Pathogenesis of in utero infection in porcine fetuses with porcine reproductive and respiratory syndrome virus.

Porcine fetuses were exposed in utero to porcine reproductive and respiratory syndrome virus (PRRSV) at stages of gestation ranging from 34 to 85 days and examined 17 to 31 days later to determine the effect of gestational age on fetal susceptibility. For each of the 8 litters tested during the study, all of the fetuses of 1 horn of the uterus were exposed to virus by intraamniotic injection; those of the other horn were exposed similarly to a sham inoculum that consisted of sterile cell culture medium. Viral infectivity titers associated with fetal tissues collected at necropsy indicated that, regardless of gestational age, the virus had replicated in fetuses exposed intraamniotically. In addition, virus had also spread and replicated in sham-inoculated littermates in 3 litters. On the basis of these findings it appears that there may be little or no temporal difference in fetal susceptibility to infection with PRRSV. If so, the lack of early fetal death as a commonly recognized feature of naturally occurring cases of PRRS may be due to a greater resistance of early gestational fetuses to the lethal effects of PRRSV, as suggested by this study, and/or a greater likelihood of transplacental infection during late gestation.     

Serologic, virologic, and histopathologic observations of encephalomyocarditis virus infection in mummified and stillborn pigs

Stillborn and mummified swine fetuses from swine farms experiencing reproductive problems were investigated for evidence of infection with encephalomyocarditis (EMC) virus by fetal serology, virus isolation, and histopathologic examination. Fetal sera or thoracic fluids of 478 abnormal fetuses collected during January through December 1987 were tested for the presence of antibody specific to EMC virus. Of 478 samples tested, 175 (36.6%) had EMC virus serum neutralizing antibody titers of 1:64 or greater. The samples positive for EMC virus antibody were obtained from 38 swine farms located in 9 states in the United States. In addition to serologic observations, tissue samples of some abnormal fetuses were examined for the presence of virus and histopathologic lesions. The EMC virus was isolated in 1 case from the fetuses of an aborted litter. The isolate was serologically identical to a reference EMC virus. Nonsuppurative encephalitis and myocarditis were observed in the fetal samples collected from 2 different herds. Thoracic fluid of 1 stillborn pig with lesions was positive for EMC virus antibody at a titer of 1:512. We suggest that a widespread reproductive problem recently experienced in several major swine-producing areas of the United States may have been caused by EMC virus infection.     

Effect of formalin fixation on the immunohistochemical detection of PRRS virus antigen in experimentally and naturally infected pigs.

The purpose of this study was to determine the effect of formalin fixation on the immunohistochemical detection of porcine reproductive and respiratory syndrome (PRRS) viral antigen in lungs of experimentally and naturally infected pigs. In separate trials, five 24-day-old pigs and six 10-day-old pigs were housed as separate groups in isolation and inoculated intranasally with 10(5.5) TCID50 of an isolate of PRRS virus (PRRSV; P129). The older and younger pigs were euthanatized at 7 and 10 days post inoculation (dpi), respectively. At necropsy, all pigs had gross and microscopic lung lesions typical of PRRS, and PRRSV was isolated from all pigs. To insure uniform fixation, lungs from each pig were cut into 1-cm-thick slices and immersed into 10% neutral-buffered formalin. After fixation in formalin for 8 hours or 1, 2, 3, 5, 6, 8, 10, and 15 days, 3 lung sections from some or all pigs were processed for histological examination using routine methods. Immunohistochemical staining for PRRSV antigen was positive at the following times (days unless otherwise stated) after fixation (percentage of pigs staining positive for PRRSV in parentheses): 8 hours (100); 1 (100); 2 (100); 3 (80); 5 (33); and 6, 8, 10, and 15 (0-all negative). To further evaluate the effects of formalin fixation on PRRSV immunodetection, 31 field cases of PRRS were selected for immunohistochemistry (IHC). Over a 3-month period, submitted cases were selected from the Purdue University Animal Disease Diagnostic Laboratory, W. Lafayette, Indiana, for IHC if 1) the clinical history included respiratory disease, 2) PRRSV was isolated from lung and/or serum from the submitted pigs or tissues, 3) at least 1 section of lung fixed in 10% neutral-buffered formalin was submitted for IHC, and 4) the duration of fixation could be accurately determined from the case history. Of the 31 PRRSV-infected pig cases meeting the selection criteria, 23 were fixed in formalin for 4 days or less. Twenty-one of these 23 (91%) were positive by IHC. Two of 8 cases fixed for greater than 4 days (25%) were positive by IHC. In practical terms, 1-day shipping of fixed samples to a laboratory followed by routine tissue processing within a laboratory should not adversely affect immunohistochemical detection of PRRS viral antigen. But a delay in shipping or processing of more than 2 days could reduce or prevent the detection of PRRS viral antigen by IHC.     

Single tube nested PCR for the detection of Toxoplasma gondii in fetal tissues from naturally aborted ewes.

A single tube nested polymerase chain reaction (PCR) assay targeting the multicopy 18S-5.8S rRNA internal transcribed spacer (ITS1) region has been developed for the diagnosis of Toxoplasma gondii-induced abortion in ovine fetal tissues. In all, 145 ovine fetal samples including brain, spleen, lung, liver, kidney, placenta and fetal fluids from 53 fetuses and stillborns of 32 farms in Northern Spain were analyzed. Thirty-six samples belonging to nine fetuses and one stillborn lamb were T. gondii PCR-positive. Although T. gondii DNA was amplified from different types of tissues, brain was the tissue with the highest detection rate. All animals that had histopathological lesions associated to T. gondii infection were positive by PCR. In addition, four fetuses whose histological examination was hindered by autolysis were PCR-positive. Results obtained by PCR and indirect fluorescent antibody test (IFAT) showed good correspondence, demonstrating the diagnostic value of the two techniques. However, PCR has the advantage over serology in its ability to diagnose T. gondii infection at earlier stages of gestation when the fetus is not yet immunocompetent and in lambs that have taken colostrum. Once other abortifacient agents are ruled out, PCR detection of the ITS1 region in fetal tissues is a valuable and relatively rapid technique for the diagnosis of ovine abortion caused by T. gondii.     

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.     

Evidence for the localization of porcine reproductive and respiratory syndrome virus (PRRSV) antigen and RNA in ovarian follicles in gilts

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) infection in ovary was studied in sexually mature, cycling, nonsynchronized gilts infected with the PRRSV 16244B, a virulent field strain. Previous studies have shown that PRRSV can be isolated from ovaries and is transplacentally passed from gilts to the fetuses. The cause of infertility following PRRSV infection is not known. In this study, we identified the tropism of PRRSV in ovarian tissue from experimentally infected gilts in samples collected between 7 and 21 days postinfection (DPI). Tissues were collected and examined by virus isolation, in situ hybridization (ISH), immunohistochemistry (IHC), and double labeling to identify PRRSV-infected cell types. PRRSV was isolated in ovarian follicles at 7 days DPI. The IHC and ISH indicated that PRRSV-positive cells in ovaries were predominantly macrophages, which were numerous in atretic follicles. No evidence of infection and/or perpetuation of PRRSV in ova was observed, indicating that the female gonad is an unlikely site of persistence. No alteration of the normal ovarian architecture that would support a possible role of PRRSV infection in porcine female infertility was observed.     

Identification of viral pathogens in aborted fetuses and stillborn piglets from cases of swine reproductive failure in Spain

The objective of the present study was to determine the presence of recognised abortifacient viruses such as porcine reproductive and respiratory virus (PRRSV), Aujeszky's disease virus (ADV), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2), in tissues from aborted fetuses and stillborn neonates in cases of late reproductive failure in swine. A total of 293 specimens (fetuses aborted in the last third of gestation and stillborn piglets) from 100 different cases of late-term abortions and premature farrowing from 15 different Spanish provinces were studied. PRRSV was detected in 9/100 cases by RT-PCR. Only 1/100 cases analysed (corresponding to a late-term aborted fetus with a negative PRRSV RT-PCR result) was positive for PCV2 by PCR. Neither ADV (monitored by viral isolation plus antigen detection) nor PPV (monitored by ELISA antigen capture test) infection was identified. The results suggest that PRRSV is one of the most important infectious agents, if not the most relevant one, associated with fetal infection leading to abortion or premature farrowing in Spain. Moreover, other viral pathogens such as ADV, PPV and PCV2 seem to have a minor impact on reproductive disease.     

Evaluation of an enzyme immunoassay for detection of Chlamydia psittaci in vaginal secretions, placentas, and fetal tissues from aborting ewes

A commercially available enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in human urogenital and conjunctival specimens was compared with isolation in cell culture for the detection of Chlamydia psittaci in vaginal and placental swabs from aborting ewes and swabs of aborted fetal tissues. The EIA on vaginal swabs collected from 10 ewes experimentally infected with C. psittaci had a sensitivity of 85.7% and a specificity of 85.7%. Vaginal swabs collected at the time of abortion or within 3 days were the best samples for detection of chlamydial infection. The 29 vaginal swabs collected during this period from experimentally infected ewes were all strongly EIA-positive, and chlamydia were isolated from 28. The EIA on vaginal swabs from 78 field cases of abortion had a sensitivity of 78.0% and a specificity of 76.8%. The EIA on swabs of cotyledons from 65 placentas had a sensitivity of 100% and a specificity of 75.0% compared with isolation in cell culture. The EIA on 57 swabs of fetal tissues or body fluids from 10 aborted fetuses or weak lambs from experimentally infected ewes had a sensitivity of 26.6% and a specificity of 88.1% compared with isolation in cell culture. Limitations of the EIA are discussed.     

Porcine reproductive and respiratory syndrome virus and Haemophilus parasuis antigen distribution in dually infected pigs

Immunohistochemical, viral and bacterial isolation techniques were used to study the distribution and localization of porcine reproductive and respiratory syndrome virus (PRRSV) and Haemophilus (H.) parasuis in experimentally infected pigs. Thirty pigs seronegative to PRRSV and H. parasuis were divided into four groups. Group A pigs (10 animals) were inoculated with both virus and bacteria; group B pigs (10 animals) were inoculated with bacteria, group C pigs (five animals) were inoculated with virus and group D pigs (five animals) were kept as negative controls. All pigs of groups A and C became infected with PRRSV, according to virological techniques used (immunohistochemistry, virus isolation and virus serology). Lung, heart and tonsils were the most frequently immunolabeled tissues, and monocyte/macrophage lineage cells were the target for PRRSV in all tissues. All pigs in groups A and B also became infected with H. parasuis based on immunohistochemical and bacterial isolation results. Serosal surfaces, lung and tonsils were the most frequently immunolabeled tissues, and bacteria were found in monocyte/macrophage lineage cells as well as within neutrophil cytoplasm. No differences in terms of bacterial distribution or localization in tissues of pigs of groups A and B were detected. These results suggest that there is no influence of the previous infection with PRRSV in the occurrence of H. parasuis infection.     

Immunohistochemical identification of Campylobacter fetus in natural cases of bovine and ovine abortions

An immunohistochemistry (IHC) procedure for the detection of Campylobacter fetus antigens using an avidin-biotin complex technique was performed on formalin fixed bovine and ovine fetal tissues from 26 natural cases of Campylobacter spp. abortion (four ovine and 22 bovine). The species of Campylobacter isolated included C. fetus ssp. venerealis from 13 bovine fetuses, C. fetus ssp. fetus from two ovine and one bovine fetus, Campylobacter jejuni from seven bovine fetuses, Campylobacter lari from two ovine fetuses and an unspeciated Campylobacter species in one bovine fetus. Histologic lesions identified in the aborted fetuses included placentitis, serositis, pneumonia, gastroenteritis, hepatitis and encephalitis. Campylobacter fetus antigens were identified by IHC in 13 of 13 bovine fetuses from which C. fetus ssp. venerealis was isolated and in two of two ovine fetuses from which C. fetus ssp. fetus was isolated. The IHC stains were negative in tissues from seven bovine fetuses from which C. jejuni was isolated, one bovine fetus infected with C. fetus ssp. fetus, one bovine fetus infected with the unspeciated Campylobacter and two ovine fetuses infected with C. lari. In positive cases, the IHC stain most frequently identified bacteria in the lung and gastrointestinal tract. The C. fetus IHC procedure performed on formalin fixed tissues is a practical tool for the diagnosis of natural cases of ovine and bovine abortion caused by C. fetus.     

Histopathological and immunohistochemical aspects of Neospora caninum diagnosis in bovine aborted fetuses

Lesions suggestive of protozoal infection such as nonsuppurative myositis, nonsuppurative myocarditis, nonsuppurative pneumonia associated with focal cellular necrosis and multifocal necrotizing nonsuppurative encephalitis were observed in 89 (34%) out of 258 aborted bovine fetuses. Immunohistochemistry (IHC) revealed tachyzoites and cysts of Neospora caninum in 55 (61.8%) of these 89 fetuses. Several fetal variables were tested by multivariate logistic regression, in which increasing odds for positivity in IHC were associated with the presence of lesion in the lung (OR=12.11) and less autolytic brain tissue (brain consistency, OR=5.73). However, autolysis in the brain precluded presumptive diagnosis made by histopathology in 53% of the fetuses examined. These results indicate that, when fetal brain is autolyzed, the lung may be adequately used for the presumptive diagnosis of N. caninum infection. The results also confirmed that, even autolyzed, the brain is the most suitable organ for anti-N. caninum IHC.     

Pathogenesis and prevention of placental and transplacental porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive problems are characterized by embryonic death, late-term abortions, early farrowing and increase in number of dead and mummified fetuses, and weak-born piglets. The virus recovery from fetal tissues illustrates transplacental infection, but despite many studies on the subject, the means by which PRRSV spreads from mother to fetus and the exact pathophysiological basis of the virus-induced reproductive failure remain unexplained. Recent findings from our group indicate that the endometrium and placenta are involved in the PRRSV passage from mother to fetus and that virus replication in the endometrial/placental tissues can be the actual reason for fetal death. The main purpose of this review is to clarify the role that PRRSV replication and PRRSV-induced changes in the endometrium/placenta play in the pathogenesis of PRRSV-induced reproductive failure in pregnant sows. In addition, strategies to control placental and transplacental PRRSV infection are discussed.     

The diagnostic sensitivity of immunohistochemistry for the detection of porcine reproductive and respiratory syndrome virus in the lung of vaccinated and unvaccinated swine.

Immunohistochemistry (IHC) is used routinely to detect porcine reproductive and respiratory syndrome virus (PRRSV) in the lung of nursery and grow/finish pigs with respiratory disease and has been reported to be highly specific (100%) but only moderately sensitive (67%). When multiple sections of lung are examined from field cases of porcine pneumonia, it is common to detect PRRSV antigen in only 1 or 2 of the sections. This study was undertaken to determine the impact of the number of lung sections evaluated on the diagnostic sensitivity of IHC for the detection of PRRSV in vaccinated and unvaccinated swine. Five anterioventral sections of lung from animals experimentally challenged with PRRSV were evaluated on a single IHC slide. Utilizing a beta binomial model, observed results were used to calculate the probability of detecting PRRSV with IHC as a function of the number of lung sections assessed. Results demonstrate that the diagnostic sensitivity of PRRSV IHC is dependent on the number of lung sections examined. In unvaccinated pigs, a beta binomial model predicts that if a single lung section were evaluated, PRRSV would likely be confirmed in only 48% of infected animals, and at least 5 sections of anterioventral lung would need to be assessed to detect >90% of PRRSV-infected pigs. Vaccination resulted in significantly lower gross and microscopic lung lesion scores and significantly fewer antigen-positive cells. In vaccinated swine, the calculated probability of detecting a PRRSV-infected pig with IHC when a single lung section is evaluated was only 14%. If PRRSV is a primary concern, diagnosticians should collect at least 5 anterioventral sections of lung from each pig to be evaluated on a single IHC slide. This approach will diminish the number of false-negative results obtained with this method of antigen detection.     

Effect of stage of gestation, litter size and uterine space on the incidence of mummified fetuses in pigs

Factors associated with incidence of mummified fetuses at 7, 9, 11, 13 and 15 wk of gestation were determined in a cross-sectional design involving 209 pregnant pigs. Percentage of observed mummies increased from 2.1 to 12.2% as gestation progressed. Incidence of mummies at 7 wk of gestation was not different between litters of nine fetuses or fewer and those of 10 fetuses or more. During the period of 7 to 15 wk of gestation, the incidence of mummies remained constant at about 1% in litters of nine fetuses or fewer, whereas in litters of 10 fetuses or more the incidence increased linearly from 1 to 12% (P less than .01). In litters of nine fetuses or fewer, a mummy occupied no less space than a live fetus until 13 wk of gestation. In litters of 10 fetuses or more, a mummy occupied less space than did a live fetus after 7 wk of gestation. An increase in the incidence of mummies in larger litters was associated with uterine space per fetus below that needed for development and survival. The 12% fetal loss in late gestation was associated with less uterine space per fetus. Length of uterine horns, litter size and stage of gestation together accounted for 12% of the variation in incidence of mummies (P less than .001). A longer uterus had greater space per fetus, a larger number of live fetuses and a lower incidence of mummies.     

The Neospora caninum-Spain 7 isolate induces placental damage, fetal death and abortion in cattle when inoculated in early gestation

The Nc-Spain 7 isolate of Neospora caninum, which was newly obtained from an asymptomatic congenitally infected calf, demonstrated a similar virulence as Nc-1 strain in mouse models. The aim of this study was to characterize the pathogenesis of Nc-Spain 7 isolate in cattle after experimental infection at 65 days of gestation. For this purpose, thirteen pregnant heifers were divided into three groups as follows: group A: 7 heifers inoculated with 1×10(8) tachyzoites of Nc-Spain 7 isolate; group B: 4 heifers inoculated with 1×10(8) tachyzoites of Nc 1 strain; and group C: 2 heifers received PBS. Serum samples were collected weekly and heparinized blood samples were collected three times (0, 28 and 42 days after inoculation) by jugular venipuncture. Placenta and fetal tissue samples were collected at time of necropsy. Specific antibody response in the dams was tested by IFAT, indirect ELISA, and rNcGRA7 and rNcSAG4 based-ELISA. Specific antibody response in fetal fluids was tested by IFAT. IFN-γ production was measured after in vitro culture of PBMC and the supernatant was assessed using a commercial kit (BOVIGAM). A significant increase in N. caninum antibody responses was detected in groups A and B by IFAT and by i-ELISA from day 14 after inoculation onwards. Besides, antibody response against rNCGra7 protein was also detected in all inoculated heifers by rNcGra7-based ELISA. Four fetuses from group A and one from group B were aborted between 3 and 5 weeks after infection. In the recovered fetuses, only 3 out of 4 fetal fluids from fetuses of group A and 1 out of 3 of group B were seropositive by IFAT, but all of them were positive by PCR. Transplacental transmission could be determined in all fetuses from groups A and B by PCR and/or IHC. Heifers of group C and their fetuses remained negative by all techniques. The results of this study demonstrate that the NC-Spain 7 isolate could be transmitted transplacentally, and produced fetal death and abortion in cattle.