Low Rho activity in hepatocytes prevents apical from basolateral cargo separation during trans-Golgi network to surface transport.

Hepatocytes, the main epithelial cells of the liver, organize their polarized membrane domains differently from ductal epithelia. They also differ in their biosynthetic delivery of single-membrane-spanning and glycophosphatidylinositol-anchored proteins to the apical domain. While ductal epithelia target apical proteins to varying degrees from the trans-Golgi network (TGN) to the apical surface directly, hepatocytes target them first to the basolateral domain, from where they undergo basolateral-to-apical transcytosis. How TGN-to-surface transport differs in both scenarios is unknown. Here, we report that the basolateral detour of a hepatocyte apical protein is due, in part, to low RhoA activity at the TGN, which prevents its segregation from basolateral transport carriers. Activating Rho in hepatocytic cells, which switches their polarity from hepatocytic to ductal, also led to apical-basolateral cargo segregation at the TGN as is typical for ductal cells, affirming a central role for Rho-signaling in different aspects of the hepatocytic polarity phenotype. Nevertheless, Rho-induced cargo segregation was not sufficient to target the apical protein directly; thus, failure to recruit apical targeting machinery also contributes to its indirect itinerary.

Breed performance in organic dairy farming in Northern Spain.

Organic farm management combines best environmental practices, a high level of biodiversity, preservation of natural resources and high animal welfare standards. To meet these criteria, farmers must have livestock well adapted to local organic conditions and information about how different breeds and crosses perform under different conditions. The objective of this study was to compare the performance of different pure breeds and cross-breeds of cattle in organic dairy systems in Northern Spain. The data analysed were obtained from monthly records kept between 2010 and 2016 on organic farms registered in the regional milk recording system. Analysis of various traits indicated that the Holstein-Friesian breed suits the organic production system in the study region. Although the reproductive performance of Holstein-Friesian cows was poorer (in terms of number of services per conception) than that of cross-breed and Brown Swiss cows, the Holstein-Friesian produced more milk and lived longer. In addition, there was no difference in calving type or calving ease between the different breed groups. The better milk fat and protein yields produced by the crosses may be useful traits for farmers interested in milk transformation. The advantage of continuing to use Holstein-Friesian cattle is that the breed is predominant worldwide, and the genealogy is well documented. If Holstein-Friesian cattle continue to be used, the main priority will be to search for well-adapted bulls (particularly for pasture-based conditions) and to elaborate a genetic merit index for organic and pasture-based systems with the aim of predicting and minimizing genotype × environment interactions.

Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes.

The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct "apicolateral" subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)-positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma.

[Bilateral chronic subdural haematoma: Analysis of a series of 190 patients]. / Hematoma subdural crónico bilateral: análisis de una serie de 190 pacientes.

OBJECTIVE: The aim of this study is to analyse the clinical findings and surgical results in a series of patients with bilateral chronic subdural haematoma (BCSDH), and compare the results with a series of patients treated for unilateral chronic subdural haematoma (UCSDH). PATIENTS AND METHODS: A retrospective study was performed on 1523 patients diagnosed and surgically treated for chronic subdural haematoma over a period of 30 years. Patients were divided into 2 groups: The study group consisting of 190 patients operated on for a BCSDH and the control group consisting of patients operated on for an UCSDH (1333 cases). RESULTS: The patient series included 126 males (66.3%) and 64 females (33.7%), with a mean age at diagnosis of 74.8±10.2. The control group consisted of 870 males (65.2%) and 463 women (34.8%), with a mean age of 73.2±12.1. The most common presenting symptoms was cognitive impairment in 63 patients (33.2%) with BCSDH and 416 (29.5%) with UCSDH. Recurrence rates were 9.4% (18 patients) and 5.7% (77 patients) in unilateral and bilateral haematomas, respectively. The mortality was 10 patients (5.2%) with BCSDH and 55(4%) with UCSDH. Factors significantly related to recurrence in the univariate analysis were being male (P=.040), anticoagulant/antiplatelet therapy (P=.032), and poor neurological status at admission (P=.039). CONCLUSIONS: This study indicates that BCSDH is more frequent in males, and the most common presentation is headache. The most important factors influencing recurrences are being male, intake of anticoagulant-antiaggregant drugs, and worse clinical status at admission.

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway.

The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER-Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER-Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

New Advances in Attention-Deficit/Hyperactivity Disorder-like Dogs.

Similar to humans, dogs could suffer an Attention-Deficit/Hyperactivity Disorder-like syndrome (ADHD-like). In fact, several studies highlight the use of dogs as a model for studying ADHD. This condition entails behavioral problems expressed through impulsivity, attention issues, hyperactivity, and/or aggression, compromising the quality of life for both the caregiver and the dog. The pathophysiology of ADHD-like is complex and is associated with dysregulation of various neurotransmitters such as serotonin and dopamine. The expression of ADHD-like behavior in dogs would appear to depend on a classical gene-environment interaction as is the case with many neurological disorders in humans. In addition to the described symptomatology, ADHD-like dogs can exhibit strong comorbidities with compulsive behaviors, aggressiveness, inappropriate elimination and fearfulness, in addition to epilepsy, foreign body ingestion, and pruritus. In spite of the fact that there is no veterinary consensus about the diagnosis of ADHD-like, some validated questionnaires could be helpful, but these cannot be used as a unique diagnostic tool. The use of drugs, such as fluoxetine, in addition to an adequate environmental enrichment, relaxation protocols, and behavior modification can achieve an adequate quality of life for both the dog and caregivers.

Actin dynamics at the Golgi complex in mammalian cells.

Secretion and endocytosis are highly dynamic processes that are sensitive to external stimuli. Thus, in multicellular organisms, different cell types utilize specialised pathways of intracellular membrane traffic to facilitate specific physiological functions. In addition to the complex internal molecular factors that govern sorting functions and fission or fusion of transport carriers, the actin cytoskeleton plays an important role in both the endocytic and secretory pathways. The interaction between the actin cytoskeleton and membrane trafficking is not restricted to transport processes: it also appears to be directly involved in the biogenesis of Golgi-derived transport carriers (budding and fission processes) and in the maintenance of the unique flat shape of Golgi cisternae.

Serotonin and Dopamine Blood Levels in ADHD-Like Dogs.

As with humans, dogs can suffer from attention deficit hyperactivity disorder-like (ADHD-like) behaviors naturally and exhibit high levels of hyperactivity/impulsivity and attention deficit problems, making the domestic dog a potential animal model for ADHD. ADHD has a very complex pathophysiology in which many neurotransmitters are involved, such as serotonin and dopamine. The aim of the study was to evaluate serum serotonin and dopamine levels in dogs with ADHD-like symptomatology. Fifty-eight dogs were studied, of which, thirty-six were classified as ADHD-like after physical and behavioral assessments. Additionally, the dogs' owners performed a series of scientifically validated questionnaires which included C-BARQ, the Dog Impulsivity Assessment Scale, and the Dog-ADHD rating scale. Serum from every animal was collected after the behavioral assessments and analyzed with commercial ELISA tests for serotonin and dopamine determination. Kruskal-Wallis tests and Lasso regressions were applied to assess the relationships between both neurotransmitters and the ADHD-like behaviors (as assessed by clinical evaluation and through the different questionnaires). The dogs clinically classified as ADHD-like showed lower serotonin and dopamine concentrations. Further, serotonin and dopamine levels were also linked to aggression, hyperactivity, and impulsivity. Decreased serotonin concentrations were also related to fear, attachment, and touch sensitivity. Finally, it must be noted that our data suggested a strong relationship between serotonin and dopamine and ADHD-like behaviors.

Factors Affecting Fatty Acid Composition of Holstein Cow's Milk.

Milk fatty acid composition has gained the interest of both manufacturers and consumers during recent years. The present paper aimed to perform an analysis of C14:0, C16:0, C18:0, C18:1, saturated (SFA), monounsaturated (MUFA), polyunsaturated (PUFA) and short chain fatty acid (SCFA) concentration in cow's milk in relation to the type of ration, parity, lactation phase and season. Cows' milk from animals being fed total mixed rations, including corn silage, had higher C14:0, C16:0 and SFA concentrations than those being fed pasture-based rations but lower concentrations of C18:0 and PUFA. Comparing to 1st parity cows, 2nd and 3rd parity animals had higher SFA and SCFA concentrations in milk. With respect to spring, C14:0, C16:0 and SFA concentrations increased in summer, autumn and winter while MUFA, PUFA and SCFA concentrations decreased. Considering the lactation phase, C14:0, C16:0 and SFA concentrations decreased in fresh cows with ketosis comparing to healthy fresh cows and increased in peak, mid and late lactation. C18:0, C18:1 and MUFA follow the opposite trend. The milk fatty acid profile varies significantly through the studied effects. The fact that the fatty acid profile is associated with animal health, organoleptic properties of milk or even methane production highlights the importance of studying factors that affect its variation.

Government veterinarians' perceptions of routine biosecurity focused on dairy cattle farms in north-western and north-eastern Spain.

The implementation of biosecurity measures in livestock production systems can be affected by the psychosocial factors of its stakeholders, which can be observed through their knowledge, attitudes and perceptions/practices. In Spain, there are no regulations per se to promote biosecurity. Of all stakeholders, farmers and veterinarians have been addressed in previous biosecurity studies, but not veterinarians belonging specifically to the government services. This study explores this particular group's perceptions of routine biosecurity in livestock production systems in north-western and north-eastern Spain, an understanding of which could help to improve the implementation of biosecurity measures on farms. Eleven interviews were conducted with veterinarians from different levels of the government services in Galicia and Catalonia, and were analyzed through content analysis. Dairy cattle farms were considered as the reference livestock production systems. The respondents stress the limited availability of staff and time resources for biosecurity. The advisory role of government veterinarians is not well recognized among farmers, who feel that their services prioritize their sanctioning role. In fact, government veterinarians consider that farmers only implement biosecurity measures to avoid being sanctioned, and not because they are aware of the importance of biosecurity. Meanwhile, the participants comment that biosecurity regulations should be flexible and need to consider the contexts of the farms where biosecurity measures are implemented. Finally, government veterinarians are willing to attend biosecurity meetings together with all farm stakeholders, at which the government services could be informed about biosecurity issues on farms. The person who could take on the biosecurity advisory role should be defined, along with further discussion of such matters as the responsibilities of each stakeholder. Government veterinary services need to be considered in studies of biosecurity operations in order to improve their implementation. It is therefore concluded that government veterinarians are seeking to balance their own institutional perspective with that of farmers and veterinarians in the routine implementation of biosecurity measures.

Metaprotein expression modeling for label-free quantitative proteomics.

BACKGROUND: Label-free quantitative proteomics holds a great deal of promise for the future study of both medicine and biology. However, the data generated is extremely intricate in its correlation structure, and its proper analysis is complex. There are issues with missing identifications. There are high levels of correlation between many, but not all, of the peptides derived from the same protein. Additionally, there may be systematic shifts in the sensitivity of the machine between experiments or even through time within the duration of a single experiment. RESULTS: We describe a hierarchical model for analyzing unbiased, label-free proteomics data which utilizes the covariance of peptide expression across samples as well as MS/MS-based identifications to group peptides-a strategy we call metaprotein expression modeling. Our metaprotein model acknowledges the possibility of misidentifications, post-translational modifications and systematic differences between samples due to changes in instrument sensitivity or differences in total protein concentration. In addition, our approach allows us to validate findings from unbiased, label-free proteomics experiments with further unbiased, label-free proteomics experiments. Finally, we demonstrate the clinical/translational utility of the model for building predictors capable of differentiating biological phenotypes as well as for validating those findings in the context of three novel cohorts of patients with Hepatitis C. CONCLUSIONS: Mass-spectrometry proteomics is quickly becoming a powerful tool for studying biological and translational questions. Making use of all of the information contained in a particular set of data will be critical to the success of those endeavors. Our proposed model represents an advance in the ability of statistical models of proteomic data to identify and utilize correlation between features. This allows validation of predictors without translation to targeted assays in addition to informing the choice of targets when it is appropriate to generate those assays.

Phospholipid synthesis participates in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex.

The lipid metabolite diacylglycerol (DAG) is required for transport carrier biogenesis at the Golgi, although how cells regulate its levels is not well understood. Phospholipid synthesis involves highly regulated pathways that consume DAG and can contribute to its regulation. Here we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking.

Live-cell Imaging of Biosynthetic Protein Transport in Hepatocytes.

Here, we describe a strategy to analyze the exit of apical and basolateral cargo from the trans-Golgi network in primary hepatocytes. The method is based on recombinant adenovirus-mediated infection combined with a pulse-chase regimen and live-cell imaging analysis of fluorescent protein-tagged dipeptidyl peptidase IV (DPPIV) and vesicular stomatitis virus G (VSVG) cargo proteins, coexpressed and accumulated in the endoplasmic reticulum via DPPIV aggregation through an engineered conditional aggregation domain and VSVG by exploiting the aggregation of the ts045 mutant at its non-permissive temperature of 40 °C.

Associations between On-farm Welfare, Milk Production, and Reproductive Performance in Dairy Herds in Northwestern Spain.

This study was conducted to evaluate the relationship between on-farm welfare, milk production, and reproductive performance in dairy herds in northwestern Spain. Data on the welfare status from 31 herds were collected using the Welfare Quality® protocol. Linear regression was applied to assess associations between WQ criteria/principle scores on farm average 305-d milk production and calving to conception interval (CCI). Results indicated that milk production was associated with the absence of prolonged hunger criterion (average increase of 22.33kg per unit score). Each one unit increase in the score for good feeding was also associated with a26.49kg increase in milk production. A10 unit increase in the scores for the absence of pain (by management procedures), expression of social behaviors, and absence of injuries was associated with reductions in CCI by 1.77, 0.8, and 0.29 days. CCI also decreased by 1.08 and 0.53 days when the principles of good health and appropriate behavior increased by 10 units. This study points out associations which can help encourage the dairy cattle sector to adopt practices that promote animal welfare.

Bovine viral diarrhea virus (BVDV) infection: Effect on reproductive performance and milk yield in dairy herds.

The impact of an outbreak of bovine viral diarrhea (BVD) on reproductive performance and milk production was investigated in 28 Spanish dairy herds using an observational study design. Mean calving to conception interval (CCI), mean number of inseminations per conception (AI/P) and mean daily milk yield were recorded from herds affected by outbreaks. Infection was confirmed by evidence of viral transmission to young stock (heifers 9-24 months) and persistently infected animals, after previous BVD-free status. Fifteen herds did not use vaccines (G1) whereas the remaining 13 received modified live BVD virus (BVDV) vaccines (G2). In G1 herds, a significant increase in CCI (13.8 days; 95% CI, 0.4-27.3 days; P = 0.050) was observed during the BVD outbreak compared to the year before the outbreak. In G2 herds, there was a not a significant increase in CCI (3.0 days; 95% CI, -1.2-7.2 days; P = 0.558). Changes in AI/P between periods were not significant in any of the herds. In G1 herds, there was an increase of 0.09 units (P > 0.05) during the outbreak, whereas in G2 herds, AI/P decreased 0.08 units (P > 0.05). Compared to milk production in the year before the BVD outbreak, there was no significant change in G1 herds throughout the study period (P > 0.05), but there were significant increases in G2 herds over the study period (P1-P2, 1.4 kg/day increase, P = 0.019; P1-P3, 2.0 kg/day increase, P = 0.007). These results indicate that BVDV infection affected reproductive performance, which is critical in maintaining dairy herd profitability.

Study of Behavioural Traits in Can de Palleiro (Galician Shepherd Dog).

The Can de Palleiro (CP) is an autochthonous canine breed from Galicia (NW Spain). Interestingly, no previous research has been published about the behaviour of this breed. Thus, the aim of the present study was to obtain a deeper understanding of CP behavioural and temperamental traits and detect any potentially problematic behaviour by using the Canine Behavioural Assessment and Research Questionnaire (C-BARQ) and the Socially Acceptable Behaviour (SAB) test. Behavioural information was obtained from 377 dogs-177 CPs and 200 general population (GP) dogs-using the C-BARQ. Additionally, 32 dogs were enrolled to perform the SAB test (19 CPs and 13 GP dogs) in order to directly evaluate their temperament. Our results indicated that CP dogs had a lower tendency to show aggressiveness towards their owners (0.18 times lower, p = 0.033) and less fear of other dogs (by 0.43 times, p = 0.001), as well as higher trainability levels (2.56 times higher, p < 0.001) when compared to GP dogs. CP dogs also had increased odds of showing chasing behaviour (3.81 times higher, p < 0.001). Conversely, CPs had reduced odds of non-social fear, separation-related problems and excitability (by 0.42, 0.35 and 0.48 times, respectively; p < 0.001, p < 0.001 and p = 0.002). The current research represents a starting point for the study of the behaviour of CPs, which appear to be a working breed, with guarding and, especially, herding characteristics.

Factors Associated with the Introduction of Mycobacterium avium spp. Paratuberculosis (MAP) into Dairy Herds in Galicia (North-West Spain): The Perception of Experts.

This study aimed at quantifying expert opinions on the risk factors involved in Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds. For this purpose, potential risk factors associated with the introduction of MAP into dairies were chosen based on a literature review and discussions with researchers and veterinarians. For each factor, a decision tree was developed, and key questions were included in each. Answers to these key questions led to different events within each decision tree. An expert opinion workshop was organized (following the recommendations of the OIE), and ordinal values ranging from 0 to 9 (i.e., a null to very high likelihood of infection) were assigned to each event. The potential risk factors were also incorporated into a structured questionnaire that was responded to by 93 farms where the sanitary status against MAP was known. Thereby, based on the values given by the experts and the information collected in the questionnaires, each farm was assigned a score based on their MAP entry risk. From these scores (contrast variable) and using a ROC curve, the cut-off that best discriminated MAP-positive and -negative farms was estimated. The most important risk factors for the introduction of MAP, according to expert opinions, involved purchase and grazing practices related to animals under six months of age. The scores obtained for each farm, also based on the expert opinions, allowed MAP positive/MAP negative farms to be discriminated with 68.8% sensitivity and 68.7% specificity. These data should be useful for focusing future training initiatives and improving risk-reduction strategies in the dairy industry.

Diacylglycerol is required for the formation of COPI vesicles in the Golgi-to-ER transport pathway.

Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.

An Evaluation of Portuguese Societal Opinion towards the Practice of Bullfighting.

Bullfighting is a controversial sport that continues to be legally permitted in a number of countries around the world, including Portugal. The spectacle has attracted significant attention from animal protectionist groups for many years because of concerns for animal distress, pain, and suffering during the fights. While there has been strong support for the sport in Portugal in the past, there is a need to study social profiles regarding the acceptability of this sport before a case can be made for changes in regional and national legislation. In this study, Portuguese attendance patterns at bullfights were assessed in addition to public opinions on welfare and ethical aspects of bullfighting, based on demographic variables. Study participants (n = 8248) were largely recruited through Portuguese social media channels (respondents may not be representative of the Portuguese population). Questionnaire data were evaluated by means of frequency tables, multiple correspondence analyses, and a two-step cluster analysis. Most respondents had a negative opinion about bullfighting and perceived that bullfighting had no positive impact on the country. However, while most respondents thought that the bull suffered during bullfighting, the opinion regarding banning bullfighting was far from unanimous. Based on the demographic analysis, the profile of individuals with more favorable responses towards bullfighting were men > 65 years old, of Roman Catholic faith, of low- or high-income levels, from more rural areas of Portugal. Somewhat surprisingly, there was a tendency to favor bullfighting amongst veterinary professionals. We conclude that there were still large pockets of individuals who desire to maintain the practice of traditional bullfighting within Portuguese society, despite recognition of animal suffering during the event.

A survey of biosecurity measures and serological status for bovine viral diarrhoea virus and bovine herpesvirus 1 on dairy cattle farms in north-west and north-east Spain.

BACKGROUND: Biosecurity is a key measure to reduce and prevent the introduction of diseases to farms and minimise spread of diseases within a herd. The aim of the study was to characterise the current application of biosecurity measures on dairy cattle farms in Spain along with their bovine viral diarrhoea and infectious bovine rhinotracheitis status. METHODS: Data on biosecurity measures for 124 dairy herds were collected using a questionnaire. The sanitary status of these farms for bovine viral diarrhoea and infectious bovine rhinotracheitis was also assessed using antibody ELISA. Data were analysed using multiple correspondence analysis and a two-step cluster analysis. RESULTS: Three main clusters of farms were identified: clusters 1 and 2 included herds of small and intermediate sizes. These, particularly cluster 1, showed the most deficiencies in the control of vehicles and visitors. However, laboratory tests were always performed on purchased animals. Cluster 3 had the largest herd sizes, with somewhat better biosecurity control of vehicles and visitors. However, farms in this cluster also purchased the most animals, sometimes without testing, and hired external workers more often. CONCLUSION: The study indicated that, in the study population, there are serious shortcomings in the application of biosecurity measures on dairy farms, exposing them to disease transmission. This survey also highlights regional and herd size-related differences in the implementation of biosecurity. Collecting data is an important first step to identification of specific weaknesses in different farm typologies, and an adequate follow-up is needed to ensure that measures are implemented correctly on farms.