Molecular genetic analysis of VRK1 in mammary epithelial cells: depletion slows proliferation in vitro and tumor growth and metastasis in vivo.

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family. VRK1, a ser/thr kinase with a nuclear localization, is the most well-studied paralog and has been described as a proproliferative protein. In lower eukaryotes, a loss of VRK1 activity is associated with severe mitotic and meiotic defects. Mice that are hypomorphic for VRK1 expression are infertile, and depletion of VRK1 in tissue culture cells can impair cell proliferation and alter several signaling pathways. VRK1 has been implicated as part of a 'gene-expression signature' whose overexpression correlates with poor clinical outcome in breast cancer patients. We present here our investigation of the role of VRK1 in the growth of normal (MCF10) and malignant (MDA-MB-231) human mammary epithelial cells, and demonstrate that shRNA-mediated depletion of VRK1 slows their proliferation significantly. Conversely, stable overexpression of a FLAG-tagged VRK1 transgene imparts a survival advantage to highly malignant MDA-MB-231 cells under conditions of nutrient and growth factor deprivation. Moreover, in a murine orthotopic xenograft model of breast cancer, we demonstrate that tumors depleted of VRK1 show a 50% reduction in size from 4-13 weeks postengraftment. The incidence and burden of distal metastases in the lungs and brain was also significantly reduced in mice engrafted with VRK1-depleted cells. These studies demonstrate that VRK1 depletion or overexpression has an impact on the proliferation and survival of cell lines derived from normal or malignant mammary tissue, and moreover show that depletion of VRK1 in MDA-MB-231 cells reduces their oncogenic and metastatic properties in vivo.

Cow-level association between serum 25-hydroxyvitamin D concentration and Mycobacterium avium subspecies paratuberculosis antibody seropositivity: a pilot study.

Vitamin D deficiency has been associated with various human diseases. Therefore, the objective of this study was to evaluate the cow-level association between serum 25-hydroxyvitamin D [25(OH)D] concentration and Mycobacterium avium ssp. paratuberculosis (MAP) seropositivity of dairy cows, adjusting for diet, breed, hair coat color, stage of lactation, reproductive status, and cow age. The sera of 80 MAP antibody ELISA-positive and 80 test-negative herd mates from 5 Minnesota dairy herds were analyzed for 25(OH)D and 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. The cows' age, production records, and hair coat color were recorded. Additionally, feed samples were obtained and analyzed for vitamin D(2) and vitamin D(3) content. A linear mixed model was used to identify potential predictors for serum 25(OH)D concentration, accounting for herd of origin. The majority of rations analyzed had over 22,000 IU of vitamin D/day (maximum: 52,000 I U/d) and the study cows' average serum 25(OH)D concentration was 62.5 ± 13.8 ng/mL. Serum ELISA-positive cows had, on average, 5.3 ng/mL lower 25(OH)D serum levels than test-negative herd mates. The reproductive status of cows was also associated with the 25(OH)D levels, with fresh cows having the lowest serum concentration. In this cross-sectional study, a temporal or causal association between MAP antibody ELISA status and serum 25(OH)D concentration could not be evaluated. In addition, the high levels of vitamin D in the rations of participating farms and the average 25(OH)D serum concentration suggest that additional supplementation with vitamin D in the ration is likely to be ineffective.

Effect of cross-fostering on transfer of maternal immunity to Mycoplasma hyopneumoniae to piglets.

The aim of this study was to assess the effect of cross-fostering on transfer of maternal Mycoplasma hyopneumoniae-specific humoral and cell-mediated immunity (CMI) from gilts to piglets. Cross-fostering, carried out within gilt pairs, was based on the gilts' M hyopneumoniae vaccination status in accordance with the following scheme: six pairs of vaccinated gilt × non-vaccinated gilt (V × N); five pairs of non-vaccinated gilt × vaccinated gilt (N × V); and five pairs of vaccinated gilt × vaccinated gilt (V × V). The piglets were cross-fostered at 0, six, 12 or 20 hours after birth. Two piglets per gilt per time point were cross-fostered (that is, eight piglets per gilt were moved) and the remaining piglets served as non-cross-fostered controls. In addition, four litters served as non-cross-fostered controls. A maximum of 10 piglets per gilt were sampled. The piglets' M hyopneumoniae-specific humoral immunity was assessed by ELISA and their CMI was assessed by delayed-type hypersensitivity testing. M hyopneumoniae-specific antibodies were detected in non-cross-fostered piglets from vaccinated dams and from piglets cross-fostered within the V × N gilt pair at six hours or more, and within the V × V gilt pair at all time points. Piglets cross-fostered within the N × V gilt pair had detectable M hyopneumoniae-specific antibodies only if they had been moved within six hours of birth. The transfer of M hyopneumoniae-specific CMI to piglets appeared to be source-dependent, and was detected only in piglets maintained on their vaccinated dams for at least 12 hours after birth.

Effect of dietary supplementation of oregano essential oils to sows on colostrum and milk composition, growth pattern and immune status of suckling pigs.

This study evaluated the effects of supplementing sow diets with oregano essential oils (OEO) during gestation and lactation on sow colostrum and milk composition and on the growth pattern and immune status of suckling pigs. A total of 70 second-parity sows were randomly assigned to 1 of 2 gestation dietary treatments within 24 h after service: control (CON) or CON + 250 mg/kg of OEO (OREG). In lactation, sows were again assigned to either the CON or OREG dietary treatment. Thus, the lactation treatments were CON-CON, CON-OREG, OREG-CON, and OREG-OREG. Colostrum and blood samples were collected from 6 sows per lactation dietary treatment. Thymus lymphocyte (T lymphocyte) subpopulations (γδ, cluster of differentiation 8, and 32 cluster of differentiation 4) were enumerated in blood and mammary secretions along with IGF-1, IgG, and IgA concentrations. Piglet growth rate were determined from 18, 17, 17, and 18 litters from the CON-CON, CON-OREG, OREG-CON, and OREG-OREG lactation dietary treatments, respectively. Growth rates were determined in 630 piglets, and piglets were individually identified and weighed on 1, 5, 9, 12, 16, and 19 d of age. Oregano essential oil supplementation during gestation or lactation had no effect (P > 0.05) on GE, CP, GE:CP, GE:fat, and IGF-1 in sow milk. Reductions of the fat percentage in milk on d 7 (P < 0.05) and d 14 (P = 0.07) were found in sows supplemented with OEO during lactation compared with those in the CON treatment. Milk from sows supplemented with OEO during lactation had the greatest number of T lymphocytes compared with those in the lactation CON treatment on d 14 of lactation (P < 0.01). The number of T lymphocytes in milk was greater for sows in the CON-OREG treatment compared with those other treatments on d 14 of lactation (P < 0.05). Energy intake was greater on d 1 to 5 in piglets from sows fed OEO during gestation than those from sows in the CON treatment (P < 0.05). A trend (P = 0.10) for greater milk intake was observed in piglets from sows supplemented with OEO during gestation compared with those from sows in the CON treatment. Similarly, a tendency for an increase in ADG on d 1 to 5 was found in piglets from sows supplemented with OEO during gestation compared with those from sows in the CON treatment (P = 0.10). Insulin-like growth factor-1 at birth and on d 7 and 14 of lactation did not differ among piglets from sows assigned to the different dietary treatments. Oregano essential oil supplementation of sow diets did not affect (P > 0.05) immunoglobulin concentrations in piglets after suckling. Supplementing sow diets with OEO during gestation or lactation did not affect (P > 0.05) the T lymphocytes, percentage of T-lymphocyte subpopulations, and natural killer cell activity of piglets during lactation. Supplementing sow diets with 250 mg/kg of OEO during gestation and lactation did not affect the growth potential of and immune responses in suckling piglets.

Effects of feeding heat-treated colostrum on passive transfer of immune and nutritional parameters in neonatal dairy calves.

The first objective of this study was to describe the effect of on-farm heat treatment of colostrum on colostral bacteria counts and IgG concentrations. The second objective was to describe the effect of feeding heat-treated (vs. raw) colostrum on passive transfer of colostral immune and nutritional parameters in neonatal calves. Pooled batches of colostrum were mixed and divided equally: one half was fed raw whereas the other half was fed after heat treatment at 60 degrees C for 60 min using a commercial on-farm batch pasteurizer. Colostrum samples were cultured for total bacteria count and total coliform count and analyzed for total IgG concentration. Forty-nine Holstein calves were fed either raw colostrum (n = 24) or heat-treated colostrums (n = 25) within 1 to 2 h after birth. Serum samples collected from calves at 0 h (precolostrum) and 24 h (postcolostrum) were assayed for serum total protein; IgG, IgA, and IgM concentrations; peripheral total leukocyte counts; neutrophil counts; lymphocyte counts; lymphocyte phenotypes; vitamin A, vitamin E, cholesterol, and beta-carotene concentrations. Serum samples collected from 2- to 5-d-old calves were tested for immunoglobulin function via a bovine viral diarrhea virus type I serum neutralization titer and for neutrophil bacterial opsonization activity. On-farm batch heat treatment of colostrum at 60 degrees C for 60 min resulted in lower colostrum bacteria concentrations while maintaining colostral IgG concentration. Calves fed heat-treated colostrum had significantly greater serum total protein and IgG concentrations at 24 h, plus greater apparent efficiency of IgG absorption (total protein = 6.3 mg/dL; IgG = 22.3 mg/mL; apparent efficiency of absorption = 35.6%) compared with calves fed raw colostrum (TP = 5.9 mg/dL; IgG = 18.1 mg/mL; apparent efficiency of absorption = 26.1%). There was no effect of treatment on serum concentrations of IgA, IgM, vitamin A, vitamin E, cholesterol, beta-carotene or vitamin E:cholesterol ratio, or on serum bovine viral diarrhea virus type I serum neutralization titers. There was no difference between treatment groups when examining calf plasma total leukocyte counts, neutrophil counts, lymphocyte counts, or neutrophil opsonization activity. However, the latter results were considered inconclusive.

Leptin increases T-cell immune response in birds.

Leptin is a 16 kDa protein hormone that besides being a satiety factor also functions as a pleiotropic molecule regulating endocrine and immune functions. The aim of this study was to investigate the role of leptin on mitogen stimulated T-lymphocyte proliferation in birds. In the first experiment (in vitro), peripheral blood was collected from turkeys and lymphocytes were isolated from samples. Recombinant chicken leptin was added at concentrations of 0, 10(-9), 10(-8), 10(-7), and 10(-6) M prior to mitogen stimulation with Concavalin A. BrdU incorporation allowed us to detect T-cell proliferation using intracellular labeling of nucleic acids. Mitogen activation induced with Concavalin A caused a proliferation of T-cells that was positively correlated with the concentration of leptin. In the second experiment (in vivo), asian blue quail were fitted with osmotic pumps releasing leptin and injected with phytohemagglutinin (PHA) in their wing-webs before, during, and after leptin administration. The response to mitogen was greater in leptin treated birds during the leptin administration, but not before or after. These findings demonstrate that leptin enhances mitogen stimulated T-cell proliferation in birds. The results correspond with previous reports on mammals.

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Diagnostic investigation of chronic porcine reproductive and respiratory syndrome virus in a breeding herd of pigs.

Forty-five sows and 15 boars were selected at random from a breeding herd known to be chronically infected with porcine reproductive and respiratory syndrome virus (PRRSV) and lymphoid, immune-privileged, and non-lymphoid/non-immune-privileged tissues were tested for the presence of the virus by PCR, virus isolation, and immunohistochemistry. The virus was isolated from the lateral retropharyngeal lymph node of one sow; the isolate was nucleic acid sequenced and determined to be of field origin, and it was inoculated into two PRRSV-naive pregnant sows (A and B) at 95 days of gestation. They were necropsied 14 days later and samples of maternal and fetal tissue and blood samples were collected. Sow A had 10 fresh, six partially autolysed, and two mummified fetuses, and sow B had six fresh and viable fetuses. Viral nucleic acid was detected by PCR in tissue pools from each sow and also from pooled fetal tissues, and the virus was isolated from fetal pools from sow A.

An evaluation of test and removal for the elimination of porcine reproductive and respiratory syndrome virus from 5 swine farms.

The objective of this field study was to evaluate the protocol of test and removal (T&R) for the elimination of porcine reproductive and respiratory syndrome virus (PRRSV) from 5 chronically infected breeding herds. The T&R protocol involved sampling the entire breeding herd in one day, testing sera by polymerase chain reaction and ELISA to detect previously exposed and/or infected animals, and subsequently removing them from the herd. Following completion of T&R, breeding herds were monitored for 12 consecutive months, using ELISA, for the presence of antibodies to PRRSV. In order to be classified as a PRRSV-negative herd, all samples collected over the 12-month monitoring period were required to be negative by ELISA (s/p ratio < 0.4). At the conclusion of the monitoring period, all 5 farms were PRRSV-negative, according to the defined testing criteria. Approximately 2.2% (74/3408) ELISA false positive samples were detected across all 5 farms during the monitoring period. The diagnostic cost required during the T&R protocol was approximately US $10.66 per animal tested. Limitations of the study were a lack of herds with large (> 2000 sows) breeding herd inventories, and herds with a history of PRRSV vaccination.

Transmission of porcine reproductive and respiratory syndrome virus from persistently infected sows to contact controls.

The objective of this study was to determine if porcine reproductive and respiratory syndrome virus (PRRSV) could persist in non-pregnant sows and if persistently infected sows could transmit virus to naive contact controls. Twelve PRRSV-naive, non-pregnant sows (index sows) were infected with a field isolate of PRRSV and housed in individual isolation rooms for 42 to 56 days postinfection. Following this period, 1 naive contact sow was placed in each room divided by a gate allowing nose-to-nose contact with a single index sow. Index sows were not viremic at the time of contact sow entry. Virus nucleic acid was detected by polymerase chain reaction, and infectious virus was detected by virus isolation in sera from 3 of the 12 contact sows at 49, 56, and 86 days postinfection. All 3 infected contacts developed PRRSV antibodies. Virus nucleic acid was detected in tissues of all of the 12 index sows at 72 or 86 days postinfection. Nucleic acid sequencing indicated that representative samples from index and infected contacts were homologous (> 99%) to the PRRSV used to infect index sows at the onset of the study. This study demonstrates that PRRSV can persist in sows and that persistently infected sows can transmit virus to naive contact animals.

Epidemiological and diagnostic observations following the elimination of porcine reproductive and respiratory syndrome virus from a breeding herd of pigs by the test and removal protocol.

The protocol of test and removal for the elimination of porcine reproductive and respiratory syndrome (PRRS) virus was applied to an 825-sow breeding herd. All the adult animals were tested and serum samples analysed by ELISA and polymerase chain reaction (PCR). Eighty-eight animals (10 x 7 per cent) were removed from the herd and, of these, three were ELISA-pOSitive and PCR-positive, and 85 were ELISA-positive and PCR-negative. They tended to be either individual sows, or groups of four to six animals housed in adjacent gestation stalls. Four of the ELISA-positive, PCR-negative sows were slaughtered and PRRS virus nucleic acid was detected in a sample of sternal lymph node from one of them. After the completion of the test and removal protocol, the breeding and finishing populations were monitored for 12 consecutive months by ELISA. The 960 samples taken were negative for PRRS virus antibodies.

Molecular responses of macrophages to porcine reproductive and respiratory syndrome virus infection.

The detailed mechanism(s) by which porcine reproductive and respiratory syndrome virus (PRRSV) impairs alveolar Mo homeostasis and function remains to be elucidated. We used differential display reverse-transcription PCR (DDRT-PCR) to identify molecular genetic changes within PRRSV-infected Mo over a 24 h post infection period. From over 4000 DDRT-PCR amplicons examined, 19 porcine-derived DDRT-PCR products induced by PRRSV were identified and cloned. Northern blot analysis confirmed that four gene transcripts were induced during PRRSV infection. PRRSV attachment and penetration alone did not induce these gene transcripts. DNA sequence revealed that one PRRSV-induced expressed sequence tag (EST) encoded porcine Mx1, while the remaining 3 clones represented novel ESTs. A full-length cDNA clone for EST G3V16 was obtained from a porcine blood cDNA library. Sequence data suggests that it encodes an ubiquitin-specific protease (UBP) that regulates protein trafficking and degradation. In pigs infected in vivo, upregulated transcript levels were observed for Mx1 and Ubp in lung and tonsils, and for Mx1 in tracheobronchial lymph node (TBLN). These tissues correspond to sites for PRRSV persistence, suggesting that the Mx1 and Ubp genes may play important roles in clinical disease during PRRSV infection.

IFN gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages.

This study reports the effect of IFN gamma on the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in macrophages. Pretreatment with IFN gamma profoundly affected PRRSV replication in porcine macrophages evaluated by reduction in titer and percentage of positive cells. The effect of IFN gamma on PRRSV replication was both dose-dependent and related to the time of exposure. The mechanism of action was not due to blocking virus attachment. The inhibitory effect on PRRSV replication in macrophages suggests that IFN gamma may play an important role in protection.

The effect of spermatozoa and seminal plasma on leukocyte migration into the uterus of gilts.

Yorkshire x Landrace gilts were used to determine the effect of spermatozoa and seminal plasma on postbreeding uterine leukocyte influx. Estrus detection was performed with a boar at 12-h intervals following synchronization with 400 IU eCG and 200 IU of hCG. All gilts were AI once, 24 h after the detection of estrus following random assignment to a 2x2x3 factorial arrangement of treatments (sperm or sperm-free AI doses), AI dose medium (seminal plasma or PBS), and lavage time following AI. Gilts were treated with sperm (5x10(9) spermatozoa; SPZ; n = 30) or sperm-free (SF; n = 30) doses containing either 100 mL of seminal plasma (SP; n = 15/treatment) or PBS (n = 15/treatment). Uterine lavage was performed once on each gilt (n = 20/time) at one of three times after AI (6, 12, or 36 h) to determine the total number of uterine leukocytes. The leukocytes consisted predominately (92 to 99%) of polymorphonuclear neutrophilic granulocytes (PMN). There was an AI x medium interaction on uterine PMN numbers. The number of uterine PMN recovered from gilts inseminated with sperm suspended in PBS was greater than the number of PMN recovered from the uterine lumen of gilts inseminated with sperm in SP, SP alone, or PBS alone (P<.05). Furthermore, SP accelerated the rate of uterine clearance when suspended with sperm cells during the first 36 h following AI (P<.05). These results indicate that seminal plasma suppresses PMN migration into the uterus following breeding and enhances the rate of disappearance of uterine inflammation.

T cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus.

The identification of antigens recognized by T cell responses has become fundamental for developing effective immunizations against viral infections. Lymphocyte proliferation and delayed-type hypersensitivity responses to porcine reproductive and respiratory syndrome virus (PRRSV) infection have been demonstrated. However, the polypeptide specificity of T cell responses to PRRSV is unknown. To identify the PRRSV polypeptides recognized by porcine lymphocytes two approaches were employed. First polypeptides of purified virions were separated by SDS-PAGE and particle suspensions obtained from nitrocellulose blots were used as antigens. Second, the polypeptides encoded by ORFs 2, 4, 5, 6, and 7 of the strain VR-2332 were expressed as fusion proteins with a histidine tag in mammalian cells, using vaccinia virus as expression system. Significant antigen-specific proliferation responses to the matrix and envelope proteins from purified virions were obtained. This finding was supported by specific and dose-dependent proliferation responses to the recombinant polypeptides encoded by ORF2, 5 and 6 detected in virus-infected but not in control pigs. These results demonstrate that T-cell responses can be detected to individual PRRSV polypeptides. The greater response to the product of ORF6 than to the other PRRSV polypeptides indicates that the viral matrix polypeptide may have a major role in cellular immunity.

Effect of porcine reproductive and respiratory syndrome virus infection on the clearance of Haemophilus parasuis by porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection in young piglets is frequently associated with secondary infection due to various pathogens, especially those of the respiratory tract. One of the most important mechanisms in respiratory diseases is related to the alteration of function of porcine alveolar macrophages (PAMs). The objective of this study was to determine how PRRS virus infection affects the capabilities of PAMs in the phagocytosis and destruction of Haemophilus parasuis. Phagocytosis percentages were determined in vitro and ex vivo, after collected PAMs were directly exposed to the virus of if PAMs were collected from piglets previously infected with PRRSV. In vitro experiments demonstrated that H. parasuis uptake by PAMs is only increased in the early stages of PRRSV infection (2 h post-infection). In contrast, in the ex vivo experiments it was shown that PAMs from PRRSV-infected piglets do not seem to change in their phagocytic rate until the later stages of infection. Together with a decrease in the phagocytic rate, a marked decrease in the functional ability of PAMs to kill bacteria was observed 7 d post-infection. It is hypothesized that when animals are exposed to PRRSV, there is a marked decrease in the functional ability of PAMs to kill bacteria through the release of superoxide anion, indicating a possible negative effect of the virus, at least at the macrophage level.

Quantitation of porcine reproductive and respiratory syndrome virus RNA in semen by single-tube reverse transcription-nested polymerase chain reaction.

Porcine reproductive and respiratory syndrome virus (PRRSV) is in boar semen for extended periods of time as determined by reverse transcription-nested polymerase chain reaction (RT-nPCR) assay. The concentration of PRRSV RNA in semen and the biological significance of the detection level, however, remain to be resolved. In order to determine the concentration of PRRSV VR-2332 (a prototypic strain of North American isolates) in semen following infection, we established a 'standard curve'-quantitative competitive (SC-QC)-RT-nPCR assay as well as an equimolar QC-RT-nPCR assay. A deletion-type competitor RNA derived from the Lelystad virus, a European strain of PRRSV, ORF-7 gene standard which shares the nested sets of primer recognition sequences with the VR-2332 ORF-7 gene was used as an internal standard. The equimolar QC-RT-nPCR assay results revealed that the number of copies of PRRSV RNA in 1 TCID50/ml of virus derived from CL-2621 cell culture supernatants varied depending upon the type of samples in which virus was added; 143 +/- 24.0 and 266.5 +/- 48.5 copies in serum and semen samples spiked with PRRSV VR-2332, respectively. For the establishment of SC-QC-RT-nPCR assay, a standard curve was generated from band intensity ratios versus a series of known initial numbers of wild-type RNA copies which were quantified by the equimolar QC-RT-nPCR assay. Various initial numbers of copies of wild-type PRRSV RNA and each band intensity ratio with 1000 copies of competitor RNA were well correlated within the range of 100 to 200,000 copies (R2 = 0.947). A 'standard curve' quantitation assay using competitive single-tube RT-nPCR will offer a rapid and reliable way to quantify low concentrations of PRRSV RNA in semen.