Two levels of MCT1 and CD147 expression in the equine red blood cells and muscle

Monocarboxylate transporters (MCTs), especially the isoforms MCT1 - MCT4, cotransport lactate and protons across the cell membranes. They are thus essential for pH regulation and homeostasis in glycolytic cells such as red blood cells (RBCs), and skeletal muscle cells during intense exercise. In 70% of the Standardbred horses the lactate transport activity (TA) in RBCs is high and transport is mediated mainly by MCTs. In the rest 30% of the Standardbreds MCT mediated transport route is not active and the TA is low. MCTs need an ancillary protein for their proper localization and functioning in the plasma membrane. The ancillary protein for MCT1 and MCT4 is a member of immunoglobulin superfamily, CD147. Here we determined the expression of MCT isoforms and CD147 in equine RBCs and gluteal muscle. We sequenced the cDNA of horse MCT1 and CD147 to achieve horse-specific antibodies and to reveal sequence variations that may affect the TA of RBCs. The amount of MCT1 and CD147 mRNA in muscle were also studied. ---- In all, 73 horses representing different breeds were used. Blood samples were drawn from the jugular vein and muscle samples were taken either from gluteal muscle using biopsy needle or during castration from expendable cremaster muscle. The TA of RBCs was studied using radiolabeled lactate and the amount of MCT isoforms and CD147 in the plasma membranes using Western blotting. The level of mRNA in muscle cells was determined using qPCR. Isoforms MCT1 and MCT2 were found in the RBCs and isoforms MCT1 and MCT4 in the muscle cells of horses. The TA of RBCs was dependent on the expression of CD147 and MCT1 in the plasma membrane. Sequence variations were found in the cDNA of both MCT1 and CD147, but they did not explain the inactivity of MCT1 mediated transport route. The single nucleotide polymorphism (SNP) Met125Val in CD147 that existed parallel with an SNP in 3´-untranslated region explained, however, attenuation in CD147 expression in Standardbreds. A single mutation Ile51Val also decreased the expression of CD147 in one Warmblood. The MCT1 and CD147 mRNA concentrations in the gluteal muscle were higher in horses with higher MCT1 and CD147 expression in RBCs and lower in horses with minor expression of CD147 and MCT1. This suggests that the bimodal distribution of TA is due to differences in transcriptional regulation that is functioning in parallel in MCT1 and CD147 gene.

Species-specific and Indication-based Use of Antimicrobials in Dogs, Cats, Cattle and Horses in Finland : Data collected using three different methods

Increasing antimicrobial resistance in bacteria has led to the need for better understanding of antimicrobial usage patterns. In 1999, the World Organisation for Animal Health (OIE) recommended that an international ad hoc group should be established to address human and animal health risks related to antimicrobial resistance and the contribution of antimicrobial usage in veterinary medicine. In European countries the need for continuous recording of the usage of veterinary antimicrobials as well as for animal species-specific and indication-based data on usage has been acknowledged. Finland has been among the first countries to develop prudent use guidelines in veterinary medicine, as the Ministry of Agriculture and Forestry issued the first animal species-specific indication-based recommendations for antimicrobial use in animals in 1996. These guidelines have been revised in 2003 and 2009. However, surveillance on the species-specific use of antimicrobials in animals has not been performed in Finland. This thesis provides animal species-specific information on indication-based antimicrobial usage. Different methods for data collection have been utilized. Information on antimicrobial usage in animals has been gathered in four studies (studies A-D). Material from studies A, B and C have been used in an overlapping manner in the original publications I-IV. Study A (original publications I & IV) presents a retrospective cross-sectional survey on prescriptions for small animals at the Veterinary Teaching Hospital of the University of Helsinki. Prescriptions for antimicrobial agents (n = 2281) were collected and usage patterns, such as the indication and length of treatment, were reviewed. Most of the prescriptions were for dogs (78%), and primarily for the treatment of skin and ear infections most of which were treated with cephalexin for a median period of 14 days. Prescriptions for cats (18%) were most often for the treatment of urinary tract infections with amoxicillin for a median length of 10 days. Study B (original publication II) was a retrospective cross-sectional survey where prescriptions for animals were collected from 17 University Pharmacies nationwide. Antimicrobial prescriptions (n = 1038) for mainly dogs (65%) and cats (19%) were investigated. In this study, cephalexin and amoxicillin were also the most frequently used drugs for dogs and cats, respectively. In study C (original publications III & IV), the indication-based usage of antimicrobials of practicing veterinarians was analyzed by using a prospective questionnaire. Randomly selected practicing veterinarians in Finland (n = 262) recorded all their antimicrobial usage during a 7-day study period. Cattle (46%) with mastitis were the most common patients receiving antimicrobial treatment, generally intramuscular penicillin G or intramammary treatment with ampicillin and cloxacillin. The median length of treatment was four days, regardless of the route of administration. Antimicrobial use in horses was evaluated in study D, the results of which are previously unpublished. Firstly, data collected with the prospective questionnaire from the practicing veterinarians showed that horses (n = 89) were frequently treated for skin or wound infections by using penicillin G or trimethoprim-sulfadiazine. The mean duration of treatment was five to seven days. Secondly, according to retrospective data collected from patient records, horses (n = 74) that underwent colic surgery at the Veterinary Teaching Hospital of the University of Helsinki were generally treated according to national and hospital recommendations; penicillin G and gentamicin was administered preoperatively and treatment was continued for a median of three days postoperatively. In conclusion, Finnish veterinarians followed well the national prudent use guidelines. Narrow-spectrum antimicrobials were preferred and, for instance, fluoroquinolones were used sparingly. Prescription studies seemed to give good information on antimicrobials usage, especially when combined with complementary information from patient records. A prospective questionnaire study provided a fair amount of valuable data on several animal species. Electronic surveys are worthwhile exploiting in the future.

Expression of lactate transporters MCT1, MCT2, MCT4 and the ancillary protein CD147 in horse muscle and red blood cells

Monocarboxylate transporters (MCTs) transport lactate and protons across cell membranes. During intense exercise, lactate and protons accumulate in the exercising muscle and are transported to the plasma. In the horse, MCTs are responsible for the majority of lactate and proton removal from exercising muscle, and are therefore also the main mechanism to hinder the decline in pH in muscle cells. Two isoforms, MCT1 and MCT4, which need an ancillary protein CD147, are expressed in equine muscle. In the horse, as in other species, MCT1 is predominantly expressed in oxidative fibres, where its likely role is to transport lactate into the fibre to be used as a fuel at rest and during light work, and to remove lactate during intensive exercise when anaerobic energy production is needed. The expression of CD147 follows the fibre type distribution of MCT1. These proteins were detected in both the cytoplasm and sarcolemma of muscle cells in the horse breeds studied: Standardbred and Coldblood trotters. In humans, training increases the expression of both MCT1 and MCT4. In this study, the proportion of oxidative fibres in the muscle of Norwegian-Swedish Coldblood trotters increased with training. Simultaneously, the expression of MCT1 and CD147, measured immunohistochemically, seemed to increase more in the cytoplasm of oxidative fibres than in the fast fibre type IIB. Horse MCT4 antibody failed to work in immunohistochemistry. In the future, a quantitative method should be introduced to examine the effect of training on muscle MCT expression in the horse. Lactate can be taken up from plasma by red blood cells (RBCs). In horses, two isoforms, MCT1 and MCT2, and the ancillary protein CD147 are expressed in RBC membranes. The horse is the only species studied in which RBCs have been found to express MCT2, and the physiological role of this protein in RBCs is unknown. The majority of horses express all three proteins, but 10-20% of horses express little or no MCT1 or CD147. This leads to large interindividual variation in the capacity to transport lactate into RBCs. Here, the expression level of MCT1 and CD147 was bimodally distributed in three studied horse breeds: Finnhorse, Standardbred and Thoroughbred. The level of MCT2 expression was distributed unimodally. The expression level of lactate transporters could not be linked to performance markers in Thoroughbred racehorses. In the future, better performance indexes should be developed to better enable the assessment of whether the level of MCT expression affects athletic performance. In human subjects, several mutations in MCT1 have been shown to cause decreased lactate transport activity in muscle and signs of myopathy. In the horse, two amino acid sequence variations, one of which was novel, were detected in MCT1 (V432I and K457Q). The mutations found in horses were in different areas compared to mutations found in humans. One mutation (M125V) was detected in CD147. The mutations found could not be linked with exercise-induced myopathy. MCT4 cDNA was sequenced for the first time in the horse, but no mutations could be detected in this protein.

Contrast-enhanced ultrasound of abdominal organs in cats

The primary aim of this thesis was the evaluation of the perfusion of normal organs in cats using contrast-enhanced ultrasound (CEUS), to serve as a reference for later clinical studies. Little is known of the use of CEUS in cats, especially regarding its safety and the effects of anesthesia on the procedure, thus, secondary aims here were to validate the quantitative analyzing method, to investigate the biological effects of CEUS on feline kidneys, and to assess the effect of anesthesia on splenic perfusion in cats undergoing CEUS. -- The studies were conducted on healthy, young, purpose-bred cats. CEUS of the liver, left kidney, spleen, pancreas, small intestine, and mesenteric lymph nodes was performed to characterize the normal perfusion of these organs on ten anesthetized, male cats. To validate the quantification method, the effects of placement and size of the region of interest (ROI) on perfusion parameters were investigated using CEUS: Three separate sets of ROIs were placed in the kidney cortex, varying in location, size, or depth. The biological effects of CEUS on feline kidneys were estimated by measuring urinary enzymatic activities, analyzing urinary specific gravity, pH, protein, creatinine, albumin, and sediment, and measuring plasma urea and creatinine concentrations before and after CEUS. Finally, the impact of anesthesia on contrast enhancement of the spleen was investigated by imaging cats with CEUS first awake and later under anesthesia on separate days. -- Typical perfusion patterns were found for each of the studied organs. The liver had a gradual and more heterogeneous perfusion pattern due to its dual blood flow and close proximity to the diaphragm. An obvious and statistically significant difference emerged in the perfusion between the kidney cortex and medulla. Enhancement in the spleen was very heterogeneous at the beginning of imaging, indicating focal dissimilarities in perfusion. No significant differences emerged in the perfusion parameters between the pancreas, small intestine, and mesenteric lymph nodes. -- The ROI placement and size were found to have an influence on the quantitative measurements of CEUS. Increasing the depth or the size of the ROI decreased the peak intensity value significantly, suggesting that where and how the ROI is placed does matter in quantitative analyses. --- A significant increase occurred in the urinary N-acetyl-β-D-glucosaminidase (NAG) to creatinine ratio after CEUS. No changes were noted in the serum biochemistry profile after CEUS, with the exception of a small decrease in blood urea concentration. The magnitude of the rise in the NAG/creatinine ratio was, however, less than the circadian variation reported earlier in healthy cats. Thus, the changes observed in the laboratory values after CEUS of the left kidney did not indicate any detrimental effects in kidneys. Heterogeneity of the spleen was observed to be less and time of first contrast appearance earlier in nonanesthetized cats than in anesthetized ones, suggesting that anesthesia increases heterogeneity of the feline spleen in CEUS. ---- In conclusion, the results suggest that CEUS can be used also in feline veterinary patients as an additional diagnostics aid. The perfusion patterns found in the imaged organs were typical and similar to those seen earlier in other species, with the exception of the heterogeneous perfusion pattern in the cat spleen. Differences in the perfusion between organs corresponded with physiology. Based on the results, estimation of focal perfusion defects of the spleen in cats should be performed with caution and after the disappearance of the initial heterogeneity, especially in anesthetized or sedated cats. Finally, these results indicate that CEUS can be used safely to analyze kidney perfusion also in cats. Future clinical studies are needed to evaluate the full potential of CEUS in feline medicine as a tool for diagnosing lesions in various organ systems.

Hevosen hankittu polyneuropatia –kirjallisuuskatsaus ja sairauden kartoitus Suomessa

Hevosen hankittu polyneuropatia (Acquired equine polyneuropathy eli AEP, ”Hattulan tauti”, ”Scandinavian knuckling syndrome”) on Skandinaviassa tavattava, uudentyyppinen hermostosairaus hevosilla. Yhteensä yli 300 hevosta on sairastunut Suomessa, Ruotsissa ja Norjassa lähes sadalla eri tallilla viimeisten kahdenkymmenen vuoden aikana. Hevosen hankitusta polyneuropatiasta tiedetään vasta melko vähän, eikä sairautta käsitteleviä julkaisuja ole kuin kolme. Hankittuun polyneuropatiaan sairastuneilla hevosilla on tyypillisenä oireena takajalkojen vuohisnivelten ylimeno eli ”knuckling”, josta seuraa kompurointia ja ataksiaa. Useimmiten vuohisnivelten ylimeno on sairastuneilla hevosilla symmetristä molemmissa takajaloissa. Ylimeno johtuu takavuohisnivelten ojentajalihasten heikkoudesta perifeeristen hermovaurioiden seurauksena. Tautitapauksia tavataan vakavuudeltaan eriasteisia. Vakavimmissa tapauksissa kliiniset oireet voivat pahentua jopa muutamassa päivässä siten, ettei hevonen kykene enää nousemaan makuulta ylös edes avustettuna ja eutanasia tulee aiheelliseksi. Lievin oirein sairastuneet hevoset parantuvat yleensä noin 5-6 kuukauden kuluessa. Hevosen hankitun polyneuropatian aiheuttaja on toistaiseksi tuntematon. Epäillään, että aiheuttaja voisi olla jonkin homeen tuottama mykotoksiini rehussa. Lisensiaatin tutkielmani keskeisin tarkoitus oli kartoittaa, miten paljon hevosen hankittua polyneuropatiaa on esiintynyt Suomessa. Suomen taudinpurkauksia ei ole aiemmin dokumentoitu muutamaa poikkeusta lukuun ottamatta. Kartoitukseen valittiin mukaan tallit, joissa epäiltiin joko hoitaneen eläinlääkärin tai tallinomistajan kuvauksen perusteella esiintyneen tyypillisiä hankittuun polyneuropatiaan viittaavia kliinisiä oireita. Polyneuropatiakartoitukseen valittuihin tallinomistajiin otettiin yhteyttä syksyllä 2009 puhelimitse soittamalla ja heille esitettiin kartoitusta varten laaditut kysymykset. Kartoituksen mukaan Suomessa on ollut 11 taudinpurkausta, jossa kyseessä on todennäköisesti ollut hevosen hankittu polyneuropatia. Taudinpurkaukset ajoittuvat vuodesta 2005 vuoteen 2009. Hevosten sairastumisprosentissa esiintyy runsaasti vaihtelua tallien välillä, mutta tyypillisesti useita saman tallin hevosia sairastuu. Sairastuneista hevosista on aineiston perusteella jouduttu lopettamaan 40 % ja 60 % on parantunut. Suomen kartoitus näyttää vahvistavan olettamusta, ettei hankitussa polyneuropatiassa ole ikä-, sukupuoli- tai rotupredilektiota. Tallit, joilla sairautta on tavattu, sijaitsevat maantieteellisesti hyvin hajanaisesti. Suomessa polyneuropatiatapauksia on ilmennyt lähes yhtä paljon syksyllä ja keväällä, kun taas Ruotsissa ja Norjassa lähes kaikki taudinpurkaukset ovat esiintyneet lopputalvella ja keväällä. Suurin osa hankittuun polyneuropatiaan sairastuneista hevosista on saanut karkearehuksi muovipaaleihin pakattua, esikuivattua säilöheinää. Kartoituksen myötä tiedetään suunnilleen, millä laajuudella hevosen hankittua polyneuropatiaa on Suomessa esiintynyt. Sairaus on toistaiseksi Suomessa melko harvinainen, mutta sitä tavataan kuitenkin lähes vuosittain. Jatkossa kartoituksen tietoja pystytään hyödyntämään epidemiologisissa tutkimuksissa sairauden aiheuttajan selvittämiseksi.

Detection, epidemiology and host spectrum of cowpox and Borna disease virus infections

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.

Effect of colostrum feeding method and presence of dam on the sleep, rest and sucking behaviour of newborn calves

"In rats, sucking milk reduces anxiety and promotes non-rapid eye movement (NREM) sleep, and in calves it induces resting but the effect on sleep is unknown. Here, we investigated how calves' sleep was affected by colostrum feeding methods. Forty-one calves were blocked by birth date and randomly allotted within blocks to the experimental treatments. Calves were housed for four days either with their dam (DAM) or individually with warm colostrum feeding (2 L four times a day) from either a teat bucket (TEAT) or an open bucket (BUCKET). DAM calves suckled their dam freely. Calves' sleeping and sucking behaviour was filmed continuously for 48 h at the ages of two and three days. Behavioural sleep (BS) was defined as calves resting at least 30 s with their head still and raised (non-rapid eye movement) or with their head against their body or the ground (rapid eye movement, REM). Latency from the end of colostrum feeding to the start of BS was recorded. We compared behaviour of TEAT calves with that of DAM and BUCKET calves using mixed models. Milk meal duration was significantly longer for TEAT calves than for BUCKET calves (mean +/- S.E.M.; 8.3 +/- 0.6 min vs. 5.2 +/- 0.6 min), but equal to that of DAM calves. We found no effect of feeding method on the duration of daily BS (12 h 59 min I h 38 min) but we found a tendency for the daily amount of NREM sleep; BUCKET calves had less NREM sleep per day than TEAT calves (6 h 18 min vs. 7 h 48 min, S.E.M. = 45 min) and also longer latencies from milk ingestion to BS (21.9 +/- 2.0 min vs. 16.2 +/- 2.0 min). DAM calves slept longer bouts than TEAT calves (10.8 +/- 1.0 min vs. 8.3 +/- 1.0 min) and less often (78 +/- 4 vs. 92 +/- 4). Sucking colostrum from a teat bucket compared with drinking from an open"