bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of an Enzyme-Linked Immunosorbent Assay for Specific Equine Neutrophil Myeloperoxidase Measurement in Blood
Authors: Franck T. , Grulke S. , Deby-Dupont G. , Deby C. , Duvivier H. , Peters F. , Serteyn D.
Published: May 2011

Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex “primary antibody-MPO-secondary antibody” was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 ± 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.
Use of Avidity Enzyme-Linked Immunosorbent Assay and Avidity Western Blot to Discriminate between Acute and Chronic Neospora Caninum Infection in Cattle
Authors: Aguado-Martínez A. , Álvarez-García G. , Arnaiz-Seco I. , Innes E. , Ortega-Mora L. M.
Published: May 2011

Avidity serological tests (avidity enzyme-linked immunosorbent assay [ELISA] and avidity Western blot) were developed and used to differentiate between acute (primary infection, reinfection, and recrudescence) and chronic Neospora caninum infection in cattle. In addition, the pattern of immunoglobulin G (IgG) avidity maturation against different specific antigens of N. caninum tachyzoites was studied. Sequential serum samples were collected from cattle naturally and experimentally infected with N. caninum. Four groups of experimentally infected cattle were included in the study and were representative of primary infection, reinfection, chronic infection, and noninfection. Serum samples were also collected from naturally infected cattle classified into nonaborting and aborting cows on the basis of clinical findings and serological profiles, and a third group composed of seronegative cows that seroconverted during the course of the experiment. All samples were tested by avidity ELISA and avidity Western blot. The IgG avidity ELISA allowed the discrimination between primary and chronic infection because all experimentally primary-infection cows showed low avidity indexes at week 4 postinfection (p.i.) compared with the high avidity values found at week 20 postinfection. However, this test did not allow the discrimination of reinfection or recrudescence from chronic infection. Regarding IgG avidity Western blot results, no antigenic markers correlating with acute (primary infection, recrudescence, and reinfection) or chronic infection were recognized. However, the 17-kD immunodominant antigen was mostly responsible for high avidity values obtained by avidity ELISA because it was intensively recognized by high-avidity antibodies in all chronically infected animals after urea treatment.
Distribution of Crenosoma Vulpis and Eucoleus Aerophilus in the Lung of Free-Ranging Red Foxes (Vulpes Vulpes)
Authors: Nevárez Alicia , López Alfonso , Conboy Gary , Ireland William , Sims David
Published: May 2011

Crenosoma vulpis and Eucoleus aerophilus are nematode parasites that can cause verminous pneumonia in wild carnivores. There is a paucity of information regarding the distribution of parasites in the lungs and the relationship between histopathological and parasitological diagnoses in naturally infected foxes. The objectives of this study were: first, to study the lobar and airway distribution of C. vulpis and E. aerophilus in wild red foxes and second, to investigate the relationship between fecal and histopathological diagnoses. Samples from 6 sites of the lung and fecal contents were obtained from 51 wild foxes in Prince Edward Island. By fecal examination, 78.4% of wild foxes tested positive for C. vulpis and 68.6% for E. aerophilus. In contrast, 66.6% and 49% of foxes had histopathological evidence of C. vulpis and E. aerophilus in the lungs, respectively. Anatomically, C. vulpis was observed in the small bronchi and bronchioles of all pulmonary lobes whereas E. aerophilus was restricted to the large bronchi and the caudal lobes. Affected airways exhibited severe epithelial glandular hyperplasia and bronchiolar mucous metaplasia. It was concluded that C. vulpis is widely distributed in airways of all pulmonary lobes, whereas E. aerophilus is mainly restricted to the bronchi of caudal lobes. Also, this study showed that histological examination of lung underestimates the infection with E. aerophilus.
Erysipelothrix Rhusiopathiae Septicemia in a Laughing Kookaburra (Dacelo Novaeguineae)
Authors: Opriessnig T. , Vance R. K. , Halbur P. G.
Published: May 2011

Erysipelothrix rhusiopathiae (E. rhusiopathiae) septicemia was demonstrated in a captive Laughing kookaburra ( Dacelo novaeguineae). The bird died after a 2-week period of weakness and weight loss. At necropsy, the bird was emaciated and had reddened and wet lungs. Microscopic lesions were limited to hepatic and pulmonary congestion with focal thrombosis. Erysipelothrix rhusiopathiae was isolated by routine bacterial culture from several organs. Further characterization of the isolate by pulsed-field gel electrophoresis indicated that the isolate has a new genotype pattern 3A(III), which is 91.7% homologous to an E. rhusiopathiae that was isolated from a pig in 2001 and 88% homologous to an isolate recovered in 2000 from a turkey with septicemia. This is the first report of E. rhusiopathiae–induced septicemia in a kookaburra.
Isolation of Equine Herpesvirus–2 from the Lung of an Aborted Fetus
Authors: Galosi Cecilia Mónica , de la Paz Viviana Cid , Fernández Leandro Carlos , Martinez Javier Pablo , Craig Mariá Isabel , Barrandeguy María , Etcheverrigaray María Elisa
Published: May 2011

This study describes the isolation of equine herpesvirus–2 (EHV-2) from the lung of an aborted equine fetus in Argentina. The isolated virus was confirmed as EHV-2 by indirect immunofluorescence using a rabbit anti–EHV-2 polyclonal antiserum and by virus-neutralization test using an equine polyclonal antibody against EHV-2. Restriction endonuclease DNA fingerprinting with BamHI also confirmed the identity of the virus as EHV-2. Furthermore, viral nucleic acid was detected by polymerase chain reaction from the original lung sample and from the DNA obtained from cells infected with the virus isolate. This work constitutes the first reported isolation of EHV-2 from an aborted equine fetus. The presence of EHV-2 in the lung of the aborted fetus would indicate that this virus is capable of crossing the placental barrier. However, no cause–effect relationship was established between the EHV-2 isolate and the abortion.
Use of a Multiplex Polymerase Chain Reaction to Rapidly Differentiate Neospora Caninum from Toxoplasma Gondii in an Adult Dog with Necrotizing Myocarditis and Myocardial Infarct
Authors: Meseck Emily K. , Njaa Bradley L. , Haley Nicholas J. , Park Edward H. , Barr Stephen C.
Published: May 2011

This report describes a 3-year-old male castrated Mastiff dog that died unexpectedly with locally extensive, acute, necrotizing myocarditis and myocardial infarction. Intralesional protozoal tachyzoites in the affected myocardium were confirmed to be Neospora caninum by a novel multiplex polymerase chain reaction (PCR) and immunohistochemistry. Protozoal organisms were not identified in other tissues by histology, immunohistochemistry, or PCR. The multiplex PCR assay was used to quickly provide preliminary results on fresh myocardium to differentiate N. caninum and Toxoplasma gondii. Neosporosis is an uncommon cause of myocarditis in adult dogs and differential diagnoses for myocarditis in this population of dogs are reviewed.
Canine Coronavirus-Associated Puppy Mortality without Evidence of Concurrent Canine Parvovirus Infection
Authors: Evermann J. F. , Abbott J. R. , Han S.
Published: May 2011

This report presents 2 cases in which puppy fatalities were associated with canine coronavirus (CCV), but no evidence of concurrent canine parvovirus (CPV-2) disease was observed. Case 1 involved a 7-week-old, male short-haired Chihuahua, which had become lethargic 24 hours after purchase from a pet store. Within 72 hours, the puppy began to vomit, had diarrhea, and was admitted to the veterinary clinic, where it was placed on IV fluids. The parvovirus Cite® test was negative. The puppy died within 12 hours of admission and was submitted for diagnostic workup. Gross pathology revealed an enteritis suggestive of CPV-2. Histopathology on intestines showed scattered dilated crypts with necrotic cellular debris and neutrophils. There was moderate depletion and necrosis of lymphoid follicles. Electron microscopy (EM) on intestinal contents was positive for coronavirus and negative for parvovirus. Immunohistochemistry (IHC) on gut sections was positive for CCV and negative for CPV-2. Case 2 was an 8-week-old, male Shih Tzu, which was admitted to the veterinary clinic exhibiting symptoms of severe gastroenteritis with abdominal pain. The referring veterinarian euthanized the puppy, and the entire body was submitted for diagnostic evaluation. Necropsy revealed a severe ileo-cecal intussusception and segmental necrotic enteritis of the small intestine. Electron microscopy of the intestinal contents was positive for coronavirus and negative for parvovirus. Immunohistochemistry on sections of affected gut were positive for CCV and negative for CPV-2. These cases emphasize the importance of pursuing a diagnosis of CCV in young puppies when CPV-2 disease has been ruled out by IHC.
Intrarenal Pelvic Nephroblastoma in a Meerkat (Suricata Suricatta)
Authors: Singh Bhanu P. , Patterson-Kane Janet C. , Redrobe Sharon P. , Chapman Jennifer L.
Published: May 2011

Nephroblastoma is the most common primary renal tumor in children and has also been reported in domestic and nondomestic animal species. Intrapelvic renal nephroblastoma is a rare variant of this tumor type in human patients. Postmortem examination of a captive meerkat ( Suricata suricatta), which was found dead, revealed enlargement of the pelvis of the left kidney by a tumor mass. Gross, histological, and immunohistochemical findings were consistent with a diagnosis of triphasic intrapelvic renal nephroblastoma. This is the first reported spontaneous case of intrapelvic renal nephroblastoma in a nonhuman species.
Molecular and Antigenic Characterization of a Mycoplasma Bovis Strain Causing an Outbreak of Infectious Keratoconjunctivitis
Authors: Alberti Alberto , Addis Maria Filippa , Chessa Bernardo , Cubeddu Tiziana , Profiti Margherita , Rosati Sergio , Ruiu Angelo , Pittau Marco
Published: May 2011

An unusually high incidence of infectious keratoconjunctivitis followed by pneumonia and arthritis was observed in beef calves of a managed herd. No Moraxella spp. or bacteria other than Mycoplasma spp. were obtained from conjunctival and nasal swabs. A strategy was designed for characterization of bovine mycoplasmas at species and strain level on the basis of a combination of molecular tools and the immunoblotting method. The strategy made it possible to rapidly assign the bacterium responsible for this outbreak to one of the phylogenetic clusters of bovine mycoplasmas delineated in this study and then to identify it as Mycoplasma bovis. The strain, designated Sar 1, showed a 100% 16S rDNA sequence identity with two European strains (120/81 and MC3386) isolated in Germany and Ireland, respectively, and hosts a vsp gene analog to the vspA, vsp422-4, and vsp422-8 genes of the M. bovis reference strain PG45T and of the field strain 422. The use of a cross-reactive rabbit serum developed against the Mycoplasma agalactiae immunodominant antigen P48 confirmed the molecular findings. The immunological response of calves against M. bovis was also investigated. This is the first report on the occurrence of M. bovis on the Island of Sardinia (Italy).
Assessment of Some Blood Parameters as Potential Markers of Hepatic Copper Accumulation in Cattle
Authors: López-Alonso Marta , Crespo Antonio , Miranda Marta , Castillo Cristina , Hernández Joaquín , Benedito José Luis
Published: May 2011

The increasing frequency of copper (Cu) toxicosis episodes in cattle in recent years, mainly associated with excess Cu supplementation, underscores the need to identify animals in the clinically silent phase of hepatic Cu accumulation. The aim of the study reported here was to evaluate the suitability of various blood parameters as potential early markers of hepatic Cu accumulation in cattle. Paired liver and blood samples from 70 calves aged 6 to 10 months were obtained at slaughter in a region in northwestern Spain where animals usually have hepatic Cu concentration higher than safe values. Neither serum Cu concentration nor ceruloplasmin (CP) concentration, the 2 parameters most commonly used for diagnosis of Cu deficiency, were significantly associated with hepatic Cu concentration. However, whole-blood Cu concentration had a slight but significant correlation with hepatic Cu concentration ( r = 0.269, P = 0.026). The use of calculated blood parameters, such as the serum or whole-blood non-CP Cu fraction, or the CP-to-serum Cu ratio, increased the correlation with the hepatic Cu concentration ( r = 0.393, P = 0.001, in the case of whole-blood, non-CP Cu concentration), but the strength of the association remained insufficient for accurate prediction of hepatic Cu values. Likewise, hepatic enzyme (aspartate transaminase and γ-glutamyltransferase) activities were significantly or nearly correlated significantly with hepatic Cu concentration, but the strength of the association remained too low for accurate prediction. It is concluded that direct analysis of Cu concentration in liver biopsy specimens—referably postmortem liver samples obtained at slaughter—the besttechniquecurrentlyavailablefor detecting chronic subclinical Cu accumulation in cattle at risk for supraoptimal Cu exposure.
Evaluation of Antigen-Capture ELISA and Immunohistochemical Methods for Avian Surveillance of West Nile Virus
Authors: Godhardt Jennifer A. , Beheler Kerry , O'Connor Michael J. , Whyte T. J. , Reisdorf Erik S. , Ubl Susan J. , Bochsler Philip N. , Toohey-Kurth Kathy L.
Published: May 2011

Accurate detection of West Nile virus (WNV) in corvids is essential for monitoring the spread of virus during the mosquito season. Viremia in corvids is very high, with titers approaching 108 viral particles/ml. In the presence of such marked viremia, the sensitivity of real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis is unnecessary, and more cost-effective methods should be assessed. To this end, antigen-capture ELISA (ACE) and immunohistochemical (IHC) assays were evaluated. Skin, cloacal swab specimens, and feathers from corvids were tested by use of ACE, and results were compared with results obtained from use of real-time RT-PCR analysis. Of the 3 sample types, skin gave the best sensitivity (98%) and specificity (100%). Skin, brain, kidney, and spleen from corvids were analyzed by IHC, and results were compared with real-time RT-PCR results. Kidney and spleen were more often positive by use of IHC than were brain and skin tissue; however, IHC did not perform as well as ACE in the identification of virus-positive birds. Results of this study support the use of a skin sample in an ACE format as an effective surveillance method for corvids.
A Devastating Outbreak of Malignant Catarrhal Fever in a Bison Feedlot
Authors: Li Hong , Taus Naomi S. , Jones Cevin , Murphy Brian , Evermann James F. , Crawford Timothy B.
Published: May 2011

In early 2003, an outbreak of malignant catarrhal fever (MCF) occurred in a bison feedlot in southern Idaho. The outbreak resulted in a 51.2% ( n = 825) mortality rate among bison, which had been exposed to sheep for 19 days. Diagnosis was made by detection of ovine herpesvirus 2 (sheep-associated MCF virus) DNA in tissues or peripheral blood by polymerase chain reaction (PCR), and by histological examination of tissue lesions. Peak losses occurred between 41 and 55 days postmean exposure time (PME), and reached a maximum of 41 head per day. No known cases of MCF were observed among the 177 head of bison that arrived in the lot 3 1/2 weeks after the departure of the sheep. Of the several thousand head of beef cattle in the lot during the outbreak, only a single case of MCF was identified. This outbreak illustrates the devastating impact the MCF virus can have on bison under certain exposure conditions, the high threat posed by adolescent lambs to susceptible species, the significantly greater susceptibility of bison than beef cattle to MCF, and the lack of horizontal transmission from clinically affected bison to herdmates.
Disseminated Transmissible Venereal Tumor in a Dog
Authors: Park Mi-Sun , Kim Yongbaek , Kang Min-Soo , Oh Sang-Yeon , Cho Doo-Youn , Shin Nam-Shik , Kim Dae-Yong
Published: May 2011

Transmissible venereal tumor (TVT) is a well-documented transplantable tumor in dogs, with no breed or sex predilection and a low metastatic rate. In this report, a 2-year-old intact female Mastiff that had numerous, rapidly growing masses throughout the subcutis mainly at the dorsal body plane, the caudal half of the ventral abdomen, and around the vulva was euthanized due to poor prognosis. Neoplastic nodules similar to those seen in the subcutis were also noted in the lung, anterior mediastinum, liver, spleen, kidney, and superficial and deep lymph nodes in both abdominal and thoracic cavities. The neoplastic nodules from the subcutis as well as metastatic foci revealed similar cytologic and histologic features, which were consistent with canine TVT. By immunohistochemical staining, the neoplastic cells were positive for lysozyme and vimentin but were negative for cytokeratin, desmin, CD3, and CD79a. The diagnosis of the TVT was further supported by the identification and analysis of long interspersed nuclear elements (LINE) from paraffin-embedded tumor tissue. This case is a rare example of TVT with multiorgan metastasis. In this case, the polymerase chain reaction technique was useful in differential diagnosis of canine round cell tumors because this technique can be applied in retrospective as well as future study.
Evaluation of Five Enzyme Immunoassays Compared with the Cytotoxicity Assay for Diagnosis of Clostridium Difficile-Associated Diarrhea in Dogs
Authors: Chouicha Nadira , Marks Stanley L.
Published: May 2011

Clostridium difficile-associated-diarrhea (CDAD) is a nosocomial infection in dogs. Diagnosis of this infection is dependent on clinical signs of disease supported by laboratory detection of C. difficile toxins A or B, or both, in fecal specimens via enzyme-linked immunosorbent assay (ELISA). Unfortunately, to the authors' knowledge, commercially available ELISAs have not been validated in dogs to date. We evaluated 5 ELISAs done on 143 canine fecal specimens (100 diarrheic and 43 nondiarrheic dogs) and on 29 C. difficile isolates. The results of each ELISA were compared with the cytotoxin B tissue culture assay (CTA). Clostridium difficile was isolated from 23% of the fecal specimens. Eighteen of the 143 fecal specimens were toxin positive (15 diarrheic and 3 nondiarrheic dogs). On the basis of multiplex polymerase chain reaction (PCR) analysis for toxin-A and -B genes, 72% of the isolates were toxigenic. The carriage rate of toxigenic isolates in diarrheic dogs was higher than that in the nondiarrheic dogs; however, these differences were not statistically significant. A good correlation was found between CTA, PCR, and culture results. The ELISAs done on fecal specimens collected from diarrheic dogs had low sensitivity (7–33%). In contrast, ELISA for toxin A or B, or both, performed on toxigenic isolates had high sensitivity (93%). These results suggest that commercially available human ELISAs are inadequate for the diagnosis of canine C. difficile-associated diarrhea when tested on fecal specimens. In contrast, the Premier ToxinA/B and Techlab ToxinA/B ELISAs may be useful for the diagnosis of canine CDAD when used on toxigenic isolates.
Internal Restriction Sites: Quality Assurance Aids in Genotyping
Authors: O'Rourke Brendon A. , Dennis Julie A. , Healy Peter J.
Published: May 2011

Improvements to restriction fragment length polymorphism (RFLP)-based genotyping assays currently used for detection of mutations responsible for bovine ferrochelatase and myophosphorylase deficiencies, and equine hyperkalemic periodic paralysis (HYPP) are described. Reports of sporadic inhibition of restriction enzyme activity suggest a critical factor in RFLP-based genotyping assays should be assurance that restriction enzymes perform to specification with every sample. The RFLP genotyping assays that use either a mismatched recognition sequence in one or both of the oligonucleotides, or incorporate a second native site within the PCR amplicon, provide the mechanism by which efficiency of restriction enzymes can be assessed with every sample. The outcome is confirmation of the activity of the discriminating enzyme regardless of genotype.
Western Blot Immunoassay as a Confirmatory Test for the Presence of Anti-Mycoplasma Hyopneumoniae Antibodies in Swine Serum
Authors: Ameri Mehrdad , Zhou En-Min , Hsu Walter H.
Published: May 2011

A Western blot immunoassay (WBI) was developed as a confirmatory test for 2 commercial Mycoplasma hyopneumoniae (Mhyo) ELISAs. The WBI detected at least 5 antigen bands (150, 130, 74, 70, and 30 kDa) from Mhyo whole membrane proteins that were not present in the antigens prepared from M. hyorhinis and M. hyosynoviae. Among discrepant sera from vaccinated pigs ( n = 17) and field samples ( n = 91) assayed by WBI: 1) 2 of the ELISA-positive samples reacted with all 5 antigens bands; 2) all blocking ELISA-positive samples ( n = 53) bound 150-, 130-, 74-, and 70-kD antigen bands; and 3) all indirect ELISA-positive samples ( n = 55) bound 150-, 130-, and 30-kD antigens. We conclude that the WBI targeting the top 4 antigen bands is a useful confirmatory test for samples initially screened using the commercial Mhyo ELISAs.
Relationship between DNA Ploidy and Proliferative Cell Nuclear Antigen Index in Canine Hemangiopericytoma
Authors: Kang Seong-Kwi , Park Nam-Yong , Cho Ho-Sung , Shin Sung-Shik , Kang Mun-Il , Kim Sang-Ki , Hyun Changbaig , Park In-Chul , Kim Jong-Tack , Jeong Cheol , Park Sung-Hee , Park Su-Jin , Jeong Jae-Ho , Kim You-Jung , Ochiai Kenji , Umemura Takashi , Cho Kyoung-Oh
Published: May 2011

The mitotic index is reported to be correlated with recurrence, mean patient survival, and metastasis of canine hemangiopericytoma (CHP). However, to the authors' knowledge, studies investigating the parameters that can predict recurrence or metastasis of CHP with low mitotic index have not been done. To evaluate growth kinetics of CHP with low mitotic index, a retrospective analysis of the proliferative activity by antiproliferative cell nuclear antigen monoclonal antibody and DNA contents by flow cytometry (FCM) was performed with 21 formalin-fixed and paraffin-embedded CHP samples. Of the 21 tumors evaluated by FCM, 6 (26.6%) were aneuploid tumors, and 15 (71.4%) were diploid tumors. There was significant correlation between the PCNA index and ploidy pattern. The diploid group had 39.1 ± 9.2 PCNA index, whereas the aneuploid group's proliferative cell nuclear antigen (PCNA) index was 63.1 ± 8.2. The diploid group had mean mitotic index value of 1.140 ± 0.855, and the aneuploid group had a mean value of 1.067 ± 0.767. From these results, the CHP samples with low mitotic index were classified into either the aneuploid group with higher PCNA index or the diploid group with lower PCNA index, suggesting that DNA ploidy and proliferative activity may give an indication about malignancy of CHPs with a low mitotic index.
Identification of Pathogenic Leptospira Strains in Tissues of a Premature Foal by Use of Polymerase Chain Reaction Analysis
Authors: Léon Albertine , Pronost Stéphane , Tapprest Jackie , Foucher Nathalie , Blanchard Béatrice , André-Fontaine Geneviève , Laugier Claire , Fortier Guillaume , Leclercq Roland
Published: May 2011

Studies were carried out to determine the cause of death in a prematurely born Thoroughbred foal that died 24 hours after birth. Necropsy revealed gross lesions suggestive of septicemia. A commercial Leptospira polymerase chain reaction (PCR) assay designed to specifically amplify the hemolysis-associated protein 1 ( hap1) gene present only in pathogenic Leptospira strains detected the presence of Leptospira DNA in various tissues of the foal. Histologic examination of lung, liver, kidney, and myocardium revealed numerous spirochetes in Warthin–Starry-stained tissue sections. Results of PCR analysis and histologic examination suggested a leptospiral infection in the newborn foal. At the moment of death, the infection coexisted with a streptococcal-associated aspiration bronchopneumonia and postpartum septicemia. These findings indicate that the PCR assay based on the amplification of the hap1 gene represents a useful tool for specific detection of pathogenic leptospira in field samples taken from horses.
Extraskeletal Osteosarcoma Associated with Retained Surgical Sponge in a Dog
Authors: Miller Margaret A. , Aper Rhonda L. , Fauber Amy , Blevins William E. , Ramos-Vara José A.
Published: May 2011

Retained surgical sponges are usually discovered in the abdominal cavity, sometimes years after the surgical procedure, and the typical reaction is formation of a foreign-body granuloma, often called gossypiboma or textiloma. In this instance, an extraskeletal osteosarcoma, associated with the granulomatous reaction to a retained surgical sponge adjacent to the stifle, was diagnosed in an 11-year-old Labrador Retriever 9 years after repair of a ruptured cranial cruciate ligament. Radiographic detection of linear foreign material in the soft tissue mass was the basis for a diagnosis of gossypiboma. The mass was surgically excised. Histologically, fibers consistent with those of cotton gauze were associated with the granulomatous inflammation and the osteosarcoma. Amputation or radiation therapy was declined; the dog was treated conservatively with doxycycline and deracoxib. Three months after surgical excision, the dog was euthanized because of local recurrence of the mass along with lameness and decreased appetite.
Peliod Hepatocellular Carcinoma in a Domesticated Ferret (Mustela Putorius Furo)
Authors: Jones Y. , Wise A. , Maes R. , Kiupel M.
Published: May 2011

Peliod hepatocellular carcinoma was diagnosed in a domesticated ferret ( Mustela putorius furo). The diagnosis was made using immunohistochemical analysis, histologic examination, and the accepted classification schemes based on histomorphologic features. Bilateral, adrenocortical hyperplasia also was evident. Speculation about a possible association between the variant of hepatocellular neoplasia diagnosed in this animal and its adrenal pathologic changes was done.
Use of Pooled Samples for the Detection of Salmonella in Feces by Polymerase Chain Reaction
Authors: Singer Randall S. , Cooke Cara L. , Maddox Carol W. , Isaacson Richard E. , Wallace Richard L.
Published: May 2011

Many epidemiological studies of Salmonella rely on conventional bacteriological culture methods to detect Salmonella in fecal samples. These culture-based methods are inefficient for epidemiological studies in populations with a low prevalence of Salmonella. The objective of this study was to optimize a protocol that uses pooled Salmonella enrichment broth cultures of bovine feces and polymerase chain reaction (PCR) for the detection of the invA gene of Salmonella in feces. In one field trial, 196 animals were sampled, and all samples were tested by culture, invA PCR on individual samples, invA PCR on pools of 5 samples, and BAX PCR on individual samples. All assays showed a high agreement on individual samples (kappa ≥ 0.75). The invA PCR was run on each of 40 pools and detected 19 of 22 culture-positive pools. In another field trial, 152 samples were taken from 4 dairies, and the invA PCR was performed on pools of 5 samples in addition to bacteriological culture of individual samples. Salmonella was detected in 5 of the 32 pools (7 total positive samples) by both PCR and culture. One pool was PCR-positive but culture-negative. Pooling did not dramatically affect the performance of the invA PCR; most of the culture-positive samples were detected, including all of the samples when there were 4 or more Salmonella colonies on the agar plate. Based on these field trials, invA PCR on pooled samples appears to be an efficient method of Salmonella detection as long as Salmonella loads are not extremely low.
Detection and Quantification of Equine Herpesvirus-1 Viremia and Nasal Shedding by Real-Time Polymerase Chain Reaction
Authors: Hussey Stephen B. , Clark Rodney , Lunn Katharine F. , Breathnach Cormac , Soboll Gisela , Whalley J. Millar , Lunn D. Paul
Published: May 2011

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% ± 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.
Detection and Quantification of Renibacterium Salmoninarum DNA in Salmonid Tissues by Real-Time Quantitative Polymerase Chain Reaction Analysis
Authors: Chase Dorothy M. , Elliott Diane G. , Pascho Ronald J.
Published: May 2011

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon ( Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.
Isolation of Vibrio Cholerae from the Brain of a Feedlot Heifer with Meningoencephalitis
Authors: Bush Jamie M. , Hyatt Doreene R. , Bolte Denise , Pandher Karamjeet
Published: May 2011

A 700-pound, 9-month-old Angus heifer from a feedlot presented with acute neurologic signs, characterized by circling, posterior weakness, and nonresponsiveness, followed by death. Histologically, the frontal lobe and the thalamus contained multiple foci of liquefaction that contained numerous degenerative neutrophils and foamy macrophages. Some of these foci were centered on blood vessels that contained fibrin thrombi and exhibited varying degrees of fibrinoid necrosis of the vessel wall. There was adjacent axonal degeneration and neuronal necrosis characterized by pronounced cytoplasmic eosinophilia, peripheralization of the nuclei, and loss of Nissl substance. Aerobic culture of the brain yielded moderate growth of Vibrio species, which was determined to be Vibrio cholerae by polymerase chain reaction analysis of a 438-base pair fragment of the 16 S ribosomal RNA gene. V. cholerae are motile, gram-negative, curved rod-shaped bacteria. Some strains of V. cholerae are important food- and water-borne bacterial pathogens that produce an often fatal diarrhea in humans. This is the first known case report of V. cholerae meningoencephalitis and cerebral abscessation in a bovine.
Degenerative Myelopathy and Vitamin a Deficiency in a Young Black-Maned Lion (Panthera Leo)
Authors: Maratea Kimberly A. , Hooser Stephen B. , Ramos-Vara José A.
Published: May 2011

Degenerative myelopathy and vitamin A deficiency were diagnosed in a 1-year-old, female, black-maned lion ( Panthera leo). Diffuse white matter degeneration characterized by dilated myelin sheaths, Wallerian degeneration, and reactive astrocytosis was present at all levels of the spinal cord. With luxol fast blue–cresyl echt violet stain, bilaterally symmetrical demyelination was observed in the fasciculus cuneatus of the cervical spinal cord and in peripheral white matter of cervical, thoracic, and lumbar segments. Additionally, the ventral gray columns and brain stem nuclei contained rare chromatolytic neurons with abnormal neurofilament accumulation. Leptomeninges of the cervical spinal cord were focally adhered to the dura and thickened by fibrosis and osseous metaplasia. Vitamin A deficiency was diagnosed based on hepatic vitamin A concentration of 1.71 μg/g dry weight. Adequate hepatic vitamin A concentration for yearling to adult domestic animals ranges between 150 and 1,000 μg/g dry weight. Lesions were distinct from those previously described in young captive lions with vitamin A deficiency, which had thickened skull bones and cerebellar herniation. The pathogenesis of vitamin A–associated myelopathy in this lion may be similar to that described in adult cattle, which is believed to result from spinal cord compression secondary to elevated pressure of cerebrospinal fluid.
Performance of a Foot-and-Mouth Disease virus Reverse Transcription-Polymerase Chain Reaction with Amplification Controls between Three Real-Time Instruments
Authors: Moniwa Mariko , Clavijo Alfonso , Li Mingyi , Collignon Brad , Kitching R. Paul
Published: May 2011

The foot-and-mouth disease virus (FMDV) is a member of the picornavirus family, possessing an 8-kb single-stranded RNA genome of positive polarity. It is highly contagious among several livestock species and can lead to severe economic consequences, as evidenced by the UK outbreak in 2001. The usage of real-time polymerase chain reaction has facilitated rapid detection of FMDV. Several real-time PCR instruments are available with various capabilities, such as portability and high sample volume analysis. Primers and a dual-labeled TaqMan probe were optimized to detect a single, highly conserved 88-bp segment of the FMDV 3D (RNA polymerase) gene. To increase the confidence of the RT-PCR result, a positive amplification control was synthesized to detect potential false-positive results due to contamination if a wildtype virus is used as positive control. In addition, a preventative measure against false-negative results was developed in which endogenous beta actin mRNA is coamplified by RT-PCR. Assay performance was compared on the LightCycler1.2 (Roche), the SmartCyclerII (Cepheid), and the SDS 7900HT (ABI). These assays successfully identified the FMDV genome and beta actin mRNA from several sources of infected nasal and oral swabs, as well as probang samples.
Comparison of Five Diagnostic Methods for Detecting Bovine Viral Diarrhea Virus Infection in Calves
Authors: Hilbe Monika , Stalder Hanspeter , Peterhans Ernst , Haessig Michael , Nussbaumer Marlies , Egli Christoph , Schelp Christian , Zlinszky Kati , Ehrensperger Felix
Published: May 2011

Five diagnostic techniques performed on skin biopsies (shoulder region) and/or serum were compared for detection of bovine viral diarrhea virus infection in 224 calves 0–3 months of age, 23 calves older than 3 months but younger than 7 months, and 11 cattle older than 7 months. The diagnostic methods used were immunohistochemistry (IHC), 2 commercial antigen ELISAs, 1 commercial antibody ELISA, and realtime RT-PCR. Results of 249 out of 258 skin and serum samples were identical and correlated within the 3 antigen detection methods and the real-time RT-PCR used. Twenty-six of these 249 samples were BVDV-positive with all antigen detection methods and the real-time RT-PCR. Nine out of 258 samples yielding discordant results were additionally examined by RT-PCR, RT-PCR Reamplification (ReA), and antigen ELISA I on serum and by immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies. Virus isolation and genotyping was performed as well on these discordant samples. In 3 cases, transiently infected animals were identified. Two samples positive by real-time RT-PCR were interpreted as false positive and were ascribed to cross-contamination. The antigen ELISA II failed to detect 2 BVDV-positive calves due to the presence of maternal antibodies; the cause of 2 false-positive cases in this ELISA remained undetermined. Only persistently infected animals were identified in skin samples by IHC or antigen ELISA I. The 3 antigen detection methods and the real-time RT-PCR used in parallel had a high correlation rate (96.5%) and similar sensitivity and specificity values.
Application and Field Validation of a PCR Assay for the Detection of Mycoplasma Hyopneumoniae from Swine Lung Tissue Samples
Authors: Cai Hugh Y. , van Dreumel Tony , McEwen Beverly , Hornby Geoff , Bell-Rogers Patricia , McRaild Pat , Josephson Gaylan , Maxie Grant
Published: May 2011

A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.
Detection of Pathogens in Ovine and Caprine Abortion Samples from Sardinia, Italy, by PCR
Authors: Masala Giovanna , Porcu Rosaura , Daga Cinzia , Denti Stefano , Canu Giuliana , Patta Cristiana , Tola Sebastiana
Published: May 2011

During 2003–2005, 399 abortion samples (315 fetuses and 84 placentae) were collected from 107 ovine and caprine farms in northern Sardinia. Tissues from aborted fetuses and placentae were examined by PCR assay to detect DNA from Coxiella burnetii, Chlamydophila abortus, Salmonella enterica Serovar abortusovis, Toxoplasma gondii, and Neospora caninum. The DNA from at least 1 of these 5 infectious agents was amplified in 41% of ovine fetuses, while only 17% of the caprine fetuses yielded a positive amplification result for at least 1 of the 5 agents. Out of a total of 366 ovine aborted samples, T. gondii DNA was detected most frequently (18.1% of fetuses and 13.1% of placentae), followed by S. abortusovis (13% of fetuses and 14.4% of placentae), C. burnetii (10.9% of fetuses, of 9.2% placentae), C. abortus (2.4% of fetuses, 6.5% of placentae), and N. caninum (2% of placentae). In 33 fetuses and 9 placentae, the simultaneous presence of pathogens with different associations was detected. Out of a total of 31 caprine aborted samples, T. gondii was detected most frequently (13% of fetuses and 25% of placentae), followed by C. abortus (12.5% of placentae), C. burnetii (12.5% of placentae), and N. caninum (8.6%).
Use of a Polymerase Chain Reaction for Detection of Mycoplasma Dispar in the Nasal Mucus of Calves
Authors: Marques L. M. , Buzinhani M. , Yamaguti M. , Oliveira R. C. , Ferreira J. B. , Mettifogo E. , Timenetsky J.
Published: May 2011

A polymerase chain reaction (PCR) assay was developed for the detection of Mycoplasma dispar in nasal mucus samples collected from calves. The target DNA sequence was the 16S rRNA gene, and the fragment was selected within a region of high polymorphism. The specificity and detection limit of the method were determined. This method was then used for the detection of M. dispar in nasal swabs collected from 301 calves, including 155 clinical samples from animals showing signs of respiratory disease and 146 samples from healthy animals. PCR with generic primers was applied to the detection of Mollicutes, followed by the detection of M. dispar. Mollicutes were detected in 52.05% of clinical samples from healthy animals and in 90.96% of samples from sick animals. Mycoplasma dispar was detected in 6.16% of healthy animals and in 34.84% of sick animals. The PCR assay was useful in verifying the presence of M. dispar in calves and may be a useful tool in monitoring this mycoplasma in cattle herds.
Necrotizing Encephalitis of Unknown Cause in Fennoscandian Arctic Foxes (Alopex Lagopus)
Authors: Berg Anna-Lena , Gavier-Widén Dolores , Nilsson Kristina , Widén Frederik , Berg Mikael , Gregorius Sune , Ågren Erik , Erlandsson Maria , Mörner Torsten
Published: May 2011

A neurologic disease affected a colony of endangered Fennoscandian arctic foxes ( Alopex lagopus) kept in captivity for breeding purposes. Several outbreaks of disease occurred between 1994 and 2004. The clinical signs included ataxia, indications of anosmia, blindness, and abnormal behavior. The disease was characterized by severe necrotizing encephalitis affecting mostly the cranial cerebrum, basal ganglia, and olfactory bulbs. Investigations to identify the etiology of the disease included testing for several infectious agents known to cause encephalitis in carnivores. Tests for Toxoplasma gondii, Encephalitozoon cuniculi, Neospora caninum, canine distemper virus, rabies, adenovirus type 1, Borna disease virus, and Listeria monocytogenes were negative. The colony was closed, and the cause of the disease remains undetermined.
Hypertrichosis in a Horse with Alimentary T-Cell Lymphoma and Pituitary Involvement
Authors: Mitsui Ikki , Jackson L. Paige , Couëtil Laurent L. , Lin Tsang L. , Ramos-Vara José A.
Published: May 2011

A 13-year-old Quarterhorse mare had a 6-month history of diarrhea, progressive weight loss, and lethargy. At presentation the mare was hirsute, had hyperhidrosis, and abnormal fat distribution in addition to severe diarrhea. A presumptive clinical diagnosis of protein-losing enteropathy and pituitary pars intermedia dysfunction was made. T-cell lymphoma was diagnosed in a rectal biopsy specimen. The owner elected to euthanize the mare because of poor prognosis and the severity of the disease. At necropsy, the mare had hypertrichosis and the pituitary gland was diffusely enlarged. Histologically, neoplastic lymphocytes infiltrated the gastrointestinal mucosa, mesenteric lymph nodes, and the pituitary gland. In addition, there was hyperplasia of the pituitary gland pars intermedia. Pituitary adenoma was not present. Hypertrichosis in this case could have been triggered by a combination of adenomatous hyperplasia of pars intermedia and lymphoma resulting in disruption of the hypothalamic dopaminergic tone or disruption of the hypothalamic thermoregulatory center.
A Novel Method for the Detection of Porcine Circovirus Type 2 Replicative Double Stranded Viral DNA and Nonreplicative Single Stranded Viral DNA in Tissue Sections
Authors: Hamberg Alexander , Ringler Susan , Krakowka Steven
Published: May 2011

Porcine circovirus type 2 (PCV2), an economically important pathogen of swine, is the necessary cause of post weaning multisystemic wasting disease (PMWS); PCV2 infection is associated with porcine dermatitis and nephritis syndrome (PDNS). Current immunohistochemical (IHC) methodologies identify PCV2 antigens but are not capable of differentiating replicating virus from nonreplicating virion particles in tissue sections. In this paper, a combination of IHC using commercial monoclonal antibodies specific for single stranded (ss) and double stranded (ds) DNA and PCV2 specific in situ hybridization (ISH) was used to show the specificity of the former for PCV2 DNA in tissue sections from PCV2-infected gnotobiotic pigs. Cold-ethanol-fixed tissue sections were superior to formalin-fixed tissues for detection of PCV2 DNA, presumably due to the lack of protein cross-linking in the latter. These data demonstrate that conventional IHC detects PCV2 DNA forms in experimentally infected PCV2-positive gnotobiotic porcine tissue sections that are minimally compromised by either formalin fixation or the hybridization conditions needed for ISH.
A Latex Agglutination Test for the Rapid Detection of Avian Influenza Virus Subtype H5N1 and its Clinical Application
Authors: Chen Jianfeng , Jin Meilin , Yu Zhengjun , Dan Hanbing , Zhang Anding , Song Yunfeng , Chen Huanchun
Published: May 2011

A rapid and simple latex agglutination test (LAT) for the detection of avian influenza virus (AIV) subtype H5N1 in chicken allantoic fluids, tracheal swabs, and tissues was developed. Monoclonal antibodies against the hemagglutinin glycoprotein of H5N1 were covalently coupled onto the surface of carboxylated latex bead using a water-soluble carbodiimide to obtain sensitized latex particles (SLP). These SLPs strongly agglutinated in the presence of allantoic fluid containing H5N1, but not fluids containing other AIV sub-types such as H1N1, H3N2, H4N6, and H9N2. Using this LAT, the virus was detectable in tracheal swabs 24 hours to 30 days after inoculating chickens with H5N1, with detection rates ranging from 45.5 to 79.2%. Much higher rates of detection were obtained from tissues collected postmortem from H5N1 experimentally infected chickens; lung tissue yielded the highest detection rate (96.7%), followed by kidney, spleen, brain, and liver tissues (90%). Lower detection rates were achieved with heart (41.7%) and cloacal tissues (26.8%). When the LAT was compared with other detection methods, the agreement with the viral isolation, H5 antigen immunochromatographic test, and H5 real-time RT-PCR test was 93.97, 95.18, and 87.95%, respectively. The test was highly specific for H5N1 in chickens and water fowls and had sensitivity comparable to other diagnostic tests evaluated.