Maternal immunization and vitamin A sufficiency impact sow primary adaptive immunity and passive protection to nursing piglets against porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV) causes a highly contagious enteric disease with major economic losses to swine production worldwide. Due to the immaturity of the neonatal piglet immune system and given the high virulence of PEDV, improving passive lactogenic immunity is the best approach to protect suckling piglets against the lethal infection. We tested whether oral vitamin A (VA) supplementation and PEDV exposure of gestating and lactating VA-deficient (VAD) sows would enhance their primary immune responses and boost passive lactogenic protection against the PEDV challenge of their piglets. We demonstrated that PEDV inoculation of pregnant VAD sows in the third trimester provided higher levels of lactogenic protection of piglets as demonstrated by >87% survival rates of their litters compared with <10% in mock litters and that VA supplementation to VAD sows further improved the piglets' survival rates to >98%. We observed significantly elevated PEDV IgA and IgG antibody (Ab) titers and Ab-secreting cells (ASCs) in VA-sufficient (VAS)+PEDV and VAD+VA+PEDV sows, with the latter maintaining higher Ab titers in blood prior to parturition and in blood and milk throughout lactation. The litters of VAD+VA+PEDV sows also had the highest serum PEDV-neutralizing Ab titers at piglet post-challenge days (PCD) 0 and 7, coinciding with higher PEDV IgA ASCs and Ab titers in the blood and milk of their sows, suggesting an immunomodulatory role of VA in sows. Thus, sows that delivered sufficient lactogenic immunity to their piglets provided the highest passive protection against the PEDV challenge. Maternal immunization during pregnancy (± VA) and VA sufficiency enhanced the sow primary immune responses, expression of gut-mammary gland trafficking molecules, and passive protection of their offspring. Our findings are relevant to understanding the role of VA in the Ab responses to oral attenuated vaccines that are critical for successful maternal vaccination programs against enteric infections in infants and young animals.

Histo-Blood Group Antigen-Producing Bacterial Cocktail Reduces Rotavirus A, B, and C Infection and Disease in Gnotobiotic Piglets.

The suboptimal performance of rotavirus (RV) vaccines in developing countries and in animals necessitates further research on the development of novel therapeutics and control strategies. To initiate infection, RV interacts with cell-surface O-glycans, including histo-blood group antigens (HBGAs). We have previously demonstrated that certain non-pathogenic bacteria express HBGA- like substances (HBGA+) capable of binding RV particles in vitro. We hypothesized that HBGA+ bacteria can bind RV particles in the gut lumen protecting against RV species A (RVA), B (RVB), and C (RVC) infection in vivo. In this study, germ-free piglets were colonized with HBGA+ or HBGA- bacterial cocktail and infected with RVA/RVB/RVC of different genotypes. Diarrhea severity, virus shedding, immunoglobulin A (IgA) Ab titers, and cytokine levels were evaluated. Overall, colonization with HBGA+ bacteria resulted in reduced diarrhea severity and virus shedding compared to the HBGA- bacteria. Consistent with our hypothesis, the reduced severity of RV disease and infection was not associated with significant alterations in immune responses. Additionally, colonization with HBGA+ bacteria conferred beneficial effects irrespective of the piglet HBGA phenotype. These findings are the first experimental evidence that probiotic performance in vivo can be improved by including HBGA+ bacteria, providing decoy epitopes for broader/more consistent protection against diverse RVs.

Vitamin A deficiency and vitamin A supplementation affect innate and T cell immune responses to rotavirus A infection in a conventional sow model.

Rotavirus A (RVA) causes ~200,000 diarrheal deaths annually in children <5yrs, mostly in low- and middle-income countries. Risk factors include nutritional status, social factors, breastfeeding status, and immunodeficiency. We evaluated the effects of vitamin A (VA) deficiency/VA supplementation and RVA exposure (anamnestic) on innate and T cell immune responses in RVA seropositive pregnant and lactating sows and passive protection of their piglets post-RVA challenge. Sows were fed VA deficient (VAD) or sufficient (VAS) diets starting at gestation day (GD)30. A subset of VAD sows received VA supplementation from GD|76 (30,000IU/day, VAD+VA). Sows (6 groups) were inoculated with porcine RVA G5P[7] (OSU strain) or Minimal Essential Medium (mock) at GD~90: VAD+RVA; VAS+RVA; VAD+VA+RVA; VAD-mock; VAS-mock; and VAD+VA-mock. Blood, milk, and gut-associated tissues were collected from sows at several time points to examine innate [natural killer (NK), dendritic (DC) cells], T cell responses and changes in genes involved in the gut-mammary gland (MG)-immunological axis trafficking. Clinical signs of RVA were evaluated post inoculation of sows and post-challenge of piglets. We observed decreased frequencies of NK cells, total and MHCII+ plasmacytoid DCs, conventional DCs, CD103+ DCs and CD4+/CD8+ and T regulatory cells (Tregs) and NK cell activity in VAD+RVA sows. Polymeric Ig receptor and retinoic acid receptor alpha (RARα) genes were downregulated in mesenteric lymph nodes and ileum of VAD+RVA sows. Interestingly, RVA-specific IFN-γ producing CD4+/CD8+ T cells were increased in VAD-Mock sows, coinciding with increased IL-22 suggesting inflammation in these sows. VA supplementation to VAD+RVA sows restored frequencies of NK cells and pDCs, and NK activity, but not tissue cDCs and blood Tregs. In conclusion, similar to our recent observations of decreased B cell responses in VAD sows that led to decreased passive immune protection of their piglets, VAD impaired innate and T cell responses in sows, while VA supplementation to VAD sows restored some, but not all responses. Our data reiterate the importance of maintaining adequate VA levels and RVA immunization in pregnant and lactating mothers to achieve optimal immune responses, efficient function of the gut-MG-immune cell-axis and to improve passive protection of their piglets.

Immune Impairment Associated with Vitamin A Deficiency: Insights from Clinical Studies and Animal Model Research.

Vitamin A (VA) is critical for many biological processes, including embryonic development, hormone production and function, the maintenance and modulation of immunity, and the homeostasis of epithelium and mucosa. Specifically, VA affects cell integrity, cytokine production, innate immune cell activation, antigen presentation, and lymphocyte trafficking to mucosal surfaces. VA also has been reported to influence the gut microbiota composition and diversity. Consequently, VA deficiency (VAD) results in the imbalanced production of inflammatory and immunomodulatory cytokines, intestinal inflammation, weakened mucosal barrier functions, reduced reactive oxygen species (ROS) and disruption of the gut microbiome. Although VAD is primarily known to cause xerophthalmia, its role in the impairment of anti-infectious defense mechanisms is less defined. Infectious diseases lead to temporary anorexia and lower dietary intake; furthermore, they adversely affect VA status by interfering with VA absorption, utilization and excretion. Thus, there is a tri-directional relationship between VAD, immune response and infections, as VAD affects immune response and predisposes the host to infection, and infection decreases the intestinal absorption of the VA, thereby contributing to secondary VAD development. This has been demonstrated using nutritional and clinical studies, radiotracer studies and knockout animal models. An in-depth understanding of the relationship between VAD, immune response, gut microbiota and infections is critical for optimizing vaccine efficacy and the development of effective immunization programs for countries with high prevalence of VAD. Therefore, in this review, we have comprehensively summarized the existing knowledge regarding VAD impacts on immune responses to infections and post vaccination. We have detailed pathological conditions associated with clinical and subclinical VAD, gut microbiome adaptation to VAD and VAD effects on the immune responses to infection and vaccines.

Rotavirus A Inoculation and Oral Vitamin A Supplementation of Vitamin A Deficient Pregnant Sows Enhances Maternal Adaptive Immunity and Passive Protection of Piglets against Virulent Rotavirus A.

The aim of this study was to determine the impact of vitamin A deficiency (VAD)/supplementation (±VA) and group A RV (RVA) maternal immunization of RVA seropositive multiparous pregnant sows, on their immune responses (anamnestic response) and on passive protection of their piglets against RVA challenge. Our results showed that VAD- mock sows had increased RVA RNA shedding at 1-5 days post piglet RVA challenge, and their litters had increased RVA shedding and diarrhea frequency throughout the experiment. VAD decreased memory B cell frequencies while VA supplementation increased RVA specific IgA/IgG antibody (Ab) secreting cell (ASC) numbers in blood, milk, and tissues of RVA inoculated VAD sows. The increased numbers of RVA specific IgA/IgG ASCs in blood, milk/colostrum, intestinal contents, and tissues in VA supplemented VAD sows, suggest a role of VA in B cell immunity and trafficking to tissues. We also observed that RVA inoculated sows had the highest viral neutralizing Ab titers in serum and milk while VA supplementation of VAD sows and RVA inoculation increased IgA+ B cell frequencies in sow colostrum. In summary, we demonstrated that daily oral VA-supplementation (2nd trimester-throughout lactation) to RVA inoculated VAD sows improved the function of their gut-mammary-IgA immunological axis, reducing viral RNA shedding, diarrhea, and increasing weight gain in suckling piglets.

The Combined Escherichia coli Nissle 1917 and Tryptophan Treatment Modulates Immune and Metabolome Responses to Human Rotavirus Infection in a Human Infant Fecal Microbiota-Transplanted Malnourished Gnotobiotic Pig Model.

Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.

Mechanisms of Kwashiorkor-Associated Immune Suppression: Insights From Human, Mouse, and Pig Studies.

Malnutrition refers to inadequate energy and/or nutrient intake. Malnutrition exhibits a bidirectional relationship with infections whereby malnutrition increases risk of infections that further aggravates malnutrition. Severe malnutrition (SM) is the main cause of secondary immune deficiency and mortality among children in developing countries. SM can manifest as marasmus (non-edematous), observed most often (68.6% of all malnutrition cases), kwashiorkor (edematous), detected in 23.8% of cases, and marasmic kwashiorkor, identified in ~7.6% of SM cases. Marasmus and kwashiorkor occur due to calorie-energy and protein-calorie deficiency (PCD), respectively. Kwashiorkor and marasmic kwashiorkor present with reduced protein levels, protein catabolism rates, and altered levels of micronutrients leading to uncontrolled oxidative stress, exhaustion of anaerobic commensals, and proliferation of pathobionts. Due to these alterations, kwashiorkor children present with profoundly impaired immune function, compromised intestinal barrier, and secondary micronutrient deficiencies. Kwashiorkor-induced alterations contribute to growth stunting and reduced efficacy of oral vaccines. SM is treated with antibiotics and ready-to-use therapeutic foods with variable efficacy. Kwashiorkor has been extensively investigated in gnotobiotic (Gn) mice and piglet models to understand its multiple immediate and long-term effects on children health. Due to numerous physiological and immunological similarities between pigs and humans, pig represents a highly relevant model to study kwashiorkor pathophysiology and immunology. Here we summarize the impact of kwashiorkor on children's health, immunity, and gut functions and review the relevant findings from human and animal studies. We also discuss the reciprocal interactions between PCD and rotavirus-a highly prevalent enteric childhood pathogen due to which pathogenesis and immunity are affected by childhood SM.

Escherichia coli Nissle 1917 Enhances Efficacy of Oral Attenuated Human Rotavirus Vaccine in a Gnotobiotic Piglet Model.

Human rotavirus (HRV) infection is a major cause of viral gastroenteritis in young children worldwide. Current oral vaccines perform poorly in developing countries where efficacious vaccines are needed the most. Therefore, an alternative affordable strategy to enhance efficacy of the current RV vaccines is necessary. This study evaluated the effects of colonization of neonatal gnotobiotic (Gn) pigs with Escherichia coli Nissle (EcN) 1917 and Lacticaseibacillus rhamnosus GG (LGG) probiotics on immunogenicity and protective efficacy of oral attenuated (Att) HRV vaccine. EcN-colonized pigs had reduced virulent HRV (VirHRV) shedding and decreased diarrhea severity compared with the LGG-colonized group. They also had enhanced HRV-specific IgA antibody titers in serum and antibody secreting cell numbers in tissues pre/post VirHRV challenge, HRV-specific IgA antibody titers in intestinal contents, and B-cell subpopulations in tissues post VirHRV challenge. EcN colonization also enhanced T-cell immune response, promoted dendritic cells and NK cell function, reduced production of proinflammatory cytokines/Toll like receptor (TLR), and increased production of immunoregulatory cytokines/TLR expression in various tissues pre/post VirHRV challenge. Thus, EcN probiotic adjuvant with AttHRV vaccine enhances the immunogenicity and protective efficacy of AttHRV to a greater extent than LGG and it can be used as a safe and economical oral vaccine adjuvant.

Whole Genome Sequence Analysis of Porcine Astroviruses Reveals Novel Genetically Diverse Strains Circulating in East African Smallholder Pig Farms.

Astroviruses (AstVs) are widely distributed and are associated with gastroenteritis in human and animals. The knowledge of the genetic diversity and epidemiology of AstVs in Africa is limited. This study aimed to characterize astroviruses in asymptomatic smallholder piglets in Kenya and Uganda. Twenty-four samples were randomly selected from a total of 446 piglets aged below 6 months that were initially collected for rotavirus study and sequenced for whole genome analysis. Thirteen (13/24) samples had contigs with high identity to genus Mamastrovirus. Analysis of seven strains with complete (or near complete) AstV genome revealed variable nucleotide and amino acid sequence identities with known porcine astrovirus (PoAstV) strains. The U083 and K321 strains had nucleotide sequence identities ranging from 66.4 to 75.4% with the known PoAstV2 strains; U460 strain had nucleotide sequence identities of 57.0 to 65.1% regarding the known PoAstV3; and K062, K366, K451, and K456 strains had nucleotide sequence identities of 63.5 to 80% with the known PoAstV4 strains. The low sequence identities (<90%) indicate that novel genotypes of PoAstVs are circulating in the study area. Recombination analysis using whole genomes revealed evidence of multiple recombination events in PoAstV4, suggesting that recombination might have contributed to the observed genetic diversity. Linear antigen epitope prediction and a comparative analysis of capsid protein of our field strains identified potential candidate epitopes that could help in the design of immuno-diagnostic tools and a subunit vaccine. These findings provide new insights into the molecular epidemiology of porcine astroviruses in East Africa.

Evidence for the presence of African swine fever virus in apparently healthy pigs in South-Kivu Province of the Democratic Republic of Congo.

African swine fever (ASF) is the most important disease constraining smallholder pig production in the Democratic Republic of Congo, causing high mortality in domestic pigs with severe impacts on the livelihoods of local populations. This study was conducted with the aim of determining the prevalence of ASF and circulating virus genotypes in asymptomatic pigs raised on smallholder farms in the South Kivu province to understand the transmission dynamics of ASF and ultimately improving disease control. A cross-sectional survey was carried out in 5 districts where 267 pig blood were screened for both antibody and viral genome using indirect Enzyme Linked Immunosorbent Assay (ELISA) and polymerase chain reaction (PCR) respectively. Additionally, amplicons from PCR positive samples were sequenced by Sanger method for genetic analysis of ASF virus (ASFV) based on the C-terminal region of the B646L gene that encodes the major capsid protein p72 and the gene E183L encoding the p54 protein. The overall seroprevalence obtained based on antibody to p30 protein was 37 % and was significantly higher (P < 0.05) in adult (>1 year) animals (44.7 %) than in younger (<1 year) ones (33.5 %). Moreover, the seropositivity varied significantly (P < 0.05) according to the pig husbandry system practiced within the districts investigated with Uvira (55 %) and Mwenga (42.2 %) having the highest ASFV antibodies, while the lowest (10.5 %) were in Kalehe. Free-range pigs exhibited a higher level of seropositivity to ASFV antibody (68.9 %) than pigs kept in the pigsty housing system (21.6 %). However, no statistically significant differences (P > 0.05) were observed when sex of the animal and breed were factored. PCR detection of ASFV amplified a specific band of expected size (257 bp) in 61 out of 267 blood samples, confirming the presence of the viral DNA in 22.8 % of asymptomatic domestic pigs. Statistical analysis revealed that ASFV infection in domestic pigs varied significantly (p < 0.001) according to geographical location and breed, with the highest infection rate found in Walungu district (33.7 %) while the lowest was registered in Kalehe (15.8 %). Local pigs (27.2 %) were more infected than crosses (9.2 %). Phylogenetic analyses based on partial sequences of the p72 and p54 genes revealed that all the ASFV detected belonged to genotype IX, which has previously been reported in other parts of DR Congo, and was clustered together with isolates from Kenya, Uganda and Republic of Congo. This study avails the first evidence of the presence of ASF virus in domestic pigs in the absence of outbreaks in South Kivu province, eastern DR Congo, indicating a need to raise awareness among pig farmers and veterinary authorities on the application of biosecurity measures and good husbandry practices to control the disease.

Performance evaluation of five commercial assays in assessing seroprevalence of HEV antibodies among blood donors.

INTRODUCTION: Although hepatitis E virus (HEV) is mainly transmitted via the faecal-oral route, the rate of HEV transmission via blood donation is on the rise. However, the seroprevalence of HEV among blood donors is not well established and is thought to be affected by the type of diagnostic assay used. We aimed to evaluate performance and correlation among widely used commercial diagnostic assays for the seroprevalence assessment of HEV-IgM/IgG among blood donors. METHODOLOGY: A total of 1049 blood donor samples were tested for HEV IgG and IgM using different enzyme immunoassays (Wantai, Eruoimmune, MP diagnostics, Mikrogen immunoblot, HEV-IgM rapid test). The performance of each assay was evaluated according to our established silver standard value based on three or more IgG concordant assay results. RESULTS: HEV seroprevalence varied considerably using these assays, ranging from 10.1 % (Euroimmune-ELISA) to 18.0 % (Wanti-ELISA) for HEV-IgG, and from 0.2 % (Wanti-ELISA) to 2.6 % (MP Rapid test) for HEV-IgM. A total of 155 of 216 (71.6%) samples tested positive for HEV-IgG by three or more concordant assays. On the other hand, IgM assays showed poor agreement as only 7.6 % (4/52) of the specimens were positive according to three or more concordant assay test results. All HEV-IgG assays revealed high sensitivity and specificity (ranging 96.5-100 %),and excellent Kappa concordance (0.88-0.95), except for Euroimmun ELISA (sensitivity=61.5 %, kappa=0.63). MP ELISA showed the highest levels of sensitivity (100 %) and specificity (98.5 %). CONCLUSIONS: Due to discrepancies in the performance of various IgG and IgM assays, seroprevalence studies should be based on furher confirmatory testing for decisive conclusions to be reached.

Milk Composition for Admixed Dairy Cattle in Tanzania.

It is well established that milk composition is affected by the breed and genotype of a cow. The present study investigated the relationship between the proportion of exotic genes and milk composition in Tanzanian crossbred dairy cows. Milk samples were collected from 209 animals kept under smallholder production systems in Rungwe and Lushoto districts of Tanzania. The milk samples were analyzed for the content of components including fat, protein, casein, lactose, solids-not-fat (SNF), and the total solids (TS) through infrared spectroscopy using Milko-Scan FT1 analyzer (Foss Electric, Denmark). Hair samples for DNA analysis were collected from individual cows and breed composition determined using 150,000 single nucleotide polymorphism (SNP) markers. Cows were grouped into four genetic classes based on the proportion of exotic genes present: 25-49, 50-74, 75-84, and >84%, to mimic a backcross to indigenous zebu breed, F1, F2, and F3 crosses, respectively. The breed types were defined based on international commercial dairy breeds as follows: RG (Norwegian Red X Friesian, Norwegian Red X Guernsey, and Norwegian Red X Jersey crosses); RH (Norwegian Red X Holstein crosses); RZ (Norwegian Red X Zebu and Norwegian Red X N'Dama crosses); and ZR (Zebu X GIR, Zebu X Norwegian Red, and Zebu X Holstein crosses). Results obtained indicate low variation in milk composition traits between genetic groups and breed types. For all the milk traits except milk total protein and casein content, no significant differences (p < 0.05) were observed among genetic groups. Protein content was significantly (p < 0.05) higher for genetic group 75-84% at 3.4 ± 0.08% compared to 3.18 ± 0.07% for genetic group >84%. Casein content was significantly lower for genetic group >84% (2.98 ± 0.05%) compared to 3.18 ± 0.09 and 3.16 ± 0.06% for genetic group 25-49 and 75-84%, respectively (p < 0.05). There was no significant difference (p < 0.05) between breed types with respect to milk composition traits. These results suggest that selection of breed types to be used in smallholder systems need not pay much emphasis on milk quality differences as most admixed animals would have similar milk composition profiles. However, a larger sample size would be required to quantify any meaningful differences between groups.

Porcine Rotaviruses: Epidemiology, Immune Responses and Control Strategies.

Rotaviruses (RVs) are a major cause of acute viral gastroenteritis in young animals and children worldwide. Immunocompetent adults of different species become resistant to clinical disease due to post-infection immunity, immune system maturation and gut physiological changes. Of the 9 RV genogroups (A-I), RV A, B, and C (RVA, RVB, and RVC, respectively) are associated with diarrhea in piglets. Although discovered decades ago, porcine genogroup E RVs (RVE) are uncommon and their pathogenesis is not studied well. The presence of porcine RV H (RVH), a newly defined distinct genogroup, was recently confirmed in diarrheic pigs in Japan, Brazil, and the US. The complex epidemiology, pathogenicity and high genetic diversity of porcine RVAs are widely recognized and well-studied. More recent data show a significant genetic diversity based on the VP7 gene analysis of RVB and C strains in pigs. In this review, we will summarize previous and recent research to provide insights on historic and current prevalence and genetic diversity of porcine RVs in different geographic regions and production systems. We will also provide a brief overview of immune responses to porcine RVs, available control strategies and zoonotic potential of different RV genotypes. An improved understanding of the above parameters may lead to the development of more optimal strategies to manage RV diarrheal disease in swine and humans.

Metagenomic analysis demonstrates the diversity of the fecal virome in asymptomatic pigs in East Africa.

Pigs harbor a variety of viruses that are closely related to human viruses and are suspected to have zoonotic potential. Little is known about the presence of viruses in smallholder farms where pigs are in close contact with humans and wildlife. This study provides insight into viral communities and the prevalence and characteristics of enteric viral co-infections in smallholder pigs in East Africa. Sequence-independent amplification and high-throughput sequencing were applied to the metagenomics analysis of viruses in feces collected from asymptomatic pigs. A total of 47,213 de novo-assembled contigs were constructed and compared with sequences from the GenBank database. Blastx search results revealed that 1039 contigs (>200 nt) were related to viral sequences in the GenBank database. Of the 1039 contigs, 612 were not assigned to any viral taxa because they had little similarity to known viral genomic or protein sequences, while 427 contigs had a high level of sequence similarity to known viruses and were assigned to viral taxa. The most frequent contigs related to mammalian viruses resembling members of the viral genera Astrovirus, Rotavirus, Bocavirus, Circovirus, and Kobuvirus. Other less abundant contigs were related to members of the genera Sapelovirus, Pasivirus, Posavirus, Teschovirus and Picobirnavirus. This is the first report on the diversity of the fecal virome of pig populations in East Africa. The findings of the present study help to elucidate the etiology of diarrheal diseases in pigs and identify potential zoonotic and emerging viruses in the region. Further investigations are required to compare the incidence of these viruses in healthy and diseased pigs in order to better elucidate their pathogenic role.

Molecular detection and genetic characterization of kobuviruses and astroviruses in asymptomatic local pigs in East Africa.

In this study, swine fecal specimens (n = 251) collected from nursing and weaned piglets raised under smallholder production systems were screened for the presence of kobuviruses by RT-PCR. Porcine kobuviruses were detected in 13.1 % (33/251) of the samples. We demonstrated that porcine kobuvirus infections exist in indigenous pigs in Kenya and Uganda and that the prevalence was higher in young piglets than older pigs: nursing piglets (15 %), post-weaning (3-month-old) pigs (17 %), 4-month-old pigs (10 %). Genetic analysis of the partial RNA-dependent RNA polymerase (RdRp) region (690 nt) revealed that kobuviruses circulating in East Africa are diverse, sharing nucleotide sequence identities ranging from 89.7 to 99.1 % and 88 to 92.3 % among them and with known porcine kobuviruses, respectively. The nucleotide sequence identities between our kobuvirus strains and those of human, bovine and canine kobuviruses were 69.4-70.7 %, 73.1-74.4 % and 67-70.7 %, respectively. Additionally, upon sequencing selected samples that showed consistent 720-bp RT-PCR bands while using the same primer set, we detected porcine astroviruses in our samples belonging to type 2 and type 3 mamastroviruses. To our knowledge, this study reports the first detection and molecular analysis of both porcine kobuviruses and astroviruses in an African region. Further studies are required to determine the role of these viruses in gastrointestinal infections of pigs in this region and to determine the genetic diversity of the circulating strains to develop accurate diagnostic tools and implement appropriate control strategies.