Staphylococcus aureus adlb gene is associated with high prevalence of intramammary infection in dairy herds of northern Italy: A cross-sectional study.

Staphylococcus aureus is a major mastitis pathogen in dairy cattle worldwide, responsible for substantial economic losses. Environmental factors, milking routine, and good maintenance of milking equipment have been described as important factors to prevent intramammary infections (IMI). Staphylococcus aureus IMI can be widespread within the farm or the infection can be limited to few animals. Several studies have reported that Staph. aureus genotypes differ in their ability to spread within a herd. In particular, Staph. aureus belonging to ribosomal spacer PCR genotype B (GTB)/clonal complex 8 (CC8) is associated with high within-herd prevalence of IMI, whereas other genotypes are generally associated with individual cow disease. The adlb gene seems to be strictly related to Staph. aureus GTB/CC8, and is a potential marker of contagiousness. We investigated Staph. aureus IMI prevalence in 60 herds in northern Italy. In the same farms, we assessed specific indicators linked to milking management (e.g., teat condition score and udder hygiene score) and additional milking risk factors for IMI spread. Ribosomal spacer-PCR and adlb-targeted PCR were performed on 262 Staph. aureus isolates, of which 77 underwent multilocus sequence typing. In most of the herds (90%), a predominant genotype was identified, especially Staph. aureus CC8 (30%). In 19 of 60 herds, the predominant circulating Staph. aureus was adlb-positive and the observed IMI prevalence was relevant. Moreover, the adlb gene was detected only in genotypes of CC8 and CC97. Statistical analysis showed a strong association between the prevalence of Staph. aureus IMI, the specific CCs, and carriage of adlb, with the predominant circulating CC and presence of the gene alone explaining the total variation. Interestingly, the difference in the odds ratio obtained in the models for CC8 and CC97 suggests that it is carriage of the adlb gene, rather than the circulation of these CCs per se, that leads to higher within-herd prevalence of Staph. aureus. In addition, the model showed that environmental and milking management factors had no or minimal effect on Staph. aureus IMI prevalence. In conclusion, the circulation of adlb-positive Staph. aureus strains within a herd has a strong effect on the prevalence of IMI. Thus, adlb can be proposed as a genetic marker of contagiousness for Staph. aureus IMI in cattle. However, further analyses using whole-genome sequencing are required to understand the role of genes other than adlb that may be involved in the mechanisms of contagiousness of Staph. aureus strains associated with high prevalence of IMI.

Incidence of milk leakage after dry-off in European dairy herds, related risk factors, and its role in new intramammary infections.

The incidence of milk leakage (ML) after dry-off (DO) and related risk factors was studied in 1,175 dairy cows from 41 commercial herds in 8 European countries: Belgium, Czech Republic, Denmark, France, Germany, Italy, the Netherlands, and Spain. Milk leakage was assessed twice for 30 s each during 3 visits at 20 to 24 h, 30 to 34 h, and 48 to 52 h after DO. Information related to dry-cow management and udder health was collected at herd and cow level, including individual somatic cell count (ISCC) from test-day controls and occurrence of clinical mastitis cases from DO until 30 d in lactation. Mixed-effect logistic regression analyses were used to identify possible risk factors for ML and to study the association between ML and new intramammary infections. Intramammary infections were defined as clinical mastitis cases during the dry period and in the first 30 d in lactation or a rise in ISCC from before to after the dry period (threshold: 200,000 cells/mL) or both. Milk leakage was observed in 24.5% of the cows between 20 and 52 h after DO, where the herd incidence varied between 0.0 and 77.8%. The reduction in number of milkings in the weeks before DO had statistically significant effect on the ML incidence. When the milking frequency was reduced from 3 times/d to 2 or maintained at twice a day, cows had 11 (95% CI = 3.43-35.46) or 9 (95% CI = 1.85-48.22) times higher odds of leaking milk, respectively, compared with cows where the milking frequency was reduced from twice to once a day. Also, the milk production 24 h before DO was associated with ML incidence. Hence, cows with a milk production between 13 and 21 L or above 21 L had 2.3 (95% CI = 1.48-3.53) and 3.1 (95% CI = 1.79-5.3) times higher odds of leaking milk, respectively, compared with cows with a milk production below 13 L. A higher ML incidence was present in the group of cows with an average ISCC in the last 3 mo before DO ≥200,000 cells/mL (odds ratio = 1.7; 95% CI = 1.13-2.41) compared with cows with an average ISCC <100,000 cells/mL. Quarters with ML tended to have 2.0 times higher odds of developing clinical mastitis compared with quarters not leaking milk. Cows with ML tended to have 1.5 times higher odds of intramammary infections (i.e., an increase of ISCC or clinical mastitis) compared with cows without ML.

Short communication: In vitro antimicrobial susceptibility of Mycoplasma bovis isolates identified in milk from dairy cattle in Belgium, Germany, and Italy.

The objective of this study was to assess the in vitro antimicrobial susceptibility of 73 isolates of Mycoplasma bovis isolated from milk of dairy cattle herds of Belgium, Germany, and Italy. Minimal inhibitory concentration (MIC) values were determined by the microbroth dilution method for the following antimicrobials: erythromycin, spiramycin, tilmicosin, tylosin, lincomycin, enrofloxacin, doxycycline, oxytetracycline, florfenicol, and tiamulin. Macrolides, florfenicol, oxytetracycline, and enrofloxacin, were chosen because they represent antimicrobials families commonly used in several countries for treatment of M. bovis, and their MIC values in cattle population are reported in several studies, allowing a comparison with previous data. Doxycycline and tiamulin were selected to assess the susceptibility of M. bovis to new antimicrobials, because they are not registered in the European Union for the treatment of dairy cattle. Among the agents of the different antimicrobial classes, the macrolides showed the highest concentration to inhibit 90% of isolates (MIC90), all above the highest concentration tested: >8µg/mL for erythromycin, >16µg/mL for spiramycin, and >32µg/mL for tilmicosin and tylosin. Also the MIC90 of lincomycin was above the highest concentration tested (>32µg/mL), but the distribution of the MIC values was almost perfectly bimodal: 41 isolates had a MIC ≤0.5µg/mL and 30 isolates >32µg/mL. Oxytetracycline had a 2-fold higher concentration to inhibit 50% of isolates (2 vs. 0.5µg/mL) and 1-fold higher MIC90 (4 vs. 2µg/mL) than doxycycline. Enrofloxacin and florfenicol had both a MIC90 of 2µg/mL, whereas tiamulin had a MIC90 of 0.5µg/mL. Significant differences on the MIC values were found among the 3 countries for several antimicrobials: compared with Germany, Belgium and Italy showed significantly higher MIC for lincomycin, spiramycin, and tylosin, and lower for oxytetracycline and florfenicol. The Belgian isolates showed the lowest MIC for enrofloxacin compared with Germany and Italy. The MIC results obtained in our study suggest the presence of a high level of resistance of M. bovis isolates originating from milk to macrolides in all countries involved in this study. On the contrary, a low level of resistance was found against the antimicrobials that are not used in cattle, such as tiamulin and doxycycline, highlighting a possible link between antimicrobial treatments and development of resistance in the studied M. bovis population.

Short communication: Methicillin-resistant Staphylococcus aureus in bulk tank milk of dairy cows and effect of swine population density.

The methicillin-resistant Staphylococcus aureus (MRSA) has recently frequently been reported in dairy cattle, usually with low prevalence. The livestock-associated MRSA (LA-MRSA) ST398 is especially involved in cases of subclinical and clinical mastitis. Swine carry LA-MRSA without clinical symptoms and are considered its reservoir and shedder. People exposed to swine are particularly at risk of LA-MRSA colonization. Environments with relevant livestock density are a demonstrated risk factor for humans to be carriers of a LA-MRSA. This work investigated dairy farms located in an area with a high livestock density, mainly represented by swine. Bulk tank milk samples from 224 dairy farms were collected, and their status was defined as MRSA-positive or MRSA-negative based on culture on chromogenic medium. The number of fattening swine and of fattening swine herds was calculated in an area of 3 km around each dairy farm through georeferencing. The probability of a Staphylococcus aureus-positive dairy farm to be MRSA positive based on the extent of potential infective pressure due to swine density was calculated. Both the number of swine herds and the number of swine were associated with the MRSA status of dairy herds. The 9 MRSA isolated were typed by multi-locus sequence typing and spa-typing, and characterized for their virulence factors and antimicrobial resistance profiles. The ST and spa-types detected are consistent with those present in the Italian swine population. Virulence and resistance profiles are mostly consistent with the types detected. This work provides the first evidence of the epidemiological challenge exerted by the density of the swine population on MRSA in dairy cows.

The effect of heat waves on dairy cow mortality.

This study investigated the mortality of dairy cows during heat waves. Mortality data (46,610 cases) referred to dairy cows older than 24mo that died on a farm from all causes from May 1 to September 30 during a 6-yr period (2002-2007). Weather data were obtained from 12 weather stations located in different areas of Italy. Heat waves were defined for each weather station as a period of at least 3 consecutive days, from May 1 to September 30 (2002-2007), when the daily maximum temperature exceeded the 90th percentile of the reference distribution (1971-2000). Summer days were classified as days in heat wave (HW) or not in heat wave (nHW). Days in HW were numbered to evaluate the relationship between mortality and length of the wave. Finally, the first 3 nHW days after the end of a heat wave were also considered to account for potential prolonged effects. The mortality risk was evaluated using a case-crossover design. A conditional logistic regression model was used to calculate odds ratio and 95% confidence interval for mortality recorded in HW compared with that recorded in nHW days pooled and stratified by duration of exposure, age of cows, and month of occurrence. Dairy cows mortality was greater during HW compared with nHW days. Furthermore, compared with nHW days, the risk of mortality continued to be higher during the 3 d after the end of HW. Mortality increased with the length of the HW. Considering deaths stratified by age, cows up to 28mo were not affected by HW, whereas all the other age categories of older cows (29-60, 61-96, and >96mo) showed a greater mortality when exposed to HW. The risk of death during HW was higher in early summer months. In particular, the highest risk of mortality was observed during June HW. Present results strongly support the implementation of adaptation strategies which may limit heat stress-related impairment of animal welfare and economic losses in dairy cow farm during HW.

Short communication: comparing real-time PCR and bacteriological cultures for Streptococcus agalactiae and Staphylococcus aureus in bulk-tank milk samples.

For more than 30 yr, a control plan for Streptococcus agalactiae and Staphylococcus aureus has been carried out in more than 1,500 dairy herds of the province of Brescia (northern Italy). From 2010 to 2011, the apparent prevalence of Strep. agalactiae has been relatively stable around 10%, but the apparent prevalence of Staph. aureus has been greater than 40% with an increasing trend. The aim of this paper was to estimate and compare the diagnostic accuracy of 3 assays for the detection of Strep. agalactiae and Staph. aureus in bulk-tank milk samples (BTMS) in field conditions. The assays were a qualitative and a quantitative bacteriological culture (BC) for each pathogen and a homemade multiplex real-time PCR (rt-PCR). Because a gold standard was not available, the sensitivities (Se) and specificities (Sp) were evaluated using a Bayesian latent class approach. In 2012 we collected one BTMS from 165 dairy herds that were found positive for Strep. agalactiae in the previous 2-yr campaigns of eradication plan. In most cases, BTMS collected in these herds were positive for Staph. aureus as well, confirming the wide spread of this pathogen. At the same time we also collected composite milk samples from all the 8,624 lactating cows to evaluate the within-herd prevalence of Strep. agalactiae. Streptococcus agalactiae samples were cultured using a selective medium Tallium Kristalviolette Tossin, whereas for Staph. aureus, we used Baird Parker modified medium with added Rabbit Plasma Fibrinogen ISO-Formulation. In parallel, BTMS were tested using the rt-PCR. Regarding Strep. agalactiae, the posterior median of Se and Sp of the 2 BC was similar [qualitative BC: Se=98%, posterior credible interval (95%PCI): 94-100%, and Sp=99%, 95%PCI: 96-100%; quantitative BC: Se=99%, 95%PCI: 96-100%, and Sp=99%, 95%PCI: 95-100%] and higher than those of the rt-PCR (at 40 cycle threshold, Se=92%, 95%PCI: 85-97%; Sp=94%, 95%PCI: 88-98%). Also in case of Staph. aureus, the posterior medians of BC were generally higher than those of rt-PCR. In fact, although the Se of BC was slightly lower (rt-PCR at 40 cycle threshold, median Se=99%, 95%PCI: 97-100%, and qualitative BC, median Se=94%, 95%PCI: 87-99%), the Sp was much higher (rt-PCR at 40 cycle threshold, median Sp=67%, 95%PCI: 38-97%; qualitative BC, median Sp=95%; 95%PCI: 76-100%). Our study confirms that BC and rt-PCR are reliable diagnostic tools to detect Strep. agalactiae and Staph. aureus, and rt-PCR results should be confirmed by BC carried out on BTMS and possibly on composite milk samples.

Exploring potential risk factors of antimicrobial use in beef cattle.

Livestock species are major contributors to the increase of antimicrobial (AM) resistance which is a worldwide concern for both human and animal health. The over-use of AM is widely acknowledged, however, unlike pigs, poultry and dairy cattle, knowledge on potential risk factors affecting AM usage (AMU) in beef industry is limited. Hence, this study aimed to investigate the impact of farm, breed, sex and season of arrival of purchased beef cattle on AMU in Italian beef cattle. Data on 1063 batches were collected from January 2016 to April 2019 from specialised beef fattening farms located in the north of Italy. Information on breed, sex, date of arrival, performance traits and AM agents used on farm was collected, and the treatment incidence 100 (TI100) indexes per batch were calculated using the defined daily dose animal estimated according to Italian summaries of product characteristics. Factors affecting TI100 indexes were investigated using a cross-classified multilevel model. Farms largely differed in terms of AMU. Males had greater AMU than females (P < 0.001), likely due to their higher susceptibility to disease. Statistically significant differences were observed between seasons of arrival with summer and spring having lower TI100 indexes than winter and autumn (P < 0.001). Indeed, winter is commonly linked to an increase in respiratory diseases in beef cattle. Finally, the TI100it indexes tended to be different among breeds with Blonde d'Aquitaine and Limousine having greater AMU compared to the other breeds. Results of this study provided valuable information on potential risk factors of AMU in beef production which may be useful to address its reduction. For instance, the development of tailored management strategies for specific breeds, targeted approaches to improve the health of males as well as greater care towards batches purchased in winter are possible advice to implement on-farm for a more responsible AM stewardship.

Seasonal pattern of mortality and relationships between mortality and temperature-humidity index in dairy cows.

The 2 studies described investigated seasonal variations of mortality and temperature-humidity index (THI)-mortality relationships in dairy cows. Mortality data were extracted from the Italian Bovine Spongiform Encephalopathy databases, which contain records on cows older than 24 mo that died on a farm from all causes (98% of total records), were slaughtered in an emergency state, or were sent for normal slaughter but were sick in the preslaughter inspection (2% of total records). Both studies evaluated mortality data during a 6-yr period (2002 to 2007). The seasonal pattern study was conducted throughout Italy and was based on 320,120 deaths. An association between season and deaths was found for all 6 yr. Summer and spring were the seasons with the highest and lowest frequency of deaths (15,773.3 +/- 2,861 and 11,619.3 +/- 792.3), respectively, and within summer months, the number of deaths in July and August (5,435 +/- 284 and 5,756 +/- 676.2, respectively) was higher than in June (4,839 +/- 344.8). The THI-mortality relationships study was carried out only for deaths (51,240) reported for the Lombardia and Emilia Romagna regions. For this study, the mortality databases were integrated with THI data, which were calculated by using data from 73 weather stations. Each farm where deaths were recorded was assigned the THI values (maximum and minimum) calculated at the closest weather station for each day the events (deaths) were reported. Analysis of data indicated that approximate THI values of 80 and 70 were the maximum and minimum THI, respectively, above which the number of deaths in dairy farms starts to increase. Maximum and minimum THI values of 87 and 77 were the upper critical THI above which the risk of death for dairy cows becomes maximum. This study defined quantitative relationships between mortality risk and THI in dairy cows and may help to provide emergency interventions and mitigation measures, which may ensure survival of dairy cows and reduce replacement costs associated with heat stress-related mortality.

Short communication: isolation of Prototheca species strains from environmental sources in dairy herds.

Composite milk samples from 548 cows, and samples from feces, feed, bedding, water, liners (before and after milking), and the postdipping product were aseptically collected from 2 Italian dairy herds from February to November of 2006. Prototheca zopfii was isolated from 11.9% of milk samples, 15% of feces, and 33.3% of bedding samples. No viable cells of P. zopfii were observed in water before washing procedures, whereas 25 to 28.6% of samples from water used for washing both refrigeration tanks and milking equipment were contaminated with this yeast-like microalga. Analogously, the presence of P. zopfii was detected only on swabs collected from the liners after milking. Interestingly, in 1 of the 2 herds, water from the drinking trough was contaminated by viable cells of both P. zopfii and the related environmental species Prototheca stagnora. No viable cells were observed in cow feed. On the basis of the results presented herein, P. zopfii seemed to be widespread throughout the environments of dairy herds where outbreaks of bovine mastitis had occurred.

Disease risk assessment by clinical immunology analyses in periparturient dairy cows.

A disease prediction system was investigated in a case-control study in the dry period of high-yielding dairy cows. Blood samples of 75 cows from 26 herds were collected before calving between -23 and -33 days (T1) and also between -2 and -6 days (T2) to investigate a panel of clinical immunology and chemistry parameters. Cows with abnormal serum lysozyme and interleukin-6 concentrations showed a greater disease prevalence until the 60th day in milk compared with non-responder cows (P<0.05 and lower at T1). Differences in disease prevalence were observed on the basis of T1 data, and also by combining the results at T1 and T2. The other laboratory parameters under study were not predictive of a disease risk. Results indicate that environmental stressors in the dry period may cause a negative imprinting of the innate immune response, underlying predisposition to later disease occurrence.

Definition of a solvent system for spherical crystallization of salbutamol sulfate by quasi-emulsion solvent diffusion (QESD) method.

In this paper we describe how the spherical crystallization process by QESD method can be applied to a water-soluble drug, salbutamol sulfate. The type of solvent, antisolvent, and emulsifier and the concentration of emulsifier to be used for the production of spherical particles with a size range 80-500 microm are determined. Furthermore, the solvent/antisolvent ratio and the temperature difference between them (Delta T) are studied. It was observed that, in the case of salbutamol sulfate, the Delta T value has no influence on the formation of spherical particles. A very large metastable zone of salbutamol sulfate in water could explain this phenomenon. Finally, the influence of emulsifier concentration and of maturation time on the size of spherical particles is studied. The results show that these two parameters must be fixed to control the size of the recovered particles.

Seasonal variations in the composition of Holstein cow's milk and temperature-humidity index relationship.

A retrospective study on seasonal variations in the characteristics of cow's milk and temperature-humidity index (THI) relationship was conducted on bulk milk data collected from 2003 to 2009. The THI relationship study was carried out on 508 613 bulk milk data items recorded in 3328 dairy farms form the Lombardy region, Italy. Temperature and relative humidity data from 40 weather stations were used to calculate THI. Milk characteristics data referred to somatic cell count (SCC), total bacterial count (TBC), fat percentage (FA%) and protein percentage (PR%). Annual, seasonal and monthly variations in milk composition were evaluated on 656 064 data items recorded in 3727 dairy farms. The model highlighted a significant association between the year, season and month, and the parameters analysed (SCC, TBC, FA%, PR%). The summer season emerged as the most critical season. Of the summer months, July presented the most critical conditions for TBC, FA% and PR%, (52 054 ± 183 655, 3.73% ± 0.35% and 3.30% ± 0.15%, respectively), and August presented higher values of SCC (369 503 ± 228 377). Each milk record was linked to THI data calculated at the nearest weather station. The analysis demonstrated a positive correlation between THI and SCC and TBC, and indicated a significant change in the slope at 57.3 and 72.8 maximum THI, respectively. The model demonstrated a negative correlation between THI and FA% and PR% and provided breakpoints in the pattern at 50.2 and 65.2 maximum THI, respectively. The results of this study indicate the presence of critical climatic thresholds for bulk tank milk composition in dairy cows. Such indications could facilitate the adoption of heat management strategies, which may ensure the health and production of dairy cows and limit related economic losses.

Investigation of the origin of prednisolone in cow urine.

After a two-year period of the frequent detection of prednisolone-positive bovine urine samples in the Italian region of Lombardy, studies were initiated to investigate the source. Because the majority of positive samples were detected at the slaughterhouse, researchers hypothesised that, together with increased cortisol and cortisone, a small quantity of prednisolone could be produced by the cows in stressful situations. In the present study, three dairy cows underwent intramuscular treatments with tetracosactide hexaacetate, a synthetic analogue of adrenocorticotropic hormone, to simulate stress. The animals were slaughtered at the end of the study. The results indicated that prednisolone could be detected occasionally in the non-stressful state, but was consistently found in the urine of stressed cows (concentrations ranged from 1.01 to 4.08 ng/mL). To confirm the stress condition, urinary cortisol and cortisone were also detected at high concentrations in the urine, typically at concentrations of hundreds of nanograms per millilitre. The results of this preliminary study did not reveal the metabolic pathway responsible for prednisolone but suggested that this corticosteroid could be produced endogenously.