Michael J. Yaeger

Persistence of Porcine Reproductive and Respiratory Syndrome Virus in Serum and Semen of Adult Boars

Four seronegative adult boars were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332. Serum and semen were collected 2-3 times weekly for over 100 days postinoculation (DPI). Serum samples were assayed for PRRSV by virus isolation (VI) and a polymerase chain reaction (PCR) and screened for antibodies to PRRSV using the indirect fluorescent antibody (IFA) and virus neutralization (VN) tests. Semen was assayed for PRRSV RNA by PCR. Virus or viral RNA was detected in the serum of all boars within 1 DPI by VI and/or PCR. However, VI results indicated that viremia was transient and occurred from 1 to 9 DPI. Viral RNA was detected in serum from 1 to 31 DPI. In the acute stage of the infection, PRRSV RNA was detected in serum by PCR prior to the presence of viral RNA in semen. The PRRSV RNA was detected in semen as early as 3 DPI and persisted for 25 DPI in 2 of the boars and 56 and 92 DPI in the remaining 2 boars. Detection of PRRSV RNA in semen occurred 2-8 and 28-35 days prior to the detection of antibodies by IFA and VN, respectively. PRRSV was isolated from the bulbourethral gland of the boar that shed viral RNA in semen for 92 DPI. These results suggest that PRRSV RNA can be detected by PCR in boar serum and semen, and may persist for variable periods of time. Viremia and the serologic status of the boar are not adequate indicators of when PRRSV or PRRSV RNA is being shed in the semen. Preliminary findings also indicated that neither shipping stress nor reinoculation with homologous PRRSV resulted in viremia or viral RNA shedding in semen.

A Prospective, Case Control Study Evaluating the Association between Clostridium Difficile Toxins in the Colon of Neonatal Swine and Gross and Microscopic Lesions

Clostridium difficile infection in swine has most often been described in suckling pigs, where it has been associated with mesocolonic edema and typhlocolitis. This prospective study was designed to assess the correlation between the presence of C. difficile toxins (TCd) in the colon contents of neonatal pigs and a number of parameters, including gross evidence of diarrhea, mesocoloninc edema, typhlitis, and colitis. C. difficile was isolated from 51% (66/129) of large intestines and TCd was detected in the colon contents of 50% (65/129) of the piglets. Fifty-eight percent (38/65) of TCd-positive piglets had normal to pelleted colon and rectal contents, whereas 75% (48/64) of TCd-negative pigs had gross evidence of diarrhea. Clostridium difficile toxin-positive animals were significantly more likely to have normal to pelleted feces. Edema of the mesocolon was observed in 38/65 (59%) of TCd-positive piglets. Because a high number of TCd-positive piglets (41%) lacked edema of the mesocolon and a high number of TCd-negative pigs had mesocolonic edema (51%), a statistically significant association between TCd and mesocolonic edema was not identified. Seventy-five percent (49/65) of TCd-positive piglets had colitis and 47/65 (72%) had typhlitis. The association between TCd and both colitis and typhlitis was statistically significant. Apparently healthy piglets were obtained from 5 separate sites. Because TCd was detected in the colon contents of 23/29 (79%) apparently healthy piglets obtained from 5 separate sites, and 70% of TCd-positive control pigs had colitis, C. difficile may represent an important subclinical issue in neonatal swine.

A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus

This survey was undertaken to determine the relative frequency of agents that are currently associated with neonatal diarrhea in swine, including Clostridium difficile and porcine reproductive and respiratory syndrome virus (PRRSV). The subjects for this study were the first 100 live 1–7-day-old piglets submitted to the Iowa State University Veterinary Diagnostic Laboratory with a clinical signalment of diarrhea, beginning on January 1, 2000. The evaluation of each pig included bacterial culture of a section of ileum, 2 sections of jejunum, and a single section of colon; a fluorescent antibody test (FAT) or immunohistochemistry (IHC) for transmissible gastroenteritis virus (TGEV); ELISAs for rotavirus and C. difficile toxins; IHC for PRRSV; and microscopic examination of ileum, midjejunum, spiral colon, liver, spleen, and lung. Survey results demonstrate a decline in the relative number of diagnoses of TGEV, Escherichia coli, and Clostridium perfringens type C compared with retrospective data. The combined case frequency rate for these 3 pathogens dropped from 70% in 1988 to 21% in 2000. This survey also demonstrated the emergence of C. difficile as an important pathogen of neonatal swine. Clostridium difficle toxin was detected in the colon contents of 29% of the piglets, and at least 1 toxin-positive animal was identified in 55% of the cases. All 29 C. difficile toxin-positive piglets had mesocolonic edema, and colitis was observed in 21 of 29 toxin-positive animals. PRRSV-positive macrophages were detected in the lamina propria of intestinal villi by IHC in 10 piglets with diarrhea. In 6 of these cases, PRRSV was the only pathogen detected. Gross and microscopic lung lesions were not a reliable indicator of PRRSV infection in these neonatal pigs with diarrhea. The addition of tests for C. difficile and PRRSV to a routine neonatal diarrhea diagnostic protocol resulted in a significant increase in the diagnostic success rate on both individual animal and case bases.

Neospora -like protozoan infection as a cause of abortion in dairy cattle

In 1989,29 of 240 drylot Holstein cows from a New Mexico dairy aborted over a 5-month period. Seven of the 9 fetuses examined had multifocal necrotizing encephalitis and multifocal myocarditis. Neospora-like protozoa were found in tissues from 3 of the fetuses. More recently, a protozoan that reacted positively with anti-N. caninum serum was identified as the most common cause of abortion in California dairy cattle. 1,2 Many of these abortions were epizootic, with multiple abortions over a 1 or 2-month period. 1 Abortions and congenital infections in calves infected by a Neosporalike protozoan have been reported in the United States, but these were isolated cases involving only a single calf in each herd. This protozoan has never been isolated, so its exact identity remains unknown. Recent studies indicate that the organism is closely related to N. caninum, but some ultrastructural and antigenic differences exist between the bovine parasite and N. caninum isolated from dogs. Except for a single case of abortion in a beef cow from Maryland, the cases of fetal and congenital neosporosis previously reported from the United States are from the western states of New Mexico, 11 Califomia and Washington. Nineteen additional cases of bovine abortion associated with Neosporalike infection that were submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory (SDADRDL) are herein reported. Specimens from cases of bovine abortions that are submitted to the SDADRDL undergo a routine protocol of testing to determine the cause of the abortion. Entire animals are necropsied and samples of brain, heart, lung, liver, kidney, placenta, tissues with gross lesions, and, since February 1991, skeletal muscle are fixed for histopathologic examination. Abomasal fluid is cultured for aerobic and microaerophilic bacteria and fungi. Scrapings of placenta are digested with potassium hydroxide and examined for fungal hyphae by a direct, nonspecific fluorescent antibody (FA) technique. If hyphae are found, the placenta is cultured for fungi. Impression smears of kidney are examined by a direct FA technique for 6 serovars of Leptospira. Direct FA tests are performed on frozen sections of kidney and spleen for infectious bovine rhinotracheitis (IBR) virus and bovine viral diarrhea (BVD) virus. Pools of brain, lung, liver, kidney, and spleen are homogenized and inoculated onto primary cultures of fetal bovine lung and turbinate cells for virus isolation. When received, a homogenate of placenta is inoculated by itself onto the same cell lines. Formalin-fixed paraffin-embedded

Neospora abortion storm in a midwestern dairy.

ing intestinal displacement, is also a possible cause of the colitis. An unusual finding in this case is the vegetative endocarditis caused or at least contributed to by the Aspergillus infection. Although fungal cultures of these lesions were not done, the organisms were morphologically similar to the organisms from the lung, which were identified as Aspergillus spp. by culture. These lesions were in the left ventricle, and embolic lesions, particularly in the kidney, are common with such cardiac lesions. Kidney lesions in this horse were suggestive of possible embolic disease; however, no fungal or bacterial organisms could be identified by histopathologic examination. Thrombosis of pulmonary blood vessels was seen histologically, and this is the most likely origin for the organisms causing the endocarditis.

West Nile Virus Meningoencephalitis in a Suri Alpaca and Suffolk Ewe

The first confirmed cases of West Nile virus (WNV) in the Western Hemisphere were reported in the state of New York in 1999. Since then, the virus has spread throughout the eastern and central United States and continues to extend westward. This report describes clinical signs and microscopic lesions associated with WNV infection in a Suffolk ewe and an alpaca, 2 species in which the disease has not been reported previously. In late August 2002, a 4.5-year-old female alpaca developed an acute onset of clinical signs characterized by torticollis, hyperesthesia, ataxia, recumbency, and altered mentation. The animal died 3.5 days after the onset of clinical signs. Microscopic examination of the brain revealed a mild to moderate, diffuse, lymphoplasmacytic meningoencephalitis. In early September 2002, a 3-year-old Suffolk ewe developed a rapidly progressive illness characterized by ataxia and convulsions. The apparent duration from onset of clinical signs until death was less than 8 hours. The ewe had a moderate, diffuse, lymphoplasmacytic meningoencephalitis with focal gliosis. Reverse transcription—polymerase chain reaction assays and immunohistochemistry on the brain were positive for WNV in both animals. These cases demonstrate that WNV is capable of causing sporadic, fatal, nonsuppurative meningoencephalitis in alpacas and sheep.

The effect of subclinical selenium toxicosis on pregnant beef cattle

A field investigation conducted by the South Dakota Animal Disease Research and Diagnostic Laboratory suggested that subclinical selenium toxicosis in pregnant cows may have contributed to an outbreak of aborted/stillborn calves in a high-selenium region of South Dakota. This study was undertaken to evaluate the relationship between abortion and subclinical selenium toxicosis in the dam and to assess the effects of subclinical selenium toxicosis on the bovine immune system. Fifteen pregnant cows were fed diets containing 0.25 (control), 6.0, and 12.0 ppm selenium beginning at 80–110 days gestation. Although selenium toxicosis has been reported to cause abortion, this study failed to reproduce abortions. A single cow in the 12-ppm selenium treatment group gave birth to a weak calf, which subsequently died. This calf had myocardial lesions consistent with those described for selenium toxicosis and had hepatic selenium levels of 9.68 ppm (wet weight). Elevated dietary selenium resulted in the depression of several leukocyte function parameters in pregnant cows. A statistically significant depression in forced antibody response was identified in both selenium-supplemented groups. A significantly diminished mitogenic response to concanavalin A and pokeweed mitogen was also observed in the 12-ppm selenium group. Although a similar pattern of depression was also observed with phytohemagglutinin, differences were not significant. These findings indicate that even in the absence of clinical alkali disease, elevated selenium levels may adversely affect both pregnancy outcome and the bovine immune system.

A comparison of virus isolation, immunohistochemistry, fetal serology, and reverse-transcription polymerase chain reaction assay for the identification of porcine reproductive and respiratory syndrome virus transplacental infection in the fetus

Virus isolation (VI), immunohistochemistry (IHC), fetal serology, and reverse-transcription polymerase chain reaction assay (RT-PCR) were performed on samples from 107 fetuses comprising 10 litters taken from sows experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). In addition to comparing the relative sensitivity and specificity of each test, RT-PCR was evaluated with respect to the relative suitability of thoracic fluids and tissues as samples, the effects of autolysis, and the effects of pooling of fetal specimens. VI, IHC, and fetal serology identified PRRSV infection in 48.6%, 23.4%, and 14.9% of 107 fetuses, respectively, and identified at least 1 infected fetus in 10, 10, and 5 of 10 litters, respectively. In utero death with autolysis reduced the test efficacy of all 3 methods. Fetal thoracic fluid and tissues were equally suitable for RT-PCR detection of PRRSV Pooling fetal tissues or fluids from VI-positive animals with comparable material from negative controls had no detrimental effect on RT-PCR results when evaluated at dilutions of 1:1, 1:2, 1:4, and 1:8. The results of RT-PCR testing were positive in 100%, 94.4%, and 83.3% of VI-positive specimens allowed to autolyze at 4, 21, or 37 C, respectively, for 24, 48, and 96 hours. Compared with the other testing modalities, RT-PCR appeared to be impacted the least by the adverse effects of autolysis.

Immunohistochemical Characterization of Rat Central and Peripheral Nerve Tumors Induced by Ethylnitrosourea

Ethylnitrosourea-induced central and peripheral nerve tumors in Sprague-Dawley rats were tested for GFAP (Glial Fibrillary Acidic Protein), S-100 protein, NSE (Neuron Specific Enolase) and Anti-Leu 7 (HNK-1) immunoreactivity utilizing the ABC method (avidin-biotin-complex) for GFAP, S-100 protein and NSE, and the PAP method (peroxidase-antiperoxidase) for Anti-Leu 7. Peripheral nerve neurinomas were consistently positive for S-100 protein and consistently negative for GFAP and Anti-Leu 7. Neurinomas would occasionally exhibit positive staining for NSE (2 of 55 tumors). The staining intensity for S-100 protein varied from strongly positive in differentiated neurinomas to weakly positive in anaplastic tumors. Neoplastic and reactive astrocytes exhibited positive staining for both S-100 protein and GFAP. Variation in the GFAP staining intensity of glial tumors correlated with the degree of differentiation as anaplastic tumors did not stain with the same intensity as their more differentiated counterparts. Oligodendrogliomas exhibited occasional immunoreactivity to S-100 protein (3 of 36 tumors). NSE reactivity in oligodendrogliomas was rarely observed (1 tumor in 36) and immunoreactivity against GFAP or Anti-Leu 7 was consistently absent. Anti-Leu 7 and NSE proved to be of little value in the classification of ENU-induced neural tumors.

Vitamin E and Selenium Concentrations in Livers of Pigs Diagnosed with Mulberry Heart Disease

During a 2-year period from January 1, 1999, to December 31, 2000, 77 diagnoses of mulberry heart disease (MHD) were documented at the Iowa State University Veterinary Diagnostic Laboratory. Mean (±SD) liver vitamin E concentrations were lower (P < 0.05) in pigs with MHD (3.12 ±1.12 ppm, wet weight) than in pigs that died of causes other than MHD (4.80 ± 3.2 ppm, wet weight). The majority of the pigs affected with MHD ranged in age from 3 to 7 weeks. Statistical influence of age was found on the concentration of vitamin E (P < 0.01) but not on concentration of selenium in liver in pigs with MHD. Concentrations of vitamin E below 2 ppm were considered deficient. Hepatic vitamin E concentrations below 2 ppm were measured in 25% of the pigs with gross and microscopic lesions of MHD. In contrast, liver selenium concentrations were adequate in all pigs.