Nutritional profile of rodent diets impacts experimental reproducibility in microbiome preclinical research.

The lack of reproducibility of animal experimental results between laboratories, particularly in studies investigating the microbiota, has raised concern among the scientific community. Factors such as environment, stress and sex have been identified as contributors, whereas dietary composition has received less attention. This study firstly evaluated the use of commercially available rodent diets across research institutions, with 28 different diets reported by 45 survey respondents. Secondly, highly variable ingredient, FODMAP (Fermentable Oligo-, Di-, Mono-saccharides And Polyols) and gluten content was found between different commercially available rodent diets. Finally, 40 mice were randomized to four groups, each receiving a different commercially available rodent diet, and the dietary impact on cecal microbiota, short- and branched-chain fatty acid profiles was evaluated. The gut microbiota composition differed significantly between diets and sexes, with significantly different clusters in ß-diversity. Total BCFA were highest (p = 0.01) and SCFA were lowest (p = 0.03) in mice fed a diet lower in FODMAPs and gluten. These results suggest that nutritional composition of commercially available rodent diets impact gut microbiota profiles and fermentation patterns, with major implications for the reproducibility of results across laboratories. However, further studies are required to elucidate the specific dietary factors driving these changes.

Blood naltrexone levels over time following naltrexone implant.

AIMS: Oral naltrexone is used in the management of both heroin and alcohol dependence. However, poor compliance has limited its clinical utility. The study's objective was to determine the period of therapeutic coverage (>or=2 ng/ml) provided by a 3.3 g naltrexone subcutaneous implant compared with existing data on 1.1 g and 2.2 g implants. METHODS: We assessed free blood naltrexone levels following treatment with a 3.3 g naltrexone implant in heroin dependent patients (n=50) in Perth, Western Australia. Results were compared with previously collated data for patients treated with either a 1.1 g (n=10) or 2.2 g (n=24) implant. RESULTS: Following 3.3 g naltrexone implant treatment, free blood naltrexone levels remained above 2 ng/ml for 145 days (95% CI 125-167). In comparison, 1.1 g or 2.2 g implant treatment resulted in 95 days (95% CI 69-121) and 136 days (95% CI 114-158) coverage, respectively. CONCLUSIONS: The 3.3 g implant provides longer therapeutic coverage than the 1.1 g implant but not significantly longer than the 2.2 g implant.

Stress increases descending inhibition in mouse and human colon.

BACKGROUND: A relationship between stress and the symptoms of irritable bowel syndrome (IBS) has been well established but the cellular mechanisms are poorly understood. Therefore, we investigated effects of stress and stress hormones on colonic descending inhibition and transit in mouse models and human tissues. METHODS: Stress was applied using water avoidance stress (WAS) in the animal model or mimicked using stress hormones, adrenaline (5 nM), and corticosterone (1 µM). Intracellular recordings were obtained from colonic circular smooth muscle cells in isolated smooth muscle/myenteric plexus preparations and the inhibitory junction potential (IJP) was elicited by nerve stimulation or balloon distension oral to the site of recording. KEY RESULTS: Water avoidance stress increased the number of fecal pellets compared to control (p < 0.05). WAS also caused a significant increase in IJP amplitude following balloon distension. Stress hormones also increased the IJP amplitude following nerve stimulation and balloon distension (p < 0.05) in control mice but had no effect in colons from stressed mice. No differences were observed with application of ATP between stress and control tissues, suggesting the actions of stress hormones were presynaptic. Stress hormones had a large effect in the nerve stimulated IJP in human colon (increased >50%). Immunohistochemical studies identified alpha and beta adrenergic receptor immunoreactivity on myenteric neurons in human colon. CONCLUSIONS & INFERENCES: These studies suggest that WAS and stress hormones can signal via myenteric neurons to increase inhibitory neuromuscular transmission. This could lead to greater descending relaxation, decreased transit time, and subsequent diarrhea.

Remission of FeLV-associated lymphosarcoma and persistent viral infection after extracorporeal immunoadsorption of plasma using staphylococcal protein A columns: details of immune response.

Sixteen feline leukemia virus (FeLV)-infected cats with lymphosarcoma (LSA) were treated by extracorporeal immunoadsorption using staphylococcal protein A columns in order to remove immunoglobulin G (IgG) and circulating immune complexes (CIC) from plasma. Complete viral clearance and long-lasting tumor regression were achieved in nine of the cats and tumor regression without virus clearance was observed in two other cats. Since LSA cats rarely go into spontaneous remission, and since other forms of therapy are ineffective, these cats offered a unique system for analyzing details of the immune response to LSA and FeLV as they are cleared. Immunological parameters associated with the FeLV and LSA responses were assessed in detail in three responder cats and three nonresponders during the treatment and follow-up periods. Two serological parameters that always correlated with complete clearance of LSA were development of precipitating antibodies against FeLV-C gp70 and development of cytotoxic antibodies that kill cultured FL74 LSA cells in the presence of complement. The precipitating antibodies were detected prior to the clearance of LSA and prior to the detection of free cytotoxic antibodies. One serological parameter that always correlated with complete clearance of. FeLV was development of free antibodies to FeLV-AB gp70. Quantitative levels of FeLV-specific CIC and feline oncornavirus-associated cell membrane antigen (FOCMA)-specific CIC correlated well with fluctuating levels of the corresponding antigens and antibodies. These results suggest that the staphylococcal protein A treatment columns remove CIC "blocking factors" directly or indirectly and thereby stimulate existing antibody responses. These antibodies mediate clearance of FeLV and LSA.

Electrophoretic protein transfer applied to immunoelectrophoresis.

A method for electrophoretic transfer of a crossed immunoelectrophoretic (CIE) pattern from an agarose gel onto a nitrocellulose sheet is described. The CIE pattern to be transferred was formed using Nonidet P-40 solubilized, bovine herpes virus-1 (BHV-1) induced antigens and bovine hyperimmune serum. An autoradiogram of a CIE pattern, formed using [35S]methionine-labeled NP-40 solubilized, BHV-1 induced antigens and bovine hyperimmune serum, was used as a control pattern. Both antigen and antibody were transferred to the nitrocellulose sheet. This was demonstrated by the ability of transferred antigen to react with specific antibody detected by the addition of 125I-labeled anti-species antibody and transferred antibody to react with both [35S]methionine-labeled antigen and 125I-labeled protein A. This method has application for determining the specificity of monospecific sera for individual arcs in CIE patterns.

Converging and diverging cholinergic inputs from submucosal neurons amplify activity of secretomotor neurons in guinea-pig ileal submucosa.

The organization of synaptic connections between guinea-pig ileal submucosal neurons was examined using intracellular recordings from single or pairs of submucosal neurons. Synaptic inputs were elicited by stimulating cholinergic neurons using pressure-pulse application of 5-hydroxytryptamine (5-HT) in ganglia adjacent to those where intracellular recordings were obtained. In addition, when pairs of intracellular recordings were obtained, one neuron was activated by intracellular stimulation and synaptic responses were recorded in the other neuron. Neurobiotin-filled microelectrodes were employed to characterize cells electrophysiologically and immunohistochemically. Recordings were obtained from 176 (173 S-type and three AH-type) neurons; 81% of cells were classified as vasoactive intestinal peptide (VIP) neurons. No fast excitatory postsynaptic potentials and only rare slow excitatory postsynaptic potentials were recorded following intracellular stimulation of paired S-type neurons. However, when paired intracellular recordings were obtained from neurons within the same ganglion and 5-HT was applied to an adjacent ganglion, this stimulation evoked synchronized fast excitatory postsynaptic potentials in 94% of pairs. In contrast, when cell bodies of VIP-VIP pairs were located in different ganglia, fast synaptic activation evoked by 5-HT stimulation was not synchronized in 87% of pairs. When intracellular recordings were obtained from a single neuron and two separate ganglia were stimulated by 5-HT pressure-pulse activation, fast excitatory postsynaptic potentials originating from both sources were recorded in the same VIP neuron. Morphological study of 34 S-type and three AH-type horseradish peroxidase-labeled neurons was conducted. AH-type neurons had multiple axonal branches with dense arborization of collaterals containing numerous varicosities in three to nine ganglia, whereas axons of S-type neurons exhibited relatively rare collaterals and varicosities within adjacent ganglia. These results demonstrate that cholinergic neurons provide both diverging and converging inputs to VIP neurons, providing a mechanism to enhance activation of VIP secretomotor neurons. The axonal projections of AH-type neurons suggest they are likely candidates to provide diverging inputs to multiple VIP neurons.

Atrial natriuretic factor stimulates in-vivo and in-vitro secretion of cortisol in teleosts.

Atrial natriuretic factor (ANF) has been shown to increase circulating cortisol levels in cannulated, free-swimming seawater (SW)-adapted flounders. Increases were apparent within 30 min of i.v. injection of human ANF (hANF;10 micrograms/kg bw) and the increase in plasma cortisol was maintained throughout the 5h experimental period. No such increase was observed in vehicle-injected controls. This apparent steroidogenic effect of ANF was supported by an ANF-induced increase in in-vitro secretion of cortisol by interrenal tissue from SW-adapted trout. By contrast hANF had no significant effect on tissue derived from freshwater adapted trout. An ANF-induced increase in plasma cortisol by a direct effect on interrenal steroidogenesis in SW teleosts would be an appropriate response for survival in hypertonic media.

Urotensin I and its N-terminal flanking peptide from the flounder, Platichthys flesus.

The caudal spinal cord region of teleost fish terminates in a neurosecretory organ, the urophysis. Two peptides have been purified to homogeneity from an extract of the urophysis of a teleost fish, the flounder. The primary structure of one peptide, Ser-Glu-Asp-Pro-Pro-Met-Ser-Ile-Asp-Leu10-Thr-Phe-His-Met-Leu-Arg- Asn-Met-Ile- His20-Met-Ala-Lys-Met-Glu-Gly-Glu-Arg-Glu-Gln30-Ala-Gln-Ile- Asn-Arg-Asn-Leu-Leu - Asp-Glu40-Val, indicates identity with urotensin I. By analogy with other urotensins, the COOH-terminal residue is probably alpha-amidated. A second peptide was present in the extract in a concentration that was approximately equimolar with that of urotensin I. The amino acid composition of this peptide indicated a total of approximately 65 residues. The amino acid sequence of a fragment produced by digestion with trypsin was established as: Ala-Ala-Ala-Ala-Gly5-Asp-Ser-Ala-Ala-Ser10-Asp-Leu-Leu-Gly-Asp1 5-Asn-Ile-Leu- Arg. This sequence shows partial homology to carp prepro-urotensin I(41-59)-peptide as deduced from the nucleotide sequence of a cloned cDNA. It is concluded that the second peptide probably represents the N-terminal flanking peptide of pro-urotensin I which, it has previously been suggested, may function as a urotensin-binding peptide (urophysin) analogous to the neurophysins.

Identification of bovine herpesvirus-1 polypeptides involved in serum neutralization.

Bovine herpesvirus (infectious bovine rhinotracheitis virus)-infected cell antigens were solubilized with Nonidet P-40. The crude antigen extract was separated by reaction with bovine hyperimmune serum in line immunoelectrophoresis; individual immunoprecipitates were used to immunize rabbits. Rabbit sera possessing serum neutralizing activity were analyzed by reaction with crude antigen extract in immunoprecipitation sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Four virus-specified glycopeptides, with molecular weights of 69-75K, 77-81K, 82-92K and 108-115K, appeared to be involved in inducing serum neutralizing antibody.

Comparisons between two isolates of stomatitis papulosa virus.

The cytopathology and length of latency in single-step growth curves of two isolates of stomatitis papulosa virus are compared in this report. Isolate 721 was obtained from a calf with oral ulcers and isolate 8665 was obtained from a calf with respiratory disease and oral ulcers. In single-step growth curves, the latency period of isolate 721 was 8 h while that of isolate 8665 was 6 h. The cytopathic effect produced by isolate 721 in bovine lung cells was characterized by enlargement of the cell, cell-to-cell adherence and large intracytoplasmic accumulations of viral inclusion material. Isolate 8665 caused rapid cell degeneration and detachment, with small accumulation of viral inclusion material. Neither of the two strains grew in bovine alveolar macrophage cultures or in the respiratory epithelium of fetal bovine tracheal explants. Intragingival inoculation of these isolates in cattle resulted in oral lesions without clinical signs of respiratory of systemic involvement. Virus was recovered from the oral lesions and from nasal secretions for as long as 10 days. Inoculation of dexamethasone-treated cattle resulted in a similar clinical condition although virus was recovered for 20 days from oral lesions and nasal secretions. Seroconversions from negative to 1 : 2560 were detected in inoculated cattle by indirect immunofluorescence.

Identification of Mycoplasmatales in pneumonic calf lungs.

Lungs from 153 calves with clinical signs of pneumonia were examined post-mortem (PM) for the presence of mycoplasmas and ureaplasmas during a 38-month period. Sixty-two percent of the cases were submitted during the months when wide fluctuations in climatic conditions occur. Using indirect fluorescent antibody tests (IFAT) and culture, mycoplasmas and/or ureaplasmas were detected in 63% of the lungs examined. Mycoplasma dispar was detected in 39%, M. bovis in 36%, Ureaplasma spp. in 22% and M. bovirhinis in 8.5% of the lungs. Thirty percent of the lungs were infected with more than one species; the most frequent combination was M. bovis, M. dispar and Ureaplasma spp. (10.5%). M. arginini, M. bovigenitalium and acholeplasmas were not cultured. M. dispar was shown to remain viable for up to 15 days PM in apical and cardiac lobes held at 4 degrees C and also was detected by IFAT in the same tissues for 49 days.

The effect of some common inactivation procedures on the antigens of bovine herpesvirus 1.

Bovine embryonic kidney cells were infected with bovine herpesvirus 1 (BHV1) or were sham-inoculated. When cytopathic effect was apparent, the cells were treated with beta-propiolactone, formalin, heat (56 degrees C), or ultraviolet irradiation until the virus was inactivated. Infected-treated, infected-untreated (IU) and sham-inoculated cultures were solubilized using Triton X-100 detergent. Resulting preparations were tested by 2-dimensional- and fused rocket-immunoelectrophoresis and were evaluated for their ability to inhibit virus neutralization by BHV1 antiserum. Eleven viral antigens were detected consistently in IU preparations, which strongly inhibited virus neutralization. Eight or more IU antigens were detected in beta-propiolactone-treated, formalin-treated and heat-treated preparations; these inhibited virus neutralization less strongly than the IU preparations. No IU antigens were detected in ultraviolet-treated preparations, nor did this material inhibit virus neutralization. One of the IU antigens was reduced preferentially by all treatments. The selective destruction of antigens by the various treatments might allow antigen-specific serological testing to distinguish vaccinated from naturally-exposed cattle.

Cross-reactions of bovine herpesvirus 1 antigens with those of other cattle herpesviruses.

Bovine embryonic kidney cells were infected with bovine herpesviruses (BHV1, 2, or 3), suid herpesvirus 1 (SHV1), or were sham-inoculated. When cytopathic effect was apparent, the cells were solubilized using Triton X-100 detergent. Resulting antigen preparations were tested by 2-dimensional immunoelectrophoresis using bovine fetal serum and antisera directed against BHV1, BHV2, BHV3, SHV1 or a restricted spectrum of BHV1 antigens. Interaction of BHV1 antiserum with BHV1 antigen preparations resulted in 11 precipitation arcs. The same antiserum produced 3 arcs with BHV2, none with BHV3, and 5 with SHV1. The interaction of BHV1 antigen preparations with BHV2, BHV3, or SHV1 antisera failed to produce demonstrable arcs. However, when heterologous antigen or antibody preparations were added to BHV1 homologous 2-dimensional immunoelectrophoresis tests, all 11 BVH1 arcs were modified by BHV1, 2 by BHV2, 4 by BHV3 and 4 by SHV1 preparations. Two antigens were common to the 4 herpesviruses. Antigen preparations were tested for their ability to inhibit virus neutralization by BHV1 antiserum; only the BHV1 preparation was active. Sera were tested for BHV1 neutralizing activity; only BHV1 antiserum and a serum specific for a restricted spectrum of BHV1 antigens were active. A glycoprotein antigen associated with BHV1 neutralization was identified which may be important in the protection of animals against disease.

Experimental infection of rabbits with bovine herpesvirus-4: acute and persistent infection.

A strain of bovine herpesvirus-4 (BHV-4) isolated from bovine cases of mammary pustular dermatitis was used for experimental infection of rabbits. The strain is serologically indistinguishable from the group prototype Movar 33/63 and from the American isolate DN599. Groups of rabbits were inoculated by various routes. Intravaginal and conjunctival inoculations resulted in vulvovaginitis and conjunctivitis, respectively, and in shedding of virus. The rabbits seroconverted for the virus, with high titers of antibodies (indirect fluorescent antibody test) that persisted throughout the experiment. Treatment with dexamethasone, beyond the acute infection, did not produce recrudescence of disease or shedding of the virus. Rabbits were killed at various times, from 3 to 6 months post-infection, and the virus was recovered from explant cultures of spleen and by cocultivation of spleen cells with bovine lung cells. These results demonstrate the usefulness of the rabbit as a model for studying the pathogenesis of BHV-4 infection in cattle.

Studies with an unclassified virus isolated from diarrheic calves.

A transmissible agent (Breda agent) was isolated from a calf with diarrhea and shown to be infectious by inoculation orally into gnotobiotic and conventionally reared calves. The "Breda" agent had the morphology of a virus and possessed a hemagglutinin. Antigenic studies showed the virus to be antigenically different from bovine coronavirus, parainfluenza 3 virus, bovine rotavirus, bovine parvovirus and bovine pestivirus (BVD). Attempts to culture the virus in cell or organ cultures or in embryonated eggs, were unsuccessful. The virus was either spherical or kidney shaped, with 7-9 nm peplomers on the surface. A few particles possessed coronavirus processes of 17-20 nm, but these were arranged irregularly and were thought to be tissue debris. Three out of eight experimental calves developed severe diarrhea and the lesions in the small and large intestines were similar to those reported for coronavirus. The virus replicated in the jejunal and ileal regions of the small intestine and in the spiral colon, as judged by immunofluorescence. The virus multiplied in all experimental calves and was excreted in the feces; excretion correlating with the onset of diarrhea or a change in the appearance of the feces. There was little or no malabsorption measured by the uptake of D-xylose and the fact that infection of both the crypt and villus epithelial cells was observed, suggests that the pathogenesis may be different from rotavirus and coronavirus. Fourteen of forty seven calves in the outbreak were infected with the virus, virus was not identified in other farm outbreaks of the disease.

Experimental infection of eastern cottontail rabbits Sylvilagus floridanus) with infectious bovine rhinotracheitis virus.

Experimental infection of eastern cottontail rabbits (Sylvilagus floridanus) with infectious bovine rhinotracheitis virus caused acute keratoconjunctivitis and a fatal systemic infection. The clinical syndrome was characterized initially by blepharospasm and ocular discharge. The rabbits were markedly depressed on post-exposure day (PED) 5 and were dead or moribund on PED 6. The virus was readily recovered from liver and adrenal gland tissue on PED 6 and from conjunctival swabs on PED 1 to 6. Histopathologic studies revealed a few necrotic foci in the liver and multiple focal to diffuse necrosis of the adrenal glands. Viral isolation and immunofluorescence tests were used to demonstrate a direct association between infectious bovine rhinotracheitis viral antigens and the lesions.

Reaction of convalescent bovine antisera with strain-specific antigens of parapoxviruses.

Two strains of papular stomatitis (PS) virus, 1 of milker's nodules (MN) virus and 1 of contagious ecthyma (CE) virus possessed 2 distinct external structures when examined by electron microscopy. The innermost, designated coat was closely apposed to the tubular surface, whereas the outer envelope loosely surrounded the virion. When convalescent sera from cattle infected with PS virus were used for immunoelectron microscopy, antibody reacted with coats and envelopes of the PS virus strains, but only with coats of MN and CE viruses. Convalescent sera from cattle infected with PS or MN virus contained complement-dependent antibodies cytolytic to cells infected with the homologous virus. In an indirect immunofluorescence test, the sera reacted with homologous strains to higher titers than with heterologous strains.

Experimental inoculation of cattle with bovine herpesvirus-4: evidence for a lymphoid-associated persistent infection.

A strain of bovine herpesvirus-4 isolated from cows with mammary pustular dermatitis was used for experimental inoculation of cattle. This strain is serologically indistinguishable from the group prototype Movar 33/63 and from strain DN-599. Seronegative cattle were inoculated IV or by simultaneous intranasal, IV, intramammary (via teat channel), and intradermal inoculations. All inoculated cattle seroconverted. Clinical signs of disease or lesions were not evident, except for a dermal lesion corresponding with one intradermal inoculation site. Virus was recovered from the dermal lesion and was excreted in the milk for 17 days. Virus was recovered from esophagopharyngeal fluid at 9 and 13 days after inoculation. At different times of euthanasia (2 to 14 months after inoculation), virus was recovered from cocultures with bovine lung cells and/or explant cultures of lymph nodes, spleen, tonsils, and, in one case, kidney. In 2 animals, the virus was recovered repeatedly during 1 year from peripheral blood leukocytes by cocultivation with bovine lung cells. The number of infectious leukocytes, as determined by infectious center assay, ranged from less than 1 to 6 infectious cells/10(7) leukocytes.

Evaluation of experimental subunit vaccines for infectious bovine rhinotracheitis.

Subunit infectious bovine rhinotracheitis (IBR) antigens were prepared by solubilization of IBR-virus infected cell cultures with nonionic detergents Triton X-100 and NP-40. When used with Freund's incomplete adjuvant, Triton X-100 and NP-40 subunit antigens induced high serum-neutralization titers in vaccinated calves. Two doses of Triton X-100 and NP-40 subunit vaccines prevented the appearance of clinical signs of disease in all vaccinated calves exposed 30 days after the second vaccinal dose to intranasal challenge exposure with Cooper strain of IBR virus. Furthermore, two doses of NP-40 subunit vaccine prevented shedding of the virus in all vaccinated calves after challenge exposure.

Clearance and shedding of infectious bovine rhinotracheitis virus from the nasal mucosa of immune and nonimmune calves.

Infectious bovine rhinotracheitis virus was rapidly cleared from the nasal mucosa of calves after intranasal aerosol exposure. Nonimmune calves (experiment 1) cleared 10(9) plaque-forming units (PFU) of virus from the nasal mucosa in less than 4 hours and 10(6) PFU of virus in 1 hour. An eclipse phase followed the clearance of viral inoculum. Replicating virus was first detected at 9 hours. Viral titers increased stepwise until maximum was attained on postinoculation day 4. Virus persisted in the nasal mucus until day 12. Clinical signs of disease corresponded with the shedding of virus. In contrast to nonimmune calves, immune calves (experiment 2; same calves as in experiment 1, but 30 days after initial exposure) cleared 10(9) PFU of virus in 1 hour and 10(6) PFU of virus in less than 5 minutes. An abortive reinfection occurred after exposure of immune calves with 10(9) PFU of virus. Virus was first detected in these calves at 14 hours after exposure and was not detected beyond 24 hours after inoculation. Immune calves given 10(6) PFU of virus did not shed virus after clearance of inoculum. Clinical signs of infection were not observed in immune calves after viral challenge exposure. The date indicated that there was no detectable residual virus beyond 3 hours after the exposure.