Pathogenesis of porcine reproductive and respiratory syndrome in growing pigs

A new reproductive and respiratory syndrome was recognized in the United States swine population in 1986. Tissue filtrates from affected pigs submitted to the Iowa State University Veterinary Diagnostic Laboratory were used to experimentally inoculate gnotobiotic pigs and reproduce respiratory disease and lesions similar to what was observed on the farms of origin. Cytopathic effects and virus particles consistent with porcine reproductive and respiratory syndrome virus (PRRSV) were observed in cell culture. Virus isolation and serology techniques were standardized. Sixteen U.S. PRRSV isolates from herds with varying disease severity and the European Lelystad virus were plaque-purified. A cesarean-derived-colostrum-deprived pig model was developed to study and compare the pathogenicity of a selected subset of these isolates. Significant differences were detected in severity of clinical disease and gross and microscopic lung lesions. Isolates were grouped into low and high virulence categories. An immunohistochemistry technique for detection of PRRSV antigen in formalin-fixed tissues was developed and standardized for diagnostic and research purposes. PRRSV antigen was detected in alveolar macrophages, macrophages throughout the lymphoid system, dendritic-like cells in tonsil, thymus, spleen and lymph nodes, endothelial cells in the heart, and Kupffer cells in the liver. Temporal analysis of the distribution of virus by isolation, and antigen by immunohistochemistry, suggests that oronasal inoculation results in infection of the tonsil and viremia in 12 to 24 hours with subsequent widespread distribution throughout the respiratory and lymphoid systems. PRRSV was found to persist in tonsils and lung for more than 28 days. Pneumonia likely results from the lysis of alveolar macrophages, altered alveolar macrophage function, and the inflammatory response to the enzymes and cytokines released by PRRSV-induced damage to alveolar macrophages. The prolonged viremia, the persistent infections, and the ineffective immune response may be the result of widespread damage to antigen presenting cells within which PRRSV antigen was consistently demonstrated by immunohistochemistry

Characterization of a novel porcine parvovirus tentatively designated PPV5

A new porcine parvovirus (PPV), provisionally designated as PPV5, was identified in U.S. pigs. Cloning and sequencing from a circular or head-to-tail concatemeric array revealed that the PPV5 possesses the typical genomic organization of parvoviruses with two major predicted open reading frames (ORF1 and ORF2), and is most closely related to PPV4 with overall genomic identities of 64.1-67.3%. The amino acid identities between PPV5 and PPV4 were 84.6%-85.1% for ORF1 and 54.0%-54.3% for ORF2. Unlike PPV4, but similar to bovine parvovirus 2 (BPV2), PPV5 lacks the additional ORF3 and has a much longer ORF2. Moreover, the amino acid sequences of ORF1 and ORF2 of BPV2 showed higher homologies to PPV5 than to PPV4. The conserved motifs of the Ca(2+) binding loop (YXGXG) and the catalytic center (HDXXY) of phospholipase A2 (PLA2) were identified in VP1 (ORF2) of PPV5, as well as in BPV2, but were not present in PPV4. Phylogenetic analyses revealed that PPV5, PPV4 and BPV2 form a separate clade different from the genera Parvovirus and Bocavirus. Further epidemiologic investigations of PPV4 and PPV5 in U.S. pigs of different ages indicated a slightly higher prevalence for PPV5 (6.6%; 32/483) compared to PPV4 (4.1%; 20/483), with detection of concurrent PPV4 and PPV5 in 15.6% (7/45) of lungs of infected pigs. Evidence for potential vertical transmission or association with reproductive failure was minimal for both PPV4 and PPV5. The high similarity to PPV4 and the lack of ORF3 may suggest PPV5 is an intermediate of PPV4 during the evolution of parvoviruses in pigs

Application of Immunohistochemistry and ELISA for the Diagnosis of Neospora-Infected Cattle

Studies were undertaken to adapt diagnostic methods for use in our laboratory for detection of Neospora sp. infection in cattle. An immunohistochemical (IHC) test was used for detection of Neospora sp. antigen in tissues of aborted bovine fetuses. Neospora sp. antigen was detected most frequently in fetal brain tissue. Polyclonal antibodies were tested for specificity and sensitivity of the IHC. Sera were obtained from Neospora sp. infected dairy herds for use as positive and negative controls in the continuing development of an enzyme-linked immunosorbent assay (ELISA)

Lack of reproduction of the hallmark porcine circovirus type 2-associated lesions in a mouse model

BALB/c, C57BL6, and C3H/HeN mice were experimentally-infected with porcine circovirus type 2 (PCV2). The mice were tested for their ability to become infected with porcine circovirus type 2 (PCV2) and to develop the hallmark PCV2-associated lymphoid depletion and histiocytic replacement of lymphoid follicles characteristic of postweaning multisystemic wasting syndrome. Since immunostimulation has been shown to increase PCV2-replication in the pig, half of the mice were immunostimulated with keyhole limpet hemocyanin in incomplete Freund’s adjuvant (KLH/ICFA) at the time of PCV2-inoculation. PCV2 inoculation was done twice at 4 and 5 weeks of age by using intramuscular and intranasal routes. Necropsies were performed in 5-day-intervals at 12, 17, 22, 27, 32, and 37 days post PCV2 inoculation. None of the mice developed clinical disease and none of the mice developed PCV2-associated lymphoid lesions. Immunohistochemistry (IHC) and in-situ-hybridization (ISH) for PCV2-antigen/nucleic acids was performed on all tissues of all mice and was negative. PCR was done on pooled tissues and serum samples obtained at necropsy. The majority of the mice (101/111 PCV2 infected mice) were positive for PCV2-nucleic acids in tissue samples. Forty-one percent of the mice (46/111 PCV2 infected mice) were positive for PCV2-nucleic acids in serum samples. There was no difference between treatment groups or lines. This study confirms that mice can be infected with PCV2 and could be important in the epidemiology of PCV2; however, the mouse model may not be useful to understand the pathogenesis of PCV2- associated lesions

Postweaning Multisystemic Wasting Syndrome (PMWS) Surveillance Study

PMWS is characterized by a clinical history of wasting or poor performance in weaned pigs and by severe lymphoid depletion and histiocytic replacement of follicles in lymphoid tissues. The detection of porcine circovirus type 2 (PCV2) antigen or nucleic acids within characteristic microscopic lesions is required for the diagnosis of PMWS. Swine veterinarians submitted a specified set of samples from one hundred field cases that they felt fit the clinical definition of PMWS. All these cases were further analyzed for the presence or absence and scored for severity of the hallmark microscopic lesions (lymphoid depletion) of PMWS, the amount of PCV2 antigen associated with the lesions, and identification of concurrent bacterial and viral infections. Fifty-four of the 100 field cases were confirmed to be PMWS, whereas, no association with PCV2 was found in 46 of the cases. This highlights the need for further diagnostic testing, specifically histopathology and antigen detection, for confirmation of cases clinically suspected to be PMWS. This will become particularly important as vaccines for PCV2-associated diseases become approved for use

Fox Squirrels (Sciurus niger) Develop West Nile Virus Viremias Sufficient for Infecting Select Mosquito Species

The West Nile virus (WNV) viremia and shedding profiles of 11 adult fox squirrels (Sciurus niger) infected by needle inoculation or mosquito bite were characterized. Daily mean titers (95% confidence intervals) for all squirrels on days 1 through 6 postexposure (p.e.) were: 10(1.7 (1.32.1)), 10(4.4 (4.04.8)), 10(5.3 (5.05.6)), 10(4.4 (3.94.9)), 10(2.7 (2.03.4)), and 10(1.1 (0.52.1)) plaque-forming units (PFU)/mL. The highest WNV serum titers of individual squirrels infected by needle inoculation or mosquito bite ranged from 10(4.5) to 10(6.1) and from 10(5.1) to 10(5.3) PFU/mL, respectively. Nine (82%) squirrels, including all 4 squirrels infected by mosquito bite, had WNV serum titers \u3e or =10(5.1) PFU/mL that persisted on average for 1.6 +/- 0.3 days. Infection and dissemination rates of Culex pipiens (L.) that fed on squirrels with serum titers of 10(4.4 +/- 0.1) PFU/mL were 56% and 13%, respectively. Both of these rates increased to over 80% when fed on squirrels with a mean WNV titer of 10(5.5 +/- 0.1) PFU/mL. Infection and dissemination also occurred in Aedes triseriatus (Say) but at a much lower rate. WNV was isolated from the oral and rectal cavities of all squirrels and from urine that was opportunistically collected from 5 squirrels. The largest quantity of WNV recovered from swabs of the oral cavity and urine was 10(3.1) PFU. The longest periods after exposure that WNV was isolated from the oral cavity and urine from a squirrel were 22 and 17 days p.e., respectively. WNV RNA was also detected in kidney tissue in 1 squirrel 29 days p.e., suggesting that fox squirrels can be persistently infected. Collectively these observations provide further evidence that squirrels can contribute to the natural history and epidemiology of WNV, especially in peridomestic environments

Effect of different adjuvants on PCV2-associated lesions

Ninety, 12-14 day old pigs were randomly assigned to five groups. Group 1 (n=19) pigs were vaccinated with a Mycoplasma hyopneumoniae (M. hyopneumoniae) vaccine with an oil-in-water adjuvant (RespiSure®; Pfizer Animal Health, Inc.). Group 2 (n=17) pigs were vaccinated with a commercial M. hyopneumoniae vaccine with an aqueous adjuvant (Carbopol) (Suvaxyn® Respifend® MH; Fort Dodge Animal Health, Inc.). Group 3 (n=18) pigs were vaccinated using an oil-in-water adjuvanted vaccine containing the same amount and type of M. hyopneumoniae antigen as in group 2. Group 4 (n=18) pigs were vaccinated using an aluminum hydroxide adjuvanted vaccine containing the same amount and type of M. hyopneumoniae antigen as in group 2. Group 5 (n=18) pigs served as the controls and were sham-vaccinated with saline. Pigs were injected with 2 mL of one of the four M. hyopneumoniae vaccines at four and again at six weeks of age. PCV2 was inoculated intranasally on the day of the second vaccination at 6 weeks of age. Half of the pigs were necropsied at 21 days post inoculation (DPI). The remaining pigs were necropsied at 35 DPI. There were no differences among groups in clinical disease scores. At 21 DPI all vaccinated groups had significantly (p\u3c0.05) more severe lymphoid depletion than the saline injected group. At 35 DPI group 1 pigs had significantly (p\u3c0.05) higher amounts of PCV2 DNA in serum than pigs in groups 2, 4, and 5 as determined by quantitative real-time PCR. There was a significant (p\u3c0.05) increase in the severity of lymphoid depletion in the lymph nodes, tonsil, and spleen in groups 1 and 3 compared to groups 2, 4, and 5. Group 3 had significantly (p\u3c0.05) higher amounts of PCV2 antigen within lymph nodes, tonsil, and spleen compared to groups 2, 4 and 5. The results confirm that all adjuvants tested enhanced PCV2-induced lesions and oil-in-water products used in this study had a more severe effect

Effect of PRRSV Infection on MHC Expression by Macrophages and Monocytes

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recent and widespread pathogen in the U.S. swine population. PRRSV infects cells of the macrophage/ monocyte/dendritic lineages which are important antigen presenting cells (APCs) of the immune system. Using flow cytometric (FACs) analysis, we demonstrated that PRRSV infection decreases the expression of the major histocompatibility complex (MHC) glycoproteins on the cell surface of infected macrophages. This decrease in MHC protein expression may reduce the ability of the macrophages to present viral antigens to the appropriate lymphocytes. The potential lack of viral antigen presentation may play a crucial role in the persistent viremia observed in PRRSV-infected pigs

A commercial porcine circovirus(PCV)type 2a-based vaccine reduces PCV2d viremia and shedding and prevents PCV2d transmission to naïve pigs under experimental conditions.

Porcine circovirus type 2 (PCV2) vaccination has been effective in protecting pigs from clinical disease and today is used extensively. Recent studies in vaccinated populations indicate a major PCV2 genotype shift from the predominant PCV2 genotype 2b towards 2d. The aims of this study were to determine the ability of the commercial inactivated PCV2a vaccine Circovac® to protect pigs against experimental challenge with a 2013 PCV2d strain and prevent transmission. Thirty-eight pigs were randomly divided into four groups with 9–10 pigs per group: NEG (sham-vaccinated, sham-challenged), VAC (PCV2a-vaccinated, sham-challenged), VAC + CHAL (PCV2a-vaccinated and PCV2d-challenged), and CHAL (sham-vaccinated, PCV2d-challenged). Vaccination was done at 3 weeks of age using Circovac® according to label instructions. The CHAL and VAC + CHAL groups were challenged with PCV2d at 7 weeks of age and all pigs were necropsied 21 days post-challenge (dpc). The VAC-CHAL pigs seroconverted to PCV2 by 21 days post vaccination (dpv). At PCV2d challenge on 28 dpv, 3/9 VAC and 1/9 VAC + CHAL pigs were seropositive. NEG pigs remained seronegative for the duration of the study. Vaccination significantly reduced PCV2d viremia (VAC + CHAL) at dpc 14 and 21, PCV2d fecal shedding at dpc 14 and 21 and PCV2d nasal shedding at dpc 7, 14 and 21 compared to CHAL pigs. Vaccination significantly reduced mean PCV2 antigen load in lymph nodes in VAC + CHAL pigs compared to CHAL pigs. When pooled serum or feces collected from VAC + CHAL and CHAL pigs at dpc 21 were used to expose single-housed PCV2 naïve pigs, a pooled fecal sample from CHAL pigs contained infectious PCV2 whereas this was not the case for VAC + CHAL pigs suggesting reduction of PCV2d transmission by vaccination. Under the study conditions, the PCV2a-based vaccine was effective in reducing PCV2d viremia, tissue loads, shedding and transmission indicating that PCV2a vaccination should be effective in PCV2d-infected herds