​Assessment of the Sanitary and Hygienic Quality of Raw Milk Marketed in the Urban Area of Mostaganem, Algeria

Study area and period
 
The study was conducted in the capital of the Wilaya of Mostaganem, located in the northwest region of Algeria. 20 milk samples were collected monthly (from a set of 20 shops), in the spring period (April-May, 2019), during the high season of production and consumption of raw milk and its derivatives by the local population. The distribution of the shops was done according to their distribution in the different districts of the city: North, East, West and South.
 
Samples
 
Samples are taken in the morning in their packaging presented for sale. Raw milk is aseptically removed after homogenization, in sterile 250 ml vials and then transported in a cooler at 4°C to the Laboratory of Sciences and Technics of Animal Production. The assessment of the hygienic situation of the premises, the equipment, the conditions of preservation and presentation for the sale of raw milk is carried out through a questionnaire. The hygiene of the staff (clothing and medical certificates), the origin of the raw milk and the presence or not of the health certificate of the livestock accompanying the sale of milk are noted.
 
Bacteriological and serological analyses
 
Bacteriological research has involved aerobic flora at 30°C (AG), thermotolerant coliforms (TTC), positive coagulase Staphylococcus (PCS) and Salmonella. The tests are carried out in accordance with official Algerian standards (JORA, 2017). The indirect search for tweezers in raw milk is carried out by the Ring-test. The reaction involves a colored antigen consisting of a suspension of killed brucella and milk antibodies (JORA, 1996).
 
Methods
 
Microbiological analyses are carried out under strict aseptic conditions with media and reagents from the Pasteur Institute, Algeria. For the research and counting of these germs, a series of dilutions (from 10^-1 to 10^-5) is carried out beforehand with physiological water from a homogenized raw milk stock solution (Guiraud, 2012).
 
Search for aerobic germs at 30°C
 
AG enumeration is performed after appropriate dilutions by inoculation on Plate Count Agar followed by incubation at 30°C for 72 hours.
 
Search for thermotolerant coliforms
 
TTC counts are conducted on Violet Red Bile Lactose Agar medium. Enumeration of red colonies with a diameter of at least 0.5 mm is performed after 24 hours incubation at 44°C.
 
Search for Staphylococci with positive coagulase
 
This research is carried out by Chapman’s medium enrichment method for incubation at 37°C for 48 hours.  Fluorescent golden colonies are retained for the count. For orientation to Staphylococcus aureus, one colony is placed in a tube containing Brain-Heart Infusion Broth and then incubated at 37°C for 18 hours. Then, 1 ml of this solution is mixed with 1 ml of rabbit plasma and incubated at 37°C for 4 hours. The presence of Staphylococcus aureus results in the coagulation of the solution.
 
Salmonella testing
 
Their search requires pre-enrichment in buffered peptone water for incubation at 37°C for 18 hours. Enrichment of this solution is carried out on a sodium acid selenite medium and then incubated at 37°C for 24 hours. Isolation is then carried out on Salmonella-Shigella medium for incubation at 37°C for 24 hours. Salmonella that does not ferment lactose have colourless, transparent colonies with or without a black center.

Serological research of brucellosis
 
It is a qualitative immunological reaction that indirectly highlights the anti-brucella antibodies present in bovine milks (Pal et al., 2020). The Ring-milktest (MRT) consists of a suspension of a strain of Brucella abortus killed by phenol and colored by hematoxylin. After a60 minutes incubation at 37°C, the test is positive when the agglutinates adhere to the lipid globules that gather with the cream ring by staining it blue-violet.
 
Hygienic situation of the premises
 
A survey using a questionnaire is carried out to assess the hygienic situation of the premises, equipment, staff and the way in which milk is sold.
 
Interpreting of results
 
Interpretation of the analyses is carried out on the values set by Algerian normative references for brucellosis (JORA, 1996) and microbiology (JORA, 2017). The decision rule will be conditioned by the level of quality: satisfactory, acceptable, unsatisfactory or toxic. The statistical analysiswas made possible by the use of SYSTAT SOFTWARE MYSTAT 12. The threshold of statistical significance is estimated at P<0.05.

Bacteriology
 
The classification of the overall estimate of the germs studied according to the standards setting the microbiological criteria of foodstuffs (JORA, 2017) is presented in (Table 1). There is a significant level of contamination of the sampled milks.
 
Taking into account the different regions of the urban area of Mostaganem, contamination is much more important for aerobic germs at 30°C and for thermotolerant coliforms.
 

 
Aerobic germs at 30°C
 
The microbiological analysis of the collected milks shows that there is a significant difference in the estimated number of AG at 30°C between the different geographical regions studied (p>0.05). The average obtained for all samples is 8.10⁹ cfu/ml, which is largely higher than the acceptability thresholds required by Algerian regulations (JORA, 2017) and the estimates made by different authors.
 
These results reflect a poor state of freshness of these raw milks since 100% of the samples are of unacceptable quality at the level of the 04 sampling areas (Table 1). This is probably related to poor practices of milking and packaging of raw milk in cattle farms (Guiraud, 2012 and Sing and Gupta, 2015) or to uncontrolled situations during their transport and sale in stores, especially in this period when the ambient temperature is quite high. We note, a great variability in the enumeration of these bacteria for all samples as described in various researches (Gupta et al., 2020 and Hamiroune et al., 2016). The region has the highest rate, as the farms that supply this region with milk are located in mountainous areas far from the urban area.
 
Positive coagulase Staphylococcus
 
PCS is detected in samples from the Eastern region (10⁴ cfu/ml), with a low count and in samples from the Southern region (3.10³ cfu/ml), qualifying it as a health risk (JORA, 2017). Positive samples represent 10% of the cases studied. PCS is an indicator of direct contamination by human pathogenic strains from healthy carriers or dirty hands of untreated sick animal workers and failure to follow good hygiene practices (Baazize-Ammi et al., 2019) but also of indirect contamination, by animal infections (subclinical mastitis) (Hamiroune et al., 2016) and by the various manipulations of objects used during milking accentuated by the dusty environment of the barn and rearing areas of the producing herd (Sing and Gupta, 2015). PCS causes inflammation of the udder which has an economic impact on milk production (Pal et al., 2020). Baazize-Ammi et al., (2019) report in the same context significantly higher average loads in milks from small-scale farms collected (9.10² cfu/ml) compared to milks from large-scale production (8´10⁴ cfu/ml). This can be explained by the high prevalence of staphylococcal mastitis on small, uncontrolled farms and by the lack of hygiene observed during milking.
 
Thermo tolerants coliforms (TTC)
 
The estimation of the number of TTC shows that there is a significant difference between the different milk samples studied in the urban area of Mostaganem (p>0.05). It is observed a high count in the eastern region with an average of 12.10⁴ cfu/ml (Table 1).
 
Samples of unsatisfactory quality represent more than 50% and come from the 4 regions studied. TTCs being commensal bacteria of the gut can survive as sapprophytes outside and contaminate the animal’s body during uncontrolled milking operations by transfer of fecal material from soil, manure and dust containing microorganisms (Guiraud, 2012). Comparable results exceeding the contamination threshold are obtained by Baazize-Ammi et al., (2019) who find that 40% of the milk analyzed on the farm is loaded with TTC. In approximately 54.5% of cases, removal of first sprays of milk before milking is lacking, milking machines are not clean and 63.6% of milkers wear inappropriate and soiled clothing.
 
Salmonella
 
Salmonella were noted to be absent in all samples (JORA, 2017). Because their numbers are generally low in products, they are often difficult to find and identify. According to Singh et al., (2018), since the animal gut is the most important reservoir of Salmonella, animal feces are the main sources of milk contamination during milking operations. Due to their consistent pathogenicity, Salmonella are not tolerated in a food such as milk (Guiraud, 2012). The results of this research corroborate with those of Bousbia et al., (2018) who found milk free of this germ. The investigations of Singh et al., (2018) revealed the presence of Salmonella spp, resistant to many antibiotics with a higher prevalence in raw milk (11.9%) compared to other products (7.4% in milk collected from vendors and 14.2% in dairies and retail stores).
 
Indirect search for brucellosis
 
Anti-brucella antibodies were revealed in two samples see (Table 2) (10% of the cases studied). This suggests that milk came from brucellosis-infected cattle farms. In Algeria, CFP due to the ingestion of raw milk or its fresh derivatives is the cause of several cases of human brucellosis (Rechidi-Sidhoum, 2019). In Sidi Bel-Abbès, 190 people were affected in 2019 compared to 211 cases detected in 2018; in Ghardaïa 198 cases in 2017 compared to 1547 in 2016 with 36 cases of bovine brucellosis in seven outbreaks across the wilaya during the latter year. For Mostaganem, only 3 cases were reported in 2020, this figure does not reflect the true incidence values given the high number of bovine brucellosis cases in the region(DSPRH, 2016-2021).
 

 
In developed countries, indirect, digestive contamination through consumption of raw milk or artisanal cheeses, perhaps observed in tourists who travel sharing the way of life of local populations or in those returning from a country where animal brucellosis is endemic, 84% of recorded cases were imported (Kamwine et al., 2017).
 
Survey of the state of shops
 
The survey on the situation of the premises showed for all shops (100%), the lack of control of milk, insufficient control of the rules of hygiene of the premises and equipment. The storage and sales equipment does not meet standards and equipment is inadequate or defective (plastic cans, storage bags used for the sale of milk as it is), milk is stored at room temperature (no refrigeration) (Baazize-Ammi et al., 2019). It was noted the absence of sanitary facilities, running water and the wearing of gowns by traders who did not present a health certificate and documents relating to the origin of the milk. The investigation revealed that the amount of milk retailed was that which was refused by the dairies.

In view of the results obtained from microbiological and serological criteria, it appears that the quality of the milks studied does not comply with the required national standards, with a potential danger associated with CFP. The non-conformity of these milks is related to poor storage conditions and sale in unsuitable stores and not subject to the commercial standards required in Algeria, accentuated by those of production practices on the farm and transport.  The presence of anti-brucella antibodies in milk indicates the lack of rigorous control of cattle farms that are infected with brucellosis. This preliminary assessment should alert all players in the informal sector to the need to implement a strategy to improve the quality of milk, both by respecting good production practices up to the sale and by establishing a rigorous control protocol at all levels.