Article Feeding Spray-Dried Porcine Plasma to Pigs Reduces African Swine Fever Virus Load in Infected Pigs and Delays Virus Transmission—Study 1 Elena Blázquez 1,2 , Joan Pujols 1,3,4 , Fernando Rodríguez 1,3,4 , Joaquim Segalés 3,4,5 , Rosa Rosell 3,6 , Joy Campbell 7 and Javier Polo 2,7, * 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA), 08193 Barcelona, Spain; elena.blazquez@irta.cat (E.B.); joan.pujols@irta.cat (J.P.); fernando.rodriguez@irta.cat (F.R.) 2 APC Europe, S.L. 08403 Granollers, Spain 3 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Barcelona, Spain; joaquim.segales@irta.cat (J.S.); rosa.rosell@irta.cat (R.R.) 4 WOAH Collaborating Centre for Emerging and Re-Emerging Pig Diseases in Europe, IRTA-CReSA, 08193 Barcelona, Spain 5 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Barcelona, Spain 6 Departament d’Acció Climàtica, Alimentació i Agenda Rural, Generalitat de Catalunya, 08193 Barcelona, Spain 7 APC LLC, Ankeny, IA 50021, USA; joy.campbell@apcproteins.com * Correspondence: javier.polo@apc-europe.com Abstract: The objective of this study was to evaluate the potential benefits of feeding spray-dried porcine plasma (SDPP) to pigs infected with African swine fever virus (ASFV). Two groups of twelve weaned pigs each were fed with CONVENTIONAL or 8% SDPP enriched diets. Two pigs (trojans)/group) were injected intramuscularly with the pandemic ASFV (Georgia 2007/01) and comingled with the rest of the pigs (1:5 trojan:naïve ratio) to simulate a natural route of transmission. Citation: Blázquez, E.; Pujols, J.; Trojans developed ASF and died within the first week after inoculation, but contact pigs did not Rodríguez, F.; Segalés, J.; Rosell, R.; develop ASF, viremia, or seroconversion. Therefore, three more trojans per group were introduced Campbell, J.; Polo, J. Feeding to optimize the ASFV transmission (1:2 trojan:naïve ratio). Blood, nasal, and rectal swabs were Spray-Dried Porcine Plasma to Pigs weekly harvested, and at end of the study ASFV-target organs collected. After the second exposure, Reduces African Swine Fever Virus rectal temperature of conventionally fed contact pigs increased >40.5 ◦ C while fever was delayed in Load in Infected Pigs and Delays the SDPP contact pigs. Additionally, PCR Ct values in blood, secretions, and tissue samples were Virus Transmission—Study 1. significantly lower (p < 0.05) for CONVENTIONAL compared to SDPP contact pigs. Under these Vaccines 2023, 11, 824. https:// study conditions, contact exposed pigs fed SDPP had delayed ASFV transmission and reduced virus doi.org/10.3390/vaccines11040824 load, likely by enhanced specific T-cell priming after the first ASFV-exposure. Academic Editor: François Meurens Keywords: African swine fever; ASFV; spray-dried porcine plasma; challenge; nutritional intervention Received: 25 January 2023 Revised: 3 April 2023 Accepted: 6 April 2023 Published: 10 April 2023 1. Introduction African swine fever virus (ASFV) is an enveloped DNA virus of the Asfarviridae family that infects domestic pigs and wild boar of all ages causing African swine fever (ASF), Copyright: © 2023 by the authors. a disease that must be reported to the World Organization for Animal Health (WOAH, Licensee MDPI, Basel, Switzerland. formerly OIE). This article is an open access article In January 2014, Lithuania made the first notification of acute ASF cases in wild boar distributed under the terms and in the European Union (EU), and Poland followed in February 2014. In June and September conditions of the Creative Commons 2014, Latvia and Estonia also reported ASFV. To date, Ukraine, Romania, Estonia, Hungary, Attribution (CC BY) license (https:// Bulgaria, Czech Republic, Slovakia, Belgium, Greece, Germany, and, recently, Italy, have creativecommons.org/licenses/by/ reported cases of ASF in wild boar and some countries in domestic pigs. All epidemiological 4.0/). Vaccines 2023, 11, 824. https://doi.org/10.3390/vaccines11040824 https://www.mdpi.com/journal/vaccines Vaccines 2023, 11, 824 2 of 15 data indicate that the EU has undergone repeated introduction of ASFV from the Eastern neighboring infected countries. The EU Reference Laboratory confirmed, through genetic studies that there was a 100% homology with the genotype II virus that entered Georgia in 2007, imported from Western Africa [1]. Furthermore, from August 2018, ASFV has spread in most pig producing countries in Asia, including China, Vietnam, South and North Korea, Philippines, Laos, Myanmar, Mongolia, Cambodia, Timor-Leste, Indonesia, Malaysia, India, Papua New Guinea, and Thailand. In addition, genotype II ASFV has recently spread to the Caribbean, and new cases in Haiti and Dominican Republic have been reported in wild and domestic pigs [2]. However, the pattern spread and severity of ASF outbreak in domestic pigs in different regions varies significantly, ranging from up to 70% reduction of total swine herd in China, Vietnam, Philippines, and other Asia countries to limited spot outbreaks in EU countries and South Korea. Biosecurity could be the main factor influencing the spread and outcome of the disease, as countries with less severe outbreaks had better biosecurity programs. The health and immune status of the herd may also perform a key role. Pietschmann et al. [3] reported that immune compromised runt and gastrointestinal challenged unhealthy pigs are more susceptible for ASF infection. Radulovic et al. [4] reported that domestic pigs with a very high hygienic and health status experienced a milder and shorter form of the illness followed by complete recovery when inoculated with an attenuated field ASFV strain. Therefore, improving health and hygienic status of the herd may be a key for ASF prevention and control. Spray-dried plasma (SDP) is a dry functional feed ingredient that contains a diverse mixture of many functional components, such as immunoglobulins, albumin, growth fac- tors, biologically active peptides, transferrin, and other molecules, that have biological activity independent of their nutritional value [5–11]. SDP either from porcine (SDPP) or bovine (SDBP) origin are extensively used in pig starter diets and consistently provide improvements in growth performance, feed efficiency, and animal survival, especially under stressful conditions, such as a pathogen challenge [6,12]. The beneficial effects of SDP are related to its mode of action that supports an efficient immune system re- sponse [8,9,13]. In addition, feeding SDPP has been shown to promote a more favorable gut microflora [14–16]. Thus, SDPP helps improve the integrity of the intestinal barrier by modulating the functional and structural properties of the intestinal mucosa in pigs and other animal models [17]. In addition, dietary SDPP can favorably affect intestinal morphol- ogy and immune cell subsets of gut tissues and blood in weaned pigs [18]. Furthermore, SDPP exerts a systemic effect, as demonstrated by the reduction of lung inflammation and the presence of monocytes and cells involved in the inflammatory events under an acute pulmonary inflammation model with LPS in mice [19,20]. Moreover, in an epidemiologic study conducted in Manitoba, Canadian researchers found that farms with less pulmonary lesions associated with the presence of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV-2), and Mycoplasma hyopneumoniae were well correlated with supplementation of SDPP in their feed [21]. Good intestinal health translates into a more efficient immune system and overall better growth performance [22,23]. Following this principle, an improved intestinal barrier function through gut microbiota has been demonstrated to influence ASFV susceptibil- ity. Fecal transplantation from warthogs (African pigs resilient to ASF) to domestic pigs improved mucosal immunity and protected pigs against experimental challenge with a live attenuated ASFV strain [24]. Although mechanisms involved in ASFV protection are not fully understood, ASFV specific antibodies [25] and cytotoxic CD8 T-cell (CTL) responses [26], together with an appropriate innate immune response [27], have been demonstrated to perform an important role. The activation of Th1-like responses, including specific CD8 T-cells, is a strategy currently pursued to obtain efficient vaccines against ASFV [28]. The fact that dietary SDPP can protect mucosal integrity and also promote opti- mal immune responses, including Th1-like responses and CTL induction [29,30], prompted Vaccines 2023, 11, 824 3 of 15 us to hypothesize that SDPP could be used as a potential nutritional intervention against ASFV transmission and progression of the infection. Therefore, the objective of this study was to assess if supplementing SDPP in diets of healthy pigs reduced ASF virus load of infected animal after an experimental ASFV pig-to-pig contact challenge. 2. Materials and Methods 2.1. Ethical Statement The study was approved by the committee of ethics and welfare “Comitè d’Experimentació Animal de la Generalitat de Catalunya” with the protocol approval number CEA-OH/11025/1. For this study, 24 Landrace × Large White male pigs (5 weeks of age) were obtained from a commercial farm with high sanitary status. The clinical state of the animals and the end-point criteria was evaluated by scoring the ASF-compatible clinical signs following a previously reported guide [31]. A score from 0 to 5 according to severity was applied as follows: 0: no clinical signs, 1: mild pyrexia (39.6–40.0 ◦ C), 2: mild pyrexia (39.6–40.0 ◦ C) and mild clinical signs (skin, digestive), 3: moderate pyrexia (40.0–40.5 ◦ C) and mild-moderate clinical signs (distal ear spots, mild limp, lying down, but remaining alert), 4: moderate-high pyrexia (40.5–41 ◦ C) and moderate clinical signs (remains dormant, only stands up when touched, hesitant step, subcutaneous bleeding <10%, diarrhea, mild tremors), and 5: pyrexia higher than 41 ◦ C and moderate-severe clinical signs (generalized subcutaneous bleeding, ataxia, spasticity, clouding, prostration, and bloody diarrhea). 2.2. Study Design The 24 pigs were divided randomly into 2 groups of 12 pigs each and were placed in 2 separate rooms at the IRTA-CReSA biosecurity level 3 animal facility. Each group of 12 pigs represented an experimental treatment group: CONVENTIONAL: ASFV challenged pigs fed a conventional diet with soy protein concentrate, and SDPP: ASFV challenged pigs fed a diet with SDPP. The SDPP used in this study was randomly selected from a manufactured lot of AP920 produced by APC Europe S.L.U. (Granollers, Spain). The CONVENTIONAL group diet was formulated with soy protein concentrate (Soycomil® P, ADM Animal Nutrition™, Quincy, IL, USA) at 100.9 g/kg and the SDPP group was fed a diet formulated with SDPP at 80 g/kg, which entirely replaced Soycomil with small adjust- ments in other ingredients and synthetic amino acids. Both diets were iso-energy (3340 kcal ME/kg) and iso-nutrient balanced (Lysine SID 12.50 g/kg feed). The experimental diets (Table 1) were prepared in mash form and offered ad libitum during the adaptation period and the 31-day study. The rooms contain slated floor and the environmental conditions for both rooms were set at 22 ± 2 ◦ C and relative humidity of 60 ± 5%. The air renewal was es- tablished to be 12 times/hour. The feed was provided each morning between 7:30–9:30 a.m. All procedures involving clinical data taking, necropsies, and laboratory analyses were performed in a blind fashion in regards the treatments. After a brief adaptation period to the diets and facility, on day 4 of the experiment (d-2) two randomly selected animals (for use as trojan pigs) per room were separated from the other pigs (10 contact pigs per room) by placing them in two subdivisions to avoid direct contact with the other pigs. Immediately thereafter, the two trojans per room were intramuscularly (IM) injected with a lethal dose of 103 genome equivalent copies (GEC) of the Georgia 2007/1-ASFV strain. Two days after virus inoculation, the trojan and contact pigs in each room were re-grouped to begin the contact exposure (day 0 post- exposure, d0pe). The ratio of trojan to naïve contact pigs was 1:5, mimicking a potential field scenario of slow ASFV transmission. As expected, trojans developed ASF signs by day 4 after inoculation and were sacrificed for humane reasons by day 6 after inoculation when showing acute ASF clinical signs. Vaccines 2023, 11, 824 4 of 15 Table 1. Composition and estimated nutritive composition of the basal experimental diet (g/kg feed). Ingredients Conventional Diet SDPP Diet Barley 298.7 313.3 Maize 295.7 303.4 Sweet milk whey 137.2 137.2 Soy protein concentrate 100.9 - Spray-dried porcine plasma (SDPP) - 80.0 Soybean meal (48% CP) 107.4 110.6 Animal fat 26.7 30.0 Dicalcium phosphate 14.4 16.1 Calcium carbonate 1.2 0.5 Salt 4.7 0.2 L-Lysine-HCl 4.2 2.3 L-Threonine 1.9 0.5 DL-Methionine 2.1 1.4 L-Tryptophan 0.7 0.2 L-Valine 0.3 - Noxyfeed * 0.2 0.2 Vitamin-Mineral complex ** 4.0 4.0 Nutritive composition Crude Protein 195.0 195.0 Crude Fiber 28.0 25.2 Fat 45.5 50.3 Ash 58.2 56.6 Energy (kcal ME/kg) 3340 3340 Total sodium 2.8 2.8 Total chlorine 6.7 4.5 Total calcium 7.5 7.5 Total phosphorous 6.7 7.4 Digestible phosphorous 3.8 3.8 Lysine (SID) 12.50 12.50 Threonine (SID) 8.13 8.13 Methionine (SID) 4.62 3.75 Met + Cys (SID) 7.38 7.78 Tryptophan (SID) 2.50 2.50 Isoleucine (SID) 7.36 6.75 Valine (SID) 8.50 9.26 Leucine (SID) 13.52 14.89 Phenylalanine (SID) 8.02 8.52 Phe + Tyr (SID) 13.41 14.60 Histidine (SID) 4.25 4.76 * ITPSA, Barcelona, Spain. Contains BHT+ propyl galate (56%) and citric acid (14%). ** Provides per kg feed: vitamin A (E-672) 10,000 UI; vitamin D3 (E-671) 2000 UI; vitamin E (alfa-tocopherol) 25 mg; vitamin B1 1.5 mg; vitamin B2 3.5 mg; vitamin B6 2.4 mg; vitamin B12 20 µg; vitamin K3 1.5 mg; calcium panthotenate 14 mg; nicotinic acid 20 mg; folic acid 0.5 mg; biotin 50 µg; Fe (E-1) (from FeSO4 ·H2 O) 120 mg; I (E-2) (from Ca(I2 O3 )2) 0.75 mg; Cu (E-4) (from CuSO4 ·5H2 O) 6 mg; Mn (E-5) (from MnO) 60 mg; Zn (E-6) (from ZnO) 110 mg; and Se (E-8) (from Na2 SeO3 ) 0.37 mg. However, none of contact pigs in either group developed fever, ASF specific signs, or viremia after 17 days of observation (d17pe). In view of these results, a modification of the protocol was obtained from the ethical committee to increase the infection pressure by providing a second exposure of ASFV infected trojan pigs to contact pigs. Accordingly, three pigs from each group were chosen randomly and inoculated IM with 103 GEC (acting as trojan seeders) while remaining inside the same pen with the six remaining contact pigs to have a 1:2 (trojan to contact pig) ratio. To maintain this optimal contact challenge ratio [32], one pig per each contact group was excluded from the study before the second ASFV- exposure. The study ended at d29pe (day 12 post-second exposure, d12pse), following the originally approved protocol (Figure 1). providing a second exposure of ASFV infected trojan pigs to contact pigs. Accordingly, three pigs from each group were chosen randomly and inoculated IM with 103 GEC (act- ing as trojan seeders) while remaining inside the same pen with the six remaining contact pigs to have a 1:2 (trojan to contact pig) ratio. To maintain this optimal contact challenge Vaccines 2023, 11, 824 ratio [32], one pig per each contact group was excluded from the study before the second5 of 15 ASFV-exposure. The study ended at d29pe (day 12 post-second exposure, d12pse), fol- lowing the originally approved protocol (Figure 1). Figure 1. Schematic representation of the study design. Figure 1. Schematic representation of the study design. During the whole study animals were daily observed for ASF-compatible clinical signs Duringrectal (including the whole study animals temperature) wereofdaily and samples bloodobserved (EDTA 15for mL ASF-compatible tubes) were taken clinical from signs (including all animals at −2rectal daystemperature) and samples of post-contact immediately blood before IM(EDTA 15 mL injection tubes) to the were taken 2 trojans, d2pe, from d5pe,all animals d12pe, at −2 d19pe days post-contact (d2pse), immediately and d26pe (d9pse). before IM In addition, injection nasal to the and rectal 2 trojans, swabs were d2pe, d5pe, d12pe, d19pe (d2pse), and d26pe (d9pse). In addition, nasal collected at d2pe, d5pe, d9pe, d12pe, d16pe, d19pe (d2pse), d23pe (d6pse), and d26pe and rectal swabs were collected (d9pse). at d2pe, Necropsies wered5pe, done d9pe, d12pe,(d12pse), on d29pe d16pe, d19pe (d2pse), following d23pe (d6pse), the schedule in Figureand1. d26pe (d9pse). Necropsies were ◦ done on d29pe (d12pse), following the schedule All samples were stored at −75 C until processed. Blood, nasal, and rectal swabs were in Figure 1. All samples analyzed were by real stored time at −75 °CPCR quantitative until processed.toBlood, (qRT-PCR) detect nasal, andAtrectal viremia. swabslesions necropsy, were analyzed by real and were registered, timesamples quantitative PCR tonsil, of spleen, (qRT-PCR) to detect viremia. gastro-hepatic At necropsy, node, submaxillary lesions node, and were registered, and retropharyngeal nodesamples were takenof spleen, tonsil, gastro-hepatic and analyzed for virus detectionnode,bysubmaxillary qRT-PCR. node, and retropharyngeal ELISPOT analysis node[33]were wastaken and analyzed conducted in frozenfor virus cells detection where viabilityby was qRT-PCR. >90% after ELISPOT thawing from analysis [33] was blood obtained conducted with EDTA atind9pe, frozen cells(d6pse) d23pe where viability and d29pe was >90% after (d12pse). thawing from blood obtained with EDTA at d9pe, d23pe (d6pse) and d29pe (d12pse). 2.3. Laboratory Analyses 2.3. Laboratory Samples of Analyses ASFV inoculum were analyzed by the qRT-PCR described by [34]. Blood samples were taken Samples of ASFV in inoculum tubes withwereEDTA on specified analyzed days. by the DNA extraction qRT-PCR describedwas doneBlood by [34]. using the Indimag Pathogen Kit (Indical Biosciences, Leipzig, Germany). Viremia samples were taken in tubes with EDTA on specified days. DNA extraction was done us- was determined by the ing qRT-PCR Indimaganalysis PathogenusingKitthe primers (Indical described Biosciences, by Fernández-Pinero Leipzig, Germany). Viremiaet al.was [35], and deter- the probe mined ASF-VP72P1 by qRT-PCR analysisdescribed in the using the current primers OIE ASF described chapter (Terrestrial by Fernández-Pinero et Manual al. [35], OIE, Section 3.9, Chapter 3.9.1 African Swine Fever Virus pages 1–18) and the probe ASF-VP72P1 described in the current OIE ASF chapter (Terrestrial Manualwith the following modification OIE, in the Section 3.9, thermoprofile Chapter made 3.9.1 African by the Swine Spanish Fever Virus National pages 1–18)Reference with theLaboratory following for ASF: 10 min at 95 ◦ C, 5 cycles 1 min at 95 ◦ C + 30 seg at 60 ◦ C, 40 cycles 10 min at modification in the thermoprofile made by the Spanish National Reference Laboratory for 95 ◦ C10+ min ASF: 30 seg at at 9560 ◦ °C, C5 with cyclesfluorescence 1 min at 95acquisition °C + 30 seginatthe 60 FAM °C, 40channel at the cycles 10 minend of °C at 95 each + PCR cycle. According to these amplification settings results were considered as positive 30 seg at 60 °C with fluorescence acquisition in the FAM channel at the end of each PCR when Ct values were ≤30, inconclusive Ct values between 30 to 35, and Ct > 35 were cycle. According to these amplification settings results were considered as positive when considered negative. Seroconversion was determined by ELISA (Ingezim PPA COMPAC, INGENASA; Madrid, Spain) with O.D. ≤ 0.061 as negative. Nasal and rectal swabs were analyzed for the presence of ASFV genome using the same procedures previously mentioned. Once the nasal and rectal swabs arrived at the laboratory, the end of the swab was cut and placed in a tube with 1 mL of PBS. The tubes were stored at −75 ◦ C until DNA extraction and analysis by qRT-PCR. At necropsy, samples of tonsil, spleen, and retropharyngeal, submaxillary, and gastro- hepatic lymph nodes were taken for subsequent qRT-PCR analysis. For each sample, 0.1 g of tissue was diluted 1:10 and homogenized using sterile PBS and TyssueLyser II (Qiagen, Hilden, Germany). DNA extraction and qRT-PCR were completed as detailed above. Vaccines 2023, 11, 824 6 of 15 Correlation Ct vs. Log10 HAD50/mL of the different analyzed tissues was carried out using ASFV Georgia 2007 viral stock at 6.99 Log10HAD50/mL as a standard. qRT-PCR were carried out as detailed above (Supplementary Table S1). Peripheral blood mononuclear cells (PBMCs) were purified from EDTA blood samples by density-gradient centrifugation with Histopaque 1077 (Sigma-Aldrich, St. Louis MI, USA). Cells were frozen at −80 ◦ C, but the viability was tested to be ≥90% after thawing and before conducting the analysis. Commercial antibody Porcine IFN-γ P2G10 and biotin P2C11, from BD Biosciences (Pharmingen, San Diego, CA, USA) at 5 µg/mL was used to quantify by ELISPOT assay the number of IFNγ secreting cells. PBMC were stimulated against ASF Georgia/01 strain virus at a MOI 0.2 to measure specific T-cell responses by ELISPOT in real time with fresh cells [35]. Phytohemagglutinin (PHA; Roche Diagnostics, Barcelona, Spain) was used as a positive control and the mock was carried out using only cells and RPMI medium as a negative control. Plates were revealed using detection antibody from BD Pharmingen (BD Biosciences Pharmingen, San Diego, CA, USA), Strep-HRP from Life Technologies (South San Francisco, CA, USA) and TMB substrate for ELISPOT assay (MABTECH, Stockholm, Sweden). ELISPOTs were read under a magnifying glass. The value obtained for each animal was obtained subtracting the corresponding values of mock-stimulated cells. 2.4. Statistical Analysis Data were analyzed as a completely randomized design using the GLM procedures of SAS (SAS Inst., Inc., Cary, NC, USA). An analysis of variance was conducted to detect differences among treatments. The independent variable was treatment. Dependent variables were body temperatures, blood, nasal, and rectal swab, and tissue Ct values. The LSMEANS procedure was used to calculate the mean values by treatment. If treatment effects were detected, least squares means were separated using the PDIFF option in SAS. Pigs were considered the experimental unit. Means are considered significantly different if p < 0.05 while trends are reported as p = 0.05 to 0.10. 3. Results 3.1. First Exposure After the first exposure to ASFV-inoculated trojans (1:5 ratio of trojans:contact pigs), none of the contact pigs developed temperatures above 40.4 ◦ C, had ASF compatible signs or viremia after 16 days of monitoring (Supplementary Table S2). One CONVENTIONAL contact pig on d2pe, and 5 CONVENTIONAL and 3 SDPP contact pigs on d5pe displayed an ASFV qRT-PCR positive result in the nasal swab, but thereafter to d16pe, all nasal swab results were negative. All blood and rectal swab samples for contact pigs were negative. Despite the nasal swab results for contact pigs, this did not result in a disseminated infection (Supplementary Table S3), even though trojan pigs in both groups had developed high fever and were euthanized d4pe per protocol requirements. 3.2. Second Exposure Second exposure: Conversely, after the second contact exposure (1:2 ratio of tro- jans:contact pigs), contact pigs from the CONVENTIONAL group became infected by ASFV, following the expected kinetics [36]. Thus, all six contact pigs from the CONVEN- TIONAL group developed acute ASF-compatible clinical signs, showing high fever (rectal temperature > 41 ◦ C) by d29pe (d12pse) with one contact pig (#15) having >41 ◦ C starting at d26pe (d9pse; Figure 2A). Interestingly, all contact pigs from the SDPP group had a delay in the onset of ASF clinical signs with only two of the six pigs (#4 and 7) having rectal temperatures >41 ◦ C the last day of the study (Figure 2B; Supplementary Table S2). jans:contact pigs), contact pigs from the CONVENTIONAL group became infected by ASFV, following the expected kinetics [36]. Thus, all six contact pigs from the CONVEN- TIONAL group developed acute ASF-compatible clinical signs, showing high fever (rectal temperature > 41 °C) by d29pe (d12pse) with one contact pig (#15) having >41 °C starting at d26pe (d9pse; Figure 2A). Interestingly, all contact pigs from the SDPP group had a Vaccines 2023, 11, 824 7 of 15 delay in the onset of ASF clinical signs with only two of the six pigs (#4 and 7) having rectal temperatures >41 °C the last day of the study (Figure 2B; Supplementary Table S2). A TEMPERATURES CONVENTIONAL GROUP Animal #13 Animal #14 42.5 Rectal Temprature (ºC) 42.0 Animal #15 41.5 Animal #16 41.0 40.5 Animal #17 40.0 Animal #18 39.5 39.0 Animal #20 NT 38.5 D17 D18 D19 D20 D21 D22 D23 D24 D25 D26 D27 D28 D29 Animal #21 NT (second Trojans) Animal #22 NT B TEMPERATURES SDPP GROUP Animal #1 Animal #2 42.5 Rectal Temprature (ºC) 42.0 Animal #3 41.5 41.0 Animal #4 40.5 Animal #5 40.0 39.5 Animal #7 39.0 Animal #8 NT 38.5 D17 D18 D19 D20 D21 D22 D23 D24 D25 D26 D27 D28 D29 Animal #9 NT (second Trojans) Animal #10 NT Figure 2. Rectal Figure 2. Rectal temperature temperatureover overtime timeafter after second second ASFV ASFV exposure. exposure. (A):(A): animals animals fed fed withwith CON- CONVEN- VENTIONAL diet and (B): animals fed with spray dried porcine plasma (SDPP) diet. In red color TIONAL diet and (B): animals fed with spray dried porcine plasma (SDPP) diet. In red color are the are the trojans animals in each group. trojans animals in each group. Average body temperature Average body temperature was was higher higher (p (p < < 0.05) 0.05) for for CONVENTIONAL CONVENTIONAL contact pigs contact pigs on d23pe (d6pse), and d26pe to d29pe (d9pse to d12pse) compared to SDPP contact on d23pe (d6pse), and d26pe to d29pe (d9pse to d12pse) compared to SDPP contact pigs pigs (Figure 3). (Figure 3). All three trojan pigs in the CONVENTIONAL group died between 4 to 7 days post- inoculation. However, two of the three trojan pigs in the SDPP group survived until the last day of the study with RT below 41 ◦ C, even though these pigs displayed severe signs of disease. Therefore, the exposure time of contact pigs to trojans displaying fever was longer in the SDPP group than in the CONVENTIONAL group due to the longer survival rate of the trojan pigs in the SDPP group. Viremia was detected in both groups at d29pe (d12pse). ASFV qPCR positive nasal swab was presented in all pigs for both groups from d23pe (d6pse) probably confirming the contact of these pigs with the second group of trojans. ASFV qPCR positive rectal swab was found in one pigs of the non-treated group on d23pe although at very low level (Ct 33); in contrast, the presence of ASFV virus in feces of pigs in the SDPP group was not found until d26pe (d9pse). Figure 4; Supplementary Tables S3 and S4. 39.0 Rectal Te 38.0 37.0 Vaccines 2023,11, Vaccines 2023, D17 11, 824 D18REVIEW x FOR PEER D19 D20 D21 D22 D23 D24 D25 D26 D27 D28 88 of D29of 15 15 (second CONVENTIONAL SDPP Trojans) Figure 3. Average rectal temperature over time between treatments after second ASFV exposure. # AVERAGE TEMPERATURES = p < 0.1; * = p < 0.05; ** = p < 0.01 SDPP = spray-dried porcine plasma. * Rectal Temperature (ºC) 42.0 ** All three trojan pigs in the CONVENTIONAL group ** 41.0 * died between 4 to 7 days post- * * # inoculation. ** However, two of the three trojan pigs in the SDPP group survived until the 40.0 last day of the study with RT below 41 °C, even though these pigs displayed severe signs 39.0 of disease. Therefore, the exposure time of contact pigs to trojans displaying fever was 38.0 longer in the SDPP group than in the CONVENTIONAL group due to the longer survival rate of the trojan pigs in the SDPP group. 37.0 Viremia was detected in both groups at d29pe (d12pse). ASFV qPCR positive nasal D17 D18 D19 D20 D21 D22 D23 D24 D25 D26 D27 D28 D29 (second swab was presented in all pigs for both groups from d23pe (d6pse) probably confirming the contact of theseCONVENTIONAL pigs with the second group SDPP of trojans. ASFV qPCR positive rectal swab Trojans) was found in one pigs of the non-treated group on d23pe although at very low level (Ct Figure 33); Figure 3. Average in contrast, 3. Average rectal the rectal temperature presence over overtime of ASFV temperature between virus time treatments in feces between of pigsafter treatments in second afterthe ASFV SDPP second exposure. group ASFV was# not exposure. = p < 0.1; * = p < 0.05; ** = p < 0.01 SDPP = spray-dried porcine plasma. found # = p until < 0.1; *d26pe (d9pse). = p < 0.05; ** = p
35). value In gastro-hepatic and submaxillary lymph nodes there was a tendency (p < 0.10) to have > 35). In gastro-hepatic and submaxillary lymph nodes there was a tendency (p < 0.10) to less average amount of virus genome copies (higher Ct values) for animals in the SDPP to the CONVENTIONAL group. In addition, pigs in the SDPP group had lower (p < 0.05) ASFV load in tonsil and retropharyngeal lymph nodes. Vaccines 2023, 11, x FOR PEER REVIEW 10 of 15 have less average amount of virus genome copies (higher Ct values) for animals in the Vaccines 2023, 11, 824 SDPP to the CONVENTIONAL group. In addition, pigs in the SDPP group had lower 10 of (p 15 < 0.05) ASFV load in tonsil and retropharyngeal lymph nodes. To understand the mechanisms explaining the differential outcome of both ASF clin- ical sign evolution and To understand virus transmission, the mechanisms explaining thethe kinetics of both differential the specific outcome antibody of both and ASF clinical sign evolution cellular and were responses virus transmission, followed. None theofkinetics of both the contact thefrom pigs specific antibody either groupand cellular developed responses detectablewere followed. anti-ASFV None ofafter antibodies the the contact firstpigs ASFV from either contact group developed challenge, which is detectable consistent anti-ASFV antibodies with the absence after theviremia. of detectable first ASFVEven contact challenge, after the which is to second exposure consistent with the ASFV inoculated absence of detectable viremia. Even after the second exposure to trojans, contact pigs did not have time to develop significant amounts of anti-ASFVASFV inoculated trojans, anti- contact pigs did not have time to develop significant amounts of anti-ASFV bodies and by d12pse remained below the threshold limit of the technique (Supplemen- antibodies and by taryd12pse Table remained below S6). In clear the threshold contrast with thelimitlack ofofthe technique antibody (Supplementary responses, Table S6). ASFV-responding In clearsecreting IFN-γ contrast cells withwere the lack of antibody detectable responses, by ELISPOT ASFV-responding as soon as 9 days afterIFN-γ secreting first ASFV en- cells were counter detectable (d9pe); thus,by ELISPOT 8 of 9 contactaspigs sooninasthe9 days SDPP after firstshowed group ASFV encounter low, albeit(d9pe); thus, detectable 8ASFV-responding of 9 contact pigs in the SDPP cells, while group 2 of 7 showed low, albeit contact pigs in thedetectable CONVENTIONAL ASFV-responding group had cells, a while 2 of 7 contact pigs in the CONVENTIONAL group had a detectable detectable response. On d23pe of the experiment (d6pse), most pigs in both groups devel- response. On d23pe oped aofdetectable the experiment cellular (d6pse), responsemost pigs in against both measured ASFV groups developed by ELISPOT a detectable (Figure cellular 6; Sup- response against ASFV plementary Table S7). measured by ELISPOT (Figure 6; Supplementary Table S7). IFN-γ ELISPOT in PBMC at d9pe 60 Spots/500,000 PBMCs 50 40 30 20 10 0 Figure 6. Figure 6. ASFV-specific ASFV-specificIFN-γ IFN-γsecreting secretingcells cellsanalyzed analyzed byby ELISPOT ELISPOTat 9atdays after 9 days firstfirst after ASFV en- ASFV counter (d9pe). PBMC from d9pe were stimulated in vitro with Georgia 2007/1 at MOI 0.2 encounter (d9pe). PBMC from d9pe were stimulated in vitro with Georgia 2007/1 at MOI 0.2 and the and the number of ASFV-responding IFN-γ-secreting cells was quantified by ELISPOT. Values shown are number of ASFV-responding IFN-γ-secreting cells was quantified by ELISPOT. Values shown are individual values subtracting the corresponding values of mock-stimulated cells. C-#: Animals in individual values subtracting the corresponding values of mock-stimulated cells. C-#: Animals in CONVENTIONAL diet; SDPP-#: animals in spray-dried porcine plasma (SDPP) diet. CONVENTIONAL diet; SDPP-#: animals in spray-dried porcine plasma (SDPP) diet. 4. Discussion 4. Discussion Despite the dramatic consequences that ASF represent for the swine industry and for Despite the dramatic consequences that ASF represent for the swine industry and global trading, ASFV cannot be considered as a highly transmissible virus, at least com- for global trading, ASFV cannot be considered as a highly transmissible virus, at least pared with other pig viruses of obligatory declaration to the WOAH [37], such as classical compared with other pig viruses of obligatory declaration to the WOAH [37], such as swine fever virus (CSFV) and foot and mouth disease virus (FMDV). In agreement with classical swine fever virus (CSFV) and foot and mouth disease virus (FMDV). In agreement this observation, naïve pigs fed daily for 14 consecutive days with feed blended with with this observation, naïve pigs fed daily for 14 consecutive days with feed blended with liquid porcine plasma inoculated with 104.3 or 105.0 TCID50/g ASFV remained ASF-free [38]. The fact that ASFV recovered from the feed remained infectious when administered intra- muscularly or intra-gastric with a probe, confirmed that infection with ASFV through feed Vaccines 2023, 11, 824 11 of 15 consumption is not as efficient as for other pathogens. Other studies feeding commercial inoculated ASFV contaminated feed not containing plasma resulted in infection of the naïve pigs [39–41]). These results suggest that unprocessed liquid porcine plasma or SDPP may contain inherent properties that diminish the infectious capacity of ASFV and may contribute to the survival of two of the three SDPP trojans until end of the present study. This limited capability of ASFV transmission might explain the lack of evident viral transmission observed between trojan and contact pigs at a ratio of 1:5 (infected vs. non- infected animal ratio). However, during the first exposure the contact pigs were probably exposed to the virus at low levels for a short time, which was adequate to trigger the induction of a mild ASFV-responding cellular response but was not sufficient to induce detectable ASFV-specific antibodies or viremia at the times tested. This finding was defini- tively confirmed after showing that some contact pigs in both groups had low amounts of ASFV genome detectable by qPCR in the nasal swab by d2pe and d5pe. The trojans in contact ASFV challenge model used in this study are as reproducible as the direct IM ASFV inoculation model. This is a reliable challenge model [32] if the optimal ratio (1:2) of trojans versus contact pigs is kept. Moreover, we consider that this is a more natural model of infection compared to the IM model. The difference between both models (IM vs. in contact) is the kinetics of the infection. Thus, the onset of infection is faster for IM injected pigs with fever and ASFV being detectable from day four post- challenge. Conversely, in contact challenge pigs develop similar clinical signs, including fever, viremia, and shedding with 3 days of delay, reaching similar maximum virus titers and ASF compatible signs than those injected IM [32]. The presence of low levels of ASFV in the nostrils of contact pigs without causing viremia or ASF clinical signs might be further explained by the induction of efficient innate immune responses capable of controlling ASFV infection [27] at the site of entry. The fact that positive nasal swab samples were detected in a lower number of contact pigs in SDPP versus the CONVENTIONAL group, can be related with either less virus excretion from the trojan pigs fed SDPP diet during first exposure or related to our original hypothesis of innate response improvement by SDPP feeding [18,29]. These results fit with the fact that at later time points (d9pe), most contact pigs fed SDPP showed ASFV-responding IFN-γ secreting cells, compared with only two animals fed the CONVENTIONAL diet. The positive effect of SDPP on Th1-like memory responses was also confirmed by others [29,30], including the induction of cytotoxic CD8+ T cells [29,30,42]. Independently of this, as mentioned earlier, Th1-like responses and CD8+ T cells perform a key role in ASFV protection [19,21] and dietary SDPP in other challenge studies has demonstrated increases in the percentage of CD8+ T cells [29,30,42]. The potential presence of ASFV specific CD8+ T-cells in contact pigs fed SDPP after the first exposure with the trojan pigs, may partially explain the delay of the ASF onset observed in this group of pigs after the second ASFV encounter [26,28,32], but also the unique feature observed in two of the trojan pigs used for the second challenge that survived 12 days after IM ASFV challenge. Despite that average viremia and nasal shedding genome values were similar between both groups of trojan by day 6 post challenge (Supplementary Table S3), these two pigs (#8, #9) in the SDPP group continued secreting ASFV to the environment until the end of the experiment (d29pe or d12pse). SDPP is a functional ingredient that is demonstrated to improve the integrity of the intestinal barrier, enrich the gut microbiota, and modulate the immune system of pigs and other animal species [13–17,22,23,43–45]. Improving integrity of intestinal barrier function may reduce the potential infectivity of ASFV and microbiota seems to have an important role for controlling ASFV infection [24,46]. In addition, SDPP supplemented in feed was shown to modulate the immune system and balance the ratio between activated and regulatory T cells [13,17], which are essential to protect against most pathogens, including ASFV [27]. As above mentioned, albeit little is known about the mechanisms involved in ASFV protection, innate and both memory B and T cells responses perform a role, most probably in a regulated way to avoid excessive inflammation. Diets with SDPP may Vaccines 2023, 11, 824 12 of 15 contribute to this regulation by providing an optimal cytokine environment [13,17,18,44]. The molecular mechanisms explaining why the contact-exposed pigs fed SDPP showed lower virus load compared to conventional pigs have not been elucidated. We believe that the accelerated cellular response (IFN-γ secreting cells) detected in this group of pigs after the first ASFV suboptimal encounter, might partially explain the protection afforded against the second challenge, this time using the optimal ratio of trojans. Recently, a study conducted by Radulovic et al., [4] demonstrated that pigs can regulate immune activation more efficiently after vaccination with attenuated ASFV strains; these animals showed milder production of proinflammatory cytokines after virus inoculation, similar to the results in previous studies when animals are fed diets supplemented with SDPP [13,17,44]. In Asia-Pacific regions, new, less virulent strains and attenuated strains of ASFV com- pared with the original Georgia strain have appeared, probably due to genetic evolution the virus and the use of non-authorized vaccine prototypes [47,48]. Such scenarios complicate the epidemiological ASFV picture and control of ASF. Based on the results from this study using a virulent ASFV strain we speculate that healthier pigs fed diets with SDPP may improve resilience to ASFV strains because diets with SDPP improves the health status of pigs, especially under stressful conditions [12]. Although the length of time the donor pigs were in contact with the other animals was greater for the SDPP pigs, the data suggests viraemia and shedding was lower which may also contribute to explain the differences observed, rather than the feed itself. Further work should be performed in the future with a larger group of animals to confirm these observations. However, the accelerated specific T-cell responses observed in SDPP-fed animals upon the first encounter with the ASFV, the lower ASFV loads found in the same group after the second challenge, together with the beneficial effect that SPPP seems to exert upon experimental ASFV vaccination [49] (back-to-back submitted manuscript) would support the hypothesis that nutritional interventions may be useful to help ameliorating the impact of ASF in endemic regions. 5. Conclusions In conclusion, feeding SDPP reduced virus load in the different target tissues and may delayed in pen transmission in pigs exposed to ASFV inoculated trojans. Therefore, supplementation of SDPP in feed may be a strategic nutritional intervention to improve the degree of protection against ASFV. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/vaccines11040824/s1, Table S1: Correlation Ct vs. Log10 HAD; Table S2: Body temperature throughout the duration of the study; Table S3: PCR results in blood, nasal and rectal swab; Table S4: Summary results of PCR in blood, nasal and rectal swab; Table S5: PCR results of different tissues; Table S6: Antibodies against ASFV in serum at different days during the study; Table S7: ELISPOT results at different days during the study. Author Contributions: Conceptualization, E.B., J.P. (Javier Polo), J.P. (Joan Pujols), F.R. and J.S.; methodology, E.B., J.P. (Joan Pujols), F.R., R.R. and J.S.; software, J.P. (Javier Polo), J.P. (Joan Pujols) and J.C.; validation, J.P. (Joan Pujols), F.R., J.S., R.R. and J.P. (Javier Polo); formal analysis, J.C., F.R., J.P. (Joan Pujols) and J.P. (Javier Polo); investigation, J.P. (Joan Pujols), F.R. and J.S.; resources, J.P. (Joan Pujols) and F.R.; data curation, J.P. (Joan Pujols), E.B., F.R. and J.P. (Javier Polo); writing—original draft preparation, J.P. (Javier Polo), E.B., J.P. (Joan Pujols), F.R. and J.S.; writing—review and editing, F.R., J.P. (Javier Polo), E.B. and J.S.; visualization, E.B. and J.P. (Javier Polo); supervision, J.P. (Joan Pujols) and J.P. (Javier Polo); project administration, J.P. (Joan Pujols); funding acquisition, J.P. (Javier Polo). All authors have read and agreed to the published version of the manuscript. Funding: Funding for this study was provided by APC Europe, S.L.U., Granollers, Spain, and APC LLC, Ankeny, US. Both companies manufacture animal blood products for animal consumption. The companies provided support in the form of salaries for authors EB, JC, and J. Polo retrospectively, but none of these companies did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors Vaccines 2023, 11, 824 13 of 15 are articulated in the ‘author contributions’ section. We also acknowledge support of the Spanish Ministry of Science and Innovation (grant reference PID2019-107616RB-I00). Institutional Review Board Statement: The study was approved by the committee of ethics and welfare “Comitè d’Experimentació Animal de la Generalitat de Catalunya” with the protocol approval number CEA-OH/11025/1. Informed Consent Statement: Not applicable. Data Availability Statement: All data from this study is provided in the manuscript and Supplemen- tary Tables. Acknowledgments: We thank the technicians and PhD students of IRTA-CReSA helping in the experimental setup. Conflicts of Interest: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: EB, and J. Polo are employed by APC Europe, S.L.U. Gra- nollers, Spain. JC and J. Polo are employed by APC LLC, Ankney, US. Both companies manufacture and sells spray-dried animal plasma; however, the companies had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. J. Pujols, FR, RR, and JS declare no conflict of interest. This does not alter the authors’ adherence to all journal policies on sharing data and materials. References 1. Śmietanka, K.; Woźniakowski, G.; Kozak, E.; Niemczuk, K.; Fraczyk, ˛ M.; Bocian, Ł.; Kowalczyk, A.; Pejsak, Z. African Swine Fever Epidemic, Poland, 2014–2015. Emerg. Infect. 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