Treponeme-Associated Hoof Disease of Free-Ranging Elk ( Cervus elaphus) in Southwestern Washington State, USA.

A novel foot disease in free-ranging elk ( Cervus elaphus) in southwestern Washington State emerged in 2008 and spread throughout the region. Initial studies showed adult elk had chronic hoof overgrowth, sole ulcers, and sloughed hoof capsules, but no cause was determined. To identify possible causes and characterize the earliest lesions, 9-, 7-, and 3-month-old elk were collected. Nine-month-old elk had sole ulcers (3/9 elk) and sloughed/overgrown hoof capsules (4/9 elk) similar to adults. Histologically, lesions consisted of coronary, heel bulb, and interdigital ulcers with suppurative inflammation, epithelial hyperplasia, deeply invasive spirochetes, and underrunning of the hoof capsule and heel-sole junction. Spirochetes were identified as Treponema via immunohistochemistry and polymerase chain reaction (PCR). Seven-month-old elk had similar underrunning foot ulcers (6/8 elk) with Treponema identified in all lesions but no chronic overgrowth or sloughed hoof capsules. Three-month-old calves had superficial coronary erosions with no inflammation or identifiable spirochetes (3/5 elk) but were culture/PCR positive for Treponema, suggesting possible early lesions. Lesions from 9- and 7-month-old elk included aerobic and anaerobic bacteria, many of which are associated with infectious foot disease in livestock. Antibody enzyme-linked immunosorbent assay of 7- and 3-month-old elk from the enzootic region showed a trend toward increased Treponema antibody titers compared to normal control elk from outside the region, further supporting the significance of Treponema in the pathogenesis of foot disease. Treponeme-associated hoof disease (TAHD) in elk, a debilitating and progressive condition, shares similarities to bovine digital dermatitis and contagious ovine digital dermatitis.

Effect of vaccination against pneumonia on the survival of bighorn sheep (Ovis canadensis) commingled with carrier animals.

Leukotoxin producing (lkt+) members of Pasteurellaceae, particularly Mannheimia haemolytica and Bibersteinia trehalosi are important pathogens of pneumonia in bighorn sheep (BHS; Ovis canadensis), causing fatal disease. Predisposing or concurrent infection with Mycoplasma ovipneumoniae enhances the severity of the disease, resulting in increased morbidity and mortality. Several studies have investigated the effectiveness of vaccines against lkt+ members of Pasteurellaceae in preventing fatal pneumonia in BHS. In all of these studies, however, vaccinated animals were challenged experimentally, by direct inoculation of the pathogens, rather than by natural challenge. Moreover, none has investigated the efficacy of the vaccines under conditions of concurrent infection with M. ovipneumoniae. We immunized three bighorn rams and one pregnant ewe with an experimental multivalent vaccine along with a commercial vaccine. The immunized animals were then commingled with two bighorn ewes known to be carriers of lkt+ members of Pasteurellaceae, to simulate natural infection or disease transmission. All vaccinated animals remained healthy. We then inoculated the two carrier ewes with nasal washings from domestic sheep containing M. ovipneumoniae. Within a week, all animals developed mild to moderate signs of pneumonia. While the rams died within two-three months post-inoculation (p.i.), the vaccinated ewe and her lamb died five and eight months p.i., respectively. Taken together, these results suggest that vaccination of BHS against lkt+ members of Pasteurellaceae alone can protect them from natural challenge by these pathogens. However, it may not be adequate to protect them against pneumonia compounded by concurrent infection with M. ovipneumoniae.

Immunization of bighorn sheep against Mannheimia haemolytica with a bovine herpesvirus 1-vectored vaccine.

Mannheimia haemolytica is an important pathogen of pneumonia in bighorn sheep (BHS), consistently causing 100% mortality under experimental conditions. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, a vaccine containing leukotoxin and surface antigens of M. haemolytica induced 100% protection in BHS, but required multiple booster doses. Vaccination of wildlife is difficult. BHS, however, can be vaccinated at the time of transplantation, but administration of booster doses is impossible. A vaccine that does not require booster doses, therefore, is ideal for vaccination of BHS. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation which obviates the need for booster administration. The objective of this study was to evaluate the potential of bovine herpesvirus 1 (BHV-1) as a vector encoding M. haemolytica immunogens. As the first step towards this goal, the permissiveness of BHS for BHV-1 infection was determined. BHS inoculated with wild-type BHV-1 shed the virus following infection. The lytic phase of infection was superseded by latency, and treatment of latently-infected BHS with dexamethasone reactivated the virus. A recombinant BHV-1-vectored vaccine encoding a leukotoxin-neutralizing epitope and an immuno-dominant epitope of the outer membrane protein PlpE was developed by replacing the viral glycoprotein C gene with a leukotoxin-plpE chimeric gene. Four of six BHS vaccinated with the recombinant virus developed significant leukotoxin-neutralizing antibodies at day 21 post-vaccination, while two of six BHS developed significant surface antigen antibodies at day 17 post-vaccination. These antibodies, however, were inadequate for protection of BHS against M. haemolytica challenge. These data indicate that BHV-1 is a suitable vector for immunization of BHS, but additional experimentation with the chimeric insert is necessary for development of a more efficacious vaccine.

Abortion in a Mediterranean miniature donkey (Equus asinus) associated with a gammaherpesvirus similar to Equid herpesvirus 7.

Fetal tissues and placenta from a third trimester Mediterranean miniature donkey (Equus asinus) abortion were submitted to the Washington State University, Washington Animal Disease Diagnostic Laboratory for abortion diagnosis. Microscopic examination of formalin-fixed tissues revealed multifocal necrotizing placentitis. Several cells within the necrotic foci contained large, eosinophilic, intranuclear inclusions. Virus isolation from fresh, frozen placenta identified a cytopathic, syncytia-forming virus. Polymerase chain reaction (PCR) from the cultured virus using degenerate universal herpesvirus primers amplified a 699-base pair portion of the DNA polymerase gene. The PCR amplicon had 96.7% nucleotide identity with the DNA polymerase gene of Equid herpesvirus 7 (EHV-7; asinine herpesvirus 2), a gammaherpesvirus. An identical sequence was obtained when the same degenerate herpesvirus primers were used for PCR on the formalin-fixed placenta. Additionally, the amplicon had complete identity with short sequences of asinine herpesviruses that have been published in association with interstitial pneumonia in donkeys. EHV-7 has previously been isolated from nasal secretions of normal donkeys and mules. Our report describes a case of abortion associated with EHV-7 or a similar virus.

The Intestinal Microbiota Influences Campylobacter jejuni Colonization and Extraintestinal Dissemination in Mice.

Campylobacter jejuni is a leading cause of human foodborne gastroenteritis worldwide. The interactions between this pathogen and the intestinal microbiome within a host are of interest as endogenous intestinal microbiota mediates a form of resistance to the pathogen. This resistance, termed colonization resistance, is the ability of commensal microbiota to prevent colonization by exogenous pathogens or opportunistic commensals. Although mice normally demonstrate colonization resistance to C. jejuni, we found that mice treated with ampicillin are colonized by C. jejuni, with recovery of Campylobacter from the colon, mesenteric lymph nodes, and spleen. Furthermore, there was a significant reduction in recovery of C. jejuni from ampicillin-treated mice inoculated with a C. jejuni virulence mutant (ΔflgL strain) compared to recovery of mice inoculated with the C. jejuni wild-type strain or the C. jejuni complemented isolate (ΔflgL/flgL). Comparative analysis of the microbiota from nontreated and ampicillin-treated CBA/J mice led to the identification of a lactic acid-fermenting isolate of Enterococcus faecalis that prevented C. jejuni growth in vitro and limited C. jejuni colonization of mice. Next-generation sequencing of DNA from fecal pellets that were collected from ampicillin-treated CBA/J mice revealed a significant decrease in diversity of operational taxonomic units (OTUs) compared to that in control (nontreated) mice. Taken together, we have demonstrated that treatment of mice with ampicillin alters the intestinal microbiota and permits C. jejuni colonization. These findings provide valuable insights for researchers using mice to investigate C. jejuni colonization factors, virulence determinants, or the mechanistic basis of probiotics.

Francisella tularensis infection without lesions in gray tree squirrels (Sciurus griseus): a diagnostic challenge.

Fifteen cases of Francisella tularensis infection (tularemia) were identified in western gray (Sciurus griseus) and eastern gray (Sciurus carolinensis) squirrels submitted to the Washington Animal Disease Diagnostic Laboratory between 2008 and 2011. All of the squirrels originated in Washington State, a geographical area with endemic tularemia in wildlife. Nine of the 15 squirrels with F. tularensis infection had gross (2/15) or microscopic (9/15) multifocal necrotizing lesions in the spleen, liver, or lymph nodes, typical of tularemia. Special stains did not reliably identify intralesional bacteria microscopically. Six of the 15 squirrels infected with F. tularensis lacked gross and microscopic lesions typical of tularemia. All 15 squirrels with F. tularensis infection were identified by polymerase chain reaction tests on the spleen, liver, or lymph node (including all 6 squirrels without typical tularemia lesions); 8 out of 9 squirrels were positive by direct fluorescent antibody test of tissues, and 5 out of 15 squirrels were positive by culture of tissues. The findings underscore the importance of considering tularemia as a possible cause of death when no lesions of tularemia can be identified at necropsy. Furthermore, the findings suggest the possibility of subclinical infections in gray squirrels, and the importance of molecular diagnostics for definitive diagnosis of F. tularensis infection in wild squirrels.

Presence of Arp specifically contributes to joint tissue edema associated with early-onset Lyme arthritis.

Antiserum to the Borrelia burgdorferi arthritis-related protein, Arp, has been shown to prevent or reduce arthritis in immunodeficient mice. To directly investigate the requirement for this lipoprotein in the generation of Lyme arthritis, we utilized targeted deletion to generate a B. burgdorferi clone that lacked only the arp gene locus. Infection of Lyme disease-susceptible C3H/HeN mice with the arp deletion mutant demonstrated significantly reduced tibiotarsal joint swelling during the first 6 weeks of infection compared to a wild-type control. The severity of joint swelling was restored to wild-type levels in mice infected with an arp mutant clone complemented in cis. Interestingly, the reduced swelling of joint tissues exhibited by mice infected with the arp deletion mutant did not directly correspond to reduced underlying arthritis. Histopathology data at 2 weeks postinfection showed some reduction in arthritis severity caused by the arp mutant clone; however, by 8 weeks, no significant difference was observed between joint tissues infected by the wild-type or arp mutant clones. The spirochete load in the joint tissues of mice infected with the arp mutant was found to be greater than that exhibited by the wild-type control. Our findings demonstrate that this lipoprotein contributes to the generation of early-onset joint swelling and suggests that arp expression has a negative secondary effect on total spirochete numbers in joint tissues.

Role of Bibersteinia trehalosi, respiratory syncytial virus, and parainfluenza-3 virus in bighorn sheep pneumonia.

Pneumonic bighorn sheep (BHS) have been found to be culture- and/or sero-positive for Bibersteinia trehalosi, respiratory syncytial virus (RSV), and parainfluenza-3 virus (PI-3). The objective of this study was to determine whether these pathogens can cause fatal pneumonia in BHS. In the first study, two groups of four BHS each were intra-tracheally administered with leukotoxin-positive (Group I) or leukotoxin-negative (Group II) B. trehalosi. All four animals in Group I developed severe pneumonia, and two of them died within 3 days. The other two animals showed severe pneumonic lesions on euthanasia and necropsy. Animals in Group II neither died nor showed gross pneumonic lesions on necropsy, suggesting that leukotoxin-positive, but not leukotoxin-negative, B. trehalosi can cause fatal pneumonia in BHS. In the second study, two other groups of four BHS (Groups III and IV) were intra-nasally administered with a mixture of RSV and PI-3. Four days later, RSV/PI-3-inoculated Group IV and another group of four BHS (Group V, positive control) were intra-nasally administered with Mannheimia haemolytica, the pathogen that consistently causes fatal pneumonia in BHS. All four animals in group III developed pneumonia, but did not die during the study period. However all four animals in Group IV, and three animals in Group V developed severe pneumonia and died within two days of M. haemolytica inoculation. The fourth animal in Group V showed severe pneumonic lesions on euthanasia and necropsy. These findings suggest that RSV/PI-3 can cause non-fatal pneumonia, but are not necessary predisposing agents for M. haemolytica-caused pneumonia of BHS.

A novel papillomavirus isolated from proliferative skin lesions of a wild American beaver (Castor canadensis).

Cutaneous papillomatosis was diagnosed in an adult American beaver (Castor canadensis). Gross lesions included numerous exophytic, roughly circular, lightly pigmented lesions on hairless areas of fore and hind feet and the nose. The most significant histopathologic findings were multifocal papilliform hyperplasia of the superficial stratified squamous epithelium, with multifocal koilocytes, and multiple cells with large, darkly basophilic intranuclear inclusion bodies. A virus with properties consistent with papillomavirus (PV) was recovered by virus isolation of skin lesions, utilizing rabbit and feline kidney cell lines. The presence of the virus was confirmed by PV-specific polymerase chain reaction. The partial sequences of E1 and L1 genes did not closely match those of any PVs in GenBank, suggesting that this might be a new type of PV. Partial E1 and L1 nucleotide sequences of the beaver papillomavirus (hereafter, ARbeaver-PV1) were used to create a phylogenetic tree employing the complete E1 and L1 open reading frame nucleotide sequences of 68 PVs. The phylogenetic tree placed the ARbeaver-PV1 in a clade that included the Mupapillomavirus (HPV1 and HPV63) and Kappapapillomavirus (OcPV1 and SfPV1) genera. The present article confirms the papillomaviral etiology of cutaneous exophytic lesions in the beaver.

Effect of tocopherol supplementation during last trimester of pregnancy on mRNA abundances of interleukins and angiogenesis in ovine placenta and uterus.

BACKGROUND: Interleukins (IL) play an important role in angiogenesis. Tocopherol possesses immunomodulating effect in addition to antioxidant property. The objective of this study was to determine whether gamma tocopherol's (gT) angiogenic activity in placental network is enhanced via promoting interleukins. METHODS: Pregnant ewes (N=18) were supplemented, orally, with 500 mg of alpha tocopherol (aT; N=6) or 1,000 mg of gT (N=7) or placebo (CON; N=5) once daily from 107 to 137 days post breeding. Uterine and placental tissue samples were obtained at the end of supplementation to evaluate relative mRNA expressions of IL-1b, IL-6, IL-8, Tumor Necrosis Factor (TNF) alpha, Vascular Endothelial Growth Factor (VEGF), kinase insert domain receptor (KDR; VGFR2; a type III receptor tyrosine kinase), and soluble fms-like tyrosine kniase-1 (sFlt1 or sVEGFR1) in uterus, caruncle and cotyledon. RESULTS: Oral supplementation of gT increased IL-6, IL-8, KDR and VEGF mRNA abundances whereas sFlt1 mRNA abundance was suppressed in uterus, caruncle and cotyledon, compared to aT and placebo treated ewes (P<0.05). The TNF alpha and IL-1b mRNA abundances were suppressed in uterus, caruncle and cotyledon but TNF alpha is higher in gT group compared to aT group (P<0.05), whereas IL-1b was similar between treatment groups (P>0.1). CONCLUSIONS: Gamma tocopherol supplementation increased IL-6, IL-8, and KDR mRNA abundances and suppressed sFlt1 and TNFalpha mRNA abundances thereby increased VEGF mRNA expression and angiogenesis in placental vascular network during late gestation. It is plausible that the angiogenic effect of gamma tocopherol in placental vascular network is exerted via an alternate path by enhancing IL-6 and IL-8.

Fatal hepatic sarcocystosis in a captive black bear (Ursus americanus) associated with Sarcocystis canis-like infection.

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.

Molecular genetic basis for fluoroquinolone-induced retinal degeneration in cats.

OBJECTIVES: Distribution of fluoroquinolones to the retina is normally restricted by ABCG2 at the blood-retinal barrier. As the cat develops a species-specific adverse reaction to photoreactive fluoroquinolones, our goal was to investigate ABCG2 as a candidate gene for fluoroquinolone-induced retinal degeneration and blindness in cats. METHODS: Feline ABCG2 was sequenced and the consensus amino acid sequence was compared with that of 10 other mammalian species. Expression of ABCG2 in feline retina was assessed by immunoblot. cDNA constructs for feline and human ABCG2 were constructed in a pcDNA3 expression vector and expressed in HEK-293 cells, and ABCG2 expression was analyzed by western blot and immunofluorescence. Mitoxantrone and BODIPY-prazosin efflux measured by flow cytometry and a phototoxicity assay were used to assess feline and human ABCG2 function. RESULTS: Four feline-specific (compared with 10 other mammalian species) amino acid changes in conserved regions of ABCG2 were identified. Expression of ABCG2 on plasma membranes was confirmed in feline retina and in cells transfected with human and feline ABCG2, although some intracellular expression of feline ABCG2 was detected by immunofluorescence. Function of feline ABCG2, compared with human ABCG2, was found to be deficient as determined by flow cytometric measurement of mitoxantrone and BODIPY-prazosin efflux and enrofloxacin-induced phototoxicity assays. CONCLUSION: Feline-specific amino acid changes in ABCG2 cause a functional defect of the transport protein in cats. This functional defect may be owing, in part, to defective cellular localization of feline ABCG2. Regardless, dysfunction of ABCG2 at the blood-retinal barrier likely results in accumulation of photoreactive fluoroquinolones in feline retina. Exposure of the retina to light would then generate reactive oxygen species that would cause the characteristic retinal degeneration and blindness documented in some cats receiving high doses of some fluoroquinolones. Pharmacological inhibition of ABCG2 in other species might result in retinal damage if fluoroquinolones are concurrently administered.

Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia.

Mycoplasma ovipneumoniae has been isolated from the lungs of pneumonic bighorn sheep (BHS). However experimental reproduction of fatal pneumonia in BHS with M. ovipneumoniae was not successful. Therefore the specific role, if any, of M. ovipneumoniae in BHS pneumonia is unclear. The objective of this study was to determine whether M. ovipneumoniae alone causes fatal pneumonia in BHS, or predisposes them to infection by Mannheimia haemolytica. We chose M. haemolytica for this study because of its isolation from pneumonic BHS, and its consistent ability to cause fatal pneumonia under experimental conditions. Since in vitro culture could attenuate virulence of M. ovipneumoniae, we used ceftiofur-treated lung homogenates from pneumonic BHS lambs or nasopharyngeal washings from M. ovipneumoniae-positive domestic sheep (DS) as the source of M. ovipneumoniae. Two adult BHS were inoculated intranasally with lung homogenates while two others received nasopharyngeal washings from DS. All BHS developed clinical signs of respiratory infection, but only one BHS died. The dead BHS had carried leukotoxin-positive M. haemolytica in the nasopharynx before the onset of this study. It is likely that M. ovipneumoniae colonization predisposed this BHS to fatal infection with the M. haemolytica already present in this animal. The remaining three BHS developed pneumonia and died 1-5 days following intranasal inoculation with M. haemolytica. On necropsy, lungs of all four BHS showed lesions characteristic of bronchopneumonia. M. haemolytica and M. ovipneumoniae were isolated from the lungs. These results suggest that M. ovipneumoniae alone may not cause fatal pneumonia in BHS, but can predispose them to fatal pneumonia due to M. haemolytica infection.

Escherichia albertii in wild and domestic birds.

Escherichia albertii has been associated with diarrhea in humans but not with disease or infection in animals. However, in December 2004, E. albertii was found, by biochemical and genetic methods, to be the probable cause of death for redpoll finches (Carduelis flammea) in Alaska. Subsequent investigation found this organism in dead and subclinically infected birds of other species from North America and Australia. Isolates from dead finches in Scotland, previously identified as Escherichia coli O86:K61, also were shown to be E. albertii. Similar to the isolates from humans, E. albertii isolates from birds possessed intimin (eae) and cytolethal distending toxin (cdtB) genes but lacked Shiga toxin (stx) genes. Genetic analysis of eae and cdtB sequences, multilocus sequence typing, and pulsed-field gel electrophoresis patterns showed that the E. albertii strains from birds are heterogeneous but similar to isolates that cause disease in humans.

Canine ABCB4: Tissue expression and cDNA structure.

The ABCB gene subfamily of ABC (ATP-binding cassette) transporters is responsible for transporting a wide spectrum of molecules including peptides, iron, bile salts, drugs, and phospholipids. In humans, ABCB4 appears to be exclusively expressed on the apical membrane of hepatocytes where it translocates phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane. Functional alterations in the ABCB4 transporter are associated with a number of cholestatic syndromes in humans. Because of its role in biliary lipid homeostasis in humans, investigation of the ABCB4 gene in dogs is warranted. Thus, the full cDNA sequence of canine ABCB4 was elucidated and its mRNA and protein expression levels in tissues were determined. Canine ABCB4 consists of 3804 nucleotides spanning 26 exons and is 89% identical to human ABCB4. Expression of ABCB4 in canine liver supports a potential role for the protein in normal biliary function similar to that in humans. The function of ABCB4 expressed in brain tissue has yet to be determined.

Hemodynamic changes during laparoscopic radiofrequency ablation of normal adrenal tissue in dogs.

OBJECTIVE: To determine the hemodynamic response to radiofrequency ablation (RFA) of normal adrenal tissue in dogs. STUDY DESIGN: Experimental study. ANIMALS: Healthy adult mixed-breed dogs (n=6). METHODS: During general anesthesia a Swan-Ganz thermodilution catheter was flow directed into the pulmonary artery and used to quantify cardiac output. An arterial catheter was used for direct blood pressure measurements. An RFA device was introduced into the left adrenal gland under observation through laparoscopic instrumentation. Blood samples were collected and hemodynamic variables studied after a stable surgical anesthetic depth was achieved (time 1), during CO(2) insufflation of the abdomen (time 2), during adrenal RFA (time 3), and after completed RFA (time 4). Catecholamine determinations were performed with a human enzyme immunoassay. Histopathology was performed to verify medullary necrosis. RESULTS: Arterial, pulmonary arterial and central venous pressure, and plasma norepinephrine increased more during RFA than during abdominal insufflation. Heart rate and cardiac index did not differ between time points. High baseline epinephrine was present and significant differences between time points were not detected. Systemic vascular resistance had very high individual variation and differences were not detected. CONCLUSIONS: RFA of normal adrenal tissues is associated with severe hemodynamic alterations. Further studies of the optimal blockage of catecholamine-induced hypertension in dogs are warranted. CLINICAL RELEVANCE: Clinicians should prepare for potential hypertensive crisis during RFA of adrenal masses, especially if treating a margin of normal tissue.

Immunomagnetic isolation of canine circulating endothelial and endothelial progenitor cells.

BACKGROUND: Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells. OBJECTIVES: The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood. METHODS: Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti-CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146-positive cells were stained with fluorescein-conjugated Ulex europaeus agglutinin 1 (UEA-1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA-anticoagulated whole blood samples from 10 healthy client-owned dogs. RESULTS: The anti-CD146-coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA-1-positive cells were obtained from whole blood, while >85-90% of cultured canine aortic endothelial cells were UEA-1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10-40 microm in size. Using immunomagnetic isolation, 43.4 +/- 15.6 CECs/mL (range 24-70/mL) were isolated from canine whole blood samples. CONCLUSIONS: Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.

What is your diagnosis? Intracranial mass in a cat.

A 14-year-old female spayed cat was presented with a 3-4-month history of circling to the left and intermittent head pressing. Neurologic examination findings localized the lesion to the left supratentorial region. Using magnetic resonance imaging, an extra-axial mass was found on the dorsal aspect of the brain at the level of the frontal and parietal lobes, compressing and displacing the brain ventrally and caudally. Craniectomy was performed and the mass was submitted for cytologic and histopathologic evaluation. Impression smears revealed abundant cholesterol crystals and loose clusters of mildly pleomorphic spindle cells, compatible with a meningioma. The histopathologic diagnosis was meningioma with psammoma bodies and numerous cholesterol clefts. Abundant cholesterol crystals within meningiomas in cats may present a diagnostic challenge when nucleated cells are scant. Other differential diagnoses for abundant cholesterol crystals in an intracranial mass include cholesterol granulomas and keratinizing cysts.

Campylobacter jejuni invade chicken LMH cells inefficiently and stimulate differential expression of the chicken CXCLi1 and CXCLi2 cytokines.

Campylobacter jejuni is a major food-borne bacterial pathogen, which is capable of causing diarrhoea containing blood and leukocytes. C. jejuni invasion of the intestinal epithelial cells and the release of proinflammatory molecules contribute to the pathophysiology of campylobacteriosis. Given the commensal relationship of C. jejuni with chickens, we hypothesized that C. jejuni invasion of chicken cells and the release of host cell cytokines would be significantly less than with human cells. To test our hypothesis, we examined the interactions of C. jejuni with chicken LMH cells, and performed in vivo experiments with chickens. The binding and internalization assays revealed that C. jejuni was significantly less invasive of LMH cells relative to human INT 407 cells, even though the bacteria bound to each host cell species equally. We also assessed interleukin-8 (IL-8) transcript, IL-8 secretion, and the release of chemoattractant molecules from the inoculated cells. Inoculation of LMH cells with C. jejuni stimulated expression of both chicken IL-8 orthologues, chCXCLi2 and chCXCLi1, but at levels significantly less than human IL-8 (huCXCL8) expressed from human INT 407 cells inoculated with C. jejuni. Moreover, the supernatant fluids of the C. jejuni-inoculated LMH cells resulted in little heterophil migration. In vivo, C. jejuni were observed bound to the cells lining the glandular crypts, but overt signs of cell invasion or pathology were not observed. These results indicate that cytokine expression in chicken LMH cells in response to C. jejuni is distinct from that of Salmonella typhimurium.