Anne Kummeling

Infectious agents associated with diarrhoea of calves in the canton of Tilarán, Costa Rica.

Abstract A case-control study of calves under 3 months of age was carried out by weekly visits to 15 farms in the canton of Tilarán, Costa Rica. Most farms were dedicated to beef or dual-purpose (DP) production. Faecal samples were collected over a 6-month period from a total of 194 calves with clinical signs and from 186 animals without clinical signs of diarrhoea as assessed by a scoring system. The samples were investigated for the presence of viruses, bacteria and parasites. Torovirus was detected for the first time in Costa Rica and was present in 14% of calves with diarrhoea and in 6% of the controls. Coronavirus and Rotavirus were less frequently encountered in either one of the groups (in 9 and 7% of scouring calves and in 1 and 2% of controls, respectively). Escherichia coli was detected in 94% of all the faecal samples, but isolates from only three samples from calves with diarrhoea contained the K99 antigen. Similarly, Salmonella was found only in scouring calves. Cryptosporidium oocysts were detected in animals with signs of diarrhoea, while other coccidia oocysts, Strongylida and Strongyloides eggs were frequently found in animals both with and without diarrhoea. A conditional logistic regression (CLR) analysis to compare healthy and scouring calves showed a significant difference with regard to the presence of Torovirus, Rotavirus and Coronavirus.

Aberrant Gene Expression in Dogs with Portosystemic Shunts

Congenital portosystemic shunts are developmental anomalies of the splanchnic vascular system that cause portal blood to bypass the liver. Large-breed dogs are predisposed for intrahepatic portosystemic shunts (IHPSS) and small-breed dogs for extrahepatic portosystemic shunts (EHPSS). While the phenotype resulting from portal bypass of the liver of the two types of shunt is identical, the genotype and molecular pathways involved are probably different. The aim of this study was to gain insight into the pathways involved in the different types of portosystemic shunting. Microarray analysis of mRNA expression in liver tissue from dogs with EHPSS and IHPSS revealed that the expression of 26 genes was altered in either IHPSS or EHPSS samples compared with that in liver samples from control dogs. Quantitative real-time PCR of these genes in 14 IHPSS, 17 EHPSS, and 8 control liver samples revealed a significant differential expression of ACBP, CCBL1, GPC3, HAMP, PALLD, VCAM1, and WEE1. Immunohistochemistry and Western blotting confirmed an increased expression of VCAM1 in IHPSS but its absence in EHPSS, an increased WEE1 expression in IHPSS but not in EHPSS, and a decreased expression of CCBL1 in both shunt types. Regarding their physiologic functions, these findings may indicate a causative role for VCAM1 in IHPSS and WEE1 for IHPSS. CCBL1 could be an interesting candidate to study not yet elucidated aspects in the pathophysiology of hepatic encephalopathy.

Distribution of extrahepatic congenital portosystemic shunt morphology in predisposed dog breeds

BackgroundAn inherited basis for congenital extrahepatic portosystemic shunts (EHPSS) has been demonstrated in several small dog breeds. If in general both portocaval and porto-azygous shunts occur in breeds predisposed to portosystemic shunts then this could indicate a common genetic background. This study was performed to determine the distribution of extrahepatic portocaval and porto-azygous shunts in purebred dog populations.ResultsData of 135 client owned dogs diagnosed with EHPSS at the Faculty of Veterinary Medicine of Utrecht University from 2001 – 2010 were retrospectively analyzed. The correlation between shunt localization, sex, age, dog size and breed were studied. The study group consisted of 54 males and 81 females from 24 breeds. Twenty-five percent of dogs had porto-azygous shunts and 75% had portocaval shunts. Of the dogs with porto-azygous shunts only 27% was male (P = 0.006). No significant sex difference was detected in dogs with a portocaval shunt. Both phenotypes were present in almost all breeds represented with more than six cases. Small dogs are mostly diagnosed with portocaval shunts (79%) whereas both types are detected. The age at diagnosis in dogs with porto-azygous shunts was significantly higher than that of dogs with portocaval shunts (P < 0.001).ConclusionThe remarkable similarity of phenotypic variation in many dog breeds may indicate common underlying genes responsible for EHPSS across breeds. The subtype of EHPSS could be determined by a minor genetic component or modulating factors during embryonic development.

Altered Subcellular Localization of Heat Shock Protein 90 Is Associated with Impaired Expression of the Aryl Hydrocarbon Receptor Pathway in Dogs

The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting protein (AIP) and aryl hydrocarbon receptor nuclear translocator (ARNT). The resulting intrahepatic portosystemic shunts (IHPSS) are frequently diagnosed in specific dog breeds, such as the Irish wolfhound. We compared the expression of components of the AHR pathway in healthy Irish wolfhounds and dogs with IHPSS. To this end, we analyzed the mRNA expression in the liver of AHR,AIP, ARNT, and other genes involved in this pathway, namely, those for aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), hypoxia inducible factor 1alpha (HIF1A), heat shock protein 90AA1 (HSP90AA1), cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1), vascular endothelial growth factor A (VEGFA), nitric oxide synthesase 3 (NOS3), and endothelin (EDN1). The observed low expression of AHR mRNA in the Irish wolfhounds is in associated with a LINE-1 insertion in intron 2, for which these dogs were homozygous. Down regulation in Irish wolfhounds was observed for AIP, ARNT2, CYP1A2, CYP1B1 and HSP90AA1 expression, whereas the expression of HIF1A was increased. Immunohistochemistry revealed lower levels of AHR, HIF1A, and VEGFA protein in the nucleus and lower levels of ARNT and HSP90AA1 protein in the cytoplasm of the liver cells of Irish wolfhounds. The impaired expression of HSP90AA1 could trigger the observed differences in mRNA and protein levels and therefore explain the link between two very different functions of AHR: regulation of the closure of the ductus venosus and the response to toxins.

Urinary oxalate and calcium excretion by dogs and cats diagnosed with calcium oxalate urolithiasis.

Urine concentrations of oxalate and calcium play an important role in calcium oxalate (CaOx) urolith formation in dogs and cats, with high excretions of both substances increasing the chance of CaOx urolithiasis. In 17 CaOx-forming dogs, urine calcium:creatinine ratio (Ca:Cr) was found to be increased compared with healthy control dogs, whereas urine oxalate:creatinine ratio (Ox:Cr) did not differ (Stevenson and others 2004). In six CaOx-forming miniature schnauzers, urinary calcium excretion (in mg/kg/24 hours) was increased compared with healthy controls, while urinary oxalate excretion was not affected (Lulich and others 1991). However, in these studies, the urine samples were not always collected at the time CaOx uroliths were actually present in the urinary tract. Moreover, in these studies, individual urinary oxalate and calcium excretions were mostly not provided. The objective of this study was to determine the urinary oxalate and calcium excretion rates, and the urinary calcium to oxalate ratio (Ca:oxalate), at the time CaOx uroliths were present in the urinary tract of dogs and cats. Data for this observational study were collected through urine sampling and questionnaires from May 2010 to January 2012. Participation in this study was voluntary, and informed consent was obtained from dog and cat owners who consulted the University Clinic for Companion Animals of the Utrecht University because of urolithiasis. Dogs and …

Closure of the rectus sheath with a continuous looped suture and the skin with staples in dogs: speed, safety, and costs compared to closure of the rectus sheath with interrupted sutures and the skin with a continuous subdermal suture.

In dogs requiring a midline celiotomy, closure of the rectus sheath with a continuous polyglyconate looped suture and closure of the skin with stainless steel staples (group 1, 20 dogs) were compared with closure of the rectus sheath with interrupted polyglyconate sutures and closure of the skin with a continuous subdermal polyglactin suture (group 2, 20 dogs) with respect to speed, safety, and costs. The subcutis was closed with a continuous polyglactin suture in all dogs. The use of a looped suture saved 3.4 minutes and the use of staples saved 5.6 minutes. Total mean time saved for all layers was 7.7 minutes (38%). Wound healing was not significantly different between group 1 and group 2. There was one case of dehiscence of the rectus sheath in group 2 and two cases of wound infection in group 1. It was concluded that closing the rectus sheath with a continuous looped suture and the skin with staples is equally safe and significantly faster than closing the rectus sheath with simple interrupted sutures and the skin with a continuous subdermal suture. Additional costs were balanced by the reduction in surgery time.

Hepatic gene expression and plasma albumin concentration related to outcome after attenuation of a congenital portosystemic shunt in dogs

In dogs with a congenital portosystemic shunt (CPSS), the outcome after CPSS attenuation is difficult to predict but is most likely related to hepatic and vascular proliferation that follows the attenuation. The aim of this study was to evaluate the prognostic value of shunt localization (extrahepatic vs. intrahepatic), plasma albumin concentration and hepatic mRNA expression of 19 genes involved in hepatic and vascular growth. The study population consisted of 48 dogs that were referred for surgical ligation of a single intrahepatic or extrahepatic CPSS. Gene expression was measured in intraoperatively sampled hepatic tissue with quantitative real-time PCR. Albumin, methionine adenosyltransferase 2α (MAT2α) and HGF activator (HGFac) were positively associated with complete recovery after CPSS attenuation using multivariate statistical analyses. Individual outcome could be correctly predicted in 83% of dogs using albumin, MAT2α and HGFac as high or low values compared to cut-off values of 19.5 g/L, 0.457 and 0.974, respectively. These variables predicted outcome after CPSS ligation better than shunt localization or albumin alone. Other evaluated gene products were not correlated with outcome.

Aberrant Expression and Distribution of Enzymes of the Urea Cycle and Other Ammonia Metabolizing Pathways in Dogs with Congenital Portosystemic Shunts

The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase.

Comparison of terazosin and prazosin for treatment of vesico-urethral reflex dyssynergia in dogs

Nineteen dogs with vesico-urethral reflex dyssynergia (VURD) were treated with prazosin or terazosin 0.5 mg/kg twice daily to compare efficacy and side effects. Dogs were referred because of signs of (partial) urethral obstruction. Physical examination, abdominal ultrasonography, urinalysis and a radiographic contrast study of bladder and urethra (urethrocystography) were routinely performed. If no mechanical causes of obstruction or disease of the distal urinary tract were observed, the diagnosis VURD was presumed and the dogs were included in our study. Follow-up information was obtained from owners or referring veterinarians. Significantly more side effects were seen in the dogs treated with terazosin (n=14; 93 per cent) compared with the dogs treated with prazosin (n=5; 20 per cent; P=0.002). Effects of the treatment were comparable between prazosin and terazosin. Labradors and dogs that were castrated surgically had a significant better survival (P<0.01) compared with other breeds and animals that were castrated chemically. There was a moderate to good effect in 60 per cent of the dogs treated with prazosin, and in 64 per cent of the dogs treated with terazosin.

No up-regulation of the phosphatidylethanolamine N-methyltransferase pathway and choline production by sex hormones in cats.

BackgroundFeline hepatic lipidosis (FHL) is a common cholestatic disease affecting cats of any breed, age and sex. Both choline deficiency and low hepatic phosphatidylethanolamine N-methyltransferase (PEMT) activity are associated with hepatic lipidosis (HL) in humans, mice and rats. The PEMT expression is known to be upregulated by oestrogens, protecting the females in these species from the development of HL when exposed to choline deficient diets. The aim of the present study was to evaluate the influence of sex hormones on choline synthesis via the PEMT pathway in healthy male and female cats before and after spaying/neutering, when fed a diet with recommended dietary choline content.ResultsFrom six female and six male cats PEMT activity was assayed directly in liver biopsies taken before and after spaying/neutering, and assessed indirectly by analyses of PEMT–specific hepatic phosphatidylcholine (PC) species and plasma choline levels. Hepatic PEMT activity did not differ between intact female and male cats and no changes upon spaying/neutering were observed. Likewise, no significant differences in liver PC content and PEMT-specific polyunsaturated PC species were found between the sexes and before or after spaying/neutering.ConclusionThese results suggest that choline synthesis in cats differs from what is observed in humans, mice and rats. The lack of evident influence of sex hormones on the PEMT pathway makes it unlikely that spaying/neutering predisposes cats for HL by causing PC deficiency as suggested in other species.