Artificial Insemination as an Alternative Transmission Route for African Swine Fever Virus.

The rapid spread of the African swine fever virus (ASFV), causing severe disease with often high fatality rates in Eurasian suids, prevails as a threat for pig populations and dependent industries worldwide. Although advancing scientific progress continually enhances our understanding of ASFV pathogenesis, alternative transmission routes for ASFV have yet to be assessed. Here, we demonstrate that ASFV can efficiently be transferred from infected boars to naïve recipient gilts through artificial insemination (AI). In modern pig production, semen from boar studs often supplies many sow herds. Thus, the infection of a boar stud presents the risk of rapidly and widely distributing ASFV within or between countries. Daily blood and semen collection from four boars after intramuscular inoculation with ASFV strain 'Estonia 2014' resulted in the detection of ASFV genomes in the semen as early as 2 dpi, in blood at 1 dpi while semen quality remained largely unaffected. Ultimately, after insemination with extended semen, 7 of 14 gilts were ASFV positive by 7 days post insemination, and all gilts were ASFV positive by 35 days post insemination. Twelve out of 13 pregnant gilts aborted or resorbed at the onset of fever. A proportion of fetuses originating from the remaining gilt showed both abnormalities and replication of ASFV in fetal tissues. Thus, we present evidence for the efficient transmission of ASFV to gilts via AI and also to implanted embryos. These results underline the critical role that boar semen could play in ASFV transmission.

Effective biosecurity to protect North American studs and clients from emerging infectious disease.

Protecting boar studs and their clients from emerging infectious disease first involves effective biosecurity measures to keep a disease out that was not present, and second, early identification and ceasing semen distribution prior to disseminating infectious disease. Experiences in the field can best guide us as to what has been effective. Circumstances in North America in the period of 1999-2004 resulted in numerous PRRS virus (Porcine Reproductive and Respiratory Syndrome) negative boar studs becoming infected and disseminating virus to sow farms. Earlier detection methods were needed, and withholding of semen pending negative test results became standard. To accomplish this, diagnostic labs complied with industry requests for same day testing. At the same time, research efforts helped clarify the major routes of PRRS virus introduction into the farms. The risk of fomites and aerosol spread became viewed as major risks. Addressing issues with people and supply entry alone did not eliminate new virus entry. The implementation of air filtration during 2005-2008 had a major impact on the rate of new virus introductions into boar studs after other measures alone were unsuccessful. Risks exposed with the introduction of PED virus (Porcine Epidemic Diarrhea) into North America further highlighted other risk factors such as feed ingredients, trailer sanitation, and the presence of clear physical barriers. The successful adaptation of testing procedures, combined with biosecurity procedures including air filtration, has made the incidence of infectious disease introduction extremely rare in North American boar studs over the last decade. While survivability of infectious disease agents can vary in different materials or in the air, successful protocols should be applied and adjusted as needed to accommodate new information or risks. Cleary defined physical barriers for people and animal entry and exit, sanitization and/or down time on incoming supplies, risk mitigation and testing of feed ingredients, and filtration have been keys to changing the incidence of emerging infectious disease introduction into boar studs.

Novel and differentially abundant microRNAs in sperm cells, seminal plasma, and serum of boars due to porcine reproduction and respiratory syndrome virus infection.

The objectives of this study were to identify and determine relative abundance of miRNAs in boar sperm, seminal plasma (SP), and serum pre- and post-viral infection. Functional enrichment analyses on predicted targets of miRNAs of interest were performed. Boars (n = 6) were inoculated with porcine reproductive and respiratory syndrome virus (PRRSv) strain 1-8-4 (Day 0). Semen and serum were collected on Day -2 and 6. Sperm and SP were separated and aliquots were flash frozen and stored at -80 °C. Serum was frozen and stored at -80 °C. Total RNA was isolated from sperm and SP samples and subjected to RNA sequencing. Microarray analysis was performed using the Day -2 and 6 RNA samples from serum, sperm and SP. Potential miRNA targets were predicted using miRanda 3.3a and targets were then analyzed for enrichment of Gene Ontology) and InterPro terms and were considered to be enriched if P < 0.01 using the Bonferroni correction. Microarray analyses resulted in 83, 13, and 10 miRNAs with differences in abundances in sperm, serum, and SP, respectively, when comparing Day -2 and 6. Results from enrichment analyses indicated that the predicted targets of 35, nine, and five miRNAs with differences in abundances for sperm, SP, and serum, respectively, that have functions and/or conserved protein domains that are enriched when compared to the pig genome. Enriched terms for P2X purinoceptors were identified for sperm, SP and serum. Enriched terms for cell adhesion were identified for sperm and serum transcripts. Enriched terms for cell signaling were identified for sperm and SP transcripts.

Evaluation of the long-term effect of air filtration on the occurrence of new PRRSV infections in large breeding herds in swine-dense regions.

Airborne transmission of porcine reproductive and respiratory syndrome virus (PRRSV) is a risk factor for the infection of susceptible populations. Therefore, a long­term sustainability study of air filtration as a means to reduce this risk was conducted. Participating herds (n = 38) were organized into 4 independent cohorts and the effect of air filtration on the occurrence of new PRRSV infections was analyzed at 3 different levels from September 2008 to January 2012 including the likelihood of infection in contemporary filtered and non-filtered herds, the likelihood of infection before and after implementation of filtration and the time to failure in filtered and non-filtered herds. Results indicated that new PRRSV infections in filtered breeding herds were significantly lower than in contemporary non-filtered control herds (P < 0.01), the odds for a new PRRSV infection in breeding herds before filtration was 7.97 times higher than the odds after filtration was initiated (P < 0.01) and the median time to new PRRSV infections in filtered breeding herds of 30 months was significantly longer than the 11 months observed in non-filtered herds (P < 0.01). In conclusion, across all 3 levels of analysis, the long-term effect of air filtration on reducing the occurrence of new PRRSV infections in the study population was demonstrated.

Evaluation of surveillance protocols for detecting porcine reproductive and respiratory syndrome virus infection in boar studs by simulation modeling.

Because porcine reproductive and respiratory syndrome virus (PRRSV) can be transmitted through semen, PRRSV-free boar studs need to be routinely monitored to rapidly detect any potential PRRSV introduction. However, current protocols for monitoring PRRSV in boar studs are diverse, sometimes very costly, and their effectiveness has not been quantified. The objective of this study was to evaluate the ability of different monitoring protocols to detect PRRSV introduction into a negative boar stud by using a simulation modeling approach. A stochastic transmission model was constructed to simulate the spread of PRRSV in a typical negative boar stud in the USA (herd size of 200 boars, 60% annual replacement) and the performance of monitoring protocols by using different sample sizes (10, 30, and 60 samples), sampling frequency (3 times a week, weekly, and biweekly), and diagnostic procedures (PCR on semen, PCR on serum, ELISA on serum, and both PCR and ELISA on serum). The monitoring protocols were evaluated in terms of the time from PRRSV introduction into the boar stud to PRRSV detection. Protocols that used PCR on serum detected the PRRSV introduction earlier than protocols that used PCR on semen, and these were earlier than those that used ELISA on serum. The most intensive protocol evaluated (testing 60 boars 3 times a week by PCR on serum) would need 13 days to detect 95% of the PRRSV introductions. These results support field observations, suggesting that an intensive monitoring protocol needs to be in place in a boar stud to quickly detect a PRRSV introduction.

Evaluation of the sensitivity of reverse-transcription polymerase chain reaction to detect porcine reproductive and respiratory syndrome virus on individual and pooled samples from boars.

Boar studs are continuously monitored for the presence of porcine reproductive and respiratory syndrome virus (PRRSV) by testing different biological samples by reverse-transcription polymerase chain reaction (RT-PCR). In most cases, samples are run in pools, even though the impact of pooling on the sensitivity of RT-PCR is unknown. The objective of this study was to evaluate the feasibility of using PCR on pooled samples through the estimation of the sensitivity of RT-PCR on different biological samples run individually, in pools of 3 and in pools of 5. Twenty-nine boars were inoculated with a low virulent PRRSV isolate. Serum, blood swab, and semen samples were obtained from each boar every 2 to 3 days for 2 weeks. Each sample was tested by RT-PCR undiluted or diluted 1:3 and 1:5 with negative samples. Eleven of the 29 boars did not appear to get infected from the inoculum, as evidenced by no seroconversion 15 days after inoculation. Data from the other 18 boars showed that serum was the best sample to detect PRRSV during acute infection, with the blood swab sample performing almost as well. Semen samples failed to detect PRRSV infection in most of the cases. Pooling samples at pool sizes of 3 and 5 resulted in a decrease in the sensitivity of RT-PCR. Sensitivity was reduced by 6% and 8%, respectively, when serum or blood swab samples were run in pools of 5. The impact of pooling on the sensitivity of PCR was higher in samples taken during the beginning of the viremic period.

Assessment of sexual behavior and effect of semen collection pen design and sexual stimulation of boars on behavior and sperm output--a review.

The importance of sexual behavior and factors influencing sexual behavior of AI boars has received minimal study. The majority of studies reviewed used a very small number of boars. A sexual behavior index (SBI) has been developed for naturally mating boars but not for AI boars. Some studies have reported significant correlations between sexual behavior traits and semen characteristics; while other studies did not find significant correlations. A new semen collection pen design (Reicks Design) has reduced the duration of time a boar requires to mount a dummy sow after entering the collection pen and the duration of time needed to exit the collection pen after ejaculation. In general, the observation of another boar mounted on the dummy sow prior to collection, releasing the penis after extension, exposing boars to non-estrous gilts for 2 days before collecting semen, placing a non-estrous gilt underneath a dummy, and removing the boar for 2 min after first mount did not enhance the number of sperm cells collected. Treatment of boars with PGF2alpha has facilitated the training of sexually experienced boars to mount a dummy sow but not that of sexually inexperienced boars. In general, the treatment of boars with PGF2alpha did not increase the total number of spermatozoa ejaculated.