The aim of this cross-sectional study was to evaluate the prevalence of subclinical mastitis (SCM) and associated risk factors in dairy cows in peri-urban areas of Kigali, Rwanda, and identify causative udder pathogens. A sample of 256 cows from 25 herds was screened with the California Mastitis Test (CMT), and udder quarters with CMT score ≥ 3 (scale 1–5) were milk sampled for culture and final bacteriological identification with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). All resultant staphylococci species were tested for beta-lactamase production with the clover leaf method. In parallel, herd bulk milk somatic cell count (SCC) of each herd was analysed using a portable device, the DeLaval cell counter. The prevalence of SCM was 43.1% at quarter level and 76.2% at cow level based on CMT test. Multiparous, Holstein cows were 2.50 (C.I = 1.32–4.71) and 10.08 (C.I = 1.54–66.13) times more likely to contract SCM infection than primiparous animals or cows of other breeds, respectively. The median and mean SCC of all herds were 1108 × 10 3 cells/mL and 1179 × 10 3 cells/mL, respectively. The most prevalent pathogens were non-aureus staphylococci (NAS; 40.2%) followed by Staphylococcus aureus (22%) and less prevalent pathogens (6%). Samples with no growth or contamination constituted 30.4% and 1.4% of the diagnoses, respectively. The most prevalent species within NAS were S. epidermidis (38.2%) followed by S. sciuri (19.5%), S. chromogenes (9.8%), and nine less prevalent NAS species (32.5%). Out of 209 staphylococci isolates, 77% exhibited beta-lactamase production. The study shows that there is high prevalence of SCM and high herd bulk milk SCC in herds in Kigali, indicating udder health problems in dairy cows. Additionally, beta-lactamase production among staphylococci species was common. Improved milking hygiene and application of biosecurity measures, or a complete mastitis control plan, is required to lower the prevalence of SCM and minimize the spread of pathogens among dairy cows.

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The study was conducted to evaluate the attachment of three lactic acid bacteria (LAB) strains and their combination in a cocktail, to stainless steel coupons from a deli slicer, and their ability to inhibit the attachment of Listeria monocytogenes. In a previous study, three LAB strains, Pediococcus acidilactici, Lactobacillus amylovorus, and Lactobacillus animalis, were isolated from ready-to-eat meat and exhibited antilisterial effect. In the study reported here, hydrophobicity tests were determined according to the method of microbial adhesion to solvent. The attachment of the cells was evaluated on stainless steel coupons from deli slicers. Extracellular carbohydrates were determined with a colorimetric method. Based on these tests, L. animalis exhibited the greatest hydrophobicity (26.3%), and its adherence increased sharply from 24 to 72 h, whereas L. amylovorus yielded the lowest hydrophobicity (3.86%) and was weakly adherent. Although P. acidilactici had moderate hydrophobicity (10.1%), it adhered strongly. The attached LAB strains produced significantly (P < 0.05) higher total carbohydrates than their planktonic counterparts did, which is an important characteristic for attachment. Three conditions were simulated to evaluate the ability of the LAB cocktail (10(8) CFU/ml) to competitively exclude L. monocytogenes (10(3) CFU/ml) on the surface of the coupons. The coupons were pretreated with the LAB cocktail for 24 h prior to the addition of L. monocytogenes, simultaneously treated with the LAB cocktail and L. monocytogenes, or pretreated with L. monocytogenes 24 h prior to the addition of the LAB cocktail. The LAB cocktail was able to reduce the attachment L. monocytogenes significantly (P < 0.05). The LAB cocktail indicated potential attachment on stainless steel and bacteriostatic activity toward L. monocytogenes attached on stainless steel, which indicates a possible role for LAB as a biosanitizer in the food industry.

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Whole-genome sequencing was carried out on 30 Staphylococcus (S.) aureus isolates from dairy cows with subclinical mastitis from all five provinces of Rwanda. Twenty-five of the isolates produced enough sequence to be analyzed using core genome multilocus sequence typing (cg-MLST). The isolates group into three main clusters. The largest cluster contain isolates of sequence type (ST) 152 (n = 6) and the closely related ST1633 (n = 2). These sequence types have previously mainly been encountered in humans. The isolates of the second-largest cluster belong to ST5477 (n = 5),so far exclusively isolated from cows in Rwanda. The third cluster consists of isolates of ST97 (n = 4), which is a well-known bovine-adapted sequence type. These three clusters were all widespread over the country. Isolates of the usually human-adapted sequence types 1 (n = 2) and 5 (n= 1) were found and a single isolate of ST2430, previously found among humans in Africa. Finally, four isolates of novel sequence types were found: ST7108 (n = 2), ST7109 (n = 1), and ST7110 (n = 1). The blaZ penicillin resistance gene was found in 84% of the isolates and was in all cases corroborated by phenotypic resistance determination. Five (20%) of the isolates carried a tetracycline resistance gene, tet(K) or tetM, and three of these five also displayed phenotypic resistance while two isolates carried a tetM-gene but were yet tetracycline susceptible. Seven (28%) isolates carried the dfrG gene conferring resistance to trimethoprim. Four of these isolates indeed were resistant to trimethoprim while three isolates were sensitive. The str gene conferring resistance to aminoglycosides was found in three isolates; however, none of these displayed resistance to gentamycin. Our data revealed a high diversity of the sequence types of S. aureus isolates from cows with subclinical mastitis in Rwanda. Two major clusters of ST97 and ST5477 are likely to be bovine adapted and cause mastitis while the third cluster of ST152 usually have been found in humans and may signify a recent transmission of these types from human to cows, for example from hand milking. The high prevalence of this sequence type among dairy cows may pose zoonotic threat. The sequence types were widely distributed without any geographic correlation. Penicillin resistance, the most common type of resistance with a prevalence over 80%, but also tetracycline and trimethoprim resistance were displayed by several isolates.