Association between rainfall and Escherichia coli in live bivalve molluscs harvested in Sardinia, Italy.

Rainfall is generally accepted as one of the most important factors associated with an increased level of E. coli in bivalve molluscs. Performing microbiological risk assessment is relevant to official control authorities to determine the sanitary status of harvesting areas and, therefore, develop monitoring strategies and identify management practices that could be used to improve the quality and safety of the final product. The present study aimed to investigate the impact of rainfall on the content of E. coli in bivalve molluscs farmed in Sardinia (Italy). Enumeration of E. coli was performed according to the Most Probable Number (MPN) method (ISO 16649-3) on 1,920 bivalve samples collected from 7 regional counties between 2018 and 2020. Bivalve molluscs samples included 955 mussels (Mytilus galloprovincialis), 500 oysters (Crassostrea gigas), 325 clams (Ruditapes decussatus), 94 warty venus (Venus verrucosa), and 46 lagoon cockles (Cerastoderma glaucum). Rainfall data were obtained by the Department of Meteorology of the ARPA Sardegna. For each sampling site, GPS coordinates were used to identify gauge stations within catchment areas. Cumulative rain (mm) was recorded 1, 3, 5, 7, and 15 days before sampling, among which the 7-day cumulative rain was the strongest predictor of E. coli counts. Several thresholds of 7-day cumulative rain (from <10 mm up to >300 mm) before sampling were used to estimate the chances of a non-compliant sample (E. coli levels above the limit for sanitary class A; 230 MPN/100 g). The 7-day cumulative rain was positively associated with the chances of non-compliance. When the 7-day cumulative rain before sampling was >300 mm, 80.5 % of the samples were non-compliant, and the odds of a non-compliant sample were 23.6 times higher, as compared to samples harvested when the 7-day cumulative rainfall was <10 mm. Precipitation data could be a useful tool for interpreting anomalous results from official control authorities and reduce the costs that originate from closure of production areas.

Prevalence of bovine mastitis-related pathogens, identified by mass spectrometry in flies (Insecta, Diptera) captured in the milking environment.

Diptera (Insecta) are able to transmit approximately 200 pathogenic microorganisms to humans and animals, causing more than 65 diseases, including bovine mastitis, which constitutes a major cause of economic losses in the dairy industry. In this scenario, 217 adult specimens of Diptera were collected from nine farms and identified to the family and/or species level. Of the 11 families recorded, Muscidae was the most prevalent (152 out of 217; 70%). All Diptera specimens were subjected to microbiological culture using conventional and selective media, and isolates were then identified at the species level by mass spectrometry. In total, 275 microorganisms were identified, with a predominance of pathogens related to environmental bovine mastitis (166/275 = 60·4%), that is, Enterococcus species (70/275 = 25·4%) and Escherichia coli (49/275 = 17·8%). Nontraditional agents related to bovine mastitis (called miscellaneous) were detected in 28% (77/275), as well as microorganisms with well-known zoonotic behaviour (e.g. Bacillus cereus). This is study contributes with knowledge of diversity of microorganisms carried by Diptera in the dairy environment, including pathogens associated with environmental and contagious bovine mastitis, and agents with human relevance. To our knowledge, a three-part chromogenic selective medium used to microbial culture of milk on farms was used for the first time to identification of pathogens in Diptera.

Species diversity and antimicrobial resistance patterns of Enterococcus spp. isolated from mastitis cases, milking machine and the environment of dairy cows.

The objectives were to study the diversity of Enterococcus spp. isolated from mastitis cases, milking equipment and the environment of dairy cows; and to determine in vitro resistance of isolates to antimicrobials that are relevant to human and animal health. Ten dairy farms were visited to collect samples from mastitis cases, faeces, bedding, aisles, water and milking equipment. Identification of Enterococcus at the species level and antimicrobial resistance testing was performed by MALDI-TOF and the disk-diffusion method, respectively. Of 365 isolates, Enterococcus hirae was the most prevalent, being more likely to be isolated from faeces than from milk (odds ratio (OR) = 39·2), liners (OR = 5·4) or bedding (OR = 2·2). Enterococcus saccharolyticus was the most prevalent in milk samples. The chances of isolating Enterococcus faecalis from milk were higher than from aisles (OR = 12·5), faeces (OR = 5·3), bedding (OR = 3·6) or liners (OR = 3·0). The odds of isolating Enterococcus faecium from faeces were higher than from liners (OR = 7·3), bedding (OR = 2·5) or aisles (OR = 2·4). Of 360 tested isolates, 1·9, 0·3 and 0·6% were resistant to penicillin, vancomycin and teicoplanin, respectively. Our results suggest that Enterococcus species can occupy specific ecological niches on dairy farms and pose a risk to public and animal health.

Effects of magnesium sulfate and dioctyl sodium sulfosuccinate on fecal hydration, output and systemic hydration in healthy horses / [Efeitos do sulfato de magnésio e do dioctil-sulfossuccinato de sódio na hidratação e eliminação fecal e sobre a hidratação sistêmica de cavalos saudáveis]

Magnesium sulphate (MS) and dioctyl sodium sulphosuccinate (DSS) are laxative drugs frequently used for the treatment of impactions. The aim of this study was to compare the effects of MS and DSS in fecal hydration, output and systemic hydration in healthy horses. Five healthy horses received 3 treatments with a 21-day interval. Treatment 1 was performed with administration of 4 liters of warm water; treatment 2: administration of 4 liters of warm water associated with 1g/kg of MS; and treatment 3: administration of 4 liters of warm water associated with 20mg/kg DSS. General and specific physical examination of the digestive system were performed, alongside with packed cell volume and total plasma protein measurement, abdominal ultrasonography and quantification of the amount and hydration of feces, before and 6, 12, 24, 36 and 48 hours after the treatments. No adverse effects were observed. The administration of laxatives promoted greater fecal output and hydration without resulting in systemic dehydration, yet no differences were observed between treatments. The absence of adverse effects of DSS demonstrates the safety of its use as a laxative drug at a dose of 20mg/kg. Studies comparing the effects of the laxative drugs in horses with large colon impaction are needed.(AU)

O sulfato de magnésio (SM) e o dioctil-sulfossuccinato de sódio (DSS) são drogas laxativas frequentemente utilizadas no tratamento da compactação. O objetivo deste estudo foi comparar os efeitos do SM e do DSS na hidratação e eliminação fecal, e na hidratação sistêmica de cavalos saudáveis. Cinco cavalos receberam três tratamentos, com intervalo de 21 dias. O tratamento 1 consistiu na administração de quatro litros de água morna; o tratamento 2: administração de quatro litros de água morna associada a 1g/kg de SM; e o tratamento 3: administração de quatro litros de água morna associada a 20mg/kg de DSS. Foram realizados exames físicos gerais e específicos do trato digestivo, hematócrito e proteína plasmática total, ultrassonografia abdominal e mensuração da quantidade e hidratação das fezes antes e após seis, 12, 24, 36 e 48 horas dos tratamentos. Não foram observados efeitos adversos. A administração de ambos os laxantes promoveu maior produção fecal e hidratação, sem resultar em desidratação sistêmica, entretanto não houve diferença entre os tratamentos. A ausência de efeitos adversos do DSS demonstra a segurança de seu uso como medicamento laxante na dose de 20mg/kg. São necessários estudos que comparem o efeito dos laxantes em equinos portadores de compactação de cólon.(AU)

Carboxymethylcellulose and psyllium effects in sand output of horses with asymptomatic sand accumulation / [Efeitos da carboximetilcelulose e do psyllium sobre a eliminação de areia de cavalos com sablose assintomática]

The aim of this study was to evaluate the effects of Psyllium (PSY) and Carboxymethylcellulose (CMC) administration on fecal elimination of sand in horses with asymptomatic sand accumulations. Eight horses were selected from sandy areas and randomly divided into 2 groups of four animals. The subjects were treated either with CMC or PSY. The presence of intestinal sand was confirmed through radiography and glove sedimentation test. The study was performed in two phases, with a 7-day interval. In phase I, all the animals received 8 liters of warm water; in phase II, the CMC group received 8 liters of water + 1g/kg of CMC, whereas the PSY group received 8 liters of water + 1g/kg of PSY. All administrations were performed through nasogastric intubation and fractionated in 2 equal volume administrations with an interval of two hours. General and specific physical examination of the digestive system were performed in conjunction with abdominal ultrasonography before the administrations and after 6, 12, 24, 36 and 48 hours, aiming to evaluate intestinal motility and presence of sand. All the feces eliminated by the animals within the 72 hours following the administrations were quantified, diluted and sedimented in order to calculate the sand output (g/kg of feces). All the animals were also subjected to radiographic examination to quantify sand accumulation prior to phase I and after 72 hours of phases I and II. No adverse effects were observed after the treatments. It was possible to notice higher sand elimination in both groups during the phase I, whereas no difference was observed in sand elimination rates between the groups in phase II. The radiographic scores presented differences between the initial timepoint and 72h in phases I and II for both groups. Based on the sand elimination rates and radiographic score, this study demonstrated that sand output was greater after administration of water alone, compared to CMC and Psyllium, leading to the inference that removal of the sandy environment and prevention of sand re-ingestion are effective measures for the elimination of sand from the colon of horses with asymptomatic sand accumulations.(AU)

O objetivo deste estudo foi avaliar os efeitos da administração do psyllium (PSY) e da carboximetilcelulose (CMC) sobre a eliminação fecal de areia em equinos com sablose assintomática. Oito equinos com confirmação radiográfica de sablose assintomática foram divididos em dois grupos (grupo CMC e grupo PSY). O estudo foi realizado em duas fases, com intervalo de sete dias. Na fase 1, todos os animais receberam 8L de água; na fase 2, o grupo CMC recebeu 8L de água + 1g/kg de CMC e o grupo PSY recebeu 8L de água + 1g/kg de PSY. Antes da administração de cada solução e após seis, 12, 24, 36 e 48 horas, foram realizados exame físico e ultrassonografia abdominal. Todas as fezes eliminadas em 72 horas foram avaliadas para quantificar a eliminação de areia (g/kg de fezes). Antes da fase 1 e após 72 horas das fases 1 e 2, o exame radiográfico foi realizado para quantificar o escore de acúmulo de areia. Houve maior eliminação de areia após a administração de água em comparação com a administração de CMC, e não se observou diferença entre a CMC e o PSY. Uma redução significativa nos escores radiográficos de acúmulo de areia foi observada após a administração de água, bem como a manutenção dos escores após a administração da CMC e do PSY. Com base na produção de areia e no escore radiográfico, este estudo sugere que a remoção do ambiente arenoso, impedindo a reingestão de areia, é uma medida eficaz para a eliminação da areia do cólon de cavalos com acúmulos de areia assintomáticos.(AU)

Efficacy of ketoprofen for treatment of spontaneous, culture-negative, mild cases of clinical mastitis: A randomized, controlled superiority trial.

The objective of this randomized, controlled superiority trial was to assess the efficacy of ketoprofen for the treatment of spontaneous, culture-negative clinical mastitis cases that were not treated with antimicrobials. Holstein cows from 3 herds were eligible for inclusion if they had mild or moderate culture-negative clinical mastitis cases in 1 quarter or more. Upon detection of clinical mastitis, farm personnel performed on-farm culture (OFC) using commercially available bi-plates. Samples used for OFC were also cultured in a research laboratory. Cows with culture-negative clinical mastitis that met the inclusion criteria were randomly allocated to 1 of 2 experimental groups: in the ketoprofen (KET) group, cows received an intramuscular injection of 3 mg/kg of ketoprofen upon confirmation of a negative OFC result; and in the control (CON) group, cows received no treatment or placebo. Milk samples were collected 14 and 21 d after detection of clinical mastitis for microbiological examination and somatic cell counting. Study outcomes were clinical cure (within 7 d after inclusion in the study), relapse (within 14 d after inclusion) and recurrence of clinical mastitis (15 to 90 d after inclusion), risk of new intramammary infection, and quarter milk somatic cell count at 14 and 21 d. We used Cox proportional hazards, logistic regression, and repeated-measures models to compare each outcome between groups. After exclusion of moderate cases (n = 6), a total of 123 clinical mastitis cases (CON = 58 and KET = 65) were used for analyses. Risks of clinical cure [83.08% (54/65) and 91.23% (52/57); hazard ratio = 1.20, 95% confidence interval (CI) = 0.82-1.76], relapse [19.23% (10/52) and 18.00% (9/50); hazard ratio = 1.09, 95% CI = 0.45-2.62], and recurrence of clinical mastitis [17.31% (9/52) and 18.00% (9/50); hazard ratio = 1.26, 95% CI = 0.49-3.38] were not different between the KET and CON groups, respectively. The odds of a new intramammary infection at 14 d [20.75% (11/53) and 29.79% (14/47); odds ratio = 1.76, 95% CI = 0.66-4.73] or 21 d [28.57% (12/42) and 15.22% (7/46); odds ratio = 0.45, 95% CI = 0.16-1.30] were not different between the KET and CON groups, respectively. Mean somatic cell count was not different between the groups at 14 or 21 d. The results of this study suggest that a single intramuscular injection of ketoprofen as sole treatment for OFC-negative, mild clinical mastitis did not reduce time to clinical cure, relapse or recurrence of clinical mastitis, risk of subsequent intramammary infection, or milk somatic cell count compared with untreated controls.

Association between teat-end hyperkeratosis and mastitis in dairy cows: A systematic review.

A systematic review was conducted to elucidate the role of teat-end hyperkeratosis (THK) as a risk factor for clinical mastitis (CM) or subclinical mastitis (SCM). Scientific papers on the subject were identified by means of a database search. All types of peer-reviewed analytical studies, observational or experimental and published in English, could be included in the review, regardless of publication year. Of 152 identified records, 18 articles were selected, of which 8 were prospective cohort studies, 9 were cross-sectional, and 1 was a hybrid case-control study. Internal validity of studies was assessed using a score system ranging from 0 to 6, based on design, risk of bias, and statistical methods. The most frequent study limitation was improper use of statistical methods to avoid confounding of associations between THK and CM or SCM. The 3 studies that used CM as outcome (all with high validity scores) showed positive associations with THK (especially severe), although the magnitude and statistical significance of the estimates differed among them. Most studies that used SCM as the primary outcome (based on microbiological examination of milk) reported that only severe THK was associated with SCM. Two studies with high validity scores reported moderate to strong associations between severe THK and incidence or prevalence of Staphylococcus aureus intramammary infection. Two studies with high validity scores reported that only severe THK was associated with the risk of somatic cell count (SCC) ≥200,000 cells/mL and increased mean SCC, respectively. Although 4 cross-sectional studies reported positive associations between THK and SCC, these associations were possibly spurious because confounding factors, such as parity, were not considered in the analyses. Results of the reviewed studies suggest that severe THK is a risk factor for both CM and SCM, as defined by microbiological examination of milk, SCC thresholds, or mean SCC. The effect of severe THK on both contagious (especially Staphylococcus aureus) and environmental CM or SCM emphasizes the importance of teat health for mastitis control. Four studies demonstrated that quarters with mild THK had lower prevalence of intramammary infection or lower mean SCC than quarters with no THK, suggesting that development of mild THK, as a physiological response to milking, can have a protective effect. Dairy consultants should focus on monitoring and minimizing occurrence of severe THK to prevent CM and SCM.

Genotyping of long term persistent Staphylococcus aureus in bovine subclinical mastitis.

Bovine mastitis affects dairy cattle worldwide and Staphylococcus aureus is one of the most common microorganisms involved in subclinical and chronic disease. Superantigens, such as enterotoxins contribute to S. aureus persistence and pathogenicity in this disease. Subclinical and chronic mastitis cases were diagnosed and S. aureus isolates from sub-clinical cases were investigated for carriage of virulence and antibiotic resistance genes that may contribute to long-term carriage and infection. Over a 12-month period, 116 S. aureus strains were recovered from 68 cows with subclinical mastitis. Classical enterotoxin genes (sea-see) were detected in 24.1% of isolates, and pvl and tsst-1 were identified in 3.4% and 46.6% the isolates, respectively. 18.1% that were persistent isolates were identified and characterized by pulsed field gel electrophoresis (PFGE), MLST, spa typing. Four isolates were methicillin-resistant S. aureus (MRSA) and belonged to SCCmec type I. Molecular typing showed that the agrI group was the most frequent, and a rare isolate was positive for both agrI and agrIII groups. Molecular characterization revealed the persistence of the spa type t10856 (ST133, clonal complex CC133, agr I), in a single animal for nine months and the persistence t605 (ST126, CC126) colonizing four animals for four months. These strains have been described recently in other herds in the same region, indicating their transmissibility and clonal expansion. We conclude that animals with subclinical mastitis are an important and somewhat overlooked reservoir for transmission within and between herds, and may carry virulence and antibiotic resistance genes contributing to persistent colonization, hinder the control of mastitis and may cause risks to the public health.

Putative virulence factors of extra-intestinal Escherichia coli isolated from bovine mastitis with different clinical scores.

We investigated the genes kpsMTII, iucD, sfaDE, afaBC, papA and papC, (proposed to be involved in extra-intestinal pathogenic Escherichia coli-ExPEC), phylogroup classification and the in vitro swimming and swarming motility in 50 E. coli isolated from bovine mastitis with different clinical severity scores (mild, moderate and severe). The aforementioned genes were detected in 12 (n = 12/50; 24·0%) isolates. kpsMTII and iucD were the most frequent genes identified in six (n = 6/50; 12·0%) and four (n = 4/50; 8·0%) of the isolates, respectively. In only one (n = 1/50; 2·0%) isolate, more than one gene was simultaneously identified: iucD and kpsMTll were detected whereas sfaDE and afaBC were not detected. Mild, moderate and severe clinical signs were observed in 40·0% (n = 20/50), 28·0% (n = 14/50) and 32·0% (n = 16/50) of the cases. Commensal phylogroups B1 (n = 19/50; 38·0%) and A (n = 19/50; 38·0%) were prevalent; whereas pathogenic phylogroups B2 and D were observed in only 10·0% (n = 5/50). Swarming and swimming motilities were observed in 90·0% (n = 45/50) and 68·0% (n = 34/50) of the isolates, respectively; and there was a significant association (P = 0·0036) between swarming motility and severe clinical cases (score 3). To the best of our knowledge, this is the first study where clinical severity of bovine mastitis cases and the genes proposed to classify ExPEC were assessed in relation to swarming and swimming motility. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli is classified as extra-intestinal (ExPEC) when strains contain at least two of the genes kpsMTII, iucD, sfaDE, afaBC and papA and/or papC. We investigated in vitro motility and the presence of these genes in 50 E. coli isolated from bovine mastitis with different clinical scores (mild, moderate and severe). Clinical severity was not associated with the genes studied. Swarming motility was associated with severe cases (score 3) of clinical mastitis. Results of this study contribute to a better understanding of the factors that determine the severity of clinical mastitis.

Diagnostic accuracy of Somaticell, California Mastitis Test, and microbiological examination of composite milk to detect Streptococcus agalactiae intramammary infections.

The objectives of this study were to estimate the accuracy of Somaticell (Idexx Laboratories Inc., Westbrook, ME), California Mastitis Test (CMT), and microbiological examination of composite milk (MEC) to diagnose Streptococcus agalactiae intramammary infections (IMI), and to assess the agreement between Somaticell and CMT to detect these infections. A secondary objective was to estimate quarter- and cow-level prevalence of S. agalactiae IMI in the herds included in the study. Seven farms were included in the study. The CMT was performed and aseptic milk samples were collected from all quarters of all lactating cows. Composite milk samples were produced in the laboratory by mixing milk from all quarters of each sampled cow. The Somaticell test was performed on a subset of S. agalactiae-positive (n = 167) and S. agalactiae-negative (n = 152) quarter milk samples. Microbiological examination of quarter milk samples (MEQ) was considered the reference test for diagnosing S. agalactiae IMI. The accuracy of all tests at various thresholds was estimated using Bayesian latent class models. Apparent prevalence of S. agalactiae IMI was 15.8% (n = 184/1,164) at the quarter level (based on MEQ) and 28.5% (n = 83/291) at the cow level (based on MEC). True prevalence, as determined by Bayesian models, was 13.0% [95% credible interval (CR): 6.4-24.4%] at the quarter level, and 25.6% (95% CR: 15.3-39.5%) at the cow level. At the cow level (n = 285), sensitivity and specificity of MEC were 95.6 and 99.5%, respectively. The accuracy of Somaticell (n = 319 quarters) to identify S. agalactiae-infected quarters was 75.4, 86.4, 88.9, 89.4, and 91.0% at thresholds of 98,000, 147,000, 205,000, 244,000, and 282,000 cells/mL, respectively. The accuracy of CMT was 87.6, 90.7, 90.8, and 87.4% at thresholds of trace, 1, 2, and 3, respectively. The areas under the receiver operating characteristic curve for Somaticell and CMT were 94.5% (95% confidence interval: 91.8-97.2%) and 92.0% (88.6-95.4%), respectively. At the tested thresholds, the sensitivity of Somaticell ranged from 94.9 to 99.5% to detect S. agalactiae IMI, and specificity ranged from 48.1 to 87.1%. The sensitivity of Somaticell at the lowest threshold (69,000 cells/mL; sensitivity = 99.9%; 95% CR: 98.2-100%) was higher than that of CMT at any tested threshold. Results of this study could be used at the farm level to reduce the use of antimicrobials and reach specific goals in S. agalactiae eradication programs.

Influence of two ovulation-inducing agents on the pituitary response and follicle blood flow in mares.

The objective of the current study was to evaluate the effects of deslorelin and hCG, two ovulation-inducing therapies, on LH surge and follicle vascularity in mares. Thirty mares were either treated with 1.5 mg IM of deslorelin, 2,500 IU IV of hCG or 2 mL IM of NaCl 0.9% (GnRH, hCG and Saline groups, respectively). Power-flow Doppler examination and blood collection were performed every hour during the first 12 hours after treatment (H0) and every six hours between hours 12 (H12) and 30 (H30) after treatment. Moreover, endpoints were evaluated every hour through the last six hours before ovulation (OV-6 to OV-1). In GnRH group, plasma LH concentration progressively increased (P < 0.001) during the first 6 hours after treatment and remained high (P > 0.1) until OV-1. A significant increase in LH concentrations was first detected (P < 0.05) at 24 hours after treatment in hCG group, while no changes (P > 0.1) on LH levels were found during H0-H30 and between OV-6 and OV-1 in the Saline group. Independent of the treatment, significant variations on the percentage of the follicle wall with Doppler signals were not observed (P > 0.1) throughout the entire experiment. A weak correlation between the preovulatory follicle vascularity and the plasma LH concentration was found in GnRH, hCG and Saline groups (r = +0.29, +0.29 and -0.23, respectively; P ˂ 0.0001). These results described for the first time the immediate and continuous pituitary response to ovulation-inducing therapy with injectable deslorelin. Moreover, spontaneous and induced ovulations were not preceded by an increased follicle vascularity, which differs from previous reports in large animals.

Administration of 2.5 mg of estradiol followed by 1,500 mg of progesterone to anovulatory mares promote similar uterine morphology, hormone concentrations and molecular dynamics to those observed in cyclic mares.

To test the hypothesis that the administration of 2.5 mg of estradiol benzoate (EB) followed by 1500 mg of long acting progesterone (LA P4) causes similar uterine changes and molecular dynamics in anovulatory mares to those observed in cyclic ones, we evaluated the changes of estrogen (ERα and ERß) and progesterone receptors (PR) in anestrous, transitional and cyclic mares by RT-qPCR and immunohistochemistry. In addition, we evaluated uterine edema, tonus and estrogens and progesterone plasma profile. Endometrial biopsies were taken from anestrous and transitional mares immediately before EB injection, 48 h after EB administration and five days after LA P4 was given. In cyclic mares, biopsies were collected at estrus and at five days after ovulation. Similar estrogen peaks were achieved after the injection of the single EB dose between treated and cyclic groups, as well as maximum uterine edema. Uterine tone was increased to diestrus levels after administration of 1500 mg of LA P4. Changes in relative abundance of transcripts for PR, ERα and ERß when progesterone stimulated endometrium was compared to estrogen stimulated endometrium were similar between cyclic and non-cyclic treated mares. However, apparent decreased PR in the endometrial glandular epithelium was not observed in non-cyclic mares five days after LA P4 administration as observed at five days after ovulation in cyclic mares. The protocol produced similar endometrial edema, uterine tonus and changes in relative abundance of PR, ERα and ERß transcripts to those observed in cyclic mares during late estrus and early diestrus, as well as similar estradiol and estrogen conjugate plasma concentrations.

Virulence markers associated with Trueperella pyogenes infections in livestock and companion animals.

Trueperella pyogenes is an opportunistic pathogen that causes diverse pyogenic infections in livestock. The genes that encode the exotoxin pyolysin (plo) and other putative factors that promote adhesion of pathogen to host cells (fimbriae fimA, fimC, fimE, fimG, neuraminidases nanH, nanP, and collagen-binding protein cbpA) have been associated with virulence, particularly in mastitis and uterus infections of dairy cows. However, the role of these virulence markers in the pathogenicity of the agent in domestic animals infections still is incompletely understood. The genes plo, fimA, fimC, fimE, fimG, nanH, nanP, and cbpA were investigated in 71 T. pyogenes strains recovered from cattle, sheep, goats, dogs, equines, and a pig, recovered from mastitis (n = 35), and non-mastitis (n = 36) cases (abscesses, reproductive tract diseases, pneumonia, lymphadenitis, encephalitis). The most common genes harboured by the isolates were: plo (71/71 = 100·0%), fimA (70/71 = 98·6%), nanP (56/71 = 78·9%), fimE (53/71 = 74·6%), fimC (46/71 = 64·8%) and nanH (45/71 = 63·4%), whereas cbpA (6/71 = 8·4%) and fimG (4/71 = 5·6%) were uncommon. The most frequent genotypes were plo/fimA/fimE/fimC/nanH/nanP (17/71 = 23·9%), plo/fimA/fimE/nanH/nanP (13/71 = 18·3%), and plo/fimA/fimE/fimC/nanP (11/71 = 15·5%). No association was observed between the presence of genes vs clinical signs or host species. To the best of our knowledge, this is the first report on aforementioned virulence factors of pathogen detected in diseased horses and dogs. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of particular virulence factors of Trueperella pyogenes that determine different pyogenic infections among domestic animals is poorly understood. Eight putative virulence genes and genotype profiles of 71 isolates were investigated among different clinical manifestations in domestic animals. The most common genes were plo (71/71 = 100·0%), fimA (70/71 = 98·6%), nanP (56/71 = 78·9%), fimE (53/71 = 74·6%), fimC (46/71 = 64·8%) and nanH (45/71 = 63·4%), whereas plo/fimA/fimE/fimC/nanH/nanP (17/71 = 23·9%), plo/fimA/fimE/nanH/nanP (13/71 = 18·3%), and plo/fimA/fimE/fimC/nanP (11/71 = 15·5%) were the most frequent genotypes. Studies involving virulence factors are critical in the investigation of molecular epidemiology, pathogenicity, and hypothetical differences in the virulence among T. pyogenes strains from different geographical areas.

In vitro algicidal effect of guanidine on Prototheca zopfii genotype 2 strains isolated from clinical and subclinical bovine mastitis.

Prototheca species have increasingly been reported to be opportunistic pathogens that cause mastitis in dairy herds, and it poses an emergent problem because at present, there are no effective therapies for the treatment of protothecal mastitis. This study investigated the in vitro algicidal effect of guanidine on 75 Prototheca zopfii genotype 2 strains isolated from 75 cases of clinical and subclinical bovine mastitis. All strains were susceptible to guanidine in vitro with minimal algaecide concentrations ranging from 0·001 to 0·035%. Guanidine is known to have a high microbicidal effect and is considered to be a new generation microbicidal compound. It is not toxic to human mucous membranes and conjunctivas at low concentrations and has been used as a disinfectant in swimming pools and as an antiseptic for human wounds. The algicidal action of guanidine at low concentrations indicates that it could be an alternative disinfectant or antiseptic for cleaning of the dairy environment and milking equipment, in pre- and postdipping solutions, in the chemical dry therapy of bovine teats and even in the intramammary therapy of P. zopfii infections. This is the first report of the in vitro algicidal effect of guanidine on P. zopfii strains of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prototheca zopfii genotype 2 is an opportunistic pathogen of bovine mastitis. To date, no effective therapies against protothecal mastitis have been developed. The in vitro algicidal effect of guanidine on 75 P. zopfii genotype 2 strains isolated from cows revealed that all of the isolates were susceptible to the compound at low concentrations, which indicates that guanidine may be used as an antiseptic/disinfectant for dairy milking equipment, in pre- and postdipping solutions, and as a chemical dry therapy or an intramammary therapy. This study describes the in vitro algicidal effect of guanidine on P. zopfii for the first time.

Pharmacokinetics and pharmacodynamics of the injectable formulation of methadone hydrochloride and methadone in lipid nanocarriers administered orally to horses.

We investigated the thermal, electrical and mechanical antinociceptive and physiological effects (heart rate, respiratory rate, arterial blood pressure, head height and abdominal auscultation score), and pharmacokinetics, of 0.5 mg/kg of the injectable formulation (ORAL) or nanoparticulated methadone (NANO) given orally, in six adult mares, using a crossover, blind and prospective design. Repeated-measure models were used to compare parametric data between and within treatments, followed by Tukey's test. Nonparametric data were analysed with Wilcoxon signed-rank, adjusted by Bonferroni tests. Blood samples were also collected up to 6 h after dosing for plasma drug quantification by LC-MS/MS. Methadone pharmacokinetic parameters were determined by noncompartmental and compartmental approaches. There were no differences in pharmacodynamic parameters. No statistical differences were observed in the pharmacokinetic parameters from noncompartmental analysis for both groups, except a significant decrease in peak plasma concentration, increase in apparent volume of distribution per fraction absorbed (Vdss /F) and increased mean residence time (MRT) for NANO. One-compartment open model with first order elimination best described the pharmacokinetic profiles for both groups. Neither ORAL nor NANO administered orally to horses produced antinociception. The nanoencapsulated formulation of methadone given orally to horses did not improve methadone pharmacokinetic parameters or increased systemic body exposure to methadone.

Associação da ocorrência do ectima contagioso (ORF vírus) em ovinos com os cuidadores desses animais / Occurrence of the Association contagious ecthyma (ORF virus) in sheep with these animals' caregivers

A enfermidade ectima contagioso está difundida em todo o estado de São Paulo. Foram amostrados 42 (8,64%) cuidadores de animais e 444 (91,36%) ovinos (n=486). A prevalência de reagentes para vírus-neutralização foi de 67% (IC95%=62-71%) nos ovinos, e em seus cuidadores de 76% (IC95%=63-89%), sendo P=0,22, ou seja, não houve diferença estatística significativa entre as espécies. A distribuição dos títulos teve diferença estatística significativa entre as espécies, com P=0,0048. As variações de titulação foram de 0,6 a 2,1 tanto nos ovinos quanto nos seus cuidadores. Dentre os 42 cuidadores de ovinos participantes do estudo, 32 apresentaram títulos de anticorpos expressos por log10 acima de 0,6.(AU)

These diseases are all widespread in the State of São Paulo. 42 (8.64%) animal caregivers and 444 (91.36%) sheep (n=486) were sampled. The reagents Prevalence paragraph virus neutralization was 67% (95% CI = 62-71%) in sheep and 76% (95% CI = 63-89%) for caregivers, with P=0.22 not being a statistically significant difference between the species. One of the distribution titles had significant difference between statistics as species with P=0.0048. The titration variations were 0.6 to 2.1, both in sheep and their caregivers. Among the 42 sheep caregivers participating in the study, 32 had antibody securities denominated in log10 above 0.6.(AU)

Comparison of different regimens of estradiol benzoate treatments followed by long-acting progesterone to prepare noncycling mares as embryo recipients.

The present study evaluated the influence of different regimens of estradiol benzoate (EB) treatments followed by a single dose of long-acting progesterone (LA P4) on plasma estrogen and P4 concentrations in noncyclic mares prepared as embryo recipients. Twenty-one anestrous mares were distributed into three groups (n = 7 mares per group), according to the EB dose received (single dose of 2.5 mg, total of 5 mg in decreasing doses, and total of 10 mg in decreasing doses), which was followed by a single administration of 1500 mg of LA P4 in all groups. Mares were reevaluated during the ovulatory phase and seven of them became part of the cyclic nontreated control group. Ultrasonography was performed to monitor endometrial edema, and blood samples were collected to measure estradiol (E2), estrogen conjugate (EC), and P4 by RIA. Maximum uterine edema was achieved 24 hours after administration of EB in all treated groups. Maximum E2 concentrations were observed 24 hours after the first EB injection in treated groups and there were no differences (P > 0.05) among treatments. Maximum EC concentration was observed 24 hours after the single EB injection in the 2.5-mg group, whereas in the 5- and 10-mg groups EC peaks were observed 48 hours after the first EB administration. Maximum P4 concentrations were detected 24 hours after LA P4 injection, although higher P4 concentrations were observed in the group treated with 2.5 mg of EB than in that treated with 10 mg of EB (P < 0.05). Because P4 concentrations were reduced after administration of high doses of EB, we also measured 17α-hydroxyprogesterone (17-OH-P) to test the hypothesis that high concentrations of EB would accelerate the conversion of P4 to 17-OH-P. However, 17-OH-P concentrations paralleled P4 profile in all groups, irrespective of EB doses. In summary, the three EB treatment regimens induced similar E2 peaks, although the observation of EC peaks 24 hours after E2 peaks in the 5- and 10-mg groups indicate that an excess of E2 was given, which was converted into EC to be inactivated. Administration of 10 mg of EB reduced P4 concentrations 24 hours after LA P4 was given. We demonstrated that the mechanism by which this reduction occurred was not by an increase in P4 metabolism to 17α-OH-P. In conclusion, the use of 2.5 mg of EB followed by 1500 mg of LA P4 appears to be a more appropriate regimen to treat noncyclic mares, although additional studies are needed to verify embryo survival with this treatment dose.

Qualitative and Quantitative Determination and Resistance Patterns of Salmonella from Poultry Carcasses.

Poultry products are important in the transmission of zoonotic pathogens, mainly Salmonella. This genus causes millions of foodborne diseases worldwide every year. Cross-contamination by food sources in human cases of salmonellosis and the increase in resistant strains have become important issues. A qualitative and quantitative Salmonella detection method was utilized in a poultry slaughterhouse in São Paulo State, Brazil. We collected 33 samples from different batches of carcasses. Each sample was analyzed at three process points: postbleeding, postdefeathering, and postchilling. A fourth point, retail simulation, was added to simulate retail market storage at 5°C for 72 h. The qualitative methods revealed 100% (33 samples) contamination at postbleeding, 39% (13 samples) contamination at postdefeathering, 58% (19 samples) contamination at postchilling, and 30% (10 samples) contamination at the retail simulation. The quantitative results, determined by the most-probable-number (MPN) technique, ranged from <0.03 to >2,400 MPN/g. We identified 23 Salmonella serovars; the most prevalent were Mbandaka, Senftenberg, and Enteritidis. Resistance to nalidixic acid was significantly more common (P < 0.05) than resistance to other antimicrobial agents. Five multidrug-resistant strains were identified. This study contributes important epidemiological data and demonstrates the need to improve sanitary conditions in slaughterhouses.

Antinociceptive effects of methadone combined with detomidine or acepromazine in horses.

REASONS FOR PERFORMING STUDY: To investigate two protocols to provide antinociception in horses. OBJECTIVES: To evaluate the antinociceptive effects of intravenous methadone combined with detomidine or acepromazine in adult horses. STUDY DESIGN: Randomised, blinded, crossover study. METHODS: Mechanical, thermal and electrical stimuli were applied to the dorsal left and right metacarpus and coronary band of the left thoracic limb, respectively. A thermal stimulus was applied caudal to the withers. The horses were treated with saline (C), a combination of methadone (0.2 mg/kg bwt) and detomidine (10 µg/kg bwt) (MD) or methadone (0.2 mg/kg bwt) and acepromazine (0.05 mg/kg bwt) (MA) at 1 week intervals. Nociceptive thresholds were measured before and at 15 min intervals until 150 min after treatment. Wilcoxon rank-sum and Wilcoxon signed rank tests were used to compare data between groups at each time point and over time within each group, followed by the Bonferroni method to adjust the P value. RESULTS: The mechanical stimulus was the most sensitive test to differentiate the antinociceptive effects of the treatments. Mechanical thresholds were greater after MD than MA between 15 and 30 min and with both MD and MA these thresholds were greater than C from 15 to 60 min. Electrical and thermal limb thresholds were greater after MD than C at 15 and 45 min and at 15, 30, 45, 75 and 105 min, respectively. Thermal limb thresholds were greater with MA than C at 30 min. Thoracic thermal threshold in MD and MA were higher than C at 45, 75, 90 and 120 min and from 30 to 75 min, respectively. CONCLUSIONS: Methadone and acepromazine produced less pronounced mechanical antinociception than MD.

Validation of mechanical, electrical and thermal nociceptive stimulation methods in horses.

REASONS FOR PERFORMING STUDY: To validate a model for investigating the effects of analgesic drugs on mechanical, thermal and electrical stimulation testing. OBJECTIVES: To investigate repeatability, sensitivity and specificity of nociceptive tests. STUDY DESIGN: Randomised experiment with 2 observers in 2 phases. METHODS: Mechanical (M), thermal (TL) and electrical (E) stimuli were applied to the dorsal metacarpus (M-left and TL-right) and coronary band of the left thoracic limb (E) and a thoracic thermal stimulus (TT) was applied caudal to the withers in 8 horses (405 ± 43 kg). Stimuli intensities were increased until a clear avoidance response was detected without exceeding 20 N (M), 60°C (TL and TT) and 15 V (E). For each set of tests, 3 real stimuli and one sham stimulus were applied (32 per animal) using a blinded, randomised, crossover design repeated after 6 months. A distribution frequency and, for each stimulus, Chi-square and McNemar tests compared both the proportion of positive responses detected by 2 observers and the 2 study phases. The κ coefficients estimated interobserver agreement in determining endpoints. Sensitivity (384 tests) and specificity (128 tests) were evaluated for each nociceptive stimulus to assess the evaluators' accuracy in detecting real and sham stimuli. RESULTS: Nociceptive thresholds were 3.1 ± 2 N (M), 8.1 ± 3.8 V (E), 51.4 ± 5.5°C (TL) and 55.2 ± 5.3°C (TT). The level of agreement after all tests, M, E, TL and TT, was 90, 100, 84, 98 and 75%, respectively. Sensitivity was 89, 100, 89, 98 and 70% and specificity 92, 97, 88, 91 and 94%, respectively. CONCLUSIONS: The high interobserver agreement, sensitivity and specificity suggest that M, E and TL tests are valid for pain studies in horses and are suitable tools for investigating antinociceptive effects of analgesics in horses.