bluuebunny/crossref_metadata_2025 huggingface dataset:
A PCR-based Test for the Specific Identification of Mycoplasma Mycoides Subspecies mycoides SC
Authors: Bashiruddin J. B. , Taylor T. K. , Gould A. R.
Published: May 2011

The polymerase chain reaction (PCR) was used to develop a test for the detection of Mycoplasma mycoides subspecies mycoides SC in the tissues of animals infected with contagious bovine pleuropneumonia (CBPP). Two sets of primers were designed; one set (MC323/MC358) to amplify a ∼1.5-kbp DNA fragment from all the members of the M. mycoides ‘Cluster’ and the other set (MM450/MM451) specifically amplified a 574-bp DNA fragment from M. mycoides subspecies. The PCR products could be differentiated further by digestion with the restriction enzyme AsnI. Enzyme digestion of amplification products from M. m. mycoides SC produced 2 fragments, whereas the other 2 M. mycoides subspecies, M. m. mycoides LC and M. m. capri, produced 3 fragments. This test was shown to be very sensitive, being able to detect between 10 and 100 organisms. Cattle were experimentally infected with the Gladysdale strain of M. m. mycoides SC, and samples of serum and mucus were taken periodically, as were postmortem samples of lung, lymph node, pleural fluid, synovial fluid, and tracheal swabs. Complement fixation test on serum samples, culture of postmortem tissues, and histopathologic examination confirmed disease. DNA was extracted from postmortem samples and amplified by PCR using primers MM450 and MM451. Digestion of products using AsnI allowed the specific identification of M. m. mycoides SC. This test could confirm CBPP in 48 hours and was thus capable of giving a more rapid result than the traditional methods of culture, isolation, and identification using biochemical and serological techniques.
In Situ Analysis of Cellular Proliferation in Canine, Feline and Equine Tumors by Immunohistochemistry: A Comparison of Bromodeoxyuridine, Proliferating Cell Nuclear Antigen, and Interchromatin-Associated Antigen Immunostaining Techniques
Authors: Théon Alain Pierre , Metzger Loretta , Griffey Stephen
Published: May 2011

Cell proliferation in canine, feline, and equine tumors was evaluated using immunohistochemical detection of in vitro 5–bromodeoxyuridine (BrdU) incorporation, proliferating cell nuclear antigen (PCNA), and interchromatin-associated antigen (p105). Ten tumors in each species were analyzed. The tumor proliferative fraction (PF) was defined as the percentage of labeled nuclei for 5,000 tumor nuclei counted. Immunoreactivity was observed with all techniques in all species. A good correlation was observed between the proliferative fractions measured with the BrdU (PFBrdU) and PCNA (PFPCNA) techniques ( rs = 0.523, P = 0.0026). There was no correlation between the PFs measured with the BrdU (PFBrdU) and p105 (PFP105) techniques. Using the median values obtained from the different approaches as cutoff points to define slowly and rapidly proliferating tumors, there was an 80% agreement ( P = 0.009) between PFBrdU and PFPCNA and no agreement between PFBrdU and PFP105 The results of this study indicate that both BrdU and PCNA labeling methods can be used reliably for identifying proliferating cells in animal tumors. In addition, PCNA could be used to replace the BrdU method to assess tumor proliferative fraction because it does not require pretreatment of tissues.
African Swine Fever Interference with Foot-and-Mouth Disease Infection and Seroconversion in Pigs
Authors: Gregg Douglas A. , Mebus Charles A. , Schlafer Donald H.
Published: May 2011

Initial oral infection of pigs with either highly virulent (L-60) or moderately virulent (DR-2) African swine fever virus (ASFV), followed in 3 days with exposure to foot-and-mouth disease virus (FMDV) (tongue inoculation and contact), failed to cause FMDV infection or seroconversion in 18 of 22 L-60-infected pigs and 13 of 34 DR-2-infected pigs. Of the 13 DR-2-infected pigs remaining free of foot-and-mouth disease (FMD), 2 pigs survived to 24 days without antibody to FMDV, despite constant contact with clinically infected pigs with FMD. Three other DR-2-infected pigs never developed FMD lesions but did develop low levels of antibody to FMDV by day 17. A group of larger pigs (in which DR-2 is less virulent) infected with DR.2 and then FMDV had a rapid but suppressed immune response to FMDV. Contact pigs introduced 3 days postinoculation and inoculated with FMDV only all became infected with ASFV by contact and died. This remarkably long lasting 1-way interference with FMD infection during acute and subacute African swine fever was not anticipated. Infection with ASFV may have blocked the initial target cells (possibly dendritic cells) necessary for establishment of FMDV infection.
African Swine Fever Virus Infection of Skin-Derived Dendritic Cells in Vitro Causes Interference with Subsequent Foot-and-Mouth Disease Virus Infection
Authors: Gregg Douglas A. , Schlafer Donald H. , Mebus Charles A.
Published: May 2011

Highly purified skin-derived dendritic cells (SDDCs) isolated from swine skin by a simple novel method were cultured for 24 hours before independent or sequential inoculation with African swine fever virus (ASFV) and foot-and-mouth disease virus (FMDV). By avidin-biotin immunohistochemical staining, ASFV antigen was detected in 50% of SDDCs as early as 1.5 hours postinfection (HPI) and in 80% by 3 HPI when cytopathic effect was noted. Cell lysis was detected with FMDV infection as early as 8 HPI; immunostaining for FMDV antigen was found in 10% of the cells. African swine fever virus replication was detected by transmission electron microscopy in a high percentage of SDDCs by 11 HPI. Sequential infection with FMDV 3 hours after ASFV inoculation blocked FMDV infection. These findings demonstrate that both ASFV and FMDV infect dendritic cells of Langerhans cell type in vitro and ASFV interferes with FMDV infection.
Survival of Pseudorabies Virus on Swabs Maintained under Standard Field Sample Shipping Conditions
Authors: McCaw Monte B. , Xu Jinsheng , Correa Maria Teresa
Published: May 2011

Pseudorabies virus survival was compared using three different types of applicator swabs in Eagle's minimum essential medium held under shipping conditions (packed with frozen gel packs) for up to 96 hours. Virus titer decay rates for dacron-tipped applicators were not statistically different from those of controls. Titer decay rates were statistically different from controls for cotton- and calcium alginate-tipped applicators. With the lowest input virus titer, virus was detectable up to 96, 72, or 24 hours after inoculation for dacron-, cotton-, and calcium alginate-tipped applicators, respectively. Dacron-tipped applicators were chosen to evaluate pseudorabies virus survival on tonsil swabs collected from experimentally challenged or contact control pigs to simulate field sampling and shipping conditions. Virus was still detectable in 20 of 24 swab samples after 72 hours in cell culture medium under shipping conditions.
Evaluation of a Blocking ELISA Using Monoclonal Antibodies for the Detection of Porcine Epidemic Diarrhea Virus and Its Antibodies
Authors: Carvajal Ana , Lanza Ignacio , Diego Rafael , Rubio Pedro , Cámenes Pedro
Published: May 2011

Two blocking enzyme-linked immunosorbent assays (ELISAs) involving the use of monoclonal antibodies (MAb) as capture and detecting agents and an indirect fluorescence test (IFT) were used for the detection of porcine epidemic diarrhea virus (PEDV) antigen or its antibodies. In the ELISA for viral antigen detection, the blocking step is accomplished by incubation of fecal samples, in duplicate wells, with PEDV-specific positive or negative serum, whereas in the ELISA for antibody detection the blocking step is accomplished by incubation of serum samples with a gut-origin virus suspension. All the methods developed were used to monitor an experimental infection in piglets with the CV-777 strain of PEDV and a natural PED outbreak in a swine fattening unit. The antigen-detection ELISA was able to detect PEDV shedding for a longer time than have previously described methods. The blocking ELISA for antibody detection was able to detect the serum antibody response sooner after PEDV infection than did IFT.
Prevalence and Serovars of Leptospira Involved in Equine Abortions in Central Kentucky during the 1991–1993 Foaling Seasons
Authors: Donahue James M. , Smith Barbara J. , Poonacha Kockanda B. , Donahoe Judy K. , Rigsby Cindy L.
Published: May 2011

In this study, the prevalence of leptospira-induced abortions/stillbirths for the past 3 foaling seasons (1991–1993) was determined, and fetal tissues and/or the mare's urine from positive cases were cultured in an attempt to isolate and identify the leptospira serovars responsible for the abortions. The sensitivity and specificity of the primary diagnostic tests, the fluorescent antibody test (FAT) and the microscopic agglutination test (MAT), used for the diagnosis of leptospirosis were also determined. For the 3 years, 74 (3.3%) of 2,264 abortion/ stillborn submissions were diagnosed as leptospirosis. Twelve cases occurred in the 1991, 19 in the 1992, and 43 in the 1993 foaling seasons. Leptospires were isolated from 45 (60.8%) of the 74 cases, and they were identified as serovar kennewicki (43 cases), serovar grippotyphosa (1 case), and a serovar similar to pomona (1 case). Of the 29 culture-negative cases, serologic results indicated that leptospires in the Pomona serogroup (kennewicki is a member of this serogroup) were responsible for 25 abortions, leptospires in the Grippotyphosa serogroup for 1 abortion, and leptospires in the Sejroe serogroup for 1 abortion. The specificities of the FAT on fetal tissues and mare's placenta and of the MAT on fetal fluid were 100%. The sensitivity of the FAT was 98.7%, and that of the MAT was 81.3%.
Rapid Detection of Pseudorabies Virus by the Shell Vial Technique
Authors: Tahir Rafique A. , Goyal Sagar M.
Published: May 2011

Conventional 24-well microtiter plates and shell vials were seeded with pig kidney (PK-15) and bovine turbinate (BT) cells. The monolayers were inoculated with 244 clinical specimens from pigs suspected of having pseudorabies virus (PRV) infection. The results of a shell vial assay (SVA) were compared with those obtained in a 24-well plate cell culture assay in terms of sensitivity and speed of virus isolation. All samples were passaged only once in cell cultures in both assays. Samples producing cytopathic effects (cpe) in 1 or both assay systems and showing positive fluorescence in a direct fluorescent antibody assay were considered to be positive for PRV. Of the 244 samples examined, 118 (48.4%) and 121 (49.2%) were positive by the 24-well plate assay and SVA, respectively. Of the 118 samples positive in 24-well plates, 113 (95.8%) were positive in BT cells and 117 (99.2%) were positive in PK-15 cells. The SVA detected 121 positive samples of which 121 (100%) were positive in PK-15 cells and 113 (93.4%) were positive in BT cells. Virus-specific cpe appeared earlier in the SVA than in the 24-well assay. At 24 hours postinoculation, 91 (75.2%) samples were cpe positive by SVA, whereas only 15 (12.7%) were positive in 24-well plates. All but 2 of the 121 (98.3%) SVA-positive samples were positive within 48 hours postinoculation, whereas only 56 of 118 (47.5%) were positive in 24-well plates during the same time period. These results indicate that the SVA is comparable in sensitivity to 24-well plate assay but yields virus isolation results more quickly. Also, PK-15 cells appeared to be more sensitive than BT cells for PRV isolation.
Identification of Ehrlichia Risticii as the Causative Agent of two Equine Abortions Following Natural Maternal Infection
Authors: Long M. T. , Goetz T. E. , Whiteley H. E. , Kakoma I. , Lock T. E.
Published: May 2011

Two pregnant mares diagnosed as having equine monocytic ehrlichiosis based on history, clinical signs, and high serum antibody titers to Ehrlichia risticii aborted subsequent to recovery from illness. Mare 1 and mare 2 experienced clinical illness at 120 and 143 days of gestation and aborted at 203 and 226 days of gestation, respectively. The fetuses were expelled in fresh condition, and both mares retained their placentas upon abortion. Gross findings for the fetuses included meconium staining and petechiation of external surfaces. Internally, there was increased volume of feces within the small and large intestines and liver discoloration with enlargement. Microscopic findings included lymphohistiocytic enterocolitis, hepatitis, and myocarditis. Lymphoid hyperplasia and depletion were present in spleen, thymus, and lymph nodes. Ehrlichia risticii was recovered from bone marrow, spleen, lymph node, colon, and liver of the first fetus and bone marrow and colon of the second fetus. Electron microscopic evaluation of the organism isolated in cell culture revealed morphology consistent with E. risticii. The isolated organism was inoculated into a naive pony, and this pony developed high levels of antibody against E. risticii, became ehrlichemic, and developed clinical signs of depression, anorexia, and mild diarrhea. These findings confirm that E. risticii is an abortifacient under conditions of natural infection and should be considered as a differential diagnosis of equine abortions.
Evaluation of an Enzyme-Linked Immunosorbent Assay for the Detection of Sheep Infected and Vaccinated with Brucella Melitensis
Authors: Delgado Silvia , Fernández Máximo , Cármenes Pedro
Published: May 2011

An indirect enzyme-linked immunosorbent assay (ELISA), using the lipopolysaccharide of the cell wall as an antigen, was used to detect Brucella melitensis antibodies in ovine serum. The test was carried out on 703 samples of field serums, which were also analyzed by the complement fixation (CF) test and the rose Bengal (RB) test. The ELISA results were more similar to those of the CF test (kappa = 0.89) than to the results of the RB test (kappa = 0.73). The ELISA also had high sensitivity (94.7%) and a somewhat lower specificity (90.4%). One group of 139 young brucellosis-free animals 3-6 months of age were vaccinated with B. melitensis rev. 1 at a dose of 1.2 × 109 live organisms. The ELISA detected a significantly lower number of reactors than the CF and RB tests ( P < 0.001). The ELISA values remained below the cutoff level during the 9 months following vaccination.
Identification and Characterization of Immunodominant Antigens during the Course of Infection with Brucella Ovis
Authors: Kittelberger Reinhold , Hilbink Frans , Hansen Mike F. , Ross Gail P. , de Lisle Geoffrey W. , Cloeckaert Axel , de Bruyn Jaqueline
Published: May 2011

The seroresponse against Brucella ovis of 8 intrapreputially and 6 intravenously infected rams and 9 ewes infected through mating was analyzed by electrophoretic immunoblotting. Additionally, 87 sera from chronically infected rams that were shedding B. ovis in their semen, 226 sera from rams belonging to infected flocks, and 324 sera from false-positive complement fixation test (CFT) reactors were examined. In all infected animals, antibody reactivity was predominantly found against 5 B. ovis components of 8–12, 17, 19, 29, and 63 kD, of which the 29-kD antigen was most dominant in the seroresponse. Antibodies to the 29-kD component were present in 93-100% of the infected sheep in each infected group, whereas the frequency of antibodies to the 4 other components varied considerably among and within the different groups. No reactivity against the 29-kD antigen was found in the false-positive CFT reactors. By using monoclonal antibodies against known bacterial macromolecules, the immunodominant antigens were identified as rough lipopolysaccharide (8-12 kD), outer membrane proteins (17, 19, 29 kD), and a heat-shock protein (63 kD).
Detection of Salmonella Enteritidis in Equine Feces using the Polymerase Chain Reaction and Genus-Specific Oligonucleotide Primers
Authors: Cohen Noah D. , Wallis Deeann E. , Neibergs Holly L. , Hargis Billy M.
Published: May 2011

Salmonella was identified in feces from horses, using the polymerase chain reaction (PCR) and genus-specific oligonucleotide primers. Feces from healthy horses were determined to be culture negative and PCR negative for Salmonella. Fecal samples were inoculated with known numbers of colony-forming units (CFU) of S. enteritidis. The fecal samples were enriched overnight in tetrathionate broth, and then DNA was extracted and amplified by PCR using genus-specific primers. Sensitivity of the assay extended to 100 CFU Salmonella enteritidis/g feces; sensitivity of microbiologic culture with enrichment extended to 100 CFU Salmonella enteritidis/g feces. Feces that were not inoculated with S. enteritidis were negative by the PCR. Detection of salmonellae in feces was possible using the PCR within 24 hours from the time of submission of samples. Because samples were enriched, isolates were available for determining antibiograms and serologic grouping or typing.
Prevalence of Cattle Persistently Infected with Bovine Viral Diarrhea Virus in 20 Dairy Herds in Two Counties in Central Michigan and Comparison of Prevalence of Antibody-Positive Cattle among Herds with Different Infection and Vaccination Status
Authors: Houe H. , Baker J. C. , Maes R. K. , Wuryastuti H. , Wasito R. , Ruegg P. L. , Lloyd J. W.
Published: May 2011

All cattle in 20 dairy herds randomly selected from herds participating in the Dairy Herd Improvement Association program in 2 counties in central Michigan were tested for the presence of bovine viral diarrhea virus (BVDV). Virus-positive animals were retested to ascertain persistent infection with the virus. A total of 5,481 animals were tested for presence of BVDV. In 9 of the herds, all animals were also tested for virus neutralizing antibody titer. Based on infection and vaccination status, these 9 herds were divided into 3 different herd categories: A, 5 herds with currently no cattle persistently infected (PI) with BVDV and without any vaccination program against BVDV in recent years; B, 2 herds with no current PI cattle but using killed BVDV vaccines; and C, 2 herds with PI cattle. PI cattle were detected in 3 out of 20 herds (15%). A total of 7 of 5,481 animals (0.13%) were PI. The mean prevalences of antibody carriers in herd categories A, B, and C were 28.8%, 76.4% and 90.6%, respectively. For one herd in category A, antibody analyses indicated that mostly young stock was seropositive, suggested recent BVDV infection in a previously closed and naive herd. Cattle in category B herds were vaccinated with killed vaccine from the age of 15 months. These herds had several antibody negative animals among the younger cows, suggesting incomplete protection against BVDV infection. In the 3 herds in which PI animals were detected, all cattle had been vaccinated with killed vaccine. The antibody-positive animals had antibody titers that were significantly different both among herds and among herd categories. The antibody titers of animals exposed to PI animals were significantly higher than those of animals vaccinated with killed vaccine.
Development and Application of an Immunohistochemical Staining Technique to Detect Avian Polyomaviral Antigen in Tissue Sections
Authors: Fitzgerald Scott D. , Reed Willie M. , Fulton Richard M.
Published: May 2011

An immunohistochemical staining technique was developed to detect polyomaviral antigens of budgerigar fledgling disease in formalin-fixed tissue sections. This technique used an indirect avidin-biotin, alkaline phosphatase labeling system with a mixture of monoclonal antibodies developed against the virus major capsid protein. The staining technique was applied retrospectively to 24 avian accessions which were originally diagnosed as budgerigar fledgling disease or avian polyomavirus infection based on microscopic findings including typical intranuclear inclusions. Immunohistochemical staining resulted in positive reactions in some tissues from 17 of 24 cases. The tissues most frequently containing typical intranuclear inclusions or positive immunohistochemical staining were the spleen, liver, and kidney. Neither of the 2 nonpsittacine cases was positive immunohistochemically. This technique may be used either as a rapid test on routinely processed diagnostic samples to confirm the presence of avian polyomavirus or for pathogenesis research studies.
A Pseudorabies Virus Mutant with Deletions in the Latency and Early Protein O Genes: Replication, Virulence, and Immunity in Neonatal Piglets
Authors: Wesley Ronald D. , Cheung Andrew K.
Published: May 2011

The pathogenicity of a double mutant of pseudorabies virus (PRV) with deletions in the latency gene and the early protein O gene was examined. In comparison to the parent Indiana-Funkhauser virus, the ability of this mutant to replicate and to cause disease in piglets is greatly reduced. At an infection dose that caused no clinical signs in 5-day-old neonatal piglets, this mutant was capable of eliciting solid protective immunity against a lethal PRV challenge. Thus, the double-gene deletion attenuates PRV but does not affect its immunogenicity. These features may be desirable for inclusion into future PRV vaccines.
Clinicopathologic Variation in Raccoons Infected with Different Street Rabies Virus Isolates
Authors: Hamir Amir N. , Moser Gale , Rupprecht Charles E.
Published: May 2011

Ten raccoons were divided into two random groups (groups 1 and 2) of five animals each. Group 1 raccoons were inoculated intramuscularly in the masseter muscle with a raccoon rabies virus isolate obtained from a natural case of raccoon rabies from the northeastern USA. Group 2 raccoons were infected by a similar route with a Latin American canine isolate of rabies virus. Raccoons either died suddenly or developed neurologic signs compatible with rabies. Clinical signs of rabies in group 1 raccoons were more severe than in group 2. Raccoons in group 1 either died acutely or were euthanized within 25 days (x ± SD = 20.6 ± 2.7 days) postinfection, whereas all group 2 raccoons showed neurologic signs and were euthanized within 17 days (14.2 ± 2.2 days) postinfection. Light microscopic findings revealed extensive nonsuppurative encephalitis predominantly located in the cerebrum and brain stem of raccoons in group 1, whereas in group 2 raccoons the lesions were confined to the brain stem regions. In group 1 raccoons, Negri bodies were commonly seen on hematoxylin and eosin (HE)-stained sections of brain and in ganglion cells of 5 other tissues (trigeminal nerve, salivary glands, duodenum, pancreas, adrenal gland). Negri bodies, however, were either absent or were only occasionally observed in corresponding tissues of raccoons infected with the canine strain (group 2). Paraffin-embedded tissue sections were also examined for Negri bodies by an immunoperoxidase test, which revealed results similar to the HE findings. Results of this study are compared with histopathologic and immunohistochemical findings in raccoons naturally infected with rabies.
Use of Species-Specific Oligonucleotide Probes to Detect Mycoplasma Gallisepticum, M. Synoviae, and M. Iowae PCR Amplification Products
Authors: Garcia Maricarmen , Jackwood Mark W. , Head Marcus , Levisohn Sharon , Kleven Stanley H.
Published: May 2011

Three digoxigenin-labeled oligonucleotide probes, complementary to the variable region of the 16S ribosomal RNA (rRNA) gene of Mycoplasma gallisepticum, M. synoviae, and M. iowae were designed. The oligonucleotides were used in a dot blot hybridization assay. The target DNA is a 780-bp fragment of the 16S rRNA gene of avian mycoplasmas amplified by a single set of primers (multispecies polymerase chain reaction [PCR]). The oligonucleotide probes were specific for their corresponding PCR products at hybridization conditions of 56 C and 50% formamide. The detection limit of the dot blot hybridization assay was approximately 70, 50, and 30 colony-forming units for M. gallisepticum, M. synoviae, and M. iowae, respectively, per 4 μl of PCR. In general, the oligonucleotide probe dot blotting assay was a more sensitive and effective method of detecting PCR products than detection by gel electrophoresis.
Histologic Evaluation of the Crop for Diagnosis of Proventricular Dilatation Syndrome in Psittacine Birds
Authors: Gregory Christopher R. , Latimer Kenneth S. , Campagnoli Raymond P. , Ritchie Branson W.
Published: May 2011

Histologic sections of crop tissue were evaluated for the presence of lymphoplasmacytic infiltrates within mesenteric ganglia. All birds with proventricular dilatation syndrome that had lymphoplasmacytic infiltrates in crop ganglia had similar infiltrates in the proventricular and/or ventricular ganglia. False-negative crop biopsy results occurred approximately 24% of the time. More invasive procedures, such as proventricular or ventricular biopsy, may be necessary if the crop biopsy is nondiagnostic in a bird with clinical signs of proventricular dilatation syndrome.
Epitheliocystis Infection in Cultured White Sturgeon (Acipenser transmontanus): Antigenic and Ultrastructural Similarities of the Causative Agent to the Chlamydiae
Authors: Groff Joseph M. , LaPatra Scott E. , Munn Robert J. , Anderson Mark L. , Osburn Bennie I.
Published: May 2011

A mild to moderate branchial epitheliocystis infection was diagnosed in subyearling (11 months old, 250–300 g) white sturgeon ( Acipenser transmontanus) from a private culture facility with a 4–8% mortality in the population. Infected branchial epithelial cells contained the coccoid to coccobacillary epitheliocystis organisms, which appeared as cytoplasmic inclusions composed of a fine, homogeneous, dense, basophilic, granular material. The infected cells were variably enlarged with spherical to oval profiles and were randomly distributed throughout the branchial epithelium. The cytoplasmic inclusions stained positive with Macchiavello stain but negative with Brown and Brenn, periodic acid-Schiff, and Gimenez stains. Expression of chlamydial antigen was demonstrated within the cytoplasmic inclusions using a standard peroxidase-antiperoxidase immunohistochemical technique. Three stages of coordinated intracellular development were recognized by electron microscopy. The reticulate bodies were oval to spherical and 0.4–0.8 × 0.5–1.4 μm but often exhibited a pleomorphic and convoluted appearance because of variable membrane invaginations and evaginations suggestive of uneven fission and budding. Separate host cells contained intermediate bodies that were spherical to oval and 0.2–0.4 × 0.3–0.6 μm although often observed in the process of apparent uneven division. The presence of a cap or plaque composed of hexagonally arrayed fibrillar surface projections was initially recognized in this stage. A homogeneous population of 0.3–0.4 μmoval elementary bodies were observed separately in individual host cells. This developmental stage had a single, dense, compact, eccentrically located cytoplasmic condensation that occurred opposite to the location of the cap of hexagonally arrayed fibrillar surface projections. Morphologic characteristics of the epitheliocystis organism in these white sturgeon were similar to those previously described in other teleosts and expands the species catalogue of epitheliocystis infection. Furthermore, the ultrastructural similarities to the chlamydiae and the immunohistochemical detection of chlamydial antigen provides further evidence that the epitheliocystis agent is related to members of the Chlamydiales. Although the infection was considered mild to moderate and could not be definitively attributed to the mortality in this population, the potential adverse impact of epitheliocystis infection on sturgeon culture should be considered especially in intensive fish culture operations.
Specific, Nonradioactive Detection of the NHP Bacterium in Penaeus Vannamei by in Situ Hybridization
Authors: Loy James K. , Frelier Paul F.
Published: May 2011

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.
Diagnosis of Oleander Poisoning in Livestock
Authors: Galey Francis D. , Holstege Dirk M. , Plumlee Konstanze H. , Tor Elizabeth , Johnson Bill , Anderson Mark L. , Blanchard Patricia C. , Brown Frank
Published: May 2011

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.
Rabies among African Wild Dogs (Lycaon Pictus) in the Masai Mara, Kenya
Authors: Kat P. W. , Alexander K. A. , Smith J. S. , Richardson J. D. , Munson L.
Published: May 2011

A pack of African wild dogs ( Lycaon pictus) ranging to the north of the Masai Mara National Reserve in southwestern Kenya was monitored from 1988 to 1989. During a 6-week period (August 1-September 13, 1989), 21 of 23 members of this pack died. Seven carcasses were retrieved, of which 4 were suitable for necropsy and histopathologic examination. Gross findings varied among individuals and included multiple bite wounds, synovitis, lymphadenopathy, submandibular, cervical, and vocal cord edema, blood in bronchi, bronchioles, stomach, and intestine, and anterioventral lung lobe consolidation. Histologic examination of 2 available brain samples revealed nonsuppurative encephalitis with eosinophilic intracytoplasmic inclusions (Negri bodies). An additional brain sample tested positive for rabies via a fluorescent antibody test. Other histologic features included severe suppurative bronchopneumonia, myocarditis, and lymphoid depletion of the lymph nodes, tonsils, and spleen. A 304-base pair (bp) nucleotide sequence from the N gene and a 310-bp sequence from the G gene from rabies isolates of 4 wild dogs indicated that infection was with a rabies variant common among domestic dogs in Kenya and Tanzania.
Comparison of Pharyngeal, Fecal, and Cloacal Samples for the Isolation of Chlamydia Psittaci from Experimentally Infected Cockatiels and Turkeys
Authors: Andersen Arthur A.
Published: May 2011

Direct comparisons were made of Chlamydial isolation rates from pharyngeal swabs, fecal samples, and cloacal swabs from cockatiels and pharyngeal and cloacal swabs from turkeys experimentally infected with Chlamydia psittaci. During pathogenesis studies, 133 paired specimens were collected from cockatiels and 118 paired specimens were collected from turkeys. Of the 51 cockatiel Chlamydial infections detected, 80.4% were positive by the pharyngeal swab sample, 45.1% were positive by the fecal swab sample, and 37.3% were positive by the cloacal swab sample. Of the 87 turkey infections detected, 93.1% were positive by the pharyngeal swab sample and 77.0% were positive by the cloacal swab. The pharyngeal swabs were the most reliable sample for isolation of chlamydia from live birds. However, no single sampling site yielded positive results from all infected birds. Specimens from multiple sites are recommended because a number of infected birds were identified by isolation from only 1 sample.
A PCR-Based Method of Detection and Differentiation of K88+ Adhesive Escherichia Coli
Authors: Franklin Mark A. , Francis David H. , Baker Diane , Mathew Alan G.
Published: May 2011

The objective of this study was to develop a polymerase chain reaction (PCR)-based method to detect and differentiate among Escherichia coli strains containing genes for the expression of 3 antigenic variants of the fimbrial adhesin K88 (K88ab, K88ac, and K88ad). Five primers were designed that allowed detection of K88+ E. coli, regardless of antigenic variant, and the separate detection of the ab, ac, and ad variants. Primers AM005 and AM006 are 21 base pair (bp) oligomers that correspond to a region of the K88 operon that is common to all 3 antigenic variants. Primers MF007, MF008, and MF009 are 24-bp oligomers that matched variable regions specific to ab, ac, and ad, respectively. Using primers AM005 and AM006, a PCR product was obtained that corresponds to a 764-bp region within the large structural subunit of the K88 operon common to all 3 antigenic variants. Primer AM005 used with MF007, MF008, or MF009 produced PCR products approximately 500-bp in length from within the large structural subunit of the K88 operon of the 3 respective antigenic variants. Fragments were identified by rates of migration on a 1% agarose gel relative to each other as well as to BstEII-digested lambda fragments. This PCR-based method was comparable to the enzyme-linked immunosorbent assay and western blot test in the ability to differentiate between the antigenic variants. K88+ E. coli were differentiated from among laboratory strains and detected in ileal samples taken from cannulated pigs challenged with a known K88+ variant. K88+ E. coli were also detected from fecal swabs taken from newly weaned pigs, thus confirming that this PCR-based test could provide a convenient clinical assay for the detection of K88+ E. coli. Detection and differentiation of K88+ E. coli using general and specific primers was successful. PCR methods of detection should permit identification of K88+ antigenic variants regardless of the level of expression of the antigen.
Standardization of Enzyme-Linked Immunosorbent Assays (ELISAs) for Quantitative Estimation of Antibodies Specific for Infectious Bovine Rhinotracheitis Virus, Respiratory Syncytial Virus, Parainfluenza-3 Virus, and Bovine Viral Diarrhea Virus
Authors: Graham D. A. , Mawhinney K. A. , McShane J. , Connor T. J. , Adair B. M. , Merza M.
Published: May 2011

Commercial enzyme-linked immunosorbent assays (ELISAs) for detection of serum antibodies to bovine viral diarrhea virus (BVDV), parainfluenza-3 virus (PI3V), respiratory syncytial virus (RSV), and infectious bovine rhinotracheitis virus (IBRV) were standardized to give a quantitative result when testing was performed at a single optimum dilution. For each test, serum samples were titrated and their end point titers calculated by an algebraic method directly from a plot of each titration series and also from a regression line fitted to this plot. The corrected optical density (COD) of each sample when tested at dilutions of 1/25, 1/50, and 1/100 was expressed as a percentage of the COD of a positive reference serum included on each plate, this value was the sample/positive (S/P) ratio. For each test, the linear relationship between the S/P ratio obtained at a dilution of 1/25, 1/50, and 1/100 and the end point titer calculated by each method was determined. In each case, the best linear relationship existed when samples were tested at a dilution of 1/100 ( r = 0.973 for BVDV, 0.962 for PI3V, 0.961 for RSV, 0.947 for IBRV). From the equation of these lines, an increase in the S/P ratio between acute and convalescent serum samples of 31%, 23%, 21%, and 35% would correspond to a 4-fold rise in ELISA titer to BVDV, PI3V, RSV, and IBRV, respectively. ELISA titers calculated from S/P ratios at 1/100 were significantly related to virus neutralization titers to BVDV, RSV, and IBRV and to hemagglutination inhibition titers to PI3V ( P ≪ 0.001 in all cases). Samples with low S/P ratios had the greatest intraassay and interassay variation. Intraassay reproducibility ranged from 3.5% to 22.3% (coefficient of variation), with a median value of 9.5%. Interassay reproducibility was lower, ranging from 6.0% to 50.6%, with a median of 17.4%.
Use of a Nonradioactive DNA Probe for Detection of Anaplasma Marginale Infection in Field Cattle: Comparison with Complement Fixation Serology and Microscopic Examination
Authors: Ge Nie-Lin , Kocan Katherine M. , Ewing Sidney A. , Blouin Edmour F. , Edwards Wanda L. , Murphy George L. , Dawson Lionel J.
Published: May 2011

A sensitive Anaplasma marginale-specific 409-base pair DNA probe was developed in a previous study for detection of A. marginale infection in experimentally infected cattle with a test that employed slot-blot and in situ hybridization. To test the suitability of the probe to detect A. marginale in the blood of naturally infected carrier cattle, slot-blot hybridization was used to determine the infection rate of A. marginale in cattle from 3 geographic areas in Oklahoma. For comparison, blood samples from the same cattle were also examined by light microscopy and were tested by the complement fixation test. For the DNA hybridization assay, the probe was labeled with digoxigenin 11-dUTP by polymerase chain reaction (PCR). DNA was extracted from blood using the QIAamp blood kit and then applied to a nylon membrane and hybridized with the probe. The study herds consisted of 31 beef cows in Harper County, OK, and 42 and 70 dairy cows from Payne and Pittsburg counties, OK, respectively. In the 3 herds, 80.6%, 92.8%, and 57.1% of the cows were positive for A. marginale as assessed with the DNA hybridization assay. In contrast, only 25.8% and 2.86% were complement fixation positive in 2 herds, and no complement fixation positives were found in 1 herd. Uncountable parasitemia that was too low to accurately determine (< 0.01%) from 29.0%, 4.8%, and 11.4% of the samples, respectively, was demonstrated by microscopic examination. All samples positive by complement fixation and microscopic examination had positive probe reactions in the DNA hybridization assay. Therefore, the PCR-mediated nonradioactive DNA probe described here may be useful in epidemiologic investigations and in identification of carrier cattle. This assay could be adapted for use in diagnostic laboratories because it is sensitive, specific, nontoxic, quickly executed, and inexpensive.
Serologic Diagnosis of Canine Leishmaniasis by Dot-ELISA
Authors: Fisa R. , Gállego M. , Riera C. , Aisa M. J. , Valls D. , Serra T. , de Colmenares M. , Castillejo S. , Portús M.
Published: May 2011

A dot-enzyme-linked immunosorbent assay (ELISA) using protein A-peroxidase was evaluated as a diagnostic test for canine leishmaniasis. The test results were in agreement with parasitologic diagnosis and indirect immunofluorescence assay results. The sensitivity of the test calculated on 31 dogs with positive parasitologic examination was 90% when a titer of 1/800 was established as a cutoff and 100% when a titer of 1/400 was established. The specificity calculated on the canine population from nonendemic areas was 100% when both titers were established. Nevertheless, in endemic areas titers near the cutoff need careful interpretation. The results of this study demonstrate that dot-ELISA protein A using a bio-dot apparatus is highly suitable for seroepidemiologic field work.
Detection of Mycobacterium Bovis in Formalin-Fixed, Paraffin-Embedded Tissues of Cattle and Elk by PCR Amplification of an IS6110 Sequence Specific for Mycobacterium Tuberculosis Complex Organisms
Authors: Miller Janice , Jenny Allen , Rhyan Jack , Saari Dennis , Suarez David
Published: May 2011

A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk ( Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex ( M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria ( M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.
Multiplex Polymerase Chain Reaction for Simultaneous Detection of Lawsonia Intracellularis, Serpulina Hyodysen Teriae, and Salmonellae in Porcine Intestinal Specimens
Authors: Elder Robert O. , Duhamel Gerald E. , Mathiesen Michelle R. , Erickson E. Denis , Gebhart Connie J. , Oberst Richard D.
Published: May 2011

Proliferative enteritis, swine dysentery, and porcine salmonellosis are the most common enteric bacterial diseases affecting pigs in the growing and finishing stages of production. Currently, diagnoses of these diseases by standard cultural techniques of intestinal specimens can be laborious, time consuming, and expensive (swine dysentery, porcine salmonellosis) or impossible (proliferative enteritis). Amplification by polymerase chain reaction (PCR) of DNA sequences specific for each bacterial agent is a highly sensitive and specific method that overcomes the limitations associated with standard detection methods. A multiplex PCR (M-PCR) assay was developed for simultaneous detection and identification of the etiologic agents associated with proliferative enteritis, swine dysentery, and porcine salmonellosis in a single reaction using total DNA obtained directly from intestinal specimens. Purified DNA obtained from pure cultures of each bacterial agent alone or mixed in different combinations and concentrations and total DNA from intestinal specimens were amplified using the Lawsonia intracellularis-, Serpulina hyodysenteriae-, and salmonellae-specific M-PCR assay. Intestinal specimens consisted of feces and mucosal scrapings obtained from field cases of each disease alone or in combinations and feces obtained from pigs challenged with S. hyodysenteriae. The banding pattern of the amplified PCR products, after agarose gel electrophoresis and staining, indicated the presence of individual or combinations of etiologic agents in each specimen. Results from this study indicated that simultaneous amplification of L. intracellularis-, S. hyodysenteriae-, and salmonellae-specific DNA sequences by M-PCR can be used for specific detection and identification of three major enteric bacterial pathogens associated with proliferative enteritis, swine dysentery, and porcine salmonellosis occurring alone or in combinations. Also, the M-PCR assay can be done using DNA obtained directly from intestinal specimens submitted for diagnostic investigation.
Distribution of Cytopathic and Noncytopathic Bovine Viral Diarrhea Virus Antigens in Tissues of Calves following Acute Experimental Infection
Authors: Spagnuolo-Weaver M. , Allan G. M. , Kennedy S. , Foster J. C. , Adair B. M.
Published: May 2011

The distribution of cytopathic and noncytopathic biotypes of bovine viral diarrhea virus (BVDV) in the tissues of colostrum-fed and colostrum-deprived calves was investigated. Colostrum-fed (group A) and colostrum-deprived (group B) calves were experimentally infected with the BVDV isolate 80/1, which contains both BVDV biotypes. Colostrum-deprived calves were also experimentally infected with a noncytopathic BVDV (group C) or with a cytopathic BVDV (group D) cloned from the 80/1 isolate. All calves were sequentially euthanized, and a wide range of tissue samples were processed for immunofluorescent and virus isolation studies. In group A, consistent immunofluorescent staining for BVDV was detected in vascular smooth muscle of numerous blood vessels in the tissues examined, mainly at 11 and 13 days postinoculation. A predominance of samples containing cytopathic BVDV was observed in the calves of this group, following virus isolation studies. Both cytopathic and noncytopathic BVDV were detected/recovered from a larger range of specimens in the calves in group B than from the calves in group A. In the calves in all the experimental groups, large amounts of BVDV antigen were detected mainly in tissue samples from the lymphoid and gastrointestinal systems, whereas only minimal amounts of BVDV were detected in the respiratory tract. Abundant noncytopathic BVDV antigen was also detected in pituitary gland and in Langerhans islets in pancreases of colostrum-deprived calves infected with the cloned noncytopathic BVDV. Noncytopathic BVDV was isolated from a wider range of tissues from calves in group C than in the colostrum-deprived calves infected with both BVDV biotypes. A cytopathic BVDV was isolated/detected in retropharyngeal, mesenteric, and abomasal lymph nodes and in thymus of 2 calves in group C. Cytopathic BVDV was detected/isolated mainly from mesenteric lymph nodes and Peyer's patches of the calves in group D.
Detection of Bovine Immunodeficiency Virus Antibodies in Cattle by Western Blot Assay with Recombinant Gag Protein
Authors: Zhang Shucheng , Xue Wenzhi , Wood Charles , Chen Qi-Min , Kapil Sanjay , Minocha Harish C.
Published: May 2011

A western blot assay using purified recombinant bovine immunodeficiency virus gag protein has been developed for detection of bovine immunodeficiency virus antibodies in bovine serum samples. The test was standardized with known bovine immunodeficiency virus positive and negative bovine serum samples and the monoclonal antibody to gag protein. Both naturally and experimentally infected cattle sera demonstrated positive test results. The result of western blot assay was compared with polymerase chain reaction test results in 134 blood samples collected from Kansas. Twenty-six samples tested positive for bovine immunodeficiency virus DNA with polymerase chain reaction (18.7%) and 25 were positive for the antibody to gag protein by western blot analysis (17.9%). Of 26 cattle testing positive using the polymerase chain reaction assay, 24 were antibody-positive by western blot assay, thus establishing a strong correlation between the two tests. The sensitivity and specificity of western blot relative to polymerase chain reaction are 0.92 and 0.99, respectively. The western blot assay proved to be a specific and sensitive test.