Autoregressive model for genetic evaluation of longitudinal reproductive traits in Brazilian Holstein cattle.

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.

Autoregressive single-step model for genomic evaluation of longitudinal reproductive traits in portuguese holstein cattle.

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.

Autoregressive repeatability model for genetic evaluation of longitudinal reproductive traits in dairy 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 and random regression test-day models for multiple lactations in genetic evaluation of Brazilian Holstein cattle.

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.

Canine parvovirus: the worldwide occurrence of antigenic variants.

The most important enteric virus infecting canids is canine parvovirus type 2 (CPV-2). CPV is the aetiologic agent of a contagious disease, mainly characterized by clinical gastroenteritis signs in younger dogs. CPV-2 emerged as a new virus in the late 1970s, which could infect domestic dogs, and became distributed in the global dog population within 2 years. A few years later, the virus's original type was replaced by a new genetic and antigenic variant, called CPV-2a. Around 1984 and 2000, virus variants with the single change to Asp or Glu in the VP2 residue 426 were detected (sometimes termed CPV-2b and -2c). The genetic and antigenic changes in the variants have also been correlated with changes in their host range; in particular, in the ability to replicate in cats and also host range differences in canine and other tissue culture cells. CPV-2 variants have been circulating among wild carnivores and have been well-documented in several countries around the world. Here, we have reviewed and summarized the current information about the worldwide distribution and evolution of CPV-2 variants since they emerged, as well as the host ranges they are associated with.

Porcine hokovirus in wild boar in Portugal.

Porcine hokovirus (PHoV), also referred to as porcine parvovirus 4 (P-PARV4), a recently discovered parvovirus of swine that is closely related to human parvovirus 4/5 (H-PARV4/5), was first described in Hong Kong. To evaluate the occurrence of P-PARV4 in Portuguese wild boars in the hunting season of 2011/2012, liver and serum samples were tested. P-PARV4 was detected in 24 % of the wild boars analyzed. Phylogenetic analysis showed a close relationship between the P-PARV4 isolates and other P-PARV4 reference strains. This virus appears to be emerging, with yet unknown implications for public health.

Detection of RHDV strains in the Iberian hare (Lepus granatensis): earliest evidence of rabbit lagovirus cross-species infection.

Rabbit hemorrhagic disease virus (RHDV) is a highly lethal Lagovirus, family Caliciviridae, that threatens European rabbits (Oryctolagus cuniculus). Although a related virus severely affects hares, cross-species infection was only recently described for new variant RHDV in Cape hares (Lepus capensis mediterraneus). We sequenced two strains from dead Iberian hares (Lepus granatensis) collected in the 1990s in Portugal. Clinical signs were compatible with a Lagovirus infection. Phylogenetic analysis of the complete capsid gene positioned them in the RHDV genogroup that circulated on the Iberian Peninsula at that time. This is the earliest evidence of RHDV affecting a species other than European rabbits.

Presence of Alphacoronavirus in Tree- and Crevice-Dwelling Bats from Portugal.

Coronaviruses (CoVs) are RNA viruses capable of infecting a wide range of hosts, including mammals and birds, and have caused significant epidemics such as the ongoing COVID-19 pandemic. Bats, the second most diverse mammalian order, are hosts for various CoVs due to their unique immune responses and ecological traits. This study investigates CoV prevalence in crevice- and tree-dwelling bats in Portugal, a country with limited prior research on bat CoVs. Using nested RT-PCR and sequencing, we screened 87 stool samples from bats, identifying one sample (1.15%) that was positive for Alphacoronavirus, belonging to Pipistrellus pipistrellus. Phylogenetic analysis revealed close genetic relationships with Alphacoronavirus strains from the same bat species in Europe. The low prevalence suggests habitat-specific differences in viral transmission, with cave-dwelling bats exhibiting higher CoV prevalence due to population density and behaviour. These findings underscore the necessity for sustained surveillance efforts aimed at comprehending CoV dynamics within bat populations, especially concerning the risk of spillover events and viral evolution. Vital to this understanding is the monitoring of bat migration patterns, which serves as a crucial tool for elucidating CoV ecology and epidemiology. Such efforts are essential for ongoing research endeavours aimed at mitigating the potential for future zoonotic disease outbreaks.

First Report of Alphacoronavirus Circulating in Cavernicolous Bats from Portugal.

The emergence of novel coronaviruses (CoVs) has emphasized the need to understand their diversity and distribution in animal populations. Bats have been identified as crucial reservoirs for CoVs, and they are found in various bat species worldwide. In this study, we investigated the presence of CoVs of four cavernicolous bats in six locations in the centre and south of Portugal. We collected faeces, anal, and buccal swab samples, as well as air samples from the locations using a Coriolis air sampler. Our results indicate that CoVs were more readily detected in faecal samples compared to anal and buccal swab samples. No CoVs were detected in the air samples. Phylogenetic analysis showed that the detected viruses belong to the Alphacoronavirus genus. This study represents the first report of Alphacoronaviruses circulating in bats in Portugal and highlights the importance of continuous surveillance for novel CoVs in bat populations globally. Ongoing surveillance for CoVs in bat populations is essential as they are a vital source of these viruses. It is crucial to understand the ecological relationships between animals, humans, and the environment to prevent and control the emergence and transmission of infectious diseases. Further ecological studies are needed to investigate the factors contributing to the emergence and transmission of zoonotic viruses.

Multicentre Etest evaluation of in vitro activity of conventional antifungal drugs against European bovine mastitis Prototheca spp. isolates.

OBJECTIVES: Bovine mammary protothecosis is a serious pathology that entails high economic losses in the dairy industry. The disease, the frequency of which has recently been increasing worldwide, is caused by unicellular, achlorophyllous, yeast-like algae of two species: Prototheca zopfii and Prototheca blaschkeae. The objective of this study was to investigate the in vitro activity of a panel of conventional antifungal drugs against Prototheca spp. isolates. METHODS: A total of 144 P. zopfii genotype 2 and P. blaschkeae strains isolated from milk of mastitic cows were subjected to drug susceptibility testing by Etest methodology. RESULTS: Five out of ten antifungal drugs tested exhibited no activity against Prototheca spp. isolates. The best activity against Prototheca spp. was demonstrated by amphotericin B (MIC90 of 1.5 mg/L). The MICs differed significantly (P < 0.01) between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to amphotericin B and azoles. Marked differences (P < 0.05) in azole and amphotericin B activities were noted among Prototheca spp. isolates originating from different European countries. Based on the correlation coefficients, a considerable cross-interaction was found among MICs of azoles and between MICs of azoles and amphotericin B for Prototheca spp. (P < 0.03). CONCLUSIONS: This study represents the largest, cross-European evaluation of antifungal activity against Prototheca spp. to date. The activity of amphotericin B against Prototheca spp. validates its potential use as a therapeutic agent against bovine protothecosis. For laboratory testing of drug activity against Prototheca spp., the Etest method is encouraged, due to its technical simplicity, rapidity and high intra- and inter-laboratory reproducibility.

Molecular Detection and Characterization of Coronaviruses in Migratory Ducks from Portugal Show the Circulation of Gammacoronavirus and Deltacoronavirus.

Coronaviruses (CoVs) are part of the Coronaviridae family, and the genera Gamma (γ) and Delta (δ) are found mostly in birds. Migratory birds have an enormous potential for dispersing pathogenic microorganisms. Ducks (order Anseriformes) can host CoVs from birds, with pathogenic expression and high economic impact. This study aimed to identify and characterize the diversity of CoVs in migratory ducks from Portugal. Duck stool samples were collected using cloacal swabs from 72 individuals (Anas platyrhynchos, Anas acuta, and Anas crecca). Among the 72 samples tested, 24 showed amplicons of the expected size. Twenty-three were characterized as Gammacoronavirus and one as Deltacoronavirus (accession numbers ON368935-ON368954; ON721380-ON721383). The Gammacoronaviruses sequences showed greater similarities to those obtained in ducks (Anas platyrhynchos) from Finland and Poland, Anas crecca duck from the USA, and mute swans from Poland. Birds can occupy many habitats and therefore play diverse ecological roles in various ecosystems, especially given their ability to migrate exceptional distances, facilitating the dispersal of microorganisms with animal and/or human impact. There are a considerable number of studies that have detected CoVs in ducks, but none in Portugal. The present study assessed the circulation of CoVs in wild ducks from Portugal, being the first description of CoVs for these animals in Portugal.

Draft Genome Sequence of a Leptospira kirschneri Serovar Mozdok Type 2 Strain Isolated from a Horse in Portugal.

Leptospira kirschneri is an agent causing leptospirosis in animals and humans. We report the draft genome sequence of Leptospira kirschneri serovar Mozdok type 2 strain Horse 112, comprising 485 contigs and having a genome size of 4,301,784 bp. This genome will facilitate studying important mechanisms for clinical outcomes.

In vitro susceptibility of Prototheca to pH and salt concentration.

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.

GWAS and gene networks for milk-related traits from test-day multiple lactations in Portuguese 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.

Genetic diversity of pathogenic leptospires from wild, domestic and captive host species in Portugal.

Leptospirosis is a neglected zoonotic disease of worldwide distribution with a significant veterinary and public health impact. It is caused by pathogenic bacteria of the genus Leptospira. The availability of effective tools to accurately identify and type leptospires is of utmost importance for the diagnosis of the disease and for assessing its epidemiology. Several multi-locus sequence typing (MLST) approaches were described for the typing of worldwide isolates of Leptospira but an extensive agreement towards the adoption of a unique consensus scheme for this agent is still lacking. Most genotyped strains originate from Asian and South American countries, with a minority originating from Europe (being most countries represented only by one or a few isolates). The knowledge of the diversity of circulating leptospires is the key to understanding the disease transmission and its zoonotic implications. In this study, we revisited the taxonomy of several isolates of pathogenic Leptospira obtained from domestic, wild and captive animals in Portugal, between 1990 and 2012. A selection of these isolates was genotyped using two previously published MLST schemes. A total of seven distinct sequence types (STs) were detected among the Portuguese isolates with two STs representing L. borgpetersenii (ST149 and ST152), two STs representing L. kirschneri (ST117 and ST100) and three STs representing L. interrogans (ST17, ST24 and ST140). Global widespread (and maybe more virulent) Leptospira genotypes seem to circulate in Portugal, particularly the L. interrogans ST17 isolates which are associated with several outbreaks of leptospirosis among humans and animals in different regions of the world. This study contributes to the enrichment of the global MLST databases with a new set of allele and sequence type information also providing novel data on circulating Leptospira serovars in Portugal.

Algaemia in a dairy cow by Prototheca blaschkeae.

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.

Bornaviruses in naturally infected Psittacus erithacus in Portugal: insights of molecular epidemiology and ecology.

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.

Detection and phylogenetic analysis of parrot bornavirus 4 identified from a Swedish Blue-winged macaw (Primolius maracana) with unusual nonsuppurative myositis.

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.

Bovine mastitis associated with Prototheca blaschkeae.

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.

Detection and quantitative analysis of human herpesvirus in pilocytic astrocytoma.

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 (<100/100 ng DNA). A second approach was to search for novel HHVs, using consensus-degenerated hybrid oligonucleotide primers (CODEHOP) PCR: no sequence indicative of a new HHV was detected. In summary, EBV was the most frequent HHV detected in pilocytic astrocytoma, but at very low levels. According to the actually accepted threshold the results suggest that EBV cannot be considered responsible for tumorigenesis of pilocytic astrocytoma.