Prevalence, hygienic status and exposure assessment of Salmonella spp. in milk and dairy chain in Rwanda

Milk and dairy products play a vital role in human diet. In developing countries, milk is among the major sources of income for many (rural) farmers and is an affordable source of animal proteins that can fulfill the rising demand in those countries, as a response to the rapid population growth. For decades, governments have been encouraging milk production increase by setting up a number of development projects and significant achievements were accomplished. In Rwanda, several interventions contributed to increase milk yield and this is expected to continue in the framework of the 2020 Vision, a national development plan aiming to transform the socio-economic conditions of Rwandans by the year 2020, and make Rwanda a middle-income country. Besides the significant increase of milk production, the aspects related to its safety and quality must be considered in parallel, since milk is a highly perishable foodstuff, requiring appropriate handling to prevent food-borne illnesses for consumers. In the African countries including Rwanda, milk increase is not followed by appropriate quality and safety management measures, and there is a situation in which the formal dairy industry coexists with the informal market channeling the major part of milk commercialized raw, boiled or processed towards traditional products mostly fermented. The present PhD study was undertaken in order to contribute to this situation, and its overall objective was the assessment of microbiological quality of milk and dairy products and hygienic status using a food chain approach from farm to fork, leading to an exposure assessment of Salmonella spp. The informal market is predominant in Rwanda, and is estimated to concern more than 90% of the whole milk from farmers grouped in 3 categories (open-grazing, semi-grazing and zero-grazing). The modernization of the chain is ongoing, with around 120 collection centers installed all over the country, and which are the interconnection between the two routes namely the formal and the informal. Processing companies are still few (5 in the time frame of this study) and process milk towards various products such as pasteurized milk, fermented pasteurized milk, cream, butter, etc. Gouda cheese is also processed mostly in the North-West of the country (Gishwati milk basin) in traditional conditions but benefitted from development program supports and have been modernized by receiving trainings on food hygiene and modern and adapted equipment. Fermented milk, boiled milk and cheese (till 2012) from the informal market are commercialized in milk shops, localized in towns and cities and serve as source of milk for a high number of consumers, whereas processed dairy products are sold in supermarkets via wholesalers directly connected to processing plants (formal market). According to most international and local standards, the raw milk quality in farms and milk collection centers in Rwanda was almost acceptable. After pasteurization, the total mesophilic count (TMC) and coliforms in milk were only slightly decreased, which indicates ineffective pasteurization and/or post-contamination, resulting in unacceptable microbiological quality of pasteurized milk and derived dairy products according to the international and local standards. Increasing bacterial counts were observed along the retail chain and could be attributed to insufficient temperature control during storage. Milk shops sold boiled and fermented milk of poor and variable microbiological quality in comparison with the pasteurized milk sold in supermarkets. The microbiological load of cheese was the highest of all dairy products, suggesting post-processing contaminations due to unhygienic handling practices. Listeria monocytogenes was absent in all samples, possibly due to competition and growth suppression exerted by the indigenous milk and/or fermenting microbiota. Staphylococcus aureus was omnipresent, with the highest concentrations found in cheese and fermented milk sold in milk shops, but there was no statistical difference between the different steps of the milk chain. The presence of S. aureus is linked with lack of animal health monitoring and/or personnel hygiene. Salmonella spp. was isolated from 7.6 % of all samples (n = 330) spread over the whole milk chain, from farm to retail. Retail cheese in Rwandan milk shops contained no L. monocytogenes and < 3 log CFU/g S. aureus, but a critical 50 % was contaminated with Salmonella, presenting a serious food safety issue. In parallel, the status of hygienic practices was assessed and the level of compliance with hygienic requirements was established, and further linked to microbiological data. It was confirmed that modern farms with appropriate equipment were more complying with hygienic requirements. At collection level, the concerned centers scored similarly with only 26% of compliant answers, due to the lack of maintenance and calibration and trainings of the personnel, among others. A big difference was observed among processing plants with one outstanding company certified against ISO 22000:2005 and HACCP which complied totally to all internationally demanded requirements of food safety management systems, whereas smaller companies scored with 33% and 29% of compliant answers. In small cheese plants, all plants did not comply with any hygienic requirement, and the personnel were not even aware of basic hygienic principles, making this level of the chain critical. In wholesale points 55% of hygienic requirements were fulfilled and the major issues were regarding the absence of a cleaning and disinfection plan, training of personnel, unfit design, etc. Supermarkets showed more satisfactory results (62%) but the lack of temperature control was the main bottleneck at this stage. In milk shops, which appeared to be another weak point along the chain, only 26% of positive answers were obtained due to the lack of temperature control, personnel hygiene, inappropriate design of the premises, etc. This study was further directed on the previously identified weak points of the chain, namely small cheese plants and milk shops, in order to get deeper information on the origin of contaminations. In cheese plants, the production was followed from milk reception to final products, and microbiological parameters were analyzed at each step. This study was conducted just after interventions on behalf of the government (2013) and its international development partners and showed noticeable improvements regarding microbiological safety of cheese, and revealed that the personnel hygiene and the processing environment are probably at the origin of occasional Staphylococcus aureus and Salmonella contamination during processing. In milk shops a similar methodology was used and this study confirmed that the applied heating treatments enable total elimination of Salmonella, revealing that the observed bottlenecks result from post-contamination from the environment, an inadequate refrigeration during storage, or insufficient hygiene by the food handlers serving boiled milk in cups in the shops. All the obtained data on the prevalence of Salmonella were completed by additional sampling of cheese and boiled milk in milk shops, and a consumer survey was conducted for both milk and cheese and provided information on consumer behavior and consumption data. It was shown that in milk shops milk is mainly consumed by men (79.7 %) and the daily consumption was the most in practice (33.3 %) with a volume of 400 mL (61.2 %). Among milk buyers in milk shops, 40.0% is also buying boiled milk for their families resulting in consumption by mainly adults aging between 19-29 years (32.3 %, n=136), and a daily consumption frequency reported by 62.5 % of the survey participants. Among the concerned households (n=36), 66.7 % is re-boiling the milk and 33.3 % is storing it refrigerated before consumption. Gouda cheese is usually sold in supermarkets belonging to the formal market, after modernization of the cheese sub-sector. A cheese portion with a weight of 62.5 g is mainly consumed (80.0 % , n=370), and weekly frequency is most in practice (38.1 %). Finally, a probabilistic exposure model for Salmonella was build up by taking into account prior collected prevalence data in milk and Gouda cheese, assumed concentration data, information of the milk chain gathered by observation, and collected consumption data and consumer behavior information. The risk of infection of Salmonella by consumption of boiled milk was estimated at 11.08% per serving in milk shops and 4.75% in households. The interventions carried out in the cheese sub-sector by the government after the high prevalence of Salmonella in cheese (2012), allowed the reduction of the risk of salmonellosis per year by 30% in case of cheese consumption by adults. The information provided by this study is of great importance for Rwandan decision makers in food safety and quality. It suggests that the ongoing interventions in milk and dairy products quality and safety should be continued and especially extended to milk shops, since a high number of consumers with a highly consumption frequency is involved. Future research is recommended in what regards the absence of Listeria monocytogenes in dairy products and the supposed role played by the indigenous microbiota flora, the effectiveness of food safety management systems (FSMS) being implemented in the Rwandan dairy sector, and the microbiological quality and safety of newly introduced types of cheese

Exposure assessment to Salmonella by consumption of informally marketed milk and Gouda cheese in Musaner town, Rwanda

This study assesses the exposure to Salmonella by consumption of boiled milk and Gouda cheese in Musanze town, Rwanda. A consumer survey (n = 90) revealed that among milk buyers in milk shops, 40.0% were also buying boiled milk for their families resulting in consumption by 136 consumers, mainly adults aging between 19-29 years (32.3%). Among those interviewed consumers in households, 66.7% were re-boiling the milk and 33.3% were storing it refrigerated before consumption. Gouda cheese is usually sold in supermarkets belonging to the formal market. A cheese portion with a weight of 62.5 g is mainly consumed (80.0 %, n=370), and the weekly consumption frequency is most in practice (38.1%). A probabilistic exposure model was built up by taking into account prevalence data of Salmonella contamination in boiled milk and Gouda cheese in Rwanda, assumed concentration data of Salmonella, information of the milk chain gathered by observation, and collected consumption data and consumer behavior information. Finally, the risk of infection per day and per year were calculated for different categories of consumers. It appears that even though Salmonella is eliminated by milk boiling, the risk of infection still persists due to post-contamination in the milk shops (8.4% of consumers infected per year), but is decreased by additional boiling in households to 4.9% (children) and 4.6% (adults) per year. For cheese consumption, the risk of infection was lowered by multiple food safety interventions for cheese producers such as trainings on food hygiene and provision of modern equipments improving thus the situation from before to after 2012 by 30.0%% per year in case of adults. Those calculations demonstrate clearly that the additional efforts to reduce the occurrence of Salmonella in the Rwandan dairy chain will benefit the Rwandan population and decrease the burden of food borne disease

Genetic analysis of African swine fever virus from the 2018 outbreak in South-Eastern Burundi

African swine fever (ASF) is a contagious viral disease that causes high mortality, approaching 100%, in domestic pigs and wild boars. The disease has neither a cure nor a vaccine, and it is caused by an ASF virus (ASFV), the only member of the family Asfarviridae, genus Asfivirus, and the only known DNA arbovirus. Twenty-four genotypes of ASFV have been described to date, and all of them have been described in Africa. ASF is endemic in Burundi, and several outbreaks have been reported in the country; the disease continues to economically impact on small-scale farmers. This study aimed at genetic characterization of ASFV that caused an ASF outbreak in the Rutana region, Burundi, in the year 2018. Tissue samples from domestic pigs that died as a result of a severe hemorrhagic disease were collected in order to confirm the disease using polymerase chain reaction (PCR) and to conduct partial genome sequencing. Nucleotide sequences were obtained for the B646L (p72) gene, the intergenic fragment between the I73R and I329L genes, and the central variable region (CVR) of the B602L gene. Phylogenetic analysis of the Burundian 2018 ASFV grouped the virus within B646L (p72) genotype X and clustered together with those reported during the 1984 and 1990 outbreaks in Burundi with high nucleotide identity to some ASFV strains previously reported in neighboring East African countries, indicating a regional distribution of this ASFV genotype. Analysis of the intergenic fragment between I73R and I329L genes showed that the Burundian 2018 ASFV described in this study lacked a 32-base pair (bp) fragment present in the reference genotype X strain, Kenya 1950. In addition, the strain described in this study had the signature AAABNAABA at the CVR (B602L) gene and showed 100% amino acid sequence identity to viruses responsible for recent ASF outbreaks in the region. The virus described in this study showed high genetic similarities with ASFV strains previously described in domestic pigs, wild suids, and soft ticks in East African countries, indicating a possible common wild source and continuous circulation in domestic pigs in the region

Use of High Density Single Nucleotide Polymorphism (SNP) Arrays to Assess Genetic Diversity and Population Structure of Dairy Cattle in Smallholder Dairy Systems: The Case of Girinka Programme in Rwanda

In most smallholder dairy programmes, farmers are not fully benefitting from the genetic potential of their dairy cows. This is in part due to the mismatch between the available genotypes and the environment, including management, in which the animals perform. With sparse performance and pedigree records in smallholder dairy farms, the true degree of baseline genetic variability and breed composition is not known and hence rendering any genetic improvement initiative difficult to implement. Using the Girinka programme of Rwanda as an exemplar, the current study was aimed at better understanding the genetic diversity and population structure of dairy cattle in the smallholder dairy farm set up. Further, the association between farmer self-reported cow genotypes and genetically determined genotypes was investigated. The average heterozygosity estimates were highest (0.38 ± 0.13) for Rwandan dairy cattle and lowest for Gir and N’Dama (0.18 ± 0.19 and 0.25 ± 0.20, respectively). Systematic characterization of the genetic variation and diversity available may inform the formulation of sustainable improvement strategies such as targeting and matching the genotype of cows to productivity goals and farmer profile and hence reducing the negative impact of genotype by environment interaction

Microbiological quality and safety assessment of the Rwandan milk and dairy chain

Milk is a valuable and nutritious food product that can partially fulfill the rising food demand of the growing African population. The microbiological status of milk and derived products was assessed throughout the milk and dairy chain in Rwanda by enumeration of the total mesophilic count, coliforms, and Staphylococcus aureus and detection of Salmonella and Listeria monocytogenes. The quality of raw milk was satisfactory for the majority of samples, but 5.2% contained Salmonella. At the processing level, the total mesophilic count and coliform numbers indicated ineffective heat treatment during pasteurization or postpasteurization contamination. Increasing bacterial counts were observed along the retail chain and could be attributed to insufficient temperature control during storage. Milk and dairy products sold in milk shops were of poor and variable microbiological quality in comparison with the pasteurized milk sold in supermarkets. In particular, the microbiological load and pathogen prevalence in cheese were unacceptably high

African swine fever virus circulation between Tanzania and neighboring countries : a systematic review and meta-analysis

For over 100 years after the description of the first case of African swine fever (ASF) in Kenya, ASF virus (ASFV) cross-border spread in eastern and southern Africa has not been fully investigated. In this manuscript, we reviewed systematically the available literature on molecular epidemiology of ASF in Tanzania and its eight neighboring countries in order to establish the transmission dynamics of ASFV between these countries. Data were retrieved from World Animal Health Information System (WAHIS), Google Scholar, PubMed, Scopus, and CrossRef databases, using the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and reviewed to document ASF outbreaks and ASFV genotypes distribution. Using phylogeographic approach applied to ASFV p72 sequence dataset, the evolutionary history and the dispersal pattern of the ASFV strains were assessed. From 2005 to 2019, a total of 1588 ASF outbreaks affecting 341,742 cases that led to 302,739 domestic pig deaths were reported. The case fatality rates (CFR) varied from 15.41% to 98.95% with an overall CFR of 88.58%. Fifteen different p72 ASFV genotypes were reported and the time to the most recent common ancestor (TMRCA) for ASFV strains dated back to 1652.233 (1626.473, 1667.735) with an evolutionary rate of 4.805 x 10(-5) (2.5857 x 10(-5), 9.7789 x 10(-5)). Phylogeographic dispersal analysis revealed several transboundary spread events of ASFV strains between these countries. These results suggest persistent circulation of ASFV in these countries and advocate for more research to improve our understanding of the transmission dynamics of the virus and for a regional approach to mitigate the spread of ASFV

Complete genome analysis of African swine fever virus responsible for outbreaks in domestic pigs in 2018 in Burundi and 2019 in Malawi

Several African swine fever (ASF) outbreaks in domestic pigs have been reported in Burundi and Malawi and whole-genome sequences of circulating outbreak viruses in these countries are limited. In the present study, complete genome sequences of ASF viruses (ASFV) that caused the 2018 outbreak in Burundi (BUR/18/Rutana) and the 2019 outbreak in Malawi (MAL/19/Karonga) were produced using Illumina next-generation sequencing (NGS) platform and compared with other previously described ASFV complete genomes. The complete nucleotide sequences of BUR/18/Rutana and MAL/19/Karonga were 176,564 and 183,325 base pairs long with GC content of 38.62 and 38.48%, respectively. The MAL/19/Karonga virus had a total of 186 open reading frames (ORFs) while the BUR/18/Rutana strain had 151 ORFs. After comparative genomic analysis, the MAL/19/Karonga virus showed greater than 99% nucleotide identity with other complete nucleotides sequences of p72 genotype II viruses previously described in Tanzania, Europe and Asia including the Georgia 2007/1 isolate. The Burundian ASFV BUR/18/Rutana exhibited 98.95 to 99.34% nucleotide identity with genotype X ASFV previously described in Kenya and in Democratic Republic of the Congo (DRC). The serotyping results classified the BUR/18/Rutana and MAL/19/Karonga ASFV strains in serogroups 7 and 8, respectively. The results of this study provide insight into the genetic structure and antigenic diversity of ASFV strains circulating in Burundi and Malawi. This is important in order to understand the transmission dynamics and genetic evolution of ASFV in eastern Africa, with an ultimate goal of designing an efficient risk management strategy against ASF transboundary spread

Vsx1 Transiently Defines an Early Intermediate V2 Interneuron Precursor Compartment in the Mouse Developing Spinal Cord

Spinal ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities. Interneurons arise during embryonic development from distinct progenitor domains distributed orderly along the dorso-ventral axis of the neural tube. A single ventral progenitor population named p2 generates at least five V2 interneuron subsets. Whether the diversification of V2 precursors into multiple subsets occurs within the p2 progenitor domain or involves a later compartment of early-born V2 interneurons remains unsolved. Here, we provide evidence that the p2 domain produces an intermediate V2 precursor compartment characterized by the transient expression of the transcriptional repressor Vsx1. These cells display an original repertoire of cellular markers distinct from that of any V2 interneuron population. They have exited the cell cycle but have not initiated neuronal differentiation. They coexpress Vsx1 and Foxn4, suggesting that they can generate the known V2 interneuron populations as well as possible additional V2 subsets. Unlike V2 interneurons, the generation of Vsx1-positive precursors does not depend on the Notch signaling pathway but expression of Vsx1 in these cells requires Pax6. Hence, the p2 progenitor domain generates an intermediate V2 precursor compartment, characterized by the presence of the transcriptional repressor Vsx1, that contributes to V2 interneuron development