INCIDENCE OF TRUEPERELLA PYOGENES IN RAW

ABSTACT One hundred and eighty raw milk samples were collected randomly from dairy farms, farmers` houses and dairy shops in Assiut city. These samples represented by 60 milk samples from cows and buffaloes of dairy farms (30 for each) , 90 samples from farmers` houses including cows, buffaloes and sheep milk (30 for each) and 30 raw dairy shops` milk samples. The samples were examined for the incidence of T.pyogenes and prevalence of Subclinical Mastitis (SCM) in raw milk. The prevalence of SCM in milk samples of dairy farms were 90% for both cows and buffaloes` milk based on California Mastitis Test (CMT) and 50% and 46.7% in cows and buffaloes` milk based on White Side Test (WST), respectivey. In the contrary, prevalence of SCM in farmers` houses milk samples based on CMT and WST were 53.3%, 73.3% &33.3% and 33.33%, 46.7% & 50% in cow, buffalo and sheep, respectively. The incidences of T.pyogenes in dairy farms milk samples were 60% in both cows and buffaloes` milk, while 63.33%, 60% and 36.67% in cows, buffaloes and sheeps` milk of farmers` houses, respectively. Additionally, the incidence of T.pyogenes in dairy shops` raw milk samples was 43.3%. PCR for 18 strains of T.pyogenes revealed that 15 strains gave positive results for pyolysin virulence gene. The public health hazards of the organism and the suggestive measures were also discussed.


INTRODUCTION
T. pyogenes is a worldwide known bacterium of genus Trueperella belonging to the family Actinomycetaceae in the order Actinomycetales of the class Actinobacteria.Genus Trueperella was named after Hans Georg Trüper, a German microbiologist (Yassin et al., 2011).It is a Gram-positive, pleomorphic, non-spore-forming, non-motile, non-capsulated, facultatively anaerobic rod, which is characterized by a fermentative Metabolism and strong proteolytic activity (Abdallah, 2016).Growth of the bacteria occurs on ordinary media but is enhanced on media containing blood or serum.Colonies of T. pyogenes on sheep blood agar were described as pinpoint, convex, slightly translucent and circular and were surrounded by a zone of β-hemolysis (Schaal, 1986;Lämmler and Hartwigk, 1995).T. pyogenes is considered to be a part of the biota of skin and mucous membranes of the gastrointestinal, upper respiratory and urogenital tracts of animals (Rzewuska et al., 2006;Silva et al., 2008).Moreover, this bacterium was also isolated from the udders of clinically healthy cows (Spittel and Hoedemaker, 2012).This opportunistic bacterium causes mastitis which is associated with a wide range of physical, chemical and microbiological changes of milk.(Radiostasis et al., 2007) These changes are accompanied with clots, discoloration of milk and high number of leukocytes in the affected milk and it contributes to remarkable reduction of milk production, lower milk quality, affect fertility of cattle,in addition to high costs of control and treatment.In human, the infections are connected with occupational exposure (Kavitha et al., 2010), including endocarditis (Jooter et al., 1978), pneumonia (Amejeiras et al., 2004), arthritis (Nicholson et al., 1998), various purulent lesions and abcesses (Gahrn-Hansen and Frederiksen.1992)and sepsis (Levy et al., 2009).T.pyogenes is armed with known numerous virulence markers, contribute to increase its pathogenic potential (Cohen et al., 2015)

Milk samples:
A total number of 180 random milk samples were collected from dairy farms, farmers` houses and dairy shops in Assuit city.Collected samples were transferred to laboratory as soon as possible to be examined for the incidence of T.pyogenes and presence of SCM.Storch test is applied on the samples to exclude the heat treated samples according to Lampert (1975).

2-Screening test to detect SCM for dairy farms and farmers` houses milk samples:
A-CMT according to Schalm et al. (1971).
B-WST according to Kahir et al. (2008).Farah and Kaldes (1999) and Sadek (2008), as their results were 28.63% and 36.6%,respectively.The prevalence of SCM based on WST revealed an incidence rate of 50% at the cow level, nearly similar result of (54%) was obtained by Singh and Baxi (1988).Lower findings were estimated by Sayed and Abdelhafeez (2009) and Islam et al. (2012) as they revealed animal-wise incidence of 33.64% and 25.5%, respectively.While, and Sadek (2008) obtained higher incidence of 60.95%.Regarding to buffaloes` milk samples out of 30, 14 (46.7%)samples were WST positive.Similar result 48.93% was detected by El-Balkemy et al. (1997).Lower finding 33.33% was obtained by Sadek (2008).However, Farah and Kaldes (1999) Sadek (2008).However, Farah and Kaldes (1999) revealed a higher incidence of 63.3%.Regarding sheep milk samples of farmers` houses, the recorded results based on CMT were 10 (33.33%) of 30 milk samples were positive.A lower incidence was obtained by Sadek (2008) 9.60%.However a higher incidence of 55.6% was shown by Moawad and Osman (2005).In addition, the positive sheep milk samples based on WST were 15 (50%) out of 30 samples.From the results illustrated in Table .2,data revealed the incidence of T.pyogenes in the dairy farm.Out of 30 cow`s milk samples, there were 18 (60%) positive for T.pyogenes.Lower incidences were estimated by Ribeiro et al. (2015) 45.1%;Alkasir et al. (2016) 28.6%; Momtaz et al. (2016) 36.50%;Tamai et al. (2018) 32.5% andRezanejad et al. (2019) who detected that 32 (14.15%) out of 226 mastitic milk samples were positive for T.pyogenes.However, higher incidences were described by Hijazin et al. (2011) in Germany, they revealed that all 71 isolates (100%) have been identified as T.pyogenes and Mbindyo et al. (2020) 74.4%.With regard to buffaloes` milk samples of dairy farms in Table .2,the incidence of T.pyogenes was 18 (60%) out of 30 milk samples.Concerning the examined farmers` houses milk samples, it`s clear that 19 (63.33%) out of 30 in cow`s milk samples.A higher incidence was detected by Abdallah.( 2016), who detected also that all 57 isolates (100%) were T.pyogenes and exhibited complete zone of hemolysis.Regarding buffalo`s millk samples of farmers` houses, results detected 18 (60%) positive samples out of 30 examined milk samples.In addition, 11 (36.67%)out of 30 sheep milk samples showed in Table .2have the phenotypic properities of T.pyogenes.Data in Table .2showed that 13 out of 30 dairy shops` raw milk samples were positive with incidence of 43%.The high incidence of T.pyogenes and mastitis in the present study may be due to inefficiency of milking personnel, and inadequate hygienic and sanitary measures applied during the milking process.Findings in Table .3revealed that out of 30 milk samples in dairy farm cows` milk samples there were 27 positive samples for CMT, 15 positive milk samples for WST and 18 positive milk samples for T.pyogenes.In the contrary of buffaloes` milk samples of dairy farms, it was shown that out of 30 milk samples, the positive milk samples were 27 for CMT, 14 for WST and 18 for T.pyogenes .The obtained results in Table .3illustrated that out of 30 cows` milk samples of farmers` houses, the positive samples were 17, 13 and 19 for CMT, WST and T.pyogenes, respectively.As regarding to buffaloes` milk samples, data showed that out of 30 milk samples, the positive milk samples were 22 for CMT, 14 for WST and 18 for T.pyogenes.In case of sheep`s milk samples, it is obvious that out of 30 milk samples, there were 10 positive CMT samples, 15 positive WST samples and 11 positive for T.pyogenes .In the present study, it was clear from photos 1 and 2 that, plo virulence gene was detected in 15 of 18 of examined strains in a percentage of 83.33 of the isolates.One of the most important virulence genes is pyolysin (plo), an extracellular toxin, which is one of the first pathogenic factors detected in T. pyogenes.Although pyolosin was primarily identified as a hemolysin of red blood cells in a variety of animal species, its cytolytic effect has been demonstrated in several different host cells such as polymorphonuclear leukocytes (PMNS) and macrophages.Several studies have reported that all T. pyogenes strains encode pyolsin gene but the frequency of its expression is higher in pathogenic strains.From the results in photo.1, it is clear that 11 strains were isolated from dairy farms` and farmers` houses cow and buffalo milk samples which gave a positive results.The results illustrated in photo.2revealed that 4 strains of dairy shops and sheep`s milk samples showed positive results for plo virulence gene.While, 1 buffalo and 2 sheep milk samples gave negative results.In comparison with the current study, a study was done by Bradely et al. (2015), all T.pyogenes isolates encoded plo with percentage of 100%.Another study performed by Abdallah ( 2016) revealed that all the 57 isolates were positive for plo encoding gene.In conclusion, raw milk samples of the present study revealed that most of the examined samples were contaminated with T.pyogenes and harbor plo gene which is responsible for the pathogenicity of the bacterium, so strict hygienic measures should be taken in consideration to improve the quality of raw milk and protect the economy of dairy industry.

REFERENCES
Photo.1: Results of PCR for testing gene encoding virulence factor of T.pyogenes (pyolysin) using primer (Pyolysin-F) for cows & buffaloes from dairy farms & farmers` houses Lane M = Ladder marker Lane 1 -6 = Positive PCR products of DNA from T.pyogenes isolates from cows milk in this study with specific primer (Pyolysin-F) Lane 7 -11 = Positive PCR products of DNA from T.pyogenes isolates from buffaloes milk in this study with specific primer (Pyolysin-F) Lane 12 = Negative control

Table 2 :
Incidence of T.pyogenes in the examined raw milk samples.

Table 3 :
Correlation between the positive CMT, WST and incidence of T.pyogenes in the examined raw milk samples.