Immunohistochemical localization and quantitative assessment of GnRH-, FSH-, and LH-receptor mRNA Expression in canine skin: a powerful tool to study the pathogenesis of side effects after spaying

It has been proposed that gonadotropins and/or gonadotropin releasing hormone (GnRH) could be involved in the pathophysiology of the side effects after spaying in bitches, such as urinary incontinence and an increased production of a woolly undercoat. In order to provide tools to investigate the role of these hormones in dogs we developed immunohistochemical techniques and real-time RT-PCR to study whether GnRH-, LH-, and FSH-receptors exist in canine skin and urinary bladder. Tissue samples from the skin of the flank region and the ventral midline of the urinary bladder from euthanised dogs were examined. We were able to quantify mRNA expression of GnRH-, FSH-, and LH-receptors in canine skin and bladder biopsies with a high primer efficacy. Immunohistochemical studies showed that GnRH-, FSH-, and LH-receptors are expressed in vessel walls, the epidermis, the hair follicle and in sebaceous and sweat glands in canine skin and in transitional epithelium, and smooth muscle tissue in the urinary bladder. Our data provide the fundamentals to examine the distribution of FSH-, LH-, and GnRH-receptors in canine skin and urinary bladder and to assess gene activity at the transcriptional level by real-time RT-PC

Luteal and placental function in the bitch: spatio-temporal changes in prolactin receptor (PRLr) expression at dioestrus, pregnancy and normal and induced parturition

Background: Endocrine mechanisms governing canine reproductive function remain still obscure. Progesterone (P4) of luteal origin is required for maintenance of pregnancy. Corpora lutea (CL) are gonadotrop-independent during the first third of dioestrus; afterwards prolactin (PRL) is the primary luteotropic factor. Interestingly, the increasing PRL levels are accompanied by decreasing P4 concentrations, thus luteal regression/luteolysis occurs in spite of an increased availability of gonadotropic support. PRL acts through its receptor (PRLr), the expression of which has not yet been thoroughly investigated at the molecular and cellular level in the dog. Methods: The expression of PRLr was assessed in CL of non-pregnant dogs during the course of dioestrus (days 5, 15, 25, 35, 45, 65 post ovulation; p.o.) as well as in CL, the utero/placental compartments (Ut/Pl) and interplacental free polar zones (interplacental sites) from pregnant dogs during the pre-implantation, post-implantation and mid-gestation period of pregnancy and during the normal and antigestagen-induced luteolysis. Expression of PRLr was tested by Real Time PCR, immunohistochemistry and in situ hybridization. Results: In non-pregnant CL the PRLr expression was significantly upregulated at day 15 p.o. and decreased significantly afterwards, towards the end of dioestrus. CL of pregnancy showed elevated PRLr expression until mid gestation while prepartal downregulation was observed. Interestingly, placental but not interplacental expression of PRLr was strongly time-related; a significant upregulation was observed towards mid-gestation. Within the CL PRLr was localized to the luteal cells; in the Ut/Pl it was localized to the fetal trophoblast and epithelial cells of glandular chambers. Moreover, in mid-pregnant animals treated with an antigestagen, both the luteal and placental, but not the uterine PRLr were significantly downregulated. Conclusions: The data presented suggest that the luteal provision of P4 in both pregnant and non-pregnant dogs may be regulated at the PRLr level. Furthermore, a role of PRL not only in maintaining the canine CL function but also in regulating the placental function is strongly suggested. A possible functional interrelationship between luteal P4 and placental and luteal PRLr expression also with respect to the prepartal luteolysis is implied

Endocrine control of canine mammary neoplasms: serum reproductive hormone levels and tissue expression of steroid hormone, prolactin and growth hormone receptors

BACKGROUND: Neoplasms of the mammary gland are among the most common diseases in female domestic dogs (Canis familiaris). It is assumed that reproductive hormones influence tumorigenesis in this species, although the precise role of the endocrine milieu and reproductive state is subject to continuing discussion. In line with this, a recent systematic review of available data on the development of mammary neoplasms revealed weak evidence for risk reduction after neutering and an effect of age at neutering. Investigation of several hormone receptors has revealed decreased expression of estrogen receptor-alpha (ERα, ESR1), progesterone (P4) receptor (PGR), prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) associated with neoplastic differentiation of mammary tissues. In other studies, increased levels of estrogens, progesterone and prolactin were found in serum and/or tissue homogenates of dogs with malignant neoplasms. However, the association between these entities within one animal population was never previously examined. Therefore, this study investigated the association between circulating serum concentrations of estradiol-17β, progesterone and prolactin, and gene expression of ERα (ESR1), ERβ (ESR2), PGR, PRLR, PRL and GHR, with respect to reproductive state (spayed vs. intact) and cycle stage (anestrus vs. diestrus). Additionally, the expression of E-cadherin (CDH-1) was evaluated as a possible indicator of metastatic potential. RESULTS: For all receptors, the lowest gene expression was found in malignant tumors compared to normal tissues of affected dogs. Steroid levels were not influenced by their corresponding receptor expression in mammary neoplasms, but increased PRL levels were negatively associated with low PRLR gene expression in malignant tumors. The expression of CDH-1 was influenced by tumor malignancy and cycle stage, i.e., the highest gene expression was found in benign mammary tumors in diestrous dogs compared to normal and malignant mammary tissues of anestrous and spayed dogs. CONCLUSIONS: Herein, it has been confirmed that transformation towards malignant neoplasms is associated with significant reduction of gene expression of particular hormone receptors. Only PRLR in malignant tumors seems to be influenced by circulating PRL levels. In dogs, CDH-1 can be used as a prognostic factor; its expression, however, in benign tumors is influenced by cycle stage

Brucella canis infection in a young dog with epididymitis and orchitis

The following case report describes the clinical and diagnostic procedure for suspected brucellosis infection in a dog. A 21 month old intact male Border Collie was presented with an enlarged right testicle and epididymis. The dog was imported to Switzerland from Germany at the age of three months, but was never abroad since then. Clinical and laboratory diagnostic investigation included bacteriology and histology. An initial serological evaluation by means of rapid slide agglutination test (RSAT) was negative. Repeated examination of the same serum by a chromatographic immunoassay (ICT) revealed a positive result. Brucella canis infection was confirmed by culture. The present case is intended to underline the importance of the suspected diagnosis of 'brucellosis' in the presence of reproductive tract problems in dogs. In addition, Brucella canis has zoonotic potential and it is imperative to comply with strict hygiene management

Ergebnis der selektiven Verpaarung beim Entlebucher Sennenhund zur Reduktion der ureteralen Ektopie

Outcome of selective mating in the Entlebucher Mountain Dog for reduction of ureteral ectopia The Entlebucher Mountain Dog is predisposed to ureteral ectopia and associated diseases of the urinary tract as well as the kidneys, which can have severe to lethal consequences. Due to the clustered occurrence of clinical signs in 11 % of Entlebucher Mountain dogs in the absence of a genetic test for ureteral ectopia, screening was introduced in 2008 to allow phenotype-based breeding selection. The ureteral orifices of the dogs are visualized by ultrasound and existing urinary retention or urinary incontinence is documented. The diagnostic findings were evaluated centrally with assignment to one of five phenotypes depending on the localization of the ureteral orifices and the renal and ureteral shape. Breeding approval and mating restrictions are the responsibility of the respective breeding associations and predominantly Entlebucher Mountain Dogs with extravesical ectopic ureters and/or clinical signs were excluded from breeding. The effect of phenotype-based selective mating on the incidence of ureteral ectopia and its clinical signs, as well as possible factors influencing the expression of the phenotype, were determined in the birth cohorts after the introduction of screening. Analysis of the data set of 1456 phenotyped Entlebucher Mountain Dogs showed, that at 11 % versus 5 %, males were more frequently assigned to the extravesical phenotype than females. The effect of phenotype-based breeding selection was examined in a subpopulation consisting of phenotyped parents and their offspring (n = 876). The prevalence of the extravesical phenotype decreased from 24 % in the 2005 to 2007 birth cohorts to 1,4 % in the 2015 to 2017 birth cohorts. Since 2015 almost no Entlebucher Mountain Dogs with incontinence, hydroureter or hydronephrosis have been recorded. It was feared that the additional selection measures to control ureteral ectopia in the small Entlebucher Mountain Dog population would intensify the inbreeding increase. However, this has so far remained absent. Therefore, as long as no genetic test is available, it is recommended to continue phenotype-based breeding selection with exclusion of dogs with extravesical ureteral ectopia and/or hydroureter/hydronephrosis/urinary incontinence, while keeping an eye on the development of the inbreeding coefficient. Keywords: Ectopic ureter, Dog, Inbreeding, Phenotype, Breeding selection Deutsch Ergebnis der selektiven Verpaarung beim Entlebucher Sennenhund zur Reduktion der ureteralen Ektopie Der Entlebucher Sennenhund ist prädisponiert für die ureterale Ektopie und damit für assoziierte Erkrankungen der Harnwege sowie der Nieren, was schwerwiegende bis letale Folgen haben kann. Aufgrund des gehäuften Auftretens klinischer Symptome bei 11 % der Entlebucher Sennenhunde wurde in Ermangelung eines Gentests auf ureterale Ektopie 2008 ein Screening eingeführt, um eine Phänotyp-basierte Zuchtselektion zu ermöglichen. Die Uretermündungen der Hunde werden in der Regel mittels Ultraschall lokalisiert und bestehender Harnrückstau oder Harninkontinenz wird dokumentiert. Die Befundung erfolgte zentral mit einer Zuordnung zu einem von fünf Phänotypen in Abhängigkeit von der Lokalisation der Uretermündungen sowie der Nieren – und Ureterengestalt. Die Zuchtzulassung und Verpaarungsbeschränkungen obliegen den jeweiligen Zuchtverbänden, wobei überwiegend Entlebucher Sennenhunde mit extravesikal ektopischen Ureteren und/oder klinischen Symptomen von der Zucht ausgeschlossen wurden. Die Auswirkung der Phänotyp-basierten selektiven Verpaarung auf das Auftreten der ureteralen Ektopie und deren klinischen Symptome sowie mögliche Einflussfaktoren auf die Ausprägung des Phänotyps wurden in den Geburtsjahren nach Einführung des Screenings ermittelt. Die Analyse des Datensatzes mit 1456 phänotypisierten Entlebucher Sennenhunden zeigte, dass mit 11 % versus 5 % Rüden häufiger als Hündinnen dem extravesikalen Phänotyp zugeteilt wurden. Die Auswirkung der Phänotyp-basierten Zuchtselektion wurde an einer Teilpopulation, bestehend aus phänotypisierten Elterntieren und ihren Nachkommen (n = 876), untersucht. Die Prävalenz des extravesikalen Phänotyps nahm von 24 % bei den Geburtsjahren 2005 bis 2007 auf 1,4 % bei den Geburtsjahren 2015 bis 2017 ab. Seit 2015 wurden nahezu keine Entlebucher Sennenhunde mehr mit Inkontinenz, Hydroureter oder Hydronephrose erfasst. Befürchtet wurde, dass die zusätzlichen Selektionsmassnahmen zur Bekämpfung der ureteralen Ektopie in der kleinen Entlebucher Sennenhundepopulation den Inzuchtanstieg verstärken würde. Dies blieb bisher jedoch aus. Daher wird, solange kein genetischer Test zur Verfügung steht, empfohlen, die Phänotyp-basierte Zuchtselektion mit Ausschluss von Hunden mit extravesikaler ureteraler Ektopie und/oder Hydroureter/Hydronephrose/Harninkontinenz vorerst weiterzuführen und gleichzeitig die Entwicklung des Inzuchtkoeffizienten im Auge zu behalten. Schlüsselwörter: Ektopischer Ureter, Hund, Inzucht, Phänotyp, Zuchtselektion Français Résultat de l’accouplement sélectif chez le bouvier de l’Entlebuch pour réduire l’ectopie urétérale Le Bouvier de l’Entlebuch est prédisposé à l’ectopie urétérale et aux maladies associées des voies urinaires ainsi que des reins, ce qui peut entraîner des conséquences fatales. En raison de l’apparition de signes cliniques chez 11 % des chiens et en l’absence d’un test génétique pour l’ectopie urétérale, un dépistage a été introduit en 2008 pour permettre une sélection d’élevage basée sur le phénotype. Les orifices urétraux des chiens ont été visualisés par échographie et la rétention ou l’incontinence urinaire existante documentée. Les résultats du diagnostic ont été évalués de manière centralisée avec attribution à l’un des cinq phénotypes en fonction de la localisation des orifices urétéraux ainsi que de la forme des reins et des uretères. L’approbation pour la reproduction et les restrictions d’accouplement relèvent de la responsabilité des associations d’élevage respectives et les bouviers de l’Entlebuch présentant des uretères ectopiques extravésicaux et/ou des signes cliniques ont majoritairement été exclus de la reproduction. L’effet de cet accouplement sélectif basé sur le phénotype sur l’incidence de l’ectopie urétérale et de ses signes cliniques ainsi que les facteurs possibles influençant l’expression du phénotype ont été déterminés dans les cohortes de naissance après l’introduction du dépistage. L’analyse de l’ensemble des données de 1456 Bouviers de l’Entlebuch phénotypés a montré que, à 11 % contre 5 %, les mâles étaient plus fréquemment affectés au phénotype extravésical que les femelles. L’effet de la sélection d’élevage basée sur le phénotype a été examiné dans une sous-population composée de parents phénotypés et de leur progéniture (n = 876). La prévalence du phénotype extravésical est passée de 24 % dans les cohortes de naissance de 2005 à 2007 à 1,4 % dans les cohortes de naissance de 2015 à 2017. Depuis 2015, presque aucun bouvier d’Entlebuch présentant une incontinence, un hydrouretère ou une hydronéphrose n’a été enregistré. Une possible augmentation de la consanguinité due aux mesures de sélection supplémentaires visant à contrôler l’ectopie urétérale ne s’est pas produite. Par conséquent, tant qu’aucun test génétique n’est disponible, il est recommandé de poursuivre la sélection d’élevage basée sur le phénotype avec exclusion des chiens présentant une ectopie urétérale extravésicale et/ou une hydrouretère/hydronéphrose/incontinence urinaire, tout en surveillant l’évolution du coefficient de consanguinité. Mots-clés: auretère ectopique, chien, consanguinité, phénotype, sélection d’élevage Italiano Risultato dell'accoppiamento selettivo nel cane da montagna Entlebuch per ridurre l'ectopia ureterale Il bovaro dell’Entlebuch è predisposto all’ectopia ureterale e quindi alle malattie che sono correlate al tratto urinario e renale con possibili conseguenze letali. A causa della frequente comparsa di segni clinici nell’11 % dei cani e in assenza di un test genetico per l’ectopia ureterale, nel 2008 è stato introdotto uno screening per consentire la selezione fenotipica dei riproduttori. Gli orifizi ureterali dei cani sono solitamente localizzato tramite ecografia e viene documentata la presenza di ritenzione o incontinenza urinaria. I risultati sono stati valutati in modo centralizzato e si è assegnato il risultato a uno dei cinque fenotipi a seconda della localizzazione degli orifizi ureterali e della forma dei reni e degli ureteri. L’autorizzazione all’allevamento e le restrizioni all’accoppiamento sono di competenza delle rispettive associazioni di allevatori e i bovari dell’Entlebuch con ureteri ectopici extravescicali e/o con segni clinici sono stati prevalentemente esclusi dalla riproduzione. L’effetto dell’accoppiamento selettivo, basato sul fenotipo per rapporto all’incidenza dell’ectopia ureterale e dei suoi segni clinici, nonché dei possibili fattori che influenzano l’espressione del fenotipo, è stato determinato nelle coorti di nascita dopo l’introduzione dello screening. L’analisi del set di dati di 1456 bovari dell’Entlebuch fenotipizzati ha dimostrato che, con una percentuale dell’11 % rispetto al 5 %, i maschi erano più frequentemente assegnati al fenotipo extravescicale rispetto alle femmine. L’effetto della selezione riproduttiva basata sul fenotipo è stato esaminato in una subpopolazione composta da genitori fenotipizzati e dalla loro discendenza (n = 876). La prevalenza del fenotipo extravescicale è diminuita dal 24 % nelle coorti di nascita dal 2005 al 2007 all’1,4 % nelle coorti di nascita dal 2015 al 2017. Dal 2015 non sono stati registrati quasi più bovari dell’Entlebuch con incontinenza, idrouretere o idronefrosi. Non si è riscontrato un possibile aumento della consanguineità dovuto alle misure di selezione aggiuntive per controllare l’ectopia ureterale. Pertanto, finché non è disponibile un test genetico, si raccomanda di continuare la selezione fenotipica con l’esclusione dei cani con ectopia ureterale extravescicale e/o idrouretere/idronefrosi/incontinenza urinaria, facendo particolare attenzione all’andamento del coefficiente di consanguineità. Parole chiavi: Uretere ectopico, cane, consanguineità, fenotipo, selezione riproduttiv

First report of Cytauxzoon sp. infection in domestic cats in Switzerland: natural and transfusion-transmitted infections

BACKGROUND: Cytauxzoonosis is an emerging tick-borne disease of domestic and wild felids. Cytauxzoon felis induces severe and often fatal disease in domestic cats. In Europe, clinical and subclinical infections caused by Cytauxzoon sp. are described. We report the first cases of Cytauxzoon sp. infection in domestic cats in Switzerland. METHODS: Clinical and laboratory data and results of PCR analyses were collected from Cytauxzoon sp. PCR-positive cats and the cats followed for up to 851 days. RESULTS: The cases were three two-month old kittens from the same litter (Cases 1-3) and two adult domestic shorthair cats (Cases 4 and 5). The cats originated from the north-west and west of Switzerland. Cases 1-3 presented with moderate to severe regenerative anaemia and intraerythrocytic inclusions. Cytauxzoon sp. was confirmed by PCR and sequencing. The kittens made a clinical and haematological recovery after blood transfusion and/or treatment with azithromycin and atovaquone, but erythroparasitaemia persisted. Case 4 presented with severe non-regenerative anaemia. Case 5 was healthy and used as a blood donor for Case 4. Following blood transfusion, Case 4 showed intraerythrocytic inclusions, and Cytauxzoon sp. was confirmed in both Cases 4 and 5 using PCR and sequencing. Case 4 achieved clinical and haematological remission after treatment with azithromycin, atovaquone and immunosuppressive drugs. Eight months later, Case 4 was presented again with anaemia but tested Cytauxzoon sp. PCR-negative. Sequencing of 1637 bp of the 18S rRNA gene of Cytauxzoon sp. revealed 100% nucleotide sequence identity among isolates of Cases 1-3 and between isolates of Cases 4 and 5, and 99% sequence identity between isolates of all cases. Phylogenetic analysis revealed the closest relationship of the Swiss isolates to Cytauxzoon sp. isolates from domestic cats and wild felids from France, Spain and Romania and to Cytauxzoon manul from a Pallas's cat. CONCLUSIONS: This is the first report of Cytauxzoon sp. infection in domestic cats in Switzerland. It is also the first report of infection in very young kittens and transmission of Cytauxzoon sp. to an adult cat by transfusion of blood from an asymptomatic cat. The cats recovered but some developed chronic asymptomatic erythroparasitaemia for up to 28 months. Domestic cats may act as reservoirs for Cytauxzoon sp. in Europe and blood donor cats should be screened for this agent by PCR