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Reproductive efficiency is major determinant of the dairy herd profitability. Thus, reproductive traits have been widely used as selection objectives in the current dairy cattle breeding programs. We aimed to evaluate strategies to model days open (DO), calving interval (CI) and daughter pregnancy rate (DPR) in Brazilian Holstein cattle. These reproductive traits were analysed by the autoregressive (AR) model and compared with classical repeatability (REP) model using 127,280, 173,092 and 127,280 phenotypic records, respectively. The first three calving orders of cows from 1,469 Holstein herds were used here. The AR model reported lower values for Akaike Information Criteria and Mean Square Errors, as well as larger model probabilities, for all evaluated traits. Similarly, larger additive genetic and lower residual variances were estimated from AR model. Heritability and repeatability estimates were similar for both models. Heritabilities for DO, CI and DPR were 0.04, 0.07 and 0.04; and 0.05, 0.06 and 0.04 for AR and REP models, respectively. Individual EBV reliabilities estimated from AR for DO, CI and DPR were, in average, 0.29, 0.30 and 0.29 units higher than those obtained from REP model. Rank correlation between EBVs obtained from AR and REP models considering the top 10 bulls ranged from 0.72 to 0.76; and increased from 0.98 to 0.99 for the top 100 bulls. The percentage of coincidence between selected bulls from both methods increased over the number of bulls included in the top groups. Overall, the results of model-fitting criteria, genetic parameters estimates and EBV predictions were favourable to the AR model, indicating that it may be applied for genetic evaluation of longitudinal reproductive traits in Brazilian Holstein cattle.

This study focused on the identification of QTL regions, candidate genes, and network related genes based on the first 3 lactations (LAC3) of milk, fat, and protein yields, and somatic cell score (SCS) in Portuguese Holstein cattle. Additionally, the results were compared with those from only first lactation (LAC1) data. The analyses were performed using the weighted single-step GWAS under an autoregressive test-day (TD) multiple lactations model. A total of 11,434,294 and 4,725,673 TD records from LAC3 and LAC1, respectively, including 38,323 autosomal SNPs and 1338 genotyped animals were used in GWAS analyses. A total of 51 (milk), 5 (fat), 24 (protein), and 4 (SCS) genes were associated to previously annotated relevant QTL regions for LAC3. The CACNA2D1 at BTA4 explained the highest proportion of genetic variance respectively for milk, fat, and protein yields. For SCS, the TRNAG-CCC at BTA14, MAPK10, and PTPN3 genes, both at BTA6 were considered important candidate genes. The accessed network refined the importance of the reported genes. CACNA2D1 regulates calcium density and activation/inactivation kinetics of calcium transport in the mammary gland; whereas TRNAG-CCC, MAPK10, and PTPN3 are directly involved with inflammatory processes widely derived from mastitis. In conclusion, potential candidate genes (TRNAG-CCC, MAPK10, and PTPN3) associated with somatic cell were highlighted, which further validation studies are needed to clarify its mechanism action in response to mastitis. Moreover, most of the candidate genes identified were present in both (LAC3 and LAC1) for milk, fat and protein yields, except for SCS, in which no candidate genes were shared between LAC3 and LAC1. The larger phenotypic information provided by LAC3 dataset was more effective to identify relevant genes, providing a better understanding of the genetic architecture of these traits over all lactations simultaneously.

Mammary tumors represent the second most common neoplasia in the canine species, where more than 50% of the cases are classified as malignant. The histological classification is used as a prognostic tool. Cadherins and catenins are responsible for cell adhesion and are intrinsic connected with the process of metastasis. E-cadherin expression in canine mammary tissues have been extensively studied. However, the studies with catenins are still scarce in the canine species. This study evaluated 74 canine mammary tissues by assessing the expression of E-cadherin and α, β and P-120 catenin molecules using the immunohistochemistry technique and their relationship with clinicopathological parameters. Three patterns of expression were identified in this study: membranous, cytoplasmic and both (membranous and cytoplasmic). In benign tumors, more than 80% of the cases had preserved expression and in malignant tumors 20% of the cases had reduced expression. A correlation between E-cadherin and P-120-catenin expression was found as well as a significant relationship between the histological type and the expression of α-catenin in malignant tumors.

Test-day milk, fat and protein yields and somatic cell score (SCS) of Brazilian Holstein cattle were used to study the effect of fixed (HTDF) vs random (HTDR) contemporary groups (herd-test-date) with or without unknown parent groups (UPG) using an autoregressive test-day animal model for genetic evaluations. Therefore, four models were used in this study to evaluate these effects: HTDF with or without UPG and HTDR with or without UPG. A total of 4,142,740 test-day records from the first three lactations were used in this study. The data was provided by the Brazilian Holstein Cattle Breeders Association and obtained from 1994 to 2016. UPG were defined by geographic region, sex and birth year of the animals with missing parents resulting in a total of 133 unknown sire groups and 153 unknown dam groups. The additive genetic variances estimated with HTDR decreased in all traits (except for SCS), but also did the estimated phenotypic variances, leaving the heritabilities almost the same in both models and ranging from 0.13 for fat (3rd lactation) fitted with HTDR to 0.24 for milk (1st lactation) fitted with HTDF. The recovery of information performed on the HTDR models increased the lower reliabilities from 0.50 to 0.75, 0.54 to 0.66, 0.64 to 0.71 and 0.25 to 0.67 for milk, fat and protein yields, and for SCS, respectively (considering only bulls with 10 or more daughters). The difference in annual genetic gains between models (HTDR vs HTDF) was for sires (cows) of 30.66 (38.59) kg, 1.18 (1.35) kg, 1.26 (1.22) kg and -0.001 (-0.03) scores for milk, fat and protein yields and SCS, respectively. The contemporary groups as a random effect in the AR model is more relevant than considering just the UPG effect, but the combination of both may provide higher annual genetic gains. The rank correlations between HTDF without UPG and the HTDR with UPG models were higher for cows (from 0.77 to 0.85) than for bulls (from 0.73 to 0.82). The HTDR with or without UPG, were the models with the best results indicating that there was no significant bias in the genetic trends using Interbull's methods 1 and 2. In general, the HTDR model with UPG was the procedure that best fitted these traits and should be the model of choice for genetic evaluations and genetic trend analysis of longitudinal traits in Brazilian Holstein cattle.

The progenies of international bulls in diverse climatic conditions and management levels may lead to different expressions of their genetic potential resulting in a re-ranking of these bulls. Therefore, evaluate the presence of genotype by environment interaction (G × E) within and across countries is important to guide the decision-making on alternative selection strategies. Thus, a two-step reaction norm (RN) approach was used to investigate the presence of G × E in Portuguese and Brazilian Holstein cattle. In step 1, we performed a within-country genetic evaluation using an autoregressive model to obtain precorrected phenotypes and environmental gradients (herd test-day solutions, HTD levels). In step 2, the precorrected phenotypes were considered as two distinct traits in a bi-trait RN model to estimate variance components across HTD levels, genetic correlation between HTD levels in Portugal and Brazil, and RN of the estimated breeding values. Additionally, the genetic correlation between countries using a bi-trait random regression (RR) sire model was obtained. In step 1, genetic additive variance for milk yield (MY) in Portugal was 14.1% higher than in Brazil. For somatic cell score (SCS), the genetic additive variance in Portugal was 12.7% lower than in Brazil. Although similar heritability estimates for SCS were observed in both countries, MY heritabilities were 0.31 for Portugal and 0.23 for Brazil. Genetic correlations (SD) between both countries obtained using RR sire model were 0.78 (0.051) for MY and 0.75 (0.062) for SCS. In step 2, MY genetic correlations among HTD levels within countries were higher than 0.94 for Portugal and 0.98 for Brazil. Somatic cell score genetic correlations among HTD levels ranged from 0.70 to 0.99 for Portugal and from 0.84 to 0.99 for Brazil. The average (SD) of genetic correlation estimates between Portuguese and Brazilian HTD levels were 0.74 (0.009) for MY and 0.57 (0.060) for SCS. These results suggest the presence of G × E for MY and SCS of Holstein cattle between both countries. Although there was no indication of G × E between Brazilian herd environments, the low genetic correlation for SCS indicates potential re-ranking of bulls between extreme environmental gradient in Portugal. Overall, the results of this study evidence the importance of national and international genetic evaluation systems to assist dairy farmers in the selection of the best genotypes to obtain the expected returns from investments in imported semen and to realize genetic progress in dairy populations under local environmental conditions.

Superovulation protocols have been described for different mouse strains, however the numbers of animals used are still high and still little information is known about hormone administration schedules and estrous cycle phases. In this study, we aimed to optimize a superovulation protocol by injecting 5 IU of pregnant mare serum gonadotropin followed by 5 IU of hCG 48 h later, using three different schedules related to the beginning of the dark cycle (3, 5 and 7 pm) in a light cycle of 7 am to 7 pm, with light on at 7 am. C57BL/6J mice at 3, 4 and 5 weeks of age were used and the estrous cycle phase for times of PMSG and hCG injections was also analyzed. Total oocyte number was counted in the morning after hCG injection. Hormones given at 3 weeks of age at 3 pm (59 ± 15 oocytes) and 7 pm (61 ± 10 oocytes) produced a significantly higher oocyte number compared with oocytes numbers collected from females at the same age at 5 pm (P = 0.0004 and

Background : Studies pointed out that the tumor-infiltrating lymphocytes (TILs) have considerable importance in canine mammary tumor (CMT). On the other hand, cancer cells sometimes find ways to use immune checkpoint proteins as a shield to avoid being identified and attacked by the immune system as programmed death 1 ligand 1 (PD-L1). In this study, it was investigated the relationship between PD-L1 expression, stromal tumor-infiltrating lymphocytes (TILs) in canine mammary tumor (CMT), and the association with clinical and pathological characteristics of the tumors. Materials, Methods & Results : PD-L1 expression and TILs were assessed in 23 female dogs with CMT. The tumors were grouped into simple carcinoma (CA, n = 8) and complex carcinoma (CC, n = 15). Stromal TILs were assessed using two thresholds as TILs-Low representing < 50% of infiltrate within stromal area and TILs-High representing ≥ 50% of stromal area. Clinicopathological data of CMT was characterized according to key parameters, as well as survival rates. TILs evaluation within tumor stroma revealed that 65.2% (n = 15) of tumors had TILs-Low. PD-L1 expression and stromal TILs were significantly associated (P = 0.009). PD-L1 expression was observed in 39% (n = 9) of all tumors of which 17.4% (n = 4) were from CA group and 21.7% (n = 5) were from CC group. PD-L1 expression within TILs was observed in 39% (n = 9) of the tumors. PD-L1 in malignant epithelium was present in all lymph node metastasis (n = 5). PD-L1 was associated with involvement of regional lymph nodes ( P = 0.034). Survival curves demonstrated TILs-Low had higher ( P = 0.010) overall survival (OS) compared with TILs-High, and PD-L1 + and PD-L1 – ( P = 0.06) did not differed. The clinicopathological variables significantly correlated with OS by univariate analysis were the histological grade ( P = 0.009), lymph node involvement ( P = 0.004), stromal TILs ( P = 0.016), and PD-L1 + /TILs-High vs. PD-L1 – /TILs-Low ( P = 0.010). Multivariate analysis revealed that group of tumors with grade II-III was independent and negative prognostic factors for OS. Discussion : In this study, PD-L1 was differently expressed according to the histologic subtypes of TMC. Currently, has been showed the presence of PD-L1 in several canine cancer. Nevertheless, only a few studies have described PD-L1 protein expression in dog tumors and showed PD-L1 was constitutively expressed on canine tumor cell lines, although the levels of basal expression were very variable. This expression can be modulated by IFN-γ exposure. In the present study, it was found a strong PD-L1 expression on TILs. The increase in PD-L1 cell surface expression by tumor cells can lead to decreased T-cell proliferation and increased apoptosis. In human breast cancer (BC) the PD-L1 expression was expressed in TILs and tumor epithelium. It has been reported the association of stromal TILs and PD-L1 expression with aggressive types and stages of BC. In this study, it was detected PD-L1 expression in malignant epithelium in all lymph node metastasis. PD-L1 overexpression was significantly associated with a series of clinicopathological parameters. It was demonstrated that PD-L1+/TILs-High had higher risk of overall survival (OS) than another group of interaction. High PD-L1 expression may be a prognostic indicator for reduced OS, while tumor PD-L1+ was associated with poorer disease-free survival. The presence of TILs has shown to be potentially predictive and a prognostic factor in BC subtypes. In CMT, it has been reported that a high proportion of TILs was correlated to several malignancy characteristics. In relation to PD-L1, further research is necessary to clarify this immune checkpoint as a potential therapeutic target and its application in clinical practice in CMT.

We investigated the applicability of ssGBLUP methodology under the autoregressive model (H-AR) for genomic evaluation of longitudinal reproductive traits in Portuguese Holstein cattle. The genotype data of 1,230 bulls and 1,645 cows were considered in our study. The reproductive traits evaluated were interval from calving to first service (ICF), calving interval (CI) and daughter pregnancy rate (DPR) measured during the first four parities. Reliability and rank correlation were used to compare the H-AR with the traditional pedigree-based autoregressive models (A-AR). In addition, a validation study was performed considering different scenarios. Higher genomic estimated breeding values (GEBV) reliabilities were obtained for genotyped bulls when evaluated under the H-AR model, with emphasis on bulls with less than 9 daughters. For this group, the averages of GEBV reliabilities corresponded to 0.62, 0.69 and 0.62 for ICF, CI and DPR, respectively, while the averages obtained by the A-AR model were 0.27, 0.15 and 0.16. The validation study was favourable to H-AR. The best results were observed in the scenario where genotyped cows were combined with contributing bulls (genotyped bulls with daughter or relationship information in the population). Overall, the results suggest that ssGBLUP methodology under the autoregressive model is a feasible and applicable approach to be used in genomic analyses of longitudinal reproductive traits in Portuguese Holstein cattle.

We investigated the efficiency of the autoregressive repeatability model (AR) for genetic evaluation of longitudinal reproductive traits in Portuguese Holstein cattle and compared the results with those from the conventional repeatability model (REP). The data set comprised records taken during the first four calving orders, corresponding to a total of 416, 766, 872 and 766 thousand records for interval between calving to first service, days open, calving interval and daughter pregnancy rate, respectively. Both models included fixed (month and age classes associated to each calving order) and random (herd-year-season, animal and permanent environmental) effects. For AR model, a first-order autoregressive (co)variance structure was fitted for the herd-year-season and permanent environmental effects. The AR outperformed the REP model, with lower Akaike Information Criteria, lower Mean Square Error and Akaike Weights close to unity. Rank correlations between estimated breeding values (EBV) with AR and REP models ranged from 0.95 to 0.97 for all studied reproductive traits, when the total bulls were considered. When considering only the top-100 selected bulls, the rank correlation ranged from 0.72 to 0.88. These results indicate that the re-ranking observed at the top level will provide more opportunities for selecting the best bulls. The EBV reliabilities provided by AR model was larger for all traits, but the magnitudes of the annual genetic progress were similar between two models. Overall, the proposed AR model was suitable for genetic evaluations of longitudinal reproductive traits in dairy cattle, outperforming the REP model.

Autoregressive (AR) and random regression (RR) models were fitted to test-day records from the first three lactations of Brazilian Holstein cattle with the objective of comparing their efficiency for national genetic evaluations. The data comprised 4,142,740 records of milk yield (MY) and somatic cell score (SCS) from 274,335 cows belonging to 2,322 herds. Although heritabilities were similar between models and traits, additive genetic variance estimates using AR were 7.0 (MY) and 22.2% (SCS) higher than those obtained from RR model. On the other hand, residual variances were lower in both traits when estimated through AR model. The rank correlation between EBV obtained from AR and RR models was 0.96 and 0.94 (MY) and 0.97 and 0.95 (SCS), respectively, for bulls (with 10 or more daughters) and cows. Estimated annual genetic gains for bulls (cows) obtained using AR were 46.11 (49.50) kg for MY and -0.019 (-0.025) score for SCS; whereas using RR these values were 47.70 (55.56) kg and -0.022 (-0.028) score. Akaike information criterion was lower for AR in both traits. Although AR model is more parsimonious, RR model assumes genetic correlations different from the unity within and across lactations. Thus, when these correlations are relatively high, these models tend to yield to similar predictions; otherwise, they will differ more and RR model would be theoretically sounder.

Background: The genus Orthobornavirus comprises non-segmented, negative-stranded RNA viruses able to infect humans, mammals, reptiles and various birds. Parrot bornavirus 1 to 8 (PaBV-1 to 8) causes neurological and/or gastrointestinal syndromes and death on psittacines. We aimed to identify and to produce epidemiologic knowledge about the etiologic agent associated with a death of two female Psittacus erithacus (grey parrot). Methods and Results: Both parrots were submitted for a complete standardised necropsy. Tissue samples were analysed by PCR. The findings in necropsy were compatible with bornavirus infection. Analysis revealed PaBV-4 related with genotypes detected in captive and in wild birds. The N and X proteins of PaBV-4 were more related to avian bornaviruses, while phosphoprotein was more related to variegated squirrel bornavirus 1 (VSBV-1). Within the P gene/phosphoprotein a highly conserved region between and within bornavirus species was found. Conclusions: Portugal is on the routes of the intensive world trade of psittacines. Broad screening studies are required to help understanding the role of wild birds in the emergence and spread of pathogenic bornaviruses. PaBV-4 phosphoprotein is closer to VSBV-1 associated with lethal encephalitis in humans than with some of the avian bornaviruses. The highly conserved P gene/phosphoprotein region is a good target for molecular diagnostics screenings.

Background: The genus Orthobornavirus comprises RNA viruses infecting humans, mammals, birds and reptiles, where parrot bornavirus 1 to 8 causes fatal neurological and/or gastrointestinal syndromes in psittacines. There is, to the best of our knowledge, no publication describing avian bornaviruses in pet parrots in Sweden. We aimed to identify and to produce epidemiologic knowledge about the etiologic agent associated with a history of severe weight loss and death of a Primolius maracana.Methods and results: The results of histopathology, immunohistochemistry and real-time RT-PCR were compatible with avian bornavirus infection. Sequencing indicated infection by parrot bornavirus 4 (PaBV-4). The genotype reported shared high identity with PaBV-4 identified from pet psittacines and from wild birds in several countries. The N gene and X protein showed genotype clusters formation. P protein revealed to be more conserved within and between species of bornaviruses. Findings suggest horizontal transmission within and between avian orders and species.Conclusion: There seems to be a worldwide trading without biosafety measures, hence, further disease transmission could be avoided. For screening purposes, the P gene is a good candidate as a universal target in molecular diagnostics. Wild birds may be key pieces in the puzzle of bornavirus epidemiology.

The multiple-lactation autoregressive test-day (AR) model is the adopted model for the national genetic evaluation of dairy cattle in Portugal. Under this model, animals' permanent environment effects are assumed to follow a first-order autoregressive process over the long (auto-correlations between parities) and short (auto-correlations between test-days within lactation) terms. Given the relevance of genomic prediction in dairy cattle, it is essential to include marker information in national genetic evaluations. In this context, we aimed to evaluate the feasibility of applying the single-step genomic (G)BLUP to analyze milk yield using the AR model in Portuguese Holstein cattle. In total, 11,434,294 test-day records from the first 3 lactations collected between 1994 and 2017 and 1,071 genotyped bulls were used in this study. Rank correlations and differences in reliability among bulls were used to compare the performance of the traditional (A-AR) and single-step (H-AR) models. These 2 modeling approaches were also applied to reduced data sets with records truncated after 2012 (deleting daughters of tested bulls) to evaluate the predictive ability of the H-AR. Validation scenarios were proposed, taking into account young and proven bulls. Average EBV reliabilities, empirical reliabilities, and genetic trends predicted from the complete and reduced data sets were used to validate the genomic evaluation. Average EBV reliabilities for H-AR (A-AR) using the complete data set were 0.52 (0.16) and 0.72 (0.62) for genotyped bulls with no daughters and bulls with 1 to 9 daughters, respectively. These results showed an increase in EBV reliabilities of 0.10 to 0.36 when genomic information was included, corresponding to a reduction of up to 43% in prediction error variance. Considering the 3 validation scenarios, the inclusion of genomic information improved the average EBV reliability in the reduced data set, which ranged, on average, from 0.16 to 0.26, indicating an increase in the predictive ability. Similarly, empirical reliability increased by up to 0.08 between validation tests. The H-AR outperformed A-AR in terms of genetic trends when unproven genotyped bulls were included. The results suggest that the single-step GBLUP AR model is feasible and may be applied to national Portuguese genetic evaluations for milk yield.

Canine parvovirus (CPV) is the most important enteric virus infecting canids worldwide. The purpose of this study was to detect CPV in naturally infected dogs from several veterinary clinics distributed throughout Portugal between 2012 and 2014 and to identify risk factors associated with CPV infection. From 209 dogs suspected of being infected with CPV, historical data and clinical signs were collected. Fecal samples were screened for CPV by PCR assay and those positive were confirmed by sequencing. The data was analyzed using logistic regression to investigate associations between each of the predisposing factors and CPV status. Of the samples collected, 77.5% tested CPV-positive. Statistical analysis showed that animals in the three age categories (p

Glycerol-based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post-thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol-based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio® ). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio® in the automatic freezer had higher (p

The objective of this study was to evaluate the effect of some management practices on the prevalence of Mycoplasma spp. in Northwestern Portuguese dairy farms from bulk tank milk (BTM) samples. Additionally, the within-herd prevalence of Mycoplasma spp. was also determined, but only in BTM positive herds. From May 2007 to November 2008, 492 BTM samples from 164 dairies randomly chosen in a population of 1234 dairy farms were analyzed. Five herds (3.0%) had positive mycoplasmal culture results, from which 4 out of 164 (2.4%) were Mycoplasma bovis, with simultaneous presence of Mycoplasma bovigenitalium or Mycoplasma canadense in two of those samples. In one out of 164 (0.6%) herds Mycoplasma capricolum subsp. capricolum was also found. In BTM positive Mycoplasma spp. herds, the apparent intra-herd prevalence was low and varied between 2.5% and 4.5%. Multiple locus variable-number of tandem-repeat analysis was conducted in order to compare the genetic relationship between the isolates. Mycoplasma spp. was found to be present in cows with subclinical mastitis with or without California Mastitis Test positive results, hence all cows should be tested when the agent is isolated from bulk tank rather than selecting suspected cows. A multivariable logistic regression using the Firth's penalized maximum likelihood estimation was performed showing that increasing number of lactating cows (OR=1.05; P

Selection for lower somatic cell count has been included in the breeding objectives of several countries in order to increase resistance to mastitis. Genetic parameters of somatic cell scores (SCS) were estimated from the first lactation test day records of Brazilian Holstein cows using random-regression models with Legendre polynomials (LP) of the order 3-5. Data consisted of 87,711 TD produced by 10,084 cows, sired by 619 bulls calved from 1993 to 2007. Heritability estimates varied from 0.06 to 0.14 and decreased from the beginning of the lactation up to 60 days in milk (DIM) and increased thereafter to the end of lactation. Genetic correlations between adjacent DIM were very high (>0.83) but decreased to negative values, obtained with LP of order four, between DIM in the extremes of lactation. Despite the favorable trend, genetic changes in SCS were not significant and did not differ among LP. There was little benefit of fitting an LP of an order >3 to model animal genetic and permanent environment effects for SCS. Estimates of variance components found in this study may be used for breeding value estimation for SCS and selection for mastitis resistance in Holstein cattle in Brazil.

Dairy cow mastitis associated with microalgae of the genus Prototheca has been reported worldwide. This alga is extremely resistant to most antimicrobials commonly used in mastitis therapy. In milk processing, different thermal treatments are generally efficient at inactivating and eliminating microorganisms. Until recently, no reports on Prototheca blaschkeae susceptibility to heat treatment have been described. Thus, considering the potential zoonotic risk that Prototheca may represent, the objective of this study was to test the susceptibility of P. blaschkeae field isolates retrieved from bovine mastitis to different temperature/time ratios that are generally used in the milk processing industry: 62°C/15 min and 30 min; 70°C/20 s, 15 min, and 30 min; 75°C/20 s; 90°C/1 s; and 100°C/1 s. The results showed a growth reduction of all isolates after the heat treatments, but only at 100°C was a total growth inhibition observed.

Bovine mastitis associated with Prototheca is considered a rare pathology, but is increasing in prevalence all over the world and therefore becoming more relevant to the dairy industry. The biochemical characterization of 47 Prototheca isolates retrieved from mastitic milk was performed in this study using API 20C Aux and two BBL Crystal Kits, followed by an analysis with InforBio software. The usage of this methodology, allowed the identification of discriminative phenotypic characteristics for the strains tested. The differential-character-finding algorithm used by this software permitted the identification of new phenotypic characteristics to discriminate between Prototheca zopfii, P. blaschkeae and P. wickerhamii, such as, citrate, phosphorycholine and arabinoside. The main objective of this study was to determine new phenotypic characteristics that allowed a better characterization of Prototheca spp. Usage of recent bioinformatic tools improved the analyses of several features that are important for a better characterization of Prototheca spp.

Dilated cardiomyopathy (DCM) is the second-most-important acquired cardiovascular disease in dogs (excluding heartworm disease in some geographic regions) and a major cause of morbidity and mortality in Estrela Mountain Dogs. The objective of this study is to describe the histologic features of DCM in Estrela Mountain Dogs, with special attention to the localization and quantification of attenuated wavy fibers (AWFs), fibrosis, and fatty infiltration. Myocardial samples from 10 areas were collected from the hearts of 10 dogs with DCM and 7 dogs without signs of cardiac disease—namely, the basal, middle, and apical portions of the free wall of both cardiac ventricles and the interventricular septum, as well as the left ventricular papillary muscle. In each sample, the presence or absence of AWFs was noted, and fatty infiltration and fibrosis were quantified. Fatty infiltration, fibrosis, and AWFs were observed in the myocardium of all dogs with DCM, in contrast to what has been described in other breeds. The left ventricular myocardium was the best tissue for diagnosis of DCM, based on these histologic features. The authors concluded that quantification of fibrosis and observation of AWFs in the left ventricular myocardium are useful in the histologic diagnosis of DCM in Estrela Mountain dogs.

Random regression models (RRM) differ in terms of the functions used to describe the shape of lactation curves. The aim was to compare random regression models under different functions to describe the lactation curves from Holstein cows in herds of the state of Minas Gerais. A database of 28,118 production records was analyzed using the test-day records of 4,230 first parity cows from five herds. The Wilmink, Ali & Schaeffer and Legendre polynomial (orders 4, 5 and 6) functions were adjusted in RRM to model the mean production trend (fixed) and genetic and permanent environmental (random) effects. The residual variances were assumed to be constant throughout lactation. Analyses were performed using the AIREMLF90 program. Except for the model with the polynomial function of order 5, all models converged. The Wilmink function showed lower values for criteria based on the -2log (L), AIC and BIC. The model with the Legendre polynomial of order 6 showed lower residual variance. Heritability estimates were similar between functions, ranging from 0.07 to 0.18 and were higher from 215 days of lactation. From 155 days of lactation, genetic and permanent environmental correlations between successive controls are of high magnitude. The Wilmink function is the most suitable for the study of milk yields from primiparous Holstein cows. The selection of animals is possible from 155 days of lactation on. Permanent environmental effects have greater influence on the milk production at the end of lactation of primiparous cows and should be considered since they are important and may be cumulative throughout lactation.

Toxoplasmosis is one of the most common food borne disease world-wide. Among food animals, sheep seems to having higher prevalence of Toxoplasma gondii infection. However, there is no consensus about the best cut-off for serodiagnosis in sheep. To estimate the more adequate cut-off value of Modified Agglutination Test (MAT) for serodiagnosis in sheep, a commercial ELISA kit was used as a golden standard. Evaluation of the optimal sensitivity and specificity was calculated using Youden's J-statistics. Values obtained were used to estimate the prevalence of sheep toxoplasmosis. One thousand four hundred and sixty seven blood samples were collected randomly from 160 farms from northern Portugal, representing approximately 10% of the ovine population from the region. All sera were tested for anti-T. gondii antibodies using the MAT. One hundred nine sheep (7.4%) presented a MAT titer > or = 1:80; 45 (3.0%) a MAT titer of 1:40; 97 (6.6%) a MAT titer of 1:20 and 1216 (83.0%) a MAT titer < or = 1:20. The best Youden's J-statistic was obtained at 1:20 titer (0.752), with 86.15% of sensitivity and 89.09% of specificity with negative and positive predictive values of 90.32% and 84.48% respectively, suggesting that the 1:20 was the most appropriate cut-off for serodiagnosis of toxoplasmosis in sheep. Assuming this cut-off, the prevalence of toxoplasmosis in the studied population was 17.1% and 92 (57.5%) of the 160 studied flocks having one or more positive sheep. Those results indicate that toxoplasmosis in Portugal should be considered in the differential diagnosis of abortions in sheep and neurological signs in lambs. Furthermore, while Portugal produces ovine meat for internal consumption and for exportation, isolation of T. gondii from ovine meat and further characterization of the isolates will be needed to understand the risk that ovine toxoplasmosis may represent for human health.

We describe the first known case of an algaemia by Prototheca blaschkeae in a dairy cow, which occurred after a chronic episode of mastitis caused by this pathogen. The organism was isolated from milk, joint fluid and blood samples, and microbiologic and molecular methods were performed to obtain a definitive identification of the algae. The affected cow was culled only after confirmation of a systemic infection by Prototheca.

We investigated the hypothetical role of human herpesviruses (HHVs) in tumour formation of the cerebellum. Thirty-five samples of pilocytic astrocytoma and 10 control samples of cerebellum from patients who died of unrelated diseases were examined. Presence of the 8 known HHVs was first studied using specific real-time quantitative Polymerase Chain Reaction (qPCR) targeting viral DNA polymerase. HHV's DNA polymerase was found present in 20 samples (7 controls, 13 astrocytomas) and was absent in 25 samples (3 controls, 22 astrocytomas). DNA polymerase of Epstein-Barr Virus (EBV) was present in 16 samples, 7/10 controls (70%) and 9/35 astrocytomas (26%). HHV-1 and Varicella-Zoster virus were detected only twice and HHV-2, Cytomegalovirus, HHV-7 and HHV-8, only once. HHV-6 was not detected. In all cases, the gene copy numbers of DNA polymerase were low (

Bovine mastitis is an important and complex disease responsible for economic losses in the dairy industry. Biotype II strains of the green alga Prototheca zopfii can be involved, most often resulting in chronic mastitis of difficult treatment associated with reduced milk production. This type of infection is rare, but the number of reported cases is increasing worldwide. In order to determine the kind of species involved in mastitis by Prototheca in northwest Portugal, 41 Prototheca isolates were genetically characterized. The algae are part of Prototheca isolates that were collected during a 6-year period, isolated from the milk of 41 dairy cows in a total of 22 herds with a history of increasing somatic cell counts, mild clinical signs of udder infection, and unsuccessful response to the usual therapy. PCR amplification of the 18S ribosomal DNA (rDNA), amplified rDNA restriction analysis, and phylogenetic analyses of the 18S rDNA sequences were performed. Thirty-seven isolates were identified as P. zopfii var. hydrocarbonea and four as Prototheca blaschkeae . These data suggest a high incidence of P. zopfii var. hydrocarbonea mastitis in the region and demonstrate for the first time the involvement of P. blaschkeae with bovine mammary gland infections.

Most test-day models used in genetic evaluations of dairy cattle define contemporary groups (CG) as the herd-test-date effect. Fitting this effect as fixed may minimize prediction bias, but requires a minimum number of observations per CG to simultaneously maximize the effective number of observations and minimize the residual error and prediction error variance. Nearly 4 million test-day records from the Portuguese Holstein database of 238,271 cows calving in 1,330 herds from 1994 through 2006 were used to evaluate the effect of clustering CG from small herds based on the similarity of their production environments. Principal component analysis was used to summarize 14 descriptive variables in 5 eigenvectors that explained 88% of the total variation. Based on the distance matrix, 2 different approaches were applied to group the herds. For each approach, 4 data sets were built having at least 3, 5, 10, or 15 observations per CG, respectively. For the data sets of group A, all herds, with or without the required number of observations per CG, were used in the clustering process. For the data sets of group B, only herds without the minimum number of observations were candidates to form clusters. All data sets were analyzed by an autoregressive test-day animal model fitting a fixed herd test date in a multiple-lactation setting, and results were compared with the current clustering procedure used in the Portuguese genetic evaluations. The data set from group B, with a minimum of 3 records per CG, was the one that provided the highest accuracy of prediction and the smaller within-CG variance, revealing a better fit for the data. This procedure also preserved the original herd structure of the database, better maximizing the number of herd groups. Correlations among EBV, rank, prediction error variance, and accuracies of prediction for this data set were high (0.97, 0.97, 0.85, and 0.82, respectively), suggesting that no major reranking is to be expected.

Recent studies of canine malignant mammary tumours suggest that reduction of E-cadherin and/or beta-catenin correlates with invasive behaviour and lymph node metastasis. The aims of this study were to examine the interrelationships between the expression of E-cadherin and beta-catenin, and the relationship between the expression of E-cadherin and/or beta-catenin and the mode of growth and metastatic capacity of canine malignant mammary tumours. 90 spontaneous malignant tumours and local and regional lymph nodes were studied. A significant relationship was evidenced between membranous expression of E-cadherin and beta-catenin (p=0.0027), but not between E-cadherin and cytoplasmic beta-catenin. Only E-cadherin as a separate factor was significantly related to tumour invasion (p=0.0072) and lymph node metastasis (p=0.0001). Neither membranous nor cytoplasmic beta-catenin expression was significantly related to either of these phenomena.

Summary Feline panleukopenia virus (FPV) and canine parvovirus (CPV) are two closely related viruses, which cause acute gastroenteritis in carnivores, and cats may be infected by strains of both viruses. The viruses are found worldwide and may have changing host ranges and genetic variation that can be found around the world in some cases. Here, we screened a Portuguese population of cats by a conventional PCR assay for the presence of FPV/CPV viruses in faecal samples and tissues between 2006–2008 and 2012–2014. The sequence analysis of the complete VP2 gene showed that 18 of 31 animals tested were positive for FPV DNA, and no case of CPV infection was detected. The analysis of specific DNA detected three new non-synonymous substitutions in the VP2 gene that were found in single groups and were related to viruses reported elsewhere by phylogenetic analysis – some were related to Italian isolates, one was closely related to isolates from Vietnam and China, and two were related with older strains from the USA. The results of our study show that FPV circulated in the Portuguese cat population and as expected the found strains are slightly divergent from those reported previously.

Abnormal catenin expression has been related to mammary carcinogenesis in both human and canine species and they are considered tumor- and invasion-suppressor molecules; however, in feline mammary tissues they have been scarcely studied.The immunohistochemical expression of α-, β- and p120-catenin was studied in a series of normal feline mammary glands, hyperplastic/dysplastic lesions and benign and malignant mammary tumors. Their relationship with clinicopathological parameters and with E- and P-cadherin expression was assessed.Normal tissues, hyperplastic/dysplastic lesions and benign tumors expressed α-, β- and p120-catenin in the membrane of more than 75% of the luminal epithelial cells, while in malignant tumors, there was a reduction in their membranous expression and a p120-catenin cytoplasmic expression in 40%. Reduced α-catenin expression was related to tumor features with prognostic value, namely tumor size (p=0.0203) and necrosis (p=0.0205). The expression of α-, β- and p120-catenin were individually related to each other and collectively associated with E-cadherin expression.The results demonstrate a relationship between feline mammary carcinogenesis and decreased expression of catenins, suggesting that they may represent a valuable tool in the diagnosis of feline mammary neoplasms.

Differential genetic expression in high and low opportunity Sicilian Holstein-Friesian and Brown Swiss herd environments was investigated using endogenous and exogenous variables in a set of three definitions. Results of genetic by environmental interaction were compared using alternative environmental definitions: within herd-year standard deviation for mature equivalent milk yield (HYSD), detectable incidence of normal vs. abnormal (peakless) lactation and herds clustered by causal relationships from high and low frequency use of nutrition, milking, health and animal handling practices. Data for genetic analysis consisted of first-lactation standardized yields of milk, fat and protein, and weighted somatic cell score for 8897 daughters of 825 Holstein-Friesian sires and 1143 daughters of 220 Brown Swiss sires. Components of covariance, heritabilities, and genetic correlations were estimated using bivariate and multivariate sire models for average and contrasting environments for each definition. Sire variances for yields were consistently smaller in the low opportunity environments of both breeds. Except for differential incidence of abnormal lactation in Friesian herds, correlated yield response in less privileged environments was 0.41 to 0.81 as much as in high opportunity environments, a substantial loss. Genetic correlations between HYSD environments for yield traits of Friesian were 0.48 to 0.66 but exceeded 0.80 for other definitions. Less correlated response in somatic cell score was also predicted for environments with low use of yield-enhancing practices (0.66 for Friesian and 0.61 for Brown Swiss), which may have resulted from less health care and poorer milking management. Therefore, unfavorable management interactions likely foster unequal gains from selection in contrasting environments defined exogenously or by incidence of peakless lactation. Conversely, greater genetic as well as phenotypic response is expected from additional inputs of nutrition, health care and milking management.

The objectives of this study were to estimate from test day records the genetic and environmental (co)variance components, correlations, and breeding values to increase genetic gain in milk yield of Lucerna and US Holstein cattle. The effects of repeated observations (within cow) were explained by first-order autoregressive processes within and across lactations using an animal model. Estimates of variance components and correlation coefficients between test days were obtained using derivative-free REML methodology. The autoregressive structure significantly reduced the model error component by disentangling the short-term environmental effects. The additional information and the more heterogeneous environmental variances between lactations in the multiple-lactation test day model than in the first lactation model provided substantially larger estimates of additive genetic variance (0.62 kg2 for Lucerna; 14.73 kg2 for Holstein), heritability (0.13 for Lucerna; 0.42 for Holstein), and individual genetic merit. Rank correlations of breeding values from multiple lactations and from first lactations ranged from 0.18 to 0.37 for females and from 0.73 to 0.89 for males, respectively. Consequently, more selection errors and less genetic gain would be expected from selection decisions based on an analysis of first lactation only, and greater accuracy would be achieved from multiple lactations. Results indicated that substantial genetic gain was possible for milk yield in the Lucerna herd (34 kg/yr). Estimates of genetic variance for Holsteins were larger than previously reported, which portends more rapid genetic progress in US herds also; under our assumptions, increases would be from 173 to 197 kg/yr.

Mycoplasma bovis has been considered an important cause of mastitis, pneumonia, and arthritis in bovines with a highly detrimental economic impact in the livestock industry. Previous epidemiological studies, using different typing techniques showed that the isolates were usually heterogeneous and results were difficult to compare between different laboratories. Reliable and comparable molecular typing techniques using geographically and temporal distinct isolates have never been used. The objective of this study was to use multiple-locus variable-number tandem-repeat analysis (MLVA) for the differentiation of M. bovis isolates. This typing scheme was developed using the sequenced genome of the M. bovis PG45 type strain. Nine tandem-repeat sequences were selected and the genetic diversity of 37 isolates of wide geographic and temporal origins was analyzed. The results were compared to those obtained with random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) for the same isolates. Cluster concordance between techniques was evaluated using Adjusted Rand and Wallace coefficients, and different cutoff levels of similarity were tested. Acceptable values of ≥0.5 for all techniques for the Wallace coefficient were only observed at the most relaxed cutoff level, i.e., 52% for MLVA, 29% for PFGE and 18% for RAPD. The Simpson's index of diversity was 0.91 for MLVA, 0.99 for RAPD analysis and 0.99 for PFGE. This MLVA assay is compatible with simple PCR and agarose gel-based electrophoresis steps as well as with high-throughput automated methods. The molecular typing scheme presented in this study provides a fast, reliable, and cost-effective typing method for surveillance of M. bovis epidemiology.

Overexpression of mucins is known to decrease cell-to-cell adhesion and thus to facilitate the invasion of cancer cells through the extracellular matrix. Mucin 6 (MUC6) is overexpressed and aberrantly O-glycosylated in human breast cancer, serving as a carrier for one of the most specific cancer-associated antigens, Tn antigen. Despite its relevance in breast cancer, MUC6 expression has not yet been characterized in canine mammary tumours (CMTs). The aims of this study were to assess the expression of MUC6 and Tn antigen in 55 benign and 77 malignant CMTs of different histological types and to investigate possible correlations with pathological features. MUC6 and Tn antigen were found to be significantly overexpressed in malignant compared with benign CMTs. MUC6 was significantly overexpressed in simple and complex carcinomas compared with simple and complex adenomas, respectively. When considering only the epithelial population, significant MUC6 overexpression was observed in carcinosarcomas when compared with benign mixed tumours. In addition, MUC6 was significantly overexpressed in simple compared with complex carcinomas. Finally, double-labelling immunofluorescence performed on seven malignant CMTs showed MUC6 and Tn co-expression. Therefore, MUC6 and Tn antigen overexpression is associated with malignant phenotypes of CMTs.

Contents The economics of dairy production are in great part dictated by the reproductive efficiency of the herds. Many studies have reported a widespread decrease in fertility of dairy cows. In a previous work (Rocha et al. 2001), we found a very poor oestrus detection rate (38%), and consequently a delayed calving to 1st AI and calving to conception intervals. However, a good conception rate at 1st AI was noted (51%) resulting in a low number of inseminations per pregnancy (IAP) (1.4). Here, results from a subsequent fertility time trend assessment study carried out in the same region for cows born from 1992 to 2002 are reported. Statistical linear models were used to analyse the data. Estimate linear contrasts of least square means were computed from each model. The number of observations per studied index varied from 12 130 (culling rate) to 57 589 (non-return rate). Mean age at first calving was 28.9 ± 0.14 months, without (p > 0.05) variation over time. There was a small, but significant (p

Prototheca sp. can assume high economic significance in the dairy industry and pose a potential risk for the public health. We investigated the in vitro susceptibility of Prototheca isolates retrieved from mastitic milk (P. zopfii and P. blaschkeae) to different pH buffers and salt concentrations using a microbroth assay adapted from the Clinical Laboratory Standards Institute guidelines. Different pH buffer solutions ranging from pH 1 to pH 12 and different sodium chloride concentrations, 4.5, 9 and 18%, were tested. P. zopfii strains presented an optimal growth between pH 5 and 9, a complete growth inhibition at pH 3, and limited growth at pH 1 and 12, whereas P. blaschkeae strains showed higher susceptibility to all pH values except for pH 3 where it demonstrated a moderate growth when compared to P. zopfii strains. When salinity was incremented, P. blaschkeae was more resistant than P. zopfii, although a reduction in growth for all strains of Prototheca was observed. This study demonstrated differences in the in vitro susceptibilities of P. zopfii and P. blaschkeae to different pH and salt concentrations and intend to be a contribution on the understanding of some of the physiologic features that can be associated with the survival of these microalgae in the environment.

To study genetic changes underlying myxoma virus evolution in its new host, the European rabbit (Oryctolagus cuniculus), we sequenced selected genomic regions of nine recent virulent field strains and a live attenuated vaccine strain ("MAV", Germany). DNA was extracted from cell culture passaged myxoma virus. A total of 4863 bp (approximately 3% of the genome) of 10 regions spanning 12 genes of the myxoma viruses was sequenced and compared to the original virulent strain "Lausanne" and its attenuated field derivative strain "6918". The field strains displayed a maximum of three (strains C43, C95) and a minimum of one (strains CD01, CD05) nucleotide substitutions. These were distributed through all analysed coding regions, except gene M022L (major envelope protein), where all strains were identical to "Lausanne" and "6918". Two new single nucleotide insertions were observed in some of the field strains: within the intergenic region M014L/M015L and within gene M009L, where it leads to a frameshift. These insertions were located after homopolymeric regions. The vaccine strain displayed 37 nucleotide substitutions, predominantly (95%) located in genes M022L and M036L. Interestingly, regions M009L and M014L/M015L of the vaccine were not amplified successfully, suggesting major genomic changes that could account for its attenuated phenotype. Our results support a high degree of genetic stability of myxoma virus over the past five decades. None of the analysed genome regions by its own seems sufficient for the genetic characterisation of field strains.

Mycoplasma capricolum subspecies capricolum ( M capricolum ) has been associated with cases of arthritis, mastitis, pneumonia, keratitis and septicaemia in small ruminants ([Monnerat and others 1999][1]). The infection is often chronic, and response to antimicrobial therapy is poor. Experimentally

MALIGNANT catarrhal fever (mcf) is a lymphoproliferative disease, caused by members of the genus Rhadinovirus , sub-family Gammaherpesvirinae (family Herpesviridae), which affects various species belonging to the order Artiodactyla ([Reid 2000][1]). The disease, which causes high mortality and low

Canine parvovirus (CPV) has been evolving, generating new genetic and antigenic variants throughout the world. This study was conducted to determine the types of CPV circulating in dogs in Figueira da Foz, Portugal. Thirty fecal samples, collected between 2006 and 2007 from dogs with clinical signs of CPV infection, were tested for CPV by a rapid, in-clinic, enzyme-linked immunosorbent assay (ELISA)/immunomigration test, by conventional real-time polymerase chain reaction (PCR), and by minor-groove binding TaqMan PCR. Of the 29 PCR-positive samples, 15 were identified as CPV-2b and 14 as CPV-2c. No CPV-2a was detected. The sensitivity of the ELISA test was 82.76% compared with the PCR assays. No significant associations were found between CPV type, clinical outcome, breed, vaccination status, or age.

Background Sialyl Lewis x (sLex) antigen is a carbohydrate antigen that is considered not only a marker for cancer but also implicated functionally in the malignant behaviour of cancer cells. Overexpression of sLex is associated with enhanced progression and metastases of many types of cancer including those of the mammary gland. Canine mammary tumours can invade and give rise to metastases via either lymphatic or blood vessels. E-Cadherin is specifically involved in epithelial cell-to-cell adhesion. In cancer, E-Cadherin underexpression is one of the alterations that characterizes the invasive phenotype and is considered an invasion/tumour suppressor gene. Partial or complete loss of E-Cadherin expression correlates with poor prognosis in canine malignant mammary cancer. The aim of this study was to analyse the sLex expression in canine malignant mammary tumours and to evaluate if the presence of sLex correlates with the expression of E-Cadherin and with clinicopathological features. Methods Fifty-three cases of canine mammary carcinomas were analysed immunohistochemically using monoclonal antibodies against sLex (IgM) and E-Cadherin (IgG). The clinicopathological data were then assessed to determine whether there was a correlation with sLex tumour expression. Double labelled immunofluorescence staining was performed to analyse the combined expression of sLex and E-Cadherin. Results sLex expression was consistently demonstrated in all cases of canine mammary carcinomas with different levels of expression. We found a significant relationship between the levels of sLex expression and the presence of lymph node metastases. We also demonstrated that when E-Cadherin expression was increased sLex was reduced and vice-versa. The combined analysis of both adhesion molecules revealed an inverse relationship. Conclusion In the present study we demonstrate the importance of sLex in the malignant phenotype of canine malignant mammary tumours. Our results support the use of sLex as a prognostic tumour marker in canine mammary carcinomas. Furthermore, we showed that sLex and E-Cadherin expression were inversely correlated. Future studies are warranted to clarify the molecular mechanism underlying the relation between sLex and E-Cadherin in canine mammary carcinoma cells which represents an important comparative model to woman breast cancer.