous natural products from marine invertebrates show striking structural similarities to those of microbial origins, suggesting that microorganisms are at least involved in the biosynthesis of the targeted bioactive compound or they even represent the sole producer of the respective metabolites. Those findings supported efforts to isolate invertebrate associated bacteria as the real producer of the bioactive compounds to overcome the supply problem as harvest from the wild is not sustainable for most bioactive marine invertebrates.
Bioprospecting for bioactive marine bacteria recognizes the noticeable capacity of marine bacteria as a source of new natural products which can be utilized to overcome the antimicrobial resistance crisis. (Multi-)drug-resistant bacteria are becoming the global challenge leading to the strong demand for new antibiotics, either in chemical structures or mode of actions. In addition, spreading of slow progressing but deadly virus, such as Hepatitis C (HCV) menace human population particularly in developing countries. Therefore, the detection and development of new anti-infective drugs is urgently needed.
There has been an increasing number of publications focusing on the isolation of invertebrate associated bacteria for the discovery on new bioactive compounds, with sponges being the invertebrate phylum that has received most attention for isolation of the associated microbiome. However, holothurians likely present another very interesting target for the isolation of bioactive bacteria. Since they are being (a) used in traditional Chinese medicine and (b) exposed to, ingesting and reworking marine sediments, which have been shown to be a promising source for the isolation of bioactive bacteria. Even more so, if Actinobacteria, which are prolific producers of bioactive compounds, are the target bacteria phylum, since Actinobacteria have been isolated repeatedly from terrestrial soil and marine sediments. Thus, sediment bioturbating holothurians with their associated microbiome should be a promising target to isolate novel Actinobacteria. A recent publication by Gao, et al. (2014) showed that Actinobactria were enriched in the gut of four deep-water holothurian, accentuating that also shallow water holothurians could be a promising target for the isolation of bioactive Actinobacteria.
In this study, we reported the potential of bioprospecting underexplored marine associated bacteria derived from the sea cucumbers H. leucospilota and S. vastus.
None of the eight targeted infectious agents beside P. multocida and E. rhusiopathiae (Coronavirus, Paramyxovirus, Influenza A, Flavivirus, Rickettsia, Leucocytozoon, Pasmodium, or Haemoproteus) was detected on the 170 tested birds. However, strong temporal dynamics has been reported for these infectious agents in wild birds, suggesting that the detection failure may reflect an absence of targeted agents at time of bird sampling. Hence, although the circulation of these agents cannot be definitively ruled out on the basis of this study, there is no evidence suggesting that they may be involved in massive mortalities of Indian yellow-nosed and sooty albatross chicks. In contrast, PCR screening confirmed the circulation of P. multocida and E. rhusiopathiae, both previously reported in 1995–1996. At least one individual of each study species carried one of these two bacteria, but no coinfection was detected. In Amsterdam albatrosses, both bacteria were detected although with low prevalence (9.5%, 95% confidence interval: 0–22.1). One live chick tested positive for P. multocida and one other for E. rhusiopathiae. Surprisingly, no adult Indian yellow-nosed albatross (out of 50 tested) tested positive for any of these bacteria at the time of sampling. In contrast, a high prevalence of shedding adult brown skuas was found for P. multocida, although this may be affected by our limited sample size (16 tested birds). Skuas are predator and scavenger birds that frequently visit the Indian yellow-nosed albatross, sooty albatross and northern rockhopper penguin colonies in search of unattended eggs and chicks. Moreover, skuas and Amsterdam albatrosses breed in the same area (Plateau des Tourbières, Fig 2). Predation or scavenging of infected animals can play a role in the epizootiology of avian cholera. Several scavenger species have been reported to become chronically infected with P. multocida, which could greatly facilitate their ability to spread the bacteria to new sites. It is thus conceivable that skuas are involved in P. multocida inter-species transmission on Amsterdam Island and represent a risk for the Critically Endangered Amsterdam albatross, although such hypothesis requires thorough investigation.
Amsterdam Island is a critical breeding ground for several threatened seabird species. Our study shows that the demographic situation has worsened substantially for three species since previous reports published 13 years ago. In Indian yellow-nosed albatrosses, the overall breeding success has remained below 15% for the past 15 years, and the occasional higher success years observed during the 1980’s and 1990’s have become scarcer. Sooty albatrosses have suffered an abrupt decline in reproductive success between 1995 and 2005 with total breeding failure since 2011. The situation has also worsened for the northern rockhopper penguin, with zero fledging since 2013 and a nearly 82% population decrease over the past 22 years. In contrast, the Amsterdam albatross breeding population has increased and breeding success has been high on average, apart from three years (1990, 2000 and 2001) of high chick mortality. As P. multocida was detected in this species and their nests are situated only 2–3 km away from the cliffs where Indian yellow-nosed albatrosses, sooty albatrosses and northern rockhopper penguin breed, we would expect a higher impact of outbreaks on the Amsterdam albatross population. In this species, nests are widely interspersed, with very low density over the plateau des Tourbières (~0.2 nest/ha) contrary to high nest densities in colonies of northern rockhopper penguin (~3,500 nests/ha) or Indian yellow-nosed albatrosses (~3,100 nests/ha). It is possible that this low nest density limits the spreading of infectious diseases and hence its impact on the population. However, a comprehensive exploration of the epizootiology of outbreaks occurring on Amsterdam Island is required to estimate the intensity of threat for this Critically Endangered species.
Emerging infectious diseases affecting humans, wildlife, and agriculture are often the result of a pathogen jumping from its original host into a novel host species. This can take the form of spillover events that result in dead end infections or short stuttering transmission chains, or a host shift with successful infection and sustained transmission in the new host (Box 1). Host shifts have resulted in multiple human pandemics, such as HIV from chimps and the H1N1 “Spanish flu” from birds, which have both killed tens of millions of people. Other important human pathogens have originated from other host species, including Plasmodium falciparum
, SARS coronavirus, Hendra and Nipah viruses, and the measles virus. Past host shifts can be detected when the phylogenies of hosts and their pathogens are different (phylogenetic incongruence—Box 1). This is very common, with a survey of the published literature finding 93% of studies comparing host and pathogen phylogenies showed evidence of host shifts, and there are relatively few cases where the pathogen phylogeny mirrors that of its host completely.
Here we examine the evolutionary factors that affect a pathogen's ability to infect a novel host and then discuss how the ability of a pathogen to adapt to be transmitted efficiently by a novel host can allow its long-term persistence. Following a host shift, selection will favour mutations that allow a pathogen to (a) enter a host cell with greater efficiency and (b) “fine tune” or optimise their fitness in the new host, for example by better utilising cellular machinery, enhancing immune avoidance, optimising virulence, and maximising transmission potential. Our focus is on viruses, owing to a wealth of recent studies, and because RNA viruses are the most likely group of pathogens to jump between hosts, possibly because of their ability to rapidly adapt to new hosts–. While we focus on genetics in this review, behaviour and ecological processes are clearly a hugely important factor in determining whether a novel host is exposed to a novel pathogen, and whether onward transmission occurs,–.
To assess AaHV1 infectivity in other Ascomycota fungal species, AaHV1 was introduced (transfected) to B. dothidea, F. graminearum and C. parasitica, which are the fungal pathogens of apple white rot (or ring rot), wheat head blight and chestnut blight diseases, respectively (Vasilyeva and Kim, 2000; Tang et al., 2012; Rigling and Prospero, 2018). AaHV1 accumulation was detected in B. dothidea (family Botryosphaeriaceae, belonging the same class Dothideomycetes with Alternaria spp.), but not in F. graminearum PH-1 (family Nectriaceae, class Sordariomycetes) and C. parasitica EP155 (family Cryphonectriaceae, class Sordariomycetes) (Figure 7A and data not shown). It is interesting to note that AaHV1 infection markedly reduced B. dothidea growth on PDA medium and apples (Figure 7B,C). At present, four RNA mycoviruses are known to infect the B. dothidea strains, two of them (a chrysovirus and proposed polymycovirus) are associated with conferring hypovirulence-associated traits to fungal hosts (Wang et al., 2014; Zhai et al., 2015, 2016). Our data indicates that AaHV1 could confer hypovirulence in a heterologous fungal host (unnatural host). This observation also implies that AaHV1 has potential for use as a biocontrol agent for other fungal crop diseases.
In this study, a total of 275 bacterial colonies were isolated in Indonesia from the internal and external parts of the two sea cucumbers, Holothuria leucospilota (HL) and Stichopus vastus (SV). Back in Germany, bacterial colonies were re-grown on marine agar (MA) resulting in 127 different strains based on 16S rRNA gene sequence analyses (Table 1). A detailed compilation of all isolated bacterial strains from HL and SV are shown in Table S1. Our results of the identified phyla are in line with previous studies that attempted to isolate cultivable bacteria from marine macroorganisms and environments. However, as determined by next generation sequencing studies, the cultivable bacteria still only represent about 1% of the estimated microbial diversity.
From the identification result, some Actinobacteria from H. leucospilota (HL 108, HL 111, HL 255, HL 66 and HL 268) show less than 98% similarity to the next type strains when compared to those in the NCBI database (http://www.ncbi.nlm.nih.gov/; using the Basic Local Alignment Search Tool (BLAST)) and thus are representatives of a putative new bacterial species. HL 108 is related to Glutamicibacter nicotianae (96.38% sequence similarity), HL 111 to Nocardioides exalbidus (97.96% sequence similarity), HL 255 to Kytococcus sedentarius (97.58% sequence similarity), and the others to Kocuria palustris (97.64% and 97.45% sequence similarity), respectively (Table S1).
Two Actinobacteria from S. vastus, SV 16 and SV 203, showed less than 98% similarity to the next type strain. These bacteria are related to Serinicoccus profundi (97.91% sequence similarity) and Mariniluteicoccus endophyticus (96.26% sequence similarity), respectively (Table S1). In addition, an Actinobacteria from S.vastus showed less than 95% similarity the next type strains. Isolated from the external part of S. vastus, SV 17 is putatively a member of a new genus of the Propionibacteriaceae (93.3% sequence similarity with Pseudopropionibacterium rubrum). The phylogenetic position, based on 16S rRNA-gene analyses, of selected isolated Actinobacteria is presented in Figure S1.
A total of 33 strains of Firmicutes that related to the genera Staphylococcus and Bacillus could be isolated from both, H. leucospilota and S. vastus (cf. Figure S2). Bacterium HL 79 showed only 92.88% similarity to the next type strain Bacillus sonorensis, and thus probably represents a new genus (Table 2). Isolates that had more than 99% sequence similarity with Staphylococcus cohnii subsp. urealyticus were found in all samples.
We identified 30 strains belonging to 9 different genera of Proteobacteria (Phylogeny tree see Figure S3). The genera Vibrio and Paracoccus were found in both H. leucospilota and S. vastus. Vibrio alginolyticus was isolated from both sea cucumbers (Table 2). Both Vibrio alginolyticus and Vibrio harveyi have caused diseases in aquatic animals including sea cucumbers. Pathogenicity of the genus Vibrio is not only caused by suitable conditions (i.e., temperature, low host immunity and nutrition) but also by the presence of the vibriolysin-like protease.
Analyses of sequences resulted in several Proteobacteria showing less than 98% similarity to the next type strains: HL 125 showed 96.23% sequence similarities to Vibrio harveyi and HL 28 showed 97.33% similarity to Paracoccus koreensis. These bacteria probably represent new species. Bacterium SV155 is putatively a member of a new genus of the Rhodobacteraceae which showed closest relationship to the genus Paracoccus (93.08% sequence similarity with Paracoccus beibuensis, Table 2).
We tested all 127 bacterial strains with the agar plug diffusion assay against environmental bacteria in a preliminary screening. Subsequently the 69 active strains were further cultured and extracted for additional bioassays. There were 19 bacterial strains that active in the preliminary test from 39 bacterial strain from internal part of H. leucospilota extracted and further assayed against microorganisms. About 47.4% (9 out of 19) of the bacterial extracts from the internal part of the H. leucospilota showed activity against Gram-positive Bacillus subtilis and 15.8% (3 out of 19) were active against Staphylococcus aureus. Strain HL 55, identified as Kocuria flava (99.21% sequence similarity), displayed potent activity against the two Gram-positives strains and additional activity against the Gram-negative E. coli (Table 2). Only one strain from the internal parts of the H. leucospilota showed activity against filamentous fungi M. hiemalis (5.3%, 1 out of 19). However, fungal activity was markedly higher against filamentous fungi M. hiemalis and R. glutinis when bacterial extracts from the external parts of H. leucospilota were tested (22.2%, 6 out of 27 tested).”
Antimicrobial testing on 27 bacterial strains from the external part of the H. leucospilota which active in the preliminary screening showed as much as 44.4% (12 out of 27) were active against B. subtilis, 51.9% were active against S. aureus, 3.7% were active against M. smegmatis, 7.4% active against M. hiemalis, and 25.9% active against R. glutinis. Strains with the highest activity were HL 22, HL 63, HL 67, HL 111, which related to Vibrio alginolyticus, Bacillus safensis, Staphylococcus cohnii subsp. urealyticus, and Nocardioides sp., respectively (Table 2). The observed antimicrobial activities are in line with previous studies, but bioactivities on S. cohnii have not been reported so far.
From 23 different bacterial strains that showed already activity in the preliminary screening, bioactive compound producing strains isolated from S. vastus were identified as Streptomyces cavourensis (SV 21), Bacillus safensis (SV 147), and a putatively new genus of the Rhodobacteraceae which closely related to genus Paracoccus (SV 155, Table 2). Streptomyces cavourensis has been reported to strongly inhibit plant pathogenic fungi. The antimicrobial activity of Paracoccus spp. has been recorded against Salmonella sp., Proteus sp., and MRSA. In addition, recent studies reported algicidal activities of Paracoccus sp. against harmful algal blooms of Prorocentrum donghaiense.
The toxic risk is implicated on many levels in the issues surrounding the “One Health” concept because of direct harmful effects of contaminants and their impact on the physiology, immune, and endocrine responses of organisms, biodiversity, and the transmission of pathogens. Contaminants and toxins can also impact host–pathogen interactions, by directly affecting the pathogens (62). However, toxins and pollutions are to a certain extent part of nature, and toxicity does not mean the same for all organisms. For example, Lake Natron (Kenya) is an unhospitable place for most species, but some have adapted to this environment (like flamingos, Spirulina, and invertebrates adapted to caustic waters they live on). As a consequence, the occurrence of toxicants per se might not be problem, and there is certainly a lot to learn from the adaptive mechanisms evolved by species living in such “toxic” environments.
Environmental pollution is a worldwide concern. The toxic risk is particularly high in environments where the human population is very dense, such as coastal areas, where species are subjected to multiple toxins and pollutants including natural toxins (e.g., paralytic shellfish poisoning toxins synthesized by certain harmful microalgae), emerging pollutants (e.g., micro- and nanoplastics) and diffuse pollution linked to multiple anthropogenic releases (63, 64). However, even remote areas without high anthropogenic activities such as polar areas are also contaminated, with a long list of legacy or emerging organic and inorganic compounds involved (65). The recent and global nature of environmental pollution is even reflected by marked differences in Holocene signatures in stratigraphic records showing unprecedented combinations of various anthropogenic substances (66). Wildlife and domestic animals are currently exposed to numerous contaminants at levels endangering their survival and health, their ability to reproduce and capability to cope with other stressors such as pathogens, and this represents a threat on biodiversity and ecosystem functioning which is now acknowledged (67–71).
The widescale development of multifactorial diseases affecting both invertebrates (bees, corals, and oysters) (72–75) and vertebrates (amphibians, cetaceans, and chiropterans) (76–79) is increasingly recognized thanks to the development of tools in genomic medicine and epidemiology that facilitate their study. As a consequence, diseases of complex etiologies are receiving increasing attention. Multifactorial diseases often emerge in organisms whose defense capacities have been reduced by changes in nutrition, temperature, salinity, pH, exposure to pollutants, toxins, radiations, etc. Through cumulative and long-term effects, toxins have significant impact on morbidity caused by both pathogens and other toxic substances (cocktails). Toxicants increase the risk of infectious diseases when the immune system is directly or indirectly affected (67, 71, 80–83). Immunotoxic effects do not only have a direct effect on human health and the viability of human and animal populations, but also affect the broader functioning of ecosystems and promote the transmission of zoonotic diseases by increasing the prevalence of pathogens in animal reservoirs or intermediary hosts. Therefore, the major threat posed by pollutants to biodiversity has currently undetermined consequences on biotic interactions (Figure 3). As a result of changes in species abundance and food web topology (extinction of “regulatory” predators, role of “super-predator,” consumptive competition, effects on keystone species, biological invasion, increase in resistant disease reservoir species, density effects dependent on emergence of epizootics or zoonotic diseases, etc.), pollution further significantly increases the risk of disease.
The occurrence of some chronic non-communicable diseases is currently soaring in southern countries, highlighting the globalization of sanitary risks (84). Part of it is due to significant advances in combating infectious diseases, which have greatly reduced mortality and as a consequence modified the occurrence of non-infectious diseases. However, environmental changes, and particularly exposure to toxic substances, were shown to play an important role in the occurrence of serious chronic non-infectious diseases in humans (respiratory, cardiovascular, neurological, and metabolic diseases, obesity, diabetes, and cancer), the prevention of which is a major challenge for our society, both for the present and the future generations. Transgenerational effects of environmental stress (85) transmitted by epigenetic mechanisms (86) have been described in various species. There is no reason to think that humans should be exception to this rule, and indeed a comparable picture emerges for wildlife from many case reports worldwide (70). This indicates the importance of the man–animal–ecosystem interface in determining the evolution and emergence of chronic diseases in humans, just as in other species. For this reason, human and veterinary medicine is often developing a reductionist and frequently reductive approach that needs reviewing in the context of the current situation. Prevention and control, which are increasingly accessible, have a great potential for tackling such complex disease dynamics.
The target of human and veterinary medicine is first the treatment and prevention of diseases. In both cases, liposomes contributed major part in controlled release and targeted to specific sites without any side effects using liposomal formulations. Sallovitz et al. described very extensively about bacterial diseases in human and veterinary medicine. Liposomal formulations have been addressed in several bacteria including Staphylococcus aureus, Salmonella species, Brucella species, and Mycobacterium species. MacLeod and Prescott demonstrated that liposomally entrapped gentamycin was used to kill Staphylococcus aureus mastitis in bovine. Liposomes containing dipalimitoyl phosphatadylcholine, and dimyristodylphosphatadyl glycerol encapsulating tobramycin showed considerable antibacterial effects against Gram positive and negative bacteria. The nisin-based formulation showed better efficacy in the treatment of clinical mastitis in lactating dairy cows caused by several different mastitis pathogens on a commercial dairy farm. Liposomes entrapped phage shows effective targeting against multidrug resistant bacterial infections. The causative agent of chronic pulmonary bacterium called Mycobacterium avium intrinsic resistance conventional chemotherapeutic agents, however when treated with aminoglycosides shows more effective.
Fungi are known to cause diseases, including candidiasis, cryptococcosis, and histoplasmosis. Liposomal AmB (LAmB) is known to be effective and less toxic than conventional antimicrobial agents, which was demonstrated in several animal models. Krawiec et al. demonstrated the high efficacy of LAmB in dogs infected with various degree of severity of blastomycosis. The treatment of fungal pneumonia is tedious process and it is very difficult to treat animals. However, when the animals administered to LAmB showed more efficient and reduced level of disseminating. The incorporation of photosensitizers (PSs) into liposomes, micelles, or nanoparticles seems to alternative and it is a promising approach to reduce the PS self-aggregation and to enhance the targeted delivery of the PS in various microbial diseases. Khan et al. formulated liposomes containing amphotericin B in tuftsin showed enhanced efficacy against systemic cryptococcosis in leucopenic mice.
Viral diseases are the major source of morbidity, mortality, and economic loss in human and animals. Liposomes are reasonably increasing the drug efficacy without inducing toxicity to the host cells; it could target only replication of virus by delivering the drugs at specific sites. Liposomal entrapped ribavarin and 2’3’-dideoxycitidine have shown promising formulation for viral diseases. Rhee et al. formulated hemagglutinin-derived synthetic peptide with a CpG-DNA-liposome complex, which induce the protection against lethal influenza virus. The toxicity of a novel liposome formulated of meglumine antimoniate in dogs with visceral leishmaniasis, the results indicated that the newly formulated compounds exhibited significant cytotoxicity. Sadozai and Saeidi dedicated exclusive review on recent developments in Liposome-based veterinary therapeutics.
Risk analysis is a key management approach for both applied epidemiologists and invasion biologists. In this section, we focus primarily on risk assessment and return to discuss risk management later. Risk assessment is applied to help develop policies in anticipation of, and in response to, disease emergence events and biological invasions. To support these risk assessments, both disciplines aim to identify qualities (traits or syndromes) that (i) make species ‘invaders’ or ‘emergers’ (e.g. [72–74]), (ii) make source environments more likely to yield them (e.g.), and (iii) render receiving environments susceptible or resistant to invaders or emerging pathogens. Modelling is used in both invasion science and epidemiology to elucidate biological processes, predict establishment and spread, to support risk assessment and to assess effectiveness of interventions. The same ‘top-down’ (correlative, e.g. statistical models, ecological niche models and machine learning) and ‘bottom-up’ (mechanistic, e.g. dynamic simulation models, network analysis, individual-based models) methods are used for predicting the possible current and future extent of EIDs and invasive species. Disease modelling methods used by epidemiologists would, of course, be directly relevant to modelling all types of infectious diseases, including those that affect species other than vertebrates, including plant pathogens. Methods for monitoring invasive species, including active field surveillance and citizen science-based passive surveillance, have much in common with methods used to monitor risks from emerging zoonoses and vector-borne diseases in the environment [78–81]. Similar sampling designs are used and their implementation in target regions or sentinel sites is often determined by similar criteria, such as likely spread patterns predicted by species distribution and spread models, and occurrence of locations where impact may be greatest (e.g.). In both disciplines, molecular approaches are used to confirm species identities and for source attribution, and both are exploring Earth observation data as proxies for potential occurrence of invaders, or risk from EIDs.
The metal oxide nanoparticles, particularly Zinc oxide nanoparticles are most commonly used in catalysis, sensors, and environmental remediation and personal care products. Recently, ZnO-NPs that were used in various applications of veterinary sciences due to their antibacterial, antineoplastic, wound healing, and angiogenic properties, and further ZnO-NPs have been used in tissue repair, as food preservative and as feed additive. ZnO-NPs found to inhibits the viability wide range of bacteria by strong interaction with bacterial cells and it could induce microbial cell injury by the generation of hydrogen peroxide from the surface of ZnO and finally it can enter into the bacteria by interacting with phosphorus and sulphur containing compounds, like DNA of bacteria. Mastitis is a disease of high yielding animals that is commonly caused by Staphylococcus, Streptococcus, and E. coli, which leads to economic consequences by reducing the yield of milk. To treat various causative agents of mastitis the usage of antibiotics has been increased and eventually leads to antibiotic resistance. ZnO-NPs have been found to be effective against biofilm inside the udder tissue causative agents, such as S. aureus and E. coli. Arabi et al. found that the bactericidal effects on both Gram-positive and Gram-negative bacteria and also effective against spores that are resistant to high temperature and high pressure by the mechanism of increase the permeability and inhibition of membrane transport and eventually leads to cell death. To determine the effect of Zinc oxide nanoparticles on various mastitis causing bacteria, including Staphylococcus epidermis, Streptococcus agalactiae, Klebsiella pneumoniae, and E. coli, the prepared ZnO-NPs with an average size of 20 nm (Figure 6). While the pathogenic bacteria treated with ZnO-NPs shows that pronounced decreased level of cell viability in all the tested bacterial strains (Figure 7).
The peculiar properties of ZnO NPs such as selective toxicity of preferential killing of cancer cells with minimal toxicity to normal primary immune cells and cancer cells, it can be used anti-cancer agent in both humans and veterinary animals, and also it is more sensitive to detect cancer biomarkers, therefore it can be easily used in ZnO-NPs based diagnostic devices. Due to all these, salient features of ZnO-NPs leads to significant usage in diagnostic and therapeutic purposes in common neoplastic conditions of animals, like lymphoma, cutaneous cancer, transmissible veneral tumor, and equine sarcoids. Recently, neoplastic disorder was most frequently found in in domestic animals, for example, haematopoietic tumours, canine transmissible veneral tumor in canines, and equine sarcoid is the most common fibroblastic skin tumor affecting horses, mules, and donkeys. These neoplastic conditions could be treated by ZnO NPs. Zinc oxide was used as feed additive in weaner piglets to overcome the effect of post-weaning diarrhoea (PWD) caused by enterotoxigenic E. coli, which causes an increase in morbidity and mortality and decrease growth rate during the weaning period in piglets. Similarly, the addition of zinc (zinc oxide) at the concentration of 2500 to 3500 ppm in feed modulated the microbial status of the digestive tract and reduced the incidence of post-weaning diarrhoea in piglets and increased productive performances. Zn is mainly used in human and livestock foods and feeds for normal physiological functions, as well as to meet the daily requirement. Salama et al. studied the effects of dietary supplements of zinc-methionine on milk production, udder health and zinc metabolism in dairy goat, the results showed that addition of Zn enhanced resistance to udder stress in dairy goats to Zn supplementation. When supplemented to poultry leads to increase the level of ADFI, ADG, DM, and intramuscular fat contents of the breast muscle, percentage of eviscerated yield, redness value in breast muscle, and pH values in thigh muscle and decreased shear force in thigh muscle, drip loss in breast and thigh muscle. ZnO-NPs enhanced growth performance, improve feed utility and provide economic benefits in weaning piglets and growth, production, and dress performance in poultry. The supplementation of increased doses of Zn up to 3000 mg/kg increases the growth and reduces diarrhoea in pigs. Rajendran et al. found that the application of Nano Zn reduced the level of the somatic cell counts in cows with subclinical mastitis and improve milk production when compared with other conventional ZnO sources. Zn deficient diets are a cause of high incidence of abortions and stillbirths and supplementation in the form of Zn nanoparticle to animals increase the reproduction.
The beneficial effects and toxic effects of any nanomaterials depend on size, shape, surface charge, dose response, aggregation, type of solvent, and type of host cells. ZnO-NPs not only causes beneficial effects but also causes the toxicity effect in several veterinary animals. ZnO-NPs causes severe toxicity in the pancreas; kidney, liver, rumen, abomasum, small intestine, and adrenal gland were observed in sheep. Liver, spleen, heart, pancreas, and bone are the target organs of ZnO-NPs on oral exposure. Najafzadeh et al. observed the mild liver toxicity and severe renal damage in lambs.
The ecotoxicological impact of diffuse pollution, phycotoxins, and contaminants of emerging concern, as well as their modulation by environmental factors, needs assessing today using an integrated approach that encompasses the different scales of organization of living beings (macroscopic, cellular, biochemical, and molecular) and includes studies in both controlled environments and in situ. The study of population response to multiple stress factors and the genetic and epigenetic bases of their capacity to adapt to these stress factors are currently priority fields of research in order to anticipate future sanitary crises that may influence the fate of species.
Mycoviruses (fungal viruses) are widely distributed across all major groups of phytopathogenic fungi (Ghabrial and Suzuki, 2009; Pearson et al., 2009). Until recently, most reported mycoviruses were known to have double-stranded RNA (dsRNA) genomes, however, a number of single-strand RNA (ssRNA) mycoviruses have also been discovered (Ghabrial et al., 2015). Mycoviruses with ssRNA genomes are currently grouped into seven families: Hypoviridae, Narnaviridae, Barnaviridae, Alphaflexiviridae, Gammaflexiviridae, Deltaflexiviridae and Mymonaviridae, and one floating genus, Botybirnavirus (Amarasinghe et al., 2018; King et al., 2018), but many other mycoviruses have not been classified.
The family Hypoviridae contains a sole genus Hypovirus with four assigned virus species. Its members (Cryphonectria hypovirus 1–4, CHV1–4) infect a phytopathogenic fungus, Cryphonectria parasitica, the causal agent of chestnut blight disease (Suzuki et al., 2018). Hypoviruses are known as capsid-less viruses and they have large ssRNA genomes of 9.1–12.7 kb that possess either a single long open-reading frame (ORF) or two ORFs that encode polyproteins with a cis-acting papain-like cysteine protease at the N-terminus (Suzuki et al., 2018). In addition, many unclassified hypoviruses (or hypo-like viruses) have recently been discovered from other filamentous fungi, e.g., Sclerotinia sclerotiorum (Xie et al., 2011; Hu et al., 2014; Khalifa and Pearson, 2014; Marzano et al., 2015) Valsa ceratosperma (Yaegashi et al., 2012), Fusarium graminearum (Wang et al., 2013; Li et al., 2015), Phomopsis longicolla (Koloniuk et al., 2014), Macrophomina phaseolina (Marzano and Domier, 2016), Botrytis cinerea (Hao et al., 2018), and Rosellinia necatrix (Arjona-Lopez et al., 2018). Recent large-scale meta-transcriptomic analysis also uncovered that the hypo-like viruses are identified from non-fungal eukaryotes (invertebrates) (Shi et al., 2016). Based on phylogenetic analyses and genomic characteristics, CHV1–4 together with several unclassified hypoviruses were proposed to be classified into three genera, namely “Alphahypovirus” (including CHV1 and CHV2), “Betahypovirus” (including CHV3 and CHV4) and “Gammahypovirus” (Yaegashi et al., 2012; Hu et al., 2014; Khalifa and Pearson, 2014).
Mycoviruses are commonly associated with latent infections, while some are able to alter of fungal host phenotypes and/or attenuate the pathogenicity of fungal phytopathogenic hosts (Ghabrial and Suzuki, 2009; Pearson et al., 2009; Xie and Jiang, 2014). Mycovirus-associated hypovirulence has been studied extensively in CHV1-infected C. parasitica (Nuss, 2005; Eusebio-Cope et al., 2015; Rigling and Prospero, 2018). In addition, CHV2, CHV3, Botrytis cinerea hypovirus 1, Fusarium graminearum hypovirus 2 and the two strains of Sclerotinia sclerotiorum hypovirus 2 infections have also been associated with the hypovirulence of their fungal hosts (Hillman et al., 1994; Smart et al., 1999; Hu et al., 2014; Khalifa and Pearson, 2014; Li et al., 2015; Hao et al., 2018). In contrast, CHV4 and several other unclassified hypoviruses have no or a limited effect on fungal host virulence (Linder-Basso et al., 2005; Yaegashi et al., 2012; Wang et al., 2013; Koloniuk et al., 2014).
Apple leaf blotch disease caused by Alternaria alternata (family Pleosporaceae, class Dothideomycetes), has been a world-wide issue in apple production for decades (Johnson et al., 2000; Abe et al., 2010). A. alternata is generally considered a weak and opportunistic pathogen that infects a broad range of plants through various routes, such as wounds. Once infecting the plant, A. alternata is able to induce blackish spots on apple leaves in late spring or early summer, causing serious defoliation and declines in fruit quality (Filajdic and Sutton, 1995; Jung, 2007). The conventional use of fungicides to control apple leaf blotch disease is inefficient, therefore biocontrol methods are regarded as potential alternative means to control the disease. To date, several mycoviruses have been identified from Alternaria spp., including potential members of the genera Chrysovirus, Partitivirus, Victorivirus, Botybirnavirus, Mitovirus, and Endornavirus (Shang et al., 2015; Komatsu et al., 2016; Chen et al., 2017; Xiang et al., 2017; Okada et al., 2018; Xavier et al., 2018; Shamsi et al., 2019), as well as proposed the genus “Alternavirus,” family “Fusariviridae” and an undescribed novel taxon (Aoki et al., 2009; Lin et al., 2015; Zhong et al., 2016). It was reported that certain mycoviruses identified from A. alternata appear to impair the colony growth of their host fungus (Aoki et al., 2009; Fuke et al., 2011). Mycoviruses are also associated with the cyclic tetrapeptide tentoxin production in A. alternata, which causes chlorosis in seedlings of sensitive plants (Shepherd, 1988). Recently, Alternaria alternata chrysovirus 1 has been described to alter host fungus growth, while it also enhances the pathogenicity of fungal host on plants, most likely through the induction of host-specific toxin production (Okada et al., 2018). However, to date, no mycovirus has been reported to attenuate the pathogenicity of Alternaria spp.
In this study, we identified and characterized a novel hypovirulence-inducing hypovirus from A. alternata designated as Alternaria alternata hypovirus 1 (AaHV1). We also showed that AaHV1 produces defective RNA (D-RNA) during fungal culture in the laboratory. In addition, we demonstrated that AaHV1 confers hypovirulence in other plant phytogenic fungi.
The fields of invasion science and EID epidemiology share the challenge of the increasing numbers of invasions and EIDs with no evidence of saturation. Invasions and EIDs involve similar biological processes, may be intrinsically linked biologically and by human activity, are addressed by scientists with similar skills and objectives, and are being driven by the same global changes. Invasions by non-pathogenic organisms can also have important impacts on human health. They are therefore both part of the One Health concept and require a One Health approach to minimize their negative impacts on humanity. We have identified exciting opportunities for synergies between the fields of invasion science and EID epidemiology and call for greater collaboration to benefit humanity.
“Moving prevention and control strategy forward” is a national macrohealth policy, which well adapted to the new medical model, “physiological-psychological-social-environmental” model. It means that the focal point of medicine will be transferred from treating disease to health care, and disease prevention will be paid more attention to. Therefore, the policy of “prevention first” will be carried out instead of traditional ideological concept “treatment is more major than prevention.” It is similar to the TCM theory of “preventive treatment of disease,” including principles of “preventing measure taken before the occurrence of disease” and “preventing measure taken after the occurrence of disease” in Canon of Internal Medicine. Concrete measures of “moving prevention and control strategy forward” include concept forward, funding forward, emphasis of the researches forward, and measures to be carried out forward. It could reduce the incidence of the major diseases from the origin and effectively control the medical expense and save resources in medicine and health. Integrative medicine researches should also observe the principles above and pay more and more attention to improve the curative effect of common and frequent diseases.
Taking cardiovascular disease, for example, there are about 30% of the population in the world died from cardiovascular and cerebrovascular events, among which 62% of stroke and 49% of cardiovascular events were directly caused by hypertension. According to the China cardiovascular reports (2008-2009), the occurrence and mortality of cardiovascular disease is still increasing in our country, and it is estimated that the number of patients with cardiovascular disease is at least 230 million. It also demonstrated that there were about 200 million hypertensive patients in China with more than 10 million patients increased annually. As the primary cardiovascular risk factor, the risk level of hypertension is equivalent to three other cardiovascular risk factors together. That is why more emphasis should be taken on prevention and intervention of earlier-stage hypertension in clinical researches of integrative medicine. Additionally, hyperlipidemia, hyperglycemia, obesity, and other risk factors also should be paid more attention to. It is reported by World Health Organization (WHO) that if risk factors were controlled as early as possible, 80% of the disease can be prevented effectively, such as coronary heart disease, stroke, and diabetes. Furthermore, paying 1 yuan in prevention will save 7-8 yuan in treatment.
As the frontier field and hot issue of cardiovascular diseases, restenosis after percutaneous coronary intervention and myocardial ischemia reperfusion injury (MIRI) during open heart surgery of cardiopulmonary bypass has become the best innovative points of clinical studies in integrative medicine. Researches showed that restenosis after percutaneous coronary intervention was closely related to blood stasis syndrome. Predominantly evaluated by restenosis (RS) rate estimated by coronary angiography (CAG), a prospective randomized controlled study was carried out on RS after PCI to observe the intervention effect of Xiong Shao Capsule (XSC). Compared with the control group, the incidence of RS rate in the XSC group was significantly lower (24.1% versus 48.5%, P < 0.05) and the extent of angiostenosis and diameter of the culprit arteries, determined by CAG, also significantly reduced after patients had been treated for 6 months with [(2.21 ± 0.85) mm versus (1.72 ± 0.99) mm, P < 0.05], and [(26.58 ± 20.72) % versus (41.19 ± 30.92) %, P < 0.05], respectively. The incidence of clinical end-point event was significantly lower in the XSC group than that in the control group (11.7% versus 27.6%) and the P value was close to statistical significance (P = 0.051). Comparing with the control group, the blood-stasis syndrome score in the XSC group was also significantly lower (P < 0.01). The results showed that XSC had a wide range of therapeutic effects including effectively preventing RS after PCI in combination with conventional western medical treatment, decreasing the attack of angina pectoris and improving the blood stasis syndrome. Experimental researches on blood activating herbs showed that it can significantly inhibit pathological vascular remodeling after balloon injury, thus reduce late lumen loss and prevent restenosis [32–36].
As the establishing the cardiopulmonary bypass of open heart surgery is key point of successful operation, myocardial ischemia reperfusion injury (MIRI), which is very obvious during the recovery of circulation, has become the hot issue needed to be resolved. Some scholars found that the pathogenesis of MIRI during open heart surgery of cardiopulmonary bypass is deficiency of heart qi in the origin and excess of heart blood stasis and internal turbid toxin in the superficiality and the therapeutic principles are boosting qi and nourishing heart, activating blood circulation and resolving toxin simultaneously. It was proposed that of astragalus injection and tetramethylpyrazine injection for boosting qi and activating blood circulation should be given by vein injection during operation and Hu Xin Bao (compatibility of extracts of ginseng and panax notoginseng with taurine) for boosting qi, activating blood circulation, and resolving toxin should be given by oral administration before operation. The research showed that astragalus injection combined with tetramethylpyrazine injection could reduce the content of MDA and myocardial enzymes' release and improve the activity of SOD, NO, and NOS. Serial studies demonstrated that boosting qi combined with activating blood circulation have significantly synergetic effects, and boosting qi, activating blood circulation, combined with resolving toxin were superior to those simple boosting qi, activating blood circulation, resolving toxin, and boosting qi combined with activating blood circulation [37, 38].
Multiple organ dysfunction syndrome (MODS) is one of the difficult problems in the field of the critical care medicine, which is characterized by acute onset, rapid progress, and extremely high mortality. Since the 1970s of 20th century, some scholars began to take vigorous action to explore a new way of preventing and treating MODS by integrative medicine and a new theory of “bacteria and bacterial toxin treated simultaneously” was presented ultimately. They also perfected schemes for the diagnosis procedure and treatment standard of MODS by both TCM and integrative medicine. And four therapeutic principles for the main types of syndromes were put forward, such as activating blood circulation to dissipate blood stasis therapy on blood stasis syndrome, clearing heat and toxin therapy on heat toxin syndrome, reinforcing the vital energy and consolidating the constitution therapy on acute deficient syndrome, and dispelling interior pathogenic factors and purgation therapy on Yangming fu-organ syndrome. Integrative medicine therapy can effectively improve the clinical efficacy and shorten the course of the disease thus reducing mortality. A famous injection of Chinese medicine, “Shen Nong 33,” with the effect of activating blood circulation to dissipate blood stasis and antiendotoxin, was developed, which has reduced the mortality of international recognized infectious four or more organs failure from 100% to 50% and reached the international advanced level. Furthermore, a new strategy of “bacteria, bacterial toxin, and inflammatory mediator treated simultaneously” was put forward on the basis of the theory of “bacteria and bacterial toxin treated simultaneously.” Xue Bi Jing injection, the first Chinese medicine preparation in emergency medicine, was developed, which have made great contributions to the advancement of critical care medicine [39–43].
Chronic hepatitis B is the common disease in China, as well as in the world, causing great affliction to patients. It has become the major issue in the treatment of chronic liver disease. The progression of chronic hepatitis B may lead to liver cirrhosis and hepatocellular carcinoma. Hepatic fibrosis is the common pathological end stage of various chronic liver diseases regardless of the etiology, and blocking the occurrence and development of fibrosis of liver is very important in chronic hepatic diseases' treatment and prognosis. TCM has become the important therapy in treating chronic hepatitis, liver fibrosis, and liver cirrhosis. Some scholars put forward the hypothesis that liver fibrosis and early liver cirrhosis can be reversed. They found out that the basic pathogenesis of liver fibrosis is weakened body resistance and blood stasis, so therapeutic method of strengthening body resistance and dispelling stasis was established, and “Fu Zheng Hua Yu Capsules,” a new drug for treating liver fibrosis, was developed. Predominantly evaluated by liver tissue fibrosis, clinical researches were carried out to observe the curative effect of the therapeutic method of strengthening body resistance and dispelling stasis. The total inversion rate of liver tissue fibrosis was 52% to 58.3% compared before and after treatment, which also confirmed that liver fibrosis can be reversed and treated. The mechanism includes significantly inhibiting lipid peroxidation, the proliferation of hepatic stellate cell and activation of collagen expression, reducing inflammation of hepatocytic injury model, increasing the activity of matrix metalloproteinases, promoting the degradation of pathological liver collagen, and so on [44–46].
Combining the macroscopic view with microscopic view, syndrome differentiation with disease differentiation, regional with global, taking stopgap measures with taking radical measures, supporting healthy aspects with eliminating pathogens, tumor treatment model by integrative medicine emphasizes contriving individual treatment plan and evaluation standard on the basis of biological characteristics and the course of disease. Malignant tumors could be treated by TCM therapies such as reinforcing the vital energy and consolidating the constitution, supplementing qi and nourishing yin, and clearing away heat and toxic materials, combined with conventional therapies such as radiotherapy, chemotherapy, and surgery. TCM treatment has significances in decreasing toxicity and increasing efficacy on radiotherapy and chemotherapy. Integrative medicine theory has a remarkable effect in alleviating symptoms such as dry mouth in hyperpyrexic consumption of yin syndrome and deficiency of both qi and yin syndrome caused by head and neck cancer after radiotherapy, relieving symptoms such as cough caused by acute radiation pneumonitis, improving immune function, and survival quality of postoperative patients, preventing the tumor from recurrence or metastasis and prolonging survival time. The new model of combining TCM and modern cancer treatment has attracted widespread attention in the world, which is known as “China Model for Cancer Treatment”. In addition, screening of tumor inhibition from more than 3,000 species of Chinese herbs and nearly 300 Chinese herbal compound, effective components having directly killing effect on cancer cell such as indirubin, camptothecin, vinblastine, matrine, and aclitaxel were extracted. Some Chinese herbs, having the effect of immunological enhancement and biological response modifier-like action such as polyporus, poria cocos, and mushroom, were also found out.
APL is a special type of acute leukemic (AL). TCM suggests that the pathogenesis of APL is weakened body resistance and excessiveness of pathogen, so therapeutic method of eliminating pathogenic factors and strengthening body resistance was established. Some scholars developed the Compound Realgar Natural Indigo Tablets (Realgar, Indigo Naturals, Salvia and Radix pseudostell) on the basis of clearing away heat and toxic materials and supplementing qi with activating blood circulation and promoting hemogenesis method. 155 cases of APL patients were treated by the Compound Realgar Natural Indigo Tablets and the remission rate was 97.42% after treating for 6 months. No side effect, serious infection, bleeding, and DIC were found during the treatment course. It was also characterized by higher negative conversion rate of PML—RARα fusion gene and simple application. The results demonstrated that the complete remission rate of treatment of the Compound Realgar Natural Indigo Tablets were 10–15% higher than that of all-trans retinoic acid (ATRA). On this basis, the effect of post-remission therapy mainly with Compound Realgar Natural Indigo Tablets on long-term survival of 74 cases patients with APL showed that the median remission time was 48 months with recurrence rate only 14.86% and 10-year survival probability was 75.38% [48–50].
Since the 1970s of 20th century, the basic syndrome of type 2 diabetes included yin deficiency with internal excessive heat, deficiency of both qi and yin, and deficiency of both yin and yang, therefore, III-type differentiation of type 2 diabetes was established and developed. It had already been adopted by national guidelines for new drug in the late 1980s. As deficiency of both qi and yin was the important basic syndrome of the disease, “Jiang Tang Jia tablets,” a new Chinese herb of supplementing qi and nourishing yin, could improve insulin resistance, islet β-cell function, and the level of glucose and lipid metabolism, the total effective rate of which was 76.54%. In addition, researches of Tang Wei Kang capsule treating early diabetic nephropathy and Tang Xin Ping treating diabetic cardiopathy have gotten progress [51, 52]. Some scholars also found out that blood stasis was another significant pathogenesis of type 2 diabetes due to the changes of hemorheology with different degree were found. So they advocated treating the disease by promoting blood circulation and removing blood stasis principally. Based on this idea, promoting blood circulation by removing blood stasis recipes, such as nourishing yin and activating blood recipes and Xian Zhen tablet of reinforcing kidney and activating blood, were developed. Those recipes have multilevel and multitarget effects, including improving symptoms, reducing blood glucose, improving blood rheology and blood flow, lowering triglycerides (TGs), and malondialdehyde (MDA), enhancing activity of erythrocyte SOD, Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme, and so forth. The experimental studies showed that the effect of Xian Zhen tablet includes lowering blood glucose and glycosylated hemoglobin, decreasing urine protein excretion, improving renal function, reducing the pathological changes of glomerular mesangial expansion and basement membrane thickening, decreasing AGEs amounts of renal cortex, and downregulating RAGE-mRNA expression in renal cortex and endothelia of heart vessel. It provided a new idea for preventing and treating diabetic and chronic vascular complications [53, 54].
Severe pancreatitis, namely, acute hemorrhagic necrotizing pancreatitis, is characterized by acute onset, rapid progress, high mortality, and poor prognosis. 65% of the death cases are due to complicating with acute respiratory distress syndrome (ARDS). According to the theoretical basis that “the six fu-viscera function well when unobstructed” and “the lung and the large intestine are interior-exteriorly related,” acute pancreatitis is treated by expelling pathogens by purgation, and the average cure rate reached to 97%, while the average cure rate of severe pancreatitis was 80%. Compared with our country and abroad, the mortality has reached the lowest level. Qing Yi decoction, a famous antipyretic and purgative prescription, protected the lung from injury in many aspects, by preserving the damage of gut barrier function, reducing or eliminating endotoxemia derived from the gut, inhibiting the production, and release of TNF, IL-6, and the translocation of bacteria. The results may fully show the superiority of integrative medicine in treating serious diseases [55, 56].
A certain progress was also made on dermatosis and burn medicine by integrative medicine therapy. Vitiligo was effectly treated by taking modified Tao Hong Si Wu decoction, external application of compound tar traditional Chinese rubbing-drugs and melagenine extracted from placenta. 243 patients with vitiligo were treated by modified Tao Hong Si Wu decoction and the total effective rate was 68.2%, the mechanism of which was related to upregulation of tyrosinase activity, increasing the melagenine content, and promoting melanocyte proliferation. Moist exposed burn therapy (MEBT), a new therapeutic system of burn medicine in integrative medicine, has become the leading enabler throughout the world. It is found out that the burn wound should be kept in a moist but not macerated environment in order to promote in nature recovery and generation of the skin rather than in traditional dry environment. And the exact curative effect was obtained by MEBT and moist exposed burn ointment (MEBO).
Severe acute respiratory syndrome (SARS) has aroused international attention for strong infectiousness, rapid progression, poor prognosis, and high mortality, which has no special effective therapy yet. 524 patients of SARS in China were divided into integrative medicine treatment group (n = 318) and western medicine treatment group (n = 206). The existence rates for the symptoms of weakness, short breath, dyspnea in the first group were significantly lower than that in the second group after treatment. The duration of weakness was averagely shortened by 1.5 days in the first group. And short breath, dyspnea, and muscle aching pain were averagely shortened by 2 days, 1 day, and 2 days, respectively. Researches showed that the effect of integrated therapy of TCM and WM for treating SARS was superior to WM treatment alone, and the integrative medicine could improve clinical symptoms such as weakness, short breath, and dyspnea [59–61]. The exact clinical curative effect was also recognized by World Health Organization (WHO).
There is a growing awareness of the increasing threats presented to humans by emerging infectious diseases (EIDs) [1–3], with the majority of human EIDs being zoonotic—originating especially from wildlife reservoirs. Emerging diseases have a huge impact on human societies across the world, affecting both current and future generations. Changes in human living patterns, along with environmental and climate changes, pose unprecedented challenges to the global health of people, animals and ecosystems. Ecosystem health correlates with human health, but the precise relationships remain poorly understood. Understanding and responding to the ecological, social and economic conditions facilitating disease emergence and transmission represent one of the major challenges for humankind today. The risk is not uniform: 53 per cent of global EID outbreaks from 1996 to 2009 were in Africa, yet the continent lags behind severely in infectious disease detection and emerging epidemic warnings.
With increasing encroachment of people and livestock into wildlife habitats, a growing movement of wildlife from environmentally degraded areas into urban and peri-urban regions, massive aggregations of people (some at increased risk for severe infectious diseases because of AIDS, malnutrition, malaria and a variety of chronic infections) moving into densely populated cities, and rapid global movement of humans, animals and their products, there is a justifiable concern about the emergence and spread of novel, highly infectious diseases. Some of the most threatening emerging pathogens are RNA viruses due to their unparalleled ability to adapt to new hosts and environments. Many RNA viral EIDs, including HIV-1, have emerged from wildlife, and an important implication of this is that the most effective place to address such zoonotic threats is at the wildlife–human interface. A key challenge in doing this is to simultaneously protect wildlife and their habitats, thereby preserving vital ecosystem structures and functions that have local and broader implications for human wellbeing and environmental sustainability, and to prevent the spillover of pathogens from wild animals into human beings.
In this multifaceted context, bats offer a critically important focus for study at the human–wildlife interface. Bats are an important reservoir and vector for spread of EIDs. Bats perform major ecological functions by pollinating plants and dispersing their seeds, as well as regulating insect populations that are critical for maintaining ecosystems; some have been recognized as ‘keystone species’. Yet bats are associated with zoonoses of potentially great global public health impact and are the source of lyssa viruses, Hendra virus, Nipah virus, severe acute respiratory syndrome (SARS)-like coronaviruses, and Ebola and Marburg viruses [16–19]; all are RNA viruses that can cause currently untreatable diseases in people, often with high case fatality rates. Bats frequently live in very close proximity to humans, often in large numbers. They often interact closely with livestock and other domestic animals that are potential intermediate hosts for viruses that can infect humans, thus effectively expanding the wildlife–human interface. These interactions are shaped by environmental, social and politico-economic drivers at multiple scales, yet these processes and interrelationships are poorly characterized and understood. Bats epitomize growing challenges associated with human–wildlife–disease interactions, and thus offer a valuable model for building a new, holistic, policy-engaged paradigm to address these, now and in the future.
A clear institutional framework has recently been proposed for responses to emerging zoonotic diseases that require a multidisciplinary, ‘one health’ approach for their management. Such an approach recognizes the interdependence of human health, animals and ecosystems, but provides little guidance for researching these in an integrated manner. More specific inclusion of the drivers of spillover events is essential if we are ever to use our research to develop long-term programmes and strategies that reduce the future likelihood or frequency of spillover events. Further, approaches for the study of these complex ecological events must include study of the relevant institutions themselves, as their policies shape local and larger scale responses and perceptions.
Vitally needed for the full, long-term addressing of the risks of bat (and other wildlife) derived zoonoses is therefore an approach that gains detailed interdisciplinary understanding, combining cutting-edge perspectives from both natural and social sciences, linked to policy impacts on public health, land use and conservation. There needs to be greater support for new approaches that cross disciplines and combine quantitative and qualitative methods, and that also directly address the politics of policy processes. Such an integrated approach will be critical to future efforts that address disease challenges at the human–wildlife interface. Here, we propose such a framework, using bat-related disease threats as an example.
This theme encompasses the framework elements that focus on Old World fruit bat ecology and population dynamics, and their interactions with ecological structure and function.
Critical questions include:
— What are the distributions, abundances, behaviours and feeding ecologies of the focal bat species?— How do anthropogenic impacts, such as habitat alteration, urbanization and hunting, affect the distribution, abundance, behaviour and feeding ecology of focal fruit bat species?— How do life histories, including quantitative population dynamics, and feeding behaviours of focal bat species influence the potential for viral spillover into human and domestic animal populations?Bat species differ markedly in their ecologies, which may influence spillover. For example, in sub-Saharan Africa, Eidolon helvum and Rousettus aegyptiacus are the most widespread and possibly the most abundant fruit bats, often living in colonies of up to several million individuals. E. helvum often roosts in trees in urban settings, whereas R. aegyptiacus roosts predominantly in caves and in more rural areas. E. helvum is migratory, probably following the burst of fruits and flowers with the onset of the wet season, but where they go during this time is largely unknown. While some individuals have been shown to migrate over 2500 km, not all individuals migrate. In West Africa, E. helvum colonies can be very large (with roosts of more than one million individuals), while in East Africa E. helvum colonies appear to be smaller and more fragmented with reportedly less-pronounced migratory behaviour. In Southeast Asia, pteropodid fruit bats also may be highly mobile, though are sometimes perceived to be sedentary, living in small, fragmented colonies [59–61]. In Australia, flying foxes often live in very large, shifting colonies. All these species have one pup per year during a synchronized birth pulse. In Bangladesh, where Nipah virus spillover occurs annually, and in Asia, there is a temporal association between bat reproduction and potential zoonotic spillover events. In West Africa, E. helvum bats probably birth and mate during migration [65–67], which might be linked to, or even driven by, the nutritional needs of the females and their offspring, but the timing and place of these remain largely unknown. It is possible, therefore, that spillover events occur on the migratory route of this species. Knowledge about migration, time and place of the reproductive cycle in conjunction with the number of animals at any given time and place, and resource availability will provide crucial information about these ecological keystone species, and point to where and when potential spillover to humans should be researched. Alternatively, bats may use migration to escape from areas with high disease load, or lower pathogen prevalence during migration.
The ecology and distributions of fruit bats in many countries in which spillover may occur are not very well characterized, particularly quantitatively. This is the case even in Australia, although huge advances have been made there in recent years. The study of zoonotic pathogens has stimulated the study of a number of species, including Pteropus giganteus and Pteropus vampyrus in India, Malaysia and Bangladesh and E. helvum in Ghana. In Australia, however, where Hendra virus spillover could come from any of four fruit bat species, the role of sympatry and cross-species virus transmission in driving spillover has not been elucidated at all. A first necessary focus in many regions is the development of national schemes to locate, count and monitor bat colonies of focal bat species, to determine migratory patterns and to assess the reproductive cycle and efficiency. Quantification of social interactions between bats (e.g. mother–offspring, mating, fighting, allogrooming, etc.) would provide information on possible virus transmission routes. A second essential focus should be to understand feeding behaviour and ecology, as undertaken already in Bangladesh. The use of novel high-resolution GPS data loggers allows detailed and quantitative studies of ranging behaviours of bats and their environmental determinants. Such methods would also underpin the identification of food plants and allow resource use to be quantified through faecal analyses. The importance of fruit bats to the structures and functions of local ecosystems is often very poorly characterized; improving our understanding of this will inform how bats influence ecosystems and how land use change might influence bat population—and consequently infection—dynamics (see below).
The lack of longitudinal population data for most bat populations limits our understanding of the impacts of anthropogenic change on the ecology and behaviours of bats, but comparisons of single species living in both urban and rural sites, particularly where there are variable exposures to different degrees of hunting pressures, can help to evaluate these. Studies tracking movement patterns (Dechmann and Fahr, unpublished data) can enable detection of temporary stopover roosts and allow resource availability to be linked to movement, reproduction and local bat population size. Importantly, identification of feeding sites can facilitate the determination of interactions with other wildlife species (especially other species of bat) and with livestock and humans (directly and through partially eaten fruit and fruit spats).
While biologists, climatologists, geographers and oceanographers define the Arctic differently, for the purpose of this article, the circumpolar region consists of 27 regions wholly or partly located above 60°N and includes approximately 44 million square kilometres. The countries of this region include Canada, the Kingdom of Denmark (specifically, Greenland and the Faroe Islands), Finland, Iceland, Norway, Russia, Sweden and the United States. The region is home to diverse environments and populations of plants, animals and people living in some of the most extreme conditions on the planet. The physical and biological environments are diverse and include temperate rainforest, boreal forest, tundra, polar desert and cold oceans. There are approximately 10 million human inhabitants in the region that are ethnically diverse with dozens of Indigenous groups (1). Many of these people still have traditional subsistence economies based upon gathering wild plants, hunting fishing and herding of reindeer (2, 3).
The region is known as being both rugged and resilient, due in part to the persistent cold temperatures and the largely frozen condition of the land and sea. However, as the Arctic warms and the lands and ice thaw, the region is increasingly fragile. Arctic temperatures have risen at twice the rate of other parts of the world resulting in decreased sea ice, coastal erosion, changes in precipitation magnitude and frequency, permafrost thawing and altered distribution of plant and animal species (4). The associated health risks for humans and animals include potential changes in pathogen and vector demographics affecting disease patterns; degradation of drinking water quality and availability, food quality and availability, and changes in animal and plant species health, among others (5–7). Rapid change and recognition of the emerging health threats have resulted in a concerted effort to enhance regional and international partnerships to share best practices in disease surveillance and prevention strategies (8, 9). Understanding the evolving health threats and anticipating and managing risks influenced by the dynamic impacts of climate change in the Arctic will require innovative science, novel tools and even greater integration of efforts. The implications of health risks – to Arctic populations and those beyond – calls for broad and diverse stakeholder collaborations to advance the fundamental understanding of emerging health threats, and the development of shared initiatives that decrease vulnerabilities of human and animal communities and the environment. An integrated and holistic approach will be essential for providing the evidence of links between climate change and health risks to support sound policy development.
LJD, MBF, and SIS conceived and designed the experiments. LJD carried out the reverse transcription, PCR, and cloning. JML contributed reagents and manpower for sequencing. LJD, THV, and JML conceived and performed statistical and phylogenetic analyses of the sequence data. LJD, THV, and SIS wrote the manuscript. All authors read and approved the final manuscript.
Both SFV and herpesviruses were detected in the nonhuman primate bushmeat samples. All positive NHP samples are presented in Table 1. All NHP samples were negative for SIV and STLV sequences. All rodent samples were negative for leptospira, anthrax, herpesviruses, filoviruses, paramyxoviruses, coronaviruses, flaviviruses, and orthopoxviruses.
The susceptibility of potential hosts varies enormously, and an important predictor of susceptibility is how closely related a novel host is to a pathogen's natural host (Figure 1A). This “phylogenetic distance effect” has been repeatedly found using experimental cross infections in all major pathogen groups, including studies of fruit flies and viruses, plants and fungi,, beetles and Spiroplasma bacteria, insects and Wolbachia
, and fruit flies and nematodes. This is presumably because close relatives of the natural host offer a similar environment to that which the pathogen is adapted to. This is likely to be especially important for pathogens because of the myriad of molecular interactions pathogens have with their hosts to infect cells, utilise resources, and avoid or suppress the host immune response.
Reconstructions of host shifts in nature have confirmed that pathogens are more likely to shift between closely related species. By reconstructing the phylogeny of rabies viruses isolated from various species of bat in North America, it has been possible to look at the patterns of cross species transmission in the wild. The rate of cross species transmission was greatest for closely related species whether looking at spillover events (recent infections that might not persist long-term) or host shifts that successfully became established. Similarly, viruses and other parasites of mammals are most likely to be shared by more closely related hosts,,,,. Additionally, the phylogenies of hantaviruses and their rodent and insectivore hosts show evidence for host switching, with data suggestive of preferential shifts between closely related species. However, within these examples there are cases of pathogens transferring successfully over great phylogenetic distances,.
Closely related species may also have similar levels of susceptibility, regardless of their distance from the pathogen's natural host (Figure 1B), which we call the “phylogenetic clade effect.” Such effects could be due to certain host clades having lost or gained immune or cellular components that affect susceptibility to a given pathogen. This may mean that the host phylogeny is a patchwork of clades with varying levels of susceptibility, with clades of susceptible hosts scattered across the tree, sometimes in taxa distantly related to the pathogen's natural host. This has been demonstrated in experimental cross infections of fruit flies and sigma viruses, where after accounting for distance from the viruses' natural hosts, the effect of the host phylogeny explained almost all of the remaining variation in viral load. If this pattern is common, it may explain cases where viruses and other pathogens recurrently shift between distantly related taxa, such as transmission of influenza viruses among birds, pigs, and humans, or human to bovid transmission of Staphylococcus aureus
, although host ecology likely also plays a role in these instances.
The strength of these effects of the host phylogeny varies between pathogen groups, with RNA viruses and pathogens that already have a broad host range being particularly prone to jumping between distantly related species,,.
At the molecular level, the availability of suitable cell surface receptors to allow viruses to enter cells may be a cause of phylogenetic effects on host shifts. For example, the ability of an avian influenza virus to infect a host is initially, at least partly, determined by the presence and within-host distribution of α2,3-linked host sialic acid (SA) receptors.
Permission was obtained from the New York Department of Agriculture and Markets to transfer the frozen specimens from JFK Airport to CDC National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NCHHSTP), and/or Columbia University's Center for Infection and Immunity (CII) for testing. When an assured gross identification of species could not be made, samples were genetically identified by phylogenetic analysis of mtDNA genes, including cytochrome c oxidase subunits I and II (COX1/2), and/or cytochrome b (CytB)–.
Nucleic acids were extracted from 10–30 mg of tissue using mechanical disruption (Qiagen tissue lyser II or Next Advance Inc Bullet Blender), followed by proteinase K treatment until complete digestion of the tissue was achieved. Purification of subsequent homogenates was performed using the Qiagen All-Prep DNA and RNA extraction kit or DNeasy Blood and Tissue kits according to the manufacturer's instructions. Nucleic acid quality was determined using the Agilent BioAnalyser (Agilent RNA nano 6000) or ß-actin PCR as previously described.
In a one-year prospective study, intake of tomatoes and all fruits and citrus fruit alone had a protective effect on shortness of breath. Similarly, a cohort study that followed children from birth up to 18 years of age reported that daily consumption of fruit and vegetables over the last 12 months was inversely associated with current asthma at 18 years. Another birth cohort study showed that intakes of fresh fruit were inversely associated with asthma symptoms, while, no significant association was observed between cooked vegetable intake and asthma symptoms. No cohort studies in children reported associations between fruit and vegetable intake and immune function in asthma
Four cohort studies addressed the association between maternal fruit and vegetable intake during pregnancy and risk of asthma-related outcomes in their children. One study reported an inverse association between asthma incidence in children and maternal fruit and vegetable intake in pregnancy. Another study reported that maternal apple intake had protective effects on ever wheeze, ever asthma, and doctor-confirmed asthma in the children; however, no consistent associations were observed between childhood outcomes and maternal vegetable consumption. In contrast, a study conducted by Chatzi et al. showed that consumption of vegetables more than eight times per week was inversely correlated with persistent wheeze, while, no association was found regarding fruit intake and wheeze. Two studies found no significant association regarding maternal fruit or vegetable intake and risk of wheeze in the offspring. Willers et al. demonstrated that fruit intake had a borderline significant association with wheeze. This study also reported that vegetable intake was positively associated with asthma symptoms in children.
SIV, simian immunodeficiency virus; SM, sooty mangabey; RM, rhesus macaque; nt, nucleotide; aa, amino acid; Ab, antibody; NAb, neutralizing antibody.
The “one health” concept defines a fundamental principle of biology: it recognizes that the health of people is connected to the health of animals and the environment (39). The inter-species transmission of pathogens is primarily a matter of the number of encounters between two species over time. To understand this process, it is necessary to keep in mind the long co-evolution of microorganisms and their hosts, the history of species evolution, the adaptation of pathogens to hosts in which they persist without seriously affecting their health, eventually if the transmission of the pathogen requires its transfer via an intermediate vector insect, the nature of the mutations that can allow the infectious pathogens to change host when they meet a new host, and the dynamics of encounters between different ecosystems. When entering more deeply in the dynamics of infectious diseases, the first challenge is to identify what are the microorganisms that are present in humans and those that colonize the wildlife and could cross the species barriers. Although many microorganisms (almost 15,000 bacteria and 2,000 viral species) have been identified so far (40, 41), the classification of microbes is still a source of debate (42) and many more unknown microorganisms could be at the origin of pathologies of NHP and human. According to molecular clock analyses, viruses and bacteria already populated the planet a few billion years ago (43), long before mammals appear on Earth. Cyanobacteria are considered as the source of oxygen found in Earth's atmosphere and several microorganisms have then contributed to the evolution of life until becoming essential to biological functions in humans (44, 45). It was estimated that the divergence time between Archeabacteria and Eubacteria (prokaryotic group comprising all bacteria excluding Archebacteria) was 3 to 4 billion years ago (46). The divergence of Old World monkeys (OWM) and hominoid primates (apes, humans) was an estimated 23 million years (My) ago (Figure 2). Heritable individual differences contributing to change for survival are likely to have played a crucial role in human differentiation from ape-like ancestors (53). Homo sapiens emerged 0.15 My ago and have spread and evolved through the entire planet while being subject to natural selection. Thus, viruses and bacteria had thrived for billions of years before Homo sapiens emerged in this ecosystem and they are exquisitely adapted to host parasitization. Over time, pathogens might select host traits that reduce their impact on the host's life span (54).
Understanding the evolution of species and pathogens may be useful to better identify the current threat. An ancient biological fight between microbial pathogens and human is likely to have shaped human evolution over the millennia through selection of alleles that were advantageous in the new ecosystem (55). Natural selection includes positive selection (selection of advantageous alleles), purifying selection (removal of disadvantageous alleles), and balanced selection (maintenance of polymorphism via heterozygote statute). Remarkably, ancient trans-species polymorphisms have been described for the major histocompatibility complex (HLA in humans) believed to result from its role in the recognition of pathogens. Consequently, HLA antigens from different species share identical epitopes (56, 57). Conversely, as humans dispersed throughout the world, populations encountered new pathogens providing strong selective pressures. The transcriptional responses of macrophages to Listeria spp. or Salmonella spp., indicated that the immune response varied between African and European individuals living in America, suggesting ancestry differences in immune response to pathogens (58). The extinction of entire tribes of Native Americans was partly linked to the importation of smallpox by Europeans in the Western Hemisphere (59). After European contact, the Native American population showed a marked decrease in HLA-DQA1 alleles, likely due to gene selection (60). The distribution of ABO alleles across human and NHP reflects the persistence of an ancestral polymorphism that originated at least 20 My ago (61). These antigens are associated with an immune response produced in the gut after contact with bacteria and viruses carrying A-like and B-like antigens and are known to act as cellular receptors for pathogens (62). In countries highly exposed to Plasmodium falciparum (the agent of malaria), adaptation selected defense mechanisms preventing the most serious consequences of the disease. Persons who expressed sickle hemoglobin or those who present glucose-6-phosphate dehydrogenase deficiency, evaded the worst complications of malaria (63, 64). The chemokine receptor mutant CCR5-delta32, expected to have been selected by bubonic plague or smallpox (65), confers resistance to HIV by preventing the virus co-receptor's expression at the cells surface (66).Yet, what is true for host-pathogen interaction after a long co-evolution is generally not for EID insofar as the new human host is not supposed to have undergone genetic selection driven by this emerging pathogen.
It is likely that only some of the microorganisms that are potentially pathogenic for human have been identified to date. In the early 2000s, it was estimated that infectious diseases were responsible for 15 million of 57 million annual deaths of humans. Among the causative pathogens, the deadliest infectious disease in humans was caused by HIV, a retrovirus which found its origin in wildlife NHP (67). Each year, about 2 million people died from AIDS, but fortunately this number has more recently dropped to about 1 million and HIV is no longer the deadliest pathogen for humans (68). Currently, Mycobacterium tuberculosis, the causative agent of tuberculosis, kills 1.7 million people annually from tuberculosis, making it the leading cause of death from infectious disease (69). This pathogen is sometimes found in NHP. In addition, more than 1.6 million people die from diarrheal disease caused by infectious pathogens, and 800,000 from malaria. Rare outbreak of malaria in human found their origin in NHP (70). Contacts between species that do not meet naturally put both species at risk for infectious diseases and the risk is magnified when they are genetically close (71). As NHP and particularly great apes are our closest relatives, protein sequence homologies are very high between great apes and humans and it seems reasonable to hypothesize that their pathogens are more likely to jump and easily adapt to humans (e.g., find an appropriate cellular receptor). Therefore, it is not surprising that NHP share many diseases with humans (72). Although the risk of accidental transmission is likely very low, the NHP pathogens which have not yet crossed the species barrier represent a possible threat for humans. The fact that almost 7.5 billion people populate Earth to date and that the human population is growing steadily is probably a factor contributing to increase this risk.
Humans are nowadays very often in contact with pets or farm animals such as cattle, pigs, poultry, and horses. Although there are many infectious diseases typically found in people working with livestock, according to the microorganisms-driven genetic selection theory humans should have less to fear from livestock than from wild animals regarding the risk of deadly zoonotic diseases since they shared the same ecosystem with livestock for millennia. This does not mean humans should not remain vigilant (this is the reason why milk pasteurization exists and why meat must be cooked before consumption) and not worry about pathogens abnormally present in their livestock because of the interconnectedness of different ecosystems. Despite veterinary cares, these animals may serve as intermediate for transmission of wildlife-borne pathogens (including NHP-borne pathogens on rare occasions) to the livestock owners and thereby represent the main source of human infectious disease and pandemics (Figure 3). According to WHO (73), a zoonosis is any disease or infection that is naturally transmissible from vertebrate animals to humans. Today, as was the case centuries ago, most EID in humans originate from zoonoses after hazardous events, the occurrence of which is impossible to predict (23). EID can refer to different epidemiological situations: (i) the disease is caused by a newly identified pathogen and did not exist previously in humans (e.g., AIDS or SARS); (ii) the disease existed before but a new etiological agent was discovered (e.g., hepatitis C); or, (iii) the disease existed before and the causative agent was identified, but it appeared for the first time in a geographic area where no case had been diagnosed previously (e.g., West Nile Virus epidemic in the USA) (74). An EID is obviously unusual; it is surrounded by uncertainty and anxiety. Epidemics of Ebola Filovirus in 1977, AIDS/HIV in 1983, Hantavirus in 1993, Influenza A/H5N1 in 1997, Nipahvirus in 1998, Severe Acute Respiratory Syndrome/SARS-Coronavirus in 2003, and MERS-Coronavirus in 2012, were of zoonotic origin (23).