Surveillance of Antimicrobial Resistance of Escherichia coli Isolates from Intestinal Contents of Dairy and Veal Calves in the Veneto Region, Northeaster Italy.

This surveillance study aimed to estimate the proportion of antimicrobial resistant strains and antimicrobial resistance (AMR) profiles of E. coli isolates detected from the intestinal contents of veal and dairy calves in the Veneto Region, Northeaster Italy. Additionally, we investigated the differences in AMR profiles between dairy and veal calves over the period 2017-2022. Overall 1150 E. coli isolates were tested from calves exhibiting enteric disease, with 868 from dairy and 282 from veal calves. The percentage of resistant isolates to nine antimicrobials was notably higher in veal calves compared to dairy calves, except for ampicillin. Throughout the study period, we observed a significant increase in the proportion of resistant isolates to florfenicol, gentamycin, paromomycin, tetracycline and trimethoprim/sulfamethoxazole in dairy calves, while we did not detect any significant increase in the proportion of resistant isolates among veal calves. A substantial proportion (75.9%) of the isolated E. coli exhibited multi-drug resistance (MDR). The proportion of multi-drug resistant isolates was significantly higher in veal calves (91.7%) compared to dairy calves (74.3%) all through the surveillance period (2017-2022), with no significant variation in MDR proportion among veal calves between 2017 and 2022 but a significant increase among dairy calves.

Layer-by-layer interleukin-12 nanoparticles drive a safe and effective response in ovarian tumors.

Ovarian cancer is especially deadly, challenging to treat, and has proven refractory to known immunotherapies. Cytokine therapy is an attractive strategy to drive a proinflammatory immune response in immunologically cold tumors such as many high grade ovarian cancers; however, this strategy has been limited in the past due to severe toxicity. We previously demonstrated the use of a layer-by-layer (LbL) nanoparticle (NP) delivery vehicle in subcutaneous flank tumors to reduce the toxicity of interleukin-12 (IL-12) therapy upon intratumoral injection. However, ovarian cancer cannot be treated by local injection as it presents as dispersed metastases. Herein, we demonstrate the use of systemically delivered LbL NPs using a cancer cell membrane-binding outer layer to effectively target and engage the adaptive immune system as a treatment in multiple orthotopic ovarian tumor models, including immunologically cold tumors. IL-12 therapy from systemically delivered LbL NPs shows reduced severe toxicity and maintained anti-tumor efficacy compared to carrier-free IL-12 or layer-free liposomal NPs leading to a 30% complete survival rate.

Corrigendum: Overview of Control Programs for Twenty-Four Infectious Cattle Diseases in Italy.

[This corrects the article DOI: 10.3389/fvets.2021.665607.].

Genotyping and Antimicrobial Susceptibility Profiling of Streptococcus uberis Isolated from a Clinical Bovine Mastitis Outbreak in a Dairy Farm.

Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of ß-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

Overview of Control Programs for Twenty-Four Infectious Cattle Diseases in Italy

The cattle industry is a major driving force for the Italian agricultural sector totalling about 5. 6 million heads for dairy and meat production together. It is particularly developed in the northern part of the country, where 70% of the whole Italian cattle population is reared. The cattle industry development in the rest of the country is hampered by the hard orography of the territories and a variety of socioeconomic features leading to the persistence of the traditional rural farming systems. The differences in the farming systems (industrial vs. traditional) also affect the health status of the farms. Whereas, Enzootic Bovine Leukosis (EBL) is almost eradicated across the whole country, in Southern Italy where Bovine Tuberculosis and Brucellosis are still present and Bluetongue is endemic due to the presence of the competent vector (Culicoides imicola), less investments are aimed at controlling diseases with economic impact or at improving farm biosecurity. On the other hand, with the eradication of these diseases in most part of the country, the need has emerged for reducing the economic burden of non-regulated endemic disease and control programs (CPs) for specific diseases have been implemented at regional level, based on the needs of each territory (for instance common grazing or trading with neighboring countries). This explains the coexistence of different types of programs in force throughout the country. Nowadays in Italy, among cattle diseases with little or no EU regulations only three are regulated by a national CP: Enzootic Bovine Leukosis, Bluetongue and Paratuberculosis, while Bovine Genital Campylobacteriosis and Trichomonosis are nationwide controlled only in breeding bulls. For some of the remaining diseases (Infectious Bovine Rhinotracheitis, Bovine Viral Diarrhea, Streptococcus agalactiae) specific CPs have been implemented by the regional Authorities, but for most of them a CP does not exist at all. However, there is a growing awareness among farmers and public health authorities that animal diseases have a major impact not only on the farm profitability but also on animal welfare and on the use of antibiotics in livestock. It is probable that in the near future other CPs will be implemented.

Business intelligence tools to optimize the appropriateness of the diagnostic process for clinical and epidemiologic purposes in a multicenter veterinary pathology service.

Laboratory tests provide essential support to the veterinary practitioner, and their use has grown exponentially. This growth is the result of several factors, such as the eradication of historical diseases, the occurrence of multifactorial diseases, and the obligation to control endemic and epidemic diseases. However, the introduction of novel techniques is counterbalanced by economic constraints, and the establishment of evidence- and consensus-based guidelines is essential to support the pathologist. Therefore, we developed standardized protocols, categorized by species, type of production, age, and syndrome at the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), a multicenter institution for animal health and food safety. We have 72 protocols in use for livestock, poultry, and pets, categorized as, for example, "bovine enteric calf", "rabbit respiratory", "broiler articular". Each protocol consists of a panel of tests, divided into 'mandatory' and 'ancillary', to be selected by the pathologist in order to reach the final diagnosis. After autopsy, the case is categorized into a specific syndrome, subsequently referred to as a syndrome-specific panel of analyses. The activity of the laboratories is monitored through a web-based dynamic reporting system developed using a business intelligence product (QlikView) connected to the laboratory information management system (IZILAB). On a daily basis, reports become available at general, laboratory, and case levels, and are updated as needed. The reporting system highlights epidemiologic variations in the field and allows verification of compliance with the protocols within the organization. The diagnostic protocols are revised annually to increase system efficiency and to address stakeholder requests.

Cancer Cell Coating Nanoparticles for Optimal Tumor-Specific Cytokine Delivery.

Although cytokine therapy is an attractive strategy to build a more robust immune response in tumors, cytokines have faced clinical failures due to toxicity. In particular, interleukin-12 has shown great clinical promise but was limited in translation because of systemic toxicity. In this study, we demonstrate an enhanced ability to reduce toxicity without affecting the efficacy of IL-12 therapy. We engineer the material properties of a NP to meet the enhanced demands for optimal cytokine delivery by using the layer-by-layer (LbL) approach. Importantly, using LbL, we demonstrate cell-level trafficking of NPs to preferentially localize to the cell's outer surface and act as a drug depot, which is required for optimal payload activity on neighboring cytokine membrane receptors. LbL-NPs showed efficacy against a tumor challenge in both colorectal and ovarian tumors at doses that were not tolerated when administered carrier-free.

Tuning Nanoparticle Interactions with Ovarian Cancer through Layer-by-Layer Modification of Surface Chemistry.

Nanoparticle surface chemistry is a fundamental engineering parameter that governs tumor-targeting activity. Electrostatic assembly generates controlled polyelectrolyte complexes through the process of adsorption and charge overcompensation utilizing synthetic polyions and natural biomacromolecules; it can yield films with distinctive hydration, charge, and presentation of functional groups. Here, we used electrostatic layer-by-layer (LbL) assembly to screen 10 different surface chemistries for their ability to preferentially target human ovarian cancer in vitro. Our screen identified that poly-l-aspartate, poly-l-glutamate, and hyaluronate-coated LbL nanoparticles have striking specificity for ovarian cancer, while sulfated poly(ß-cyclodextrin) nanoparticles target noncancerous stromal cells. We validated top candidates for tumor-homing ability with a murine model of metastatic disease and with patient-derived ovarian cancer spheroids. Nanoparticle surface chemistry also influenced subcellular trafficking, indicating strategies to target the cell membrane, caveolae, and perinuclear vesicles. Our results confirm LbL is a powerful tool to systematically engineer nanoparticles and achieve specific targeting.

Evaluation of Minimum Inhibitory Concentrations for 154 Mycoplasma synoviae isolates from Italy collected during 2012-2017.

Mycoplasma synoviae (MS) is a highly prevalent bacterial species in poultry causing disease and severe economic losses. Antibiotic treatment is one of the control strategies that can be applied to contain clinical outbreaks in MS-free flocks, especially because this bacterium can be transmitted in ovo. It becomes, then, very important for veterinarians to know the antibiotic susceptibility of the circulating strains in order to choose the most appropriate first-line antibiotic molecule as a proactive role in fighting antibiotic resistance. We evaluated the Minimum Inhibitory Concentrations (MICs) of enrofloxacin, oxytetracycline, doxycycline, erythromycin, tylosin, tilmicosin, spiramycin, tiamulin, florfenicol and lincomycin for MS isolates collected between 2012 and 2017 in Italy. A total of 154 MS isolates from different poultry commercial categories (broiler, layer, and turkey sectors) was tested using commercial MIC plates. All MS isolates showed very high MIC values of erythromycin (MIC90 ≥8 µg/mL) and enrofloxacin (MIC90 ≥16 µg/mL). MIC values of doxycycline and oxytetracycline obtained were superimposable to each other with only a one-fold dilution difference. Discrepancies between MIC values of tylosin and tilmicosin were observed. Interestingly, seven isolates showed very high MIC values of lincomycin and tilmicosin, but not all of them showed very high MIC values of tylosin. Most of the MS isolates showed low MIC values of spiramycin, but seven strains showed a MIC ≥16 µg/mL. In the observation period, the frequency of the different MIC classes varied dependently on the tested antibiotic. Interestingly, tilmicosin MICs clearly showed a time-dependent progressive shift towards high-concentration classes, indicative of an on-going selection process among MS isolates. Until standardized breakpoints become available to facilitate data interpretation, it will be fundamental to continue studying MIC value fluctuations in the meantime in order to create a significant database that would facilitate veterinarians in selecting the proper drug for treating this impactful Mycoplasma.

A Longitudinal Case Study on Dissemination of ST398 Methicillin-Resistant Staphylococcus aureus Within a Dairy Cow Herd.

The study was conducted to describe the dynamics of ST398 methicillin-resistant Staphylococcus aureus (MRSA) on a dairy herd in northeastern Italy. MRSA was first identified in this herd of 120 cows in 2016, after which the herd was sampled once every 3 months for 1 year (April 2016-May 2017). Samples collected included nasal swabs and milk samples from cows and nasal swabs from farmworkers. In addition, pen fencing and teat milk liners were swabbed and air samples from cow pens and the milking parlor were collected. All samples were tested for MRSA using a selective medium; positive isolates were confirmed by mecA PCR. A representative set of MRSA isolates was genotyped using spa typing and multilocus sequence typing. Overall, 34 (mean 23%, range 16-30%) milking cows were found harboring MRSA in the mammary gland and only 6 recovered from infection or colonization. The mean incidence rate was 14% (range 8-20%), mean cure rate was 23% (range 13-43%), and estimated basic reproduction number (R0) was 1.08. The average of positive quarters found was 35.1% and most of the positive quarters (82.4%) developed subclinical mastitis. The mean duration of MRSA colonization in quarters during the study was 247 days, but quarters affected by subclinical mastitis harbored MRSA for a longer time than healthy ones (285 days vs. 131 days). After the second sampling, the farmer segregated MRSA-positive cows from the uninfected cows and milked them last. Despite segregation, 25 newly infected or colonized cows were detected. MRSA isolates from cows, environment, and two farmworkers belonged to the same sequence type (ST398) and spa type (t034). This study highlights the ability of ST398 MRSA to cause a persistent infection of the mammary gland and to survive in the farm environment.

Identification of Multidrug-Resistant Escherichia coli from Bovine Clinical Mastitis Using a Ceftiofur-Supplemented Medium.

Escherichia coli causes a significant number of clinical mastitis cases in dairy cattle worldwide. The antimicrobial susceptibility of E. coli is important for both human and animal health. Surveillance reports recorded that the efficacy of most antibiotics is substantially preserved but detection of E. coli from clinical mastitis cases producing extended-spectrum beta-lactamases and plasmid-encoded AmpC beta-lactamases has been reported. These resistance determinants have frequently been associated with multidrug resistance. The aim of this study was to determine if a MacConkey agar medium supplemented with 8 mg/L of ceftiofur (MC-CEF) could be a useful tool to identify cephalosporin-resistant and multidrug-resistant (MDR) E. coli among bovine mastitis isolates. During the period 2010-2011, 773 E. coli were isolated from bovine clinical mastitis milk samples collected in 80 dairy farms in Northern Italy. A total of 105 E. coli were selected and assigned either to group randomly selected E. coli (RSEC; n = 53), based on a random selection among the whole collection of 773 E. coli, or to group ceftiofur-resistant E. coli (CEFREC; n = 52). CEFREC isolates were identified by spreading the 773 E. coli isolates on MC-CEF. Minimum inhibitory concentration (MIC) was used to test the phenotypic antimicrobial susceptibility to 16 antibiotics. The MIC results confirmed the ceftiofur resistance in 73.1% (38/52) of CEFREC isolates, whereas all RSEC isolates were susceptible to ceftiofur. The comparison of MIC values for each antibiotic tested between the two groups revealed significantly higher frequencies of resistance to antimicrobials other than ceftiofur in the CEFREC group. Resistance profiles highlighted a significantly higher frequency of MDR isolates among CEFREC (73.1%) than RSEC (17%) E. coli. The results showed that MC-CEF may be a useful selective medium to identify cephalosporin-resistant and MDR E. coli on dairy farms, without performing MIC on all the isolates.

Relationship between Protein Oxidation Biomarkers and Uterine Health in Dairy Cows during the Postpartum Period.

High neutrophil (PMN, Polymorphonuclear neutrophil) counts in the endometrium of cows affected by endometritis, suggests the involvement of oxidative stress (OS) among the causes of impaired fertility. Protein oxidation, in particular, advanced oxidation protein products (AOPP), are OS biomarkers linked to PMN activity. To test this hypothesis, the relationship between protein oxidation and uterus health was studied in thirty-eight dairy cows during the puerperium. The animals were found to be cycling, without any signs of disease and pharmacological treatments. PMN count was performed either through a cytobrush or a uterine horn lavage (UHL). Cows were classified into four groups, based on the uterine ultrasonographic characteristics and the PMN percentage in the uterine horns with a higher percentage of high neutrophil horn (HNH). They were classified as: Healthy (H); Subclinical Endometritis (SCE); Grade 1 Endometritis (EM1); and Grade 2 Endometritis (EM2). AOPP and carbonyls were measured in plasma and UHL. UHL samples underwent Western blot analysis to visualize the carbonyl and dityrosine formation. Plasma AOPP were higher (p < 0.05) in EM2. AOPP and carbonyl group concentrations were higher in the HNH samples (p < 0.05). Protein concentration in the UHL was higher in the EM2 (p < 0.05). Carbonyl and dityrosine formation was more intense in EM1 and EM2. Protein oxidation observed in the EM2 suggests the presence of an inflammatory status in the uterus which, if not adequately hindered, could result in low fertility.

Association between passive immunity and health status of dairy calves under 30 days of age.

Calves are highly susceptible to disease and mortality occurrence within the first month of life. Even if failed transfer of passive immunity (FTPI) is commonly recognized as a main factor affecting calf health and survival, conflicting results are reported in literature about the association between passive immunity (PI) and calf health, especially regarding enteric diseases. Therefore, a prospective cohort study was conducted on 78 calves of three Italian dairy farms during winters of years 2014-2016, with the specific aim of evaluating the association between PI and health status of calves within 30 days of age under field conditions. Blood samples were collected between 1 and 5 days of age from each calf included in the study, and disease and mortality occurrence was monitored throughout the first month of life. Additionally, fecal samples were collected from calves with scours before treatment. Blood serum samples were tested by an electrophoretic method for the assessment of immunoglobulin (Ig) concentration, whereas fecal samples were submitted to ELISA test for positivity to Escherichia coli K99, rotavirus, coronavirus, and Cryptosporidium spp. Only enteric diseases occurred in calves of this study. Calves that suffered from diarrhea or died within the first month of life had lower serum Ig concentrations than those that remained healthy or survived (P < .05). Even if not significantly (P = .127), lower serum Ig concentrations were observed in sick calves that had been treated with antibiotics compared to those that had not been treated. The odds of disease and mortality occurrence were 24 (95% CI = 3-231) and 11 (95% CI = 1-111) times higher, respectively, for calves with FTPI (serum Ig concentration <10.0 g/L) than for those with an adequate PI transfer (P <  .05). Calves with adequate PI transfer had also a 6-day delay in the age at first disease onset compared to those with FTPI (P < .01). Even if estimated on a small number of calves, those with FTPI had higher risks of enteric infections by rotavirus (odds ratio = 12; 95% CI = 1-137) and Cryptosporidium spp. (odds ratio = 9; 95% CI = 1-72) (P < .05). In this study, the PI level influenced the occurrence of enteric diseases and mortality in calves under one month of age, confirming the importance of a proper colostrum provision to calf health and, consequently, to the reduction of antimicrobial use in dairy farming. However, further investigations are needed, particularly focusing on the relationship between PI and specific enteropathogen infections in calves.

RNA-Peptide nanoplexes drug DNA damage pathways in high-grade serous ovarian tumors.

DNA damaging chemotherapy is a cornerstone of current front-line treatments for advanced ovarian cancer (OC). Despite the fact that a majority of these patients initially respond to therapy, most will relapse with chemo-resistant disease; therefore, adjuvant treatments that synergize with DNA-damaging chemotherapy could improve treatment outcomes and survival in patients with this deadly disease. Here, we report the development of a nanoscale peptide-nucleic acid complex that facilitates tumor-specific RNA interference therapy to chemosensitize advanced ovarian tumors to frontline platinum/taxane therapy. We found that the nanoplex-mediated silencing of the protein kinase, MK2, profoundly sensitized mouse models of high-grade serous OC to cytotoxic chemotherapy by blocking p38/MK2-dependent cell cycle checkpoint maintenance. Combined RNAi therapy improved overall survival by 37% compared with platinum/taxane chemotherapy alone and decreased metastatic spread to the lungs without observable toxic side effects. These findings suggest (a) that peptide nanoplexes can serve as safe and effective delivery vectors for siRNA and (b) that combined inhibition of MK2 could improve treatment outcomes in patients currently receiving frontline chemotherapy for advanced OC.

Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates.

Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates.

Molecular characterization by MLVA of Coxiella burnetii strains infecting dairy cows and goats of north-eastern Italy.

Q fever is a worldwide zoonotic disease caused by Coxiella burnetii (C. burnetii), an obligate intracellular bacterium. In ruminants, shedding into the environment mainly occurs during parturition or abortion, but the bacterium is shed also in milk, vaginal mucus, stools and urine. In Italy few surveys have been conducted and reported seroprevalence values ranged between 10% and 60%, even if few human cases have been described. Genotyping of bacteria is crucial for enhancing diagnostic methods and for epidemiological surveillance. The objective of this study was to investigate genotypic differences of C. burnetii genotypes directly in 34 samples, collected during a 3-years survey among 11 dairy cattle and 11 goat farms in the north-eastern part of Italy using a 6-locus multiple loci variable number of tandem repeat analysis (MLVA) method. The samples analysed included 13 bulk tank milk (BTM), 6 individual milk, 11 vaginal swabs and 4 foetal spleens. MLVA-type 2 was determined as the most prevalent in cattle in this study. C. burnetii strains circulating in the studied cattle population are very similar to genotypes previously described, while genotypes from goats showed an important variability. Further investigation are needed to understand the reason of this pattern.

IFAT and ELISA phase I/phase II as tools for the identification of Q fever chronic milk shedders in cattle.

Q fever is a widespread zoonotic disease caused by Coxiella burnetii. In cattle the bacterial shedding can persist without symptoms for several months and the shedders identification is a critical issue in the control of the infection at herd level. Following the example of the human protocols for the assessment of Q fever infection status, the aim of this study was the evaluation of the antibody response dynamics to phase I and phase II antigens in C. burnetii shedder dairy cows by means of a phase-specific serology, to verify the suitability of the investigated tools in recognising milk shedders. A total of 99 cows were monitored during time and classified on the basis of serological and PCR results in five groups identifying different shedding patterns. The 297 sera collected in three sampling times were tested by means of ELISA IgG for differential phase I and phase II antibodies detection, while a selection of 107 sera were tested by means of phase specific IgM and IgG IFAT. Both ELISA IgG and IFAT IgG highlighted a low reactivity in non-shedder seropositive animals compared to chronic milk shedder animals. ELISA IgG seemed to perform better than IFAT IgG-IgM, showing significant serological differences among groups that allowed recognising specific serological group patterns, in particular for chronic and occasional milk shedders. These results supported the hypothesis that an animal classification based on phase patterns is reasonable, although it needs to be further investigated.

Effect of induced ruminal acidosis on blood variables in heifers.

BACKGROUND: Ruminal acidosis is responsible for the onset of different pathologies in dairy and feedlot cattle, but there are major difficulties in the diagnosis. This study modelled the data obtained from various blood variables to identify those that could indicate the severity of ruminal acidosis. Six heifers were fed three experimental rations throughout three periods. The diets were characterised by different starch levels: high starch (HS), medium starch (MS) and low starch, as the control diet (CT). Ruminal pH values were continuously measured using wireless sensors and compared with pH measurements obtained by rumenocentesis. Blood samples were analysed for complete blood count, biochemical profile, venous blood gas, blood lipopolysaccharide (LPS) and LPS-binding proteins (LBP). RESULTS: The regression coefficient comparing the ruminal pH values, obtained using the two methods, was 0.56 (P = 0.040). Feeding the CT, MS and HS led to differences in the time spent below the 5.8, 5.5 and 5.0 pH thresholds and in several variables, including dry matter intake (7.7 vs. 6.9 vs. 5.1 kg/d; P = 0.002), ruminal nadir pH (5.69 vs. 5.47 vs. 5.44; P = 0.042), mean ruminal pH (6.50 vs. 6.34 vs. 6.31; P = 0.012), haemoglobin level (11.1 vs. 10.9 vs. 11.4 g/dL; P = 0.010), platelet count (506 vs. 481 vs. 601; P = 0.008), HCO3(-) (31.8 vs. 31.3 vs. 30.6 mmol/L; P = 0.071) and LBP (5.9 vs. 9.5 vs. 10.5 µg/mL; P < 0.001). A canonical discriminant analysis (CDA) was used to classify the animals into four ruminal pH classes (normal, risk of acidosis, subacute ruminal acidosis and acute ruminal acidosis) using haemoglobin, mean platelet volume, ß-hydroxybutyrate, glucose and reduced haemoglobin. CONCLUSIONS: Although additional studies are necessary to confirm the reliability of these discriminant functions, the use of plasma variables in a multifactorial model appeared to be useful for the evaluation of ruminal acidosis severity.