Comparative evaluation of disease dynamics in wild boar and domestic pigs experimentally inoculated intranasally with the European highly virulent African swine fever virus genotype II strain "Armenia 2007".

Since the reintroduction of African swine fever virus (ASFV) in Europe in 2007 and its subsequent spread to Asia, wild boar has played a crucial role in maintaining and disseminating the virus. There are significant gaps in the knowledge regarding infection dynamics and disease pathogenesis in domestic pigs and wild boar, particularly at the early infection stage. We aimed to compare domestic pigs and wild boar infected intranasally to mimic natural infection with one of the original highly virulent genotype II ASFV isolates (Armenia 2007). The study involved euthanising three domestic pigs and three wild boar on days 1, 2, 3, and 5 post-infection, while four domestic pigs and four wild boar were monitored until they reached a humane endpoint. The parameters assessed included clinical signs, macroscopic lesions, viremia levels, tissue viral load, and virus shedding in nasal and rectal swabs from day 1 post-infection. Compared with domestic pigs, wild boar were more susceptible to ASFV, with a shorter incubation period and earlier onset of clinical signs. While wild boar reached a humane endpoint earlier than domestic pigs did, the macroscopic lesions were comparatively less severe. In addition, wild boar had earlier viremia, and the virus was also detected earlier in tissues. The medial retropharyngeal lymph nodes were identified as key portals for ASFV infection in both subspecies. No viral genome was detected in nasal or rectal swabs until shortly before reaching the humane endpoint in both domestic pigs and wild boar, suggesting limited virus shedding in acute infections.

Epidemiology and distribution of gastrointestinal parasites in fattening pig farms in northern Italy.

In Italy, pig breeding is characterised by intensive farms in which parasitic diseases often present a subclinical pattern, while being responsible for decreased animal welfare and great economic losses. The present study aimed to assess the prevalence of major parasites in pigs, and, for this purpose, 880 faecal samples of fattening pigs raised in 22 intensive farms located in northern Italy were collected in two different sampling sessions, at the beginning and end of the fattening cycle. For the detection of helminth eggs and coccidian oocysts, a quantitative flotation technique was used, whereas a conventional PCR was performed to confirm the identification of cestode eggs found by copromicroscopic analysis. Moreover, data regarding herd management were collected to assess risk factors for parasite occurrence. A total of 95 samples were positive for at least one parasitic taxon (10.8%); the most detected parasite was Ascaris suum (7.6%), followed by Trichuris suis (1.7%) and Cystoisospora suis (0.9%). Further, eggs with morphometric features compatible with those of Hymenolepis diminuta were detected in 16 samples (1.8%), and the analysis of sequences confirmed the identification of cestode eggs. Statistical analysis showed that large farms and those applying the all-in/all-out system were associated with a lower risk of nematode infection. This study provided data on prevalence and burden of gastrointestinal parasites in two different times of the fattening cycle. It was evidenced that endoparasites are persistent, albeit with low prevalences, and would need specific measures to reduce their effects on both animal health and productivity.

Isolation and molecular confirmation of Brucella suis biovar 2 from slaughtered pigs: an unanticipated biovar from domestic pigs in Egypt.

BACKGROUND: Brucella suis is a zoonotic pathogen with a serious impact on public health and the pig industry worldwide. Information regarding B. suis in pigs in Egypt is scarce. This study aimed to investigate the prevalence of B. suis in slaughtered domestic pigs at El-Basatin abattoir in Cairo, Egypt. A total of 1,116 domestic pigs slaughtered in 2020 were sampled for Brucella isolation and identification. Identified Brucella isolates were molecularly confirmed at species, and biovar levels using Bruce ladder PCR and Suis ladder multiplex PCR. Additionally, high-risk practices of 16 abattoir workers (4 veterinarians, 10 butchering and evisceration workers, and 2 scalding workers) were investigated using a pre-piloted structured questionnaire. RESULTS: Brucella isolates were recovered from 1.3% of examined pigs (n = 14) at consistently low rates (1.1-2.9%) across the year of sampling from February to December 2020. All isolates were confirmed as B. suis biovar (bv) 2. Remarkably, 92.9% (13/14) of isolates showed atypical ability to produce H2S and hence were considered as B. suis bv2 atypical phenotype. The prevalence was higher in males (1.8%) than in females (0.9). However, this difference was not significant (Odds ratio = 1.9; CI 95% 0.7 - 5.7; P = 0.2). No detectable pathological lesions were associated with B. suis bv2 infection in examined pigs. All strains were isolated from cervical lymph nodes, highlighting a potential oral transmission. High-risk practices were recorded among swine abattoir workers in this study: 75% do not wear gloves or disinfect their knives daily, and 18.8% were willing to work with open wound injuries. CONCLUSIONS: To the best of our knowledge, this is the first isolation of B. suis bv2 in Egypt. Detection of H2S producing B. suis bv2 atypical phenotype is alarming as it may result in misinterpretation of these isolates as highly human pathogenic B. suis bv1 in Egypt and possibly elsewhere. Further epidemiological tracing studies are crucial for the detection of the origin of this biovar. Including pigs in the national surveillance program of brucellosis, and an education program for swine abattoir workers about occupational risk of B. suis is a need in Egypt.

Detecting the selection signatures in Chinese Duroc,Landrace, Yorkshire, Liangshan, and Qingyu pigs.

Here we used two kinds of chips data from 5 pig breeds, Chinese Duroc (DD), Landrace (LL), Yorkshire (YY), Liangshan (LS), and Qingyu pigs (QY) in China to identify genes which show evidence of selection during domestication. Four breed pairs, LS-YY, QY-YY, DD-YY, and LL-YY pair, were performed to detect selection signatures using the Fst method. Then we identified a list of genes that played key roles in domestication and artificial selection. For example, the PTPRM gene was shared in LS-YY, QY-YY, and DD-YY pairs and it regulates a variety of cellular processes including cell growth, differentiation as signaling molecules. The HACD3 gene was shared in QY-YY and DD-YY pairs, and the HACD3 protein is involved in the production of very long-chain fatty acids of different chain lengths. Besides, the MYH11 gene that related to muscle contraction was found in LS-YY and LL-YY pair. These results suggested that genes related to immunity, disease resistance, and metabolism were subjected to strong selection pressure in Chinese domestic pigs in the progress of domestication and evolution; however, genes related to appearance, production performance, and reproduction were undergone strong artificial selection in commercial pig breeds.

Global prevalence, subtypes distribution, zoonotic potential, and associated risk factors of Blastocystis sp. in domestic pigs (Sus domesticus) and wild boars (Sus scrofa): A systematic review and meta-analysis.

The intestinal parasite Blastocystis sp. is a widely distributed protist among humans and various animal hosts, with significant prevalence in developing countries. Due to the zoonotic nature of its subtypes (STs), we aimed at global estimation of the prevalence, STs distribution, zoonotic potential, and associated risk factors of Blastocystis sp. infection in domestic pigs (Sus domesticus) and wild pigs/wild boars (Sus scrofa). The study was designed and conducted in 2021 via searching articles in PubMed, Scopus, Google Scholar, and Web of Science databases, based on the PRISMA checklist, and meta-analysis was done using a random-effects model to calculate the weighted estimates and 95% confidence intervals (95% CIs). Totally, 43 papers (47 datasets) reported data on 7977 examined pigs in 24 countries with a total prevalence of 50.9% (95% CI: 42.8-59%). In details, prevalence was higher among domestic pigs [52.4% (95% CI: 43.9-60.7%)] than wild boars [31.2% (95% CI: 11.2-62%)], but is poorly statistically supported as far as the CIs largely overlap. Out of 28 reported STs, nine (ST1-ST7, ST10, and ST15) were reported from domestic pigs, while six (ST1, ST3-ST5, ST8, and ST15) had been isolated from wild boars. Among nine zoonotic STs (ST1-ST8, and ST12), all were identified in examined swine populations, except for ST12. As well, ST1 and ST5 were probably the most frequently circulating STs among these animals. In addition, male and older pigs showed higher Blastocystis sp. infection. Altogether, Blastocystis epidemiology and the distribution of its related STs in pigs is still open to question and requires more extensive studies, especially in the neglected regions of the world.

Seroprevalence of Toxoplasma gondii and associated risk factors in domestic pigs raised from Cuba.

A cross-sectional study was carried out to determine the seroprevalence of Toxoplasma gondii and associated risk factors in pigs in the largest pork-producing region in Cuba. Serum samples from 420 pigs, including 210 sows and 210 post-weaning pigs, were tested for antibodies against T. gondii using a commercial indirect enzyme-linked immunosorbent assay. Anti-T. gondii antibodies were detected in 56 animals (13.3%, 95% CI: 10.1-16.6). A generalized estimating equations model revealed that the risk factors associated with higher seropositivity in pigs were altitude (higher in farm's location < 250 m above sea level (masl) versus ≥ 250 masl) and age (higher in sows compared to post-weaning pigs). The results indicated that this protozoan parasite is widely distributed on pig farms in the study area, which is a public health concern since the consumption of raw or undercooked pork meat products containing tissue cysts is considered one of the main routes of T. gondii transmission worldwide. Control measures should be implemented to reduce the risk of exposure to T. gondii in pigs in Cuba.

First detection of African swine fever (ASF) virus genotype X and serogroup 7 in symptomatic pigs in the Democratic Republic of Congo.

BACKGROUND: African swine fever (ASF) is a highly contagious and severe hemorrhagic viral disease of domestic pigs. The analysis of variable regions of African swine fever virus (ASFV) genome led to more genotypic and serotypic information about circulating strains. The present study aimed at investigating the genetic diversity of ASFV strains in symptomatic pigs in South Kivu province of the Democratic Republic of Congo (DRC). MATERIALS AND METHODS: Blood samples collected from 391 ASF symptomatic domestic pigs in 6 of 8 districts in South Kivu were screened for the presence of ASFV, using a VP73 gene-specific polymerase chain reaction (PCR) with the universal primer set PPA1-PPA2. To genotype the strains, we sequenced and compared the nucleotide sequences of PPA-positive samples at three loci: the C-terminus of B646L gene encoding the p72 protein, the E183L gene encoding the p54 protein, and the central hypervariable region (CVR) of the B602L gene encoding the J9L protein. In addition, to serotype and discriminate between closely related strains, the EP402L (CD2v) gene and the intergenic region between the I73R and I329L genes were analyzed. RESULTS: ASFV was confirmed in 26 of 391 pigs tested. However, only 19 and 15 PPA-positive samples, respectively, were successfully sequenced and phylogenetically analyzed for p72 (B646L) and p54 (E183L). All the ASFV studied were of genotype X. The CVR tetrameric repeat clustered the ASFV strains in two subgroups: the Uvira subgroup (10 TRS repeats, AAAABNAABA) and another subgroup from all other strains (8 TRS repeats, AABNAABA). The phylogenetic analysis of the EP402L gene clustered all the strains into CD2v serogroup 7. Analyzing the intergenic region between I73R and I329L genes revealed that the strains were identical but contained a deletion of a 33-nucleotide internal repeat sequence compared to ASFV strain Kenya 1950. CONCLUSION: ASFV genotype X and serogroup 7 was identified in the ASF disease outbreaks in South Kivu province of DRC in 2018-2019. This represents the first report of ASFV genotype X in DRC. CVR tetrameric repeat sequences clustered the ASFV strains studied in two subgroups. Our finding emphasizes the need for improved coordination of the control of ASF.

Genetic profile of African swine fever virus responsible for the 2019 outbreak in northern Malawi.

BACKGROUND: African swine fever (ASF) is an infectious transboundary animal disease which causes high mortality, approaching 100% in domestic pigs and it is currently considered as the most serious constraint to domestic pig industry and food security globally. Despite regular ASF outbreaks within Malawi, few studies have genetically characterized the causative ASF virus (ASFV). This study aimed at genetic characterization of ASFV responsible for the 2019 outbreak in northern Malawi. The disease confirmation was done by polymerase chain reaction (PCR) followed by molecular characterization of the causative ASFV by partial genome sequencing and phylogenetic reconstruction of the B646L (p72) gene, nucleotide alignment of the intergenic region (IGR) between I73R and I329L genes and translation of the central variable region (CVR) coded by B602L gene. RESULTS: All thirteen samples collected during this study in Karonga district in September 2019 were ASFV-positive and after partial genome sequencing and phylogenetic reconstruction of the B646L (p72) gene, the viruses clustered into ASFV p72 genotype II. The viruses characterized in this study lacked a GAATATATAG fragment between the I173R and the I329L genes and were classified as IGR I variants. Furthermore, the tetrameric amino acid repeats within the CVR of the B602L gene of the 2019 Malawian ASFV reported in this study had the signature BNDBNDBNAA, 100% similar to ASFV responsible for the 2013 and 2017 ASF outbreaks in Zambia and Tanzania, respectively. CONCLUSIONS: The results of this study confirm an ASF outbreak in Karonga district in northern Malawi in September 2019. The virus was closely related to other p72 genotype II ASFV that caused outbreaks in neighboring eastern and southern African countries, emphasizing the possible regional transboundary transmission of this ASFV genotype. These findings call for a concerted regional and international effort to control the spread of ASF in order to improve nutritional and food security.

Neonatal and Juvenile Ocular Development in Göttingen Minipigs and Domestic Pigs: A Histomorphological and Immunohistochemical Study.

Pigs are considered one of the relevant animal models for ocular research as they share several histological and anatomical similarities with the human eye. With the increasing interest in juvenile animal models, this study aimed to describe the postnatal development of ocular structures in 16 Göttingen minipigs and 25 F2 domestic pigs, between birth and 6 months of age, using histopathology and immunohistochemistry against Ki-67, caspase-3, calbindin, glial fibrillary acidic protein, rhodopsin, and synaptophysin. All ocular structures in both pig breeds were incompletely developed at birth and for variable periods postnatally. Noteworthy histological features of immaturity included vascularization in the corneal stroma in neonatal Göttingen minipigs, increased cellularity in different substructures, remnants of the hyaloid vasculature, short and poorly ramified ciliary body processes, and a poorly developed cone inner segment. Increased cellular proliferation, highlighted by abundant Ki-67 immunolabeling, was observed in almost all developing structures of the pig eye for variable periods postnatally. Apoptosis, highlighted with caspase-3 immunolabeling, was observed in the retinal inner nuclear layer at birth and in the regressing hyaloid vasculature remnants. Immunohistochemistry against rhodopsin, synaptophysin, and calbindin demonstrated the short size of the developing photoreceptors and the immature cone inner segment morphology. Calbindin labeling revealed significant differences in the amount of positively labeled cone nuclei between the retinal area centralis and the non-area centralis regions. The elongation of Müller cell processes in the developing retina was shown with glial fibrillary acidic protein. In both pig breeds, the eyes reached histomorphological and immunohistochemical maturity at 6 months of age.

Toxoplasma gondii genotypes circulating in domestic pigs in Serbia.

Consumption of undercooked or raw pork is considered a significant risk factor for human infection with Toxoplasma gondii. In this study, we investigated the genetic structure of 18 T. gondii strains obtained from slaughter pigs from Northern Serbia (mainly Vojvodina). The examined samples originated from eight pigs from large commercial farms, six backyard pigs and four free-range Mangalica pigs, all found to be positive for either viable T. gondii or T. gondii DNA. Genotyping was attempted from both pig tissues and mouse brains from the bio-assays using a multiplex multilocus nested polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RFLP) method with seven markers (GRA6, alt. SAG2, PK-1, BTUB, C22-8, CS3 and Apico). Identification was achieved for nine T. gondii isolates. Seven isolates were classified as type II and two as type III. These results are consistent with previous studies on animal isolates from Serbia as well as with previous reports that type III is more frequently found in samples from Southern Europe than in those from other parts of the continent.

Osteochondrosis, Synovial Fossae, and Articular Indentations in the Talus and Distal Tibia of Growing Domestic Pigs and Wild Boars.

Articular osteochondrosis (OC) often develops in typical locations within joints, and the characterization of OC distribution in the pig tarsus is incomplete. Prevalence of OC is high in domestic pigs but is presumed to be low in wild boars. Postmortem and computed tomography (CT) examinations of the talus and distal tibia from 40 domestic pigs and 39 wild boars were evaluated for the locations and frequencies of OC, synovial fossae, and other articular indentations, and frequency distribution maps were made. All domestic pigs but only 5 wild boars (13%) had OC on the talus. In domestic pigs, OC consistently affected the axial aspect of the medial trochlea tali in 11 (28%) joints and the distomedial talus in 26 (65%) joints. In wild boars, all OC lesions consistently affected the distomedial talus. On the articular surface of the distal tibia, all domestic pigs and 34 wild boars (87%) had synovial fossae and 7 domestic pigs (18%) had superficial cartilage fibrillation opposite an OC lesion (kissing lesion). Other articular indentations occurred in the intertrochlear groove of the talus in all domestic pigs and 13 wild boars (33%) and were less common on the trochlea tali. The prevalence of tarsal OC in wild boars is low. In domestic pigs and wild boars, OC is typically localized to the distomedial talus and in domestic pigs also to the medial trochlea tali. Further investigations into the reasons for the low OC prevalence in wild boars may help in developing strategies to reduce OC incidence in domestic pigs.

The silent spread of Porcine Bocavirus in Croatian pigs: should we be concerned?

A survey was conducted to evaluate the presence and prevalence of Porcine Bocavirus (PBoV) in Croatian domestic pigs by means of PCR targeting the NS1 gene fragment of PBoV. This study included testing of faecal samples collected from 10 small commercial farms and 11 small backyard holdings in Croatia. The presence of PBoV was confirmed by PCR in 24 out of 57 composite faecal samples from small commercial farms and in 12 out of 43 composite faecal samples from small backyard holdings. The PCR products of 18 positive samples were sequenced for genotyping. PBoV sequences grouped into the PBoV-a, PBoV-b and PBoV-c groups with 90.81% to 99.25% nucleotide identity. All Croatian PBoV sequences showed a high nucleotide and amino acid identity with PBoV sequences from China and Hong Kong, the United States, Sweden, and Slovenia. These results clearly show that PBoV is circulating among the domestic pig population in Croatia.

Porcine Epidemic Diarrhea Virus and Discovery of a Recombinant Swine Enteric Coronavirus, Italy.

Porcine epidemic diarrhea virus (PEDV) has been detected sporadically in Italy since the 1990s. We report the phylogenetic relationship of swine enteric coronaviruses collected in Italy during 2007-2014 and identify a drastic shift in PEDV strain variability and a new swine enteric coronavirus generated by recombination of transmissible gastroenteritis virus and PEDV.

Experimental pig-to-pig transmission dynamics for African swine fever virus, Georgia 2007/1 strain.

African swine fever virus (ASFV) continues to cause outbreaks in domestic pigs and wild boar in Eastern European countries. To gain insights into its transmission dynamics, we estimated the pig-to-pig basic reproduction number (R 0) for the Georgia 2007/1 ASFV strain using a stochastic susceptible-exposed-infectious-recovered (SEIR) model with parameters estimated from transmission experiments. Models showed that R 0 is 2·8 [95% confidence interval (CI) 1·3-4·8] within a pen and 1·4 (95% CI 0·6-2·4) between pens. The results furthermore suggest that ASFV genome detection in oronasal samples is an effective diagnostic tool for early detection of infection. This study provides quantitative information on transmission parameters for ASFV in domestic pigs, which are required to more effectively assess the potential impact of strategies for the control of between-farm epidemic spread in European countries.

Survey of Trichinella infection from domestic pigs in the historical endemic areas of Henan province, central China.

The aim of this work was to investigate the current situation of Trichinella infection from domestic pigs in the historical endemic areas of Henan province, central China. A total of 823 diaphragm samples from the indoor-raised pigs were collected in five cities of Henan during 2014-2015 and examined by artificial digestion method. The overall prevalence of Trichinella infection in pigs was 0.61 % (5/823). Trichinella larvae were detected in 0.91 % (5/550) of pigs from Nanyang city of Henan. The larval burden in infected animals was 0.03 larvae per gram (lpg) of muscles with a range from 0.02 to 0.05 lpg. The larvae were identified as Trichinella spiralis by multiple PCR. Our study confirms the existence of swine trichinellosis in Henan, but the infection level was under the minimum level for defining infectious sources for humans. However, the prevalence of swine Trichinella infection in Henan need to be further evaluated with a large scale of pork samples for ensuring meat food safety.

Evaluation of Lab-on-a-Disc Technique Performance for Soil-Transmitted Helminth Diagnosis in Animals in Tanzania.

Soil-transmitted helminth (STH) infections are caused by roundworms, hookworms, whipworms, and thread worms. Accurate diagnosis is essential for effective treatment, prevention, and control of these infections. This study evaluates a new diagnostic method called Single-image Parasite Quantification (SIMPAQ), which uses a lab-on-a-disc (LoD) technique to isolate STH eggs into a single imaging zone for digital analysis. The study evaluates the purification performance of the SIMPAQ technique for detecting STH eggs in animal samples. This was a cross-sectional study conducted among 237 pigs and 281 dogs in the Morogoro region in Tanzania. Faecal samples were collected and processed with the LoD technique, as well as flotation and McMaster (McM) methods for comparison purposes. The overall prevalence of STH infections was high as per the LoD technique (74%), followed by McM (65.44%) and flotation (65.04%). Moreover, the overall performance of the LoD technique, using McM as the gold standard, was 93.51% (sensitivity), 60.89% (specificity), 81.91% (PPV), and 83.21% (NPV). The LoD technique exhibited high prevalence, sensitivity, and NPV, which demonstrates its value for STH egg detection and its crucial role in the era of accurate STH diagnosis, promoting proper management of the infection.

Evidence of Human-designated Antiretroviral (ARV) Drug Residues in Broiler Chicken, Domestic Pigs, and Animal Feeds in Tanzania.

Background: Recent reports have indicated the use of antiretroviral (ARV) drugs to boost animal production in neighboring Uganda, with further reports of use in several African countries. Methods: This cross-sectional study was conducted in nine districts in Tanzania, and involved screening for the presence of three first-line ARVs (lamivudine, nevirapine, and efavirenz) residues in the muscle and blood of domestic pigs and broiler chickens, and in sampled animal feed and water. Residues were determined using liquid chromatography and mass spectrometry (LC-MS/MS). The method involved calibration of the lower limit of quantitation (LLOQ) and limit of detection (LOD). ARVs were detected and quantified using the Multiple Reaction Monitoring (MRM) system. Results: 131 (66.8%) of the 196 samples of muscle, blood, and animal feed were found to contain lamivudine residues, with the highest concentration detected in domestic pig blood and muscle (7.58mg/kg) and the lowest concentration (0.01 mg/kg) in broiler chicken feed. There was a significant relationship between the presence of lamivudine by sample type and sample origin (p=0.000). Nevirapine and efavirenz drugs were not detected in any of the collected samples. No ARV residues were detected in water samples (n=37). Conclusion: This study confirms the use of ARVs in animal production in Tanzania as evidenced by the presence of residues in animal feeds. We found lamivudine residues in domestic pigs and broiler chickens at concentrations higher than those recently reported in other East African studies. Farmers living with HIV may be using ARVs from their prescribed medications, which may lead to poor adherence and the emergence of drug resistance. Besides direct human and animal health issues, these residues in animal feeds and animal excreta can lead to environmental contamination leading to several negative impacts. We recommend a total ban on human-designated ARVs in animal production and advocate for comprehensive studies and monitoring systems across African countries to reveal potential societal and other reasons for their use and provide comprehensive solutions using One Health approaches.

A Biochemical and Histological Assessment of Postmortem Changes to the Eyes of Domestic Pigs: A Preliminary Study.

The Postmortem Interval (PMI) is the time from the death of an animal to its discovery. From a veterinary forensic standpoint, an accurate estimation of the PMI is of particular importance, especially with the observed increase in deaths of domestic and wild animals. A preliminary study was conducted using the eyes of domestic pigs. A biochemical analysis was conducted on the vitreous humor of the eye, whilst a histological analysis was conducted on the retina. The eyes were stored at +4 °C and changes were assessed at time intervals of 0, 12, 24, 48, and 120 h. The biochemical analysis during the PMI established a decrease in sodium, chlorine, and glucose concentrations, and a rise in potassium concentration. Accordingly, a simple linear regression showed a significant correlation between changes in concentrations of sodium (Na+), potassium (K+), chloride (Cl-), and glucose, in relation to the PMI. The histological analysis showed evident morphological changes in the retina, which included homogenization of the rod and cone cells, pyknosis of the outer nuclear layer, homogenization of the outer plexiform layer, pyknosis of the inner nuclear layer, homogenization of the inner plexiform layer, and pyknosis of the nuclei of the ganglion layer of the retina.

African swine fever virus DNA is present in non-biting flies collected from outbreak farms in Romania.

BACKGROUND: African swine fever (ASF) is a highly contagious and severe haemorrhagic disease of Suidae, with mortalities that approach 100 percent. Several studies suggested the potential implication of non-biting dipterans in the spread of ASFV in pig farms due to the identification of the ASFV DNA. However, to our knowledge, no study has evaluated the viral DNA load in non-biting dipterans collected in outbreak farms and no risk factors have been analysed. In this context, our study aimed to analyse the risk factors associated with the presence of non-biting dipterans collected from ASF outbreaks in relation to the presence and load of viral DNA. METHODS: Backyard farms (BF), type A farms (TAF), and commercial farms (CF), were targeted for sampling in 2020. In 2021, no BF were sampled. Each farm was sampled only once. The identification of the collected flies to family, genus, or species level was performed based on morphological characteristics using specific keys and descriptions. Pools were made prior to DNA extraction. All extracted DNA was tested for the presence of the ASFV using a real-time PCR protocol. For this study, we considered every sample with a CT value of 40 as positive. The statistical analysis was performed using Epi Info 7 software (CDC, USA). RESULTS: All collected non-biting flies belonged to five families: Calliphoridae, Sarcophagidae, Fanniidae, Drosophilidae, and Muscidae. Of the 361 pools, 201 were positive for the presence of ASFV DNA. The obtained CT values of the positive samples ranged from 21.54 to 39.63, with a median value of 33.59 and a mean value of 33.56. Significantly lower CT values (corresponding to higher viral DNA load) were obtained in Sarcophagidae, with a mean value of 32.56; a significantly higher number of positive pools were noticed in August, mean value = 33.12. CONCLUSIONS: Our study brings compelling evidence of the presence of the most common synanthropic flies near domestic pig farms carrying ASFV DNA, highlighting the importance of strengthening the biosecurity measures and protocols for prevention of the insect life cycle and distribution.

Characterization of the Protective Cellular Immune Response in Pigs Immunized Intradermally with the Live Attenuated African Swine Fever Virus (ASFV) Lv17/WB/Rie1.

Candidate vaccines against African swine fever virus (ASFV) based on naturally attenuated or genetically modified viruses have the potential to generate protective immune responses, although there is no consensus on what defines a protective immune response against ASFV. Studies, especially in sensitive host species and focused on unravelling protective mechanisms, will contribute to the development of safer and more effective vaccines. The present study provides a detailed analysis of phenotypic and functional data on cellular responses induced by intradermal immunization and subsequent boosting of domestic pigs with the naturally attenuated field strain Lv17/WB/Rie1, as well as the mechanisms underlying protection against intramuscular challenge with the virulent genotype II Armenia/07 strain. The transient increase in IL-8 and IL-10 in serum observed after immunization might be correlated with survival. Protection was also associated with a robust ASFV-specific polyfunctional memory T-cell response, where CD4CD8 and CD8 T cells were identified as the main cellular sources of virus-specific IFNγ and TNFα. In parallel with the cytokine response, these T-cell subsets also showed specific cytotoxic activity as evidenced by the increased expression of the CD107a degranulation marker. Along with virus-specific multifunctional CD4CD8 and CD8 T-cell responses, the increased levels of antigen experienced in cytotoxic CD4 T cells observed after the challenge in immunized pigs might also contribute to controlling virulent infection by killing mechanisms targeting infected antigen-presenting cells. Future studies should elucidate whether the memory T-cell responses evidenced in the present study persist and provide long-term protection against further ASFV infections.