The relationships between environment, diet, transcriptome and atopic dermatitis in dogs

Canine atopic dermatitis (CAD) is a multifactorial disease including genetic predisposition and other predisposing factors like living environment and diet. There is no known cure for this disease. The right and functional treatment can be hard to find, and more effort should be put into the prevention. The aim of this thesis was to find environmental factors and breeds associated with allergic skin symptoms and atopic dermatitis in pet dogs. In addition, the effect of a raw diet on gene expression, physiology and metabolism was studied with clinical diet intervention trial setup, using client-owned dogs. A population of 8643 dogs from the validated DOGRISK questionnaire was used to analyse the environmental factors and dog characteristics related to CAD. Five breeds with the most owner-reported skin symptoms as a percentage within the breed were found to be i) West Highland white terrier, ii) boxer, iii) English bulldog, iv) Dalmatian, and v) French bulldog. When FCI breed groups were compared to mixed-breed dogs, groups 3 (Terriers) and 6 (Scent hounds and related breeds) had a significantly higher risk for owner-reported skin symptoms. On the other hand, groups 5 (Spitz and primitive types) and 10 (Sighthounds) had a significantly lower risk for owner-reported skin symptoms. The environmental factors found significantly associated with less owner-reported skin symptoms and veterinary-verified CAD were i) being born in the owner family and ii) living with other dogs. In addition, significant association of less owner-reported skin symptoms was found with dogs living in a detached house. Factors that were significantly associated with more owner-reported skin symptoms included extremely clean household and over 50 % of white colour in the coat. In the clinical diet intervention study, 46 client-owned atopic and healthy Staffordshire bull terriers were fed two different kind of diets (raw and dry food). Haematological and clinical chemistry profiles, folate, B12, iron, and transforming growth factor β1 (TGF- β1) concentration were analyzed. In addition, gene expression profiles were determined from the skin samples of eight dogs. Cholesterol was significantly increased in the dry food fed dogs and decresed in the raw food fed dogs. In addition, the raw food significantly decreased, among others, alkaline phosphatase and glucose. Plasma folate and B12 and whole blood iron were significantly decreased, and TGF- β1 significantly increased by the raw food diet. The skin gene expression was also affected by the diet. There were genes related to immune defence, reactive oxygen species and antioxidants upregulated in the dogs fed raw food. Several genes were found differentially expressed between atopic and healthy dogs, some unrelated to diet fed and some differentially affected by the diet in atopic and healthy dogs. These results are preliminary and should be confirmed using more samples. Nevertheless, they give an interesting and novel information about the effect of the diet on the skin gene expression. In conclusion, this thesis presents results from different study designs all concentrating on the same disease, canine atopic dermatitis. Many results presented here are related to immune defence and exposure to microbes. Too clean environment and food might not stimulate the immunity enough and could lead to incorrect development of the immune system in young animals. The incomplete stimulation could result in CAD and hypersensitivities later in life, and should be considered when thinking of the prevention of these diseases.Koirien atooppinen dermatiitti (atopia) on multifaktoriaalinen sairaus, jota ei voi parantaa. Sen hoito on monesti hankalaa, jatkuvaa ja kallista. Lisäksi käytetyillä lääkkeillä voi olla sivuvaikutuksia, ja jatkuvat antibioottikuurit lisäävät antibioottiresistenttien bakteerien syntyriskiä. Niinpä enemmän tutkimusta tarvitaan taudin ehkäisykeinoista niin ympäristötekijöiden, perintötekijöiden kuin ravinnonkin osalta. Tämän väitöskirjan tavoitteena oli selvittää koirien atopiaan liittyviä tekijöitä epidemiologisen tutkimuksen ja kliinisen ruokintatutkimuksen keinoin. Ympäristötekijöiden ja atopian yhteyttä selvitettiin validoidun DOGRISK-kyselytutkimuksen avulla. Siinä käytettiin 8643:n koiran populaatiota, joka sisälsi sekä sairaita että atooppisista iho-oireista kärsiviä koiria. Osalla iho-oireisista koirista oli myös eläinlääkärin diagnoosi atopialle. Tämän ryhmän osalta tehtiin myös erillinen analyysi. Iho-oireita esiintyi prosentuaalisesti eniten rodun sisällä valkoisella länsiylämaanterrierillä, bokserilla, englanninbulldoggilla, dalmatialaisella sekä ranskanbulldoggilla. Roturyhmistä merkitsevästi enemmän iho-oireita sekarotuisiin verrattuna oli ryhmällä 3 (Terrierit) sekä ryhmällä 6 (Ajavat ja jäljestävät koirat) ja toisaalta merkitsevästi vähemmän taas ryhmällä 5 (Pystykorvat ja alkukantaiset koirat) sekä ryhmällä 10 (Vinttikoirat). Vähäisempään iho-oireiden riskiin merkitsevästi olivat yhteydessä se, että koira oli syntynyt omistajaperheeseen, sen kanssa asui muitakin koiria ja se asui omakotitalossa. Kaksi ensimmäistä olivat yhteydessä myös vähäisempään atopiariskiin (diagnosoidut koirat). Suurempaan iho-oireriskiin merkitsevästi olivat yhteydessä hyvin puhdas koti ja yli 50 % valkoista väriä turkissa. Kliinisessä ruokintatutkimuksessa terveet ja atooppiset staffordshirenbullterrierit ruokittiin joko kuivamuonalla tai raakaruoalla noin neljän kuukauden ajan. Koirilta analysoitiin hematologinen ja kliinisen kemian profiilit sekä folaatin, B12-vitamiinin, raudan ja transforming growth factor β1:n (TGF-β1) pitoisuudet verestä. Lisäksi kahdeksalta koiralta analysoitiin ihon geenitoimintaa. Kolesterolipitoisuus veressä laski merkitsevästi raakaruokaa syöneillä koirilla ja nousi merkitsevästi kuivamuonaa syöneillä koirilla. Lisäksi raakaruoka laski merkitsevästi mm. veren alkalisen fosfataasin aktiivisuutta sekä glukoosipitoisuutta. Raakaruokaa syöneillä koirilla veren rauta-, folaatti- ja B12-vitamiinipitoisuus laskivat ja TGF-β1 -pitoisuus nousi merkitsevästi. Ruokavaliolla oli vaikutusta myös ihon geenitoimintaan. Raakaruoka lisäsi mm. immuunipuolustukseen, antioksidantteihin ja reaktiivisiin happiradikaaleihin liittyvien geenien toimintaa. Myös atooppisten ja terveiden koirien välillä löydettiin paljon geenitoiminnan eroja, osaan atopiaan liittyvistä geeneistä vaikutti myös ruokavalio. Tulokset tulee vielä vahvistaa suuremmalla näytemäärällä, mutta tutkimus antaa mielenkiintoisia viitteitä ruokavalion vaikutuksesta ihon geenitoimintaan ja sen huomioimisen tärkeydestä tulevissa tutkimuksissa. Tässä väitöskirjassa tutkittiin koirien atopiaan liittyviä tekijöitä monen eri lähestymistavan avulla. Tuloksista monet liittyvät mm. immuunipuolustukseen ja mikrobialtistukseen. Liian puhdas ympäristö ja ruoka eivät välttämättä stimuloi kehittyvää immuunipuolustusta tarpeeksi nuorilla eläimillä. Tämä voi johtaa immuniteetin heikkoon kehittymiseen ja altistaa esimerkiksi atopialle ja yliherkkyyksille myöhemmin elämässä, ja onkin tärkeä seikka huomioitavaksi, kun halutaan ehkäistä näiden sairauksien syntyä

Koirille tarkoitetun suomalaisen peuranlihan mikrobiologinen laatu ja ravintosisältö

Tämän työn tarkoituksena on kuvata koiran ruokana käytettävän suomalaisen peuranlihan ravintoarvot, raskasmetallipitoisuudet sekä mikrobiologinen laatu. Peuranliha valitaan koiran ruokavalion pohjaksi monesti siinä tilanteessa, kun koira näyttää merkkejä ruoka-aineyliherkkyydestä tai -allergiasta, ja joudutaan etsimään uusia proteiininlähteitä. Kun omistaja itse tai ravitsemusalan ammattilainen suunnittelee koiralle kotiruokavalion tuoreista raaka-aineista, on oleellista tietää käytettävien raaka-aineiden ravintoainepitoisuudet. Lisäksi lihan mikrobiologisen laadun tunteminen on tärkeää silloin, kun omistaja päättää syöttää lihan koiralleen raakana. Jos riista on koiran pääasiallinen ravinnonlähde pitkiä aikoja, voivat riistanlihan mahdollisesti korkeat raskasmetallipitoisuudet nousta ongelmaksi koiran terveyden kannalta. Työssä tutkittiin yhteensä 50 peuranlihanäytettä kymmenestä eri erästä. Näytteistä määritettiin lihan mikrobiologinen laatu sekä viljelemällä että PCR-menetelmän avulla. Lisäksi kahdesta lihaerästä lähetettiin näytteet ravintoaine- ja raskasmetallianalyyseihin. Kahdelle koiralle ja kahdelle kissalle syötettiin tutkittavaa peuranlihaa ja eläimiltä kerättiin sylkinäytteitä, kuppisivelynäytteitä sekä ulostenäytteitä patogeenien tutkimista varten. Peuranliha sisälsi liian vähän useita kivennäis- ja hivenaineita sekä D-, E- ja B12-vitamiinia täyttääkseen koiran ravintoainetarpeet. Liha oli hyvin rasvaista. Kahden koiralle välttämättömän aminohapon määrä lihassa oli melko matala. Kadmium- ja lyijypitoisuudet olivat lihassa matalat. Yhteensä neljästä lihaerästä löydettiin L. monocytogenes PCR-menetelmän avulla ja kahdesta lihaerästä se kyettiin viljelemällä eristämään. Y. enterocolitica sen sijaan todettiin PCR-menetelmällä seitsemästä lihaerästä ja myös se eristettiin kahdesta lihaerästä. PCR-menetelmällä kampylobakteeria löytyi kolmesta lihaerästä ja STEC:ia kaikista lihaeristä. L. monocytogenes eristettiin viljelemällä yhdestä koiran ulostenäytteestä ja kampylobakteeri saman koiran ulosteesta toisella näytteenottokerralla. PCR-menetelmällä saatiin positiivinen kampylobakteeritulos kaikista kissojen ulostenäytteistä sekä kolmesti toisen koiran ulostenäytteistä ja kerran toisen koiran ulostenäytteestä. L. monocytogenes löytyi PCR-menetelmällä kerran toisen koiran ulostenäytteestä. Tutkittu peuranliha ei enterobakteerien osalta täyttänyt olemassa olevia vaatimuksia hyvälaatuisesta lihasta 80 %:ssa eristä, mutta säädettyjä raja-arvoja voi pitää liian matalina, kun niitä sovelletaan eläimille tarkoitettuihin sivutuotteisiin. Patogeeneja esiintyi PCR-menetelmällä paljon, mutta vain harvasta näytteestä saatiin patogeeni eristettyä, mikä viittaa niiden vähäiseen määrään lihassa

Environmental and phenotype-related risk factors for owner-reported allergic/atopic skin symptoms and for canine atopic dermatitis verified by veterinarian in a Finnish dog population

The aim of this cross-sectional study was to observe whether environmental factors and phenotypic traits are associated with owner-reported skin problems and with veterinary diagnosed canine atopic dermatitis (CAD). Data were collected using the validated online DOGRISK questionnaire. Out of the data that the questionnaire provides for analysis, focus was first turned towards addressing questions regarding 'Atopy/allergy (skin symptoms)' using a total of 8643 dogs: 1585 dogs with owner-reported allergic/atopic skin symptoms and 7058 dogs without. A subsequent analysis compared dogs with veterinary-verified CAD (n = 322) as a case group against the 7058 dogs without owner-reported skin symptoms. The association between 21 factors related to the environment, canine phenotypes and breed groups within both populations were analysed using univariable and multivariable logistic regression. The environmental factors that showed a significant inverse association with the risk of owner-reported allergic/atopic skin symptoms were as following: whether the dog was living in a detached house, whether there were other dogs in the household, and whether the dog was born in the current household. Having over 50% white colour in the coat and living in an extremely clean household were significantly associated with an increased risk of owner-reported allergic/atopic skin symptoms. The five breeds demonstrating the highest proportion of owner-reported allergic/atopic skin symptoms were West Highland white terrier, Boxer, English bulldog, Dalmatian and French bulldog. The Fe A de A ration Cynologique Internationale dog breed groups 3 (Terriers) and 6 (Scent hounds and related breeds) showed a significantly higher risk for owner-reported allergic/atopic skin symptoms than mixed breed dogs. In the second population, the inverse association was observed between the risk of CAD and the presence of other dogs in the household, and whether the dog had been born in the current household. The results indicate that some environmental factors and canine phenotypes are associated with CAD and owner-reported skin symptoms, but they still do not prove causality.Peer reviewe

Hair arsenic level in rice-based diet-fed Staffordshire bull terriers

Background: There have been concerns related to inorganic arsenic (iAs) in rice and the risk of chronic toxicity in human beings, especially children. Rice is a common constituent of pet food, and dogs often eat the same food on a continual daily basis for long periods of time. Therefore, the purpose of this study was to assess the risk of chronic iAs exposure in rice-based diet-fed dogs. Methods: Hair iAs level was measured in seven rice-based diet-fed dogs (mean age 3.8 years) and in nine dogs that did not consume any rice (mean age 4.4 years), using inductively coupled plasma mass spectrometry. Results: The hair iAs level was significantly higher (P=0.005) in dogs fed a rice-based diet (mean 0.143 µg/g) than in dogs that did not consume any rice (mean 0.086 µg/g), while age and sex did not show associations with hair iAs level. Conclusion: The results suggest that eating a rice-based diet for long periods of time represents a risk for chronic iAs exposure in dogs.Peer reviewe

Identification of modifiable pre- and postnatal dietary and environmental exposures associated with owner-reported canine atopic dermatitis in Finland using a web-based questionnaire

A cross-sectional hypothesis generating study was performed to investigate modifiable exposures such as whether feeding pattern (a non-processed meat based diet, NPMD, or an ultra-processed carbohydrate based diet, UPCD), certain environmental factors and their timing of exposure might be associated with the development of canine atopic dermatitis (CAD). Also, genetic and demographic factors were tested for associations with CAD. The data was collected from the validated internet-based DogRisk food frequency questionnaire in Finland. A total of 2236 dogs were eligible for the study (the owners reported 406 cases and 1830 controls). Our main interest was to analyze modifiable early risk factors of CAD, focusing on nutritional and environmental factors. We tested four early life periods; prenatal, neonatal, early postnatal and late postnatal periods. Twenty-two variables were tested for associations with CAD using logistic regression analysis. From the final models we identified novel dietary associations with CAD: the NPMD during the prenatal and early postnatal periods had a significant negative association with the incidence of CAD in adult dogs (age above 1 year). Oppositely, UPCD was associated with a significantly higher risk for CAD incidence. Other variables that were associated with a significantly lower risk for CAD were maternal deworming during pregnancy, sunlight exposure during early postnatal period, normal body condition score during the early postnatal period, the puppy being born within the same family that it would stay in, and spending time on a dirt or grass surface from 2 to 6 months. Also, the genetic factors regarding maternal history of CAD, allergy-prone breeds and more than 50% white-colored coat all showed a significant positive association with CAD incidence in agreement with previous findings. Although no causality can be established, feeding NPMD early in life seemed to be protective against CAD, while UPCD could be considered a risk factor. Prospective intervention studies are needed to establish the causal effects of the protective role of NPMD on prevalence of CAD during the fetal and early postnatal life.Peer reviewe

Owners perception of acquiring infections through raw pet food: a comprehensive internet-based survey

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Diet and dog characteristics affect major and trace elements in hair and blood of healthy dogs

Obtaining correct amounts of essential elements, and avoiding toxic metals are key factors in dog health. Through analyzing major and trace elements in hair and blood of 50 healthy companion dogs using ICP-MS, we study their associations with dog characteristics and diet, hypothesizing that eating the same diet long-term results in strong correlations between hair and blood element concentrations, and that dog characteristics and diet affect element status. The correlation between hair and blood was significant for Hg (R = 0.601, p = 0.000) and Pb (R = 0.384, p = 0.010). The following associations were significant (p < 0.05): Dark hair had higher Ca and Mg compared to light hair. Females had higher hair Zn, blood Mn, and blood As compared to males. Blood Mn and Se increased, while blood Pb decreased with age. Raw diet fed dogs had higher hair Zn and Se compared to dry or mixed diet fed dogs, and lower blood Mn compared to dry diet fed dogs. Dry and mixed diet fed dogs had higher blood Cd compared to raw diet fed dogs. Mixed diet fed dogs had higher hair Ca and Mg compared to raw or dry diet fed dogs, and higher hair Pb compared to dry diet fed dogs. Wild game consumption was associated with higher blood Pb, and rice consumption with higher blood As. In conclusion, hair provides an alternative for assessing Hg and Pb exposure, and major and trace elements status is affected by hair color, sex, age, and diet.Peer reviewe

Urban environment predisposes dogs and their owners to allergic symptoms

Our companion-animals, dogs, suffer increasingly from non-communicable diseases, analogous to those common in humans, such as allergic manifestations. In humans, living in rural environments is associated with lower risk of allergic diseases. Our aim was to explore whether a similar pattern can be found in dogs, using a nation-wide survey in Finland (n = 5722). We characterised the land-use around dog's home at the time of birth as well as around its current home, and described several lifestyle factors. The severity of owner-reported allergic symptoms in dogs was estimated with a comprehensive set of questions, developed by experts of canine dermatology. Also, the prevalence of diagnosed allergies in dog owners was recorded. The results indicate that allergic symptoms are more prevalent in urban environments both in dog owners and in dogs (accounting the effect of dog breed). Several factors related to rural living, such as bigger family size and regular contact with farm animals and other pets, were also protective against allergic symptoms in dogs. Interestingly, allergic dogs were more likely to have allergic owners than healthy dogs were. Therefore, we suggest that the mutual presence of allergic symptoms in both species indicates common underlying causal factors of allergic diseases.Peer reviewe

The effect of a raw vs dry diet on serum biochemical, hematologic, blood iron, B12, and folate levels in Staffordshire Bull Terriers

Background To date, very few studies have compared the effects of different types of feeding practices on canine physiology, such as feeding exclusively dry, raw, or homemade foods. Objectives We aimed to report the changes in hematologic, serum biochemical, plasma folate, B-12, and whole blood iron levels in dogs fed two different diets. Methods A pilot study was developed to compare the effects of a heat-processed high carbohydrate (HPHC) and nonprocessed high-fat (NPHF) diet. A total of 33 client-owned Staffordshire Bull Terriers were used; 18 had canine atopic dermatitis, seven were healthy, and eight were grouped as "borderline" dogs since they did not fulfill at least six of Favrot's criteria. The comparisons were made between the diet groups at the end visit of the diet intervention, as well as within the diet groups during the study. Results Significant differences between and within the diet groups were observed, although the majority of outcomes remained within the RIs. The median time of diet intervention was 140 days. Red blood cell counts, mean cell hemoglobin concentrations, and platelet counts were significantly higher, and mean cell hemoglobin, mean cell volume, alkaline phosphatase, inorganic phosphorus, and cholesterol were significantly lower in the dogs fed the NPHF diet compared with those fed the HPHC diet after the diet trial was completed. In addition, folate, B-12, and iron decreased significantly in the NPHF diet group. Conclusions This pilot study indicated that diet had an impact on blood values, although most remained within RIs, pointing out the need for further studies.Peer reviewe

Whole blood trace element and toxic metal concentration in dogs with idiopathic epilepsy and healthy dogs : A case-control study

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