Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) via boar semen has been documented. Since semen is widely disseminated for artificial insemination and the virus can cause significant health and economic consequences, it is essential to have well-validated, rapid diagnostic techniques to detect and quantitate the virus for diagnostic and research purposes. Previously, boar semen was tested by a nested PCR (nPCR) assay which was compared to the "gold standard" swine bioassay. A correlation of 94% was observed, indicating that, most of the time, PCR detected infectious virus. Subsequently, a real-time PCR targeting the 3 untranslated region of the PRRSV genome was compared with nPCR by testing 413 serum and semen samples from PRRSV-inoculated and control boars. There was 95% agreement between the results of the two tests, with the majority of samples with discordant results containing virus at the lower range of detection by the assays. The virus in all samples was quantitated by using a standard curve obtained by serial dilution of an in vitro transcript. By using the in vitro transcript, the lower limit of sensitivity was observed to be approximately 33 copies/ml. Reactivity with a panel of more than 100 PRRSV isolates from various geographical regions in the United States was also documented. No reactivity with nine nonrelated swine viruses was noted. A real-time PCR was also developed for the detection of the European Lelystad virus and the Europeanlike PRRSV now found in the United States. In six of six PRRSV-inoculated boars, peak levels of viremia occurred at 5 days postinoculation (DPI) and were most consistently detectable throughout 22 DPI. In five of six boars, PRRSV was shed in semen for 0 to 2 days during the first 10 DPI; however, one of six boars shed the virus in semen through 32 DPI. Therefore, in general, the concentration and duration of PRRSV shedding in semen did not correlate with the quantity or duration of virus in serum. These differences warrant further studies into the factors that prevent viral replication in the reproductive tract.Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded RNA virus belonging to the order Nidovirales, family Arteriviridae, along with lactate dehydrogenaseelevating virus of mice, equine arteritis virus, and simian hemorrhagic fever virus (4, 13). PRRSV causes significant respiratory disease in nursery and growing-finishing pigs and late-term abortions in sows (29,46). Since the etiologic agent was identified in 1991, multiple methods of virus dissemination have been discovered, including transmission via semen (2,12,18,30,47,50). Transmission of PRRSV through the semen of infected boars may not always occur, but due to the widespread use of artificial insemination in the modern swine industry, dissemination of PRRSV via semen constitutes a major risk for swine herds. Therefore, it is important to provide diagnostic testing to ensure a PRRSV-free semen supply (41). Interestingly, the duration of PRRSV shedd...

Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick’s next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.

There is a need for genetic markers or biomarkers that can predict resistance towards a wide range of infectious diseases, especially within a health environment typical of commercial farms. Such markers also need to be heritable under these conditions and ideally correlate with commercial performance traits. In this study, we estimated the heritabilities of a wide range of immune traits, as potential biomarkers, and measured their relationship with performance within both specific pathogen-free (SPF) and non-SPF environments. Immune traits were measured in 674 SPF pigs and 606 non-SPF pigs, which were subsets of the populations for which we had performance measurements (average daily gain), viz. 1549 SPF pigs and 1093 non-SPF pigs. Immune traits measured included total and differential white blood cell counts, peripheral blood mononuclear leucocyte (PBML) subsets (CD4+ cells, total CD8α+ cells, classical CD8αβ+ cells, CD11R1+ cells (CD8α+ and CD8α-), B cells, monocytes and CD16+ cells) and acute phase proteins (alpha-1 acid glycoprotein (AGP), haptoglobin, C-reactive protein (CRP) and transthyretin). Nearly all traits tested were heritable regardless of health status, although the heritability estimate for average daily gain was lower under non-SPF conditions. There were also negative genetic correlations between performance and the following immune traits: CD11R1+ cells, monocytes and the acute phase protein AGP. The strength of the association between performance and AGP was not affected by health status. However, negative genetic correlations were only apparent between performance and monocytes under SPF conditions and between performance and CD11R1+ cells under non-SPF conditions. Although we cannot infer causality in these relationships, these results suggest a role for using some immune traits, particularly CD11R1+ cells or AGP concentrations, as predictors of pig performance under the lower health status conditions associated with commercial farms.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by a positive RNA strand arterivirus. PRRS virus (PRRSV) interacts primarily with lung macrophages. Identifying the genetic components involved in host resistance/susceptibility would represent an important step forward in the design of disease control programs. In this study, alveolar macrophages derived from five commercial pig lines were used to study the innate immune response to PRRSV infection in vitro. Analysis by flow cytometry has demonstrated that bronchial alveolar lavage fluid (BALF) preparations were almost exclusively composed of alveolar macrophages and that the pigs tested were free from infection. Macrophages from the Landrace line showed significantly reduced virus replication and poor growth of PRRSV during 30 h of infection. By 72 h, PRRSV viral load was down to 2.5 log(10) TCID(50) compared with an average of 5 log(10) TCID(50) for the other breeds tested. These observations suggest that factors intrinsic to the Landrace breed may be responsible for this reduced or delayed response to PRRSV. Preliminary investigation suggests that the PRRSV coreceptor, sialoadhesin, may not be responsible for the Landrace macrophage phenotype as its abundance and localisation were comparable in all the breeds. Strikingly, we found that the reduced or delayed growth of PRRSV was temporally associated with high levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-8 mRNA accumulation and substantial reduction of secretion of IL-8, suggesting a key contributory role for cytokine synthesis and secretion during the innate immune response to PRRSV infection.

No abstract