Journal of Veterinary Diagnostic Investigation

Diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive failure in swine is difficult because of the rapid inactivation of virus in fetuses that have died prior to abortion or farrowing. In this report, we describe gross and microscopic lesions of diagnostic value found in fetuses transplacentally infected with PRRSV during late gestation. Seven sows free of PRRSV-specific antibody and 1 sow (#8) that had been previously infected with PRRSV were oronasally exposed to a PRRSV inoculum at or about 90 days of gestation (DG). One control sow (#9) was oronasally exposed to a sham inoculum at 90 DG. Sows were euthanized 21 days postexposure, and fetuses were tested for virus. Transplacental infection was detected in litters 1–7, and gross lesions of the umbilical cord were observed in some fetuses in 6 of the 7 litters. No transplacental infection or fetal lesions were found in litters 8 and 9. The gross lesions in the umbilical cords ranged from segmental hemorrhagic areas 1–2 cm in length to a full length involvement of the cord, which was grossly distended with frank hemorrhage. All live fetuses that had gross lesions in the umbilical cord were viremic, and histopathologic examination revealed a necrotizing umbilical arteritis with periarterial hemorrhage. This was the most consistent microscopic lesion in fetuses infected with PRRSV. Sows 1–7 had endometritis and myometritis of various degrees, suggesting PRRSV also may induce these lesions. Careful gross and microscopic examination of the umbilical cord may aid in the diagnosis of PRRSV-induced reproductive failure.

Morbillivirus infection was diagnosed in 35/67 bottlenose dolphins (Tursiops truncatus) from the Gulf of Mexico that stranded from October 1993 through April 1994 in Alabama, Mississippi, and Texas (USA) during periods of increased dolphin standings in each of the 3 states. Diagnosis was based on histologic lesions, immunohistochemical demonstration of morbilliviral antigen, and detection of morbilliviral RNA by a reverse transcriptase polymerase chain reaction (RT-PCR) test performed on formalin-fixed, paraffin-embedded tissue (5 dolphins), on histologic lesions and detection of morbilliviral RNA by RT-PCR performed on formalin-fixed, paraffin-embedded tissue (1 dolphin), and on detection of morbilliviral RNA by RT-PCR performed on unfixed lung samples collected from carcasses with advanced postmortem autolysis (29 dolphins). Histologic lesions included proliferative interstitial pneumonia with syncytial cells and eosinophilic intranuclear and intracytoplasmic inclusion bodies, lymphoid depletion and syncytial cells with eosinophilic intranuclear inclusion bodies in lymph nodes, eosinophilic intracytoplasmic inclusion bodies in transitional epithelium of urinary bladder, and a syncytial cell with eosinophilic intranuclear inclusion bodies in epidermis. Concomitant pulmonary aspergillosis was diagnosed histologically in 4 dolphins. This is the 5th reported morbilliviral epizootic of aquatic mammals and the 2nd involving bottlenose dolphins in the United States.

Necropsy tissues were examined from an adult wild-caught Ducorps cockatoo (Cacatua ducorpsii) with progressive neurologic signs. Of the tissue specimens selected for histologic evaluation, only the brain contained rare amphophilic, glassy intranuclear inclusions within astrocytes and some neurons. Astrocyte and neuronal degeneration and necrosis also were observed. Scattered astrocytes, with and without discernable inclusions, contained avian polyomavirus (APV) nucleic acid, as determined by DNA in situ hybridization. In addition, endothelial cells and intravascular leukocytes contained psittacine beak and feather disease viral nucleic acid, as determined by DNA in situ hybridization, indicating dual viral infection. Electron microscopic examination of formalin-fixed brain tissue revealed typical intranuclear APV particles in some astrocytes. Encephalopathy ultimately was attributed to APV infection.

Twenty-five colostrum-deprived lambs reared in isolation were inoculated with a US variant of ovine adenovirus serotype 6 (OAV-6) strain RTS-151, Pasteurella haemolytica, or a combination of the 2 agents. Although severe pulmonary lesions were caused by each agent, the lesions were more severe and lasted longer with the combined infection. Lesions induced by OAV-6 alone developed 6–9 days after inoculation and lasted for 15 days, the length of the experiment. The lesions were characterized by suppurative inflammation at the junction of the terminal bronchioles and alveoli. Air spaces were filled with neutrophils and sloughed epithelial cells, which often contained large intranuclear inclusions. Lesions induced by P. haemolytica alone developed within 1 day and persisted for no more than 10 days and were characterized by severe pulmonary edema with variable amounts of fibrin. Lesions induced by the combined infection had aspects of each infection alone and resulted in severe disease in 4 of 8 lambs that were permitted to live more than 1 day after inoculation with bacteria. Early pulmonary lesions included edema, limited fibrin deposition, and slight purulent bronchiolitis and alveolitis. Later lesions included necrosis and more fibrin. For lambs inoculated with both pathogens, resolution was incomplete 15 days after inoculation of virus (10 days after inoculation of P. haemolytica). The results presented here corroborate previous findings indicating that the RTS-151 variant of OAV-6 is common in lambs and acts in concert with P. haemolytica to cause severe and often fatal pneumonia.

A polymerase chain reaction (PCR) assay for the detection of toxigenic Pasteurella multocida in nasal and tonsillar swab specimens collected from pigs was developed. Target DNA was isolated with guanidine thiocyanate and diatomite, and 2 primer sets derived from sequences in the gene that encodes the dermonecrotic toxin-of P. multocida were used simultaneously. The method was adapted to microtiter plate format allowing large-scale use of the PCR assay. To identify false-negative test results caused by failure of amplification, a positive control template was constructed that was spiked to each DNA sample. The PCR assay was evaluated with clinical samples and compared with 2 routinely used methods for detection of toxigenic P. multocida: isolation from a selective agar and direct detection of the toxin in extracts of primary cultures by an enzyme-linked immunosorbent assay (ELISA). The sensitivity of the PCR assay was tested with 346 nasal and tonsillar swabs specimens collected from pigs of 9 herds known to be infected with toxigenic P. multocida. Toxigenic P. multocida was isolated from 22 specimens, only 28 specimens tested positive in ELISA, but 40 tested positive in the PCR assay; thus the PCR assay is the most sensitive of the 3 methods. The specificity of the PCR assay was tested with 372 swab specimens collected from pigs of 6 herds certificated to be free from toxigenic P. multocida. Toxigenic P. multocida was not isolated from any of these specimens, all tested negative in ELISA, and 370 tested negative in PCR. The 2 positive specimens came from 2 pigs of 1 litter and tested only weakly positive in the PCR assay. From these results, it was concluded that the PCR assay is not only highly sensitive but also highly specific.

A dot immunobinding assay (DIA) was developed for the detection of antibodies to Salmonella enteritidis. Western blot analysis of outer membrane proteins from SE identified 2 polypeptides of molecular masses 43 and 46 kD that were specific for S. enteritidis. These 2 polypeptides were utilized as antigens in the DIA. The DIA was tested on sera from chickens experimentally infected with S. enteritidis. Results of the DIA were compared with that of conventional microagglutination and serum plate tests. The DIA was a highly specific and sensitive test that can be useful for screening birds to determine if they are infected with S. enteritidis. Its simplicity, reliability, reproducibility, and speed in interpreting the assay results makes it a useful screening test for flock monitoring.

The antigens extracted from Dichelobacter nodosus with potassium thiocyanate were analyzed by western blotting with sera from footrot-free sheep and from sheep infected with D. nodosus to identify components specific for infection. Several components with molecular mass < 33 kD were associated with infection, particularly bands of 32.5 kD, 30.5 kD, and 28 kD. Components with molecular mass > 56 kD may be responsible for false-positive reactions observed when sera of footrot-free sheep react with the potassium thiocyanate extract in solid phase enzyme-linked immunosorbent assays (ELISAs). Bands of 62.5 kD, 56 kD, 40 kD, and 19 kD were recognized for most sheep regardless of their disease status or ELISA reactivity and therefore do not appear to be detected by the ELISA. No components of the potassium thiocyanate extract were completely specific for infection. Antigens in the extract were identified primarily on the cell envelope by immunogold electron microscopy. Labeling was concentrated in the periplasm, with minor labeling of the cell surface. The extract consisted of tangled strands of particles with electron-dense cores, and few vesicular structures were observed.

Necrotizing hepatopancreatitis (NHP) is a disease of farm-raised Pacific white shrimp (Penaeus vannamei) caused by a pleomorphic intracellular bacterium. A DNA probe that is specific for the etiologic agent of necrotizing hepatopancreatitis was devised and tested in an in situ hybridization assay. A procedure was developed for labeling a single-stranded DNA probe with digoxigenin by the polymerase chain reaction. The DNA probe encompasses the V1 and V2 variable regions of the 16S ribosomal RNA (rRNA) gene and is designed to hybridize to complementary sequences of the 16S rRNA of the NHP bacterium. The probe was tested on fixed, paraffin-embedded specimens, and an intense, specific hybridization signal was localized to the cytoplasm of hepatopancreatic epithelial cells that were infected with the NHP bacterium, as demonstrated by serial sections stained with hematoxylin and eosin or the Steiner and Steiner method. Negative results were obtained from normal shrimp and from shrimp infected with Vibrio spp. The specificity of the probe was confirmed using either mammalian or avian tissues infected with other intracellular bacteria, including Ehrlichia canis, Salmonella enteritidis, Brucella abortus, and Chlymidia spp., and using another species of shrimp (P. monodon) infected with a different rickettisa-like intracellular bacterium.

Ceftiofur (XNL) and its primary metabolite, desfuroylceftiofur (DXNL), were evaluated for in vitro activity against 539 isolates from veterinary sources. Actinobacillus pleuropneumoniae, Pasteurella spp., Haemophilus somnus, Salmonella spp., Escherichia coli, staphylococci, and streptococci were tested. Overall, XNL and DXNL were equivalent in activity against the gram-negative organisms with all minimum inhibitory concentrations (MICs) within 1 serial dilution. Against the staphylococci, MIC differences of 2–3 serial dilutions were detected with an MIC₉₀ for XNL and DXNL of 1.0 and 4.0–8.0 g/ml, respectively. Although the MIC₉₀ obtained for Streptococcus suis for each compound was within 1 dilution, the MIC values against individual strains were 2–3 dilutions greater for DXNL than for XNL. The MICs obtained with the bovine and equine streptococci for DXNL (MIC₉₀ = 0.03 g/ml) were 5 serial dilutions higher than those obtained for XNL (MIC₉₀ £ 0.0019). Although DXNL was less active than XNL against the streptococci, these differences were not clinically important because both XNL and DXNL were highly active for these bacteria. Although these differences are of little importance with the streptococci, they may have important implications for susceptibility testing of the staphylococci. In conclusion, with the exception of the staphylococci, both XNL and DXNL were highly active against the organisms tested, with MICs for both compounds several fold lower than plasma levels achieved during dosing of XNL.

Tilmicosin is a novel macrolide antibiotic developed for exclusive use in veterinary medicine. The first tilmicosin-containing product was approved to treat bovine respiratory disease associated with pasteurellae. The development of antimicrobial susceptibility testing guidelines for tilmicosin was predicated on the relationship of clinical efficacy studies that demonstrated a favorable therapeutic outcome, on pharmacokinetic data, and on in vitro test data, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The NCCLS-approved breakpoints for the MIC dilution testing are resistant ‡ 32 g/ml, intermediate 16 g/ml, and susceptible £ 8 g/ml. The zone of inhibition interpretive criteria for disk diffusion testing with a 15- g disk are resistant £ 10 mm, intermediate 11–13 mm, and susceptible ‡ 14 mm.

Peripheral blood mononuclear cells were isolated from noninfected control cows and from cows with either subclinical or clinical paratuberculosis (Johne s disease). Cells were incubated for 6, 12, 24, and 48 hours in complete medium with the following mitogens: concanavalin A (ConA), phytohemagglutinin-P (PHAP), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide. In addition, cells were incubated for the same time periods with a Mycobacterium paratuberculosis sonicate (MpS) and live and heat-killed M. paratuberculosis at 10:1 bacteria: cell ratio. After incubation, cell-free supernatants were analyzed for y-interferon (γ-IFN) production. Cells from subclinical cows produced significantly higher levels of γ-IFN than did cells from clinical animals after stimulation with mitogens ConA, PHAP, and PWM. Levels of γ-IFN produced by noninfected control animals generally followed the pattern of those of subclinical animals. After incubation with MpS, significantly greater quantities of γ-IFN were produced by cells isolated from subclinical animals than by cells from clinical cows and noninfected controls. Stimulation of cells with heat-killed or live M. paratuberculosis evoked a similar response. This study indicates that γ-IFN production by peripheral blood mononuclear cells in response to M. paratuberculosis antigen may be an important diagnostic tool for the detection of paratuberculosis in subclinically affected animals.

Detailed postmortem examinations were conducted on 30 cattle from a dairy herd with bovine tuberculosis to determine the distribution of lesions in Mycobacterium bovid-infected cattle. Twenty-four different tissue specimens from each animal were examined for gross lesions and collected for bacteriologic culturing and histologic examination. Tuberculosis was confirmed in 15 cattle with evidence of infection in 1 or more of the following tissues: medial retropharyngeal, parotid, tracheobronchial, mediastinal, caudal deep cervical, and subiliac lymph nodes; palatine tonsil; and lung. Gross and histologic lesions were present most frequently in lymph nodes of the thoracic region. Mycobacterium bovis was isolated from 3 cattle that had no gross lesions of tuberculosis. One animal had lesions only in the subiliac lymph node, which is not routinely examined during slaughter surveillance. Results of this study indicate that not all cattle infected with M. bovis have visible lesions of tuberculosis in sites that are routinely inspected. These findings are important because detection of gross lesions of tuberculosis during inspection of carcasses at slaughter is the primary method for detection of tuberculous cattle and herds in the United States.

A Holstein dairy farm suffered an abortion outbreak due to neosporosis. Abortion losses were > 18%. Cows with the highest Neospora antibody titers were at the greatest risk of aborting. Mummified fetuses were found after the 43rd day of the outbreak. The epidemic curve was suggestive of a point source exposure, which is consistent with the hypothesis that Neospora can be spread by a definitive host.

Since mid- 1989, 37 cases of oleander poisoning in livestock have been diagnosed at the California Veterinary Diagnostic Laboratory System. The most frequent source for oleander exposure was plant clippings. Sudden death was the most common presenting complaint. Other signs reported included diarrhea, pulmonary edema, tachycardia, cardiac arrhythmias, colic, and lethargy. In the past, a presumptive diagnosis of oleander poisoning could be based only on matching clinical signs with evidence of consumption of oleander. A new 2 dimensional Thin-layer chromatography analysis of ingesta for oleandrin and an awareness of lesions in heart muscle have greatly improved the ability to diagnose oleander toxicosis.