MILK Symposium review: Microbiological quality and safety of milk from farm to milk collection centers in Rwanda.

The aim of this study was to generate knowledge on the most important milk quality and safety attributes, including somatic cell count (SCC), total bacterial count (TBC), Escherichia coli, Salmonella, and Brucella spp. antibodies and antibiotic residues in milk in the chain from farm to milk collection center (MCC) in Rwanda. In addition, we investigated farm and management factors associated with high TBC, SCC, and Salmonella counts. Raw milk was sampled at the farm and MCC levels. Milk samples were taken from dairy farms linked to 2 selected MCC in each of the 4 provinces in Rwanda. In total, 406 bulk milk samples from 406 farms and 32 bulk milk samples from 8 MCC were collected and analyzed. Farm milk average SCC varied between 180 × 103 and 920 × 103 cells/mL, whereas average SCC in milk samples at MCC varied between 170 × 103 and 1,700 × 103 cells/mL. The mean milk TBC of different farms per MCC varied between 1.1 × 106 and 1.6 × 107 cfu/mL, whereas in milk samples from different MCC, the mean TBC ranged between 5.3 × 105 and 2.4 × 108 cfu/mL. The high TBC in milk from MCC suggests proliferation or recontamination of milk by bacteria during transportation. Escherichia coli was detected in 35 of 385 farm milk samples and ranged between 5 cfu/mL and 1.1 × 104 cfu/mL, whereas in milk samples from the MCC, it was detected in 20 out 32 samples varying between 5 cfu/mL and 2.9 × 103 cfu/mL. Overall farm prevalence of Salmonella in milk samples was 14%, but no milk samples from MCC were positive for Salmonella. Five out of 22 bulk milk samples from different MCC were positive for Brucella spp. antibodies, but no Brucella antibodies were detected in milk samples from farms. The prevalence of antibiotic residues as detected by the Delvotest SP NT (DSM, Delft, the Netherlands) was low: 1.3% in farm milk samples and undetected in MCC milk samples. Lack of a separate milking area was associated with high TBC, whereas offering of supplemental feeds, keeping data of past diseases, and an unhygienic milking area were associated with high SCC. Lack of teat washing before milking was the only factor associated with Salmonella contamination of milk at the farm level. This study indicated high TBC and SCC of milk samples at the farm and MCC levels, which indicates both microbial contamination of milk and poor udder health in dairy cows. Presence of E. coli, Salmonella, and Brucella antibodies in milk was common, but finding antibiotic residues in milk was uncommon.

A cross sectional study of prevalence and risk factors associated with subclinical mastitis and intramammary infections, in dairy herds linked to milk collection centers in Rwanda.

The objective of this cross-sectional study was to investigate prevalence, causative udder pathogens and their antimicrobial resistance (AMR), as well as cow and herd risk factors associated with subclinical mastitis (SCM = cows with at least one udder quarter with california mastitis test (CMT) score > 2) and intramammary infections (IMI) caused by Staphylococcus(S.) aureus or Non aureus staphylococci (NAS) in dairy cows linked to Milk Collection Centers (MCCs) in Rwanda. Screening for SCM with the CMT was done on 572 cows from 404 herds linked to two MCCs in each of four provinces. Milk from udder quarters with a CMT score ≥3 (scale 1-5) was sampled for bacteriological analysis. Antimicrobial resistance was evaluated in 60 selected S. aureus isolates. Multivariable mixed effect and ordinary logistic regression analyses were performed to identify cow and herd level risk factors associated with SCM, NAS or S. aureus IMI in cows. The prevalence of SCM was 37.3 % at quarter level and 62.0 % at cow level. Bacteria were isolated 73.7 % of the cultured milk samples, whereas 23.3 % were culture-negative and 3.0 % were contaminated. Staphylococcus aureus and NAS were the most prevalent pathogens, representing more than half of all bacteriological findings. Staphylococcus chromogenes and S. epidermidis were the most prevalent NAS species identified. Of the S. aureus strains 83.3 % were resistant for penicillin, 100 % for clindamycin and 20 % tetracycline. The risk factor analysis showed that an increased stage of lactation, dirty udder and legs in single cow herds and lack of calf suckling the dam, dirty udder and legs and lack of feeding cow after milking in multiple cow herds were significantly (P < 0.05) associated with an increased odds of SCM. Similarly, increasing stage of lactation in single cow herds, and housing cows in individual cattle kraal or on earthen floor and hand washing between cows during milking in multiple cow herds were associated with increased odds for NAS IMI. Poor hygiene of milking area in single cow herds and absence of foremilk stripping in multiple cow herds were significantly (P < 0.05) associated with increased odds for S. aureus IMI in cows. In conclusion, SCM prevalence was high across MCCs. The majority of identified pathogens were contagious in nature and they exhibited resistance to penicillin. Control of the identified risks factors and improved biosecurity through adoption of best practices, and farmer training could contribute to lowering SCM prevalence in Rwanda.

Aetiology and prevalence of subclinical mastitis in dairy herds in peri-urban areas of Kigali in Rwanda.

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 × 103 cells/mL and 1179 × 103 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.

Meat retail conditions within the establishments of Kigali city (Rwanda): bacteriological quality and risk factors for Salmonella occurrence.

Meat constitutes one of the major vehicles for human foodborne infections. This study aimed to assess the retail conditions and to determine the microbiological quality and safety of meat retailed within the establishments of Kigali (Rwanda). A questionnaire survey was carried out in 150 retail outlets to characterise meat retail conditions. Additionally, 270 retail meat samples were analysed for the enumeration of hygiene indicator bacteria (total mesophilic bacteria and Escherichia coli) and for the qualitative detection of Salmonella, using conventional culture methods. The results revealed that beef was the predominant meat sold within the retail premises of Kigali city, while meat from non-bovine animal species was mainly sold in large establishments. Salmonella was detected in 19.6% of all the retailed meat samples evaluated, whereas the mean loads for total mesophilic bacteria and E. coli were 7.3 and 3.5 log cfu/g, respectively. Three factors, namely the temperature conditions of the meat under retail, the cleanability of the used meat cutting boards, and the training of personnel in hygienic meat handling practices, were found to be significantly (p ≤ 0.05) associated with the risk of Salmonella occurrence in the retailed meat. The findings from this study highlight the need for improvements in hygienic meat handling practices, particularly, in small and medium meat retail establishments in Kigali.

Main microorganisms involved in the fermentation of Ugandan ghee.

Ghee is widely produced from a traditional fermented butter-like product named mashita in western Uganda. However, no detailed studies have been done to identify the microorganisms involved in mashita fermentation. The aim of this study was to identify the microorganisms present at the end of mashita ripening using culture-dependent and culture-independent techniques. The most commonly identified species of lactic acid bacteria (LAB) in mashita using culture-dependent techniques were Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus plantarum and Lactobacillus perolens constituting 37.3%, 10.1%, 8.1% and 7.7% of total bacterial colonies isolated respectively. L. paracasei was the only bacterial species identified in all mashita samples by culture-dependent technique. Two of the four most commonly isolated LAB species (L. helveticus and L. plantarum) were also identified in mashita by a culture-independent method (PCR-DGGE). Other main LAB species identified in mashita by PCR-DGGE were Bifidobacterium sp., Enterococcus faecium, Lactobacillus brevis, Lactobacillus acetotolerans, Lactobacillus sp., Lactococcus raffinolactis, Lactococcus lactis subsp. lactis and Streptococcus salivarius. The main species of acetic acid bacteria (AAB) identified in the mashita using PCR-DGGE method were Acetobacter aceti, Acetobacter lovaniensis, Acetobacter orientalis and Acetobacter pasteurianus. PCR-DGGE identification of yeasts showed that Brettanomyces custersianus, Candida silvae, Geotrichum sp., Issatchenkia occidentalis, Issatchenkia orientalis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Trichosporon asahii were main yeast species in mashita. A. lovaniensis, A. aceti, and I. orientalis were identified in all the six mashita samples analyzed using PCR-DGGE method. Other microbial species were only found in some samples. These results indicate that LAB and yeasts, as in similar fermentation products, but also AAB are main microbial groups involved in mashita fermentation.

Potential of selected strains of lactic acid bacteria to induce a Th1 immune profile.

Lactic acid bacteria (LAB) might switch the Th2 biased immune response in allergic patients towards a balanced Th1/Th2 immune profile, leading to amelioration of allergy. To select strains of LAB that could be of potential application for foods in controlling allergy, 35 bacterial strains were screened in vitro using murine splenocytes and peritoneal exudate cells (PECs). Streptococcus thermophilus AHU1838 (FERM AP-21009), and Lactobacillus paracasei subsp. casei AHU1839 (FERM AP-21010) enhanced the secretion of Th1 cytokines such as interferon-gamma (IFN-gamma) and interleukin-12 (IL-12). The two strains of LAB also up-regulated the expression of CD40, and CD86 in dendritic cells (DCs), and activated cytotoxic T lymphocytes (CTL). These two strains could therefore be used in producing fermented food products that can enhance the Th1 immune profile which is important in ameliorating allergy.

Yoghurt fermented by Lactobacillus delbrueckii subsp. bulgaricus H+ -ATPase-defective mutants exhibits enhanced viability of Bifidobacterium breve during storage.

Persistent acid production by Lactobacillus delbrueckii subsp. bulgaricus during refrigerated storage is a major cause of reduced viability of probiotic strains such as Bifidobacterium breve in yoghurt. It was established that H+ -ATPase-defective mutants of lactic acid bacteria have reduced growth and metabolism in low pH environments. Therefore, the aim of this study was to evaluate inhibition of post-acidification and maintenance of B. breve viability in yoghurt fermented by L. delbrueckii subsp. bulgaricus mutants with reduced membrane-bound H+ -ATPase activity during refrigerated storage. Spontaneous neomycin mutants of L. delbrueckii subsp. bulgaricus that had a significantly (P < or = 0.05) reduced H+ -ATPase activity were successfully isolated. Yoghurt fermented using L. delbrueckii subsp. bulgaricus SBT0164 No. 55-1 (mutant) starter culture had markedly reduced post-acidification and maintained viability (> or = 10(8) CFU/ml) of both Bifidobacteruim breve JCM 1192(T) and Bifidobacteruim breve JCM 7017 during storage at 10 degrees C for 21 days. These results clearly showed that yoghurt fermented by mutants of L. delbrueckii subsp. bulgaricus with reduced membrane-bound H+ -ATPase activity has reduced post-acidification that prolongs viability of B. breve in yoghurt during refrigerated storage.