Immunohistochemical Detection of Mycoplasma agalactiae in Formalin‐Fixed,Paraffin‐Embedded Tissues from Naturally and Experimentally Infected Goats

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin‐fixed, paraffin‐wax‐embedded sections and labelled by the avidin–biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody‐based immunohistochemical technique is an efficient and specific method for the post‐mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.     

Demonstration of Listeria monocytogenes by Immunohistochemistry in Formalin‐Fixed Brain Tissues from Natural Cases of Ovine and Bovine Encephalitis

In the present work, evidence of Listeria monocytogenes antigens based on the avidin–biotin complex (ABC) immunoperoxidase technique was performed on formalin‐fixed central nervous system tissues (CNS) from a total of 23 natural cases of encephalitis (four ovine and 19 bovine). Listeria monocytogenes serotype 4 was isolated from 10 of 17 cultured specimens. Meningoencephalitis characterized by focal necrosis, microabscesses, perivascular cuffing, and gliosis with presence of macrophages and/or neutrophils was observed at histological examination. Positive L. monocytogenes antigens were successfully identified by immunohistochemistry (IHC) in the CNS of all 23 cases. Paraffin‐embedded tissues assayed were stored up for 17 years. Morbidity of the outbreaks was between 0.3–3% and 0.1–1% for ovine and bovine cases, respectively. In all the ovine cases, flocks involved were under extensive grazing conditions. In nine of the 19 bovine cases (47.3%), supplementation with corn silage was used. The ABC test can help as a practical tool for the diagnosis of natural cases of L. monocytogenes encephalitis on formalin‐fixed specimens from ovine and bovine.     

A 6‐bp Deletion Variant in a Novel Canine Glutathione‐S‐Transferase Gene (GSTT5) Leads to Loss of Enzyme Function

Objectives

Glutathione‐S‐transferases (GSTs) detoxify reactive xenobiotics, and defective GST gene polymorphisms increase cancer risk in humans. A low activity GST‐theta variant was previously found in research beagles. The purpose of our study was to determine the molecular basis for this phenotype and its allele frequency in pet dogs.

Methods

Banked livers from 45 dogs of various breeds were screened for low GST‐theta activity by the substrate 1,2‐dichloro‐4‐nitrobenzene (DCNB), and were genotyped for variants in a novel canine GST gene, GSTT5. Whole‐genome sequences from 266 dogs were genotyped at one discovered variant GSTT5 locus.

Results

Canine livers ranged 190‐fold in GST‐theta activities, and a GSTT5 exon coding variant 385_390delGACCAG (Asp129_Gln130del) was significantly associated with low activity (P < 0.0001) and a marked decrease in hepatic protein expression (P = 0.0026). Recombinant expression of variant GSTT5 led to a 92% decrease in V_max for DCNB (P = 0.0095). The minor allele frequency (MAF) for 385_390delGACCAG was 0.144 in 45 dog livers, but was significantly higher in beagles (0.444) versus nonbeagles (0.007; P = 0.0004). The homozygous genotype was significantly over‐represented in Pembroke Welsh corgis (P < 0.0001) based on available whole‐genome sequence data.

Conclusions

An Asp129_Gln130del variant in canine GSTT5 is responsible for marked loss of GST‐theta enzyme activity. This variant is significantly over‐represented in purpose‐bred laboratory beagles and in Pembroke Welsh corgis. Additional work will determine the prevalence of this variant among other purebred dogs, and will establish the substrate range of this polymorphic canine enzyme with respect to common environmental carcinogens.     

Canine GM2‐Gangliosidosis Sandhoff Disease Associated with a 3‐Base Pair Deletion in the HEXB Gene

Background

GM2‐gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β‐hexosaminidase A (Hex‐A) and β‐hexosaminidase B (Hex‐B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency.

Objectives

To characterize the phenotype and genotype of GM2‐gangliosidosis disease in an affected dog.

Animals

One affected Shiba Inu and a clinically healthy dog.

Methods

Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes.

Results

A 14‐month‐old, female Shiba Inu presented with clinical signs resembling GM2‐gangliosidosis in humans and GM1‐gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex‐A and Hex‐B activities in both tissues. Genetic analysis identified a homozygous, 3‐base pair deletion in the HEXB gene (c.618‐620delCCT).

Conclusions and Clinical Importance

Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2‐gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2‐gangliosidosis seen in this dog is the Sandhoff type. Because GM1‐gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1‐ and GM2‐gangliosidosis should be considered to make a definitive diagnosis.     

Validation of a Commercially Available Human Immunoturbidimetric Assay for Haptoglobin Determination in Canine Serum Samples

Haptoglobin is a positive acute-phase protein with a valuable role as a marker of inflammation in both human and veterinary medicine. The aim of this study was to validate a commercially available immunoturbidimetric method designed for human haptoglobin determination (Izasa SA, Barcelona, Spain) for its use in canine samples. Cross-reactivity between anti-human haptoglobin antiserum and canine haptoglobin was found when agarose gel immunodiffusion and ELISA tests were performed. The use of canine pooled serum with haptoglobin concentration of 6.3 g/L as standard provided higher analytical range than commercially available standards. Intra-assay and inter-assay coefficients of variation were 2.49% and 4.60%, respectively. A linear regression model between immunoturbidimetric results and a previously validated spectrophotometric method (Tridelta Development Limited, Ireland) yielded a slope at 95% confidence interval of 0.94 (0.86, 1.02) and y-intercept at 95% confidence interval of 0.11 (−0.59, 0.82). No significant differences were produced by anticoagulants, lipaemia and bilirubinaemia, although haemolysis significantly decreased haptoglobin. A significant increase of haptoglobin concentration was detected in inflammatory conditions such as pyometra and leishmaniasis, in neoplastic conditions, and after glucocorticoid administration. Canine serum haptoglobin concentration can be reliably measured using the commercially available Izasa immunoturbidimetric method developed for human haptoglobin determination. This method is precise and accurate, provides a wider analytical range than previous reported methods, and can be easily automated and used for routine haptoglobin determination in canine samples.     

Evaluation of a Portable Meter to Measure Ketonemia and Comparison with Ketonuria for the Diagnosis of Canine Diabetic Ketoacidosis

Background: The diagnosis of canine diabetic ketoacidosis (DKA) usually is based on measurement of urinary acetoacetate (ketonuria). In humans, this test is less sensitive and specific than blood 3-β-hydroxybutyrate (ketonemia) evaluation.
Hypothesis: Ketonemia measurement using a portable meter is more accurate than ketonuria determination with a dipstick to diagnose canine DKA.
Animals: Seventy-two client-owned diabetic dogs with ketonemia, ketonuria, or both.
Methods: Prospective observational study. Based on blood bicarbonate concentration and anion gap, dogs were divided into 2 groups: patients with DKA ( n = 25); patients with diabetic ketosis ( n = 47). Sensitivity, specificity, and positive and negative likelihood ratio (LR) at different cut-off points were determined for both ketonemia and ketonuria. Receiver operating characteristic (ROC) analysis was used to assess the accuracy of each diagnostic test to diagnose DKA.
Results: With regard to ketonemia, cut-off values of 2.3 and 4.3 mmol/L revealed 100% sensitivity and 100% specificity, respectively, whereas cut-off values of 2.8 and 3.5 mmol/L showed a −LR of 0.05 and a + LR of 13.16, respectively. With regard to ketonuria, a cut-off value of 1+ revealed 92% sensitivity, 40% specificity, and −LR of 0.20, whereas a cut-off value of 3+ revealed 44% sensitivity, 94% specificity, and +LR of 6.89. The areas under the ROC curves for the ketonemia and ketonuria tests were significantly different (0.97 and 0.81, respectively, P = .003).
Conclusions and Clinical Importance: Measurement of ketonemia is accurate and more effective than measurement of ketonuria to diagnose canine DKA.     

Analytical and Clinical Validation of a Time-resolved Immunofluorometric Assay (TR-IFMA) for Canine C-reactive Protein in Serum

A time-resolved immunofluorometric assay (TR-IFMA) was developed for the determination of C-reactive protein (CRP) in canine serum. CRP was isolated from canine acute-phase serum by affinity chromatography on agarose coupled with phosphorylethanolamine. This isolated dog CRP was used as standard to calibrate the assay. Intra-assay and inter-assay coefficients of variation were in the ranges 5.3–7.1% and 4.8–13.3%, respectively. Accuracy, evaluated by adding 2 and 10 μg/ml of CRP to serum samples, provided recoveries of 99.9% and 106.8%. High correlation was found between CRP measurements by TR-IFMA and a by commercial enzyme-linked immunosorbent assay (R² = 0.98). The limit of detection for the TR-IFMA method was 0.000067 μg/ml and the measurement of CRP in serial dilutions of acute-phase dog sera generated curves with the same slope as the one constructed with purified CRP. The TR-IFMA provides a precise, accurate and highly sensitive assay for CRP determination in dog samples. CRP levels in dogs with different diseases ranged between 10.2 and 210.7 μg/ml and were significantly higher than those observed in healthy dogs (< 7.1 μmg/ml).     

Kinetics of Plasma Cell‐Free DNA and Creatine Kinase in a Canine Model of Tissue Injury

Background

Cell‐free DNA (cfDNA) comprises short, double‐stranded circulating DNA sequences released from damaged cells. In people, cfDNA concentrations correlate well with disease severity and tissue damage. No reports are available regarding cfDNA kinetics in dogs.

Objectives/Hypothesis

Cell‐free DNA will have a short biological half‐life and would be able to stratify mild, moderate, and severe tissue injury. Our study aims were to determine the kinetics and biological half‐life of cfDNA and to contrast them with those of creatine kinase (CK).

Animals

Three groups of 10 dogs undergoing open ovariohysterectomy, surgery for cranial cruciate ligament rupture (CCLR), or hemilaminectomy.

Methods

Plasma for cfDNA and CK analysis was collected at admission, at induction of anesthesia, postsurgery (time 0) and at 6, 12, 24, 36, 48, 60, and 72 hours after surgery.

Results

The biological half‐life of plasma cfDNA and CK were 5.64 hours (95% confidence interval [CI 95], 4.36–7.98 hours) and 28.7 hours (CI95, 25.3–33.3 hours), respectively. In the hemilaminectomy group, cfDNA concentrations differed significantly from admission at 6–12 hours after surgery. Creatine kinase activity differed among the surgical groups and reached a peak 6 hours after surgery. In the ovariohysterectomy and CCLR groups, plasma CK activity 72 hours after surgery did not differ from admission activity of the ovariohysterectomy group. In contrast, in the hemilaminectomy group, plasma CK activity after 72 hours did not return to the ovariohysterectomy group admission activity.

Conclusions and Clinical Importance

Plasma CK activity has a longer biological half‐life than previously thought. In contrast to plasma CK activity, cfDNA has a short half‐life and could be a useful marker for peracute severe tissue injury.     

Canine cell line,IPC‐366, as a good model for the study of inflammatory breast cancer

Inflammatory breast cancer (IBC) is an aggressive type of cancer with poor survival in women. Inflammatory mammary cancer (IMC) in dogs is very similar to human IBC and it has been proposed as a good surrogate model for study the human disease. The aim was to determine if IPC‐366 shared characteristics with the IBC cell line SUM149. The comparison was conducted in terms of ability to grow (adherent and nonadherent conditions), stem cell markers expression using flow cytometry, protein production using western blot and tumorigenic capacity. Our results revealed that both are capable of forming long‐term mammospheres with a grape‐like morphology. Adherent and nonadherent cultures exhibited fast growth in vivo. Stem cell markers expressions showed that IPC‐366 and SUM149 in adherent and nonadherent conditions has mesenchymal‐like characteristics, E‐cadherin and N‐cadherin, was higher in adherent than in nonadherent cultures. Therefore, this study determines that both cell lines are similar and IPC‐366 is a good model for the human and canine disease.     

Isolation of a Campylobacter lanienae‐like Bacterium from Laboratory Chinchillas (Chinchilla laniger)

Routine necropsies of 27 asymptomatic juvenile chinchillas revealed a high prevalence of gastric ulcers with microscopic lymphoplasmacytic gastroenteritis and typhlocolitis. Polymerase chain reaction (PCR) analysis using Campylobacter genus‐specific partial 16S rRNA primers revealed the presence of Campylobacter spp. DNA in the faeces of 12 of 27 animals (44.4%). Species‐specific partial 16S rRNA PCR and sequencing confirmed that these animals were colonized with Campylobacter lanienae, a gram‐negative, microaerophilic bacterium that was first identified on routine faecal screening of slaughterhouse employees and subsequently isolated from faeces of livestock. Campylobacter lanienae was isolated from the faeces of six PCR‐positive animals and identified with species‐specific PCR and full 16S rRNA sequencing. Phylogenetic analysis showed that these isolates clustered with C. lanienae strain NCTC 13004. PCR analysis of DNA extracted from gastrointestinal tissues revealed the presence of C. lanienae DNA in the caecum and colon of these chinchillas. Gastrointestinal lesions were scored and compared between C. lanienae‐positive and C. lanienae‐negative animals. There was no correlation between colonization status and lesion severity in the stomach, liver, duodenum, or colon. Possible routes of C. lanienae infection in chinchillas could include waterborne transmission and faecal–oral transmission from wild mice and rats or livestock. Based on these findings, the authors conclude that C. lanienae colonizes the lower bowel of chinchillas in the absence of clinical disease. This is the first report of C. lanienae in any rodent species. Campylobacter lanienae isolates from different mammalian species demonstrate heterogeneity by 16S rRNA sequence comparison. Analysis using rpoB suggests that isolates and clones currently identified as C. lanienae may represent multiple species or subspecies.     

Identification of a Plasmid‐Mediated Quinolone Resistance Gene in Salmonella Isolates from Texas Dairy Farm Environmental Samples

A recent increase in plasmid‐mediated quinolone resistance (PMQR) has been detected among Salmonella isolated from humans in the United States, and it is necessary to determine the sources of human infection. We had previously isolated Salmonella from dairy farm environmental samples collected in Texas, and isolates were tested for anti‐microbial susceptibility. Two isolates, serotyped as Salmonella Muenster, showed the discordant pattern of nalidixic acid susceptibility and intermediate susceptibility to ciprofloxacin. For this project, whole‐genome sequencing of both isolates was performed to detect genes associated with quinolone resistance. The plasmid‐mediated qnrB19 gene and IncR plasmid type were identified in both isolates. To our knowledge, this is the first report of PMQR in Salmonella isolated from food animals or agricultural environments in the United States.     

Evaluation of the Usefulness of a PCR Assay Performed at a Clinical Laboratory for the Diagnosis of Respiratory Disease Induced by Equine Herpesvirus Type 1 in the Field

A PCR assay for the diagnosis of respiratory disease induced by equine herpesvirus type 1 (EHV-1) was performed at the clinical laboratory in the Racehorse Clinic of the Ritto Training Center of the Japan Racing Association from December 2007 to March 2008. The assay was performed without the trouble of contamination throughout the study and its turnaround time was approximately 6 hr. The PCR detection rates of EHV-1 among seroconverted horses were 22.2% for nasal swabs and 33.3% for blood samples. However, EHV-1 DNA was also detected in horses without seroconversion at a low rate. These results indicated that the PCR assay should be used as an adjunct method, but would help to make an early diagnosis of EHV-1 infection.