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.

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%.

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).

Pooled sera of experimentally infected fish (see above), harvested at day 7 and day 10 after infection, were screened for presence of infectious virus. Sera were diluted 1:100 (v/v) and 1:1000 (v/v) in culture medium for inoculation of SK21 cells, which was carried out as described above. If no CPE occurred in the first passage of the virus, a second passage was performed.

No additional information is available for this paper.

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.

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.

In total 51 diarrheic and 50 non-diarrheic piglets aged 3–7 days were included in this study. The piglets were selected from four commercial Danish swine herds presenting high standards of management and housing. Diarrhea of unknown etiology (not caused by either enterotoxigenic E. coli, C. perfringens type C, rotavirus A, coronavirus or parasites) and poorly responding to antibiotic therapy was present in at least 30 % litters in each herd for a period of minimum six months. From each herd 11–14 diarrheic and 12–13 non-diarrheic piglets (age matched) from several litters (maximum two piglets per litter) were selected. The diarrheic piglets had diarrhea for at least 2 days prior to euthanasia and were selected from the litters with the highest prevalence of diarrhea. The non-diarrheic piglets did not have diarrhea at any time and were selected from the litters with no diarrhea or very low prevalence of diarrhea. For further details on the selection of herds and piglets the reader is referred to Kongsted et al.; 2013.

Virus infections are a continuous challenge in large-scale aquaculture of Atlantic salmon (Salmo salar). Environmental factors, high intensity production and infectious agents affect both welfare and production [1–3]. The two most prevalent viral diseases in Norwegian Atlantic salmon aquaculture are heart and skeletal muscle inflammation (HSMI) and pancreas disease (PD). Piscine orthoreovirus (PRV) is associated with HSMI, is ubiquitous in sea reared Atlantic salmon in Norway and often detected without any signs of disease [5, 6]. Pancreas disease is caused by Salmon pancreas disease virus, more commonly known as Salmonid alphavirus (SAV). The two viral diseases have overlapping geographic distributions [4, 7], both target heart and skeletal muscle and may co-infect Atlantic salmon [8–10].

PRV is a non-enveloped virus with a segmented, double stranded RNA genome, belonging to the genus Orthoreovirus in the family Reoviridae [5, 11]. Salmonid erythrocytes are major target cells for PRV and more than 50% of these cells may be infected in the peak phase of the infection. In later stages of the infection, PRV infects myocytes of the heart and skeletal muscles. The histopathological changes in heart and skeletal muscle gave the condition its name in the late 1990s, and later the association with PRV was established [5, 14].

SAV is an enveloped virus with a single-stranded positive sense RNA genome of the family Togaviridae. Pancreas, heart and skeletal muscle are the main target tissues. The disease is recognized by growth retardation, reduced slaughter quality and increased mortality [9, 16, 17]. Histopathological changes are characterized by acute necrosis of exocrine pancreas, myocardial and skeletal muscle necrosis with subsequent inflammation. The pancreatic lesions are a hallmark of PD and hence used for diagnostic differentiation from HSMI and cardiomyopathy syndrome (CMS). However, PRV and SAV have common target tissues in heart and skeletal muscles, making interactions between the two viral infections possible.

SAV is divided into six different phylogenetic subtypes and subtypes 2 and 3 are present in Norwegian aquaculture [19, 20]. The two subtypes show approximately 7% differences in nucleotide sequence, are endemically present in separate geographic areas and differ in virulence [20–22]. The mechanisms behind the difference in virulence are unknown. No stereotypical difference is described between subtype 2 and 3. PD outbreaks vary in duration, severity and accumulated mortality, indicating that factors other than SAV influences disease development. Interaction with other infectious agents may be such a factor.

Protection to a secondary virus infection induced by an unrelated primary virus infection has been recognized since the 1950s, and has also been described for several viruses infecting salmonid fish [26–30]. However, the duration of the protection of rainbow trout to infectious hematopoietic necrosis after primary infection with the non-virulent cutthroat trout virus was found to be no more than 4 weeks. In addition, some viral infections in terrestrial animals are shown to aggravate disease development of a secondary viral infection [31, 32].

The purpose of this study was to determine if a primary PRV infection alters the outcome of a subsequent SAV infection. Experimental infection trials were performed to compare disease development, viral kinetics and expression of disease associated genes between PRV-SAV co-infected and SAV infected fish.

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).

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

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.

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.

No pigs died or were removed from Exp. 2 during the study. Effects of treatment were significant (P < 0.0001) for BW, ADG, and ADFI (Table 5). The effect of day of study was significant (P < 0.005) for all variables and the interaction of treatment by day of study was significant (P < 0.0001) for BW.

Pigs fed SDBP had increased (P < 0.05) BW, ADG, and ADFI at d 7 and 14 compared with pigs fed all other diets. Average BW, ADG, and ADFI at d 7 and 14 did not differ among diets that did not contain SDBP. Some dietary treatments had G:F means at d 7 that were negative, therefore G:F results for d 7 are not reported. Gain:feed did not differ significantly among diets at d 14.

As done for Exp. 1, the ADG at d 7 for each pen by dietary treatment for Exp. 2 is presented in Fig. 2. Pens provided the SDBP diet had a minimum and maximum ADG result of 39 and 144 g, respectively. However, all pens fed non-SDBP diets had at least 1 negative ADG result. Maximum ADG results ranged from 69 to 122 g for non-SDBP treatments. As in Exp. 1, the experimental conditions of unsanitary pens and non-medication likely affected variation in pen results in Exp. 2, but response to dietary treatments was probably not strictly related to chance of pen location because dietary treatments were randomly allotted to pens within weaning group and by BW group.

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.

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.

In Exp. 1, the improved performance of pigs fed diets with SDPP compared to SPC during the initial 2 wk post-weaning are consistent with results of multiple experiments reviewed by others (Torrallardona, 2010; Pujols et al., 2016). Only ADG of pigs fed the diet with 5.00% SDPP compared to 2.50% SDPP was increased at d 7 and thereafter no significant differences in any performance variables were noted for diets with 2.50 or 5.00% SDPP. The observation that none of the pens provided SDPP diets had negative ADG at d 7 post-weaning, while 18 to 27% of pens provided non-SDPP diets had negative ADG results suggest that pigs fed SDPP diets were more resilient to the non-medication use and unsanitary pen conditions during the initial week post-weaning. Spray-dried porcine plasma contains a diverse mixture of proteins with biological activity including globulin, albumen, transferrin, glycoproteins, apolipoproteins, enzyme inhibitors, and proteins associated with blood clotting mechanisms (Kar et al., 2016). Improved growth of animals fed diets with SDPP has been attributed to actions of plasma globulins against luminal pathogens and toxins and reduced pro-inflammatory cytokine disruption of intestinal, respiratory, and reproductive mucosal barrier function (Pérez-Bosque et al., 2016). Other plasma proteins, such as growth factors and bioactive peptides, may also contribute to actions of plasma that beneficially influence mucosal barrier surfaces (Pérez-Bosque et al., 2016).

In Exp. 1, after all pigs were fed a common diet absent of the specialty proteins starting at d 15, only pigs previously fed the 2.5% SDPP diet had significantly higher average BW and ADFI at d 28 than pigs previously fed diets with SPC, 0.33 or 1.00% APP. By d 28 ADG did not differ significantly for pigs previously fed any of the specialty proteins. Other research has observed similar results for ADG of pigs fed diets with SDPP compared to SPC as observed in Exp. 1 (Torrallardona, 2010). Recently, Pujols et al. (2016) reported that ADG and BW were increased during the initial 2 wk post-weaning when 6% SDPP was included in the diet compared to SPC, however no differences for ADG or average BW among starter diets was observed at d 48, although mortality was reduced at d 48 and 145 and carcass weight was increased for pigs previously fed SDPP in the starter diet. The increased ADG and BW of pigs fed diets with SDPP during the initial 2 wk after weaning may or may not be maintained to the end of the nursery phase depending on the severity of post-weaning stress, ability of pigs to recover, and the incidence and degree of subsequent stress later in the nursery. The stress associated with weaning may have consequences on intestinal barrier function resulting in compromised performance in later life stages. Use of SDP in starter diets has been demonstrated to attenuate some of the effects of barrier dysfunctions associated with weaning stress (Peace et al., 2011; Boyer et al., 2015).

Performance variables in Exp. 1 did not differ significantly for pigs fed SPC and APP diets. Based on supplier information, the APP product used in Exp. 1 and 2 was produced using a proprietary process to reduce or eliminate anti-nutritional factors and activate components in porcine plasma thus requiring less mass of product to be used in formulations compared with commercial SDP. Published research with APP is limited. The provision of 0.2% APP in feed for mature gilts challenged with porcine reproductive and respiratory syndrome virus (PRRSV) reduced rectal temperature, PRRSV load (RNA copies/ml) and serum IL-1 and increased serum IL-18, suggesting that APP had immunomodulatory effects that benefit gilts with PRRSV (Song et al., 2015). Improvements in productivity have also been reported for PRRSV positive sows provided 0.5% SDPP in gestation and lactation feed (Campbell et al., 2006). Another study reported that 0.1% APP in gestation and lactation diets reduced percentage of small pigs at birth and wean to estrus interval of sows, increased weaning weight of pigs, and increased post-weaning growth of pigs to the end of the nursery period, while addition of 0.3% APP to nursery diets improved ADG of pigs only during the early post-weaning period (Musser et al., 2015). Similar productivity improvements were reported for sows fed lactation diets containing 0.5% SDP (Crenshaw et al., 2007).

Spray-dried bovine plasma was selected as a positive control protein source used in Exp. 2 because to the authors knowledge no publications have reported performance comparisons of pigs fed diets with SDBP versus APP. Bovine plasma may vary slightly in amino acid composition from porcine plasma, however each source contains similar profiles of globulin, albumen, and other proteins with biological activity and both sources of plasma have demonstrated increased performance of pigs when compared to other non-plasma protein sources (Torrallardona, 2010). Growth of pigs fed SDBP vs. SDPP was not different when each source was included at 6% of the diet, and pigs fed SDPP had higher ADFI, but lower G:F than pigs fed SDBP (Crenshaw et al., 2015). In a review of other studies where SDBP and SDPP were compared at the same dietary levels within the study (Torrallardona, 2010), performance results favoring either SDPP or SDBP were variable across studies, but most reported higher feed intake for pigs fed SDPP.

In Exp. 2, pigs provided a diet with 5.00% SDBP had higher BW, ADG and ADFI at d 7 and 14 post-weaning compared to diets containing either 8.04% SPC; 0.4% APP; 10.66% EHSY; a combination of 0.4% APP, 6.36% EHSY, and 2.5% fish meal; or 0.44% IEGG. As observed in Exp. 1 with SDPP diets, pens in Exp. 2 provided the SDBP diet did not have any pens with a negative ADG at d 7, while all other specialty protein diets had 10 to 50% of the pens with negative ADG. However, across all non-SDBP diets, some pens fed the different specialty protein diets had a maximum ADG above the minimum ADG for SDBP, suggesting that local pen environment may have influenced growth response to diets, but that the other specialty proteins did not consistently enhance growth performance compared to the SDBP diet under the experimental conditions of unsanitary pens and non-use of medication.

As in Exp. 1, performance differences were not detected between non-SDBP diets in Exp. 2. The level of 0.4% APP used in the APP and CB diets were based on supplier recommendations at the time of the experiment. Both diets containing APP resulted in lower performance than the SDBP diet and no performance differences compared to SPC, EHSY, or IEGG. One publication has indicated improved growth of pigs provided 0.30% APP in nursery diets but only in the early post-weaning period (Musser et al., 2015). The diet with a combination of EHSY, APP, and fish meal was designed to provide a complex mixture of plant and animal proteins with 0.40% APP to compare against 0.40% APP and 7.49% SPC. However, performance of pigs fed the CB diet did not differ from any of the non-SDBP diets and resulted in inferior performance compared to the SDBP diet. This observation is consistent with data indicating that PRRSV positive pigs fed a nursery diet regimen containing combinations of SPC, egg/fish pepton, highly processed poultry protein, yeast culture, and other feed additives had lower ADG and BW and higher mortality at the end of the nursery period (d 49 post-weaning) compared with pigs fed a less complex nursery diet regimen containing SDBP (Crenshaw et al., 2017).

Pig performance results for the diets with EHSY alone or in combination with fish meal and APP were inferior to the SDBP diet and did not differ from the SPC diet. One study has reported that pigs fed either simple or complex diets containing EHSY had similar or slightly better ADG and G:F compared with pigs fed diets containing SDP (Tsai et al., 2013).

In Exp. 2, performance of pigs fed the IEGG product was inferior to SDBP and did not differ from the other protein sources. The IEGG product used in Exp. 2 was spray-dried whole egg product (IEGG) derived from hens strategically vaccinated against various strains of Escherichia coli, Lawsonia intracellularis, rotavirus, coronavirus, Clostridia sp., and Salmonella sp., to produce specific IgY antibodies against these pathogens. Reasons for lack of a growth response to feeding the IEGG product are unknown but may have been related to either absence or overabundance of specific pathogens in the environment for which the specific IgY antibodies from the IEGG product could potentially impact. Pathogen-specific challenge studies have indicated similar improvements in performance for pigs fed hyper-immunized egg yolk products compared with pigs fed SDPP when pigs were challenged with pathogens common to the specific IgY antibodies contained in the egg product (Owusu-Asiedu et al., 2002). However, like Exp. 2 with SDBP, under non-specific pathogen challenge with unsanitary pens, pigs fed diets containing SDPP had improved growth performance compared with that of pigs fed a control diet, and a hyper-immunized egg yolk product did not increase performance over the control group (Torrallardona and Polo, 2016).

In conclusion, under the conditions of non-use of medication in feed or by other routes and unsanitary pens, diets with different levels of activated porcine plasma did not improve performance of pigs compared to diets with soy protein concentrate and neither soy protein concentrate or activated porcine plasma in diets provided equivalent performance to diets with spray-dried porcine plasma in Exp. 1. In Exp. 2, activated porcine plasma, enzymatically hydrolyzed soy and yeast protein, spray dried whole eggs from hyper-immunized hens, or a combination of fish meal, activated porcine plasma, and enzymatically hydrolyzed soy and yeast protein did not improve performance of pigs compared to diets with soy protein concentrate and none of the specialty proteins used in Exp. 2 provided equivalent performance to a diet containing spray-dried bovine plasma. Therefore, we were not able to confirm the hypothesis that growth performance of weaned pigs housed in unsanitary conditions and fed non-medicated diets containing the tested specialty proteins were equivalent to that of pigs fed diets containing either spray-dried porcine or bovine plasma.

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

Loop mediated isothermal amplification assay as a new diagnostic tool for diagnosis of ERM disease in salmonids was developed and evaluated. The ERM-LAMP assay is rapid, as its result appeared after one hour, and sensitive than the conventional diagnostic method of ERM disease. The ERM-LAMP assay requires only a regular laboratory water bath and is hence suitable as a routine diagnostic tool in private clinics and field applications where equipment such as thermal cycling machines and electrophoresis apparatus are not available.

D. Natarajan: conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.

T. Pratheeba: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

V. Taranath: Performed the experiments; Contributed reagents, materials, analysis tools or data.

DVR Sai Gopal: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

PHE-CoV: Porcine hemagglutinating encephalomyelitis coronavirus; HA/HI: Hemagglutination/hemagglutination inhibition; IHC: Immunohistochemistry; nested PCR: Nested-polymerase chain reaction; RT-PCR: Reverse transcriptase-polymerase chain reaction; MAb: Monoclonal antibody; GICA: Colloidal gold immunochromatography; HE: Hemagglutinin-esterase protein (HE); S: Spike glycoprotein; ELISA: Enzyme-linked immunosorbent assay; BSA: Bovine serum albumin; PBS: Phosphate Buffered Saline; NC: Nitrocellulose; TGEV: Transmissible gastro-enteritis virus; PEDV: Pig Epidemic Diarrhea Virus; PRV: Pseudorabies virus; HCV: Hog Cholera Virus; BCV: Bovine coronavirus; MHV: Mouse hepatitis virus; HCV-OC43: Human respiratory coronavirus OC43.

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.

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

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.