A cross-sectional study was carried out in Nairobi, focusing on the four primary abattoirs for beef and pork. The selection criteria for these four zones were centered on both formal and informal operations, with every two sites being representative of either beef or pork. These four sites were the study’s most preferred and relevant sites.

The study involved two sample types: stool and meat cuts. The subjects included handlers of pork and beef.

During the period from February to August 2023, 302 stool samples and 165 meat cuts were collected resulting in a total of 467 samples for the study. Simple random sampling techniques were used for the random selection of meat cuts and participants in the 4 Study sites. Data on beef/pork handlers were collected qualitatively and quantitatively using questionnaires. Dry sterile leakproof stool collections container was used to collect fecal samples. In the informal, 50 g of fresh beef/ pork cuts sections were randomly collected from different body parts of the carcasses, while in the formal setup, the same quantity of meat cuts/products were randomly selected from the production site. The 50 g of beef/pork cuts sections and fecal samples were then packaged in sterile sample packaging bags or sampling containers and placed carefully in different sterile cooler boxes with ice packs for transportation and storage. These samples were transported to KEMRI-CVR labs at temperatures between 2˚C and 8˚C for up to 5 hours after collection.

Table 1 and Table 2 show the distribution of sampling from the selected abattoirs.

Beef/pork cuts were defrosted and homogenized, and 1 g of the 50 g punched beef/pork sample were collected and macerated in 1 ml of amino acid-rich bench stable minimum essential medium (MEM, pH 7.0). The combinations were vortexed for approximately 10 seconds and centrifuged for 5 min at 10,000 rpm before Trizol RNA extraction.

The collected stool samples were diluted in (1:10) tenfold phosphate-buffered solution (pH 7.4) by adding 1 g of a stool specimen to the phosphate-buffered saline solution (PBS). Alternatively, 1 ml of loose stool samples were added to 9 ml of PBS in case samples were retrieved from those with diarrheal symptoms. The prepared suspensions were centrifuged at 3000 rpm for 30 min before rotavirus identification using immunoassays.

The prepared suspensions of diluted stool samples were subjected to indirect sandwich ELISA-serotyping using a commercial enzyme immunoassay (EIA) in accordance with the manufacturer’s instructions (Meridian Bioscience™ ImmunoCard™ STAT! Rotavirus Test Kit, Rotaclone, Cincinnati, USA).

The EIA positive stool samples and all homogenized meat suspensions underwent the procedure for nucleic acid extraction using Qiagen and Trizol RNA extraction kits respectively. The extraction procedures were conducted according to the manufacturer’s specification with some little modifications required. Detection of the RVAs dsRNA was performed by Two Step RT-PCR using ReverTrace and KOD PLUS Kits. The dsRNA was denatured to ssRNA at 97c for 2 min, the resulting ssRNA product was reverse transcribed at 42c for 30 min, 97c for 5 min, and 4c for 5 min using ReverTrace kit for complementary DNA synthesis. The resulting product, complementary DNA, was then used for primary PCR, to amplify the fragments VP4 and VP7 segments of RVA using KOD-PLUS kit. Amplification was done on a Thermocycler using the following thermocycling conditions: 95c for 2 min, (94c for 30 sec, 48c for 30 sec, 72c for 1 min) 30 cycles, 72c for 7 min, and 4c for infinity. The primary PCR products were ultimately analyzed using 1.2% agarose gel electrophoresis and then visualized using transilluminator device.

Multiplexed Semi-Nested RT-PCR

The primary PCR products positive for VP7 and VP4 were to be subjected to G and P genotyping using different sets of VP7 2^nd and VP4 2^nd primers respectively. PCR reactions were performed using a Thermocycler with the following thermocycling conditions—95c for 5 min, (94c for 30 sec, 48c for 30 sec, 72c for 1 min) 35 cycles, 72c for 7 min, and 4c for infinity ( Table 3 ).

Data analysis was conducted using the Power BI statistical software application. The results from laboratory tests and questionnaire responses were tabulated, coded, and analyzed by employing frequency distribution tables and percentages. Descriptive statistics was also used to provide a clear image of background variables. The Chi-square was to be employed to associate rotavirus with meat handlers’ hygienic practices. Eppicollect5 was used to collect and store the socio-demographic data from the questionnaire. Finally stored data in Epicollect5 was accompanied by manual documentation of A4 hard copies as backups before storage in a locked cabinet.

The prevalence of rotavirus among beef/pork handlers was 0% (n = 302), 95%CI: The Indirect Sandwich ELISA results for the stool samples analyzed were all non-reactive in the 4 study sites as shown in Table 5 below.

Additionally, the prevalence of rotavirus in meat cuts was also 0.00% (n=165), 95%CI. The Two-Step RT PCR results for Meat cuts from the 4 study sites were all non-reactive as shown in Table 5 below.

The study included 302 beef and pork handlers. The demographic characteristics examined included gender, age, education level, and marital status, as detailed in Table 5 . Their average age was 35.69 years old across all 4 abattoirs. Regarding the education level of the personnel involved in the study, the highest attained level was tertiary (university/college). In the formal establishments, most participants had attained tertiary (university/college) level education with few participants having lower levels of education. In contrast, the informal establishments had a more diverse range of education levels among their participants. While the highest level of education was also tertiary, many of the participants in the informal abattoirs had lower levels of education, unlike the formal establishments. Additionally, in formal settings, most participants, including those with tertiary education, received comprehensive hygiene training. The emergence of COVID-19 significantly increased the demand for such training, ensuring that individuals working in formal abattoirs were well-equipped with essential hygiene knowledge and practices. This heightened focus on hygiene has been crucial in maintaining health standards and preventing disease transmission within these environments [26] .

However, the situation was markedly different in informal establishments. While participants with tertiary-level education in informal abattoirs did receive some hygiene training, those with lower levels of education were often left without adequate training. This disparity in access to hygiene training based on educational background raises concerns, as it highlights a significant gap in knowledge and practices among employees. Unlike in formal abattoirs, where hygiene training was consistently implemented across all employee groups, informal establishments exhibited a troubling inconsistency that could jeopardize health and safety standards.

In the informal establishments, most of the meat handlers were observed to be working without the appropriate personal protective equipment. Many were seen wearing torn dust coats, and were missing essential PPE items such as gloves, gumboots, hair nets, and masks. This was the case despite the inherently unhygienic environment in which they were operating.

In stark contrast, the meat handlers in the formal establishments were provided with the necessary PPE to ensure their safety and maintain proper hygiene standards. The formal abattoirs ensured that their employees were equipped with the appropriate protective gear, including intact dust coats, gloves, gumboots, hair nets, and masks, enabling them to work in a more hygienic and safe manner. It was observed that the majority of participants did not exhibit symptoms of diarrhea, vomiting, or abdominal pain, with the exception of a few individuals who experienced non-rotaviral stomach complications and presented with abdominal pain.

Y—Yes, N—No, O—Other, P—Primary, S—Secondary, T—Tertiary.

The findings showed that 36 (11.9%) had primary or lower level of education, 145 (48.1%) had secondary level education while 121 (40.0%) of the study respondents had tertiary level education. Analysis of marital status revealed that 55 (18.21%) of the patients were single while 247 (81.79%) were married as shown in Table 6 .

The absence of RV in 467 samples (302 stool samples and 166 meat cuts) from the 4 selected abattoirs in Nairobi, Kenya, indicates a low risk associated with the consumption of these products. Globally, the primary sources of foodborne viral diseases are fish, fruits, and vegetables, which are often linked to water transmission [27] [28] . However, this study contributes to the limited research on viral detection in animal-derived products in Kenya, particularly fresh meat products.

Rotavirus (RV) can be transmitted between animals and humans through contaminated food, posing a public health risk. While infections are often self-limiting, studying foodborne viruses in animal products is crucial within the One Health framework. Therefore, this research aids in understanding transmission dynamics and informing public health interventions [13] .

However, the lack of detection of Rotavirus A (RVA) in beef and pork cuts does not necessarily indicate a public health concern, despite the capacity of these microorganisms to survive on various surfaces under diverse environmental conditions, including low humidity, ambient temperatures, and refrigeration [11] [28] . Recent data indicate that RVA was responsible for 3.1% of foodborne outbreaks in Brazil in the past few years [14] . Furthermore, rotavirus is recognized as a significant pathogen associated with neonatal diarrhea on a global scale [29] . This study tends to screen RVA in stool samples from symptomatic and asymptomatic participants of adult age, working in abattoirs. The study findings support the rationale for the increased prevalence of the identified etiological agent in neonates with acute gastroenteritis (AGE) compared to adults, despite adult meat handlers working in environments that are more susceptible to zoonotic diseases.

Despite the absence of documented direct links between contaminated products and outbreaks of rotavirus (RV) in the population, infections typically occur via the fecal-oral route, person-to-person contact, or through contaminated food [30] [31] . RV is recognized as an enteric virus that serves as an indicator of environmental contamination during food handling processes and can help assess the sanitary quality of water used in these procedures. This indicates that the transmission of these pathogens can happen in food-handling environments, particularly through handlers or contaminated water sources [27] .

Furthermore, certain viruses can lead to cross-contamination in both commercial and domestic settings due to their resistance to cleaning and sanitizing methods [28] . These viruses exist as small, non-enveloped virions, lacking lipid bilayers, with outer protein layers (capsids) that confer greater resistance to environmental factors, disinfectants, and common sanitizers. Notably, RVs form highly stable and resilient viral particles in the environment due to their triple-layered capsid structure [18] .

In Kenya, environmental monitoring studies have been conducted to assess the presence of enteric viruses in food, even in the absence of specific legislation. Recent evaluations revealed no detection of RV in the samples analysed [24] [32] . However, this finding does not necessarily indicate optimal hygiene conditions for products handled and sold, particularly in informal settlements. While the quantification and verification of the infectivity of viral particles were not feasible, the amplification of extracted genetic material was achieved.

The results of this study hold significant implications for health inspection and surveillance agencies, suggesting that meat products available in Nairobi pose minimal risk to consumer health. During the study, beef and pork samples showed a 0% frequency of RV detection, which is reassuring given the high consumption rates of these products in Nairobi. Nevertheless, the potential risks associated with improper handling of these products in home environments warrant consideration. Although these meats are generally not consumed raw, there are reports indicating that consumers often struggle with proper handling and storage, which could increase the risk of RV transmission in domestic settings [33] .

The negative findings regarding RV may also reflect the positive impact of vaccination programs introduced by Kenya’s national immunization program in 2014. The introduction of a two-dose monovalent vaccine, Rotarix and a three-dose pentavalent vaccine, Rotateq offered an opportunity to mitigate the spread of acute rotaviral gastroenteritis [34] . This intervention is expected to reduce a substantial amount of childhood morbidity and mortality, which could likely contribute to the reduced prevalence of viral infections.

Collectively, these factors contribute to a lower risk of contamination in meat products, thereby supporting public health outcomes in the region [35] [36] .

This study aimed to identify a specific virus; however, it is imperative to expand the research scope to encompass additional pathogens. Due to current funding constraints in our country, our investigation was limited to the aforementioned virus. Future research efforts will aim to expand the focus to include the identification of other significant viruses, including norovirus, adenovirus, hepatitis E virus, coronavirus, rhinovirus, astrovirus, and hepatitis A virus. Additionally, the research was limited to a single geographical area, which restricts our ability to evaluate the wider epidemiological context. Nonetheless, we highlight that this is the first study conducted in the selected city. Future investigations will aim to encompass multiple counties across Kenya to offer a more thorough assessment of viral presence in food sources and among their handlers.

We advocate for a comprehensive analysis of the contributions of various enteric viruses, including astroviruses, coronaviruses, hepatitis E virus (HEV), hepatitis A virus (HAV), adenoviruses, and norovirus, to outbreaks of acute gastroenteritis by integrating their testing as potential viral agents.

It is also imperative to delineate the specific roles of food handlers in the transmission dynamics of foodborne diseases across Kenya. Strengthening the food safety regulatory framework is vital for implementing policies that restrict symptomatic food workers from engaging in food handling and mandate a minimum of 48 hours off work following the resolution of symptoms. This approach effectively mitigates disease transmission risks.

Furthermore, comprehensive food safety training programs should be established in the study area, focusing on enhancing awareness of enteric diseases, understanding modes of foodborne disease transmission, and implementing effective prevention strategies, particularly emphasizing rigorous hand hygiene practices.

Additionally, thorough investigations of gastroenteritis outbreaks in the region are essential, with rotavirus considered a potential etiological agent among other pathogens. This multifaceted approach aims to enhance public health outcomes and reduce the burden of foodborne illnesses.