Ability of unsterilized recycled manure solids bedding to support growth of Klebsiella pneumoniae and Escherichia coli.

Although recycled manure solids (RMS) bedding is used on dairy farms, it could allow bacterial growth when contaminated by feces and thus increase the incidence of clinical mastitis in cows. The objective of this study was to describe bacterial growth in three different types of RMS bedding, as well as in sand, when samples were experimentally inoculated with Escherichia coli and Klebsiella pneumoniae. Two 3-day trials were conducted, during which treatments included inoculating bedding samples with E. coli and K. pneumoniae, as well as no inoculation. The trial was repeated 3 times for each bedding sample on each day. Samples were incubated at 15°C for 3 d and bacterial counts were measured every day. After inoculation, there was no significant K. pneumoniae or E. coli growth phase during the trial in those RMS samples that were prepared either in a container or in a heap. Recycled manure solids and sand samples prepared in a rotary drum, however, showed a similar active growth phase of K. pneumoniae during the first 24 h of the trial. Moreover, a significant E. coli growth phase was observed in the samples of sand bedding in the first 24 h. The 3 different types of RMS bedding samples did not react in a similar manner to coliform inoculation. No active growth phase was observed in bedding samples already containing a high bacterial concentration following inoculation with coliforms.

Bien que la litière de fumier recyclé (LFR) soit utilisée dans les fermes laitières, elle pourrait permettre la croissance bactérienne lorsqu'elle est contaminée par des matières fécales et augmenter ainsi l'incidence de mammite clinique chez les vaches. L'objectif de cette étude était de décrire la croissance bactérienne dans trois types de LFR, ainsi que dans du sable, lorsque des échantillons étaient inoculés expérimentalement avec Escherichia coli et Klebsiella pneumoniae. Deux essais de trois jours ont été réalisés, au cours desquels les échantillons de litière ont été inoculés ou non avec E. coli et K. pneumoniae. L'essai a été répété trois fois pour chaque échantillon de litière, chaque jour. Les échantillons ont été incubés à 15 °C pendant 3 jours et la numération bactérienne a été mesurée chaque jour. Après inoculation, il n'y a pas eu de phase de croissance significative de K. pneumoniae ou d'E. coli au cours de l'essai dans les échantillons de LFR préparés dans un conteneur ou en tas. Les échantillons de sable et de LFR préparés dans un tambour rotatif ont cependant montré une phase de croissance active similaire de K. pneumoniae pendant les premières 24 heures de l'essai. En outre, une phase de croissance significative d'E. coli a été observée dans les échantillons de litière de sable au cours des premières 24 h. Les trois différents types d'échantillons de LFR n'ont pas réagi de la même manière à l'inoculation de coliformes. Aucune phase de croissance active n'a été observée dans les échantillons de litière contenant déjà une concentration bactérienne élevée après l'inoculation de coliformes.(Traduit par Docteur Simon Dufour).

Diagnosing Intramammary Infection: Meta-Analysis and Mapping Review on Frequency and Udder Health Relevance of Microorganism Species Isolated from Bovine Milk Samples.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provides accurate species-level identification of many, microorganisms retrieved from bovine milk samples. However, not all those microorganisms are pathogenic. Our study aimed to: (1) determine the species-specific prevalence of microorganisms identified in bovine milk of apparently healthy lactating quarters vs. quarters with clinical mastitis (CM); and (2) map current information and knowledge gaps on udder health relevance of microorganisms retrieved from bovine milk samples. A mixed study design (meta-analysis and mapping review) was chosen. We gathered several large Canadian, US and Brazilian data sets of MALDI-TOF results for organisms cultured from quarter milk samples. For meta-analysis, two datasets (apparently healthy quarters vs. CM samples) were organized. A series of meta-analyses was conducted to determine microorganisms' prevalence. Then, each species reported was searched through PubMed to investigate whether inflammation (increased somatic cell count (SCC) or signs of CM) was associated with microorganism's recovery from milk. A total of 294 different species of microorganisms recovered from milk samples were identified. Among 50,429 quarter-milk samples from apparently healthy quarters, the 5 most frequent species were Staphylococcus chromogenes (6.7%, 95% CI 4.5-9.2%), Aerococcus viridans (1.6%, 95% CI 0.4-3.5%), Staphylococcus aureus (1.5%, 95% CI 0.5-2.8%), Staphylococcus haemolyticus (0.9%, 95% CI 0.4-1.5%), and Staphylococcus epidermidis (0.7%, 95% CI 0.2-1.6%). Among the 43,924 quarter-milk CM samples, the 5 most frequent species were Escherichia coli (11%, 95% CI 8.1-14.3%), Streptococcus uberis (8.5%, 95% CI 5.3-12.2%), Streptococcus dysgalactiae (7.8%, 95% CI 4.9-11.5%), Staphylococcus aureus (7.8%, 95% CI 4.4-11.9%), and Klebsiella pneumoniae (5.6%, 95% CI 3.4-8.2%). When conducting the PubMed literature search, there were 206 species identified by MALDI-TOF for which we were not able to find any information regarding their association with CM or SCC. Some of them, however, were frequently isolated in our multi-country dataset from the milk of quarters with CM (e.g., Citrobacter koseri, Enterococcus saccharolyticus, Streptococcus gallolyticus). Our study provides guidance to veterinarians for interpretation of milk bacteriology results obtained using MALDI-TOF and identifies knowledge gaps for future research.

Corrigendum: Association Between Recycled Manure Solids Bedding and Subclinical Mastitis Incidence: A Canadian Cohort Study.

[This corrects the article DOI: 10.3389/fvets.2022.859858.].

Association Between Recycled Manure Solids Bedding and Subclinical Mastitis Incidence: A Canadian Cohort Study.

Recycled manure solids (RMS) are increasingly used as bedding for dairy cows. However, potential impact of RMS bedding on animal health is not well described. The objective of this study was to evaluate subclinical mastitis incidence in cows housed on RMS bedding. Twenty RMS farms and a comparative group of 60 straw-bedded farms were enrolled in a 1-year longitudinal study (2018-2019). Data from 11,031 dairy cows were collected. Variations of individual somatic cell count were evaluated using three different methods. First, we compared the cow's mean lactation linear score between cows housed on the two bedding types. Then, we compared across bedding types the risk for a given cow of having a milk test with a linear score ≥ 4. Finally, we evaluated the dynamics of somatic cell count using pairs of tests within a cow. More specifically, we considered that only pairs of DHI tests where the first test yielded a linear score < 4 were at risk of an incident subclinical mastitis event. Then, we defined a newly acquired subclinical mastitis when the second test was ≥ 4. All models were adjusted for putative confounders. We could not highlight a significant association between bedding type and cow's mean lactational linear score (least square mean of 2.47 in cows from RMS farms vs. 2.37 in straw farms; 95%CI for linear score's difference: -0.20, 0.40). Furthermore, we could not find an association between bedding type and the risk of a high linear score (≥ 4). For the latter, cows housed on RMS had 0.93 times the risk of having a high linear score than straw-bedded cows (%95 CI: 0.68, 1.28). Moreover, cows on recycled manure solids farms had 0.73 time the risk of acquiring subclinical mastitis when compared to straw-bedded farms. Again, this risk was not statistically significant (%95 CI: 0.54, 1.00). In our study, RMS bedding was not associated with subclinical mastitis, as measured by somatic cell count, when compared to cows housed on a more conventional bedding, straw bedding.

Impact of recycled manure solids bedding on hygiene and odds of hock lesions in dairy cows.

The use of recycled manure solids (RMS) as bedding for dairy cows has become more popular over the last decade. Once producers own the RMS producing equipment, they are not restricted in the amount of bedding they can use in stalls, due to its large availability and low production costs. Thus, they are usually able to provide a soft lying area for their cows. Nevertheless, the potential positive impact of RMS on cows' hygiene and odds of hock lesions is not clearly demonstrated. Objectives of this research were, therefore, to compare RMS-bedded cows' hygiene level and odds of hock lesions to straw-bedded cows (comparative group). To achieve this, an observational cross-sectional study was conducted in 27 RMS- and 61 straw-bedded herds. During the farm visit, 30 cows per herd were scored for hygiene of three body regions (udder, lower legs and flank/upper legs) using a score ranging from 1 to 4 (1 being the cleanest and 4 the dirtiest). In addition, both hocks were scored (0 to 3) to evaluate the presence of lesions (0 when there was no lesion and 3 when a swelling protrusion > 2.5 cm was present). Continuation-ratio models were used to compute the odds of having a given score to the odds of having a lower score. Recycled manure solids usage was associated with lower odds of having an udder hygiene score ≥3 [odds ratio (OR): 0.43; 95% CI: 0.20, 0.95] and of having a score of 4 (OR: 0.29; 95% CI: 0.09, 0.93). It was also associated, compared to straw, with cleaner lower legs. We observed lower odds of having a score ≥2 (OR: 0.45; 95% CI: 0.21, 0.98), ≥3 (OR: 0.16; 95% CI: 0.04, 0.66), or a score of 4 (OR: 0.07; 95% CI: 0.01, 0.40) in RMS-bedded herds. No statistically significant association could be highlighted between RMS usage and flank/upper legs hygiene. Furthermore, we did not observe any statistically significant associations between bedding type (RMS vs. straw) and odds of hock lesions. In conclusion, cows bedded with RMS had generally cleaner udders and cleaner lower legs than straw-bedded ones. No association was found between bedding type and odds of hock lesions.

Comparison of microbiota of recycled manure solids and straw bedding used in dairy farms in eastern Canada.

Recycled manure solids (RMS) bedding is an alternative bedding option that is growing in popularity on Canadian dairy farms. However, the microbiological characteristics and production of RMS bedding are poorly documented under on-farm conditions in eastern Canada. This bedding could support the presence of pathogens and could have an effect on cow and human health. The aim of this study was to describe the RMS microbiota when used under dairy cows and compare it with straw bedding. Unused and used bedding from 27 RMS and 61 straw-bedded dairy farms were collected and compared using 16S amplicon sequencing, bacterial counts, and Salmonella spp. and Listeria monocytogenes detection. Microbiota composition of unused RMS and unused straw were different. After use, both bedding microbiota were similar in their bacterial composition, structure, and diversity. Unused RMS generally contained higher bacterial counts than did unused straw, except for Klebsiella spp. counts. Salmonella spp. and L. monocytogenes were more frequently detected in unused RMS (Salmonella spp.: 11%; L. monocytogenes: 30%), than in unused straw (Salmonella spp.: 0%; L. monocytogenes: 11%). Finally, 2 RMS production systems (extraction of the liquid fraction followed by maturation in an opened or enclosed container vs. in a heap) did not influence the microbiota richness and bacteria distribution (α-diversity), but did influence the microbiota structure (ß-diversity). In conclusion, animal and human pathogens were found in greater numbers and more frequently in unused RMS than unused straw, and this could eventually affect dairy cow or human health.

Clinical Mastitis Incidence in Dairy Cows Housed on Recycled Manure Solids Bedding: A Canadian Cohort Study.

Bedding can affect mammary health of dairy cows. The objectives of this study were to evaluate clinical mastitis incidence in cows housed on recycled manure solids bedding and, more specifically, to determine which pathogens were involved. We followed 26 recycled manure solids farms and 60 straw-bedded farms as a comparative group during 1 year (2018-2019). For each episode of clinical mastitis, defined as a visual alteration of the milk, with or without local or systemic signs of infection, producers sampled aseptically the affected quarter, provided some details about the animal, and sent the sample to the research team at the Université de Montréal. We received and analyzed 1,144 milk samples. The samples were cultured according to the National Mastitis Council guidelines and the different colony phenotypes were subsequently identified with mass spectrometry. In 54.6% of CM cases, a single phenotype of bacteria was cultured (pure culture), while two different phenotypes were found in 16.8% of the samples (mixed culture), and no growth was observed in 14.4% of the samples. Samples with three or more phenotypes were considered contaminated and were not included in the pathogen-specific analyses (14.3% of the submitted samples). The most frequently identified bacterial species in pure and mixed culture in farms using recycled manure solids were Streptococcus uberis (16.0%), Escherichia coli (13.8%), Klebsiella pneumoniae (13.2%), Streptococcus dysgalactiae (6.2%), and Staphylococcus aureus (3.4%). In straw farms, the most frequent species were S. aureus (16.6%), S. uberis (11.0%), E. coli (9.1%), S. dysgalactiae (8.0%), and K. pneumoniae (1.1%). The incidence of clinical mastitis (all cases together) was not higher in recycled manure solids farms (14.0 cases/100 cow-year; 95% CI: 8.3-23.7) compared with straw-bedded farms (16.3 cases/100 cow-year; 95% CI: 9.0-29.6). However, K. pneumoniae clinical mastitis episodes were 7.0 (95% CI: 2.0-24.6) times more frequent in recycled manure solids farms than in straw farms. Adjusted least square means estimates were 1.6 K. pneumoniae clinical mastitis cases/100 cow-year (95% CI: 0.8-3.4) in recycled manure solids farms vs. 0.2 cases/100 cow-year (95% CI: 0.1-0.6) in straw-bedded farms. Klebsiella pneumoniae clinical mastitis is in general severe. Producers interested in this bedding alternative need to be aware of this risk.

Refining Knowledge of Factors Affecting Vitamin B12 Concentration in Bovine Milk.

Milk is an excellent source of vitamin B12 (B12) for humans. Therefore, being able to guarantee a high and consistent concentration of this vitamin would enhance consumer perception of milk as a health food. The aim of the paper was to gather additional knowledge on factors that could explain B12 variation in cow milk through two observational studies: (1) to explore the relationship between milk B12 and ruminal conditions, such as pH and volatile fatty acid concentrations; and (2) to examine the impact of bedding on B12 concentrations in bulk tank milk. For study 1, a total of 72 milk and ruminal liquid samples were obtained from 45 Holstein cows fitted with ruminal cannula between 10 and 392 days of lactation. For study 2, bulk tank milk samples were obtained from 83 commercial herds; 26 herds used recycled manure solid bedding and 57 used straw bedding. Milk samples were analyzed for B12 using radioassay. Using principal component regression analysis, we observed that ruminal pH and the acetate:propionate ratio for cows receiving the early lactation ration were positively correlated with milk B12. Bedding did not influence milk B12 in bulk tanks, which averaged 4276 pg/mL. In conclusion, as B12 is synthesized by ruminal bacteria, optimizing ruminal conditions had a positive effect on milk B12, while bedding management had no influence.

Effect of recycled manure solids as bedding on bulk tank milk and implications for cheese microbiological quality.

The dairy farm environment influences the raw milk microbiota and consequently affects milk processing. Therefore, it is crucial to investigate farm management practices such as the bedding materials. The aim of this study was to evaluate the effect of recycled manure solids (RMS) as bedding material on bulk tank milk and microbiological implications for cheese quality. Bulk tank samples were collected from 84 dairy farms using RMS or straw bedding. The use of RMS did not influence thermophilic and mesophilic aerobic viable counts from spores. However, straw-milk samples gave higher values for mesophilic anaerobic spore-forming bacteria (0.44 log cfu/mL) than RMS-milk samples (0.17 log cfu/mL). The presence of thermoresistant lactic acid bacteria was not increased in milk from farms using RMS. Nevertheless, taxonomic profiles of thermoresistant bacteria isolated were different between the 2 types of milk. More Enterococcus faecalis and Streptococcus spp. were identified in RMS-milk samples. Thermoresistant enterococci and streptococci could easily end up in cheese. Therefore, milk proteolytic activities of these isolates were tested. Neither Streptococcus spp. nor Enterococcus faecium isolates exhibited proteolytic activities, whereas 53% of E. faecalis showed some. Also, only 1 vancomycin-resistant enterococcus was detected. Survival of selected RMS-milk samples isolates (3 E. faecalis and 1 Streptococcus thermophilus) was evaluated during a model Cheddar cheese manufacture. Although those strains survived well, they did not modify the acidification curve of milk. However, they might cause organoleptic defects during cheese maturing.

Effects of recycled manure solids bedding on the spread of gastrointestinal parasites in the environment of dairies and milk.

The primary aim of this work was to isolate common bovine digestive tract parasites in recycled manure bedding (RMS), as well as to determine the ability of current RMS preparation procedures to eliminate these pathogens. Other objectives were to assess whether any of the aforementioned parasites could be retrieved in bulk milk from dairies using RMS and to study whether the prevalence of these parasites differed among manure of cows housed on RMS versus on straw bedding. For the study, 27 RMS farms and 61 control farms were recruited. Samples of manure from the pre-pit and milk from the bulk tank were recovered from straw-bedding farms and RMS-based farms. In addition, samples from the manure solid fraction after liquid extraction, RMS before use, and RMS currently in use were recovered from RMS herds. Parasites were first detected by double centrifugation zinc sulfate flotation to enhance isolation of gastrointestinal protozoa, and by modified Wisconsin sugar flotation for the appraisal of gastrointestinal nematodes. Cryptosporidium parasites were confirmed by nested PCR amplification and sequencing of a portion of the gene encoding the small subunit rRNA. Results revealed a high prevalence of Cryptosporidium spp. (C. parvum, C. andersoni, and C. meleagridis, identified by PCR) and Eimeria spp. (mainly E. bovis and E. zuernii) parasites in both types of farms, with a larger proportion of manure samples from RMS-bedded farms testing positive for Cryptosporidium parasites compared with manure from straw-bedded farms. Both Cryptosporidium spp. and Eimeria spp. oocysts were found at every step of RMS preparation and transformation, showing that current RMS preparation strategies do not guarantee the destruction of protozoan parasites. Cryptosporidium parvum, a potential zoonotic risk for professionals in close contact with livestock, was found to be present in 32 out of 61 straw-bedded and 24 of 27 RMS farms. No protozoan parasites were found in any sample derived from bulk milk, neither by microscopy analysis nor by molecular methods.