IgY is an attractive and effective alternative approach to antibiotics due to its high specificity. It should be noted that swine are exposed to many infectious agents in commercial operations and therefore the swine industry will benefit more from IgY antibodies if they are produced against a mixture of common disease causing organisms rather than one specific disease. If this approach works well, it may help to justify, to some extent, commercial application of these antibodies.

Oral administration of specific IgY appears to have considerable potential as a means of controlling diarrhea diseases and exerting growth-promoting activity in swine. IgY technology will probably provide the best alternative to antibiotics. Some advantages of IgY in the control of swine diseases are:They are highly effective.They are highly cost-effective with only a small amount of antibody required per pig.Collection of eggs is non-invasive.The treatment is safe and live organisms are not used.The procedure is environmentally friendly.No toxic residues are produced and there is no development of resistance.The treatment can be used to control many different types of pathogens.

For sequencing experiments, the stool filtrate was proteinase K treated prior to RNA extraction. RNA was isolated from primary stool filtrate using RNA-Bee (Tel-Test, Inc.) according to manufacturer's instructions. RT-PCR and 3'RACE reactions were performed using SuperScript III and Platinum Taq (Invitrogen One-Step RT-PCR). For 5'RACE reactions cDNA was generated with Themoscript (Invitrogen) and amplified with Accuprime Taq (Invitrogen). Amplicons were either cloned into pCR4 (Invitrogen) or sequenced directly.

Two independent reviewers screened titles and abstracts for their relevance. We included publications that mentioned norovirus in the title or abstract but we excluded papers about food (oyster) and waterborne outbreaks, food surveillance or food related experiments, and oyster/seafood surveillance. We excluded papers on murine noroviruses as models. Papers describing norovirus surveillance in wild mice and papers using mice as model for human norovirus were included (Figure 5).

In a second round, we screened the papers for whether they described (1) animal surveillance studies to detect human or animal norovirus by PCR, sequencing or by serosurveillance including negative results; (2) experimental animal infections with human or animal norovirus; (3) human surveillance studies to detect animal norovirus by PCR, sequencing or by serosurveillance including negative results; (4) animal norovirus characterization including molecular assays and genome announcements.

(“Norovirus”/exp OR “norovirus infection”/exp OR (Norovirus* OR Norwalk OR “small round-structur*” OR srsv*):ab,ti) AND ([animals]/lim OR “reservoir”/exp OR (nonhuman/de NOT human/exp) OR “zoonosis”/de OR “disease model”/de OR (animal* OR reservoir* OR nonhuman* OR non-human* OR animal* OR rat OR rats OR mouse OR mice OR murine OR dog OR dogs OR canine OR cat OR cats OR feline OR rabbit OR cow OR cows OR bovine OR rodent* OR sheep OR ovine OR pig OR swine OR porcine OR veterinar* OR chick* OR baboon* OR nonhuman* OR primate* OR cattle* OR goose OR geese OR duck OR macaque* OR avian* OR bird* OR mammal* OR poultry OR bat OR porpoise* OR zoono* OR farm OR farms OR “disease model*”):ab,ti)

Several authors have tested the efficacy of immunoglobulin preparations with antibody activity against human rotavirus as a means of providing passive immunity to children. For example, children consuming a defatted colostrum preparation from cows immunized against a strain of human rotavirus had no improvement of symptoms when the infection was established (patients admitted to a hospital with rotavirus infection), however the preparation was effective in limiting diarrhea in children when consumed prior to the infection. In another study, cessation of excretion of rotavirus in the stool of infants with acute rotavirus gastroenteritis was correlated with the presence of neutralizing activity in the stool after ingestion of a bovine whey protein concentrate from rotavirus-hyperimmunized cows, although there was not a significant decrease in duration of diarrhea in that study. Other studies have found that treatment of children with hyperimmune bovine colostrum from cows immunized with human rotavirus serotypes reduces the duration and severity of diarrhea due to rotavirus, and can provide significant protection from rotavirus infection.

Enteropathogenic bacteria have also been the target for development of immune milk. Over 80% of childrens’ stools became negative for the E. coli strains used to hyperimmunize the cows that provided the source of immunoglobulin in a bovine colostrum/milk immunoglobulin concentrate consumed by children for 10 days. Interestingly, only one in nine children treated with the immunoglobulin concentrate, and having diarrhea that was associated with E. coli strains which were not used in the immunization of the cows, developed negative stools, underscoring the importance of the bacterial strain-specificity of the immune product. Consumption of a hyperimmune immunoglobulin concentrate with a high antibody titer against a lipopolysaccharide isolated from Shigella flexneri 2a also has been shown to provide protection against a challenge with the same strain. However, no difference in diarrhea or other symptoms in children with stools positive for S. dysenteriae was found whether treated with bovine colostrum from cows immunized against S. dysenteriae or with colostrum from cows not hyperimmunized.

Enterotoxigenic E. coli also is commonly associated with traveler’s diarrhea. Prophylaxis against this infection may be achieved by providing passive immunity with immune milk. A bovine whey protein concentrate from cows immunized with enterotoxigenic E. coli serotypes and consumed 3-times daily for seven days protected all of the adult volunteers from developing diarrhea after being challenged with an enterotoxigenic E. coli strain. In contrast, 90% of the volunteers who received control immunoglobulin concentrate prior to challenge developed diarrhea after the E. coli challenge. Subsequent studies using IgG isolated from bovine colostrum from cows hyperimmunized against specific E. coli colonization factor antigens also have shown protective effects in volunteers challenged with colonization factor antigen-bearing enterotoxigenic E. coli[65], however other studies by the same group did not demonstrate significant effects of similar milk immunoglobulin products.

Bovine colostrum concentrate preparations derived from cows that have not been hyperimmunized against specific antigens also may provide some benefit via passive immunization for some diseases. For example, a commercial product which is made from large standardized pools of colostrum collected from over 100 cows has been used to treat a number of diseases, including diarrhea caused by diarrheagenic E. coli. Similar preparations from non-immunized cows may provide protection against bacterial toxins that are the cause of diarrhea in AIDS patients. These studies, along with the above mentioned study comparing colostrum preparations from cows immunized against S. dysenteriae or non-immunized cows, demonstrate that bovine colostrum contains significant antimicrobial properties as a result of natural exposure of the cows to antigens of pathogens that may afflict humans.

The experiment was approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC approval number 2-14-7742-S).

Prophylactic administration of low doses of antibiotics has been historically used to promote the growth and avoid infectious diseases in livestock animals. However, due to the emergence of antibiotic resistant microbes, several governments in countries around the world have prohibited the use of antibiotics as growth promoters for animals.

One of the most important challenges of agricultural immunology therefore is to find alternatives for developing drug-independent safe food production systems by modulating the immune system of animals. The work reviewed here encourages the research of probiotics to beneficially modulate the immune system of the bovine host. This review provides comprehensive information on the innate antiviral immune response of bovine IECs against virus, which can be further studied for the development of strategies aimed to improve antiviral defenses. The analyzed data also suggest that beneficial microbes have a great potential to be used as antiviral alternatives able to reduce severity of infections in the bovine host.

The development of specific in vitro study systems for cattle such as BIE cells as well as the selection and characterization of microbes that exert beneficial functions specifically and efficiently in the bovine host are key points for the successful development of immunomodulatory feeds aimed to protect against infections and reduce or avoid the use of antibiotics.

To meet the need for a rapid, efficient and cost-effective diagnosis, multiplex RT-PCR panels, including astroviruses and other gastrointestinal pathogens, have been developed over time. Early attempts using either end-point or qPCR proved to be as efficient as singleplex PCR for the detection in stool samples of HAstVs, noroviruses, adenoviruses, sapoviruses and enteroviruses. In the latter, the analysis of melting curves allowed the determination of dual-infection by the formation of dual peaks, while being at least 10× more sensitive than end-point PCR. Since then, several multiplex assays relying on different formats of detection have been developed and used for the diagnosis of astrovirus infection in humans or animals. Remarkably, among commercially-available solutions, the FilmArray Gastrointestinal Panel (BioFire Diagnostics, Salt Lake City, UT, USA) allows for the simultaneous detection of 22 different enteric pathogens directly from stool specimens, with a reported sensitivity and specificity of 100% and 99.9%, respectively, for the detection of MAstV 1 (but not MAstV 6 and 9), and a turnaround time of around one hour.

Although no systematic evaluation of the clinical benefit of the many existing panels and methods has yet been published, several studies have pointed out the advantages to streamline the diagnosis of presumptive infectious diarrhea with the use of a comprehensive multiplex PCR panel for the detection of known pathogens, and also for the detection of pathogens not requested or unable to be tested by conventional tests. For example, the FilmArray Gastrointestinal Panel has been used to detect astrovirus infections among diarrheic patients who were initially tested negative for Clostridium difficile and/or rotavirus. In another study, the Seeplex Diarrhoea ACE Detection (Seegene, Seoul, Korea) multiplex PCR assay was used in parallel to routine assays, to detect 11 cases of astrovirus infection among 245 stool samples from pediatric patients. Other broad multiplex PCR tests, including the Luminex technology (Austin, TX, USA), have been reported to offer an unprecedented ability to diagnose gastrointestinal infections in immunocompromised patients, with assay performances comparable to the tests examined.

Molecular approaches based on the amplification of viral genome or transcripts have dramatically improved the sensitivity of detection in comparison to EM, immunoassays or virus isolation, making substantial gains. With thresholds of detection as low as 10 to 100 genome copies per gram of stool, and the ability to develop type-specific detection systems, RT-PCR has now become a very common tool for the diagnosis of astrovirus infection in clinical laboratories. However, the design of amplification systems, in particular the intrinsic properties of the primers, are key, especially with regard to the amplification efficiencies and the ability to detect variant strains. For example, among the RT-PCR systems that have been developed for the detection of MAstV 1 (serotypes HAstV 1–8), some are targeting non-coding regions of the virus in a very sensitive and specific manner, while others are designed into conserved motives of the capsid, thereby allowing subsequent typing but with a risk of sub-optimal amplification efficiencies (for a complete review including a table of the most commonly used RT-PCR systems, see).

Alternative to RT-PCR, nucleic acid sequence-based amplification (NASBA) has also shown a good concordance with RT-PCR-based methods for the detection of MAstV 1 (serotypes HAstV 1–8). After the discovery of distant HAstV strains, MLB and VA1/HMO-C, additional primers have been developed and used to describe new populations of viruses. Beyond human astroviruses, many RT-PCR systems were also developed to detect astroviruses in wildlife, livestock or pets. Although consensus primers can detect a large number of astroviruses among both animal and human strains, there is not yet a universal pan-astrovirus RT-PCR system.

In parallel to the development of PCR primers, the application of real-time PCR (qPCR) in a diagnostic setting has improved the diagnosis of astrovirus infections by reducing the risk of false positives, allowing quantitation of viral loads and shortening the time to results (a positive or negative result is usually available within 24 h of specimen collection). qPCR can be done using a nucleic acid stain (typically SYBR green) followed by melting curve analysis, or by the use of a specifically designed hydrolysis probe coupled with a fluorophore (typically Taqman). One-step RT-qPCR methods have also been developed. Further refinements have been proposed with an integrated cell culture/RT-qPCR assay that is able to detect low levels of astrovirus after an incubation of seven days or less, but this approach has remained essentially of interest for research purposes only. Of note, although such advances have brought the ability to detect precise quantification of viral loads, the interpretation of very low amounts of virus in relation to clinical symptoms, especially in asymptomatic individuals, is still not always easy.

In this study, Vero cells and IECs were used to propagate PEDV. For the propagation of PEDV using Vero cells, the confluent cell monolayer was washed once with sterile phosphate-buffered saline (PBS; pH 7.2), and incubated with 1 mL of inoculum for 1 h in a T25 flask supplemented with 21 μg/mL of trypsin (Gibco) at 37 °C under 5% CO2. Then, the inoculum was removed and the cells were washed twice with PBS, and 4 mL of maintenance medium (DMEM, Gibco) without fetal bovine serum supplemented with 5 μg/mL trypsin was added to the flask. The propagation of PEDV using IECs was performed according to the method described above, but with the use of 10 μg/mL of trypsin during adsorption. In parallel, cells mock-inoculated with DMEM were used as control. The PEDV infected cells and viral control cells were cultured at 37 °C under 5% CO2. The cytopathic effect (CPE) was monitored daily, and cells were harvested until the CPE exceeded 80%. After one freeze-thaw cycle, the supernatants were collected, packed separately, and stored at −80 °C until required. Virus titer was measured in 96-well plates by 10-fold serial dilution of samples at five-passage intervals. The 50% tissue culture infective dose (TCID50) was expressed as the reciprocal of the highest dilution showing CPE by the Reed–Muench method.

The above examples of homologous transfer of passive immunity set the stage for considering the opportunities for heterologous passive transfer. Immune milk products generally are some form of protein product derived from the colostrum and/or milk of dairy cattle. The cows typically are hyperimmunized against one or more antigens representing pathogens of bacterial or viral origin. Crude preparations of the immunoglobulin from colostrum or milk may range from essentially no alteration of the immunoglobulin concentration in the product to partial immunoglobulin isolation or concentration in a whey protein concentrate.

The primary immunoglobulin in cow colostrum and milk is IgG, whereas the primary immunoglobulin in human milk is IgA. Nevertheless, bovine IgG from colostrum or milk can be effective as a means of providing passive immunity to protect animals and humans from disease. The use of bovine colostral immunoglobulin preparations from immunized cows for disease protection of the neonate of other species has been demonstrated in swine, and experimental animal models such as mice. There also are a number of examples of the use of bovine immune milk products in the treatment or prevention of human disease, especially in cases where the pathogen acts by way of the gastrointestinal tract. When considering these studies, it should always be kept in mind that the colostrum or milk preparations potentially contain other immune modulating substances than immunoglobulins, as discussed briefly below (section 6.3).

The concept of using immune milk derived from hyperimmunized cows for treatment of human disease can be traced back to the 1950s and earlier. Some of the early efforts in this field involved using immune milk products for treatment of rheumatoid arthritis and hay fever. Immune milk preparations produced from milk from cows immunized with a heat-killed, lyophilized mixture of bacteria found to reside in the human gastrointestinal tract has been studied for the prevention and treatment of rheumatoid arthritis, high blood cholesterol, high blood pressure, and oral submucous fibrosis. On the other hand, most studies on the use of immune milk have examined the potential of immune milk for prevention and treatment of infectious diseases, particularly gastrointestinal disease.

Even milk that does not come form hyperimmunized cows may in some sense be regarded as immune milk. Bovine anti-human rotavirus IgG1 antibodies have been found in raw and pasteurized milk from cows that had not been specifically immunized against that virus. Milk from non-immunized cows also has been found to contain measurable antigen-binding activity against several human pathogenic bacteria.

All experimental protocols were approved by the Laboratory Animal Monitoring Committee of Jiangsu province and performed accordingly. Twelve commercial high-health sows (Large White) were randomly divided into four groups (Table 3) and were housed in four separate rooms. These sows were confirmed virologically negative for PEDV, TGEV and porcine deltacoronavirus by RT-PCR and serologically negative for PEDV and TGEV antibody by indirect ELISA. On 28 and 14 days before farrowing, sows in group one were immunized intramuscularly with 2 ml of inactivated CV777 (2.0 × 107 TCID50) emulsified in equal volume of Gel 01 ST adjuvant (Seppic, France), separately. Group two were immunized intramuscularly with 2 ml of inactivated DR13 (2.0 × 107 TCID50) emulsified in equal volume of Gel 01 ST adjuvant. Group three were immunized intramuscularly with 2 ml of inactivated YC2014 (2.0 × 107 TCID50) emulsified in equal volume of Gel 01 ST adjuvant. Group four were treated with 2 ml of saline emulsified in equal volume of Gel 01 ST adjuvant. After immunization, sera samples were collected from sows at 7-day intervals until 35 days after immunization (7 days after farrowing) for detection of nucleocapsid protein specific antibodies with commercial indirect ELISA kits (Biovet, Canada) and PEDV neutralizing antibodies using YC2014 as indicator virus as described previously. Briefly, 50 μl of PEDV strain YC2014 (2.0 × 103 TCID50/ml) was added to an equal volume of the sera samples and incubated for 1 h at 37 °C. The mixture was then inoculated to a 96-well plate containing confluent Vero cells. 2 h later, the culture plate was washed with D-Hank’s three times, and then added 100 μl of DMEM containing 2 % fetal bovine serum (FBS). 48 h later, the culture plate was fixed with cold methanol for 10 min at −20 °C, and incubated with mouse anti-PEDV nucleocapsid protein polyclonal antibody for 1 h at 37 °C, and then stained with FITC-labeled rabbit anti-mouse IgG (Santa Cruz Biotechnology). The serum titers were determined as the reciprocal of the last serum dilution at 70 % or greater fluorescent focus reduction in the infected cell cultures under a fluorescent microscope. The colostrum and the milk on 7th day after farrowing of each sow were also collected and used for the detection of PEDV neutralizing antibodies.

The average number of sows farrowing was 13, piglets in each group was coded and chosen randomly, respectively. On 7th day after farrowing, thirty piglets were chosen (Table 4), ten from group one (inactivated CV777 immunized group), ten from group two (inactivated DR13 immunized group), five from group three (inactivated YC2014 immunized group), and five from group four (saline treated group). These piglets were divided into six groups (Table 4), each group were housed in a separate room and were artificial feeding with milk. Piglets in group one to group four were challenged orally with 1.0 × 104 TCID50 of YC2014. Group five were challenged orally with 1.0 × 104 TCID50 of CV777. Group six were challenged orally with 1.0 × 104 TCID50 of DR13. Piglets were observed daily after challenge about the diarrhea symptoms.

PED can generally be controlled using a vaccine strategy. Vaccination with killed or attenuated PEDV vaccine has been widely carried out in China and other swine raising countries, where PED usually manifests as a mild and enzootic pattern (lower mortality) some years ago. However, severe acute diarrhea outbreaks associated with high morbidity (80–100 %) and mortality (50–90 %) were observed in suckling piglets in most areas of China since December 2010, although most sow herds had previously been vaccinated with traditional inactivated PEDV vaccines based on CV777 or DR13. In April 2013, PED was diagnosed in the eastern Midwest region of the United States, subsequently, it spread rapidly to 30 neighboring states by June 2014. Accumulative evidence indicates that this large-scale outbreak of diarrhea may be caused by highly virulent PEDV variants [7–9].

The S protein makes up the large surface projections of the virion and plays a pivotal role in determining viral-cellular fusion activity and activating the immune system [10–12]. The variations in amino acid sequence likely changed the immunogenicity of the S protein and led to immunization failure of current commercial vaccines. In this study, we successfully isolated the YC2014 PEDV strain from porcine intestinal samples in dead piglets during outbreaks of acute diarrhea. The S gene nucleotides analysis of YC2014 indicated that it was clustered with the PEDV epidemic strains, with 15 nucleotide insertion in three sites and three nucleotide deletion in one site compared to classical PEDV vaccine strains CV777 and DR13. The variations of the S gene sequence and deduced amino acid sequence of the YC2014 strain compared to traditional PEDV vaccine strains may be the reason why some swine farms had well PEDV vaccine immunizations but still had sustained epidemic diarrhea which caused huge economic losses.

Vaccination is one of the most effective ways in preventing PED infection. Immunization of sows with PEDV vaccines at 20–30 days before production will provide substantial passive immunity to the newborn piglets. In this study, the nucleocapsid protein specific antibody levels of three different inactivated PEDV strains immunized groups gradually increased at all the testing time points. However, the antibody titers of these three groups are not significantly different. Immunization with inactivated YC2014 could protect piglets from acute diarrhea against homologous strain challenge. Since YC2014 was clustered with the PEDV epidemic strains, with >99 % nucleotide identity to most of these epidemic strains, the inactivated YC2014 may protect other variant strains challenge in some extent. However, it needs further refine animal studies to assess this suppose. The results showed that, great antigenicity variation had occurred to this YC2014 PEDV strain under the selective pressure of vaccines. Therefore, it is important to investigate PEDV variants currently circulating in sow herds to assess their ability to allow for cross-protection against highly virulent PEDV strains and prevent PEDV epidemics.

RNA was used as a template to generate cDNA using Prime Script RT Reagent Kit (Takara, Biotechnology, China). Then PDCoV, PEDV, TGEV, SADS-CoV and porcine rotavirus (PoRV) were detected by RT-PCR. Primers of PDCoV, PEDV, TGEV and PoRVA/B/C were designed and preserved by the Key Laboratory for Animal-derived Food Safety of Henan Province. Primers of SADS-CoV were synthetized that targeted the mostly conserved gene of SADS-CoV. The primers were shown in Table 1.

A watery, grey diarrhea was noted in the seeder pig 48 hours-post-inoculation with the cell culture-derived challenge material and lasted for approximately 5 days. Approximately 48–72 hours post-exposure with the seeder pig, the contact pigs showed intermittent signs of mild-moderate diarrhea lasting for approximately 7 days. No other clinical signs of diarrhea were appreciated in the contact pigs throughout the remainder of the study. S1, S3, and S4 showed no signs of diarrhea, while S2 had diarrhea for approximately 1 day 24 hours-post-contact with the PG. Post-challenge, the N/C group showed clinical signs of mild diarrhea beginning approximately 48 hours-post-inoculation that lasted for approximately 5 days with intermittent diarrhea in a few pigs for 3 weeks post-challenge. In contrast, no clinical signs were noted in the PG/C pigs post-challenge. The rectal swab fluids were not tested for other enteric pathogens.

Infectious viral diseases, both emerging and re-emerging, pose a continuous health threat and disease burden to humans. Many important human pathogens are zoonotic or originated as zoonoses before adapting to humans–[4]. This is exemplified by recently emerged diseases in which mortality ranged from a few hundred people due to infection with H5N1 avian influenza A virus to millions of HIV-infected people from acquired immunodeficiency syndrome–[8]. Severe acute respiratory syndrome (SARS) coronavirus and the pandemic influenza A/H1N1(2009) virus in humans were linked to transmission from animal to human hosts as well and have highlighted this problem–[11]. An ongoing systematic global effort to monitor for emerging and re-emerging pathogens in animals, especially those in key reservoir species that have previously shown to represent an imminent health threat to humans, is crucial in countering the potential public health threat caused by these viruses.

Relatively few studies have been conducted on diseases of non-domestic carnivores, especially regarding diseases of small carnivores (e.g. mustelids). Ferrets (Mustela putorius furo) can carry bacteria and parasites such as Campylobacter, Giardia, and Cryptosporidium in their intestinal tract and potentially spread them to people,. In addition, they can transmit influenza A virus to humans and possibly on rare occasions rabies virus as well–[16]. Because of their susceptibility to several human respiratory viruses, including human and avian influenza viruses, SARS coronavirus, nipah virus, and morbilliviruses–[21], ferrets have been used as small animal model for these viruses. To further characterize this important animal model and to obtain epidemiological baseline information about pathogens in ferrets, the fecal viral flora of ferrets was studied using a metagenomics approach. Both known and new viruses were identified.

Jejunum and ileum are the primary infection sites of PDCoV, and PDCoV antigen is observed both in the small intestines and large intestines. So we chose small and large intestines for the detection of PDCoV antigen by IHC. The prepared tissue samples were formalin-fixed, and paraffin-embedded tissue sections were de-waxed in xylene and rehydrated in decreasing 95, 85, 75% concentrations of ethanol for 1 min. Antigen retrieval was performed in citrate buffer (pH 6.0) at 95°C for 20 min. Slides were blocked with 5% bovine serum albumin (BSA) (Boster, China) at 37°C for 1 h, and then incubated with rabbit anti-PDCoV-N protein polyclonal antibody overnight at 4°C in a humidified chamber. Stained sections were then incubated with biotinylated secondary antibodies (Boster, China) at 37°C in a humidified chamber for 1 h, and treated with strept avidin–biotin complex (SABC) (Boster, China) for 1 h. Slices were washed three times with PBS after each incubation step, and positive cells were visualized with the treatment of diaminobenzidine (DAB). Sections were counterstained with Hematoxylin and images were obtained using a light microscope.

The swine intestinal epithelial cell (IEC) line established by Wang et al. was kindly provided by Prof. Yanming Zhang, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China. IEC and Vero cells (ATCC CCL-81) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), and supplemented with 10% fetal bovine serum (Gibco). The clinical samples (small intestine tissues) used in this study were collected from a pig farm in Nanjing, China, at which an outbreak of acute diarrhea among piglets had been reported. The virus isolated from samples was identified as PEDV by M gene-based reverse transcription PCR (RT-PCR). The small intestine tissue was homogenized with serum-free DMEM, and then centrifuged (Thermo Scientific Sorvall Legend Micro 17, Waltham, MA, USA) at 5000× g at 4 °C for 10 min. The supernatant was filtered using 0.22-μm pore-size cellulose acetate (Merck Millipore, Darmstad, Germany), and used for virus isolation.

PEDV has become a major economic concern for North American pig producers since it was first identified in the United States in 2013 (Stevenson et al., 2013; Chen et al., 2014). The lack of highly effective vaccines and the relative ineffectiveness of common treatment methods have led to a search for alternative methods of treating and preventing outbreaks. The inclusion of SDP into the diets of young pigs has been shown to have health benefits for other diseases. This study was designed to determine whether there is any benefit to adding BovSDP to the diet during PEDV infection.

Supplementing feed rations of pigs, fish, poultry, cats, and dogs with SDP as protein source is performed on a regular basis. SDP is a protein-rich product obtained from blood from healthy animals (cattle or pigs) at slaughter (Torrallardona, 2010; Pérez-Bosque et al., 2016). In pet food, SDP is a preferred binder in canned food products due to its high-protein content and its physicochemical properties (Polo et al., 2005; Rodriguez et al., 2016). Porcine SDP was first introduced as protein source for pigs during the early 1990s (Cole and Sprent, 2001) and since has been used widely in the diet of weaned pigs (Torrallardona, 2010). Benefits of adding porcine SDP include improvement of weight gain mainly due to increased feed intake and reduction of incidence and severity of diarrhea after weaning (Adewole et al., 2016). Comprehensive information on the effect of SDP obtained in 75 trials involving over 12,000 pigs has been summarized (Torrallardona, 2010).

In this study, the PEDV challenge was performed after an acclimation period of 1 wk to minimize stress from weaning and transport to the new facility. Successful PEDV challenge was confirmed by detecting PEDV RNA in fecal swabs. All pigs (100%) from the BovSDP–PEDV group shed PEDV in fecal samples from dpi 2 to 6, whereas between 25% and approximately 87% of the animals in PEDV group shed the virus during that time. These results highlight that a higher number of animals excreted PEDV during the early stages of infection in BovSDP–PEDV group. Differences in the challenge dose or challenge process can be ruled out. The PEDV inoculum was prepared in a similar manner and at the same time for both groups by a single person and stored on ice until challenge of each group. The challenge was conducted by the same personnel for both groups with approximately 20 min between the two groups. The obtained results could have been by chance due to the group sizes. Alternatively, the BovSDP–PEDV animals could be more prone to excrete PEDV in the early stages in the infection. Unlike in suckling pigs that are very susceptible to PEDV infection, only 2 per 13 PEDV-infected pigs (both from the PEDV challenged groups) had to be killed due to the severity of clinical signs. This is expected and comparable with other trials infecting 3-wk-old pigs (Crawford et al., 2015).

In the current study, the PEDV IgG and IgA antibody responses were more rapid in the BovSDP group compared with the PEDV group. Specifically, by dpi 7, 60% of the BovSDP pigs were anti-IgG and IgA positive compared with 12.5% of the PEDV pigs. Of note, while systemic IgA antibodies were measured, mucosal IgA levels were not determined, and it is therefore unknown how the addition of the BovSDP affected the gut immunity. In a previous study, a good correlation of IgA levels in serum and feces was found (Gerber and Opriessnig, 2015), and as fecal samples and gut mucosa are more difficult to process during routine lab work, serum IgA levels are commonly tested. Explanations for the earlier humoral immune response in the BovSDP group may include acceleration of the clinical course by the dietary supplement; however, a more rapid immune response due to earlier replication of the virus in more pigs unrelated to the diet modification is also possible. Serum anti-PEDV Ig in pig serum has been demonstrated to neutralize infectivity of PEDV (Hofmann and Wyler, 1989), and bovine plasma could have a similar neutralizing activity, which was not further assessed in this study. Besides the presence of possible neutralizing antibodies in SDP, other plasma compounds such as peptides (Anderson and Anderson, 2002) could contribute to the benefits seen with SDP addition to a diet.

Virus shedding in the BovSDP group was 2.1 d shorter than in the PEDV group. The fecal PEDV RNA shedding in this group was terminated by dpi 11. A previous study has shown that PEDV-infected pigs shed infectious PEDV capable of horizontal transmission for 14 to 16 d after infection (Crawford et al., 2015). This is similar to what was observed in the PEDV-infected pigs without BovSDP in the diet in this study.

The data from this trial indicate a beneficial effect of BovSDP on acute PEDV infection, which is similar to previous reports using the PRRSV infection model (Pujols et al., 2011). Specifically, pigs fed BovSDP were able to clear virus shedding 2.1 d sooner than non-BovSDP pigs. The addition of BovSDP to the diet resulted in faster and stronger PEDV antibody responses and reduced PEDV shedding time compared with pigs with no BovSDP in the diet. Limitations of this study include the usage of one single virus titer and the low numbers of pigs tested. In addition, two PEDV pigs had to be removed early from the study, which could have impacted the outcomes. A larger study with higher numbers of pigs per group comparing porcine and bovine-derived SDP with multiple necropsy days should be conducted to further confirm the possible benefits of SDP in PEDV-infected pigs.

There were no adverse reactions noticed at the injection site or overall health of sow post-vaccination. Each day post-challenge, the piglets were evaluated by animal services veterinarian for the clinical scores. The clinical score was assigned as 0 (healthy) to 4 (dead or moribund) for each piglet based on parameters such as animal demeanor, degree of depression and willingness to nurse. The average clinical scores for the vaccinated sow’s piglets was close to zero, whereas the control sow’s piglets had average scores reaching on day 2 and 3 post-challenge (Fig. 8a). Similarly, fecal scores were recorded from 0 (normal pasty faces) to 2 (watery diarrhea) for all the piglets each day post-challenge. Diarrhea increased until day 4 post-challenge and then started to reduce for both groups of piglets (Fig. 8b).

The weight of each piglet was monitored every day post-farrowing for 10 days, and the analysis of the average weight of piglets showed no appreciable differences between two groups of piglets (Fig. 8c).

Survival of the piglets was monitored for 10 days after challenge. At 6 days post-challenge, 7 out of 8 piglets (87.5 %) of vaccinated sow survived whereas only 3 out of 7 piglets (42.9 %) of control sow survived the challenge (Fig. 8d). Statistically significant (P = 0.0002) better survival rate in the litter of vaccinated sow can be explained by the fact that these piglets were less depressed and showed more willingness to nurse than piglets of a control sow. However, more experiments with large number of sows are needed to confirm these data.

In summary, our vaccine had negligible effect in either preventing diarrhea or preventing PEDV-mediated weight loss, but did partially protect piglets in terms of severity of clinical disease and significantly reduced mortality. Future work to improve the protective efficacy of the subunit vaccine for PEDV may include testing new adjuvants. For instance, in the recently published report, oil-in-water adjuvant was used to formulate recombinant S1 protein. Another approach is use of oral immunization instead of intramuscular (IM) route. A field study demonstrated that orally vaccinated sows with live attenuated PEDV vaccine exhibited higher IgA and virus neutralizing antibody levels in the colostrum or sera compared to those of the counterparts administered the IM vaccine with the same dose. To deliver a recombinant protein orally, a live vector such as adenoviral vector might be used.

An orphaned Rothschild giraffe from Dublin Zoological Gardens was presented to the University Veterinary Hospital at 14 days of age with a history failure of passive transfer of immunity, anorexia, dehydration, hypoglycemia, acidosis and persistent profuse watery diarrhea. The animal was treated on admission with intravenous fluid therapy, antimicrobials and non-steroidal anti-inflammatory drugs. The giraffe calf died 4 days post-treatment. A post-mortem examination primarily revealed a severe abomasitis and enteritis. No significant pathogenic organisms were isolated on bacteriological culture of the spleen, rumen or abomasum presumably as a consequence of intensive antimicrobial therapy. A faecal specimen was sent to our laboratory for further investigation.

Preliminary testing of the faecal specimen obtained (by Transmission Electron Microscopy) revealed the specimen to be positive for RV infection. Husbandry issues were considered to assess the potential route of RV infection. Interestingly, the calf had not been in contact with any other ruminants' prior to admission to the University Veterinary Hospital. However indirect contact with other animals could not be out ruled. Other issues for consideration were the movements of the keepers between different animal enclosures, the feeding equipment used and the apparatus used to clean the housing areas.

The MAstV-1 species is comprised of HAstV-1–8, and surveillance has revealed that HAstV-1 is the most commonly detected type in children, followed by HAstV-2–5, whereas HAstV-6–8 have been rarely detected. HAstV-4 and HAstV-8 have been associated with infection of older children and longer duration of diarrhea (>7 days). A HAstV-4 strain was also isolated from an infant with fatal meningoencephalitis. Based upon the phylogenetic analysis of the ORF2 region, different lineages within each HAstV type have been proposed; HAstV-1 (HAstV-1a–d) and HAstV-2 (HAstV-2a–d) have been divided into four lineages, whereas HAstV-3 (HAstV-3a–b) and HAstV-4 (HAstV-4a–c) have been classified into two and three lineages, respectively.

One 10 L-sample of raw sewage was collected in an urban wastewater treatment plant in the area of Barcelona, Spain. The sample was collected in a sterile container and stored for up to 2 hours at 4°C before being processed. The viruses present in the sample were concentrated in 30 mL of phosphate buffer by organic flocculation based on the procedure previously described by Calgua et al., 2008. A second concentration step with elution of the viral particles was performed. Briefly, 10 mL of the viral concentrate were eluted with 40 mL of 0.25 M glycine buffer (pH 9,5) at 4°C, suspended solids were separated by low speed centrifugation at 7500 × g for 30 min at 4°C and the viruses present in the supernatant were finally concentrated in 1 mL of PBS by ultracentrifugation at 87500 × g for 1 h at 4°C. This was then DNase treated, and then total nucleic acid was extracted.

The ELISA test was performed using a previously published method. Briefly, each well of a 96-well microtiter plate (Nalgene Nunc International, Penfield, NY) was coated with 0.44 ng of S1 protein, incubated overnight, and blocked with 1% bovine serum. The 1/100 diluted samples of serum in PBS with 10% goat serum were reacted at 37 °C for 30 min, washed, and incubated with 20 000-fold diluted peroxidase-conjugated goat anti-porcine IgG. Using tetramethylbenzidine-hydrogen peroxide as the substrate, the reaction was visualized for 10 min at room temperature and terminated with 2.5 M sulfuric acid prior to OD measurement at 450 nm. Positive, negative, and blank samples were tested in duplicate on each plate.