bluuebunny/crossref_metadata_2025 huggingface dataset:
Canine C-Reactive Protein Measurements in Cerebrospinal Fluid by a Time-Resolved Immunofluorimetric Assay
Authors: Martínez-Subiela Silvia , Caldin Marco , Parra Maria Dolores , Ottolini Nicola , Bertolini Giovanna , Bernal Luis J. , García-Martinez Juan D. , Cerón Jose J.
Published: May 2011

In the current study, the quantification of C-reactive protein (CRP) in cerebrospinal fluid (CSF) of dogs using an adapted time-resolved immunofluorimetric assay (TR-IFMA) was investigated, as well as whether the assay could be used to detect the range of CRP concentrations found in different clinical situations. Intra- and interassay coefficients of variation were below 15% in all cases. The TR-IFMA measured the CRP values in a proportional and linear manner ( r = 0.99); also CRP concentrations measured in CSF and in serum were significantly correlated ( r = 0.80, P = 0.003). The limit of detection of the method was 7.1 × 10−6 mg/l. The assay was able to detect differences in CRP concentrations in CSF of dogs with inflammatory disorders compared with dogs with spinal cord compression or idiopathic epilepsy. In conclusion, TR-IFMA constitutes a very sensitive, precise, and accurate method for the measurement of CRP concentrations in CSF.
Dog Leukocyte Antigen Class II–Associated Genetic Risk Testing for Immune Disorders of Dogs: Simplified Approaches Using Pug Dog Necrotizing Meningoencephalitis as a Model
Authors: Pedersen Niels , Liu Hongwei , Millon Lee , Greer Kimberly
Published: May 2011

A significantly increased risk for a number of autoimmune and infectious diseases in purebred and mixed-breed dogs has been associated with certain alleles or allele combinations of the dog leukocyte antigen (DLA) class II complex containing the DRB1, DQA1, and DQB1 genes. The exact level of risk depends on the specific disease, the alleles in question, and whether alleles exist in a homozygous or heterozygous state. The gold standard for identifying high-risk alleles and their zygosity has involved direct sequencing of the exon 2 regions of each of the 3 genes. However, sequencing and identification of specific alleles at each of the 3 loci are relatively expensive and sequencing techniques are not ideal for additional parentage or identity determination. However, it is often possible to get the same information from sequencing only 1 gene given the small number of possible alleles at each locus in purebred dogs, extensive homozygosity, and tendency for disease-causing alleles at each of the 3 loci to be strongly linked to each other into haplotypes. Therefore, genetic testing in purebred dogs with immune diseases can be often simplified by sequencing alleles at 1 rather than 3 loci. Further simplification of genetic tests for canine immune diseases can be achieved by the use of alternative genetic markers in the DLA class II region that are also strongly linked with the disease genotype. These markers consist of either simple tandem repeats or single nucleotide polymorphisms that are also in strong linkage with specific DLA class II genotypes and/or haplotypes. The current study uses necrotizing meningoencephalitis of Pug dogs as a paradigm to assess simple alternative genetic tests for disease risk. It was possible to attain identical necrotizing meningoencephalitis risk assessments to 3-locus DLA class II sequencing by sequencing only the DQB1 gene, using 3 DLA class II–linked simple tandem repeat markers, or with a small single nucleotide polymorphism array designed to identify breed-specific DQB1 alleles.
Phenotypic and Genotypic Characterization of Antimicrobial Resistance in Enterohemorrhagic Escherichia Coli and Atypical Enteropathogenic E. Coli Strains from Ruminants
Authors: Medina Alberto , Horcajo Pilar , Jurado Sonia , De La Fuente Ricardo , Ruiz-Santa-Quiteria José A. , Domínguez-Bernal Gustavo , Orden José A.
Published: May 2011

Two hundred and twenty-six attaching and effacing Escherichia coli (AEEC) strains (20 enterohemorrhagic E. coli and 206 atypical enteropathogenic E. coli) isolated from calves, lambs, and goat kids with diarrhea and from healthy cattle, sheep, and goats were tested for their resistance to 10 antimicrobial agents by the disc diffusion method. Resistant and intermediate strains were analyzed by polymerase chain reaction for the presence of the major resistance genes. The overall percentage of resistant strains to tetracycline, streptomycin, erythromycin, and sulfamethoxazole was very high (>65%). Moreover, a high level of resistance (approximately 30%) to ampicillin, chloramphenicol, trimethoprim, and trimethoprim-sulfamethoxazole was also detected. The AEEC strains were very susceptible (>90%) to gentamicin and colistin. Because AEEC from ruminants can cause diseases in human beings, the high frequency of antimicrobial resistance detected in the current study is a source of concern. For each antimicrobial agent, the predominant resistance genes in the resistant strains were ampicillin, blaTEM (97.1%); tetracycline, tetA (76.7%); gentamicin, aac(3)II (80%); streptomycin, strA/strB (76.7%) and aadA (71.7%); chloramphenicol, catI (85.1%); trimethoprim, dhfrI (76.3%); and sulfamethoxazole, sul1 (60%) and sul2 (63.3%). In the majority of cases, resistance to a given antimicrobial, except for streptomycin, was caused by a single gene. A negative association between tetA and tetB, between aac(3)II and aac(3)IV, and between dhfrI and dhfrV was observed. The present study gives baseline data on frequency and molecular basis of antimicrobial resistance in AEEC strains from ruminants.
Efficacy of an Anaerobic Swab Transport System to Maintain Aerobic and Anaerobic Microorganism Viability after Storage at −80°C
Authors: Musser Jeffrey M. B. , Gonzalez Rosa
Published: May 2011

An Amies agar gel swab transport system was evaluated for its ability to maintain bacterial viability and relative quantity after freezing at −80°C. Nine American Type Culture Collection (ATCC) bacterial strains were used: 3 anaerobic strains ( Propionibacterium acnes, Peptostreptococcus anaerobius, and Clostridium sporogenes) and 6 facultative or strict aerobic bacterial strains ( Stenotrophomonas maltophilia, Escherichia coli ([ATCC 25922 and ATCC 11775], Salmonella enterica subsp. enterica serovar Typhimurium, Staphylococcus saprophyticus, and Lactobacillus casei). The bacterial species were chosen because they corresponded to bacteria identified in psittacine feces and cloacal samples. There were no significant differences between growth scores at baseline and after storage at −80°C for 40 days for any of the bacteria examined after 48 and 72 hr of incubation, with the exception of P. anaerobius. For P. anaerobius, there was a significant reduction ( P < 0.001) in the growth score after storage at −80°C for 40 days from that of the baseline; however, the bacteria were still viable. The tested swab transport system may be useful when lengthy storage and transport times necessitate freezing samples prior to culture.
Isolation and Genotyping of Toxoplasma Gondii Causing Fatal Systemic Toxoplasmosis in an Immunocompetent 10-Year-Old Cat
Authors: Spycher Andrea , Geigy Caroline , Howard Judith , Posthaus Horst , Gendron Karine , Gottstein Bruno , Debache Karim , Herrmann Daland C. , Schares Gereon , Frey Caroline F.
Published: May 2011

A 10-year-old male, neutered domestic shorthair cat was presented with fever, anorexia, vomiting, and diarrhea. Serologic testing for Feline immunodeficiency virus and Feline leukemia virus were negative. Fine-needle aspirates of mesenteric lymph nodes revealed the presence of banana-shaped apicomplexan parasites. The cat died after 4 days of hospitalization. Postmortem polymerase chain reaction (PCR) analysis confirmed the presence of Toxoplasma gondii in all examined organs. Parasites were ex vivo isolated in outbred mice and subsequently transferred into cell culture. Genotyping, using genetic markers for SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico for PCR–restriction fragment length polymorphism, revealed infection with type II T. gondii displaying type II alleles at all loci except Apico, which exhibited a type I allele. This is the most frequently identified genotype among cats acting as definitive hosts in central Europe, but to the authors' knowledge, it has never been associated with systemic toxoplasmosis in an adult, immunocompetent cat.
Reference Intervals for Total Protein Concentration, Serum Protein Fractions, and Albumin/Globulin Ratios in Clinically Healthy Dairy Cows
Authors: Alberghina Daniela , Giannetto Claudia , Vazzana Irene , Ferrantelli Vincenzo , Piccione Giuseppe
Published: May 2011

The aim of the current study was to evaluate total serum protein concentration measured by the biuret reaction as well as albumin and globulin protein fractions determined by agarose gel electrophoresis. These data were used to establish reference intervals in dairy cows of different ages. Blood was collected from 111 clinically healthy Modicana dairy cows by means of jugular venipuncture. Reference intervals (mean ± standard deviation) were determined for total protein (67.54 ± 11.53 g/l), albumin (31.86 ± 4.60 g/l), α1-globulin (5.77 ± 2.20 g/l), α2-globulin (5.84 ± 1.90 g/l), β-globulin (7.46 ± 1.94 g/l), and γ-globulin (16.73 ± 4.54 g/l) concentrations as well as for albumin/globulin (A/G) ratio (0.88 ± 0.43). Values from 2-, 3-, 4-, 5-, and 6-year-old cows were compared statistically. One-way analysis of variance showed age-related differences for α-globulin and β-globulin fractions only. The results of the current study provide reference intervals for total protein concentration as well as albumin and globulin protein fractions in 2- to 6-year-old dairy cows.
Comparison of the Vanadate Oxidase Method with the Diazo Method for Serum Bilirubin Determination in Dog, Monkey, and Rat
Authors: Ameri Mehrdad , Schnaars Henry , Sibley John , Honor David
Published: May 2011

The most widely used method for bilirubin concentration determination is the diazo method, which measures the color of azobilirubin. The vanadate oxidase method is based on oxidation of bilirubin to biliverdin by vanadate. The objective of this study was to compare total and direct bilirubin concentration ([Bt] and [Bd], respectively) determined by the diazo and vanadate oxidase methods in pooled serum samples from dogs, monkeys, and rats spiked with panels of different concentrations of bilirubin standards. Pooled serum samples from 40 dogs, 40 monkeys, and 60 rats were spiked with either ditaurine conjugates of bilirubin or a standard reference material. The results obtained from both assays were compared using Deming regression analysis. The intra- and interassay precision, expressed as a percentage of the coefficient of variation (%CV), was determined for [Bt] and [Bd], and the mean percentage of recovery was calculated. The vanadate oxidase method displayed an excellent correlation ( r = 0.99–1.00) with the diazo method. Using Deming regression, there were minimal negative or positive constant and proportional biases for [Bt] and [Bd]. The precision studies revealed that the vanadate oxidase method has comparable between-run and within-run CVs to those of the diazo method. The recovery study demonstrated that the diazo method more closely approximates the expected values of [Bt]. In conclusion, the vanadate oxidase method is a simple and rapid method that can be employed as an alternative to the diazo method when interfering substances are present in the serum samples of dog, monkey, and rat.
Frequency of the Allelic Variant of the PTPLA Gene Responsible for Centronuclear Myopathy in Labrador Retriever Dogs as Assessed in Italy
Authors: Gentilini Fabio , Zambon Elisa , Gandini Gualtiero , Rosati Marco , Spadari Alessandro , Romagnoli Noemi , Turba Maria Elena , Gernone Floriana
Published: May 2011

Centronuclear myopathy (CNM) is an autosomal recessive hereditary disease affecting Labrador Retriever dogs. The disease is characterized by muscle lesions, typically encompassing reduction in the number and atrophy of type II fibers, and is caused by a short interspersed repeat element insertion in exon 2 of the protein tyrosine phosphatase-like member A. The actual allele frequency is unknown; a study was undertaken to ascertain it using a convenience-sample population composed of 217 Labrador Retrievers. In addition to 3 subjects already diagnosed with CNM, used as positive controls for polymerase chain reaction, only 2 unrelated dogs were heterozygous wild-type/mutation (wild-type/mut). Thus, the frequency of the CNM allele observed in the present study was 1.8% and 0.47% when including and excluding the 3 mut/ mut homozygous cases, respectively. Based on the Hardy–Weinberg exact test ( P = 1.00), the genotype frequency without the CNM-affected dogs was in agreement with the Hardy–Weinberg equilibrium. Assuming the Hardy–Weinberg equilibrium law, the expected frequency of the homozygous mutated genotype was calculated to be approximately 0.00005, which corresponds to 1 case of CNM out of 20,000 dogs. In conclusion, the present study indicates that the CNM allele is present but rare in a convenience sample of Labrador Retrievers in Italy.
Pathological and Immunohistochemical Features of Subdural Histiocytic Sarcomas in 15 Dogs
Authors: Ide Tetsuya , Uchida Kazuyuki , Kagawa Yumiko , Suzuki Kazuhiko , Nakayama Hiroyuki
Published: May 2011

Subdural histiocytic sarcomas from 15 dogs (mean age: 7.8 years) were histopathologically examined. Among the 15 dogs, there was a marked breed predominance (toward Pembroke Welsh Corgi dogs, 47%), but no gender predilection. Focal solitary subdural masses were detected in the cerebrum (12 cases) and spinal cord (1 case), whereas diffuse infiltrative lesions were observed in the cerebral leptomeninges in 2 cases. All neoplastic lesions had common histological features characterized by the proliferation of pleomorphic histiocytic cells combined with various inflammatory reactions. Multinucleated giant cells, phagocytosis, and atypical mitotic figures in the neoplastic cells were commonly observed. Most of the pleomorphic neoplastic cells in the present cases were immunopositive for monocytic, histiocytic, or both markers, such as human leukocyte antigen (HLA)-DR, ionized calcium-binding adaptor molecule 1 (Iba1), cluster of differentiation (CD)163, and CD204, except for the neoplastic cells in 2 focal and 2 diffuse histiocytic sarcomas. The findings suggest that differences in cell origin, molecular expression, or both patterns are responsible for the distribution patterns of canine subdural histiocytic sarcomas.
Listeriosis Outbreak in Dairy Cattle Caused by an Unusual Listeria Monocytogenes Serotype 4b Strain
Authors: Bundrant Brittany N. , Hutchins Tony , den Bakker Henk C. , Fortes Esther , Wiedmann Martin
Published: May 2011

A listeriosis outbreak, in dairy cattle, with a high case mortality and acute death after onset of symptoms was investigated using gross pathology and bacteriologic approaches, including molecular characterization of a clinical Listeria monocytogenes isolate. In a herd of 315 animals, 9 animals showed clinical symptoms consistent with listeriosis, including 3 animals that died within 2–4 days after acute onset of clinical signs, 4 animals that were euthanized, and 2 that survived. Initial EcoRI ribotyping and serotyping indicated that this outbreak was caused by an unusual L. monocytogenes serotype 4b strain, which was classified into lineage III. Further characterization of this isolate by DNA sequencing–based subtyping methods indicated that the strain responsible for this outbreak represented a unique genotype as supported by its classification into a new sigB allelic type, which has not been identified previously among >290 isolates, and by compelling phylogenetic evidence. While lineage III isolates are generally rare, they seem to be more common among L. monocytogenes isolates from animals with clinical signs of listeriosis. This is the first report of a particularly severe clinical course of disease associated with infection by a lineage III strain. The high prevalence of Listeria spp., including L. monocytogenes, in the farm environments may favor emergence and evolution of novel, and possibly more virulent, L. monocytogenes strains. Continued monitoring of animal listeriosis cases and outbreaks may not only improve animal health but also aid in the early discovery of newly emerging L. monocytogenes strains.
Primary Meningeal Hemangiosarcoma in a Grower Pig
Authors: Spitzbarth Ingo , Peters Martin , Stan Alexandru C. , Wohlsein Peter
Published: May 2011

A juvenile female pig found dead was submitted for necropsy. A focal meningeal red mass measuring 3.5 cm in diameter was located over the left olfactory bulb. Histologically, the mass was composed of polygonal-to-spindle–shaped cells forming vascular clefts with focal invasion of the neuropil of the olfactory bulb. Immunohistochemistry revealed strong expression of vimentin, and a variable labeling of factor VIII–related antigen, smooth muscle actin, and neurotrophin receptor p75. The final diagnosis was a primary meningeal hemangiosarcoma.
Hypertrophic Osteopathy Associated with a Renal Adenoma in a Cat
Authors: Johnson Robert L. , Lenz Stephen D.
Published: May 2011

Hypertrophic osteopathy is a hyperostotic syndrome of the appendicular skeleton that is most commonly associated with intrathoracic neoplasia or inflammation. The condition is rarely associated with intra-abdominal lesions. The majority of cases have occurred in dogs and human beings, with fewer cases reported in cats, horses, and other species. A 15-year-old male neutered Domestic Shorthair cat presented for swollen limbs and difficulty in ambulation. Radiographs and gross postmortem revealed severe periosteal hyperostosis of the diaphysis and metaphysis of all 4 limbs, including the humerus, radius, ulna, carpi, metacarpi, femur, tibia, tarsi, metatarsi, and phalanges. The axial skeleton was spared. Hyperostotic lesions were characterized microscopically by lamellar bony trabeculae separated by adipocytes and scant hematopoietic tissue. In several areas, fibrovascular connective tissue, woven bone, and islands of cartilage were also present. A 2.5 cm × 2.5 cm perirenal neoplasm compressed the left kidney and adrenal gland. This mass consisted of well-differentiated tubules of cuboidal epithelial cells and was most consistent with a renal tubular adenoma, because mitotic figures were rare, and no distant metastases were found. Thoracic pathology was absent. Hyperostosis was consistent with hypertrophic osteopathy secondary to the renal adenoma. The pathogenesis of hypertrophic osteopathy is uncertain, but predominant theories point to increased peripheral circulation and angiogenesis as a key initiating event. Recent literature highlights the potential role of vascular endothelial growth factor and platelet-derived growth factor in the human condition. The mechanism by which this renal adenoma caused hypertrophic osteopathy is unknown.
Tonsillitis in a Weaner Pig Associated with Geotrichum Candidum
Authors: Lee Effie J. , Gabor Melinda , Turner Mark , Ball Mathew , Gabor Les
Published: May 2011

Geotrichum candidum was cultured from the tonsils of a free-ranging weaner pig that was presented with lethargy and inappetence. Histopathology indicated pyogranulomatous tonsillitis with intralesional fungal hyphae and arthrospores. Geotrichosis is a rare, opportunistic mycosis of immunocompromised hosts, usually human beings.
Change in Predominance of Bovine Viral Diarrhea Virus Subgenotypes among Samples Submitted to a Diagnostic Laboratory over a 20-Year Time Span
Authors: Ridpath Julia F. , Lovell Gayla , Neill John D. , Hairgrove Thomas B. , Velayudhan Binu , Mock Richard
Published: May 2011

Although the causative agent of bovine viral diarrhea was initially categorized as 1 species, phylogenetic analysis revealed that these viruses belong to 2 different species, Bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2, with 2–11 subgenotypes within each species. Distribution of species and subgenotypes has been shown to vary with geographic region. Whether distribution shifts over time is not known. Surveys conducted between 1994 and 2008 reported 3 subgenotypes circulating among cattle in the United States: BVDV-1a, BVDV-1b, and BVDV-2a. The average percent prevalence of BVDV-1a, BVDV-1b, and BVDV-2a strains reported in surveys before 2001 were 21%, 43%, and 36%, respectively. Surveys conducted on viruses isolated after 2001 reported decreasing percentages of BVDV-1a and BVDV-2a strains, with BVDV-1b strains accounting for 75–100% of samples. Comparison of these surveys is confounded by differences in geographic location, collection methods, and sample type used in the survey. The purpose of the present study was to determine whether the prevalence of BVDV subgenotypes shifted in samples collected from the same geographic region and by the same laboratory over time. BVDV strains isolated in years 1988, 1998, and 2008, at the Texas Veterinary Medical Diagnostic Laboratory, Amarillo, Texas, were genotyped, and the prevalence of BVDV-1a, BVDV-1b, and BVDV-2a strains were determined. Typing, on the basis of phylogenetic analysis, was done on 148 samples. The strongest trend detected among these samples was a pronounced decrease in the number of BVDV-1a strains over time.
A Simulation Model to Quantify the Value of Implementing Whole-Herd Bovine Viral Diarrhea Virus Testing Strategies in Beef Cow–Calf Herds
Authors: Nickell Jason S. , White Brad J. , Larson Robert L. , Renter David G. , Sanderson Mike W.
Published: May 2011

Although numerous diagnostic tests are available to identify cattle persistently infected (PI) with Bovine viral diarrhea virus (BVDV) in cow–calf herds, data are sparse when evaluating the economic viability of individual tests or diagnostic strategies. Multiple factors influence BVDV testing in determining if testing should be performed and which strategy to use. A stochastic model was constructed to estimate the value of implementing various whole-herd BVDV cow–calf testing protocols. Three common BVDV tests (immunohistochemistry, antigen-capture enzyme-linked immunosorbent assay, and polymerase chain reaction) performed on skin tissue were evaluated as single- or two-test strategies. The estimated testing value was calculated for each strategy at 3 herd sizes that reflect typical farm sizes in the United States (50, 100, and 500 cows) and 3 probabilities of BVDV-positive herd status (0.077, 0.19, 0.47) based upon the literature. The economic value of testing was the difference in estimated gross revenue between simulated cow–calf herds that either did or did not apply the specific testing strategy. Beneficial economic outcomes were more frequently observed when the probability of a herd being BVDV positive was 0.47. Although the relative value ranking of many testing strategies varied by each scenario, the two-test strategy composed of immunohistochemistry had the highest estimated value in all but one herd size–herd prevalence permutation. These data indicate that the estimated value of applying BVDV whole-herd testing strategies is influenced by the selected strategy, herd size, and the probability of herd BVDV-positive status; therefore, these factors should be considered when designing optimum testing strategies for cow–calf herds.
Interlaboratory Comparison of Porcine Circovirus-2 Indirect Immunofluorescent Antibody Test and Enzyme-Linked Immunosorbent Assay Results on Experimentally Infected Pigs
Authors: Patterson Abby R. , Johnson John K. , Ramamoorthy Sheela , Hesse Richard A. , Murtaugh Michael P. , Puvanendiran Sumathy , Pogranichniy Roman M. , Erickson Gene A. , Carman Susy , Hause Ben , Meng Xiang-Jin , Opriessnig Tanja
Published: May 2011

A blinded interlaboratory assessment of the diagnostic agreement and accuracy of serologic tests for routine detection of antibodies against Porcine circovirus-2 (PCV-2), including indirect fluorescent antibody tests (IFATs) and enzyme-linked immunosorbent assays (ELISAs) was conducted in 7 North American laboratories. Serum samples were collected weekly, on trial days 0, 7, 14, 21, 28, 35, 42, and 49, from the following groups of animals: 1) negative controls ( n = 7), 2) PCV-2a ( n = 8), 3) PCV-2b ( n = 8), 4) PCV-1 ( n = 8), 5) PCV-2 vaccine A ( n = 8; Ingelvac® CircoFLEX™), 6) PCV-2 vaccine B ( n = 8; Circumvent® PCV2), and 7) PCV-2 vaccine C ( n = 8; Suvaxyn® PCV2 One Dose). Results from each laboratory were analyzed by kappa and receiver operating characteristic (ROC) analysis. Kappa analysis indicated that, by trial day 49, IFATs had almost perfect agreement, in-house ELISAs had fair to almost perfect agreement, and commercially available anti–PCV-2 immunoglobulin G ELISAs (I or S) had moderate to substantial agreement. From trial days 14–49, the area under the ROC curve for the 2 laboratories that offered IFATs, the 4 laboratories that offered in-house ELISAs, and the 3 laboratories that used commercially available ELISAs ranged from 0.94 to 1.00, 0.72 to 1.00, and 0.95 to 1.00, respectively. However, test sensitivities varied based on laboratory-specific cutoffs that were used to dichotomize test results.
Immunomagnetic Separation Significantly Improves the Sensitivity of Polymerase Chain Reaction in Detecting Giavdia Duodenalis and Cryptosporidium spp. in Dairy Cattle
Authors: Coklin Tatjana , Farber Jeffrey M. , Parrington Lorna J. , Kingombe Cesar I. Bin , Ross William H. , Dixon Brent R.
Published: May 2011

The effectiveness of molecular methods for the detection of species of Giardia and Cryptosporidium in fecal samples is often reduced by low or intermittent cyst and oocyst shedding, and/or the presence of polymerase chain reaction (PCR) inhibitors. The present study investigates the use of immunomagnetic separation (IMS) as an additional concentration step before PCR in the detection of these common protozoan parasites in dairy cattle. The IMS-PCR assays were optimized for amplifying fragments of the 16S ribosomal RNA (rRNA), β-giardin, and glutamate dehydrogenase (GDH) genes of Giardia duodenalis, as well as fragments of the 18S rRNA, heat shock protein (HSP)-70, and Cryptosporidium oocyst wall protein (COWP) genes of Cryptosporidium spp. In all cases, IMS-PCR was more sensitive than PCR alone. A significantly greater number of Giardia–positive samples were identified using IMS-PCR of the 16S rRNA gene ( P < 0.01) and of the GDH gene ( P < 0.01), as compared with PCR without any additional concentration step. In the case of Cryptosporidium, IMS-PCR of the COWP gene ( P = 0.02) resulted in a significantly greater number of positives than did PCR without the IMS concentration step. The greatest number of positives, however, was obtained using IMS-PCR to amplify a portion of the 16S rRNA gene of Giardia and a portion of the HSP-70 gene of Cryptosporidium. A further comparison of the optimized IMS-PCR assays to immunofluorescence microscopy suggested that the IMS-PCR assays were considerably more sensitive than microscopy was in the detection of Giardia cysts and Cryptosporidium oocysts in fecal samples.
An Immunohistochemical Assay to Detect Trophoblasts in Frozen Feline Placenta
Authors: Scott Veronica L. , Wallace Kedra , Mays Stephany , Ryan Peter , Coats Karen S.
Published: May 2011

The placenta, a fetal endocrine organ, is composed of subpopulations of trophoblasts, including cytotrophoblasts, and syncytiotrophoblasts. Trophoblastic populations can be distinguished based upon their expression of cytokeratin. The purpose of the current study was to develop an immunohistochemistry (IHC) method to identify trophoblasts selectively in frozen feline placental tissue using antibodies specific for cytokeratin. The mouse monoclonal antibody anti-human pan-cytokeratin AE1/AE3 and a commercial detection system were used. Nonspecific immunoreactivity was encountered that could not be eliminated with altered blocking methods. The nonspecific reactivity was attributed to the goat anti-mouse/rabbit immunoglobulin G (IgG) peroxidase polymer included in the commercial kit. Alternatively, a polyclonal rabbit anti-cow cytokeratin wide spectrum screening antibody with goat anti-rabbit IgG polyclonal secondary antibody was used to detect cytokeratin in feline placental tissue. The IHC procedure eliminated nonspecific immunoreactivity while specifically labeling cytokeratin. This new approach provides an IHC method to identify trophoblasts specifically in feline placenta.
Validation of an Automated Method for Salivary Alpha-Amylase Measurements in Pigs (Sus Scrofa Domesticus) and its Application as a Stress Biomarker
Authors: Fuentes María , Tecles Fernando , Gutiérrez Ana , Otal Julio , Martínez-Subiela Silvia , Cerón José J.
Published: May 2011

The aim of the current study was to validate an automated spectrophotometric method for salivary alpha-amylase measurement in pigs and evaluate its possible application as a noninvasive stress biomarker. The analytical validation included intra- and interassay precision, linearity under dilution, and limit of detection. In addition, to study the possible use of salivary alpha-amylase as a possible stress marker, 12 crossbred growing pigs of 3–4 months of age were subjected to restraint stress by a nasal snare for at least 1 min, and saliva samples were obtained at different time points. The results of analytical validation indicated that the method was precise and able to measure alpha-amylase in a linear manner. The results obtained in the stress test showed a significant increase in salivary alpha-amylase activity. Although other factors influencing this enzyme activity should be studied, these preliminary results indicate that salivary alpha-amylase could be a reliable biomarker of stress in pigs.
A Functional Turbidimetric Method to Determine C-Reactive Protein in Horses
Authors: Tugirimana Pierrot L. , De Clercq Dominique , Holderbeke Astrid L. , Kint Jos A. , De Cooman Liesbeth , Deprez Piet , Delanghe Joris R.
Published: May 2011

A turbidimetric method to determine serum C-reactive protein (CRP) concentration, based on soybean oil–phosphocholine interaction, was performed on horse serum samples to evaluate its potential diagnostic value in veterinary medicine. Intralipid 20% in 0.1 M Tris–calcium buffer (pH 7.5) was added to horse serum. After 30 min of incubation at 37°C, the CRP–phosphocholine complexes were turbidimetrically, bichromatically (660 nm/700 nm) quantified on a commercial analyzer. Furthermore, comparison between CRP and other inflammatory markers, including white blood cell and neutrophil counts, was performed to evaluate the diagnostic value of both tests. Standardization of the assay was done using a commercial human CRP calibrator. The CRP measurements were performed on serum samples (296 patients and 34 controls). Reference values were found to be lower than 10 mg/l. The method was found to be linear between 1 and 400 mg/l. A moderate correlation was observed between CRP values and the relative neutrophil counts. Receiver-operating characteristics analysis demonstrated the area under the curve for CRP was 0.928, which was superior ( P < 0.001) to the neutrophil count (0.804) and the leukocyte count (0.664) in detecting the presence of inflammation. This CRP assay showed reliable results as an acute phase test in horses, confirming its species-independent capability to detect CRP in various mammals, including horses.
Assessment of Urine Solute and Matrix Effects on the Performance of an Enzyme-Linked Immunosorbent Assay for Measurement of Interleukin-6 in Dog Urine
Authors: Wood Michael W. , Nordone Sushila K. , Vaden Shelly L. , Breitschwerdt Edward B.
Published: May 2011

Measurement of cytokine concentrations within body fluids is a means of recognizing subclinical and/or unresolved, infectious and inflammatory states in patients. In the urinary tract, such information may be useful for identifying patients with pyelonephritis, asymptomatic bacteriuria, recurrent infections, and cystitis. One such cytokine, interleukin-6 (IL-6), is recognized as a primary cytokine that is produced following exposure of the urothelium to bacterial virulence factors. Complicating reliable testing for this and other cytokines is the nature of urine itself. Urine varies widely in its composition as indicated by the range of pH and urine specific gravity (USG) observed in healthy patients. An additional variable is the protein and carbohydrate matrix capable of hindering immunologic testing modalities, such as enzyme-linked immunosorbent assays (ELISAs). The purpose of the current study was to examine the role of urine pH, USG, and matrix while optimizing a canine-specific chemiluminescent ELISA for the measurement of IL-6 in the urine of dogs. Urine spiked with IL-6 obtained maximal IL-6 quantitative recoveries of only 55 ± 10% (mean ± 1 standard deviation) when an ELISA optimized for cell culture supernatants was used. The urine matrix and variations in USG were determined to by contributing to this poor IL-6 recovery. Using specific matrix inhibitors and optimal dilutions improved the IL-6 quantitative recovery to 91 ± 5%. Urine pH (5.5–9.5) had no effect. The current work underscores the importance of critically optimizing testing modalities for biomarkers, particularly if they are immunologic in origin.
Immunochromatographic Gold-Based Test Strip for Rapid Detection of Infectious Bursal Disease Virus Antibodies
Authors: Nurulfiza Isa , Hair-Bejo Mohd , Omar Abdul R. , Aini Ideris
Published: May 2011

The immunochromatographic assay is an alternative method for simple and rapid detection of Infectious bursal disease virus (IBDV) in chickens using colloidal gold—antibody conjugate. The whole-virus antigen of IBDV (UPM04190 isolate) and the high-affinity polyclonal antibodies directed against IBDV were blotted onto nitrocellulose membranes for test and control lines, respectively. Evaluation of the strip was performed using serum samples from experimentally and naturally infected chickens. The results showed that the test strip was more sensitive than the commercial enzyme-linked immunosorbent assay (ELISA) because it could detect a dilution factor up to 1:20,000 (250 ELISA units) for positive samples. It was also specific, in that it detected IBDV antibodies and did not cross-react with antibodies to other chicken viruses. The method was rapid (2 min) in both clinical and field environments with samples needing only a minimum amount (50 μl) of blood to produce an acceptable detection signal. The pen—site test strip proved successful in monitoring the immune status of chickens against the IBDV infection.
Detection and Genetic Analysis of Aquabirnaviruses in Subclinically Infected Aquarium Fish
Authors: Shin Sang Phil , Gomez Dennis Kaw , Kim Ji Hyung , Choresca Casiano Hermorpia , Han Jee Eun , Jun Jin Woo , Park Se Chang
Published: May 2011

Aquabirnaviruses (ABVs) cause serious diseases in a variety of fish species used worldwide in aquaculture and have been isolated from a variety of healthy fish and shellfish species. The type species of ABV is Infectious pancreatic necrosis virus (IPNV), which is the causative agent of a highly contagious disease in juvenile salmonid fish. Marine birnaviruses (MABVs) have been isolated from various marine fish and shellfish. In Korea, ABV infection has been identified in several fish and shellfish. The current study presents sequence data from nested polymerase chain reaction products of 3 ABV strains obtained from different species of asymptomatic aquarium fish collected from a private commercial aquarium in Korea. Phylogenetic analysis of these strains, based on the partial nucleotide sequence of the VP2/NS junction, placed them within the genogroup VII (95–99% bootstrap confidence), which also contains MABV. The subclinically infected fish may be a source of MABV infection for other susceptible fish species inside the aquarium and potentially represent a serious challenge for the management of MABV infections. Additionally, the presence of MABV in these subclinically infected aquarium fish imported from other countries indicates that there is a need for the establishment of appropriate quarantine practices.
Selective Isolation of Avian Influenza Virus (AIV) from Cloacal Samples Containing AIV and Newcastle Disease Virus
Authors: Zowalaty Mohamed E. El , Chander Yogesh , Redig Patrick T. , El Latif Hemmat K. Abd , Sayed Mona A. El , Goyal Sagar M.
Published: May 2011

Avian influenza viruses (AIVs) are important zoonotic pathogens whose natural reservoir is waterfowl. In addition to AIV, waterfowl are often coinfected with other viruses, such as the paramyxoviruses, of which Newcastle disease virus (NDV) is of particular importance because of the highly virulent nature of certain strains of this virus for domestic poultry. In routine surveillance of waterfowl for AIV, a number of cloacal samples were encountered that were positive for AIV by real-time reverse transcription polymerase chain reaction (RT-PCR), but did not yield AIV by inoculation in embryonated chicken eggs. On further testing, these samples were also positive for NDV by conventional RT-PCR. It was hypothesized that if both NDV and AIV are present in a sample, the former may overgrow AIV yielding false-negative AIV results. Such samples were treated with chicken anti-NDV polyclonal antiserum and then inoculated in embryonated chicken eggs. Several samples were found to be positive for different subtypes of AIV, indicating that, in the presence of mixed infection with NDV and AIV, it is imperative to remove the influence of NDV, so a true picture of AIV prevalence emerges. An additional benefit is that information on the circulation of NDV in these birds sheds light on their epidemiologic and ecologic significance.
Rapid and Simple Polymerase Chain Reaction—Based Diagnostic Assays for GM2 Gangliosidosis Variant 0 (Sandhoff-Like Disease) in Japanese Domestic Cats
Authors: Rahman Mohammad M. , Shoubudani Tomoaki , Mizukami Keijiro , Chang Hye-Sook , Hossain Mohammad A. , Yabuki Akira , Mitani Sawane , Higo Takashi , Arai Toshiro , Yamato Osamu
Published: May 2011

Polymerase chain reaction (PCR)-based assays combined with microchip electrophoresis were developed and evaluated for diagnosis and genotyping of GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japanese domestic cats. A preliminary genotyping survey was carried out in the population of Japanese domestic cats (1,015 cats in total) in southern Japan. Three kinds of assays including PCR primer-induced restriction analysis (PIRA) and mutagenically separated (MS)-PCR were carried out using blood-stained Flinders Technology Associates filter papers (FTA cards) as templates. The PCR products were analyzed by both agarose gel and microchip electrophoreses. All assays were sufficient to determine the genotypes of this disease, but MS-PCR offered the most rapid and simplest test, as it does not need the restriction enzyme step required in PCR-PIRA. The use of microchip electrophoresis in combination with FTA cards for sampling could shorten the time required for genotyping and simplify the procedure as well. The genotyping survey in the current study did not find any cats that possessed the mutant allele, suggesting that the prevalence of this allele is low (<0.1%) in southern Japan.
Detection of Methicillin-Resistant Staphylococcus Aureus Sequence Type 8 in Pigs, Production Environment, and Human Beings
Authors: Sunde Marianne , Tharaldsen Hanne , Marstein Lillian , Haugum Magne , Norström Madelaine , Jacobsen Trond , Lium Bjørn
Published: May 2011

Two follow-up studies of a positive methicillin-resistant Staphylococcus aureus, (MRSA) finding in the 2008 European Union baseline survey on MRSA in pig herds were performed to gain more knowledge about the epidemiology of the particular MRSA type, a known human type (ST8/t008), among pigs. Two persons on a Norwegian farm in the study were found to be MRSA carriers, and human-to-animal transmission was suspected. In the first follow-up study, all pigs ( n = 346) were sampled by taking nasal swabs. A pooled sample from 5 individual pigs housed together in a single pen, and a dust sample from the equipment in the same room, were positive. Dust samples from a building housing MRSA-negative animals were negative. The MRSA was not detected in the second follow-up, after removing positive animals from the farm and cleaning and disinfecting. A low MRSA occurrence among the animals was found, suggesting that MRSA ST8/t008 may be less able to colonize and persist in pig holdings compared with more host-adapted S. aureus strains.
An Unusual Strain of Haemophilus Parasuis that Fails to React in a Species-Specific Polymerase Chain Reaction Assay
Authors: Turni Conny , Blackall Patrick J.
Published: May 2011

A total of 30 nasal swabs from pigs preweaned and 11 nasal swabs from sick weaned pigs on a farm in Queensland, Australia, were cultured for the presence of Haemophilus parasuis. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) genotyping and indirect hemagglutination and gel diffusion serotyping were performed on the retrieved H. parasuis isolates. A total of 3 genotypes were recognized among the 42 isolates recovered, and 4 representative isolates of each genotype were found to be nontypeable in the Kielstein/Rapp-Gabrielson serotyping scheme. A total of 20 of the 22 isolates of genotype 1 did not amplify in the species-specific conventional PCR number 1 (cPCR1) based on the 16S ribosomal RNA (rRNA) gene but did give the expected PCR amplicon in 2 other species-specific PCR assays, one of which is also based on the 16S rRNA gene. Nine selected isolates representing all genotypes, both positive and negative in the cPCR1, were sequenced, and all showed a 4-base mutation occurring at the forward primer annealing site. The quadruple base pair substitution from GTGG to TGTT near the 3' end of the forward primer sequence may explain the failure of amplification. Diagnostic laboratories should be aware that such failures can occur and should consider having an alternative PCR available to confirm negative results or, alternatively, use phenotypic characteristics for the identification of suspect H. parasuis isolates.
Rapid and Reliable Identification of Ionophore Antibiotics in Feeds by Liquid Chromatography–Tandem Mass Spectrometry
Authors: Huang Min , Rumbeiha Wilson K. , Braselton W. Emmett , Johnson Margaret
Published: May 2011

Veterinary diagnostic laboratories are constantly required to be innovative to develop quick, reliable, and cost-effective methods for use in diagnosis of intoxications. Livestock intoxication with ionophore antibiotics is sometimes encountered. A rapid qualitative method based on liquid chromatography–tandem mass spectrometry was developed to screen for the presence of monensin, lasalocid, salinomycin, and narasin antibiotics in feeds. This new method has high specificity and high sensitivity, and yet minimum sample preparation was employed. The analytes were extracted in methanol–water (90:10) and analyzed without further purification. Because the test uses a single extraction procedure and no cleanup is necessary, its turnaround time is 4 hr. The method applies the concept of „identification points” (IP) score to identify suspected ionophore antibiotics. The total IP score used to identify each of the ionophore antibiotics by this method was 7.0, which exceeds the minimum score of 4.0 required for acceptable identification of unknown compounds. The specificity of the method was achieved using retention time and multiple reaction monitoring with a total IP score of 7.0.
Porcine Teschovirus Polioencephalomyelitis in Western Canada
Authors: Salles Mônica W. S. , Scholes Sandra F. E. , Dauber Malte , Strebelow Günter , Wojnarowicz Chris , Hassard Lori , Acton Andy C. , Bollinger Trent K.
Published: May 2011

Beginning in 2002, a small number of pig farms in western Canada began reporting 4–7-week-old pigs with bilateral hind-end paresis or paralysis. Low numbers of pigs were affected, some died, most had to be euthanized, and those that survived had reduced weight gains and neurological deficits. Necropsies revealed no gross lesions, but microscopic lesions consisted of a nonsuppurative polioencephalomyelitis, most severe in the brain stem and spinal cord. The lesions were most consistent with a viral infection. Tests for circovirus, Porcine reproductive and respiratory syndrome virus, coronavirus, Rabies virus, and Pseudorabies virus were negative. Using immunohistochemistry, virus neutralization, fluorescent antibody test, and nested reverse transcription polymerase chain reaction, Porcine teschovirus was identified in tissues from affected individuals. To the authors' knowledge, this is the first report of teschovirus encephalitis in western Canada and the first reported case of polioencephalomyelitis in pigs in Canada, where teschovirus was confirmed as the cause.
Beta 2 Toxigenic Clostridium Perfringens Type A Colitis in a Three-Day-Old Foal
Authors: Hazlett Murray J. , Kircanski Jasmina , Slavic Durda , Prescott John F.
Published: May 2011

Beta 2 (β2)-toxigenic Clostridium perfringens type A was recovered in large numbers from the intestine of a neonatal foal with colitis. The foal had been treated with gentamicin. Necropsy revealed marked distension of cecum and colon with watery, rust-colored homogeneous fluid and gastric infarction. Microscopic colonic lesions were superficial necrosis of 50% of the colonic mucosal surface and scattered 1–3-mm ulcers with subjacent neutrophilic infiltration and large Gram-positive bacilli in the necrotic mucosa. Beta-2 toxin was demonstrated in the lesions by immunohistochemical staining.
Fatal Hepatic Sarcocystosis in a Captive Black Bear (Ursus Americanus) Associated with Sarcocystis Canis-Like Infection
Authors: Davies Jennifer L. , Haldorson Gary J. , Bradway Dan S. , Britton Ann P.
Published: May 2011

Fatal hepatic sarcocystosis was diagnosed in a 13-year-old captive black bear ( Ursus americanus) with a history of acute onset of vomiting, polyuria, polydipsia, and bilirubinuria. Gross lesions included severe icterus, multisystemic hemorrhage, and gall bladder edema. The most significant microscopic lesion was severe necrotizing hepatitis with intralesional protozoa that reproduced by endopolygeny consistent with a Sarcocystis spp. Infrequent microglial nodules were randomly scattered within the white matter of the cerebral cortices, thalamus, and brainstem, but intralesional protozoal schizonts were not observed. In the liver, immunohistochemistry was positive for Sarcocystis spp. and negative for Toxoplasma gondii and Neospora spp. Positive staining was not observed in the brain. Genus-specific polymerase chain reaction (PCR) amplification of the 18S ribosomal RNA gene was performed on formalin-fixed, paraffin-embedded sections of liver and brain; in both tissues, PCR was positive for Sarcocystis spp. Sequence analysis of the PCR amplicons revealed 100% identity to the published sequences of Sarcocystis canis and Sarcocystis arctosi.
Pulmonary trematodosis (Pharyngostomoides sp.) in a juvenile raccoon (Procyon lotor)
Authors: Olson Erik J. , Conboy Gary A. , Stromberg Bert E. , Hayden David W.
Published: May 2011

A lethargic, wild, juvenile raccoon died and was subsequently presented for necropsy. The lungs had widely disseminated, finely granular, yellow–tan foci with moderate pulmonary, as well as, thoracic lymphadenopathy. Histopathology revealed numerous cross-sections of larval trematodes morphologically consistent with diplostomid mesocercariae and metacercariae as well as moderate alveolointerstitial pneumonia with a mixed population of eosinophils, macrophages, lymphocytes, and plasma cells. The presence of larval trematodes within lymphatics in multiple organs indicates a route of fluke migration. Live specimens of mesocercariae and metacercariae recovered from refrigerated lung tissue after 7 days were identified as Pharyngostomoides sp., an intestinal diplostomid parasite (trematode) of raccoons and other mammals that is similar in appearance and life cycle to Alaria spp.