Administration of LAB can prevent development of ruminal acidosis by creating optimal conditions for lactic acid consuming bacteria such as Megasphaera elsdenii or Propionibacterium spp. (Chaucheyras-Durand and Durand, 2010). Addition of Lb. acidophilus, Lb. salivarius, and Lb. plantarum at concentrations of 107–108 CFU/g, reduced the incidence of diarrhea in young calves (Signorini et al., 2012). LAB species including Lb. paracasei and Lb. plantarum, and those isolated from honey like Lb. kunkeei, Lb. apinorum, Lb. mellis, Lb. mellifer, Lb. apis had in vitro activities against mastitis pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus uberis, or E. coli (Piccart et al., 2016; Diepers et al., 2017). LAB-probiotics were also successfully used to relieve symptoms of diseases such as coccidiosis, an important parasitic disease of young ruminant livestock, caused by Eimeria. These LAB-probiotics minimized the impact of this disease by reducing the risk of dissemination of this parasite (Giannenas et al., 2012). On the other hand, Cao et al. (2007) reported on the effectiveness of intra-mammary infusion of nisin for treating mastitis caused by Staphylococcus aureus in dairy cows.

Synbiotics are defined as combination of probiotics and prebiotics that beneficially affect the host by improving survival and settlement of live microbial dietary supplements in the gastrointestinal tract. This happens by selectively stimulating the growth and/or by activating the metabolism of one or a limited number of health-promoting bacteria, and thus improving host welfare (Gibson et al., 2004). A prebiotic that confers gastrointestinal health benefits could support the growth of a probiotic which has activity against a potential pathogen (Allen et al., 2013).

Guerra-Ordaz et al. (2014) reported a synergistic effect when Lb. plantarum (2 × 1010 CFU) and lactulose (10 g/kg feed) were concomitantly used to treat colibacillosis in pigs, reducing diarrhea and improving the average daily weight gain. Yasuda et al. (2007) showed that Lb. casei subsp. casei (1 × 107 CFU/kg feed) and dextran (5%, w/w) in combination improved the milk yield and milk components in cows, and they hypothesize the symbiotic association had a prophylactic effect inhibiting mastitis development. Data from Naqid et al. (2015) suggested caution on the use of synbiotics because intra-interaction can occur within the combination and reduce the expected activity. Mookiah et al. (2014) showed same body weight increase in chickens treated with multi-strain Lactobacillus probiotic at 1 × 109 CFU/g or prebiotic (isomalto-oligosaccharides; 5 or 10 g/kg of feed) separately or their association as synbiotic. Szczurek et al. (2018) showed that synbiotic composed of whey lactose and Lb. agilis did not show any advantage when compared to each compound alone.

In fish, the administration of Ent. faecalis and mannan-oligosaccharides enhanced growth, immune response, and survival of Rainbow trout (Oncorhynchus mykiss) to the infection of A. salmonicida (Rodriguez-Estrada et al., 2013). In Tilapia, there is an effect against A. hydrophila when the combination of 1 × 108 CFU Lb. brevis JCM 1170 or Lb. plantarum JCM 1149 and fructo-oligosaccharides (1 g/kg feed) were added to the feed, but the same combination did not improve animal growth or feed conversion (Liu et al., 2017).

E. bieneusi and Cryptosporidium spp. are important pathogens that can cause severe watery diarrhea in humans and animals globally. To explore the role of peafowl in the epidemiology of these pathogens above, we characterized the prevalence and genotypes of E. bieneusi and Cryptosporidium spp. in peafowl from Beijing and Jiangxi Province, China.

In our study, we detected, for the first time, the prevalence (6.59%) of E. bieneusi in peafowl, which was similar to some studies performed in other birds (Bart et al., 2008; Lallo et al., 2012; Pirestani et al., 2013). In contrast to our result, much higher prevalence was found in studies conducted by Li et al. (2014) in chicken, Lobo et al. (2006) in pigeon and Tavalla et al. (2018) in exotic birds. Lower infection rates were identified in pigeon and Brazilian captive birds investigated by Slodkowica-Kowalskaet et al. (2013) and da Cunha et al. (2017) respectively. Cryptosporidium spp. has been detected in a wide range of domestic and wild avian hosts worldwide (Nakamura and Meireles, 2015). In China, Cryptosporidium spp. has been reported in ruddy shelduck (Amer et al., 2010), quails (Wang et al., 2012), ostriches (Qi et al., 2014), domestic pigeons (Li et al., 2015), parrots (Zhang et al., 2015), Java sparrows (Yao et al., 2017) and chickens (Liao et al., 2018). In the present study, we first detected the prevalence of Cryptosporidium spp. in peafowl in China. In Beijing, the infection rate was 4.58%, while in Jiangxi Province, the infection rate was 9.52%. This differences may be caused by climatic conditions, management level and differences in age composition of peafowl in the two locations. To our knowledge, there is no other reports on Cryptosporidium spp. infection in peafowl other than studies conducted by Nakamura et al. (2009). In studies performed in other birds elsewhere, infection rates of Cryptosporidium spp. vary from 0.82% to 43.9% (Baroudi et al., 2013; Li et al., 2015a; Maca and Pavlasek, 2015). Though infection rate varies with geographical location, avian species and detection methods, the number of samples may also be the causation of the differences. Moreover, different hosts are unequally susceptible to various genotypes of the parasites, which may also be responsible for the difference in infection rates. In addition, higher prevalence of E. bieneusi and Cryptosporidium spp. in the adolescent peafowl might be caused by their naive immune status.

Three genotypes of E. bieneusi were identified based on analyzing the ITS region, including two known genotype, Peru6 and D, and one novel genotype, JXP1. All the positive samples detected in Beijing belong to genotype D. In Jiangxi Province, the predominant genotype was JXP1, followed by genotype Peru 6 (Table 3). Genotype D has been widely found in mammals, including humans (Kicia et al., 2016; Prasertbun et al., 2017; Wang et al., 2017), non-human primates (Karim et al., 2014), Suidae (Nemejc et al., 2014; Zhao et al., 2014), ruminants (Huang et al., 2017; Zhao et al., 2015), canids (Li et al., 2015b; Zhang et al., 2016), felines (Li et al., 2016; Xu et al., 2016), rodents (Perec-Matysiak et al., 2015; Yang et al., 2016) and equines (Deng et al., 2016; Qi et al., 2016; Yue et al., 2017). However, genotype D was reported only in a few avian species, such as chicken (Rodrigues da Cunha et al., 2016), pigeon (Pirestani et al., 2013), common crane and red-crowned crane (Zhao et al., 2016), rook (Perec-Matysiak et al., 2017), falcon (Mueller et al., 2008) and swan goose (Anser cygnoides) (da Cunha et al., 2017), and our findings constitute the first report of the genotype in peafowl. Genotype Peru6 was mainly reported in humans (Bern et al., 2005; Lobo et al., 2012), cattle (Santin et al., 2005), sheep and goats (Zhao et al., 2015). Pigeon and lovebird also can be infected by the genotype (Luisa Lobo et al., 2006; Zhao et al., 2016). To our knowledge, this is the first report that peafowls were infected with the genotype Peru6. Together, both genotype Peru 6 and D can infect a wider range of host species, including peafowl, thus may cause microsporidiosis to the host. Moreover, the novel genotype (JXP1) of E. bieneusi identified in our study should be further explored to reveal its danger to the host, especially to human beings. Phylogenetic analysis was performed based on the ITS sequence of E. bieneusi. Both genotype Peru 6 and the novel genotype JXP1 obtained in the present study were grouped into group 1b, and genotype D was clustered into the group 1a. Both group 1b and group 1a belong to group 1, the most important human-pathogenic group. These results suggest that peafowl are potential source of human and animal microsporidiosis.

Molecular characterization of the SSU rRNA gene verified the presence of two genotypes/species, Cryptosporidium Avian genotype Ⅲ and Goose genotype Ⅰ. The former was found in peafowl from Beijing, and the latter was recovered in peafowl from Jiangxi Province. Cryptosporidium Avian genotype Ⅲ was considered specific to birds and first identified in parrots in western Australia by Ng et al. (2006). After that, the genotype was isolated from birds in other countries, such as from the families Psittacidae in Brazil (Gomes et al., 2012; Nakamura et al., 2009; Novaes et al., 2018), Japan (Abe and Makino, 2010; Makino et al., 2010), China (Qi et al., 2011) and the USA (Ravich et al., 2014), from seagulls in Thailand (Koompapong et al., 2014), and from aquatic birds in Spain (Cano et al., 2016). Though the Cryptosporidium Avian genotype Ⅲ seems to play no role in zoonotic potential, its global distribution makes it impossible to ignore its potential threat to bird health. Cryptosporidium Goose genotype I had been detected in Canada Geese (Zhou et al., 2004) and was identified in the feces of peafowl for the first time. Both Cryptosporidium Avian genotype Ⅲ and Goose genotype Ⅰ have not been found in humans up to now. Further research is needed to determine whether zoonotic Cryptosporidium species/genotypes occur in peafowl in other areas and settings.

Many countries of origin for legal and illegal wildlife imports to the USA include “hotspots” of emerging and reemerging infectious and zoonotic pathogens (Jones et al. 2008; Smith et al. 2009) such as HPAI, Middle East respiratory syndrome (MERS) coronavirus, Nipah virus and Brucella ssp., as well as economically important livestock diseases. Introducing disease purposefully or accidentally need not utilize illegal trade since regulations concerned with pathogen introduction via trade are focused mainly on domestic species (regulated by CDC and USDA) and not enforced by the agency primarily monitoring wildlife trade into the USA (USFWS).

Since the majority of regulatory oversight of the wildlife trade is not specifically aimed at prevention of disease introduction, it remains a challenge to prioritize collection of the relevant information or risk mitigation measures. The Congressional Research Service notes that while the USA is involved in CITES, and contributes to the Coalition Against Wildlife Trafficking and Association of Southeast Asian Nations (ASEAN) wildlife law enforcement network, the USA “does not participate in international efforts to regulate international wildlife trade to prevent disease transmission or invasive species, as no such international organization currently exists” (Wyler and Sheikh 2008).

In 2014, President Obama issued the National Strategy for Combating Wildlife Trafficking to guide federal agencies in the global fight against wildlife trade. Yet even after the recent Ebola outbreaks in Africa, disease has not been a priority in this fight. The USA does adhere to the World Trade Organization’s (WTO) Sanitary and Phytosanitary (SPS) Agreement, which regulates the international trade in animals, animal products and plants, and is a member of the OIE, which sets international health standards for animals and animal products, recently including wildlife. In an attempt to support this effort, EHA recently worked with the OIE to develop a comprehensive list of proven wildlife hosts of OIE-listed diseases in order to inform member countries of the broad range of potential carriers of diseases of importance and to raise awareness surrounding potential wildlife trade health risks (Smith et al., unpubl. data).

We do not yet have a comprehensive picture of the scope and associated health risks posed by the international trade of wildlife. However, it is clear that the USA is a global leader in legal and illegal wildlife consumption. The demand for wild animals for use as companion animals/pets has been responsible for the majority of the live animal trade in the Western Hemisphere. This market involves billions of individual live animals, ranging from invertebrates and corals to non-human primates, originating from all over the globe. The demand for trophies, fashion, traditional medicines and exotic foods are some of the main drivers of the importation of wildlife products. The import process provides an opportunity to reinforce “critical control points” prior to entry through US borders. This is especially pertinent given that there is very limited traceability of wildlife species once entry has been gained into the USA.

The overarching goal of this work is to mitigate risk of pathogen introduction to US agriculture via wildlife trade. To accomplish this, we must first understand and characterize trade pathways as described herein. Given the large volume of imports, limited enforcement resources and lack of surveillance tools and infrastructure for many wildlife spp., the authors believe there is great opportunity for both regulated and non-regulated diseases of importance to public, agricultural or wildlife health to enter the USA. Thus, there should be an emphasis within the US Government and wildlife disease communities on filling gaps in the data for high priority pathways in order to better characterize risk. Specifically, threats posed by (1) large volumes of live aquatic species, (2) wild animal host species not currently regulated (e.g., some rodents) and (3) species closely related to domestic agriculture (e.g., hoofstock/camels) that may enter the USA for multiple purposes were prioritized by this working group for further assessment.

The E. bieneusi genotypes were separated into 9 distinct groups on the basis of the ITS1/5.8S/ITS2 sequences, and the group 1 was further divided into 8 subgroups (group1a-group1i) (Zhang et al., 2018b). The isolate BJP-18 (MK168302) identified in Beijing was clustered into group1a, JXP1 (MK168300) and JXP-41 (MK168301), were clustered together in group1b (Fig. 1). Phylogenetic tree of Cryptosporidium spp. was constructed based on the SSU rRNA gene. The two isolates of Cryptosporidium spp. recovered in the present study were grouped with the genotype Goose I (AY504516) and Avian genotype Ⅲ (KU885387) with 99% bootstrap support respectively (Fig. 2).

Pacific salmon (Oncorhynchus spp.) species have supported coastal ecosystems and Indigenous populations surrounding the North Pacific Ocean for tens of millennia. Today, through their anadromous life history, salmon continue to transport nutrients between aquatic and terrestrial environments (Cederholm et al., 1999), supply the primary food sources for orca whales and sea lions (Wasser et al., 2017; Willson and Halupka, 1995; Chasco et al., 2017; Thomas et al., 2017) and provide economic livelihoods for local communities (Noakes et al., 2002). In the Northeast Pacific, widespread declines of Chinook (O. tshawytscha) and sockeye (O. nerka) salmon have occurred in the last 30 years, leading some populations to the brink of extirpation (Peterman and Dorner, 2012; Heard et al., 2007; Miller et al., 2011; Jeffries et al., 2014), and a cause of great concern to Indigenous groups, commercial and recreational fishers, and the general public. Although the exact number of salmon spawning in rivers is unknown, there are large declines in sockeye salmon over a large geographic area (Peterman and Dorner, 2012). Similarly, Chinook salmon stocks are at only a small percentage of their historic levels, and more than 50 stocks are extinct (Heard et al., 2007).

It is thought that infectious disease may contribute to salmon declines (Miller et al., 2011), but little is known about infectious agents, especially viruses, endemic to Pacific salmon. Infectious disease has been identified as a potential factor in poor early marine survival in migratory salmon; an immune response to viruses has been associated with mortality in wild migratory smolts and adults (Miller et al., 2011; Jeffries et al., 2014), and in unspecified mortalities of salmon in marine net pens in British Columbia (BC) (Miller et al., 2017; Di Cicco et al., 2018). For instance, immune responses to viruses such as Infectious haematopoietic necrosis virus (IHNV) and potentially undiscovered viruses, have been associated with mortality in wild juvenile salmon (Jeffries et al., 2014). This is an important observation as mortality of juvenile salmon can be as high as ~90% transitioning from fresh water to the marine environment (Clark et al., 2016). Together, these suggest that there are undiscovered viruses which may contribute to decreased survival of Pacific salmon but a concerted effort to look for viruses that may contribute to mortality has been absent.

Here, virus-discovery was implemented to screen for viruses associated with mortality. Together, sequencing of dead or moribund aquaculture salmon and live-sampled wild salmon, in-situ hybridization, and epidemiological surveys revealed that previously unknown viruses, some of which are associated with disease, infect wild salmon from different populations.

Yersiniosis or enteric red mouth disease (ERM) is a serious systemic bacterial infection of fishes which causes significant economic losses in salmonid aquaculture worldwide. Although infection with this agent has been reported in other fish species, salmonids especially rainbow trout Oncorhrynchus mykiss, are highly susceptible to ERM. The disease was first described in the rainbow trout in the United State in 1958, from Hagerman Valley, Idaho by Rucker, and later the causative organism named Yersinia ruckeri. The disease is endemic in North America and widespread elsewhere. It was also described in 1981 in France, Germany and United Kingdom and has now been reported in most of Europe, Australia and South Africa.

The causative agent, Yersinia ruckeri, is a gram-negative, non-spore-forming rod-shaped bacterium with rounded ends and like the other members of the Enterobacteriaceae family is glucose-fermentative, oxidase-negative and nitrate-reductive. ERM outbreaks usually begin with low mortality, and then escalate to result in high losses. Characteristic symptoms of ERM are haemorrhages of the mouth and gills, though these are rarely seen in acute infections but may be present in chronic infections, diffuse haemorrhages within the swim bladder, petechial haemorrhage of the pyloric caecae, bilateral exophthalmia, abdominal distension as a result of fluid accumulation, general septicaemia with inflammation of the gut, the spleen is often enlarged and can be almost black in colour. Transmission occurs by direct contact with carrier fish, other aquatic invertebrates and birds. The ability of Y. ruckeri to survive and remain infective in the aquatic environment is considered to be a major factor in spread of the disease. Furthermore, Y. ruckeri is able to form biofilms and grow on surfaces and solid supports in fish tanks, like many bacteria in aquatic environments, which lead to recurrent infections in rainbow trout farms. Although vaccination has for a decade been very successful in the control of infections caused by Y. ruckeri in trout farms, cases of yersiniosis have been reported in trout farms where vaccination didn't provide enough protection against the infection and due to carrier state. Different diagnostic methods have been developed for detection of Y. ruckeri including culturing, serological and molecular techniques. Isolation and identification using agar media and the organism's biochemical characteristics are considered the gold standard for Y. ruckeri diagnosis. Serological methods for detection of Y. ruckeri have also been developed and these include ELISA, agglutination, and the immunofluorescence antibody technique (IFAT). Molecular techniques are able to detect low levels of the bacterium and facilitate detection of asymptomatic carriers, which is very important for prevention of ERM transmission and spread. Restriction fragmentation-length polymorphism and PCR assays are widely used for detection of low levels of Y. ruckeri in infected trout tissues and blood and also for detection of asymptomatic carriers. Although PCR has been shown to be a powerful and sensitive tool in detection of Y. ruckeri, its requirements for expensive equipments, a precision thermocycler and laboratory training limit its use in the field as a routine diagnostic tool.

Alternate isothermal nucleic acid amplification methods, which require only a simple heating device, have been developed to offer feasible platforms for rapid and sensitive detection of a target nucleic acid. These include nucleic acid-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and ramification amplification. LAMP is a nucleic acid amplification method that synthesises large amounts of DNA in a short period of time with high specificity. The strand displacement activity of Bst DNA polymerase impels auto-cyclic DNA synthesis with loop-forming primers to yield long-stem loop products under isothermal conditions: 60–65°C for about 60 min. The LAMP reaction requires four or six primers that target six or eight separate DNA sequences on the target and give the assay very high specificity. LAMP amplification products can be detected by gel electrophoresis, by real time monitoring of turbidity with a turbidimeter or with the naked-eye. Visual detection can be accomplished using different methods such as detection of a white precipitate (magnesium pyrophosphate), use of an intercalating DNA dye such as SYBR Green I gel stain, use of florescent detection reagent, FDR,, or use of oligonucleotide probes labelled with different fluorescent dyes and low molecular weight cationic polymers such as polyethylenimine, PEI.

LAMP-based assays have been developed for numerous aquaculture animal pathogens, including white spot syndrome virus, yellow head virus, Edwardsiella tarda and Nocardia seriolae, Tetracapsuloides bryosalmonae, Myxobolus cerebralis, Thelohania contejeani, Koi herpes virus (CyHV-3) and viral hemorrhagic septicaemia (VHS). The objective of this study was to develop and evaluate LAMP, as a simple, rapid and sensitive diagnostic tool for ERM disease.

The relative protection percentage (RPS) in the vaccination-challenge study was calculated as follows. The ‘percentage protected control’ was calculated as the number of survivors in the control group, divided by the total number of control fish, multiplied by 100% (% protected control = [# survivors control] / [total # control fish] * 100% = x %). The ‘percentage protected in the vaccine group’ was calculated as the number of survivors in the vaccine group, divided by the total number of vaccinated fish, multiplied by 100% (% protected vaccine group = [# survivors vaccine] / [total # vaccinated fish] * 100% = y %). The RPS is formulated as RPS = 1-(% protection test / % protected control) or in a mathematical formulation RPS = 1-(x/y)* 100%.

Nearly half of all declared wildlife imports to the USA came through the ports of New York, Los Angeles and Miami, thus providing opportunities for targeted strengthening of monitoring and law enforcement efforts. The vast majority of wildlife imports through New York are commercial, and 97% of declared wildlife imports come via air cargo (US Fish and Wildlife Service 2005). Reasons for high traffic through New York include the fact that it is a fashion capital, the home of many scientific and educational facilities, and a top port of entry for tropical fish importers (United States Fish and Wildlife Service (USFWS) (2004). Accordingly, the top live imports to New York are medicinal leeches and fish, while top commodities include caviar, shell products, furs and skins (United States Fish and Wildlife Service (USFWS) (2004). Los Angeles is also a predominantly commercial port when it pertains to wildlife imports. Over 80% of imports arrived via air and most remaining imports via ocean cargo. Main imports include live aquatic species and reptiles as well as shell products, jewelry, eggs and skin/hair products (US Fish and Wildlife Service 2005). Miami, the largest port of entry from Central and South America, showed similar trends, receiving over 90% of its imports by air, comprised mainly of fish and reptile species.

Transgenic technologies can also provide and effective means for enhancing animal health. Because hosts and pathogens have coevolved, long-term selection may not be the most effective approach for the enhancement of disease resistance. Although GS has provided enhanced disease resistance to some organisms (e.g., parasites; Stear et al., 2007), it is unlikely to generate specific resistance to microorganisms (bacteria and viruses) because they evolve more rapidly than do their hosts. Precise and efficient GE tools instead provide a route to make significant, sudden improvements in general and pathogen-specific innate and humoral immunity (Table 1). The improvement of disease prevention in livestock will increase the quality of life of production animals, contribute to the needed acceleration of food production, and serve to enhance food security worldwide. Developing animals resistant to viral pandemics (Reed et al., 2009) is in the best interest of all constituencies as a means to improve animal welfare and to enhance food and human health security.

People take multivitamin and mineral supplements with an expectation to reduce the incidence rate of chronic disease or cancer as an insufficient intake of antioxidant vitamins and minerals has been identified to increase the occurrence of cardiovascular disease and cancer. The Linxial trial is the leading study that supports this statement. Multivitamin and mineral supplements were given to nutrition-deficient individuals in Linxial area based on the fact that their incidence rates of esophageal and gastric cardia cancers were high. This study was performed to examine whether esophageal and gastric cardia cancers were reduced in patients with esophageal dysplasia that appeared to be a precancerous lesion. According to the results, although multivitamin supplementation did not decrease the incidence of esophageal, gastric and other cancers and cerebrovascular disease and mortality rate in overall, mortality rate was reduced in the group administered with selenium, vitamin E and beta carotene in combination compared with other groups administered with other components.13 During the 10-year follow-up after stopping multivitamin and mineral supplementation, the effect of combined administration of selenium, vitamin E and beta-carotene persisted in reducing mortality rate, but this effect lasted only in subjects aged below 55 years.14

In contrast, the intake of antioxidant vitamins and minerals did not lower the incidence of cancer or ischemic heart disease in healthy individuals according to the SU.VI. MAX study developed in France.15,16 In the analysis of American healthy male physicians in the Physicians' Health Study, multivitamin intake did not reduce cardiovascular or coronary heart disease mortality.17

To sum up the above findings of several cohort studies, although the Nurses' Health Study suggested that multivitamin use had a weak favorable effect on colon and breast cancers, there was no impact of multivitamin use on the risk of cardiovascular disease or cancer in overall.18,19,20

In the 2012 Cochrane review, there was no evidence to support antioxidants supplements for primary or secondary prevention, and beta-carotene, vitamin E and vitamin A seem to increase mortality rate. For these reasons, antioxidants supplements need to be considered as medicinal products rather than supplements. Thus, sufficient evaluation is necessary before marketing.21 In 2013, the U.S. Preventive Services Task Force reported that there are no proofs that taking a nutritional dose of vitamins or minerals reduces the risk of cardiovascular disease or cancer and mortality rate in healthy individuals without known nutritional deficiencies.22

One promising aspect of GE is the potential for the development of functional foods that enhance food safety, human nutrition, and health (Table 1). For example, in China the nutritional value of bovine milk has been improved by GE to express human α-lactoglobulin and human lactoferrin, proteins normally found in human milk but missing from bovine milk (Wang et al., 2008; Yang et al., 2008). Given the increasing prevalence of obesity and cardiovascular disease in developed nations, changes in product composition in conjunction with improvements in dietary practices could contribute to improvements in consumer health. The amounts and type of fats in animal products are topics of frequent public discourse, and from the perspective of sustainability, improved feed conversion efficiency increases the ratio of lean-to-fat deposition in livestock. Net benefits include reduced production costs, improved product quality, reduced excretion of nitrogenous wastes into the environment, decreased grazing pressure on fragile landscapes, and reduced pressure on world feed supplies (Sillence, 2004). A decrease in the prevalence of deleterious fats and cholesterol and an increase in the prevalence of MUFA and n-3 fatty acids are consistent with dietary recommendations for cardiovascular health and an objective difficult to achieve in the absence of GE. In fact, 3 proof-of-principle studies have been published: 1) GE goats that expressed a rat stearoyl-CoA desaturase in the mammary gland and yielded milk with a reduced saturated fatty acid content and increased content of CLA, a beneficial antioxidant fatty acid (Reh et al., 2004); 2) a GE pig made transgenic for a Δ12 fatty acid desaturase gene from spinach that produced the PUFA, linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), which are essential for human (and pig) nutrition (Saeki et al., 2004); and 3) a GE pig that was developed to express an n-3 fatty acid desaturase capable of converting n-6 fatty acids to n-3 fatty acids (Lai et al., 2006). Although fish provide an excellent source of dietary n-3 fatty acids, which are important for fertility, cardiovascular health, immune system health, mental health, and cancer prevention (Prather, 2006), worldwide fisheries will be challenged to sufficiently supply n-3 fatty acids to the developing world. As the most widely consumed meat, pork logically should be considered as an alternative source of n-3 fatty acids. Consistent with this strategy, the pigs developed by Lai et al. (2006) produce increased content of n-3 fatty acids from n-6 analogs, and their tissues have a reduced ratio of n-6/n-3 fatty acids. Such animals may be useful as models for human health and for providing a dietary source that could enhance the health of consumers in developed and developing countries.

The first FDA approval of a GE animal product, the anticoagulant ATryn (recombinant human antithrombin), firmly established the importance and safety of engineered mammary gland-based protein expression systems. Additional GE projects in cattle and goats have targeted the mammary gland for the expression of proteins to enhance the welfare of animals, and the safety and stability of milk products (Table 1). Bacterial diarrhea, which is responsible for more than 2 million infant deaths per year in developing countries, results from campylobacter, salmonellae, shigellae, and some strains of E. coli infections. Transgenic goats that express the human lysozyme protein, a natural antimicrobial protein in breast milk, were developed to produce milk with an enhanced shelf life that would improve the gastrointestinal health of goat kids and children (Maga et al., 2006a,c; Brundige et al., 2008). Experiments in vitro and in vivo have established that milk from these goats has antimicrobial properties, whether pasteurized or not, and that this milk inhibits the enteric bacteria E. coli when fed to piglets (a nonruminant model for children; Maga et al., 2006b). Approval of this product could make a significant contribution to the alleviation of hunger and disease (Brundige et al., 2009).

Olive flounder is one of main marine species having high economic value in the countries of East Asia. Because of industrial importance by increasing of demand, it is serious subject to understand the pathogenic infection and production performance of olive flounder. Mass mortality of fishes is the most severe problem, accompanying vast deficit in aquaculture farm. Efforts to prevent regrettable death have especially been conducted in immunology1–3. However, so far, it is hard to figure out cause of death exactly in short time because of variables of expansive marine ecosystem. Mass mortality of olive flounder was normally caused by diseases via various sources of infection such as virus, bacteria, or parasite from external environment.

Viruses depend on host cell ribosomes to produce their proteins, and sometime use host cell DNA and RNA polymerases for replication and transcription, respectively. Many viruses encode proteins that modify the host transcription or translation apparatus to favor the synthesis of viral proteins over those of the host cell. Among viruses, viral hemorrhagic septicemia virus (VHSV) is affiliated to Novirhabdovirus genus, which is a member of the Rhabdoviridae family4. The six gene were contained in the VHSV genome of about 11 K bases and each of them coded nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), nonstructural viral protein (NV), and RNA polymerase (L) in the following order 3’-N-P-M-G-NV-L-5’4. Infection of VHSV results in contagious viral hemorrhagic septicemia (VHS) in diverse fish species regardless of their inhabitation; seawater or freshwater5. In East Asia, a lot of infection cases into olive flounder have been reported steadily, since VHSV was detected in middle of 1990s6–9.

A variety of scuticociliates have been reported as cause of scuticociliatosis in marine species including turbot, guppy, and southern bluefin tuna10–12. In olive flounder, disease has been reported to be causing from various scuticociliates; Uronema marinum, Pseudocohnilembus persalinus, Philasterides dicentrarchi, Miamiensis avidus13–16. Interestingly, judging from infection experiments using various scuticociliates plus identification outcome of 8 isolates acquired from olive flounders with symptom of ulcers and haemorrhages, Miamiensis avidus was suggested as the major aetiologic agent of scuticociliatosis because of high pathogenicity and mortality rate compared with other scuticociliates14,17.

Infection of bacteria could sustain serious damage to fish. Streptococcosis is known to be caused by a variety of streptococcic species; Streptococcus parauberis, Streptococcus iniae, Streptococcus difficilis, Lactococcus garvieae, Lactococcus piscium, Vagococcus salmoninarum, and Carnobacterium piscicola, and has become major nuisance in olive flounder farms18–21. In particular, Streptococcus iniae, Lactococcus garvieae, and Streptococcus parauberis have been introduced to be related with Streptococcosis in olive flounder19–23.

The main issue of aquaculture industry is to reduce economic loss by preventing mortality of fish from various pathogens. A large number of immunologic studies have been proceeded about various immune-related gens against pathogen infection3,24–27. A huge quantity of genomic information from next generation sequencing (NGS) technique has been gradually increasing for the last few years, indicating that researchers could approach more comprehensive understanding view about genome of organisms than when they research a single gene level. With development of wide-sized analysis methods, it is not difficult to figure out change of gene expression level after any chemical treatment or environmental change. Recently, studies to identify large-scale genes were conducted in the olive flounder genome for researches about vaccine, gonadal development, and sex determination28–30. In particular, characterizing of immune-related genes was reported in olive flounder spleen tissue31. A lot of studies reported earlier were focused on gene expression analysis of single pathogen and specifically defined the expression pattern of limited genes32–36. Further, infection by two or more pathogens were reported in the olive flounder genome37,38. In order to solve these problem, we need plentiful genomic information to respond rapidly to multiple infection of pathogens. However, researches, which were comprehensively analysed about change of gene expression pattern by different type of pathogens, have not been reported in the olive flounder genome, so far.

In this research, we identified differentially expressed genes (DEGs) by transcriptome analysis and conducted gene ontology (GO) analysis with genes identified. Then, we tried to find important genes which showed consistently meaningful expression change in the results of three infection experiments. As a result, we determined 10 up-regulated genes and 57 down- regulated genes in common after infection of three pathogens. We aimed to provide essential genome information which is related with pathogen infection and explore the various consequences related to differential infections and find out the common strategies against specific candidates involved in disease progression in natural habitat of aquaculture.

Five groups of 25 Asian seabass (60 g) were randomly assigned to treatment groups (vaccines see Table 1). Fish were vaccinated with 0.1 mL of the prototype vaccines by intraperitoneal injection on day 0. Negative control fish were injected with 0.1 mL of placebo vaccine (vaccine dilution buffer PBS + 1.5% NaCl, formulated in ISA 763A VG oil as described above). Fish were starved for at least 36 hours prior to the vaccination. Immediately before the vaccination, fish from each group were weighed together to obtain the average body weight for each group. All fish were anaesthetized using AQUI-S (AQUI-S, New Zealand) prior to the vaccination procedure. Fish were fed ad libitum from the day after vaccination. Vaccinated fish were kept in 250 L partitions of 500 L tanks that were created by installing a vertical net in the tank. On day 28 post vaccination, all fish were challenged. The challenge dose was 2.0 x 107 TCID50/fish and the fish were subsequently kept in 125 L partitions of 250 L tanks that were created by installing a vertical net in the tank. Mortalities were recorded daily up to 28 days post challenge.

japonica Thunb. Regresses Nonalcoholic Steatohepatitis in a Methionine- and Choline-Deficient Diet-Fed Animal Model

Nonalcoholic fatty liver disease (NAFLD) is a condition in which excessive fat accumulates in the liver of a patient who does not have a history of alcohol abuse. NAFLD is classified into two categories: simple steatosis, in which only steatosis is observed, and nonalcoholic steatohepatitis (NASH), in which, in addition to steatosis, lobular inflammation and liver cell injury are observed. Although the underlying mechanisms of disease progression remain poorly understood, the “two-hit” hypothesis described the pathophysiology of NASH. Steatosis (a primary insult) can sensitize the liver to secondary serious insults including reactive oxygen/nitrogen species, gut-derived endotoxins, pro-inflammatory cytokines like tumor necrosis factor-alpha (TNFα), resulting in NASH development. NASH may develop into liver cirrhosis or hepatocellular carcinoma. The available treatment options for NASH include weight loss, dietary, and lifestyle modifications, use of insulin sensitizing, and lipid lowering drugs. Furthermore, combinations of these approaches have also been tried for management of NASH. Since NASH is a multifactorial disease, single target based therapy has limited implications.

Lonicera japonica Thunb. (Caprifoliaceae), also known as Japanese honeysuckle, ‘‘Jin Yin Hua’’ or ‘‘Ren Dong’’, native in the East Asian, is recognized as edible and medicinal food. The edible buds and flowers could be made into liquor and tea in the folk diet. At the same time, it could be used as medicine, cosmetics, ornamental groundcover, and so on. The constituents of this plant have been previously investigated and shown to contain iridoid glucosides and polyphenolic compounds. The main polyphenolic components in L. japonica are chlorogenic acid, caffeic acid, flavoyadorinin-B, methyl chlorogenate, and protocatechuic acid, make great contribution to its special activities. In current Chinese Pharmacopoeia, chlorogenic acid has been officially used as the indicator compound to characterize the quality of this herb. The modern pharmacological studies showed that L.

japonica had wide pharmacological actions, such as antibacterial, anti-inflammatory, antiviral, antiendotoxin, antipyretic, and other activities. Since 1995, Lonicera japonica has been listed in the Pharmacopoeia of the People’s Republic of China, and made in some preparations to treat chronic enteritis, pneumonia, acute tonsillitis, nephritis, acute mastitis, leptospirosis in clinic. L. japonica also has been employed extensively to prevent and treat some serious viral diseases of human and veterinary, such as SARS coronavirus, H1N1 (Swine) flu virus. In Chinese Pharmacopoeia, only the flowers and flower buds have been officially used as the active parts to treat diseases.

Pharmacological studies have demonstrated that L.

japonica has anti-fibrotic effect in rats with fibrosis induced by dimethylnitrosamine. It has also been reported that an herbal formula consisting of L.

japonica attenuates carbon tetrachloride-induced liver damage in rats; hepatoprotective potential of this plant could be considered. However, the possibility that L.

japonica could prove beneficial in ameliorating NASH has not been previously explored. The use of a diet deficient in essential amino acids such as methionine and choline is a well-accepted model for inducing NASH, which recapitulates many of the features of this disease in humans, including a histologic picture that mimics that seen in human fibrotic disorders associated with hepatic lipid accumulation, and the presence of inflammation and oxidative stress. To support the application of L.

japonica in the treatment of NASH-like disorders, the study investigated the hepatotherapeutic effect of L.

japonica in an animal model of methionine- and choline-deficient diet (MCDD)-induced hepatic injury and to elucidate the underlying mechanism involved.

Ciprofibrate, a fibric acid derivative, is a commercially available drug in the treatment of hyperlipidemia. It has been documented that ciprofibrate at the daily oral dosage of 10 mg/kg was effective to ameliorate hyperlipidemia in hypertriglyceridemic mice. In the present study, ciprofibrate was thus used as positive control to compare the effects of L.

japonica on the amelioration of NASH.

Omega-3 fatty acids have the first double bond three carbons from the methyl terminal, whereas omega-6 fatty acids have their first double bond six carbons from the methyl terminal. Major types of omega-3 fatty acids are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Omega-6 fatty acids include linoleic acid (LA) and arachidonic acid (AA). Mammals require the two essential fatty acid, ALA and LA to yield more bioactive derivatives through elongation and desaturation, and longer chain derivatives, EPA and DHA as well as AA can be obtained through oral intake of diet.23 LA is elongated and desaturated to form AA which encourages the production of proinflammatory cytokines, setting the stage for inflammation. ALA is elongated to EPA and DHA which are major components of the phospholipid membranes of the brain and retina and have anti-inflammatory effect.24 Omega-3 rich foods are salmon, herring, anchovi, sablefish, whitefish, tuna and others. Common vegetable oils have a higher content of omega-6 than omega-3. Although flaxseed, canola, mustard, walnut, soybean and other vegetable oils are rich in omega-3 fatty acids, vegetable oils abundantly contain ALA and have low levels of EPA or DHA. Since the conversion of ALA contained in vegetable oil to DHA or EPA is very inefficient, there is weaker evidence that ALA intake decreases cardiovascular events compared with DHA or EPA. Thus, it is almost impossible that taking flaxseed oil may influence cardiovascular disease progression.22 The mechanisms by which omega-3 fatty acids may reduce risk for cardiovascular disease are thought to be attributable to lowered serum triglyceride levels and antithrombotic, anti-inflammatory and antihypertensive activities.24

Several clinical studies identified that intake of fish oil reduces serum triglyceride level and blood pressure in both normal individuals and patients with hypertriglyceridemia, and lowers the frequency of arrythmia and the progression of atherosclerosis. Thus, we have arrived at a conclusion that daily intake of 0.5 to 1.8 g of EPA/DHA decreases mortality rate caused by cardiovascular disease, and the sufficient amount of omega-3 fatty acids can be achieved by eating fatty fish at least twice a week.25

The American Heart Association recommends that adults eat fish (particularly fatty fish) at least twice a week, and foods rich in ALA such as tofu, soybeans, walnuts, flaxseeds and their oil, and canola oil. Moreover, 1 g of EPA and DHA daily is recommended when coronary artery disease is already present, and 2 to 4 g of EPA and DHA daily is suggested to decrease triglyceride levels by 20% to 40%.26

In the analysis results of the Nurses' Health Study on postmenopausal women, the incidence rate of coronary artery disease was significantly reduced with an increasing intake frequency of fish at once a month, 1 to 3 times a month, once a week, 2 to 4 times a week, and more than 5 times a week. In particular, mortality rate caused by cardiovascular disease was reduced at a greater rate.27 Moreover, the incidence of ischemic stroke was also decreased with increasing fish intake.28

The effect of concurrent use of EPA with lipid-lowering agent has been also proved by the Japan EPA Lipid Intervention Study (JELIS). In the study, more than 18,000 Japanese patients with hypercholesterolaemia were recruited., The incidence rate of cardiovascular disease was reduced by 19% in the group consumed 1.8 g of EPA with statins daily for 5 years compared to that of the group with statins alone.29

According to a recent meta-analysis of 20 randomized clinical trials (RCTs) performed on about 68,000 subjects, omega-3 supplements did not reduce overall mortality rate, and mortality rate caused by cardiovascular disease, and the risk of myocardial infarction or stroke.30 However, several limitations have been pointed out in the results of those studies.31

Therefore, consistent consumption of fatty fish is recommended to prevent the risk of cardiovascular disease in postmenopausal women. Taking omega-3 supplement is expected to prevent cardiovascular disease in postmenopausal women almost not eating oily fish and not taking statins.32 The results from literature reviews provide that omega-3 intake, beside cardiovascular disease, has preventive effect on osteoporosis, cognitive dysfunction, cancer and inflammation.33

The objective of this study was to elucidate the role of E. coli, Enterococcus spp., C. perfringens and C. difficile in neonatal porcine diarrhea with no previously established etiology. We used FISH method for investigation of the prevalence, abundance and location of these potentially pathogenic bacteria in the intestinal tissue because a direct visualization of microorganisms helps to determine their association with the mucosa surface and may therefore have a potential value in elucidating their role in the disease. Sensitivity of FISH method for detection of bacteria depends on many factors including metabolic activity of microbial cells and availability of target sequences in the tissue samples. It has been shown that in natural samples the fluorescence signal intensity may be too low for identification of microorganisms. However, because the intestinal microbiota are expected to express high metabolic activity and possess high content of rRNA, the FISH method seems to be an appropriate approach for in situ detection of enteric bacteria. In order to better control for eventual false negative results due to methodological problems we decided to apply a general bacterial probe simultaneously with the specific probe on all tissue specimens. As the FISH results of hybridization with the general probe were satisfactory, we consider sensitivity of FISH performed in this investigation to be high.

According to informations provided at probeBase, three oligonucleotide probes used in this study: Enterococcus spp, C. perfringens and C. difficile probes, were shown to be highly specific and reliable for detection of these particular bacteria. However, the E. coli probe used in this investigation was noted to be unable to target all E. coli strains. Additionally, the target sequence of this probe was shown to match other enterobacteria; however these were not relevant as swine pathogens.

Quantification of microbial community detected by FISH in the intestinal tissue samples is not possible when the bacteria are present in high concentration as it hinders distinction of fluorescence signals from single bacterial cell. Therefore, evaluation of the amount of bacteria was done in a semi-quantitative manner, based on the subjective judgment of the investigator.

Adhesion of bacteria to the epithelial cells is believed to be an initial and pivotal event in the pathogenesis of most bacterial enteric infections and is necessary for allowing bacteria to survive and persist in a continuously moving environment and to defeat host defense mechanisms. In this study adherent bacteria were seen in 37 % of diarrheic and 14 % of non-diarrheic piglets. The non-diarrheic piglets were collected from the same herds as the diarrheic ones. Therefore, it cannot be excluded that some of the non-diarrheic piglets were in the initial phase of infection, thus expressed similar composition of eventual pathogens as the diarrheic piglets. In situ hybridization with the specific probes identified these bacteria as E. coli and/or Enterococcus spp. and there was a significant positive correlation between adherence of these bacteria and diarrhea. Furthermore, large amounts of E. coli were seen in significantly higher number of diarrheic piglets compared to non-diarrheic animals. Overgrowth and colonization of the mucosal surface by E. coli suggest its involvement in diarrhea. The piglets involved in this study were negative for enterotoxigenic E. coli (ETEC), which are considered to be the most common cause of pig neonatal diarrhea and for which the ability to attach to and colonize the intestinal epithelium is believed to be a hallmark virulence trait. However, E. coli strains other than ETEC have also been shown to be able to adhere to the intestinal mucosa surface and cause diarrhea. In addition, attaching and effacing E. coli (AEEC) that have ability to cause attaching and effacing lesions in the gut mucosa, have been associated with diarrhea in domestic animals including pigs. Therefore, further work will be done towards identifying and defining the pathogenicity of adherent E. coli found in this study.

We observed a significant positive correlation between the presence of E. coli and histomorphological changes in the intestinal mucosa. Villous atrophy is a common condition in diarrheal diseases and in suckling piglets this is primarily associated with viral or parasitic infections. However, some reports have shown that shortening of villi and epithelial lesions can follow colonization of the mucosa by E. coli[22,23]. Since the piglets included in this study were thought to be free from infection with commonly known pathogens, at this stage of investigation it is difficult to conclude whether overgrowth and colonization of the mucosa by E. coli was a primary event in the pathogenesis of villous atrophy or was secondary to infection with other, yet unidentified microorganisms, which cause alteration in the intestinal villi. Further studies are currently being conducted in order to determine the etiology of the presently described diarrhea.

Enterococci are commensal bacteria in the intestinal tract. However, it has been reported that certain members of enterococci can sporadically cause diarrhea in neonatal animals including piglets. The pathogenic potential of enterococci seems to be associated with their ability to intimately adhere to the intestinal epithelium but the mechanisms by which these bacteria cause diarrhea remain unclear. So far, no evident mucosal damage has been reported in association with enterococci infection. In this study we also observed adhesion of Enterococcus spp. to the intestinal epithelial cells in the diarrheic piglets, which suggests pathogenic ability of these bacteria. A significant positive correlation between the presence of enterococci and histological lesions (villous atrophy and mild epithelial lesions) can be explained by the fact that the positive fluorescence signals for adherent Enterococcus spp. were seen in the small intestine of piglets that also had adherent E. coli. If these lesions were a consequence of a bacterial infection, they should be associated with E. coli rather than Enterococcus spp. as discussed above. Nevertheless, simultaneous colonization of the intestinal mucosa surface by these bacteria is an interesting finding and suggests their close interactions. Previously, a virulent synergistic effect between E. coli and E. faecalis has been described in relation to experimental polymicrobial infections and it has been suggested that E. faecalis may inhibit phagocytosis of other pathogens including E. coli, and prevent them from intracellular death.

The majority of the piglets positive for adherent E. coli and adherent enterococci belonged to the same herd, which indicates that environmental factors influence composition of intestinal microbiota and eventual pathogens. This finding emphasizes the complexity of pathogenesis of porcine neonatal diarrhea and suggests that consideration of herd related aspects may be crucial for diagnosis and control of diarrheic conditions in piglets.

C. perfringens type A and C. difficile are nowadays regarded as ones of the most common bacterial species involved in pig neonatal diarrhea worldwide. In this study, the occurrence of C. perfringens and its amount detected by FISH were similar in diarrheic and non-diarrheic piglets. Pathologically, degenerative and necrotic changes in the intestinal mucosa are commonly associated with clostridial enteritis and the bacteria are usually present among the necrotic tissue. Such lesions were observed only in one diarrheic piglet in this study and it has been confirmed by microbiological testing that this piglet was positive for C. perfringens type C, which in that case can be regarded as a cause of enteritis. However, the mechanisms that could be involved in C. perfringens type A infection, remain unclear and there is no certain evidence for an adhesion of this bacterium to not destroyed intestinal tissue. Only few studies have investigated C. perfringens type A adhesive properties, but their results were inconclusive and to date, it is generally believed that C. perfringens does not have the ability to adhere to healthy intestinal epithelium. In agreement with this, the presence of C. perfringens in close proximity to the mucosal surface was seen in similar prevalence in both groups of piglets in this study (20 % diarrheic vs. 30 % non-diarrheic) and did not correlate with histological lesions, suggesting that the localization of C. perfringens cells in the intestinal mucosa is not linked to its pathogenicity. However, the pathogenesis of clostridial enteritis is commonly associated with the ability to produce toxins and diagnosis of the infection is based on the detection of large numbers of toxigenic bacteria. Therefore, the role of C. perfringens type A should not be definitely ruled out and the determination of the importance of this bacterium in neonatal diarrhea should be supported by thorough investigation on clostridial toxins.

C. difficile infections are currently reported as one of the most common causes of pig neonatal diarrhea in some countries and whenever diagnosed, the culturing reveals heavy growth of this bacterium. Microscopically, C. difficile infection is characterized by catarrhal, fibrinous or purulent colitis, however such lesions were not observed in this study. Furthermore, there was no association between the presence of this bacteria and pathological changes in the colon. Moreover, the occurrence of C. difficile and its amount did not differ significantly between diarrheic and non-diarrheic piglets. Therefore the presence of this bacterium seems not to be linked to the investigated diarrhea and these results are in agreement with other reports. Additional studies with focus on clostridial toxins are being conducted to determine the role of both Clostridia species in the pathogenesis of presently reported neonatal diarrhea.

Recently new genetic selection and domestication programs have commenced for large marine fish1. Various groupers (subfamily Epinephelinae of the family Serranidae) including giant groupers (Epinephelus lanceolatus), tiger groupers (E. fuscoguttatus) and humpback groupers (Cromileptes altivelis), compose one important new group of large marine species for domestication and selection. However, a viral disease, “viral nervous necrosis (VNN)” has been reported across grouper production centers in Taiwan, Malaysia, Indonesia and elsewhere around Asia2, 3. The causative agent of VNN in groupers and other marine fish is the nervous necrosis virus (NNV) of the genus Betanodavirus, within the family Nodaviridae (International Committee on Taxonomy of Viruses) (Harikrishnan et al.4 for review).

Many authors believe NNV is a serious threat to future marine fish aquaculture (including groupers). Pakingking et al.5 concluded “Catastrophic mortalities inflicted by piscine betanodaviruses remain as a major deterrent in the sustainable aquaculture of several species of groupers reared in ponds and floating net-cages in the open sea”. In a similar vein, Harikrishnan et al.4 concluded “viral nervous necrosis (VNN) caused by nervous necrosis virus (NNV) is one of the most important viral diseases that cause mass mortality in more than 39 marine fish species in 10 families”. Likewise Munday et al.6 concluded “In the last decade betanodavirus infections have emerged as major constraints on the culture of marine fish in all parts of the world”. Several recent reviews recognize betanodavirus as a significant problem for marine fish farming almost world wide7–9.

Fish host NNV resistance could be a key trait for future selection and the estimated heritability for NNV resistance in Atlantic cod appears to be high10–12. Moreover QTLs for resistance to NNV in Asian Sea Bass were reported13. However, notwithstanding such promising genetic selection opportunities, there are substantial knowledge gaps about the genetic diversity of NNV among Asian grouper hosts: are there many strains within and between regions, perhaps with different virulence so that each region or NNV strain may then require different selection programs, or not? Moreover, is there evidence that NNV strains vary over time? Genetic programs are long term and expensive undertakings so selecting for resistance to the appropriate strain/s is an important consideration, but one with a relative absence of knowledge for Asian grouper NNV. Without some clarity, we may select for inappropriate NNV strains, or see new NNVs transported from a different region for which the grouper have no resistance.

On a global scale, and considering many fish host species, previous phylogenetic analyses have resolved certain geographic and other patterns for NNV. For example there appears to be clustering of VNN genotypes into four main groups that include the so called barfin flounder nervous necrosis virus (BFNNV), the tiger puffer nervous necrosis virus (TPNNV), the striped jack nervous necrosis virus (SJNNV) and the red spotted grouper nervous necrosis virus (RGNNV) based on molecular phylogenetic analysis and percent nucleotide identity14. These four clusters are somewhat associated with water temperatures (and also hosts), for example there is a tendency for BFNNV and TPNNV genotypes to occur in temperatures up to 20 °C, for SJNNV to occur from 20 to 25 °C and for RGNNV for occur from 25 up to 30 °C15.

Within the red spotted grouper nervous necrosis virus (RGNNV) strain, to which all Asian grouper NNV belong, however, no one so far has reported evidence of genetic subgrouping by region, species or year in a formal statistical manner, especially when we restrict hosts just to Asian grouper. Available Genbank data show all Asian grouper NNV RNA2 RNA sequences, from Japan to Indonesia, are very closely related, varying by just one or two percent; this closeness means there are challenges to determine phylogenetic relationships especially when using traditional DNA or RNA distance based methods. However, Lowenstein et al.16 have found for tuna that particular polymorphic nucleotide positions (characteristic attributes) may categorically discriminate groups/ species previous not previous distinguished using traditional analyses based on percent RNA or DNA similarity. Ruan et al.17 reported certain polymorphic nucleotide positions could discriminate among strains of the SARS virus while Zou et al.18 reported that “character based” bar coding methods outperformed other approaches in discriminating closely related sea snails. Within aquaculture, these approaches of identifying nucleotides that are present in one group but not others have yet to be considered to a large degree, although our group have applied these ideas of characteristic attributes to recently resolve a very long standing taxonomic issue in oysters, whether the very closely related Crassostrea gigas and C. angulata are one or two species, as a prelude to conducting selection on the now identified C. angulata in Vietnam19.

The goal of this report was to collate the most comprehensive data set to date on NNV RNA2 sequences for warm water Asian marine finfish, whether published and/or lodged in Genbank over the last 20 years, including some sequence data produced by our group for Vietnamese and Taiwanese grouper, to statistically test the data for evidence of NNV strain variation that associates with geography, host species and year and also to determine whether there are “characteristic attributes” that indicate regional (or host, year) specific differences among the strains. This knowledge will help guide future selection criteria/NNV strains to consider in future genetic selection programs.

Hymenocallis littoralis (Amaryllidaceae), known locally as “Melong kecil,” is an ornamental and bulbous perennial herb. It has been traditionally used in Philippines as a vulnerary. Plants in the Amaryllidaceae family were reported to contain alkaloids that are known to exhibit a wide range of pharmacological activities. A number of alkaloids were isolated from the Hymenocallis littoralis such as lycorine, littoraline, hippeastrine, lycorenine, tazettine, pretazettine, macronine, homolycorine, lycoramine, vittatine, and haemanthamine. These compounds were reported to possess various pharmacological effects such as antiviral, antiparasitic, anticancer, antibacterial, antioxidant, and wound healing [4–6].

Lycorine, a pyrrolophenanthridine alkaloid, is one the major alkaloids found in H. littoralis [2, 7]. It displays strong antiviral effect against poliovirus, measles, and herpes simplex type 1 viruses. Besides, lycorine also possesses potent antiretroviral, antimitotic [10, 11], and cytotoxic activities [2, 12].

Different analytical techniques have been described for the qualitative and quantitative determination of alkaloids in both wild plant and in vitro culture of Amaryllidaceae including GC-MS [2, 11, 12], spectrophotometric, HPTLC [11, 13], and enzyme immunoassay. Few HPLC methods coupled with various detection methods were described for determination of lycorine in Amaryllidaceae plants such as Galanthus species, Leucojum aestivum, and Pancratium maritimum and Sternbergia species as well as tissue culture of Pancratium maritimum, Narcissus confusus, and Leucojum aestivum.

In vitro propagation is an important tool for rapid multiplication of medicinal plants [17, 18] as well as for the production of secondary metabolites. Tissue culture will ensure that the sources of the medicinal plants will not be exhausted or overexploited for their secondary metabolites. This is because the number of wild plants will not be effected due to overharvesting of the respective plants. Moreover the medicinal properties of a plant can be retained or increased via in vitro techniques. Previously, Yew et al. have reported the effect of different cytokinins on in vitro shoot length and multiplication of H. littoralis. By adjusting phytohormones concentrations in the medium, differences in amount, rate, and growth patterns of explants were observed [19, 20].

Despite many publications on the pharmacological effects of its chemical constituents, there is very little information available on the phytochemical analysis of H. littoralis wild plants or callus culture. Likewise, there is no report on the capability of callus culture of H. littoralis to produce secondary metabolites such as lycorine which has important pharmacological effects. The establishment and quantification of lycorine via in vitro propagation technique could be a first step in mass production of any desired secondary metabolites in pharmaceutical industries. Thus, in the present study, a simple HPLC with UV detection method was developed for phytochemical analysis of different parts of H. littoralis wild plants. The method was further extended for the quantification of lycorine in callus culture obtained from various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine concentrations.

Quinoline is known for its bactericidal, antiseptic and antipyretic action. Chloroquine (CQ) belongs the quinoline group. It is a white or slightly yellow crystalline powder with bitter taste. It is a lysosomotropic weak base, soluble in water at pH 4.5 with molecular formula of C18H26CIN3. The derivatives of CQ includes chloroquine diphosphate (C18H29ClN3.2H3PO4), chloroquine phosphate (C18H29ClN3.H3PO4), chloroquine sulfate (C18H26ClN3. H2SO4) and chloroquine dihydrochloride (C18H26ClN3.2HCl). CQ has been used as a primary antimalarial drug since 1930s due to its tolerability, effectiveness against malaria and inexpensive synthesis. In addition to serving as a malarial drug, CQ is now used in cancer therapy due to its enhancement property against tumour activity. CQ is also shown to significantly improve insulin levels in type 2 diabetes (T2D). In addition CQ is used as an antifungal, it is used in the treatment of rheumatic and immune-mediated diseases, management of HIV, SARS-CoV and influenza A/H5N1 virus.

Unfortunately, CQ use could have various side effects in mammals such as cardiac arrest, blindness, arrhythmias, hypokalemia, retinopathy, renal failure and cerebral oedema. In addition, CQ treatment during pregnancy is extremely toxic to embryo and overdose could lead to death. The negative effect of CQ is due to the fact that it inhibits diastolic depolarization, which slows down conduction and alters the intracellular transport of ionic transport. It also inhibits glucose 6-phosphate dehydrogenase activity, enzyme synthesis in nucleic acids; cyclic AMP pathway and it also increase oxidative stress in the organs,. Due to its high affinity towards nucleates and nucleoproteins, CQ could accumulate in lysosomes, adrenal glands and in epithelial cells of kidney which alters the secretion of aldosterone.

Over production and extensive use of pharmaceuticals including CQ may reach the aquatic ecosystem mainly through sewage effluents, washing out of faecal materials by rain, domestic wastewater and STPs. The presence of these pharmaceutical drugs or their residues in the aquatic environment is a serious issue throughout the world. Due to their resistance to degradation and lipophilic property they persist in the aquatic environment and could have negative effects on the biota. So far, more than 100 pharmaceuticals have been identified the aquatic ecosystem, recently Ramaswamy et al. and Shanmugam et al. have detected pharmaceutical and personal care products such as carbamazepine, triclosan, parabens, diclofenac, ketoprofen, naproxen, ibuprofen and acetylsalicylic acid in Indian major rivers such as Kaveri, Vellar and Thamiraparani.

Ecological risks by manmade chemicals are a potential subject of concern. Toxicity of any chemical can be determined by using bioassay methods. Specifically fish bioassay is considered as crucial in the field of eco-toxicology. As fish are one of the most organisms of the aquatic food web, and as they are a chief sources of food all over the world and as they are highly sensitive to slight environmental changes, it is important to conduct fish bioassay. Bioassays play an important role in providing information about the impact of emerging chemicals. In addition to bioassay, biomarkers are considered as early warning signals in the field of environment risk assessment. The biomarker response reveals the health status of an organism, population and ecosystem. Biochemical and histological biomarkers are known to be sensitive tools to detect direct effects of pollutants in the specific organ. These biomarkers may provide information from the starting point of biological effects to the impact on cell physiology.

Among the biochemical biomarkers enzymes are commonly used as a marker of pathological alterations of the organ, as they rapidly respond to chemicals. Glutamate oxaloacetate transaminase (GOT or AST), glutamate pyruvate transaminase (GPT or ALT) and lactate dehydrogenase (LDH) are the enzymes found in heart, liver, kidney, skeletal muscles and erythrocytes. GOT and GPT participate in transamination reactions. Likewise, LDH is an oxidative enzyme which is important for glycolytic activity. The alterations in these enzymes are used as organ health indicators of chemical exposure. GOT, GPT and LDH are widely used enzymological parameters in toxicology and in clinical chemistry to know the status of organs. Similarly, histopathological changes provide the direct effects of the toxicant in organs and also reveal the difference between damage induced by toxicant and other factors in organs/tissues. In fish, gills are the primary site of toxicant exposure and their structural changes indicate the impact of toxicant. Liver is the second largest organ in the body and are known to be a defense organ. Antoine et al. reported that liver is the major target area of human pharmaceuticals. Likewise kidney is a target organ for many pharmaceutical drugs. Hence, histological observation of vital organs such as gill, liver and kidney are important biomarkers in determining the toxic effect of human pharmaceuticals.

The present study was carried out to evaluate the acute and sublethal toxicity of chloroquine (CQ), an antimalarial drug in a freshwater fish Cyprinus carpio using certain biomarkers. The experimental model C. carpio is a common carp cultured widely in India

Bacterial zoonoses have a major impact on global public health. Both emerging and re-emerging bacterial zoonoses have gained increasing national and international attention in recent years. The closer contact with companion animals and rapid socioeconomic changes in food production system has increased the number of animal-borne bacterial zoonoses.

Animal bite injuries in daily human-animal contact are not surprising, especially for the school-aged children. Most of these wounds are medicated by patients as first aid and not registered in health systems. In more developed countries, most of the victims with moderate to severe bite injuries will seek professional medical treatment. Regardless, all bites should be treated as serious, especially if the skin is broken. Prompt diagnostic and treatment can prevent wound complications. The possibility to form biofilms by previously mentioned wound microorganisms is quite high, may cause severe tissue damage and protect the bacteria from innate-immune response and antimicrobials. The most of the commercial topical agents and wound dressings are ineffective against the biofilm matrix. Surgical repair (for example, CO2 surgical laser techniques, Leon Cantas, personal research notes 2014), which is usually used to obtain a better cosmetic result might be needed to remove biofilm formed bite infections. This mechanical debridement is essential in the eradication of a wound biofilm. Antimicrobials may be more effective in the treatment of the wound after debridement in the prevention of biofilm reformation. Despite the use of currently optimal culturing methods, approximately 7% of infected wounds yield no bacterial growth. In such cases, some other fastidious pathogens, i.e., Chlamydia spp., Mycoplasma spp., and even viruses should be investigated. New advanced molecular diagnostic techniques are needed. Prevention strategies for animal bites include close supervision of child–animal interactions, stronger animal control laws, better reporting of animal bites, and public education for better ownership of pets. Regular nail trimming, routine oral examinations under annual health checks and comprehensive dental treatments of the companion animals (i.e., routine removal of the teeth tartar and plaques) by veterinarians will reduce the bacterial mass exposure to humans in case of direct contacts or animal bites.

It is important to realize that enteropathogenic zoonoses may be contracted from both clinically sick and apparently healthy companion animals. Feeding of pets with raw food diets is a potential source of Salmonella, Campylobacter, and other important bacterial zoonoses; however, some recalls of commercial pet food diets have also occurred as a result of contamination with those microorganisms. Pig ear dog treats, in particular, have been implicated as an important source of Salmonella infection for dogs, which can also serve as a source of infection to humans.

Nevertheless, it can be said that easy-to-use personal hygiene rules should be applied by companion animal owners. Thorough hand washing with soap after handling of a companion animal and before eating or drinking, avoiding mouth-to-mouth contact, avoiding aerosolization of dusty fecal matter will help to prevent transmission of the zoonotic disease to humans. The animals with diarrhea should be isolated immediately and veterinary advice should be sought. The household should be cleaned with agents and kept as clean as possible.

On the other hand, the prevalence of antimicrobial resistance in small animal pathogens is increasing globally due to overuse of broad spectrum antibiotics by veterinarians. There is an immediate need for worldwide smarter use of antimicrobials that have some positive effect on the recovery of animals from life threatening diseases. National veterinary antimicrobial treatment guidelines should be established by the local authorities according to the updated routine surveillance results.

Chronic diarrhea, dermatitis, ear and eye infections of pets caused by microbes demand longer durations of antimicrobial remedies at home. More frequent use of advanced laboratory tests, such as; feed/insect/mould allergy tests and differential diagnosis of the other relevant auto-immune disorders may help to investigate the main underlying cause of the such reactions which can be managed in various alternative treatment methods (i.e., hypoallergenic diets) rather than antibiotics solely. Herein, pet specific auto-immune vaccines against allergens and auto-Lactobacillales (Auto-Lac, Leon Cantas, personal research notes, 2011–2014) as dietary supplements can also be more frequently administered within the preventative veterinary practice measures. Owners should be encouraged to insure their family animals to afford such costly veterinary services contradictory to the cheaper and sometimes life-long medical (i.e., antibiotic) treatment demanding options. Veterinarians should also spear more time to educate the pet owners under consultations to handle infected-antimicrobial treated animals with precaution due to irreversible consequences of the antimicrobial resistance development and its spread in households. Proper hand washing and use of gloves are strictly recommended while handling antimicrobial in veterinary clinics. Veterinarians should prescribe broad spectrum and synthetic antimicrobials preferably after culturing with extreme precautions (i.e., dosage, dosing intervals and length of the treatment). Reduced antibiotic use will hinder the development of antibiotic resistance in animal microbiota which might cause zoonotic infections in humans (50, 52).

Food-borne zoonoses are an important public health concern worldwide and every year a large number of people affected by diseases due to contaminated animal originated food consumption. Food hygiene education of the consumers is an important competent of food-borne diseases prevention. However, main prevention of food-borne zoonoses must begin at the farm level with in the concept of “One Health.” Herein, control of the production stress especially in intensive livestock industry, with the development of better animal health management routines (i.e., routine vaccinations, immune stimulants: pre-, probiotic feed additives) and the increased animal welfare programs, will contribute eventually to an optimal production of animal health. Increased antimicrobial resistance among emerging and re-emerging farm-borne bacterial pathogens in crowded settings (i.e., poultry, pig farms) is a growing problem. Restrictive antimicrobial choice with better animal welfare managements are needed to control the spread of antibiotic resistance elements.

In the EU, the use of avoparcin was banned in 1997 and the use of spiramycin, tylosin, and virginiamycin for growth promotion were banned in 1998. All other growth promoters used in feeding of food producing animals were banned from January 1, 2006 after a few national bans the years ahead3. In the U.S., politicians are still discussing to introduce a similar ban (S-742, 109th U.S. Congress (Preservation of Antibiotics for Medical Treatment Act). Despite the ban on the use of all antibiotics as growth promoters in the EU and a ban on the use of quinolones as growth promoters in the poultry feed in the US medical, important antibiotics are still routinely fed to livestock prophylactically to increase profits and to ward-off potential bacterial infections in the stressed and crowded livestock and aquaculture environments in some parts of the world (50, 90, 91). Because stress lowers the immune system function in animals, antibiotics are seen as especially useful in intensive animal confinements (92). The non-therapeutic use of antibiotics involves low-level exposure in feed over long periods – an ideal way to enrich resistant bacterial population (93, 94). Moreover, antibiotic resistance has been detected in different aquatic environments (95). Fish pathogenic bacteria often produce devastating infections in fish farms where dense populations of fish are intensively reared. Bacterial infections in fish are regularly treated with antibiotics in medicated feed. So far, most of the fish pathogenic bacteria with a history in diseased fish farms have developed drug resistance (96). Modern fish farming relies increasingly on vaccination procedures and improved management to avoid infections (97). For example, the Norwegian aquaculture industry has produced over one million tons farmed fish4 by using improved vaccines, management techniques, and only 649 kg of antimicrobials in 2011 (98).

Vector-borne and zoonotic bacterial pathogens are a major source of emerging diseases, and since the time of Hippocrates, weather and climate are linked to the incidence of such infectious diseases. Complexity of epidemiology and adoptive capacity of microorganisms and the arthropods make the vector-borne disease almost impossible to eradicate. Insect repellants, routine tick checks after outdoor activity in risk regions, prompt-proper tick removal, use of long sleeves and trousers (light-colored), and routine insecticide treatment of pets are recommended as general preventative measures (99). Herein, Lyme disease, tick-borne illness, is vastly underestimated over past decades and clearly the urgent prevention is needed. Besides individual awareness of such vector-borne diseases, better national surveillance and reporting programs will contribute to improved the disease control strategies. Clinicians have an important role in the effective management of vector-borne zoonotic diseases, with enhanced differential diagnostic skills based on clinical symptoms and rapid molecular identification techniques (100–103). Most of the time, the clinicians are on the first line of detection of these epidemics due to large group of patients with novel sets of similar symptoms. Increased medical networking via online databases offer a broad overview to followers with regard to changes in temporal patterns of illness in real time, which helps faster detection of new epidemics (104).

Identification and control of emergent zoonotic bacterial diseases require a “One Health” approach, which demands combined efforts of physicians, veterinarians, epidemiologists, public health workers, and urban planners. Collaborative international routine surveillance strategies, prompt – reliable agent identification techniques, and optimization of the treatment regiments will ensure the prevention and management of such infections.

The bacterial strains used in this study were listed in (table 1). Y. ruckeri strains were cultured on trypticase-soy-agar. The purity of the cultures was tested with Gram stain and confirmed biochemically with the API 20E rapid identification system.

Each strain from other bacterial strains was propagated on its specific medium and then tested by Gram stain and biochemically.

Aquaculture has been ongoing for centuries, but this industry has undergone rapid and extensive expansion because of the rapid growth of the average seafood consumption per person in the last 50 years. To accommodate this demand, aquaculture companies are now breeding fish to improve traits such as their growth rate, conversion of feed into muscle, disease resistance, fertility and other features associated with food quality. Nevertheless, one of the main challenges faced by this industry is its impact on environmental sustainability where clearly a public intolerance to any potential new source of pollution or the further degradation of the natural environment may act as a drawback. Proteomics application in aquaculture is mainly focussed on nutrition, welfare and health management, as these have proven to be major constraints to an efficient production in aquaculture systems (Rodrigues et al., 2012).

With regard to the nutrition source of farmed fish, there is a recent trend to move away from the traditional use of marine-harvested resources towards a diet-containing vegetable protein and oil sources. Although this reduces the impact on the marine-based food source, the growth rates and feed efficiency are compromised. However, proteomics is contributing greatly to a better understanding of the metabolic pathways affected by these dietary changes, as demonstrated in species like rainbow trout (Martin et al., 2003; Vilhelmsson et al., 2004; Keyvanshokooh and Tahmasebi-Kohyani, 2012), Atlantic Salmon (Sissener et al., 2010; Morais et al., 2012), Gilthead seabream (Ibarz et al., 2010; Rufino-Palomares et al., 2011; Siva et al., 2012; Matos et al., 2013) or Diplodus sargus (de Vareilles et al., 2012). These studies were mainly focussed on fish liver and muscle with identified protein responses involved in glycolysis, amino-acid catabolism, energy and lipid metabolism, oxidative stress or the immune system.

Fish diseases are responsible for the main economic losses in aquaculture. These diseases are mainly caused by viral, parasitic and bacterial infections and significantly affect the production yield worldwide (Hill, 2005). Several pathogen detection methods (traditional, immunological, molecular, etc) have been extensively used, with vaccination being the main research area for disease prevention (Biering et al., 2005, Hastein et al., 2005, Sommerset et al., 2005). Proteomics techniques have been assisting with this problem, especially at the level of development of new vaccines and disease diagnostics. Recent studies describe the isolation and the proteome analysis of the envelope proteins of the pathogen Iridovirus, which is responsible for the high mortality in cultured Grouper and also present in other Southeast Asian farmed species (Zhou et al., 2011).

Proteomics is also an extremely valuable tool in assessing fish welfare through the development of new aquaculture practices that ensure that farmed marine animals can be reared in an environment that optimizes their capacity to cope with unavoidable challenges/stress, thus enhancing their state of welfare and health. The main target organ to be analysed is the liver, providing a window to their metabolic status, or body fluids like blood plasma that is easily retrievable from the live animal. Stress-related studies mostly focussed on the correlation between environmental sources of stress in aquaculture with proteome changes. They include high stock densities (Provan et al., 2006; Alves et al., 2010), handling (Alves et al., 2010; Cordeiro et al., 2012) and preslaughter stress (Morzel et al., 2006). Studies focussed on the analysis of plasma proteins have concentrated on the detection and validation of welfare markers, with several proteins like microglobulins, macroglobulins, apolipoproteins, α1-antitrypsin, transferrin, plasminogen and complement system proteins among others being identified as possible candidates (Russell et al., 2006; Brunt et al., 2008; Bohne-Kjersem et al., 2009; Kumar et al., 2009).

An interesting field of application of proteomics is also the study of the pathogenesis of infectious disease affecting the avian species. The importance of this topic ranges from the economical aspect, to reduce the impact of avian diseases on production by characterizing the pathogenesis and by identifying new biomarkers of vaccines, to the need to study some avian diseases as a zoonosis, for example, avian flu, where human host adaptation signatures have been identified (Miotto et al., 2010) and responses to the virus characterised in mice (Zhao et al., 2012) and chicken (Sun et al., 2014). Proteomics has been already utilized to study the pathogenesis of herpes viruses (Kunec, 2013), with a special focus on Marek disease (Thanthrige-Don et al., 2010; Hu et al., 2012), which is of particular interest as a model for human tumours (Buza and Burgess, 2007).