Biliary Enterocytozoon bieneusi infection in a dog under immunomodulatory therapy.

An 8-year-old female spayed Labrador retriever was presented for the evaluation of severe weight loss 10 weeks after starting an immunomodulatory treatment, including prednisolone and cyclosporine, for meningoencephalitis of unknown origin. Plasma biochemistry analysis showed mild to moderate increases in liver enzyme activities and a moderate decrease in urea concentration. Abdominal ultrasound revealed mild hepatomegaly and a large gall bladder with unremarkable wall and content. Cholecystocentesis was performed and bile was examined both cytologically and by molecular methods, which revealed the presence of Enterocytozoon bieneusi. Treatment was initiated with albendazole but was discontinued due to the development of severe neutropenia. The medical management was subsequently changed to fenbendazole and the dog made a complete recovery. This report describes the first case of clinical manifestation and successful treatment of biliary E. bieneusi infection in a dog.

Diagnostic utility and validity of 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) lipase activity in horses with colic.

Little is known about the clinical usefulness in horses of the 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) lipase assay, a biomarker used in other species for the detection of pancreatitis. The main objectives of this study were to evaluate the prevalence of increased DGGR-lipase activity in horses with signs of colic and investigate its association with, and validity to diagnose, underlying gastrointestinal diseases, treatment method (medical or surgical), and outcome (survival or non-survival). Clinical data from 192 horses presented for colic to a teaching hospital were analysed retrospectively. DGGR-lipase activity was measured in frozen plasma collected within 24 h of presentation. Non-parametric tests and Chi-squared or Fisher's exact tests were used to evaluate differences and associations between DGGR-lipase activity and continuous and categorical variables or outcomes, respectively. Measures of the validity of DGGR-lipase as a diagnostic test were also calculated. Increased DGGR-lipase activity above published reference limits was demonstrated in 30.2% of horses with signs of colic, and was above 2x the upper reference limit (URL) in 15.6%. The median DGGR-lipase activity in horses with large bowel displacement or torsion was significantly higher than the median activity for large bowel impaction and for gastric impaction, dilation, or ulceration. DGGR-lipase activity > 2x URL was significantly associated with surgical treatment, strangulating disease, and non-survival. However, as a diagnostic or screening test for these target outcomes, DGGR-lipase activity was poor to fair consequent to poor sensitivity, poor negative likelihood ratio, and an area under the receiver operating characteristic curve, with optimal cut-offs based on the Youden Index, within reference limits.

Pre-analytical stability of sorbitol dehydrogenase in equine heparinized plasma.

Sorbitol dehydrogenase (SDH) activity is one of the most sensitive and specific markers for hepatocellular injury in horses, but its reported lability makes it impractical for use in many clinical settings. To date, stability of SDH in equine samples has only been evaluated in a limited number of studies in serum samples of horses with activities within reference intervals. The objective of the study was to determine pre-analytical stability of equine SDH activity in heparinized plasma stored at different temperatures for up to 72 h. Twenty client-owned horses admitted to a veterinary teaching hospital for any reason were included in the study. Blood samples collected in lithium-heparin tubes were immediately centrifuged and SDH activity was analyzed within 1 h of collection (T0). Aliquots of plasma were stored at room temperature, 4 °C and -20 °C and SDH activity was re-analyzed after 4 h (T4), 24 h (T24) and 72 h (T72). A significant difference from values measured at T0 was found for samples stored at room temperature (P = 0.022) and -20 °C (P < 0.001), but not at 4 °C. The activity of SDH was within ±20% of that measured at T0 for all samples under all temperature conditions stored for 4 h, and for all samples stored at 4 °C for 24 h. Bland-Altman plots revealed narrow limits of agreement at T4 for all storage temperatures and at T24 for samples stored at 4 °C. The mean absolute percentage error and 95th percentile of the absolute percentage error were lower for samples stored at 4 °C than those stored at room temperature or -20 °C. The activity of SDH has adequate stability for 4 h regardless of storage temperature and 24 h if stored at 4 °C across a wide range of values. Knowledge of the pre-analytical stability of SDH may permit its broader use in assessing hepatic disorders in horses.

Histiocytic sarcoma in miniature schnauzers: 30 cases.

OBJECTIVES: To summarise the clinical presentation and outcomes in a series of miniature schnauzers diagnosed with histiocytic sarcoma. MATERIALS AND METHODS: Retrospective review of medical records of miniature schnauzers diagnosed with histiocytic sarcoma between 2008 and 2019 at two referral centres in the UK. Signalment, clinical signs at initial presentation, imaging results and clinico- and histopathological findings, treatment type and outcome were recorded. Progression-free survival and overall survival time were calculated. RESULTS: Thirty dogs were included. Twenty-four of 29 dogs undergoing imaging of the thorax had lung and/or mediastinal involvement. The median overall survival time for dogs that were not euthanased within 3 days of diagnosis was 117 days (range 10 to 790). Three dogs underwent surgery; 13 received treatment with lomustine as a sole therapy - with partial responses documented on imaging in five of six dogs and 11 of 13 showing clinical improvement. CLINICAL SIGNIFICANCE: Histiocytic sarcoma should be considered as a differential diagnosis for miniature schnauzers with pulmonary masses. Although responses to treatment were common, they were usually short-lived because of the aggressive nature of the disease.

Short communication: Pegbovigrastim treatment in vivo does not affect granulocyte ability to migrate to endometrial cells and kill bacteria in vitro in healthy cows.

In periparturient dairy cows, immune suppression, resulting in decreased neutrophil numbers and function, leads to increased susceptibility to postpartum conditions such as mastitis, retained placenta, and metritis. Administration of polyethylene glycol-conjugated bovine granulocyte colony stimulating factor (pegbovigrastim, Imrestor; Elanco Animal Health, Greenfield, IN) 7 d before and within 24 h of calving, effectively improves granulocyte production and function in vivo as well as in milk. A recently developed coculture assay was adapted for use with endometrial epithelial cells to assess the effects of pegbovigrastim application on directed granulocyte migration and bactericidal activity in vitro on a per-cell basis in endometrial cell cultures. Granulocytes from treated and untreated periparturient cows (6 and 5 per group, respectively) were evaluated for their ability to migrate to and kill bacteria after treatment, in context of the infected endometrium. We hypothesized that in addition to increasing the absolute concentration of circulating neutrophil granulocytes, pegbovigrastim treatment in vivo alters the ability of granulocytes to migrate to endometrial cells in vitro. The results clearly show a marked increase in the total concentration of granulocytes and monocytes between the 2 treatment groups as early as 2 d after the first injection, and this increased between the samples taken 2 d after calving. No migratory or killing differences were identified between granulocytes of both groups, suggesting that pegbovigrastim-induced granulocytes were as effective as non-induced cells. This may also be due to the absence of negative energy balance in the study animals and leads us to conclude that the positive effects seen in vivo are most likely based on the larger number of granulocytes present rather than a direct effect of pegbovigrastim treatment on the functionality of cells for the parameters tested in this study.

Computed tomographic findings in 12 cases of canine multi-centric lymphoma with splenic and hepatic involvement.

OBJECTIVES: To describe the CT findings in a population of dogs with multi-centric lymphoma that involved the spleen and liver. MATERIALS AND METHODS: Clinical records between January 2008 and June 2015 were reviewed. Thoracic and abdominal CT examinations of patients diagnosed with multi-centric lymphoma were evaluated by a board-certified radiologist. A diagnosis of multi-centric lymphoma with splenic and hepatic involvement was based upon cytological identification and immunophenotyping of neoplastic lymphocytes in cellular samples harvested from a peripheral lymph node, the spleen and the liver. RESULTS: Twelve dogs were included in this study, of which 11 had B-cell lymphoma; immunophenotyping was inconclusive in one dog. The spleen appeared normal in seven dogs and nodules were identified in five dogs. Splenic nodules were hypoattenuating in four of five dogs and isoattenuating in one of five. After contrast administration, three of five appeared hypoattenuating and two of five isoattenuating. The liver appeared normal in 10 dogs and hepatic nodules were identified in two dogs. All hepatic nodules were isoattenuating before contrast and hypoattenuating following contrast administration. CLINICAL SIGNIFICANCE: The CT appearance of the spleen and liver was normal in the majority of dogs with multi-centric lymphoma. Fine needle aspiration of the spleen and liver is recommended when using CT to stage dogs with multi-centric lymphoma.

Cytological Findings of 140 Bile Samples from Dogs and Cats and Associated Clinical Pathological Data.

BACKGROUND: Cholecystocentesis can be part of the diagnostic workup of hepatobiliary disease in small animals, but literature on cytological evaluation of bile is scant. OBJECTIVES: To determine the diagnostic utility of cytological assessment of bile aspirates. ANIMALS: Fifty-six and 78 client-owned dogs and cats, respectively, with bile collected by cholecystocentesis and submitted to our diagnostic laboratory between 1999 and 2014. METHODS: Retrospective study describing cytological findings of bile, concurrent bacterial culture results, hematological and serum biochemical data, gallbladder biopsy results, as well as final diagnosis and complications after cholecystocentesis. RESULTS: Infectious agents were found in 30% of canine and 22% of feline bile aspirates, and inflammation in 5% and 19% respectively. Presence of microorganisms was more often detected on cytological examination (24%) than by culture (21%). The most common bacterial isolates were Escherichia coli and Enterococcus spp., isolated from 14.8% and 6.7% of cultured samples respectively. Only increased canine pancreatic lipase immunoreactivity concentration (cPLI) was significantly associated with the presence of microorganisms, inflammatory cells, or both in bile. Clinically relevant complications of cholecystocentesis occurred in 2 dogs. The majority of the animals undergoing cholecystocentesis suffered from hepatic, pancreatic, gastrointestinal disease, or a combination thereof. CONCLUSIONS AND CLINICAL IMPORTANCE: Cytological examination of bile is inexpensive and straightforward, and yields diagnostically relevant information that precedes and complements bacterial culture.

Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine.

Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66µM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26µM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

Comparative human-horse sequence analysis of the CYP3A subfamily gene cluster.

Cytochrome P450 enzymes (CYP450s) represent a superfamily of haem-thiolate proteins. CYP450s are most abundant in the liver, a major site of drug metabolism, and play key roles in the metabolism of a variety of substrates, including drugs and environmental contaminants. Interaction of two or more different drugs with the same enzyme can account for adverse effects and failure of therapy. Human CYP3A4 metabolizes about 50% of all known drugs, but little is known about the orthologous CYP450s in horses. We report here the genomic organization of the equine CYP3A gene cluster as well as a comparative analysis with the human CYP3A gene cluster. The equine CYP450 genes of the 3A family are located on ECA 13 between 6.97-7.53 Mb, in a region syntenic to HSA 7 99.05-99.35 Mb. Seven potential, closely linked equine CYP3A genes were found, in contrast to only four genes in the human genome. RNA was isolated from an equine liver sample, and the approximately 1.5-kb coding sequence of six CYP3A genes could be amplified by RT-PCR. Sequencing of the RT-PCR products revealed numerous hitherto unknown single nucleotide polymorphisms (SNPs) in these six CYP3A genes, and one 6-bp deletion compared to the reference sequence (EquCab2.0). The presence of the variants was confirmed in a sample of genomic DNA from the same horse. In conclusion, orthologous genes for the CYP3A family exist in horses, but their number differs from those of the human CYP3A gene family. CYP450 genes of the same family show high homology within and between mammalian species, but can be highly polymorphic.

A locus for autosomal dominant progressive non-syndromic hearing loss, DFNA27, is on chromosome 4q12-13.1.

We ascertained a large North American family, LMG2, segregating progressive, non-syndromic, sensorineural hearing loss. A genome-wide scan identified significant evidence for linkage (maximum logarithm of the odds (LOD) score = 4.67 at theta = 0 for D4S398) to markers in a 5.7-cM interval on chromosome 4q12-13.1. The DFNA27 interval spans 8.85 Mb and includes at least 61 predicted and 8 known genes. We sequenced eight genes and excluded them as candidates for the DFNA27 gene.

Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23.

Genes causing nonsyndromic autosomal recessive deafness (DFNB12) and deafness associated with retinitis pigmentosa and vestibular dysfunction (USH1D) were previously mapped to overlapping regions of chromosome 10q21-q22. Seven highly consanguineous families segregating nonsyndromic autosomal recessive deafness were analyzed to refine the DFNB12 locus. In a single family, a critical region was defined between D10S1694 and D10S1737, approximately 0.55 cM apart. Eighteen candidate genes in the region were sequenced. Mutations in a novel cadherin-like gene, CDH23, were found both in families with DFNB12 and in families with USH1D. Six missense mutations were found in five families with DFNB12, and two nonsense and two frameshift mutations were found in four families with USH1D. A northern blot analysis of CDH23 showed a 9.5-kb transcript expressed primarily in the retina. CDH23 is also expressed in the cochlea, as is demonstrated by polymerase chain reaction amplification from cochlear cDNA.

The relationship of beta-glucuronidase activity in crevicular fluid to probing attachment loss in patients with adult periodontitis. Findings from a multicenter study.

Analysis of gingival crevicular fluid (GCF) offers a non-invasive means of studying the host response in periodontal disease, and may provide an early indication of the patient at risk for active periodontitis. A number of host markers have been studied for their relationship to disease activity (probing attachment loss or PAL). GCF levels of the acid glycohydrolase beta-glucuronidase (beta G), a marker of primary granule release from polymorphonuclear leukocytes (PMN), have been shown to identify patients with periodontitis at risk for additional PAL. In this multicenter trial, we evaluated (a) the short-term effect of conservative periodontal therapy on beta G activity in GCF, and (b) the relationship of persistently elevated beta G activity to PAL in patients who were monitored for 6 months. The study population included a total of 140 patients with chronic adult periodontitis. 130 patients were on a regular recall schedule, and 10 were previously untreated. After collection of baseline clinical data at all sites, and analysis of beta G in GCF from one site (mesiobuccal) per tooth, the patients received a scaling and prophylaxis. Two weeks later patients were seen for collection of GCF. If elevated enzyme activity was found at 2 weeks, the patients were seen at 3 months for a clinical exam and GCF collection. Clinical parameters were collected from all patients at 6 months. Therapy tended to reduce beta G activity in GCF.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of beta-glucuronidase activity in crevicular fluid to clinical parameters of periodontal disease. Findings from a multicenter study.

Previous reports have suggested that persistently elevated levels of the acidic glycohydrolase beta-glucuronidase (beta G) in gingival crevicular fluid (GCF) can identify patients with chronic adult periodontitis who are at risk for future probing attachment loss (PAL). To comprehensively study beta G activity in GCF, a multicenter trial examining the relationship of the enzyme in GCF to traditional clinical parameters of periodontal disease and PAL was conducted. In this report, the baseline data was used to evaluate the relationship of beta G activity in GCF to traditional parameters of periodontal disease. The study group included 130 patients who had been treated for periodontal disease and were on a regular recall schedule, and 10 patients with chronic adult periodontitis who had never received treatment. Upon entering the longitudinal trial, the patients were examined, and a standardized 30-s GCF sample was collected from the mesiobuccal crevice of all study teeth. As a control, GCF samples and clinical data were collected from 62 patients with a healthy periodontium or mild inflammatory gingivitis without loss of probing attachment. At baseline, beta G activity for the periodontitis patients ranged from 0 to 1704 Units (U), with a median of 32 U. beta G could not be detected in 0.2% of samples (activity < or = 2.0 U). The 90% cumulative relative frequency was 139 U. For the healthy/gingivitis subjects beta G activity ranged from 0 to 504 U, with a median of 22 U. Enzyme was not detectable in 0.4% of samples. Only 0.9% of samples contained greater than 139 U. beta G activity in GCF was not related to gender or age. For the periodontitis patients, elevated enzyme activity (> or = 140 U) was most often associated with molar teeth, followed by maxillary bicuspids. Maxillary central incisors, and mandibular central and lateral incisors displayed the lowest frequency of elevated enzyme activity. The relationship of beta G activity to the traditional parameters of probing depth and bleeding on probing was assessed. For shallow sites (1.0-1.5 mm, 2.0-2.5 mm probing depth), the large majority of GCF samples contained low enzyme activity (90% of samples < 50 U). Descriptive indicators demonstrated a trend of increased beta G activity with increased probing depth. The median beta G activity shifted from 15 U for the shallowest sites (1.0-1.5 mm) to 127 U for the deepest sites (5-8 mm). However, this was due to a broadening of the distribution rather than representing a shift in the distribution profile.(ABSTRACT TRUNCATED AT 400 WORDS)