Detection of Bartonella spp. in dogs after infection with Rickettsia rickettsii.

BACKGROUND: Dynamics of infection by Bartonella and Rickettsia species, which are epidemiologically associated in dogs, have not been explored in a controlled setting. OBJECTIVES: Describe an outbreak investigation of occult Bartonella spp. infection among a group of dogs, discovered after experimentally induced Rickettsia rickettsii (Rr) infection. ANIMALS: Six apparently healthy purpose-bred Beagles obtained from a commercial vendor. METHODS: Retrospective and prospective study. Dogs were serially tested for Bartonella spp. and Rr using serology, culture, and PCR, over 3 study phases: 3 months before inoculation with Rr (retrospective), 6 weeks after inoculation with Rr (retrospective), and 8 months of follow-up (prospective). RESULTS: Before Rr infection, 1 dog was Bartonella henselae (Bh) immunofluorescent antibody assay (IFA) seroreactive and 1 was Rickettsia spp. IFA seroreactive. After inoculation with Rr, all dogs developed mild Rocky Mountain spotted fever compatible with low-dose Rr infection, seroconverted to Rickettsia spp. within 4-11 days, and recovered within 1 week. When 1 dog developed ear tip vasculitis with intra-lesional Bh, an investigation of Bartonella spp. infection was undertaken. All dogs had seroconverted to 1-3 Bartonella spp. between 7 and 18 days after Rr inoculation. Between 4 and 8 months after Rr inoculation, Bh DNA was amplified from multiple tissues from 2 dogs, and Bartonella vinsonii subsp. berkhoffii (Bvb) DNA was amplified from 4 of 5 dogs' oral swabs. CONCLUSIONS AND CLINICAL IMPORTANCE: Vector-borne disease exposure was demonstrated in research dogs from a commercial vendor. Despite limitations, our results support the possibilities of recrudescence of chronic subclinical Bartonella spp. infection after Rr infection and horizontal direct-contact transmission between dogs.

Bartonella henselae in a dog with ear tip vasculitis.

BACKGROUND: Bartonella henselae, a Gram-negative, zoonotic, alpha-proteobacteria has been previously implicated in association with cutaneous vasoproliferative lesions (bacillary angiomatosis), nodular panniculitis and multifocal erythema (erythema multiforme) in dogs. OBJECTIVE: Describe clinical, microbiological and histological lesions in a dog with ear margin vasculitis and B. henselae infection. ANIMALS: A 12-month-old, specific pathogen-free intact female beagle dog maintained in a vector-free laboratory animal resource facility. METHODS AND MATERIALS: Bartonella and Rickettsia serological evaluation, Bartonella and Rickettsia PCR, Bartonella alpha-proteobacteria growth medium (BAPGM) enrichment blood culture/PCR, histopathological investigation and confocal immunohistochemical evaluation. RESULTS: Serological investigation (seroreversion) and PCR testing of aural tissue biopsies failed to support Rickettsia rickettsii as a cause of the aural vasculitis; however, B. henselae, genotype San Antonio 2 DNA was amplified and sequenced from both ear tip margins and from normal-appearing abdominal skin. Seroconversion to B. henselae was documented retrospectively by IFA testing. Bartonella henselae organisms were visualized by confocal immunostaining within all three biopsies. Histopathology revealed small vessel necrotizing vasculitis and dermal necrosis. Bartonella henselae seroreversion and complete resolution of skin lesions occurred in conjunction with administration of oral doxycycline and enrofloxacin for six weeks. CONCLUSIONS AND CLINICAL IMPORTANCE: Bartonella henselae is an emerging zoonotic pathogen that has been associated with leucocytoclastic vasculitis in humans and may have had a contributing or causative role in the development of the cutaneous aural margin vasculitis in this beagle.

Oral Transmucosal Detomidine Gel in New Zealand White Rabbits (Oryctolagus cuniculus).

Handling and restraining rabbits for routine procedures may be impossible without prior sedation, result in unnecessary stress or injury to the rabbit or handler, and increase experimental variability. Parenteral administration of sedatives can cause stress also, as well as localized pain and tissue damage, especially in fractious animals. Detomidine hydrochloride, an α2-adrenergic receptor agonist, is commercially available in an oral transmucosal (OTM) gel formulation that is FDA-approved for sedation and restraint in horses. This study investigated the efficacy and safety of detomidine gel as an alternative to injectable sedation in rabbits. Eight adult male New Zealand White rabbits each received 0.6, 1.2, or 1.8 mg/kg OTM detomidine gel. Physiologic parameters and sedation scores (SS) were assessed at 10-min intervals from before administration until 100 min afterward. Histopathology of cardiac tissue was scored through 12 d after dosing. Gel administration increased the SS in all rabbits, but none of the animals developed clinically effective sedation (SS of 10 or greater, based on 5 reflex responses on a 3- or 4-point scale). The SS did not differ among dosage groups, and the time-dose interaction was not statistically significant. Heart rate decreased rapidly in all rabbits, with no difference among dosage groups, and there was no effect of time or dosage on peripheral capillary oxygen saturation. Minimal to mild degenerative changes were seen in the myocardium of all treated rabbits, but myocyte necrosis, inflammation, fibrosis, and mural thrombi-reported previously in rabbits that had received parenteral detomidine-did not occur. OTM detomidine gel was safely and easily administered to rabbits, but the duration and level of sedation were unpredictable. The use of OTM detomidine as a sole agent to facilitate handling and restraint of rabbits does not offer advantages over existing parenteral regimens.

Co-infection of the Siberian hamster (Phodopus sungorus) with a novel Helicobacter sp. and Campylobacter sp.

We report the isolation of a novel helicobacter isolated from the caecum of the Siberian hamster (Phodopus sungorus). Sequence analysis showed 97% sequence similarity to Helicobacter ganmani. In addition, we report the co-infection of these Siberian hamsters with a Campylobacter sp. and a second Helicobacter sp. with 99% sequence similarity to Helicobacter sp. flexispira taxon 8 (Helicobacter bilis), a species isolated previously from patients with bacteraemia. Gross necropsy and histopathology did not reveal any overt pathological lesions of the liver and gastrointestinal tract that could be attributed to the Helicobacter or Campylobacter spp. infections. This is the first helicobacter to be identified in the Siberian hamster and the first report of co-infection of Helicobacter spp. and Campylobacter sp. in asymptomatic Siberian hamsters.

Abdominal lipomatosis with secondary self-strangulation of masses in an adult rhesus macaque (Macaca mulatta).

An 10-y-old, intact male rhesus macaque (Macaca mulatta) presented for bilateral scrotal swelling and a distended abdomen. A soft mass in the left upper quadrant of the abdomen was palpated. A barium study did not reveal any gastrointestinal abnormalities. Exploratory laparotomy revealed a large (1.25 kg, 15.0 × 13.0 × 9.5 cm), red and tan, soft, circumscribed, spherical mass within the greater omentum and 10 to 20 smaller (diameter, 1 to 4 cm), soft to firm masses in the mesentery and greater omentum. The resected mass was a self-strangulating abdominal lipoma, a pedunculated neoplasm composed of white adipocytes arising from peritoneal adipose tissue undergoing secondary coagulation necrosis after strangulation of the blood supply due to twisting of the mass around the peduncle. The smaller masses were histologically consistent with simple or self-strangulating pedunculated abdominal lipomas. The macaque presented again 9 mo later with a firm, 5.0-cm mass in the midabdomen, with intestinal displacement visible on radiographs. Given this animal's medical history and questionable prognosis, euthanasia was elected. Necropsy revealed numerous, multifocal to coalescing, 1.0- to 15.0-cm, pale tan to yellow, circumscribed, soft to firm, spherical to ellipsoid, pedunculated masses that were scattered throughout the mesentery, greater omentum, lesser omentum, and serosal surfaces of the gastrointestinal tract. All of the masses were pedunculated abdominal lipomas, and most demonstrated coagulation necrosis due to self-strangulation of the blood supply. To our knowledge, this report is the first to describe abdominal lipomatosis with secondary self-strangulation of masses in a rhesus macaque.

Antinociceptive effects of sustained-release buprenorphine in a model of incisional pain in rats (Rattus norvegicus).

Effective management of postoperative pain is an essential component of the care and welfare of laboratory animals. A sustained-release formulation of buprenorphine (Bup-SR) has recently been introduced to the veterinary market and has been reported to provide analgesia for as long as 72 h. Using evoked mechanical and thermal hypersensitivity tests, we here evaluated the antinociceptive effects of Bup-SR in a model of incisional pain in rats. Paw withdrawal responses were obtained before and 1 through 4 d after surgery. Rats are assigned to receive Bup-SR (0.3, 1.2, or 4.5 mg/kg SC once) or buprenorphine HCl (Bup HCl, 0.05 mg/kg SC twice daily for 3 d). Responses to mechanical and thermal stimuli in the 1.2 and 4.5 Bup-SR groups did not differ from those of rats in the Bup HCl group. Thermal latency on day 3 in rats that received 0.3 mg/kg Bup-SR was significantly different from baseline, indicating that this dose effectively decreased thermal hypersensitivity for at least 48 h. Marked sedation occurred in rats in the 4.5 Bup-SR group. Our findings indicate that Bup-SR at 0.3 or 1.2 mg/kg SC is effective in minimizing hypersensitivity with minimal sedation for at least 48 h (thermal hypersensitivity) and 72 h, respectively, in the incisional pain model in rats.

Tissue distribution of enrofloxacin in African clawed frogs (Xenopus laevis) after intramuscular and subcutaneous administration.

As part of an enrofloxacin pharmacokinetic study, concentrations of enrofloxacin and ciprofloxacin (metabolite) were measured in various tissues (brain, heart, kidney, liver, lung, and spleen) collected from treated (subcutaneous delivery, n = 3; intramuscular delivery, n = 3; untreated controls, n = 2) adult female Xenopus laevis by using HPLC. Enrofloxacin was rapidly absorbed after administration by either route and readily diffused into all sampled tissues. Enrofloxacin and ciprofloxacin were present in the tissue samples collected at 8 h. The highest average tissue concentrations for enrofloxacin were found in kidney, with the lowest concentrations in liver. Ciprofloxacin tissue concentrations paralleled but were always lower than those of enrofloxacin for all time points and tissues except brain and kidney. These results, together with previously published pharmacokinetic data and known minimal inhibitory concentrations of common pathogenic bacteria, provide a strong evidence-based rationale for choosing enrofloxacin to treat infectious diseases in X. laevis.

Carbon dioxide and oxygen levels in disposable individually ventilated cages after removal from mechanical ventilation.

Disposable individually ventilated cages have lids that restrict air exchange when the cage is not mechanically ventilated. This design feature may cause intracage CO2 to increase and O2 to decrease (hypercapnic and hypoxic conditions, respectively) when the electrical supply to the ventilated rack fails, the ventilated rack malfunctions, cages are docked in the rack incorrectly, or cages are removed from the ventilated rack for extended periods of time. We investigated how quickly hypercapnic and hypoxic conditions developed within disposable individually ventilated cages after removal from mechanical ventilation and compared the data with nondisposable static cages, disposable static cages, and unventilated nondisposable individually ventilated cages. When disposable individually ventilated cages with 5 adult mice per cage were removed from mechanical ventilation, CO2 concentrations increased from less than 1% at 0 h to approximately 5% at 3 h and O2 levels dropped from more than 20% at 0 h to 11.7% at 6 h. The breathing pattern of the mice showed a prominent abdominal component (hyperventilation). Changes were similar for 4 adult mice per cage, reaching at least 5% CO2 at 4 h and 13.0% O2 at 6 h. For 3 or 2 mice per cage, values were 4.6% CO2 and 14.7% O2 and 3.04% CO2 and 17.1% O2, respectively, at 6 h. These results document that within disposable individually ventilated cages, a hypercapnic and hypoxic microenvironment develops within hours in the absence of mechanical ventilation.

Hematologic, serologic, and histologic profile of aged Siberian hamsters (Phodopus sungorus).

Biologic samples from 18 (12 female, 6 male) Siberian hamsters (Phodopus sungorus) representing an aged colony (17 to 27 mo) were examined. Values for CBC and serum biochemical parameters were determined, and macroscopic and microscopic pathologic evaluations were performed. Blood urea nitrogen levels were significantly higher in male (54.2 ± 14 mg/dL) compared with female (35.3 ± 22 mg/dL) hamsters and correlated histologically with a higher incidence of chronic glomerulonephropathy in males (5 of 6 males; 0 of 12 females). All 18 hamsters had histologic evidence of follicular mite infestation. Half (6 of 12) of the female hamsters showed cystic rete ovarii. Other histologic findings included thymic or thyroid branchial cysts (3 of 18), focal enteritis (2 of 18), and single cases of hepatic hemangiosarcoma, renal adenoma, subcutaneous mast cell tumor, cutaneous sebaceous adenoma, cutaneous trichofolliculoma, squamous papilloma of the nonglandular stomach, epididymal cholesteatoma, pyometra, and pituitary craniopharyngeal cyst. This study is the first published report of hematologic and serum chemical values for any population of Siberian hamsters and the first published report showing a potential male predisposition for chronic progressive glomerulonephropathy and a potential female predisposition for cystic rete ovarii.

Analgesic effects of tramadol, tramadol-gabapentin, and buprenorphine in an incisional model of pain in rats (Rattus norvegicus).

Postoperative pain management in laboratory animals relies heavily on a limited number of drug classes, such as opioids and nonsteroidal antiinflammatory drugs. Here we evaluated the effects of saline, tramadol, tramadol with gabapentin, and buprenorphine (n = 6 per group) in a rat model of incisional pain by examining thermal hyperalgesia and weight-bearing daily for 6 d after surgery. All drugs were administered preemptively and continued for 2 consecutive days after surgery. Rats treated with saline or with tramadol only showed thermal hyperalgesia on days 1 through 4 and 1 through 3 after surgery, respectively. In contrast, buprenorphine-treated rats showed no thermal hyperalgesia on days 1 and 2 after surgery, and rats given tramadol with gabapentin showed reduced thermal hyperalgesia on days 2 and 4. For tests of weight-bearing, rats treated with saline or with tramadol only showed significantly less ipsilateral weight-bearing on day 1 after surgery, whereas rats given either buprenorphine or tramadol with gabapentin showed no significant change in ipsilateral weight-bearing after surgery. These data suggest that tramadol alone provides insufficient analgesia in this model of incisional pain; buprenorphine and, to a lesser extent, tramadol with gabapentin provide relief of thermal hyperalgesia and normalize weight-bearing.

Uterine leiomyoma in a Guyanese squirrel monkey (Saimiri sciureus sciureus).

An adult female squirrel monkey (Saimiri sciureus) presented with a 3.0 x 2.5 cm firm mass palpable within the caudal abdomen. Differentiation of the organs or structures involved with the mass could not be achieved with radiography or ultrasonography. Exploratory laparotomy revealed a mass within the lumen of the uterus; the mass was removed by partial hysterectomy. On gross examination, the mass was a focally extensive, unencapsulated, firm, solitary tumor. Histologic examination revealed that the mass was composed of interlacing bundles of smooth muscle cells with little fibrous stroma. The cells were elongated with poorly delineated borders and cigar-shaped nuclei, each containing a single, small nucleolus. Fewer than 1 mitosis per 20 high-power (magnification, x 400) fields were present. These gross and histologic findings supported a diagnosis of uterine leiomyoma. Although leiomyomas are the most common tumor of the reproductive tract in nonhuman primates, to our knowledge the current lesion is the first uterine leiomyoma reported to occur in a squirrel monkey.

Comparison of Gastrografin to barium sulfate as a gastrointestinal contrast agent in red-eared slider turtles (Trachemys scripta elegans).

Red-eared slider turtles (Trachemys scripta elegans) commonly develop intestinal obstruction. The gastrointestinal transit time in turtles tends to be longer than in other animals, making a rapid diagnosis of obstruction difficult. Fifteen red-eared sliders were given either Gastrografin or 30% w/v barium sulfate orally to compare ease of administration, transit time, and image quality. Each contrast medium was easy to administer but barium sulfate had to be administered more slowly (mean = 40s) than Gastrografin (mean = 20s) to prevent regurgitation. The mean transit and emptying time of Gastrografin was at least 9 h faster than barium sulfate at all time points except gastric transit. Both contrast media had a smooth, uniform appearance that outlined the mucosa with well-defined margins within the stomach and proximal small intestine. Dilution of Gastrografin occurred as it progressed through the intestines, resulting in decreased opacity in the distal small intestine and colon. Pre-administration packed cell volume and total serum protein levels of four turtles receiving Gastrografin were compared with levels at 24-, 96-, and 168-hours postadministration as well as to four control turtles not receiving contrast medium. Packed cell volume and total serum protein levels did not significantly differ among the Gastrografin and control group. From a clinical perspective, administration of Gastrografin allows for quicker results with only minor hematologic changes in red-eared sliders, but visualization of this contrast medium in the lower gastrointestinal tract may be insufficient for an accurate diagnosis.

The pharmacokinetics of enrofloxacin in adult African clawed frogs (Xenopus laevis).

Pharmacokinetics of enrofloxacin, a fluoroquinolone antibiotic, was determined in adult female Xenopus laevis after single-dose administration (10 mg/kg) by intramuscular or subcutaneous injection. Frogs were evaluated at various time points until 8 h after injection. Plasma was analyzed for antibiotic concentration levels by HPLC. We computed pharmacokinetic parameters by using noncompartmental analysis of the pooled concentrations (naive pooled samples). After intramuscular administration of enrofloxacin, the half-life was 5.32 h, concentration maximum was 10.85 µg/mL, distribution volume was 841.96 mL/kg, and area under the time-concentration curve was 57.59 µg×h/mL; after subcutaneous administration these parameters were 4.08 h, 9.76 µg/mL, 915.85 mL/kg, and 47.42 µg×h/mL, respectively. According to plasma pharmacokinetics, Xenopus seem to metabolize enrofloxacin in a manner similar to mammals: low levels of the enrofloxacin metabolite, ciprofloxacin, were detected in the frogs' habitat water and plasma. At necropsy, there were no gross or histologic signs of toxicity after single-dose administration; toxicity was not evaluated for repeated dosing. The plasma concentrations reached levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a reasonable regimen to consider when treating sick frogs. The treatment of sick frogs should be based on specific microbiologic identification of the pathogen and on antibiotic susceptibility testing.

Lipoic acid downmodulates CD4 from human T lymphocytes by dissociation of p56(Lck).

Lipoic acid is an antioxidant that suppresses and treats a model of multiple sclerosis, experimental autoimmune encephalomyelitis. We now demonstrate that treatment of human PBMC and T cell lines with LA downmodulated CD4 expression in a concentration-dependent manner. LA treatment of Con A stimulated PBMC specifically removed CD4 from the T-cell surface, but not CD3. Epitope masking by LA was excluded by using monoclonal antibodies targeting different domains of CD4. Incubation on ice inhibited CD4 removal following LA treatment, suggesting that endocytosis was involved in its downmodulation. LA is in a unique category of compounds that induce CD4 downmodulation by various mechanisms (e.g., gangliosides). We hypothesized that LA might induce dissociation of p56(Lck) from CD4, thus leading to its downmodulation. Immunoblot analyses demonstrated reduced co-precipitation of p56(Lck) from Jurkat T-cells following LA treatment and precipitation of CD4. This unique immunomodulatory effect of LA warrants further investigation.

Alpha lipoic acid inhibits human T-cell migration: implications for multiple sclerosis.

We have demonstrated previously the ability of the antioxidant alpha lipoic acid (ALA) to suppress and treat a model of multiple sclerosis (MS), relapsing experimental autoimmune encephalomyelitis (EAE). We describe the effects of ALA and its reduced form, dihydrolipoic acid (DHLA), on the transmigration of human Jurkat T cells across a fibronectin barrier in a transwell system. ALA and DHLA inhibited migration of Jurkat cells in a dose-dependent fashion by 16-75%. ALA and DHLA reduced matrix metalloproteinase-9 (MMP-9) activity by 18-90% in Jurkat cell supernatants. GM6001, a synthetic inhibitor of MMP, reduced Jurkat cell migration, but not as effectively as ALA and DHLA did. Both ALA and DHLA downmodulated the surface expression of the alpha4beta1 integrin (very late activation-4 antigen; VLA-4), which binds fibronectin and its endothelial cell ligand vascular cell adhesion molecule-1 (VCAM-1). Moreover, ALA, but not DHLA, reduced MMP-9-specific mRNA and extracellular MMP-9 from Jurkat cells and their culture supernatants as detected by relative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. ALA and DHLA inhibited Jurkat cell migration and have different mechanisms for inhibiting MMP-9 activity. These data, coupled with its ability to treat relapsing EAE, suggest that ALA warrants investigation as a therapy for MS.

FK506 and a nonimmunosuppressant derivative reduce axonal and myelin damage in experimental autoimmune encephalomyelitis: neuroimmunophilin ligand-mediated neuroprotection in a model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) in which demyelination and axonal loss result in permanent neurologic disability. We examined the neuroprotective property of the immunosuppressant FK506 (tacrolimus), FK1706 (a nonimmunosuppressant FK506 derivative) and cyclosporin A (CsA) in a chronic relapsing experimental autoimmune encephalomyelitis (EAE) model of MS. Female SJL/J mice were immunized by subcutaneous (s.c.) injection with proteolipid protein 139-151 peptide in complete Freund's adjuvant. At the onset of paralysis, 12-14 days after immunization, mice received daily s.c. injections of FK506 (0.2, 1, and 5 mg/kg), FK1706 (5 mg/kg), CsA (2, 10, and 50 mg/kg), saline or vehicle (30% dimethylsulfoxide) for 30 days. FK506 (at a dose of 5 mg/kg) reduced the severity of the initial disease and suppressed relapses. FK1706 did not significantly alter the clinical course and CsA (at a dose of 50 mg/kg) lessened the severity of the initial episode of EAE but did not alter relapses. In the thoracic spinal cord, FK506 (5 mg/kg), FK1706 (5 mg/kg), and CsA (50 mg/kg) significantly (P < 0.001) reduced the extent of damage in the dorsal, lateral, and ventral white matter by a mean of up to 95, 68, and 30%, respectively. A nonimmunosuppressant dose of FK506 (0.2 mg/kg) also significantly (P < 0.001) reduced the extent of damage in the spinal cord by a mean of up to 45%. Other dosages of these compounds were ineffective. FK506 markedly protects against demyelination and axonal loss in this MS model through immunosuppression and neuroprotection.

Alpha lipoic acid inhibits T cell migration into the spinal cord and suppresses and treats experimental autoimmune encephalomyelitis.

Oxidative injury may be important to the pathogenesis of multiple sclerosis (MS). We tested the antioxidant alpha lipoic acid (ALA) in an experimental murine model of MS, experimental autoimmune encephalomyelitis (EAE). ALA was administered to SJL mice 7 days after immunization with proteolipid protein (PLP) 139-151 peptide. Mice that received 5-100 mg/kg/day of ALA had dose-dependent reductions in their 10-Day Cumulative Disease Scores (10-Day CDS) by 23-100%. Minimal inflammation, demyelination and axonal loss occurred in the spinal cords (SC) of ALA-suppressed mice, and there was a marked reduction in CD3+ T cells and CD11b+ monocyte/macrophage cells within the SC. Mice treated with ALA (100 mg/kg/day) commencing on the first day of clinical EAE had a significant reduction in 10-Day CDS. SC of ALA-treated mice had reduced demyelination and axonal loss and a rapid reduction in CD3+ T cells. In vitro, ALA and its reduced form, dihydrolipoic acid, inhibited the activity of matrix metalloproteinase-9 (MMP-9) in a dose-dependent fashion. ALA is highly effective at suppressing and treating EAE and does so by inhibiting T cell trafficking into the SC, perhaps by acting as a matrix metalloproteinase inhibitor.