Housing and care of laboratory cats: from requirements to practice.

Increased public awareness of the welfare and well-being of laboratory animals in biomedical research and related ethical considerations inspired us to review recent developments and recommendations for the care and housing of laboratory cats. The present review focuses on the practical requirements for maintaining domestic cats as laboratory animals - from the construction of animal shelters to the termination of an experiment. An excellent standard of housing and care will reduce the bias of experimental results due to stress. To provide cats with living conditions that best meet their natural physical requirements and permit natural social behaviour, laboratories should spare no effort to achieve high housing standards. Hence, the present report not only aims to be a practical reference for those who are involved in the care and husbandry of cats, but it also aims to motivate researchers to improve their knowledge in this field and to provide humane conditions for all cats kept for scientific purposes.

Feline leukemia virus outbreak in the critically endangered Iberian lynx (Lynx pardinus): high-throughput sequencing of envelope variable region A and experimental transmission.

The Iberian lynx is the most endangered felid species. During winter/spring 2006/7, a feline leukemia virus (FeLV) outbreak of unexpected virulence killed about 2/3 of the infected Iberian lynxes. All FeLV-positive animals were co-infected with feline hemoplasmas. To further characterize the Iberian lynx FeLV strain and evaluate its potential virulence, the FeLV envelope gene variable region A (VRA) mutant spectrum was analyzed using the Roche 454 sequencing technology, and an in vivo transmission study of lynx blood to specified-pathogen-free cats was performed. VRA mutations indicated weak apolipoprotein B mRNA editing enzyme and catalytic polypeptide-like cytidine deaminase (APOBEC) restriction of FeLV replication, and variants characteristic of aggressive FeLV strains, such as FeLV-C or FeLV-A/61C, were not detected. Cats exposed to FeLV/Candidatus Mycoplasma haemominutum-positive lynx blood did not show a particularly severe outcome of infection. The results underscore the special susceptibility of Iberian lynxes to infectious diseases.

Liquid culture medium for the rapid cultivation of Helicobacter pylori from biopsy specimens.

The goal of this study was to develop a liquid culture medium for the rapid isolation, cultivation, identification and subsequent antibiotics susceptibility testing of Helicobacter pylori directly from biopsy specimens. Five liquid media were tested: Ham's F-12, Brucella broth, tryptic soybroth, brain heart infusion broth and Mueller-Hinton broth. After optimisation of the medium, it was applied in order to investigate biopsy samples from 150 patients with gastro-duodenal disorders and compared with traditional culture methods, microscopy and an H. pylori-specific TaqMan real-time polymerase chain reaction (PCR). The most reliable and rapid growth of H. pylori, even at a small inoculum size, was obtained in Ham's F-12 medium with 5% horse serum. The developed system allowed the primary isolation of H. pylori in clinical samples and provided 87% sensitivity and 100% specificity.

[Feline hemoplasmas in Switzerland: identification of a novel species, diagnosis, prevalence, and clinical importance]. / Feline Haemoplasmen in der Schweiz: Identifikation einer neuen Spezies, Diagnose, Prävalenz und klinische Bedeutung.

Two feline hemotropic mycoplasma spp. (aka hemoplasma) have previously been recognized. We recently discovered a third novel species in a cat with hemolytic anemia, designated 'Candidatus Mycoplasma turicensis', which is closely related to rodent haemoplasmas. This novel species induced anemia after experimental transmission to two SPF cats. Three quantitative real-time PCR assays were newly designed and applied to an epidemiological study surveying the Swiss pet cat population. Blood samples from 713 healthy and ill cats were analyzed. Up to 104 parameters per cat (detailed questionnaire, case history, laboratory parameters and retroviral infections) were evaluated. 'Candidatus Mycoplasma haemominutum' infection was more prevalent (8.5%) than Mycoplasma haemofelis (0.5%) and 'Candidatus Mycoplasma turicensis' (1%). Hemoplasma infections were associated with male gender, outdoor access, and old age, but not with disease or anemia. Infections were more frequently found in the South and West of Switzerland. Several hemoplasma infected cats, some acutely infected, others co-infected with FIV or FeLV, showed hemolytic anemia indicating that additional factors might play a role in the pathogenesis of the disease.

Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin.

Microcystins are toxins produced by freshwater cyanobacteria. They are cyclic heptapeptides that exhibit hepato- and neurotoxicity. However, the transport systems that mediate uptake of microcystins into hepatocytes and across the blood-brain barrier have not yet been identified. Using the Xenopus laevis oocyte expression system we tested whether members of the organic anion transporting polypeptide superfamily (rodent: Oatps; human: OATPs) are involved in transport of the most common microcystin variant microcystin-LR by measuring uptake of a radiolabeled derivative dihydromicrocystin-LR. Among the tested Oatps/OATPs, rat Oatp1b2, human OATP1B1, human OATP1B3, and human OATP1A2 transported microcystin-LR 2- to 5-fold above water-injected control oocytes. This microcystin-LR transport was inhibited by co-incubation with the known Oatp/OATP substrates taurocholate (TC) and bromosulfophthalein (BSP). Microcystin-LR transport mediated by the human OATPs was further characterized and showed saturability with increasing microcystin-LR concentrations. The apparent K(m) values amounted to 7 +/- 3 microM for OATP1B1, 9 +/- 3 microM for OATP1B3, and 20 +/- 8 microM for OATP1A2. No microcystin-LR transport was observed in oocytes expressing Oatp1a1, Oatp1a4, and OATP2B1. These results may explain some of the observed organ-specific toxicity of microcystin-LR. Oatp1b2, OATP1B1, and OATP1B3 are responsible for microcystin transport into hepatocytes, whereas OATP1A2 mediates microcystin-LR transport across the blood-brain barrier.

Localization of organic anion transporting polypeptide 4 (Oatp4) in rat liver and comparison of its substrate specificity with Oatp1, Oatp2 and Oatp3.

Organic anion transporting polypeptides (rodents: Oatps; human: OATPs) are involved in the absorption and elimination of a wide variety of structurally unrelated amphipathic organic compounds. Several members of this protein family mediate the uptake of substrates across the basolateral membrane of hepatocytes as the first step in hepatic elimination. In contrast to the well-characterized Oatp1 and Oatp2, the localization and substrate specificity of the recently cloned Oatp4 have not been investigated in detail. Therefore, we raised an antibody against the C-terminal end of Oatp4 and localized this 85-kDa protein to the basolateral membrane of rat hepatocytes. Similar to Oatp1 and Oatp2, Oatp4 is a multispecific transporter with high affinities for bromosulfophthalein, dehydroepiandrosterone sulfate, leukotriene C4, and anionic peptides. In addition, we compared the substrate specificity of Oatp4 to that of Oatp3, which so far has mainly been shown to mediate intestinal bile acid transport. Oatp3 had a similar broad substrate specificity, but in general much lower affinities than Oatp4. Thus, while Oatp4 seems to work in concert with Oatp1 and Oatp2 in the basolateral membrane of rat hepatocytes, Oatp3 is a multispecific transport system in the small intestine.

Hepatic uptake of cholecystokinin octapeptide by organic anion-transporting polypeptides OATP4 and OATP8 of rat and human liver.

BACKGROUND & AIMS: Cholecystokinin (CCK) is a major gastrointestinal peptide hormone that is released postprandially from the small intestine and exerts marked effects on gallbladder and gastrointestinal motility. The smaller isoforms CCK-8 and CCK-4 are rapidly taken up into hepatocytes, metabolized, and excreted into bile. Our aim was to identify and characterize the hepatocellular CCK-8 uptake system. METHODS: CCK-8 uptake was measured in Xenopus laevis oocytes expressing the organic anion-transporting polypeptides of rat liver (Oatp1, Oatp2, Oatp3, or Oatp4) and of human liver (OATP-A, OATP-B, OATP-C, or OATP8) and in primary cultured rat hepatocytes. RESULTS: Rat Oatp4 and human OATP8 efficiently mediated CCK-8 uptake in oocytes, with Michaelis constant (Km) values of 14.9 +/- 2.9 micromol/L and 11.1 +/- 2.9 micromol/L, respectively. CCK-8 uptake by hepatocytes was also saturable, with a Km of 6.7 +/- 2.1 micromol/L. The Km value in rat hepatocytes is consistent with Oatp4-mediated transport. CONCLUSIONS: CCK-8 is selectively transported by rat Oatp4 and human OATP8, both of which are exclusively expressed at the basolateral membrane of hepatocytes. These 2 transporters are the first and probably the predominant hepatic uptake systems for CCK-8 and may be critical for the rapid clearance of this hormone from the circulation.

Effect of phenobarbital on the expression of bile salt and organic anion transporters of rat liver.

BACKGROUND/AIMS: The hepatic clearance of drugs and cholephilic organic anions is stimulated by phenobarbital (PB). Our aim was to analyze the effects of PB on the expression of hepatocellular bile salt and organic anion transporters. METHODS: Male Sprague-Dawley rats were treated intraperitoneally with PB (80 mg/kg/d) or saline for 5 days. Transporter expression was quantified by northern and western blot analysis and initial uptake rates of bromosulphophthalein (BSP) and digoxin were measured in isolated hepatocytes. RESULTS: Compared to control rats, PB treatment increased expression of the organic anion transporting polypeptide 2 (Oatp2; Slc21aS) more than 2-fold on the RNA (P < 0.05) and protein (P < 0.001) levels. Expression of Oatpl (Slc21al), Oatp4 (Slc21a6) and the Na+-taurocholate cotransporting polypeptide (Ntcp; Slc10a1) was unaltered. At the canalicular pole, expression of the bile salt export pump (Bsep; ABCB11) and of the multidrug resistance proteins 2 (Mrp2; ABCC2) and 6 (Mrp6; ABCC6) was not significantly changed. Whereas hepatocellular BSP uptake was unaffected by PB, digoxin uptake was stimulated 4-fold. CONCLUSIONS: The induction of digoxin uptake by PB correlates with Oatp2 expression. In contrast, the lack of increase of Oatpl and Oatp4 expression is in accordance with unchanged BSP uptake. These data challenge the previously held view that PB induces hepatocellular BSP uptake systems.

Identification of organic anion transporting polypeptide 4 (Oatp4) as a major full-length isoform of the liver-specific transporter-1 (rlst-1) in rat liver.

A novel organic anion transporting polypeptide (Oatp)4(1) was isolated from rat liver that is 35 amino acids longer than the reported rat liver specific organic anion transporter (rlst)-1 and exhibits a 64% amino acid sequence identity with the human OATP-C (LST-1/OATP2; gene symbol SLC21A6). When expressed in Xenopus laevis oocytes, Oatp4 (Slc21a10) mediated polyspecific uptake of a variety of organic anions including taurocholate (K(m) approximately 27 microM), bromosulfophthalein (K(m) approximately 1.1 microM) and steroid conjugates. Based on nuclease protection analysis Oatp4 appears to be the predominant transcript in rat liver indicating that rlst-1 plays a minor role in overall hepatic organic anion uptake.

Rifamycin SV and rifampicin exhibit differential inhibition of the hepatic rat organic anion transporting polypeptides, Oatp1 and Oatp2.

The antibiotics, rifamycin SV and rifampicin, are known to interfere with hepatic bile salt and organic anion uptake. The aim of this study was to explore which transport systems are affected. In short-term-cultured rat hepatocytes, low concentrations (10 micromol/L) of both compounds inhibited mainly sodium-independent taurocholate uptake, whereas higher concentrations (100 micromol/L) also inhibited sodium-dependent taurocholate uptake. In Xenopus laevis oocytes expressing the Na(+)/taurocholate cotransporting polypeptide (Ntcp), high rifamycin SV and rifampicin concentrations were required for inhibition of taurocholate uptake. In contrast, sodium-independent taurocholate uptake mediated by the organic anion transporting polypeptides, Oatp1 and Oatp2, was already substantially inhibited by 10 micromol/L rifamycin SV. Rifampicin potently inhibited Oatp2-mediated taurocholate uptake, but did not interfere with Oatp1-mediated taurocholate uptake. Similar effects of rifamycin SV and rifampicin were found for Oatp1- and Oatp2-mediated estradiol-17beta-glucuronide transport. Dixon plot analysis yielded a pattern compatible with competitive inhibition of estradiol-17beta-glucuronide transport with K(i) estimates of 6.6 micromol/L and 7.3 micromol/L for rifamycin SV-induced inhibition of Oatp1 and Oatp2, respectively, and of 1.4 micromol/L for rifampicin-induced inhibition of Oatp2. These results demonstrate that rifamycin SV and rifampicin exhibit differential inhibition on Oatp1 and Oatp2, and identify rifampicin as a selective Oatp2 inhibitor. The data indicate that these inhibitors can be used to determine the in vivo relevance of Oatp1 and Oatp2 for the overall bioavailability and disposition of drugs and other Oatp1/2 substrates.

Localization and function of the organic anion-transporting polypeptide Oatp2 in rat liver.

BACKGROUND & AIMS: Multispecific organic anion-transporting polypeptides (Oatps) are involved in the transcellular movement of amphipathic compounds in many tissues including the liver, kidney, and blood-brain barrier. Recently, a high-affinity digoxin transporter (Oatp2) was cloned from rat brain and shown to be also expressed in the liver. METHODS: We investigated the cellular and subcellular distribution of Oatp2 in rat liver by in situ hybridization technology and immunofluorescence microscopy and compared its substrate specificity with that of Oatp1 in complementary RNA-injected Xenopus laevis oocytes. RESULTS: The results show a selective basolateral (sinusoidal) expression of Oatp2 in midzonal to perivenous hepatocytes, but not in periportal or the innermost layer of perivenous hepatocytes. Common substrates of both Oatp1 and Oatp2 include bile salts, steroid conjugates, thyroid hormones (T3, T4), ouabain, and the endothelin receptor antagonist BQ-123 (Michaelis constants: Oatp1, approximately 600 micromol/L; Oatp2, approximately 30 micromol/L). Other organic anions including sulfolithotaurocholate, bilirubin monoglucuronide, and sulfobromophthalein were transported only by Oatp1. CONCLUSIONS: These results provide definite evidence for the partially overlapping and partially selective substrate specificities of Oatp1 and Oatp2. The unique acinar distribution of Oatp2 might indicate that it represents a high-affinity "backup" system for complete hepatocellular removal of certain cholephilic substances from portal blood plasma.

Molecular cloning and functional characterization of two alternatively spliced Ntcp isoforms from mouse liver1.

To isolate the murine Na+/taurocholate cotransporting polypeptide (Ntcp), we screened a mouse liver cDNA library and identified Ntcp1, encoding a 362 amino acid protein and Ntcp2, encoding a 317 amino acid protein which had a shorter C-terminal end. Both isoforms mediated saturable Na+-dependent transport of taurocholate when expressed in Xenopus laevis oocytes. Analysis of the gene revealed that Ntcp2 is produced by alternative splicing where the last intron is retained.