Distinct Mannheimia haemolytica serotypes isolated from fatal infections in veal calves and dairy cows.

Fatal Mannheimia haemolytica (M. haemolytica) infections in cattle, which emerged in the Netherlands between 2004 and 2018, showed two distinct disease presentations: acute fibrinous polyserositis (FPS) in veal calves, and acute fibrinous pleuro-pneumonia (FPP) in adult dairy cattle. To determine whether these presentations were caused by different M. haemolytica genotypes, whole genome sequencing was performed on 96 isolates cultured after necropsy from inflamed sites of veal calves that died of M. haemolytica-associated FPS (n = 49) or with FPP lesions (n = 2), and from dairy cows that died of M. haemolytica-associated FPP (n = 45). Among the 96 M. haemolytica isolates, 93 were shown to belong to either of two large clusters, with 48/51 calf isolates belonging to one, and 43/45 cow isolates and two calf isolates from cases of FPP to the other. All M. haemolytica isolates from veal calves with FPS were of serotype A2, whereas the isolates from dairy cows and two calves with FPP were predominantly of serotypes A1 and A6. Most serotype A2 isolates from veal calves with FPS (95.6 %) contained multiple antibiotic resistance genes (ARGs) against three to five antimicrobial classes (phenicols, sulphonamides, tetracyclines, aminoglycosides or beta-lactams). In contrast, these ARGs were only present in 10.8 % of M. haemolytica A1 and A6 isolates from pneumonic adult cattle and absent in isolates from the two calves with FPP. These two disease presentations appear to be caused by genetically distinct strains with different antimicrobial resistance gene patterns. While M. haemolytica serotype A2 is generally considered to be a commensal microorganism of cattle, it was clearly associated with fatal FPS in veal calves in the Netherlands.

Emergence of fatal Mannheimia haemolytica infections in cattle in the Netherlands.

In the Dutch national surveillance system, outbreaks of fatal infections by Mannheimia haemolytica (M. haemolytica) in dairy cows and veal calves have become apparent in recent years. These observations prompted an in-depth analysis of available pathology data over the period 2004-2018 to investigate changes in the occurrence and/or expression of M. haemolytica-associated cattle disease. With multilevel logistic regression models, time trends were identified and corrected for farm, season, pathologist and region. Deaths associated with M. haemolytica infection increased over time with dairy cows and veal calves diagnosed with fatal M. haemolytica infections 1.5 and 1.4 times more frequently every following 3-year period between 2004 and 2018, respectively. M. haemolytica-associated disease showed two distinct disease presentations: acute pleuropneumonia in dairy cows and polyserositis in veal calves. The prevalence of both disease presentations with M. haemolytica confirmed increased in each 3-year time period between 2004 and 2018, with an odds ratio (OR) of 1.5 for acute pleuropneumonia in dairy cows and an OR of 1.7 for polyserositis in veal calves. No change was found for M. haemolytica-associated disease in dairy calves. Although M. haemolytica is considered an opportunist bovine pathogen, and the presence of primary pathogens such as BHV-1, BVDV and Mycoplasma species was not completely ruled out in our study, substantial evidence is provided to indicate infections with M. haemolytica were the most likely cause of death. M. haemolytica-associated diseases occurred more often in October-June than July-September, and were detected more often in necropsied animals from the North, South and East Netherlands than the West Netherlands.

Quantification of the health-status of the Dutch Labrador retriever population.

Health issues in purebred dogs are currently considered one of the biggest problems in companion animal health. The Labrador retriever (LR) is one of the most popular dog breeds. The aim of this study was to quantify LR breed health in comparison with mixed-breed dogs (MB), by using four different data sources: a veterinary practice management system (appr. 35,000 unique individuals LR + MB), data from two animal insurance companies (appr. 15,500 and 4500 individuals respectively), and a histopathological laboratory (appr. 4000 individuals). After extensive recoding of the data, health parameters utilised to quantify breed health were longevity, frequency of practice visits and insurance expense claims, and diagnostic codes. A Kaplan-Meier univariate and multivariable Cox proportional hazard model were used to evaluate longevity. A negative binomial model was used to analyse the frequency of visits, claims, and diagnostic codes in both sets of insurance data. Logistic regression was used to look into the categorical diagnostic codes in the laboratory data. The median lifespan of the LR was similar (12 years, practice data) or longer (10 versus 8 years, insurance data) than MB for individuals with a known birth and death date. When including censored individuals, survival time in the LR was comparable to MB individuals up to 10 years of age. Above 10 years of age, the LR lived a similar length as MB with a medium to large body size, but shorter than all MB. The LR visited the veterinary practice more often (risk ratio (RR) 1.2, 95% confidence interval 1.2-1.3), and also showed a higher frequency of insurance expense claims (RR 2.2 (2.1-2.3) and RR 1.2 (1.1-1.3) respectively for the two insurance data sets). The largest difference in organ systems between the LR and MB in insurance claims was related to ears (RR 5.3 (4.8-5.8) and RR 2.6 (2.3-3.1)), followed by airways (RR 2.6 (2.4-2.8)), tendons & muscles (RR 2.4 (2.2-2.6) and RR 1.4 (1.1-1.7)), and joints (RR 1.7 (1.3-2.1)), without a difference in median age at diagnosis. The data from the histopathological laboratory suggested a higher disease burden related to oncology for the LR compared to MB (OR 1.2, 95% CI 1.0-1.3). Oncological diagnoses were made at a younger age in the LR (8.8 versus 9.4 years). The disease burden was significantly higher for the LR than MB, but these results may suffer from substantial bias such as selection bias towards the database, and different behaviour of LR versus MB owners with regards to veterinary care. In the future, longer term population data can corroborate these results.

Ovine and Bovine Congenital Abnormalities Associated With Intrauterine Infection With Schmallenberg Virus.

In December 2011, a previously unknown congenital syndrome of arthrogryposis and hydranencephaly in sheep and cattle appeared in the Netherlands as an emerging epizootic due to Schmallenberg virus (SBV). Gross lesions in 102 lambs and 204 calves included porencephaly, hydranencephaly, cerebellar dysplasia and dysplasia of the brainstem and spinal cord, a flattened skull with brachygnathia inferior, arthrogryposis, and vertebral column malformations. Microscopic lesions in the central nervous system showed rarefaction and cavitation in the white matter, as well as degeneration, necrosis, and loss of neurons in the gray matter. Brain and spinal cord lesions were more severe in lambs than in calves. Ovine and bovine cases examined early in the outbreak showed encephalomyelitis. SBV infection was confirmed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in brain samples in 46 of 102 lambs (45%) and in 32 of 204 calves (16%). Immunohistochemistry, performed on tissue samples from 18 RT-qPCR-positive lambs, confirmed the presence of bunyaviral antigen in neurons of the brain in 16 cases. SBV antibodies were detected by enzyme-linked immunosorbent assay in fetal blood in 56 of 61 sampled ovine cases (92%). In a virus neutralization test, all tested dams of affected newborns, 46 ewes and 190 cows, were seropositive. Compared with other teratogenic viral infections, the pathogenesis and lesions of SBV in sheep and cattle fetuses are similar to those of other ruminant orthobunyaviruses. However, the loss of spinal ventral motor neurons and their tracts, resulting in micromyelia, distinguishes SBV infection from other viral central nervous system lesions in newborn ruminants.

Peri-articular histiocytic sarcoma and previous joint disease in Bernese Mountain Dogs.

BACKGROUND: Peri-articular histiocytic sarcoma (PAHS) occurs in dogs, including Bernese Mountain Dogs (BMD). An etiologic relationship with previous joint disease has not been documented. HYPOTHESIS: Peri-articular histiocytic sarcoma in BMD will be more frequently encountered around previously diseased joints compared with normal joints. ANIMALS: 920 European BMD. METHODS: A retrospective study, in which data were obtained through an Internet questionnaire and from 2 veterinary pathology laboratories. Archived samples of hematoxylin-eosin (H&E) staining diagnosed PAHS and synovial cell sarcoma (SCS) were immunolabeled with CD18 and pancytokeratin. Descriptive, comparative, and actuarial statistics comprise the data analysis. RESULTS: All primary synovial tumors were identified as PAHS based on their morphology, positive CD18, and negative pancytokeratin labeling. Joint disease was diagnosed in 226 BMD, of which 15 developed PAHS in a previously diseased joint and 3 in a nondiseased joint. Of the remaining 694 BMD without joint disease, 9 developed PAHS. The odds ratio for a dog with previous joint disease developing PAHS is calculated as 5.4 (95% CI: 2.3-12.5; P < .0001) compared with no previous joint problem. A significant association between previous joint disease and PAHS in the same joint was demonstrated for the left elbow (P = .016), right elbow (P = .006), right shoulder (P = .047), left and right stifle (P < .001), and left carpal joint (P = .010). CONCLUSIONS AND CLINICAL IMPORTANCE: The results of this study suggest a relation between previous joint disease and the development of PAHS in the same joint of European BMD. Owners of BMD should monitor dogs for peri-articular swellings, particularly around previously diseased joints.

Immunohistochemical evaluation of p53 expression with different antibodies in malignant canine tumours with or without p53 gene mutation.

Six monoclonal antibodies and a polyclonal antibody (CM1) were used to investigate the overexpression of p53 protein by immunohistochemistry (IHC) in six sarcomas and 21 mammary carcinomas from 27 dogs. IHC was compared with p53 gene mutation analysis performed on the same samples. Only the monoclonal PAb240, PAb421 and the CM1 antibodies were able to detect expression of canine p53 protein. CM1 was found to give the highest concordance (8/11) between positive expression of the p53 protein by IHC and the presence of a gene mutation. In the samples that were negative for p53 expression by IHC, but contained a p53 gene mutation according to DNA analysis, the mutation often affected the epitopes that could have been recognized by these antibodies. Only one out of 16 tumours without a p53 gene mutation had a weakly positive IHC result. These findings indicate that in these two types of canine tumours, IHC - particularly with CM1 - can detect many alterations in p53 expression owing to a gene mutation. False-positive results were very infrequent.

Validation of the use of proliferation markers in canine neoplastic and non-neoplastic tissues: comparison of KI-67 and proliferating cell nuclear antigen (PCNA) expression versus in vivo bromodeoxyuridine labelling by immunohistochemistry.

In order to evaluate the suitability of Ki-67 and proliferating cell nuclear antigen (PCNA) for determination of proliferative activity, the immunohistochemically determined nuclear expression of these antigens in canine non-neoplastic and neoplastic tissues was compared with the results of in vivo bromodeoxyuridine (BrdU) labelling, which - by measurement of the fraction of S-phase cells - is considered as the standard in the analysis of proliferative activity. The samples investigated consisted of non-neoplastic mammary and lymphoid tissues, and of benign and malignant (primary/metastatic) mammary tumours, and malignant lymphomas. Great regional heterogeneity prevented determination of an overall labelling index (LI) in normal lymphoid tissues. In the remaining combined group of samples, LI values were significantly ranked in the order PCNA>Ki-67>BrdU. However, the correlation of Ki-67 or PCNA as compared to BrdU LI values was only moderate in the combined group [approximately 0.5, Spearman rank test] as well as in most subgroups, whilst it was very poor in the group of primary mammary cancers. These observations indicate that Ki-67 or PCNA LIs as markers of proliferation do not evenly match in vivo BrdU labelling.

Locally produced growth hormone in canine insulinomas.

The production and release of GH has been demonstrated in a variety of extra-pituitary tissues. In this respect insulin-producing pancreatic tumours are also of interest because it has been observed that GH may promote islet cell proliferation. However, these effects have only been related to GH of pituitary origin and the possibility of local production of GH with autocrine-/paracrine effects has not been considered. In this study, a reverse transcriptase polymerase chain reaction (RT-PCR) was used to demonstrate the presence of GH mRNA in pancreatic tissue of five healthy dogs and insulinomas of 14 dogs. After Southern blotting of the RT-PCR products, blots were hybridized using a canine-specific GH-probe and quantified using phosphor imaging. GH gene expression was further demonstrated by in situ hybridization using a canine digoxigenin-labelled GH-specific cDNA probe. In addition, GH immunohistochemistry was performed. In five samples of normal pancreatic tissue a weak hybridization signal was found. This signal was significantly higher in nine of 12 primary tumours. In ten of 11 metastases there was a positive hybridization signal, and this signal was also significantly higher than in the primary tumours. In situ hybridization in one sample demonstrated that GH mRNA was only produced in the tumour cells. The local production of GH was confirmed by positive staining of tumour tissue with anti-GH antibodies in ten of 12 samples. It is concluded that canine insulinomas express the gene encoding GH mRNA. The locally produced GH may have an autocrine/paracrine effect on tumour progression. The relatively high expression levels in metastases of these tumours may be related to the low inhibitory influence of somatostatin outside the pancreas.

Canine prostate carcinoma: epidemiological evidence of an increased risk in castrated dogs.

The present retrospective study investigated the frequency of prostate carcinoma (PCA) among prostate abnormalities in dogs and determined whether castration influences the incidence of PCA in dogs. During the years 1993-1998, 15,363 male dogs were admitted to the Utrecht University Clinic of Companion Animals, and of these dogs 225 were diagnosed with prostatic disease. In addition, another 206 male dogs were diagnosed as having prostatic disease based on cytologic examination of aspiration biopsies submitted by referring veterinarians. Benign prostatic hyperplasia was diagnosed in 246 dogs (57.1%), prostatitis in 83 dogs (19.3%), and PCA in 56 dogs (13%). Dogs with PCA were significantly older (mean age=9.9 years) than dogs with other prostatic diseases (mean age=8.4 years). The Bouvier des Flandres breed had an increased risk (odds ratio (OR)=8.44; 95% CI 4.38-16.1) of having PCA. Castration (26/56) increased the risk (OR=4.34; 95% CI 2.48-7.62) of PCA. The mean age at diagnosis of PCA in castrated dogs and in intact male dogs was not significantly different. The interval between castration and onset of prostatic problems was highly variable, suggesting that castration does not initiate the development of PCA in the dog, but it does favour tumor progression.

Clinico-pathological aspects of canine cutaneous and mucocutaneous plasmacytomas.

In this study the clinico-pathological aspects of cutaneous and mucocutaneous plasmacytomas were investigated in 63 dogs (one dog with two tumours). The tumours were most commonly observed in the skin of the trunk and legs. Yorkshire Terrier (n = 8) was the most commonly affected breed and males were affected more commonly than females (36 versus 23, respectively). Plasmacytomas were histologically classified into mature, hyaline, cleaved, asynchronous, monomorphous blastic and polymorphous blastic cell types. Monomorphous blastic cell type was the most frequent type (n = 21), followed by cleaved (n = 19) and asynchronous (n = 11) cell types. Secondary amyloid depositions were observed in eight cases. Immunohistochemical staining showed monoclonal lambda light chain positivity in all cases. In the immunohistochemical staining for cyclin D1, which is a prognostic marker in human plasma cell tumours, moderate numbers of positive tumour cells were observed in only one case of (muco)cutaneous plasmacytoma. All other cases were negative or contained few positive tumour cells. On the other hand, high numbers of tumorous plasma cells reacted positively with cyclin D1 in three out of six cases of canine multiple myelomas. Prognosis of the (muco)cutaneous plasmacytomas was good, except in one dog which developed a lymphoma afterwards. No significant correlations were observed between the cell type and the location of the tumour, presence of amyloid or prognosis.

Tenascin expression in normal, hyperplastic, dysplastic and neoplastic canine mammary tissues.

Mammary tumours are the most common neoplasias of female dogs and may have a complex histological pattern with both epithelial and spindle cells participating in the transformation process. A frequent feature of these tumours is chondroid or bone metaplasia of the extracellular matrix, which mainly occurs in areas of proliferated spindle-shaped cells, probably of myoepithelial origin. The present study evaluates immunohistochemically the expression of tenascin in 186 surgical samples of canine mammary tissues, ranging from normality to neoplasia. Tenascin was present in all mammary tissues studied, with an increased expression in remodelling situations and in neoplastic lesions. Basement membrane was the most frequently labelled structure, but stromal tissue was more often and widely labelled in neoplastic lesions. The extracellular matrix was positive in solid and anaplastic carcinomas as well as in spindle cell proliferation areas. Tenascin expression in extracellular matrix was also abundant in areas of initial chondroid metaplasia and, with variable extension, in almost all cartilage islands of mixed tumours. In well differentiated secretory areas only apical granules of luminal cells were positive, suggesting a different pattern of tenascin expression during secretory differentiation. The digestion of chondroitin sulphate significantly improved the labelling for tenascin when a co-expression of these two molecules was present. Although our results suggest that tenascin cannot be used as a marker of transformation or of malignancy in canine mammary oncology, it is clear that this molecule plays an important role in proliferation and differentiation processes in the canine mammary gland.

Canine balloon and signet-ring cell melanomas: a histological and immunohistochemical characterization.

Seven tumours that were composed of balloon (n=4) or signet-ring (n=3) cells were investigated for a putative melanocytic origin. The tumours were located in the skin or the mouth cavity. In one case a sample from inguinal lymph node metastasis was available. Two antibodies used in man for the immunohistochemical diagnosis of melanomas, namely anti-Melan-A and anti-tyrosinase, were examined for their cross-reactivity with the corresponding canine antigens. The Melan-A antibody labelled all balloon cell tumours and one signet-ring cell tumour, whereas the anti-tyrosinase antibody was not reactive in any of the tumours. The Melan-A antibody also labelled a variety of canine epithelioid and spindle cell melanomas; non-melanocytic tumours were all negative. This study confirmed the occurrence of balloon and signet-ring cell melanomas in dogs. Melan-A antibody was found to be useful in the diagnosis of pigmented and non-pigmented canine melanomas.

Enhancement of polymorphonuclear cell-mediated tumor cell killing on simultaneous engagement of fcgammaRI (CD64) and fcalphaRI (CD89).

Antibodies can efficiently induce antitumor responses via recruitment of Fc receptor-bearing cytotoxic cells. Polymorphonuclear (PMN) cells represent attractive effector cells for antibody-directed immunotherapy. This, because activated PMN cells coexpress the class I receptors for IgG (FcgammaRI, CD64) and IgA (FcalphaRI, CD89), which are potent cytotoxic trigger molecules. Both receptors, however, require the FcR gamma chain for signaling. In this study, we show that FcgammaRI and FcalphaRI can trigger function independently of one another and do not cross-compete for the FcR gamma chain. FcalphaRI proved more efficient in initiating early signaling events and effector functions, such as redirected tumor cell killing and generation of superoxide. In addition, simultaneous engagement of FcgammaRI and FcalphaRI resulted in enhanced tumor cell lysis. These data support the development of concepts in which both FcgammaRI and FcalphaRI on PMN cells are targeted for tumor therapy.

Growth hormone induces tyrosyl phosphorylation of the transcription factors Stat5a and Stat5b in CMT-U335 canine mammary tumor cells.

It has now been well documented that the normal and tumorous canine mammary glands can be extra-pituitary sites of substantial growth hormone (GH) synthesis. Until now, attempts to reproduce the GH synthesis in-vitro using canine mammary explants or mammary tumor cells have not been successful. Therefore, the response of CMT-U335 canine mammary tumor cells to administered porcine GH (pGH) was investigated as an in-vitro model to study the possible effects of GH synthesis on this site. CMT-U335 cells spontaneously express the growth hormone receptor (GHR) as well as the prolactin receptor (PRLR). Twenty five minutes after administration, GH induced, in a dose-dependent manner, phosphorylation of the transcription factors Stat5a and Stat5b. Clear phosphorylation was induced by 10(-7) M and 10(-8) M pGH, with virtually no phosphorylation at 10(-9) M pGH. A similar dose-dependent phosphorylation of Stat5a by ovine prolactin was found in these cells. Although at high concentrations binding of pGH to the canine PRLR can occur (albeit with a low pKa), the similar dose-dependent effect of oPRL on Stat5a phosphorylation indicated that pGH signaled through the GHR. Remarkably, pGH induced a moderately decreased proliferation of CMT-U335 tumor cells, which may indicate that GH induces differentiation in these tumor cells. The GH-induced activation of Stat5a and Stat5b in these cells, as part of the JAK/Stat signal transduction pathway, is consistent with mammary GH playing a role in autocrine and/or paracrine stimulation of (tumorous) mammary cells.

Immune response in hormonally-induced prostatic hyperplasia in the dog.

We induced prostatic enlargement in castrated dogs using either androgen alone or androgen combined with estrogen. In addition to previously reported hyperplastic changes, marked infiltration with immune effector cells was observed. This mononuclear cell infiltrate was phenotypically characterized using CD3 as pan T-lymphocyte marker, CD79 for B-lymphocytes, MAC378 for macrophages, and antibodies against kappa- and lambda-immunoglobulin (Ig) light chains for plasma cells. The majority of inflammatory cells (>80%) in the mononuclear infiltrates were T-lymphocytes and the numbers correlated with the degree of inflammation. The B-lymphocytes were found particularly in areas with marked follicular formation and diffuse infiltration, whereas there were only a few positive cells (<10%) in areas with a moderate or slight inflammation. Macrophages were found primarily in areas with atrophic and cystic changes with and without inflammation. The expression of lambda-Ig-positive cells depended on the degree of inflammation (5-10%), whereas immunoreactivity of kappa-Ig did not correlate with the extent of inflammatory reaction. Our present findings together with the evaluation of longitudinal biopsies of hormonally-induced BPH indicate that hyperplasia preceded cell-mediated and humoral immune response.

IgA and the IgA Fc receptor.

IgA has traditionally been regarded a non-inflammatory antibody. This might indeed be true for secretory IgA (SIgA), which exerts its function at mucosal surfaces where commensal microorganisms and dietary antigens prevail. Serum IgA, however, potently triggers (pro)-inflammatory activity upon binding to the myeloid IgA receptor, FcalphaRI. Here, new insights in the roles of IgA and FcalphaRI are addressed and a model integrating the various functions of IgA in immunity is discussed.

Progestin-induced mammary growth hormone (GH) production.

Toxicity studies using beagle dogs revealed in the 1980s that synthetic progestins may induce a syndrome of growth hormone (GH) excess, known as acromegaly, and the development of predominantly benign mammary hyperplasia. In the early 1990s is was discovered that progestin-induced GH excess in the dog originates within the mammary gland. This mammary-derived GH may have endocrine, para/autocrine as well as exocrine effects. The expression of GH mRNA is also found in cats and humans indicating that mammary GH expression is not unique for the dog. The mammary gene is identical to the pituitary-expressed gene and uses the same promoter. Nevertheless a striking difference exists in the mammary gland. Pit-1, which is a prerequisite factor for pituitary GH mRNA expression, is likely not involved in the mammary gene expression. These studies shed new light on the mechanism of progesterone-induced mammary hyperplasia and urges for further research on potential adverse effects of synthetic progestins.

Growth hormone gene expression in normal lymph nodes and lymphomas of the dog.

Growth hormone (GH) is involved in the development, maturation, and function of the immune system. Recent studies have demonstrated that GH can be synthesized and secreted by lymphoid tissues, where it may act as an autocrine/paracrine growth factor. To determine whether GH may be involved in the development of hematological malignancies, GH gene expression in canine lymphomas was investigated. GH mRNA was detected in non-tumorous lymph nodes and in the majority of the lymphomas, by RT-PCR analysis. In situ and Northern blot hybridizations were negative. Analysis of the transcriptional start sites of the GH gene using 5'-RACE (rapid amplification of cDNA ends) showed that the canine lymphoid transcripts contained a 33-85 bp enlarged 5'-untranslated region compared to the pituitary and mammary GH transcripts. Part of the lymphoid GH transcripts contained intron 1, which would result in early termination of the translation due to an in-frame stopcodon. GH measurements in lymphoid tissues revealed a low content of immunoreactive GH. The results presented demonstrate that canine lymphoid tissue is an extrapituitary site of GH gene expression. However, GH production appeared to be low, indicating that lymphoid GH is probably not a major factor in the development or progression of canine lymphoma.