A new spinosaurid dinosaur species from the Early Cretaceous of Cinctorres (Spain).

A new spinosaurid genus and species is described based on the right maxilla and five caudal vertebrae of a single specimen from the Arcillas de Morella Formation (Early Cretaceous) at the locality of Cinctorres (Castellón, Spain). Protathlitis cinctorrensis gen. et sp. nov. is diagnosed by one autapomorphic feature as well as by a unique combination of characters. The autapomorphy includes a subcircular depression in the anterior corner of the antorbital fossa in the maxilla. The new Iberian species is recovered as a basal baryonychine. The recognition of Protathlitis cinctorrensis gen. et sp. nov. as the first baryonychine dinosaur species identified from the Arcillas de Morella Formation (late Barremian) from the same time as Vallibonavenatrix cani, the first spinosaurine dinosaur from the same formation in the Morella subbasin (Maestrat Basin, eastern Spain), indicates that the Iberian Peninsula was home to a highly diverse assemblage of medium-to-large bodied spinosaurid dinosaurs. It seems that spinosaurids appeared during the Early Cretaceous in Laurasia, with the two subfamilies occupying the western part of Europe during this period. Later, during the Barremian-Aptian, they migrated to Africa and Asia, where they would diversify. In Europe, baryonychines were dominant, while in Africa, spinosaurines were most abundant.

Calculating clinical mastitis frequency in dairy cows: Incidence risk at cow level, incidence rate at cow level, and incidence rate at quarter level.

The lack of standardization in reporting clinical mastitis incidence limits the ability to compare results across multiple studies without additional calculations. There is both a biological and statistical rationale for evaluating the at-risk period at the quarter level. This study aimed to: (1) to outline an applied method for calculating clinical mastitis (CM) incidence rate at the quarter level using currently available software; and (2) to present the results of three different measurements: incidence risk at cow level, incidence rate at cow level, and incidence rate at quarter level. In an open population prospective cohort of eight commercial dairy farms monitored from May 15, 2016, to May 31, 2017, all CM cases (n = 7513) were identified by trained on-farm personnel, who collected all milk samples from all quarters with visibly abnormal milk. Microbiological identification was determined by culture and MALDI-TOF. All lactating quarters were at risk for CM. A quarter was at risk for a new CM case if there was at least 14 d between a previously diagnosed case and the current case in the same quarter, or if a different pathogen was isolated in the same quarter within 14 d. A total of 17,513,429 quarters days at risk (QDAR) were estimated. A statistical software macro and Structured Query Language (SQL) were used to bring all data together. The monthly incidence rate at the cow level was 16.6 cases per 10,000 cow-days, the monthly incidence rate at the quarter level was 4.4 cases per 10,000 QDAR and the monthly incidence risk at the cow level was 4.8 cases per 100 cows. Although the evaluation of QDAR requires additional computation when compared to other methods, it might allow for a more precise evaluation of the data and a more accurate evaluation of mastitis incidence. Clearly defining the methods used to report mastitis incidence will improve our ability to discuss and learn about the differences and similarities across studies, regions, and countries.

A new styracosternan hadrosauroid (Dinosauria: Ornithischia) from the Early Cretaceous of Portell, Spain.

A new styracosternan ornithopod genus and species is described based on the right dentary of a single specimen from the Mirambell Formation (Early Cretaceous, early Barremian) at the locality of Portell, (Castellón, Spain). Portellsaurus sosbaynati gen. et sp. nov. is diagnosed by two autapomorphic features as well as a unique combination of characters. The autapomorphies include: the absence of a bulge along the ventral margin directly ventral to the base of the coronoid process and the presence of a deep oval cavity on the medial surface of the mandibular adductor fossa below the eleventh-twelfth tooth position. Phylogenetic analyses reveal that the new Iberian form is more closely related to the African taxon Ouranosaurus nigeriensis than to its synchronic Iberian taxa Magnamanus soriaensis and Iguanodon galvensis. In addition, Portellsaurus sosbaynati is less related to other Iberian taxa such as Iguanodon bernissartensis and Proa valdearinnoensis than to the other Early Cretaceous Iberian styracosternans Mantellisaurus atherfieldensis and Morelladon beltrani. A new phylogenetic hypothesis is proposed that resolves Iguanodon (I. bernissartensis, I. galvensis) with the Valanginian Barilium dawsoni into a monophyletic clade (Iguanodontoidea). The recognition of Portellsaurus sosbaynati gen. et sp. nov. as the first styracosternan dinosaur species identified from the Margas de Mirambell Formation (early Barremian-early late Barremian) in the Morella sub-basin (Maestrat Basin, eastern Spain) indicates that the Iberian Peninsula was home to a highly diverse assemblage of medium-to-large bodied styracosternan hadrosauriforms during the Early Cretaceous.

Sand bedding as a reservoir for Lactococcus garvieae dissemination in dairy farms.

Lactococcus garvieae is now recognized as a species with clinical significance for human and veterinary medicine. The aim of this study was to evaluate the presence of this pathogen in sand bedding and milk samples. Two farms in Minnesota with problems of clinical and subclinical mastitis due to streptococci-like organisms were selected. Twenty-four Lactococcus garvieae isolates from sand bedding and 18 isolates from quarter milk were comparatively studied using a genotypic approach. RAPD (random amplification of polymorphic DNA) PCR and REP (repetitive element palindromic) PCR experiments highlighted a similar electrophoretic profile. When genes belonging to the core genome of L. garvieae were tested through a MLRT (multilocus restriction typing), we again observed that all L. garvieae isolates coming from sand bedding and milk shared a common profile, distinguishable from previously studied representative L. garvieae strains. These data indicate that the L. garvieae isolated from sand bedding and milk originated from a few strains adapted to persist in the same habitat. This supports the hypothesis that sand bedding can represent a reservoir of L. garvieae strains and be a potential vehicle for their dissemination in dairy farms.

Staphylococcus aureus Isolates from Bovine Mastitis in Eight Countries: Genotypes, Detection of Genes Encoding Different Toxins and Other Virulence Genes.

Staphylococcus aureus is recognized worldwide as one of the major agents of dairy cow intra-mammary infections. This microorganism can express a wide spectrum of pathogenic factors used to attach, colonize, invade and infect the host. The present study evaluated 120 isolates from eight different countries that were genotyped by RS-PCR and investigated for 26 different virulence factors to increase the knowledge on the circulating genetic lineages among the cow population with mastitis. New genotypes were observed for South African strains while for all the other countries new variants of existing genotypes were detected. For each country, a specific genotypic pattern was found. Among the virulence factors, fmtB, cna, clfA and leucocidins genes were the most frequent. The sea and sei genes were present in seven out of eight countries; seh showed high frequency in South American countries (Brazil, Colombia, Argentina), while sel was harboured especially in one Mediterranean country (Tunisia). The etb, seb and see genes were not detected in any of the isolates, while only two isolates were MRSA (Germany and Italy) confirming the low diffusion of methicillin resistance microorganism among bovine mastitis isolates. This work demonstrated the wide variety of S. aureus genotypes found in dairy cattle worldwide. This condition suggests that considering the region of interest might help to formulate strategies for reducing the infection spreading.

Microbiota of cow's milk; distinguishing healthy, sub-clinically and clinically diseased quarters.

The objective of this study was to use pyrosequencing of the 16S rRNA genes to describe the microbial diversity of bovine milk samples derived from clinically unaffected quarters across a range of somatic cell counts (SCC) values or from clinical mastitis, culture negative quarters. The obtained microbiota profiles were used to distinguish healthy, subclinically and clinically affected quarters. Two dairy farms were used for the collection of milk samples. A total of 177 samples were used. Fifty samples derived from healthy, culture negative quarters with a SCC of less than 20,000 cells/ml (group 1); 34 samples derived from healthy, culture negative quarters, with a SCC ranging from 21,000 to 50,000 cells/ml (group 2); 26 samples derived from healthy, culture negative quarters with a SCC greater than 50,000 cells/ml (group 3); 34 samples derived from healthy, culture positive quarters, with a SCC greater than 400,000 (group 4, subclinical); and 33 samples derived from clinical mastitis, culture negative quarters (group 5, clinical). Bacterial DNA was isolated from these samples and the 16S rRNA genes were individually amplified and pyrosequenced. All samples analyzed revealed great microbial diversity. Four bacterial genera were present in every sample obtained from healthy quarters (Faecalibacterium spp., unclassified Lachnospiraceae, Propionibacterium spp. and Aeribacillus spp.). Discriminant analysis models showed that samples derived from healthy quarters were easily discriminated based on their microbiota profiles from samples derived from clinical mastitis, culture negative quarters; that was also the case for samples obtained from different farms. Staphylococcus spp. and Streptococcus spp. were among the most prevalent genera in all groups while a general multivariable linear model revealed that Sphingobacterium and Streptococcus prevalences were associated with increased 10 log SCC. Conversely, Nocardiodes and Paenibacillus were negatively correlated, and a higher percentage of the genera was associated with a lower 10 log SCC.

Microbial diversity of bovine mastitic milk as described by pyrosequencing of metagenomic 16s rDNA.

Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.

The "other" gram-negative bacteria in mastitis: Klebsiella, serratia, and more.

Mastitis caused by gram-negative infections is of increasing importance on modern and well-managed dairy farms. Without a doubt, E coli tends to be the most important cause of these gram-negative infections when the data are tallied across farms.1 However, more precise investigation of individual farms often reveals a farm-specific infection pattern where a single gram-negative bacterial species predominates. Several farms with a predominance of "other" gram-negative IMIs may be observed. We have shown the presence of outbreaks on individual dairy farms with K pneumoniae, S marcescens, and Enterobacter cloacae. On farms with a predominance of these "other" gram-negative infections, a detailed epidemiologic investigation may reveal the source of these infections. It is quite surprising to identify the difference in host immune response pattern and the associated clinical and subclinical presentations of IMIs due to the different gram-negative organisms. Experimental and field observations would suggest that among the gram-negative bacterial causes of mastitis, Klebsiella spp are causing the most severe cases, closely followed by E coli and then much less clinical severity is observed in Serratia spp and Enterobacter spp cases. The precise mechanisms that would explain the difference in clinical severity are not known, but the most likely explanation appears to be the structure of the lipid A fraction of the LPS of the bacterial species. Important differences in the lipid A fraction of LPS between and within bacterial species are observed. The prevention of IMIs with gram-negative bacteria has components that are generic across species and components that are species specific. Generic prevention may be obtained by improving hygiene and reducing exposure of teat ends to environmental contamination. Also the use of a J5 bacterin is expected to provide some reduction in severity of gram-negative IMIs across bacterial species. Specific prevention programs will depend on the actual transmission behavior of the dominant species causing IMIs in the herd. Several clonal outbreaks of gram-negative bacterial species have been described. In such situations, optimal milking procedures, segregation and culling of infected animals, and targeted treatment would be advisable. Even more specific are the prevention procedures associated with S marcescens outbreaks, where resistance against specific biocides will lead to transmission of infection through teat disinfectants. Removal of these biocides from the cow environment is than essential. Antimicrobial treatment of gram-negative bacteria has often considered to be of limited value and treatment should be more targeted toward cow survival and reduction of clinical symptoms. More recently, extended treatment with a third-generation cephalosporin was reported to be efficacious in the treatment of E coli and Klebsiella spp but not of E cloacae. Further investigations in effective treatment protocols for gram-negative IMIs are warranted.

CNS mastitis: nothing to worry about?

In this paper, we analyzed a very large field data set on intramammary infections (IMI) and the associated somatic cell count (SCC) in dairy cows. The objective of the study was to analyze the impact of coagulase-negative staphylococci (CNS) IMI on cow SCC, both mean and variability, and on the potential of these infections to have a major impact on the bulk milk SCC (BMSCC). Data and milk samples for bacterial culture were collected by Quality Milk Production Services (QMPS) between 1992 and March of 2007. The QMPS program services dairy farms in New York State and other states in the Northeastern USA and operates in conjunction with Cornell University. Only records from cows where SCC and milk production data were available, and where only one organism was isolated from bacterial cultures of milk samples (or where culture was negative) were used for this analysis. A total of 352,614 records from 4200 whole herd mastitis screening sampling qualified for this study. Within herds an average of 15% (S.D. 12%) of cows sampled were infected with CNS, ranging between 0 and 100%. Average within herd prevalence of cows with a CNS IMI and an SCC over 200,000 cells/ml was 2% (S.D. 4%) with a minimum of 0% and a maximum of 50%. Results of linear mixed models showed three distinct populations of IMI statuses: negative cultures with the lowest SCC; CNS and Corynebacterium bovis with a moderate increase in SCC, and Streptococcus agalactiae, Streptococcus spp. and Staphylococcus aureus showing an important increase in SCC. Surprisingly, milk production was slightly but significantly higher in CNS infected cows compared to culture-negative cows, whereas it was strongly reduced in cows with a major pathogen IMI. The percentage contribution of CNS infections to the BMSCC was 17.9% in herds with a BMSCC less than 200,000 cells/ml. This value decreased to 11.9 and 7.9% in herds with bulk milk SCC between 200,000 and 400,000 and over 400,000 cells/ml, respectively. We concluded that very few herds with milk quality problems would have an important increase in BMSCC that could be mostly attributed to CNS infections. On the other hand, in herds with low BMSCC, CNS infections may be an important contributor to the total number of somatic cells in the bulk milk.

A comparison of antimicrobial susceptibility patterns for Staphylococcus aureus in organic and conventional dairy herds.

Selective pressure from antimicrobial use, mutations, or acquisition of foreign resistance determinants mediate antimicrobial resistance. If antimicrobial use is the major selective pressure encouraging the development of resistance, then reduced use should result in decreased resistance. We compared antimicrobial susceptibility patterns of Staphylococcus aureus isolates obtained from milk samples from 22 organic (nonantibiotic using) dairy herds to isolates from 16 conventional dairy herds. Susceptibility testing was performed by disk diffusion, and zone diameters were recorded in millimeters for 144 isolates from organic farms and 117 isolates from conventional farms and were also classified as susceptible or not-susceptible (intermediate and resistant categories combined). Strength of association between high or low use and proportion susceptible was evaluated by Chi-square analysis and differences in mean zone diameter for isolates from organic farms versus isolates from conventional farms were compared by analysis of variance. Analysis was done for each antimicrobial and deemed significant at p < or = 0.05. Differences in antimicrobial susceptibility were observed between S. aureus isolates from organic and conventional herds for seven of the nine antibiotics studied. Herds that were certified organic had S. aureus isolates that were more susceptible to antimicrobials. Overall, S. aureus isolates from both organic and conventional herds showed good susceptibility to most commonly used bovine mastitis antimicrobials; however, isolates from organic herds were significantly more susceptible. Longitudinal studies of herds undergoing the transition to organic farming would help elucidate the dynamics of antimicrobial resistance and the potential return of antimicrobial susceptibility.