The metabolic differences of anestrus, heat, pregnancy, pseudopregnancy, and lactation in 800 female dogs.

Introduction: Reproduction causes major hormonal and physiological changes to the female body. However, the metabolic changes occurring during canine reproduction are scarcely studied. Methods: In this cross-sectional study, we assessed the metabolic effects of canine reproductive status using a 1H NMR metabolomics platform optimized and validated for canine use. The study population consisted of a total of 837 healthy, intact female dogs in breeding age, of which 663 dogs were in anestrus, 78 in heat, 43 were pseudopregnant, 15 were pregnant, and 38 were lactating. The differences in metabolite profiles between these states were studied by the Kruskal-Wallis test with post-hoc tests performed using the Dunn's test, and visualized by box plots and a heatmap. The ability of the metabolite profile to differentiate pregnant dogs from non-pregnant ones was assessed by creating a multivariate Firth logistic regression model using forward stepwise selection. Results: Lactation, pregnancy and heat all were associated with distinct metabolic changes; pregnancy caused major changes in the concentrations of glycoprotein acetyls, albumin and creatinine, and smaller changes in several lipids, citrate, glutamine, and alanine. Pseudopregnancy, on the other hand, metabolically largely resembled anestrus. Lactation caused major changes in amino acid concentrations and smaller changes in several lipids, albumin, citrate, creatinine, and glycoprotein acetyls. Heat, referring to proestrus and estrus, affected cholesterol and LDL metabolism, and increased HDL particle size. Albumin and glycoprotein acetyls were the metabolites included in the final multivariate model for pregnancy detection, and could differentiate pregnant dogs from non-pregnant ones with excellent sensitivity and specificity. Discussion: These results increase our understanding of the metabolic consequences of canine reproduction, with the possibility of improving maternal health and ensuring reproductive success. The identified metabolites could be used for confirming canine pregnancy.

Neutralizing Antibody Titers in Hospitalized Patients with Acute Puumala Orthohantavirus Infection Do Not Associate with Disease Severity.

Nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), is an acute febrile illness caused by Puumala orthohantavirus (PUUV). NE manifests typically with acute kidney injury (AKI), with a case fatality rate of about 0.1%. The treatment and management of hantavirus infections are mainly supportive, although neutralizing monoclonal antibodies and immune sera therapeutics are under investigation. In order to assess the potential use of antibody therapeutics in NE, we sought to determine the relationship between circulating PUUV neutralizing antibodies, PUUV nucleocapsid protein (N) IgG antibodies, and viral loads with markers of disease severity. The study included serum samples of extensively characterized patient cohorts (n = 116) from Tampere University Hospital, Finland. The results showed that upon hospitalization, most patients already had considerable neutralizing and anti-PUUV-N IgG antibody levels. However, contrary to expectations, neutralizing antibody titers from the first day of hospitalization did not appear to protect from AKI or correlate with more favorable disease outcomes. This indicates that further studies are needed to investigate the applicability of neutralizing antibodies as a therapy for hospitalized NE patients.

A novel canine nuclear magnetic resonance spectroscopy-based metabolomics platform: Validation and sample handling.

BACKGROUND: Metabolomics has been proven to be an invaluable research tool by providing comprehensive insight into systemic metabolism. However, the lack of scalable and quantitative methods with known reference intervals (RIs) and documented reproducibility has prevented the use of metabolomics in the clinical setting. OBJECTIVE: The objective of this study was to validate the developed quantitative nuclear magnetic resonance (NMR) spectroscopy-based metabolomics platform for canine serum and plasma samples and determine optimal sample handling conditions for its use. METHODS: Altogether, 8247 canine samples were analyzed using a Bruker's 500 MHz NMR spectrometer. Using statistical approaches derived from international guidelines, we studied method precision, measurand stability in various long- and short-term storage conditions, as well as the effect of prolonged contact with red blood cells (RBCs), and differences among blood collection tubes. We also screened interferences with lipemia, hemolysis, and bilirubinemia. The results were compared against routine clinical chemistry methods, and RIs were defined for all measurands. RESULTS: We determined RIs for 123 measurands, most of which were previously unpublished. The reproducibility of the results of the NMR platform appeared generally outstanding, and the integrity of the results can be ensured by following standard blood drawing and processing guidelines. CONCLUSIONS: Owing to the advantages of quantitative results, high reproducibility, and scalability, this canine metabolomics platform holds great potential for numerous clinical and research applications to improve canine health and well-being.

High serum creatinine concentration is associated with metabolic perturbations in dogs.

BACKGROUND: The kidneys have many essential metabolic functions, and metabolic disturbances during decreased renal function have not been studied extensively. OBJECTIVES: To identify metabolic changes in blood samples with increased serum creatinine concentration, indicating decreased glomerular filtration. ANIMALS: Clinical samples analyzed using a nuclear magnetic resonance (NMR) based metabolomics platform. The case group consisted of 23 samples with serum creatinine concentration >125 µmol/L, and the control group of 873 samples with serum creatinine concentration within the reference interval. METHODS: Biomarker association with increased serum creatinine concentration was evaluated utilizing 3 statistical approaches: Wilcoxon rank-sum test, logistic regression analysis (false discovery rate (FDR)-corrected P-values), and random forest classification. Medians of the biomarkers were compared to reference intervals. A heatmap and box plots were used to represent the differences. RESULTS: All 3 statistical approaches identified similar analytes associated with increased serum creatinine concentrations. The percentages of citrate, tyrosine, branched-chain amino acids, valine, leucine, albumin, linoleic acid and the ratio of phenylalanine to tyrosine differed significantly using all statistical approaches, acetate differed using the Wilcoxon test and random forest, docosapentaenoic acid percentage only using logistic regression (P < .05), and alanine only using random forest. CONCLUSIONS AND CLINICAL IMPORTANCE: We identified several metabolic changes associated with increased serum creatinine concentrations, including prospective diagnostic markers and therapeutic targets. Further research is needed to verify the association of these changes with the clinical state of the dog. The NMR metabolomics test is a promising tool for improving diagnostic testing and management of renal diseases in dogs.

Detection of dengue virus type 2 of Indian origin in acute febrile patients in rural Kenya.

Dengue virus (DENV) has caused recent outbreaks in coastal cities of Kenya, but the epidemiological situation in other areas of Kenya is largely unknown. We investigated the role of DENV infection as a cause of acute febrile disease in non-epidemic settings in rural and urban study areas in Kenya. Altogether, 560 patients were sampled in 2016-2017 in rural Taita-Taveta County (n = 327) and urban slums of Kibera, Nairobi (n = 233). The samples were studied for DENV IgM, IgG, NS1 antigen and flaviviral RNA. IgG seroprevalence was found to be higher in Taita-Taveta (14%) than in Nairobi (3%). Five Taita-Taveta patients were positive for flaviviral RNA, all identified as DENV-2, cosmopolitan genotype. Local transmission in Taita-Taveta was suspected in a patient without travel history. The sequence analysis suggested that DENV-2 strains circulating in coastal and southern Kenya likely arose from a single introduction from India. The molecular clock analyses dated the most recent ancestor to the Kenyan strains a year before the large 2013 outbreak in Mombasa. After this, the virus has been detected in Kilifi in 2014, from our patients in Taita-Taveta in 2016, and in an outbreak in Malindi in 2017. The results highlight that silent transmission occurs between epidemics and also affects rural areas. More information is needed to understand the local epidemiological characteristics and future risks of dengue in Kenya.

Breed differences of heritable behaviour traits in cats.

Cat domestication and selective breeding have resulted in tens of breeds with major morphological differences. These breeds may also show distinctive behaviour differences; which, however, have been poorly studied. To improve the understanding of feline behaviour, we examined whether behavioural differences exist among cat breeds and whether behaviour is heritable. For these aims, we utilized our extensive health and behaviour questionnaire directed to cat owners and collected a survey data of 5726 cats. Firstly, for studying breed differences, we utilized logistic regression models with multiple environmental factors and discovered behaviour differences in 19 breeds and breed groups in ten different behaviour traits. Secondly, the studied cat breeds grouped into four clusters, with the Turkish Van and Angora cats alone forming one of them. These findings indicate that cat breeds have diverged not only morphologically but also behaviourally. Thirdly, we estimated heritability in three breeds and obtained moderate heritability estimates in seven studied traits, varying from 0.4 to 0.53, as well as phenotypic and genetic correlations for several trait pairs. Our results show that it is possible to partition the observed variation in behaviour traits into genetic and environmental components, and that substantial genetic variation exists within breed populations.

Fearful dogs have increased plasma glutamine and γ-glutamyl glutamine.

Anxiety-related disorders, including fearfulness are common and leading welfare problems among the worldwide dog population. The etiology of anxieties is complex and affected by genetic and environmental factors. Thus, there is a need for more comprehensive approaches, such as metabolomics, to understand the causes of anxiety and to identify anxiety-related biomarkers for more efficient diagnostic and treatment options. To study metabolic alterations related to canine fearfulness, a non-targeted plasma metabolite profiling was performed in a cohort of 20 fearful and 21 non-fearful dogs. The results showed that nine metabolic features were significantly associated with fearfulness. The most prominent change included increased plasma glutamine and γ-glutamyl glutamine (γ-Glu Gln) in fearful dogs across breeds. Alterations in glutamine metabolism have previously been associated with several psychiatric disorders, indicating the relevance of this finding also in dogs. In addition, we describe a novel breed-specific association between renal biomarker symmetric dimethylarginine (SDMA) and canine fearfulness. These observed metabolic alterations may result from high levels of prolonged psychological stress in fearful dogs.

Early weaning increases aggression and stereotypic behaviour in cats.

Behaviour problems are common in companion felines, and problematic behaviour may be a sign of chronic stress. In laboratory animals, early weaning increases the risk for aggression, anxiety, and stereotypic behaviour. However, very few studies have focused on early weaning in one of the world's most popular pets, the domestic cat, although weaning soon after the critical period of socialisation is common practice. To study the effects of early weaning (<12 weeks) on behaviour, a large data set (N = 5726, 40 breeds) was collected from home-living domestic cats through a questionnaire survey. The results show that weaning before 8 weeks of age increases the risk for aggression, but not fearful behaviour. Moreover, cats weaned after 14 weeks of age have a lower probability for aggression towards strangers than early weaned cats and a lower probability for stereotypic behaviour (excessive grooming) than cats weaned at 12 weeks. The effect of weaning age on stereotypic behaviour is partially explained by the effects on aggression. These findings indicate that early weaning has a detrimental effect on behaviour, and suggest delayed weaning as a simple and inexpensive approach to significantly improve the welfare of millions of domestic cats.

Questionnaire survey of detrimental fur animal epidemic necrotic pyoderma in Finland.

BACKGROUND: In 2007, a previously unrecorded disease, fur animal epidemic necrotic pyoderma (FENP), was detected in farmed mink (Neovision vision), foxes (Vulpes lagopus) and Finnraccoons (Nyctereutes procyonoides) in Finland. Symptoms included severe pyoderma with increased mortality, causing both animal welfare problems and economic losses. In 2011, an epidemiologic questionnaire was mailed to all members of the Finnish Fur Breeders' Association to assess the occurrence of FENP from 2009 through the first 6 months of 2011. The aim was to describe the geographical distribution and detailed clinical signs of FENP, as well as sources of infection and potential risk factors for the disease. RESULTS: A total of 239 farmers (25%) returned the questionnaire. Clinical signs of FENP were observed in 40% (95% CI 34-46%) of the study farms. In addition, the survey clarified the specific clinical signs for different animal species. The presence of disease was associated with the importation of mink, especially from Denmark (OR 9.3, 95% CI 2.6-33.0). The transmission route between Finnish farms was associated with fur animal purchases. Some risk factors such as the farm type were also indicated. As such, FENP was detected more commonly on farms with more than one species of fur animal in comparison to farms with, for example, only foxes (OR 4.6, 95% CI 2.4-8.6), and the incidence was higher on farms with over 750 breeder mink compared to smaller farms (OR 3.8, 95% CI 1.6-9.0). Contact between fur animals and birds and other wildlife increased the risk of FENP on farms. Responses also indicated that blocking the entry of wildlife to the animal premises protected against FENP. CONCLUSIONS: FENP was most likely introduced to Finland by imported mink and spread further within the country via domestically purchased fur animals. Some potential risk factors, such as the type and size of the farm and contact with wildlife, contributed to the spread of FENP. Escape-proof shelter buildings block the entry of wildlife, thus protecting fur animals against FENP.

Health and Behavioral Survey of over 8000 Finnish Cats.

A comprehensive feline health survey was conducted to reveal breed-specific inheritable diseases in Finnish pedigree cats for genetic research. Prevalence of 19 disease categories and 227 feline diseases were defined in a study population of 8175 cats belonging to 30 breeds. Dental and oral diseases, with a prevalence of 28%, and dental calculus and gingivitis (21 and 8%, respectively) were the most prevalent disease category and diseases among all cats and in most of the breeds. An exception was Korats, which were more often affected by the diseases of the respiratory tract (23%) and asthma (19%). Other prevalent disease categories affected various organ systems, such as the skin (12%), the urinary system (12%), the digestive tract (11%), eyes (10%), the musculoskeletal system (10%), and genitals of female cats (17%). Prevalent health or developmental issues included repetitive vomiting (4%), tail kink (4%), feline odontoclastic resorption lesion (4%), urinary tract infections (4%), as well as cesarean section (6%) and stillborn kittens (6%) among female cats. We found 57 breed-specific conditions by Fisher's exact tests and logistic regression analyses, including 32 previously described and 19 new breed-specific diseases. The genetic defect has already been found in six of them: polycystic kidney disease, progressive retinal atrophy, hypertrophic cardiomyopathy, and three types of tail malformations. Behavioral profiling revealed breed-specific traits, such as an increased human avoidance in British Short and Longhairs and a higher level of aggression in Turkish vans. Our epidemiological study reveals the overall health profile in Finnish pure and mixed breed cats and identifies many breed-specific conditions without molecular identity for genetic research.

Environmental Risk Factors of Pediatric-Onset Primary Sclerosing Cholangitis and Autoimmune Hepatitis.

OBJECTIVES: The aim of this population-based observational case-control questionnaire study was to investigate the possible role of environmental risk factors associated with pediatric-onset autoimmune liver diseases. METHODS: Seventy-one patients with autoimmune liver diseases (<16 years) received a questionnaire with 22 items, evaluating contact with environmental factors (eg, family manners, type of housing, pets) before the diagnosis. Two age- and sex-matched control groups were used: inflammatory bowel disease (IBD; n = 91) and healthy subjects (n = 716; matched also for place of residence at birth). Univariate analysis (odds ratio [OR] and 95% confidence interval) for all variables was calculated. Fisher exact test was performed to depict associations between variables and the multivariate logistic regression to test their interactions. RESULTS: In the final analyses, the responses of 51 autoimmune liver diseases cases (n = 51/71, 72%), 59 IBD controls (n = 59/91, 65%), and 292 healthy controls (n = 292/716, 41%) were investigated. In univariate analysis only having a cat, a dog, and a cat or a dog were risk factors of autoimmune liver diseases (OR varying between 2.6-3.4); no other significant associations (eg, place of residence, number of siblings, family manners) were found. Multivariate logistic regression analyses showed that especially living with a cat in block of flats was a risk factor (OR 3.6, 1.2-10.8). CONCLUSIONS: Living in a close contact with a pet (especially a cat) was a risk factor of autoimmune liver diseases. This finding may suggest an involvement of an unidentified agent (ie, toxin/microbe) among the triggers of these diseases.