The Significance of Wild Plants in the Evolutionary Ecology of Three Major Viruses Infecting Cultivated Sweetpotato in Uganda

The studies presented in this thesis contribute to the understanding of evolutionary ecology of three major viruses threatening cultivated sweetpotato (Ipomoea batatas Lam) in East Africa: Sweet potato feathery mottle virus (SPFMV; genus Potyvirus; Potyviridae), Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus; Closteroviridae) and Sweet potato mild mottle virus (SPMMV; genus Ipomovirus; Potyviridae). The viruses were serologically detected and the positive results confirmed by RT-PCR and sequencing. SPFMV was detected in 24 wild plant species of family Convolvulacea (genera Ipomoea, Lepistemon and Hewittia), of which 19 species were new natural hosts for SPFMV. SPMMV and SPCSV were detected in wild plants belonging to 21 and 12 species (genera Ipomoea, Lepistemon and Hewittia), respectively, all of which were previously unknown to be natural hosts of these viruses. SPFMV was the most abundant virus being detected in 17% of the plants, while SPMMV and SPCSV were detected in 9.8% and 5.4% of the assessed plants, respectively. Wild plants in Uganda were infected with the East African (EA), common (C), and the ordinary (O) strains, or co-infected with the EA and the C strain of SPFMV. The viruses and virus-like diseases were more frequent in the eastern agro-ecological zone than the western and central zones, which contrasted with known incidences of these viruses in sweetpotato crops, except for northern zone where incidences were lowest in wild plants as in sweetpotato. The NIb/CP junction in SPMMV was determined experimentally which facilitated CP-based phylogenetic and evolutionary analyses of SPMMV. Isolates of all the three viruses from wild plants were genetically similar to those found in cultivated sweetpotatoes in East Africa. There was no evidence of host-driven population genetic structures suggesting frequent transmission of these viruses between their wild and cultivated hosts. The p22 RNA silencing suppressor-encoding sequence was absent in a few SPCSV isolates, but regardless of this, SPCSV isolates incited sweet potato virus disease (SPVD) in sweetpotato plants co-infected with SPFMV, indicating that p22 is redundant for synergism between SCSV and SPFMV. Molecular evolutionary analysis revealed that isolates of strain EA of SPFMV that is largely restricted geographically in East Africa experience frequent recombination in comparison to isolates of strain C that is globally distributed. Moreover, non-homologous recombination events between strains EA and C were rare, despite frequent co-infections of these strains in wild plants, suggesting purifying selection against non-homologous recombinants between these strains or that such recombinants are mostly not infectious. Recombination was detected also in the 5 - and 3 -proximal regions of the SPMMV genome providing the first evidence of recombination in genus Ipomovirus, but no recombination events were detected in the characterized genomic regions of SPCSV. Strong purifying selection was implicated on evolution of majority of amino acids of the proteins encoded by the analyzed genomic regions of SPFMV, SPMMV and SPCSV. However, positive selection was predicted on 17 amino acids distributed over the whole the coat protein (CP) in the globally distributed strain C, as compared to only 4 amino acids in the multifunctional CP N-terminus (CP-NT) of strain EA largely restricted geographically to East Africa. A few amino acid sites in the N-terminus of SPMMV P1, the p7 protein and RNA silencing suppressor proteins p22 and RNase3 of SPCSV were also submitted to positive selection. Positively selected amino acids may constitute ligand-binding domains that determine interactions with plant host and/or insect vector factors. The P1 proteinase of SPMMV (genus Ipomovirus) seems to respond to needs of adaptation, which was not observed with the helper component proteinase (HC-Pro) of SPMMV, although the HC-Pro is responsible for many important molecular interactions in genus Potyvirus. Because the centre of origin of cultivated sweetpotato is in the Americas from where the crop was dispersed to other continents in recent history (except for the Australasia and South Pacific region), it would be expected that identical viruses and their strains occur worldwide, presuming virus dispersal with the host. Apparently, this seems not to be the case with SPMMV, the strain EA of SPFMV and the strain EA of SPCSV that are largely geographically confined in East Africa where they are predominant and occur both in natural and agro-ecosystems. The geographical distribution of plant viruses is constrained more by virus-vector relations than by virus-host interactions, which in accordance of the wide range of natural host species and the geographical confinement to East Africa suggest that these viruses existed in East African wild plants before the introduction of sweetpotato. Subsequently, these studies provide compelling evidence that East Africa constitutes a cradle of SPFMV strain EA, SPCSV strain EA, and SPMMV. Therefore, sweet potato virus disease (SPVD) in East Africa may be one of the examples of damaging virus diseases resulting from exchange of viruses between introduced crops and indigenous wild plant species. Keywords: Convolvulaceae, East Africa, epidemiology, evolution, genetic variability, Ipomoea, recombination, SPCSV, SPFMV, SPMMV, selection pressure, sweetpotato, wild plant species Author s Address: Arthur K. Tugume, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Latokartanonkaari 7, P.O Box 27, FIN-00014, Helsinki, Finland. Email: tugume.arthur@helsinki.fi Author s Present Address: Arthur K. Tugume, Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Kampala, Uganda. Email: aktugume@botany.mak.ac.ug, tugumeka@yahoo.com

Rearing strategies of young dairy calves in relation to production, behaviour and welfare

Vasikoiden kasvatus yksilökarsinoissa, imemismahdollisuuden puute maitojuoton yhteydessä sekä pienet juomamäärät ovat tekijöitä, jotka mahdollisesti voivat vähentää vasikoiden hyvinvointia. Vasikoiden kasvatukseen etsitäänkin uusia tapoja, joissa eläinten käyttäytymistarpeet ja hyvinvointi otetaan entistä paremmin huomioon. Tässä väitöskirjassa vasikoiden kasvatusta on tarkasteltu sekä tuotannon että vasikoiden käyttäytymisen ja hyvinvoinnin kannalta. Väitöskirja koostuu kolmesta kokeesta, joista ensimmäisessä tutkittiin vasikoiden kasvatusta ryhmäkarsinoissa ulkona tai sisällä ja vasikoiden kasvatusta sisällä ryhmä- tai yksilökarsinoissa. Toisessa kokeessa vasikoiden annettiin imeä emiään rajoitetusti lypsyn jälkeen viiden tai kahdeksan viikon ajan ja kolmannessa selvitettiin vasikoiden veden juontia, kun vasikat saivat juomarehua vapaasti. Lisäksi kokeiden yhdistetystä aineistosta analysoitiin eri rehujen syöntimäärien suhdetta sekä rehujen vaikutusta kasvuun ennen ja jälkeen maidosta vieroituksen. Tutkimuksessa todettiin, että vasikoita voi ryhmässä kasvattaa kylmissä ja vaihtelevissa sääoloissa ulkona, kunhan ne hoidetaan ja ruokitaan erittäin huolellisesti. Kylmällä ilmalla vasikat saattavat kuitenkin syödä väkirehua vähemmän varsinkin, jos ruokailupaikka on ulkona ja makuualue sisällä. Ryhmässä kasvaneet vasikat aloittivat sekä kuivien rehujen syönnin että märehtimisen nuorempina kuin yksilökarsinassa kasvaneet. Ryhmissä esiintyvää käyttäytymisongelmaa, toisten vasikoiden imemistä, voidaan vähentää hoito- ja ruokintamenetelmillä. Annettaessa vasikoiden imeä emiään rajoitetusti lypsyn jälkeen vasikat oppivat imemään emiään hyvin nopeasti. Lypsytyö vaikeutui muutamien lehmien kohdalla, sillä ne pidättivät maitoa lypsettäessä. Saadessaan imeä rajoitetusti vasikat imivät suurehkoja maitomääriä kerrallaan. Vieroittaminen suurilta maitomääriltä viiden viikon iässä oli kuitenkin liian aikaista, koska vasikat eivät vielä syöneet riittävästi kuivia rehuja. Vieroitus emästä niin viiden kuin kahdeksankin viikon iässä aiheutti vasikoissa levottomuutta ja ääntelyn lisääntymistä. Saadessaan hapatettua juomarehua vapaasti vasikat joivat keskimäärin vain vähän vettä, olipa vesilähteenä avoin ämpäri tai vesinippa. Vasikoiden välillä oli suurta vaihtelua veden juontimäärissä. Viikkoa ennen maidosta vieroitusta vasikat joivat 0-3 l vettä päivässä. Vasikat joivat nipasta kerrallaan vähemmän vettä kuin ämpäristä, ja käyttivät enemmän aikaa päivässä veden juomiseen kuin vesiämpäristä juoneet vasikat. Suurin osa vasikoista joi vettä juomanipoista erikoisella tavalla esimerkiksi painamalla nippaa otsalla ja juomalla tippuvaa vettä. Vesinipat voivat olla siis vasikoille joko vaikeita tai epämukavia käyttää. Vasikoiden juoman maitomäärän lisääntyessä kasvu lisääntyy selvästi. Runsas maidon juominen vähentää kuitenkin kuivien rehujen syöntiä ja vieroitusvaiheessa kasvu voi hidastua. Vasikat olisikin tärkeää vieroittaa vähitellen, ettei muutoksia kasvuun tulisi. Syönti- ja kasvutulokset eivät aina anna oikeaa kuvaa kasvatusmenetelmien eroista eläinten hyvinvoinnin kannalta. Käyttäytyminen on herkkä hyvinvoinnin mittari ja se tulisikin aina huomioida eri kasvatusmenetelmiä arvioitaessa. .

Microclimate and gas emissions in dairy buildings : Instrumentation, theory and measurements

The aim of this thesis was to develop measurement techniques and systems for measuring air quality and to provide information about air quality conditions and the amount of gaseous emissions from semi-insulated and uninsulated dairy buildings in Finland and Estonia. Specialization and intensification in livestock farming, such as in dairy production, is usually accompanied by an increase in concentrated environmental emissions. In addition to high moisture, the presence of dust and corrosive gases, and widely varying gas concentrations in dairy buildings, Finland and Estonia experience winter temperatures reaching below -40 ºC and summer temperatures above +30 ºC. The adaptation of new technologies for long-term air quality monitoring and measurement remains relatively uncommon in dairy buildings because the construction and maintenance of accurate monitoring systems for long-term use are too expensive for the average dairy farmer to afford. Though the documentation of accurate air quality measurement systems intended mainly for research purposes have been made in the past, standardised methods and the documentation of affordable systems and simple methods for performing air quality and emissions measurements in dairy buildings are unavailable. In this study, we built three measurement systems: 1) a Stationary system with integrated affordable sensors for on-site measurements, 2) a Wireless system with affordable sensors for off-site measurements, and 3) a Mobile system consisting of expensive and accurate sensors for measuring air quality. In addition to assessing existing methods, we developed simplified methods for measuring ventilation and emission rates in dairy buildings. The three measurement systems were successfully used to measure air quality in uninsulated, semi-insulated, and fully-insulated dairy buildings between the years 2005 and 2007. When carefully calibrated, the affordable sensors in the systems gave reasonably accurate readings. The spatial air quality survey showed high variation in microclimate conditions in the dairy buildings measured. The average indoor air concentration for carbon dioxide was 950 ppm, for ammonia 5 ppm, for methane 48 ppm, for relative humidity 70%, and for inside air velocity 0.2 m/s. The average winter and summer indoor temperatures during the measurement period were -7º C and +24 ºC for the uninsulated, +3 ºC and +20 ºC for the semi-insulated and +10 ºC and +25 ºC for the fully-insulated dairy buildings. The measurement results showed that the uninsulated dairy buildings had lower indoor gas concentrations and emissions compared to fully insulated buildings. Although occasionally exceeded, the ventilation rates and average indoor air quality in the dairy buildings were largely within recommended limits. We assessed the traditional heat balance, moisture balance, carbon dioxide balance and direct airflow methods for estimating ventilation rates. The direct velocity measurement for the estimation of ventilation rate proved to be impractical for naturally ventilated buildings. Two methods were developed for estimating ventilation rates. The first method is applicable in buildings in which the ventilation can be stopped or completely closed. The second method is useful in naturally ventilated buildings with large openings and high ventilation rates where spatial gas concentrations are heterogeneously distributed. The two traditional methods (carbon dioxide and methane balances), and two newly developed methods (theoretical modelling using Fick s law and boundary layer theory, and the recirculation flux-chamber technique) were used to estimate ammonia emissions from the dairy buildings. Using the traditional carbon dioxide balance method, ammonia emissions per cow from the dairy buildings ranged from 7 g day-1 to 35 g day-1, and methane emissions per cow ranged from 96 g day-1 to 348 g day-1. The developed methods proved to be as equally accurate as the traditional methods. Variation between the mean emissions estimated with the traditional and the developed methods was less than 20%. The developed modelling procedure provided sound framework for examining the impact of production systems on ammonia emissions in dairy buildings.

Quantitative trait loci detection and benefits from marker-assisted selection in dairy cattle

Knowing the chromosomal areas or actual genes affecting the traits under selection would add more information to be used in the selection decisions which would potentially lead to higher genetic response. The first objective of this study was to map quantitative trait loci (QTL) affecting economically important traits in the Finnish Ayrshire population. The second objective was to investigate the effects of using QTL information in marker-assisted selection (MAS) on the genetic response and the linkage disequilibrium between the different parts of the genome. Whole genome scans were carried out on a grand-daughter design with 12 half-sib families and a total of 493 sons. Twelve different traits were studied: milk yield, protein yield, protein content, fat yield, fat content, somatic cell score (SCS), mastitis treatments, other veterinary treatments, days open, fertility treatments, non-return rate, and calf mortality. The average spacing of the typed markers was 20 cM with 2 to 14 markers per chromosome. Associations between markers and traits were analyzed with multiple marker regression. Significance was determined by permutation and genome-wise P-values obtained by Bonferroni correction. The benefits from MAS were investigated by simulation: a conventional progeny testing scheme was compared to a scheme where QTL information was used within families to select among full-sibs in the male path. Two QTL on different chromosomes were modelled. The effects of different starting frequencies of the favourable alleles and different size of the QTL effects were evaluated. A large number of QTL, 48 in total, were detected at 5% or higher chromosome-wise significance. QTL for milk production were found on 8 chromosomes, for SCS on 6, for mastitis treatments on 1, for other veterinary treatments on 5, for days open on 7, for fertility treatments on 7, for calf mortality on 6, and for non-return rate on 2 chromosomes. In the simulation study the total genetic response was faster with MAS than with conventional selection and the advantage of MAS persisted over the studied generations. The rate of response and the difference between the selection schemes reflected clearly the changes in allele frequencies of the favourable QTL. The disequilibrium between the polygenes and QTL was always negative and it was larger with larger QTL size. The disequilibrium between the two QTL was larger with QTL of large effect and it was somewhat larger with MAS for scenarios with starting frequencies below 0.5 for QTL of moderate size and below 0.3 for large QTL. In conclusion, several QTL affecting economically important traits of dairy cattle were detected. Further studies are needed to verify these QTL, check their presence in the present breeding population, look for pleiotropy and fine map the most interesting QTL regions. The results of the simulation studies show that using MAS together with embryo transfer to pre-select young bulls within families is a useful approach to increase the genetic merit of the AI-bulls compared to conventional selection.

Short-term plant-decomposer feedbacks in grassland plants

Plant species differ in their effects on ecosystem productivity and it is recognised that these effects are partly due to plant species-specific influences on soil processes. Until recently, however, not much attention was given to the potential role played by soil biota in these species-specific effects. While soil decomposers are responsible for governing the availability of nutrients for plant production, they simultaneously depend on the amount of carbon provided by plants. Litter and rhizodeposition constitute the two basal resources that plants provide to soil decomposer food webs. While it has been shown that both of these can have effects on soil decomposer communities that differ among plant species, the putative significance of these effects for plant nitrogen (N) acquisition is currently understudied. My PhD work aimed at clarifying whether the species-specific influences of three temperate grassland plants on the soil microfood-web, through rhizodeposition and litter, can feed back to plant N uptake. The methods and approach used (15N labelling of plant litter in microcosm experiments) revealed to be an effective combination of tools in studying these feedbacks. Plant effects on soil organisms were shown to differ significantly between plant species and the effects could be followed across several trophic levels. The labelling of litter further permitted the evaluation of plant acquisition of N derived from soil organic matter. The results show that the structure of the soil microfood-web can have a significant role in plant N acquisition when the structure is experimentally manipulated, such as when comparing systems consisting of microbes to those consisting of microbes and their grazers. However, despite this, the results indicate that differences in N uptake from soil organic matter between different plant species are not related to the effects these species exert on the structure of the soil microfood-web. Rather, these differences in N uptake seem to be determined by other species-specific traits of live plants and their litter. My results thus indicate that different resources provided by different plant species may not induce species-specific decomposer feedbacks on plant N uptake from soil organic matter. This further suggests that the species-specific plant effects on soil decomposer communities may not, at least in the short term, have significant consequences on plant production.

Environmental effects of thermal and radioactive discharges from nuclear power plants in the boreal brackish-water conditions of the northern Baltic Sea

During recent decades, thermal and radioactive discharges from nuclear power plants into the aquatic environment have become the subject of lively debate as an ecological concern. The target of this thesis was to summarize the large quantity of results obtained in extensive monitoring programmes and studies carried out in recipient sea areas off the Finnish nuclear power plants at Loviisa and Olkiluoto during more than four decades. The Loviisa NPP is located on the coast of the Gulf of Finland and Olkiluoto NPP on that of the Bothnian Sea. The state of the Gulf of Finland is clearly more eutrophic; the nutrient concentrations in the surface water are about 1½ 2 times higher at Loviisa than at Olkiluoto, and the total phosphorus concentrations still increased in both areas (even doubled at Loviisa) between the early 1970s and 2000. Thus, it is a challenge to distinguish the local effects of thermal discharges from the general eutrophication process of the Gulf of Finland. The salinity is generally low in the brackish-water conditions of the northern Baltic Sea, being however about 1 higher at Olkiluoto than at Loviisa (the salinity of surface water varying at the latter from near to 0 in early spring to 4 6 in late autumn). Thus, many marine and fresh-water organisms live in the Loviisa area close to their limit of existence, which makes the biota sensitive to any additional stress. The characteristics of the discharge areas of the two sites differ from each other in many respects: the discharge area at Loviisa is a semi-enclosed bay in the inner archipelago, where the exchange of water is limited, while the discharge area at Olkiluoto is more open, and the exchange of water with the open Bothnian Sea is more effective. The effects of the cooling water discharged from the power plants on the temperatures in the sea were most obvious in winter. The formation of a permanent ice cover in the discharge areas has been delayed in early winter, and the break-up of the ice occurs earlier in spring. The prolonging of the growing season and the disturbance of the overwintering time, in conditions where the biota has adjusted to a distinct rest period in winter, have been the most significant biological effects of the thermal pollution. The soft-bottom macrofauna at Loviisa has deteriorated to the point of almost total extinction at many sampling stations during the past 40 years. A similar decline has been reported for the whole eastern Gulf of Finland. However, the local eutrophication process seems to have contributed into the decline of the zoobenthos in the discharge area at Loviisa. Thermal discharges have increased the production of organic matter, which again has led to more organic bottom deposits. These have in turn increased the tendency of the isolated deeps to a depletion of oxygen, and this has further caused strong remobilization of phosphorus from the bottom sediments. Phytoplankton primary production and primary production capacity doubled in the whole area between the late 1960s and the late 1990s, but started to decrease a little at the beginning of this century. The focus of the production shifted from spring to mid- and late summer. The general rise in the level of primary production was mainly due to the increase in nutrient concentrations over the whole Gulf of Finland, but the thermal discharge contributed to a stronger increase of production in the discharge area compared to that in the intake area. The eutrophication of littoral vegetation in the discharge area has been the most obvious, unambiguous and significant biological effect of the heated water. Myriophyllum spicatum, Potamogeton perfoliatus and Potamogeton pectinatus, and vigorous growths of numerous filamentous algae as their epiphytes have strongly increased in the vicinity of the cooling water outlet, where they have formed dense populations in the littoral zone in late summer. However, the strongest increase of phytobenthos has extended only to a distance of about 1 km from the outlet, i.e., the changes in vegetation have been largest in those areas that remain ice-free in winter. Similar trends were also discernible at Olkiluoto, but to a clearly smaller extent, which was due to the definitely weaker level of background eutrophy and nutrient concentrations in the Bothnian Sea, and the differing local hydrographical and biological factors prevailing in the Olkiluoto area. The level of primary production has also increased at Olkiluoto, but has remained at a clearly lower level than at Loviisa. In spite of the analogous changes observed in the macrozoobenthos, the benthic fauna has remained strong and diversified in the Olkiluoto area. Small amounts of local discharge nuclides were regularly detected in environmental samples taken from the discharge areas: tritium in seawater samples, and activation products, such as 60Co, 58Co, 54Mn, 110mAg, 51Cr, in suspended particulate matter, bottom sediments and in several indicator organisms (e.g., periphyton and Fucus vesiculosus) that effectively accumulate radioactive substances from the medium. The tritium discharges and the consequent detection frequency and concentrations of tritium in seawater were higher at Loviisa, but the concentrations of the activation products were higher at Olkiluoto, where traces of local discharge nuclides were also observed over a clearly wider area, due to the better exchange of water than at Loviisa, where local discharge nuclides were only detected outside Hästholmsfjärden Bay quite rarely and in smaller amounts. At the farthest, an insignificant trace amount (0.2 Bq kg-1 d.w.) of 60Co originating from Olkiluoto was detected in Fucus at a distance of 137 km from the power plant. Discharge nuclides from the local nuclear power plants were almost exclusively detected at the lower trophic levels of the ecosystems. Traces of local discharge nuclides were very seldom detected in fish, and even then only in very low quantities. As a consequence of the reduced discharges, the concentrations of local discharge nuclides in the environment have decreased noticeably in recent years at both Loviisa and Olkiluoto. Although the concentrations in environmental samples, and above all, the discharge data, are presented as seemingly large numbers, the radiation doses caused by them to the population and to the biota are very low, practically insignificant. The effects of the thermal discharges have been more significant, at least to the wildlife in the discharge areas of the cooling water, although the area of impact has been relatively small. The results show that the nutrient level and the exchange of water in the discharge area of a nuclear power plant are of crucial importance.

Production of F4 Fimbrial Adhesin in Plants: a Model for Oral Porcine Vaccine against Enterotoxigenic Escherichia coli

F4 fimbriae of enterotoxigenic Escherichia coli (ETEC) are highly stable multimeric structures with a capacity to evoke mucosal immune responses. With these characters F4 offer a unique model system to study oral vaccination against ETEC-induced porcine postweaning diarrhea. Postweaning diarrhea is a major problem in piggeries worldwide and results in significant economic losses. No vaccine is currently available to protect weaned piglets against ETEC infections. Transgenic plants provide an economically feasible platform for large-scale production of vaccine antigens for animal health. In this study, the capacity of transgenic plants to produce FaeG protein, the major structural subunit and adhesin of F4 fimbria, was evaluated. Using the model plant tobacco, the optimal subcellular location for FaeG accumulation was examined. Targeting of FaeG into chloroplasts offered a superior accumulation level of 1% of total soluble proteins (TSP) over the other investigated subcellular locations, namely, the endoplasmic reticulum and the apoplast. Moreover, we determined whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, i.e. stability in gastrointestinal conditions, binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. The chloroplast-derived FaeG protein did show resistance against low pH and proteolysis in the simulated gastrointestinal conditions and was able to bind to the F4R, subsequently inhibiting the F4+ ETEC binding in a dose-dependent manner. To investigate the oral immunogenicity of FaeG protein, the edible crop plant alfalfa was transformed with the chloroplast-targeting construct and equally to tobacco plants, a high-yield FaeG accumulation of 1% of TSP was obtained. A similar yield was also obtained in the seeds of barley, a valuable crop plant, when the FaeG-encoding gene was expressed under an endosperm-specific promoter and subcellularly targeted into the endoplasmic reticulum. Furthermore, desiccated alfalfa plants and barley grains were shown to have a capacity to store FaeG protein in a stable form for years. When the transgenic alfalfa plants were administred orally to weaned piglets, slight F4-specific systemic and mucosal immune responses were induced. Co-administration of the transgenic alfalfa and the mucosal adjuvant cholera toxin enhanced the F4-specific immune response; the duration and number of F4+ E. coli excretion following F4+ ETEC challenge were significantly reduced as compared with pigs that had received nontransgenic plant material. In conclusion, the results suggest that transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against porcine F4+ ETEC infections. The findings here thus present new approaches to develop the vaccination strategy against porcine postweaning diarrhea.