Effect of pH on the incidence of listeria spp.

in silage of dairy farms in Plateau state,

Nigeria
DD Gushe1 CB Onusiriuka2 A.A Haroun2 OO Nwankiti3 & L Shedua3.
1
Federal College of Animal Health and Production Technology Vom, Plateau State;
2
Nigerian Defence Academy, Kaduna, Kaduna State; 3 National Veterinary Research Institute
Vom, Plateau State.

Abstract
Listeria spp. isolates were studied for a possible correlation between the spread of Listeria
species and silage pH in two dairy farms (LS and IS). Results show that 34 % (17/50) and 98 %
(49/50) of ‘LS’ and ‘IS ‘silage samples had pH range of 4.00 to 4.99, respectively. These yielded
41 % (7/17) and 27 % (13/48) Listeria spp. incidences. Only ‘LS’ silage recorded 40% (20/50)
pH value within the range of 5.0 - 5.99. This also showed 25% (5/20) Listeria spp.
contamination. 26% (13/50) of ‘LS’ silage samples had pH range of 6.00 and above. These
yielded 46% (6/13) isolates of Listeria spp. No ‘IS’ silage samples had pH range > 4.00; and 5.0
to 5.99. Fermentation profile of ‘LS’ silage also showed a low silage quality. Correlation
analysis between the spread of Listeria in LS silage and pH range indicates that an increase in
silage pH range increased Listeria incidence in LS which is not statistically significant r = 0.559.
The correlation between spread of Listeria in IS and pH range shows that an increase in silage
pH range decreased Listeria incidence. Data analysis further showed that the correlation is
moderately inverse r = – 0.241, meaning that there is not enough evidence to say that this
correlation exists in the population. These findings dispute the assertion that ensiled forage
possessing pH range 3.7 - 4.7 preserve silage. .. The results agree with other researchers that
Listeria grows in silage with low pH values…there is no consensuses on the pH of silage in
which Listeria are inhibited.
INTRODUCTION:
Ruminant Listeriosis was first connected to consuming contaminated silage in 1922. Reports
implicated poorly fermented silage, with improper fermentation characteristics like possessing a
pH of. > 4.0 as the source of Listeriosis in dairy farms [1]. Literature associated poor quality
silage with large numbers of both anaerobic and aerobic flora, which do not only reduce the
nutrient value of silage but constitute a risk to animal health and animal food products' safety [2]
Rapid reduction of pH-value on a stable level determines Lactic acid fermentation, microbial
stability of silage and forage preservation. L. monocytogenes especially and other Listeria spp.,
are frequently isolated from improperly fermented silage [1]. Yet, correlation concerning the
incidence of Listeria spp. in silage and silage pH in Plateau State is still grossly lacking. Facts
such as the 20 – 30 % mortality reported, silage feeding and of subsequent asymptomatic
shedding of Listeria monocytogenes in products are treats to dairy industry.
AIM: To assess the effect of pH on the incidence of Listeria spp. in silage at two dairy farms in
Plateau State, Nigeria.
Objectives:
1. To determine pH and fermentation characteristics of Silage at the respective Dairy farms’
2. To determine the incidence of L. monocytogenes and other Listeria spp of Silage at the
respective Dairy farms’ and,
3. To assess the correlation between the incidence of Listeria and Silage pH.
METHODS:
Assessment of fermentation characteristics of ensiled forage at dairy farm
The of fermentation characteristics of ensiled forage at dairy farms was determined
according to the method described by Heinrichs and Van Saun [3- 4]. The colour, odour, and
silage texture were observed for abnormalities.
Determination of silage pH:
The determination of silage pH was done according to the method described by [5].
225 ml of distilled or deionized water (pH 7.0) was added to 25 g of silage and allowed to
equilibrate at room temperature for 15 minutes The pH was then measured and recorded for each
silage sample with the aid of a standard pH meter).
Screening and Isolation of Listeria in Silage
Listeria species were screened using culture reference method for detecting Listeria in all
foods in the US FDA bacteriological and analytical methods (BAM) described by [6].
25g sample was homogenized in 225ml Listeria enrichment broth and incubated at 30oc
followed by streaking onto Oxford Listeria selective agar (Oxford CMO 865) and incubation at
37oc for 24, 48 and 7days respectively. Suspected Listeria colonies were observed as black with
a black halo on aesculin-containing media.
Since certain other bacteria can form weakly brownish black colonies, the black colour
development took longer than 2 days prior to noting the result down as suspected positive
sample. 5 or more typical colonies were transferred from Oxford agar (OXA) to Trypticase soy
agar with yeast extract (TSAYE), and streaking for purity and typically isolated colonies was
carried out. Purified isolates of the samples were sub – cultured in brain heart infusion broth
(BHIB) and store at – 80o C.
Correlation analysis of pH data and isolated Listeria in Silage samples
A total of 18 Listeria strains isolated from 50 Dairy farm A (LS) silage and 13 Listeria
isolated from Dairy farm B (IS) silage were statistically analyzed for pH – Listeria spp.
contamination correlation [7] .
Statistical analyses
The Statistical analyses were done using Statistical Package for the Social Sciences, IBM
Corp [8]
pH and fermentation characteristics of ensiled forage at dairy farms
An acidic pH range of 94% (47/50) and 100% (50/50) with 6% (3/50) and 0% (0/50)
alkaline pH range were observed in LS and IS (Dairy farms A and B) silage respectively. The pH
tests show that LS silage had good quality while IS silage had poor quality (Table 1).
The Silage fermentation assessment parameters show that LS and IS silage had 54%
(27/50) and 98% (49/50) good silage aroma respectively. 46% (23/50) and 2% (1/50) had spoilt
silage aroma. Likewise, 58% (29/50), and 98% (49/50) standard silage colour were noted in LS
and IS samples respectively. The silage texture test yielded 26% (13/50) and 98% (49/50) as
normal texture features with 74% (37/50) and 2% (49/50) as spoilt silage texture in dairy farms
A and B silage samples respectively (Table 1).
Screening and isolation of Listeria species in samples
The morphologic, phenotypic and biochemical characteristics of L. spp. which are: easculine
production; Gram stain reaction and β -Heamolysis on 5% sheep blood agar - A; B; and C
respectively (figures 1). While Figure 2 is the biochemical characteristics such as Voges -
Proskuer and Nitrate to Nitrite conversion tests (D and E respectively).

Figure 1: Morphologic & phenotypic characteristics of screened samples:

Easculine production; Gram stain reaction and β -Heamolysis on 5% sheep blood agar (A; B; and
C respectively).

Figure 2: Biochemical Characteristics such as Voges - Proskuer test and Nitrate to nitrite
conversion test (D and E respectively).
Incidence of Listeria species in silage from Dairy farms
The incidence of Listeria species in silage indicated 36% (18/50) Listeria isolates in 50
silage LS samples and 26% (13/50) isolates in IS silage samples respectively. 6% (1/18) 0%
incidence of L. monocytogenes was observed in LS and IS silage respectively. A 31 % (31/100)
incidence of Listeria isolates was observed in LS silage samples (Table 2). This test result
presents with a severe Listeriosis treat in farm A relative to farm B (Table 2).
Correlation between incidence of Listeria species and silage pH
Results show that 34 % (17/50) and 98 % (49/50) of ‘LS’ and ‘IS ‘silage samples had pH
range of 4.00 to 4.99, respectively. These yielded 41 % (7/17) and 27 % (13/48) Listeria spp.
incidences. Only ‘LS’ silage recorded 40% (20/50) pH value within the range of 5.0 - 5.99. This
also showed 25% (5/20) Listeria spp. contamination. 26% (13/50) of ‘LS’ silage samples had pH
range of 6.00 and above. These yielded 46% (6/13) isolates of Listeria spp. No ‘IS’ silage had
alkaline pH, and 5.0 to 5.99. Also, pH range < 4.00 was null (Table 3).

Table 1: Fermentation characteristics of ensiled forage at dairy farms

Dairy silage odour Silage Colour Silage Texture Silage pH Range
farms Okay Spoilt Normal Spoilt Normal Spoilt Acidic Alkaline
(straw (firmly (breaks 4.0 - < 7.11 –
colour) indestructible) slowly) 7.0 7.40
A (LS) 54% 46% 58% 42% 26% 74% 94% 6%
(27/50 (23/50) (29/50) (21/50) (13/50) (37/50) (47/5) (3/50)

B (IS) 98% 2% 98% 2% 98% (49/50) 2% 100% 0%

(49/50) (1/50) (49/50) (1/50) (49/50) (50/50) (0/50)
Keys: Spoilt silage smell: Tobacco smell, Caramel, Rancid or; Ammonia smell. Spoilt silage
colour: dark brown or dark green. Silage texture: becomes loose or firm when clutched firmly in
the palm released.
Table 2: Incidence of Listeria species in silage at Dairy farms
Dairy Farm Silage A (LS) B (IS)
Easculine hydrolysis, 36% (18/50*) 26%
Morphologic, Phenotypic and

SGPR, and Catalase (13/50)

biochemical characteristics

Positive, Oxidase
Negative and Motile
MR Test, V P Test, 36% (18/50)* 100%
& NO3 - NO2 Test 13/13)
Suspected Listeria 0% (18/18) 26%
spp. present (13/50)
L. monocytogenes 6% (1/18) *
0

L. grayi suspected 94% (17/18)

100%
(13/13)
KEYS: * β – heamolysis was noted in one (1) of the LS isolates only which showed a negative
VP test but was further confirmed in a not yet published pathogenicity and molecular studies of
the isolates.
Table 3: Correlation between incidence of Listeria species and silage pH
Total Number of Samples Range positive for Listeria (%)
Silage pH In this range (%) LS IS
LS (n: 50) IS (n: 50) (n: 50) (n: 50)
Below 4.00 0 0 0 0
4.00 --4.99 34 (17/50) 96 (48/50) 41 % (7/17) 27 (13/48)
5.00 – 5.99 40% (20/50) 0 (0) 25% (5/20) 0
Above 6.00 26% (13/50) 4 (2/50) 46% (6/13) 0
Total 100 (50) 100 (50) 18/50 13/50
Discussion and Conclusion
Discussion:
Silage is the most common feed used in Dairy farms. It has minimal loss of nutrients on
storage, serving to preserve the excess forage during wet conditions. Yet, it has been implicated
as one of the greatest forage related problems on a farm [3].
Physical, sensory characteristics and pH assessment, give insight into silage quality
[4]. Comparing the standard silage parameters requirements [ 9] against the odour;
colour, texture and pH assessment results, LS silage has poor silage quality - the number of
abnormal silage sensory parameters observed were higher than those with normal parameters.
However, the observed sensory parameters of IS silage were satisfactory [9] and imply that IS
silage is of optimal standard. .
The incidence of Listeria species in silage show 31 % (31/100) of Listeria isolates in
silage of the two Dairy Farms. Premised on the observed fermentation and, Listeria sensu strictu
and sensu lato clades features [14 and, 10 – 12], only 1% of these isolates were L.
monocytogenes (Listeria sensu strictu member).
Correlation analysis between the spread of Listeria in LS silage and pH range indicates
that an increase in silage pH range increased Listeria incidence in LS silage which is not
statistically significant (p > 0.05), r = 0.559. The correlation between the spread of Listeria in IS
silage and the pH range shows that an increase in silage pH range decreased Listeria incidence.
The observed correlation is moderately inverse r = – 0.241, without enough evidence to say that
this correlation exists in the population (since the p value( 0.759 ) > α value ( 0.05 ) ¿. These findings
dispute the assertion that properly ensiled forage have pH range between 3.7 and 4.7 (13). A pH
below 4.00 was vacant in this study. The results agree with other researchers that corn silage has
lower final pH range of 3.70 to 4.00 [14] and Listeria grows in silage samples with low pH
values. Hence there are no consensuses on the pH of silage in which Listeria are inhibited [15].
Conclusion
Based on:
i. The 31 % incidence of Listeria species in silage of the two Dairy farms in Plateau State
and,
ii. The correlation test showing no consensuses on the pH of silage in which Listeria are
inhibited,
• It was concluded that the Dairy farms studied have Listeriosis risk potentials.

ACKNOWLEDGEMENTS: We acknowledge: We acknowledge: The PG School, NDA

Kaduna; The Provost, FCAH&PT Vom, The Executive Director NVRI Vom and, Kieran Jordan
of Food Safety, Department, Teagasc Food Research Centre, Moorepark, Fermoy, County Cork,
Ireland.
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