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Distribution of salmonella contamination in ten animal feedmills
veterinary microbiology ELSEVIER
Veterinary Microbiology 5 1 (1997) 159- I69
Distribution of salmonella contamination in ten animal feedmills Robert H. Davies
*,
Clifford Wray
Bacteriology Department, Central Veterinary Laboratory, Woodham Lane, New Haw. Addlestone, Surrey. KT15 3NB, UK
Received 5 December 1996; accepted 20 March 1997
Abstract Detailed sampling of spillage and dust from milling equipment was carried out in nine animal feedmills, three of which were sampled twice. The salmonella isolation rate ranged from 1.1% to 41.7% of the samples and the most contaminated mills were those where the inside of the cooling systems for pellet or mash had been colonised by salmonella. A wide range of salmonella serotypes were isolated which included Salmonella typhimurium and S. enteritidis. Limited sampling every two weeks for an l&month period in another animal feedmill showed marked variation in the contamination rate of samples and range of salmonella serotypes found. Contamination of ingredient intake pits and outloading gantries for finished products by wild bird droppings containing salmonella was also found in four mills. Crown Copyright 0 1997 Published
by Elsevier Science B.V. Keywords: Salmonella spp.; Feeding and nutrition; Feedmills
1. Introduction Although animal feed has been recognised as an important vector of new salmonella infections for livestock farms (Shapcott, 1984; Blackman et al., 19921, there have been few recent published studies of salmonella contamination in animal feedmills. The results of monitoring bulked rations by sampling under voluntary Codes of Practice (MAFF) suggest that < 10% of finished rations are contaminated with Salmonella enteritidis and Salmonella typhimurium being infrequent isolates (Anon., 1996). A survey of poultry rations in The Netherlands in 1990/1991 found over 21% of poultry
* Corresponding author. 0378-I 135/97/$17.00 Crown Copyright 0 1997 Published by Elsevier Science B.V. PII s0378-1135(97)00114-4
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R.H. Davies, C. Wray/ Veterinary Microbiology 51 (1997) 159-169
meals to be contaminated with salmonella but no S. enteritidis or S. typhimurium was found despite a contemporaneous epidemic of S. enteritidis in poultry (Veldman et al., 1995). A similar situation was found in a recent survey in the USA where 16% of compound feeds were contaminated (Muirhead, 1995). Again, in a similar survey in Germany (Bisping, 1993), S. enteritidis and S. typhimurium were very rare. There are difficulties with monitoring animal feeds because of non-uniform distribution of contamination and the large size of loads (Jones and Ricke, 1994). The sensitivity of testing feed for salmonella depends on good sample site selection and the number of samples taken (McChesney, 1995). In Sweden, monitoring feed at key points during production rather than finished rations has increased the identification of process contamination and has identified many instances of unsuspected contamination of feed cooling systems (Malmqvist et al., 1995). This paper describes the identification of contamination points in animal feedmills by sampling feed spilled from specific storage areas and equipment and a comparison of the contamination rate of different feedmills by culturing large numbers of such samples.
2. Materials and methods Mills were selected for investigation on the basis of criteria described in Table 1. In mills where the investigation was prompted by identification of S. enteritidis or S. typhimurium in finished feeds, only one such isolate had been made during routine monitoring by the company laboratory many weeks prior to the sampling visit. Where possible mills were visited at 4:00-5:OO AM on a Monday after a weekend shutdown period so that the interior of coolers could be sampled, but in some cases (mills D and E), there was no shutdown period so not all coolers could be sampled internally. Approximately 25 g samples of fine spillage which had escaped from seals and seams in equipment and auger systems were collected in sterile plastic jars from the various areas listed in Table 2. Plastic disposable gloves were used for each sample. When spillages were sparse, a sterile hand-brush was used to gather the material. Samples of aggregated fatty material from within coolers were taken from the pellet or mash entry point in the roof of the cooler. Dust samples were also taken from beneath the lids of storage bins and from ledges around the ingredient intake pits and finished product
Table 1 Reasons for investigation
of animal feedmills
Reason for investigation
Mill code
Type of mill (species for which feed produced)
S. typhimurium in finished feed S. enteritidis in finished feed Frequent finished feed contamination No salmonella history
A B, H C D, E, F G. I, J
Mixed species Mixed species Poultry Mixed species
with other salmonella
serotypes
87/430(20.2) 6/12(50.0)
4/12(33.3)
O/38 oj40 O/46 O/58 l/24(4.2) a 18/24(75.0) a 24/28(85.7) a 22/36(61.1) a 20/28(71.4) = 2/28(7.1) O/44 O/36
Cooler
23/308(7.5)
Press
68/282(24.1) 81/637(12.7) 31/198(15.7Wl/348(11.8) Number of mills positive per sampling site 10/12(83.3) 11/12(91.7) 7/12(58.3) 10/12(83.3)
O/8 10/44(22.7) l/72(1.4)
O/8 8/2(X40.0) l/12(8.3) 10/24(41.7) l/20(5.0)
O/8 2/12(16.7)
2/12(16.7) Oj32 o/22 O/l6 7/12(58.3)
Mixer/ weigher o/20 Oj32 O/32 O/32 O/28 3/16(18.7) 4/16(25.0) O/8 12/20(60.0) O/l6 2/56(3.6) o/20
16/78(20.5) 3;68(4.4) 4/80(5.0) 4/71(5.6) 12/40(0.3) 21/52(40.4) 3/4(X7.5) 4/64(6.2) 3/20(15.0)
l/28(3.6) 4;36(11.1) O/l8 7/32(21.9) 12/16(75.0) 17/24(70.8) l/16(6.2) 9/28(32.1) 4/8(50.0) 4/8(50.0) 9/48(18.7) o/20
Grinder
of samples tested (%)
4/42(9.5) l/40(2.5) l/40(2.5) O/32 2/12(16.7) 4/24(16.7) 2/12(16.7) 10/20(50.0) 2/16(12.5) O/22 14/56(25.0) l/32(3.1)
Ingredient bins and augers
Intake pits and augers
Number of samples positive for salmonella/number
isolation from spillage samples from milling equipment
a Salmonella isolated from aggregate inside the coolers. I and 2 subscript = some mills visited on two occasions. NS = Not sampled as not present.
F G H I Total
E2
IA DZ E,
B C
A2
A,
Mill reference
Table 2 Salmonella
4/9(44.4)
8/ 12(66.7)
g/12(66.7)
O/28 Oj40 o/40 O/l2 NS NS g/14(57.1) 2/4(50.0) 3/16(18/7) 4/12(33.3) O/36 NS
Warehouse and bagging plant
22/210(10.5)17/202(8.4)
O/8 5/20(25.0) O/32
o/12 l/24(4.2) O/32 l/38(2.6) 4/12(33.3) 2/12(16.7) 7/12(58.3) l/4(25.0) l/4(25.0)
Outloading gantry
73/484(15.1)
O/32 l/44(2.3) O/52 o/44 9/40(22.5) 17/40(42.5) 21/52(40.4) g/32(25.0) 12/24(50.0) 2/32(6.2) 3/60(5.0) O/32
Finished product bins
23/290(7.9) 10/356(2.8) 5/362(1.4) 12/335(3.6) 47/184(25.5) 82/200(41 .O) 72/202(35.6) 80/204(39.2) 65/156(41.7) 13/134(9.7) 53/388(13.6) 3/264(1.1)
Total
:: ;: s L!
$$-
z
; $.
3 Y 3 z.
F .s Q @ K. 51 n
R.H. Dauies, C. Wray/ Veterinary Microbiology 51 (1997) 159-169
162
loading gantries. Wild bird droppings and rodent droppings were also collected when present. The samples were returned to the laboratory immediately after collection and added to 225 ml buffered peptone water (BPW, Oxoid: CM509). The samples were then incubated at 37°C for 16-18 h after which 0.2 ml of broth was inoculated into a 20-ml petri dish of semi-solid Rappaport (MSRV: LabM 150) containing 20 pg ml-’ Novobiocin which was incubated at 41.5”C for 48 h. Subcultures were streaked onto Rambach agar (Merck 7500) after 24- and 48-h incubation and the Rambach agar was incubated for 18-24 h at 37°C. Suspect salmonella colonies were confirmed by full serotyping. Phage typing of S. enteritidis and S. typhimurium isolates was carried out by the Central Public Health Laboratory, Colindale, UK.
3. Results The salmonella isolation results obtained from the various areas sampled in the mills are shown in Table 2. The intake pits were the most frequently contaminated sampling site (24.1% samples positive, range O-75.0%), but in two mills, no contamination was found. The second most frequently contaminated site was the cooler area (20.2% samples positive, range O-85.7%) and high levels of contamination were associated with salmonella isolation from aggregate inside the coolers indicative of cooler contamination problems. Those mills (D, E and F) which had cooler contamination problems also produced final heat-treated rations which were intermittently contaminated with the same salmonella serotypes that were found in the coolers over a long period of time. The contamination rate of pelleting press areas was low except in mill F where a low temperature ripening was used before conditioning or where there was a very high level of general contamination of the mills. With the exception of those mills (D, E and F) in which cooler contamination was present, the finished product areas were less contaminated than the ingredient processing areas, so there was a net reduction in contamination during processing in most mills. In some mills, salmonella was found in fresh wild bird droppings collected from intake pit areas, warehouses and outloading gantries (Table 3).
Table 3 Salmonella
dropping
samples in animal feedmills
Mill reference
No. of wild bird dropping samples taken
No. of wild bird dropping samples containing salmonella
Salmonella
A B
12 10
3 1
S. typhimurium DT129, S. typhimurium DT40 S. typhimurium DT104
C D E
12 1 4 12
0 0
H
isolation from wild bid
No wild bird droppings
1 5 found in mills F, G, I.
serotypes found
S. ohio S. kentucky, S. typhimurium DT99
S. tj~phimurwm
SClO-
found
S mbundrrla
S. ohio
S enrerirrdrs
PT4
t irchow s. newporr
S. rrbrrdeert
S herdelbcrg s. grr’e
s. infun tis S. thompson
S rndrnno
S. brundenberg
S. braenderap
s. ,cnnc.wee
S. ornnienhcrx
DT46
S. rvphimurism
S /okson>
S
s. ngnma
S. schwrrr;enRrrr,rd
DT I60
S. mbandaka S. cubana
S. dr.vppool
S. friednau
S. agono
S. smnftenbrrg S. 4,lZ:b. -
S. hal,ana
S. kedoaxor,
DT99
DT8
S ,aLx
DT208
S. gphlmurium
S. ryphimuriam
S alachua
S okarie
S. idikan
s. rr,&lnu
s. coeln
S. monrecideo
S. t\phiml~riam
S. senf~enberg
H
S monre~~rdeo S. kentuckv
S. ohio
s. WnHeSSeP
G
S ohio
S monrrt,idrtr
S. odeloidr
DT40
S. typhrmurium
DTI I
DT49A
S. 4,12:d: -
S. cubano
DT99 DTS
S lennence
DT I9 I
S. kedoqou
S. ohm
F
S rvphimurircm
2nd visit
s. senfrrnher#
DT I29
S. typhimurrum
S. enter,rtd,.r
S. enreriridls
S. monfe~ idea
S. cubono
2nd viw
S. ryphimurium
S. ryphimurrum 2nd v,\,,
S indiono
S. rvphimurtum
S. tvphrmurium
S. 4.12:d:m
PT6
S. monrr~~idro S. ohio
S. 4,lZ:d:-
S. cubonu
S. bergen
S. ohio
S. monte~ rdeo
S. oronienberg
S. rermrsee
RDNC
DT193
131 visit
E
S. indiona
S. mbondakn
S. fyphrmariam
DTl04L
DT193
iat VlS,,
D
s. xhwar;en~rund
S. i,phimrcrium
S. r~phimarium
C
s. cerro
DTJO
DT I29 S. lyphimurrum
0
S. lexington
S. mbonduku
s. ,enneSSee
S ryphtmurircm
nella
types
1st visit
Salmo-
A
Salmonella serotypes and phage types found in mne ammal feedmills
Table 4
S. berm
S. qiobo
S. typhimertam
I
DT IOJL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2.1
Sampling period
spillage
1 (S. 0 0 1 (S. 0 0 1 LS. 0 0 1 (S. 1 (S.
0
goldcoast)
goldcoast)
goldcoast)
goldcoast)
8,2O:i: - )
2 Ls. indiana) 0 1 (S. 16:-:-j 0 0 2 (S. goldcoast)
0
0
Intake pit spillage
sub-samples
sampling
Number of positive
Table 5 Mill J-fortnightly
from various
1 (S. 0 0 1 (S. 1 (S. 1 (S. 0 0 1 (S. 0 0 0 0 0 1 (S. 0 0 0 2 (S. 1 (S. 0
kentucky/S. mbandaka)
infantis)
CiKh0W)
16:-:-j
newbrunswick)
indiana)
tentlessee)
Mixer/weigher blending bin area spillage
cubana)
sampling
0
0 0 0
0 0 0
0 0 0 0
0 0 I (S.
0 0 0
2 ts. agonlz) 1 (S. goldconst) 0 0
1 (S. kentuck?;) 0 0
tees)
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0 0 0 0 0 1 (S. uirchmv)
0
Cooler aggregate
0 0 0 1 (S. mbandaka) 1 (S. oranienberg) 0 0 0
0
Press spillage
serotype)
1 (S. mbnndaka) 2 (S. idikan) 0 0 1 (S. h/k) 0 0 0
0
Grinder spillage
sites (Salmonella
0 2 tS. ~irchow, S. lic~ingstonr) 0 0 2 tS. agona. S. goldcoast) 0 0 0 0 0 0 0 0 0 0 0 0
0
0
0
Bagger spillage
l/l2 3/12 3/12 2/12 6/12 o/12 o/12 6/12 o/12 l/l2 o/12 o/12 l/l2 3/12 l/l2 l/l2 o/12 3/12 2/12 2/12
-//I2--
I
Totals
40 41 42
25 26 27 28 29 30 31 32 33 34 35 36 31 38 39
22 23 24
mbandoka) typhimurium ~i~ingstone)
goldcoasr)
DT104)
DT104L,
agama)
0
0 II/84 (13.1)
2 (S. Ohio/S. 26/84 (30.9)
0
1 (S. ~phimuritm 0 1 (S. rennessee) 0 0 0 0 0 0 0
0
0
0
0
0
0
0
0
0
cubana)
2 (S. typhimurium S. idikan)
I (S. indiana) 0 2 (S. goldcoast/S. 0 0
1 (S. enteritidis PT4) 2 (S. tennessee)
2 (S. 0 0 I (S. 1 (S. 1 (S. 0 DT99)
0
(4.8)
6/84 (7.1)
14,‘84 (16.7)
(7.1)
6/84
0
0
0 4/84
0
I (S. morrte~idro) 0 0
1 (S. t~irchnw) 0 0 0 0 0 0 0 0 0 _
0
0
0
0
0 0 0
2 (S. tees/S. derby) I (S. liuingstone) 0 0 0 0 0 0 0 _
0
0
0
0
0 0
0
0
0
mbandaka)
derby)
li~~ingstone) mbandaka)
1 (S. L,irchow) 0 0 0 0 0 0 0 0 0 0 0 0 0 I (S. melengridis) 1 (S. fetmssee) 0 0
0
0
0
0 0 0 0 2 (S. 1 (S. 0 1 (S. 0 0 0 0 0 0 0 2 (S. 0 0
o/12 2/12 65/504 (12.9%)
O/12
2/12
4/12 o/12
2/12 l/12
2 2 s 2
2 a% 3
z % _,
3 2 ‘L.
<
s
0
t. _?
2/12 l/12 l/12 o/12
b
2/12
3/12 o/12 o/12 l/12 4/12 4/12 l/12 l/12
166
R.H. Davies, C. Wray/
Veterinary Microbiology
51 (1997) 159-169
No salmonella was found in mouse or rat droppings collected from the mills. A very low overall prevalence of salmonella contamination was found in two of the mills (B and I). A wide range of salmonella serotypes was found in some of the mills, particularly mill H where 18 different serotypes were found (Table 4). This was also the mill with the highest level of contamination apart from mills D, E and F (in which cooler contamination was present), which generally presented with a more restricted range of serotypes, especially in finished product areas. S. typhimurium was found in six of the nine mills but the multiresistant strain of S. typhimurium DT104L was only found in three of these. S. enteritidis was found in three of the nine mills and only one of these isolates was phage type 4. The results of fortnightly monitoring of a limited number of sites in one feed mill (J) for 84 weeks are shown in Table 5. Twenty-six different serotypes (including those with incomplete antigenic structures) were isolated. Mill J was notable for frequent isolation of S. goldcoast and S. virchow. S. enteritidis was isolated on one occasion and S. typhimurium on three occasions. Salmonella isolates were occasionally made from inside the coolers but there was no persistent contamination by specific serotypes.
4. Discussion Salmonella infection in chicks via contaminated feed can be established with as little as one organism (Milner and Shaffer, 1952) and other factors such as age, production, nutritional and environmental stress, intercurrent disease and the fat content of feed may also be involved (Jones et al., 1982; Lax et al., 1995). There is also a marked serotype and strain variation in infectivity with birds acting as biological filters for the more virulent strains in feed, which may not necessarily be the most prevalent strains isolated during routine monitoring (Jones and Richardson, 1996). Acid treatment of feed has been shown to reduce flock infection in chickens (Shapcott, 1984; Humphrey and Lanning, 1988) but the predominant serotypes found in acidified feed from mills A, D and J have also been found in poultry flocks supplied with their feed (Davies, unpublished observation). Monitoring individual rations for salmonella is an inefficient process because of the need to sample large volumes of material with a heterogenous distribution of contamination (McChesney, 1995), therefore, a means of assessing contamination of the milling process is required (Malmqvist et al., 1995). This is particularly true of cereal ingredients which form the largest part of all rations and are considered a low risk ingredient as whole grains are not a natural reservoir for salmonella compared with finely processed ingredients such as oil seed residue meals (McChesney, 1995). Cross-contamination of grains with faeces of rodents, wild birds and cats containing S. typhimurium DT104L has been found frequently on cattle and pig farms where grain stores are close to infected livestock housing (unpublished data). An improved method of indirectly sampling grains as well as a risk assessment approach to grain storage is, therefore, required. As contaminated dust tends to settle locally (Schmidt and Hoy, 1996), sampling spillage samples from specific areas in the mill can provide an indication of the nature
R.H. Da&s.
C. Wray/
Veterinary Microbiology
51 (1997) 159-169
16’1
and extent of contamination problems. A high contamination rate with a wide range of salmonella serotypes in ingredient areas suggests poor ingredient selection, whereas, a high contamination rate of ingredient spillage collected after storage, but not before, suggests persistent contamination of storage bins. Contamination in spillages around the coolers suggests intrinsic contamination of the fatty aggregate which builds up at the entry point of the cooler. This can be confirmed by taking samples from inside the empty cooler, which requires the sampler to climb up inside the cooler to reach the feed entry point where the most contaminated aggregate develops. Samples of rations taken before and after cooling during the first five minutes production after an overnight shutdown period can also be used to demonstrate cooler contamination. Collection and culture of wild bird droppings by feed companies can also be used to identify the dangers of contamination of intake pits, outloading gantries and warehouses by birds and prompt more decisive action on spilled feed clearance, bird-proofing and deterrents. As well as identifying specific contamination hazards isolation of salmonella from spillage, samples collected from defined sites in the mill is a convenient way of comparing the relative contamination risks associated with a particular mill. The isolation rate may vary with time according to the level of contamination of ingredients which have recently been processed or seasonally with increases in contamination due to higher atmospheric humidity in spring and early winter (Kiilher. 1993). To take account of this, repeat sampling (as in mill J where the frequent isolation of S. uirchow and S. goldcoast, which are otherwise uncommon in feed, was noteworthy) using limited numbers of samples is desirable for ongoing monitoring of a mill. A single independent investigation in which large numbers of samples are taken should also give a good indication of the risks associated with a particular mill. This latter approach may be useful for a customer which wishes to independently evaluate the salmonella status of a potential feed supplier. Although few isolates of S. enteritidis and S. typhimurium were found during this study, their presence did indicate that ingredients contaminated by them had been used. Many mills use heat treatment for broiler rations. It is suggested that temperatures of 80°C for at least one minute are likely to be successful but there is no clear guidance of effective conditioning temperatures and times in current mill equipment where very short conditioning times are usual (McIlroy et al., 1989; Voeten and van de Leest, 1989). S. typhimurium habituated to highly dehydrated conditions has been shown to survive 60 min at 100°C (Kirby and Davies, 19901. Even when feeds are effectively heat-treated, there is still a risk of recontamination in coolers. This is a particular problem in mills where ruminant rations conditioned at relatively low temperatures share the same lines as poultry feeds, although specialist poultry mills may also be affected if salmonella survives conditioning in the first half hour before full conditioning temperatures are reached following a shutdown period and becomes established in the cooling system. Many poultry breeder and layer rations are treated with organic acid compounds in an attempt to reduce salmonella contamination and this may be helpful (Humphrey and Lanning, 1988) but these may be less effective under humid storage conditions than formaldehyde, which has recently been licensed for use in finished feeds in the USA
168
R.H. Dauies, C. Wray/
Veterinary Microbiology
51 (1997) 159-169
(Smyser and Snoeyenbos, 1979; Brown, 1996). There is a danger, however, that blanket application of anti-salmonella treatments may encourage poor ingredient selection and less stringent application of other salmonella control measures. If the level of contamination rises because of this, it may be too much for the activity of the treatment or batches of highly contaminated feed may escape treatment when application of the antibacterial fails because of blockages or faulty metering devises.
Acknowledgements This work was funded by the Ministry of Agriculture, Fisheries and Food. The authors are grateful to Mrs. S. Bedford and Mrs. K. McIntosh for technical assistance, to colleagues in the Animal Health and Welfare Group and the State Veterinary Service for help with arranging study sites and to the various feed companies involved for permission to take the samples.
References Anon., 1996. Salmonella in animal feedingstuffs and ingredients, 1995 (Jan.-Dec.) Animal Health (Disease Control). Division Report. MAFF, Tolworth, UK. Bisping, W., 1993. Salmonella in feedstuffs. Dtsch. Tiersrztl. Wschr. 100, 262-263. Blackman, J., Bowman, T., Chambers, J., Kisilenks, J., Parr, J., St. Laurent, A.M., Thompson, J., 1992. Controlling salmonella in livestock and poultry feeds. Report of Agriculture Canada and Canadian Feed Associates. Brown, R.H., 1996. FDA approves the use of formaldehyde in poultry feed. Feedstuffs 68, 15. Humphrey, T.J., Lanning, D.G., 1988. The vertical transmission of salmonellas and formic acid treatment of chicken feed. Epidemiol. Infect. 100, 43-49. Jones, F.T., Richardson, K.E., 1996. Fallacies exist in current understudy of salmonella. Feedstuffs 68, 22-25. Jones, F.T., Ricke, S.C., 1994. Researchers propose tentative HACCP plan for feed mills. Feedstuffs 66, 35-42. Jones, P.W., Collins, P., Brown, G.T.H., Aitken, M., 1982. Transmission of Salmonella mbandaka to cattle from contaminated feed. J. Hygiene 88, 255-263. Kirby, R.M., Davies, R., 1990. Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures. J. Appl. Bacterial. 68, 241-246. Kijlher, B., 1993. Demonstration of dissemination and enrichment of salmonella in the environment. Dtsch. Tieitiztl. Wschr. 100, 264-274. Lax, A.J., Barrow, P.A., Jones, P.W., Wallis, T.S., 1995. Current perspectives in salmonella% Br. Vet. J. 151, 351-377. Malmqvist, M., Jacobsson, K.G., Higgblom, P., Cerenius, F., Sjiiland, L., Gunnarsson, A., 1995. Salmonella isolated from animals and feedstuffs in Sweden during 1988-1992. Acta Vet. Stand. 36, 21-39. McChesney, D.G., 1995. FDA survey results: salmonella contamination of finished feed and the primary meal ingredient. Proceedings of the 99th Annual Meeting of the United States Animal Health Association, Nov. 2, 1995, Reno, Nevada (in press). McIlroy, S.G., McCracken, R.M., Neill, S.D., O’Brien, J.J., 1989. Control, prevention and eradication of Salmonella enteritidis infection in broilers and broiler breeder flocks. Vet. Rec. 125, 545-548. Milner, K.C., Shaffer, M.F., 1952. Bacteriologic studies of experimental salmonella infections in chicks. I. Infect. Dis. 90, 8186-8189. Muirhead, S., 1995. FDA survey shows low salmonella level in feed. Feedstuffs 67, 1-5.
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Schmidt, R., Hoy, S.E., 1996. Investigations on dust emission from chicken and layer houses. Berlin Mllnch. Tiertiztl. Wschr. 109, 95-100. Shapcott, R-C., 1984. Practical aspects of salmonella control. Progress report on a programme in a large broiler integration. In: Snocyenbos, G.H. (Ed.), Proceedings of the International Symposium on salmonella. American Association of Avian Pathology, Kennett Square, PA, USA, pp. 109-l 14. Smyser, CF., Snoeyenbos, G.H., 1979. Evaluation of organic acids and other compounds as salmonella antagonists in meat and bone meal. Poultry Sci. 58, 50-54. Veldman. A., Vahl, H.A., Borggreve, G.J., Fuller, D.C., 1995. A survey of the incidence of salmonella species and enterobacteriaceae in poultry feeds and feed components. Vet. Rec. 136, 169-172. Voeten, A.C., van de Leest, L., 1989. Influence of the pelleting temperature used for feed on salmonella infection in broilers. Arch. Gefhigelk. 53, 225-230.
Veterinary Microbiology 5 1 (1997) 159- I69
Distribution of salmonella contamination in ten animal feedmills Robert H. Davies
*,
Clifford Wray
Bacteriology Department, Central Veterinary Laboratory, Woodham Lane, New Haw. Addlestone, Surrey. KT15 3NB, UK
Received 5 December 1996; accepted 20 March 1997
Abstract Detailed sampling of spillage and dust from milling equipment was carried out in nine animal feedmills, three of which were sampled twice. The salmonella isolation rate ranged from 1.1% to 41.7% of the samples and the most contaminated mills were those where the inside of the cooling systems for pellet or mash had been colonised by salmonella. A wide range of salmonella serotypes were isolated which included Salmonella typhimurium and S. enteritidis. Limited sampling every two weeks for an l&month period in another animal feedmill showed marked variation in the contamination rate of samples and range of salmonella serotypes found. Contamination of ingredient intake pits and outloading gantries for finished products by wild bird droppings containing salmonella was also found in four mills. Crown Copyright 0 1997 Published
by Elsevier Science B.V. Keywords: Salmonella spp.; Feeding and nutrition; Feedmills
1. Introduction Although animal feed has been recognised as an important vector of new salmonella infections for livestock farms (Shapcott, 1984; Blackman et al., 19921, there have been few recent published studies of salmonella contamination in animal feedmills. The results of monitoring bulked rations by sampling under voluntary Codes of Practice (MAFF) suggest that < 10% of finished rations are contaminated with Salmonella enteritidis and Salmonella typhimurium being infrequent isolates (Anon., 1996). A survey of poultry rations in The Netherlands in 1990/1991 found over 21% of poultry
* Corresponding author. 0378-I 135/97/$17.00 Crown Copyright 0 1997 Published by Elsevier Science B.V. PII s0378-1135(97)00114-4
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R.H. Davies, C. Wray/ Veterinary Microbiology 51 (1997) 159-169
meals to be contaminated with salmonella but no S. enteritidis or S. typhimurium was found despite a contemporaneous epidemic of S. enteritidis in poultry (Veldman et al., 1995). A similar situation was found in a recent survey in the USA where 16% of compound feeds were contaminated (Muirhead, 1995). Again, in a similar survey in Germany (Bisping, 1993), S. enteritidis and S. typhimurium were very rare. There are difficulties with monitoring animal feeds because of non-uniform distribution of contamination and the large size of loads (Jones and Ricke, 1994). The sensitivity of testing feed for salmonella depends on good sample site selection and the number of samples taken (McChesney, 1995). In Sweden, monitoring feed at key points during production rather than finished rations has increased the identification of process contamination and has identified many instances of unsuspected contamination of feed cooling systems (Malmqvist et al., 1995). This paper describes the identification of contamination points in animal feedmills by sampling feed spilled from specific storage areas and equipment and a comparison of the contamination rate of different feedmills by culturing large numbers of such samples.
2. Materials and methods Mills were selected for investigation on the basis of criteria described in Table 1. In mills where the investigation was prompted by identification of S. enteritidis or S. typhimurium in finished feeds, only one such isolate had been made during routine monitoring by the company laboratory many weeks prior to the sampling visit. Where possible mills were visited at 4:00-5:OO AM on a Monday after a weekend shutdown period so that the interior of coolers could be sampled, but in some cases (mills D and E), there was no shutdown period so not all coolers could be sampled internally. Approximately 25 g samples of fine spillage which had escaped from seals and seams in equipment and auger systems were collected in sterile plastic jars from the various areas listed in Table 2. Plastic disposable gloves were used for each sample. When spillages were sparse, a sterile hand-brush was used to gather the material. Samples of aggregated fatty material from within coolers were taken from the pellet or mash entry point in the roof of the cooler. Dust samples were also taken from beneath the lids of storage bins and from ledges around the ingredient intake pits and finished product
Table 1 Reasons for investigation
of animal feedmills
Reason for investigation
Mill code
Type of mill (species for which feed produced)
S. typhimurium in finished feed S. enteritidis in finished feed Frequent finished feed contamination No salmonella history
A B, H C D, E, F G. I, J
Mixed species Mixed species Poultry Mixed species
with other salmonella
serotypes
87/430(20.2) 6/12(50.0)
4/12(33.3)
O/38 oj40 O/46 O/58 l/24(4.2) a 18/24(75.0) a 24/28(85.7) a 22/36(61.1) a 20/28(71.4) = 2/28(7.1) O/44 O/36
Cooler
23/308(7.5)
Press
68/282(24.1) 81/637(12.7) 31/198(15.7Wl/348(11.8) Number of mills positive per sampling site 10/12(83.3) 11/12(91.7) 7/12(58.3) 10/12(83.3)
O/8 10/44(22.7) l/72(1.4)
O/8 8/2(X40.0) l/12(8.3) 10/24(41.7) l/20(5.0)
O/8 2/12(16.7)
2/12(16.7) Oj32 o/22 O/l6 7/12(58.3)
Mixer/ weigher o/20 Oj32 O/32 O/32 O/28 3/16(18.7) 4/16(25.0) O/8 12/20(60.0) O/l6 2/56(3.6) o/20
16/78(20.5) 3;68(4.4) 4/80(5.0) 4/71(5.6) 12/40(0.3) 21/52(40.4) 3/4(X7.5) 4/64(6.2) 3/20(15.0)
l/28(3.6) 4;36(11.1) O/l8 7/32(21.9) 12/16(75.0) 17/24(70.8) l/16(6.2) 9/28(32.1) 4/8(50.0) 4/8(50.0) 9/48(18.7) o/20
Grinder
of samples tested (%)
4/42(9.5) l/40(2.5) l/40(2.5) O/32 2/12(16.7) 4/24(16.7) 2/12(16.7) 10/20(50.0) 2/16(12.5) O/22 14/56(25.0) l/32(3.1)
Ingredient bins and augers
Intake pits and augers
Number of samples positive for salmonella/number
isolation from spillage samples from milling equipment
a Salmonella isolated from aggregate inside the coolers. I and 2 subscript = some mills visited on two occasions. NS = Not sampled as not present.
F G H I Total
E2
IA DZ E,
B C
A2
A,
Mill reference
Table 2 Salmonella
4/9(44.4)
8/ 12(66.7)
g/12(66.7)
O/28 Oj40 o/40 O/l2 NS NS g/14(57.1) 2/4(50.0) 3/16(18/7) 4/12(33.3) O/36 NS
Warehouse and bagging plant
22/210(10.5)17/202(8.4)
O/8 5/20(25.0) O/32
o/12 l/24(4.2) O/32 l/38(2.6) 4/12(33.3) 2/12(16.7) 7/12(58.3) l/4(25.0) l/4(25.0)
Outloading gantry
73/484(15.1)
O/32 l/44(2.3) O/52 o/44 9/40(22.5) 17/40(42.5) 21/52(40.4) g/32(25.0) 12/24(50.0) 2/32(6.2) 3/60(5.0) O/32
Finished product bins
23/290(7.9) 10/356(2.8) 5/362(1.4) 12/335(3.6) 47/184(25.5) 82/200(41 .O) 72/202(35.6) 80/204(39.2) 65/156(41.7) 13/134(9.7) 53/388(13.6) 3/264(1.1)
Total
:: ;: s L!
$$-
z
; $.
3 Y 3 z.
F .s Q @ K. 51 n
R.H. Dauies, C. Wray/ Veterinary Microbiology 51 (1997) 159-169
162
loading gantries. Wild bird droppings and rodent droppings were also collected when present. The samples were returned to the laboratory immediately after collection and added to 225 ml buffered peptone water (BPW, Oxoid: CM509). The samples were then incubated at 37°C for 16-18 h after which 0.2 ml of broth was inoculated into a 20-ml petri dish of semi-solid Rappaport (MSRV: LabM 150) containing 20 pg ml-’ Novobiocin which was incubated at 41.5”C for 48 h. Subcultures were streaked onto Rambach agar (Merck 7500) after 24- and 48-h incubation and the Rambach agar was incubated for 18-24 h at 37°C. Suspect salmonella colonies were confirmed by full serotyping. Phage typing of S. enteritidis and S. typhimurium isolates was carried out by the Central Public Health Laboratory, Colindale, UK.
3. Results The salmonella isolation results obtained from the various areas sampled in the mills are shown in Table 2. The intake pits were the most frequently contaminated sampling site (24.1% samples positive, range O-75.0%), but in two mills, no contamination was found. The second most frequently contaminated site was the cooler area (20.2% samples positive, range O-85.7%) and high levels of contamination were associated with salmonella isolation from aggregate inside the coolers indicative of cooler contamination problems. Those mills (D, E and F) which had cooler contamination problems also produced final heat-treated rations which were intermittently contaminated with the same salmonella serotypes that were found in the coolers over a long period of time. The contamination rate of pelleting press areas was low except in mill F where a low temperature ripening was used before conditioning or where there was a very high level of general contamination of the mills. With the exception of those mills (D, E and F) in which cooler contamination was present, the finished product areas were less contaminated than the ingredient processing areas, so there was a net reduction in contamination during processing in most mills. In some mills, salmonella was found in fresh wild bird droppings collected from intake pit areas, warehouses and outloading gantries (Table 3).
Table 3 Salmonella
dropping
samples in animal feedmills
Mill reference
No. of wild bird dropping samples taken
No. of wild bird dropping samples containing salmonella
Salmonella
A B
12 10
3 1
S. typhimurium DT129, S. typhimurium DT40 S. typhimurium DT104
C D E
12 1 4 12
0 0
H
isolation from wild bid
No wild bird droppings
1 5 found in mills F, G, I.
serotypes found
S. ohio S. kentucky, S. typhimurium DT99
S. tj~phimurwm
SClO-
found
S mbundrrla
S. ohio
S enrerirrdrs
PT4
t irchow s. newporr
S. rrbrrdeert
S herdelbcrg s. grr’e
s. infun tis S. thompson
S rndrnno
S. brundenberg
S. braenderap
s. ,cnnc.wee
S. ornnienhcrx
DT46
S. rvphimurism
S /okson>
S
s. ngnma
S. schwrrr;enRrrr,rd
DT I60
S. mbandaka S. cubana
S. dr.vppool
S. friednau
S. agono
S. smnftenbrrg S. 4,lZ:b. -
S. hal,ana
S. kedoaxor,
DT99
DT8
S ,aLx
DT208
S. gphlmurium
S. ryphimuriam
S alachua
S okarie
S. idikan
s. rr,&lnu
s. coeln
S. monrecideo
S. t\phiml~riam
S. senf~enberg
H
S monre~~rdeo S. kentuckv
S. ohio
s. WnHeSSeP
G
S ohio
S monrrt,idrtr
S. odeloidr
DT40
S. typhrmurium
DTI I
DT49A
S. 4,12:d: -
S. cubano
DT99 DTS
S lennence
DT I9 I
S. kedoqou
S. ohm
F
S rvphimurircm
2nd visit
s. senfrrnher#
DT I29
S. typhimurrum
S. enter,rtd,.r
S. enreriridls
S. monfe~ idea
S. cubono
2nd viw
S. ryphimurium
S. ryphimurrum 2nd v,\,,
S indiono
S. rvphimurtum
S. tvphrmurium
S. 4.12:d:m
PT6
S. monrr~~idro S. ohio
S. 4,lZ:d:-
S. cubonu
S. bergen
S. ohio
S. monte~ rdeo
S. oronienberg
S. rermrsee
RDNC
DT193
131 visit
E
S. indiona
S. mbondakn
S. fyphrmariam
DTl04L
DT193
iat VlS,,
D
s. xhwar;en~rund
S. i,phimrcrium
S. r~phimarium
C
s. cerro
DTJO
DT I29 S. lyphimurrum
0
S. lexington
S. mbonduku
s. ,enneSSee
S ryphtmurircm
nella
types
1st visit
Salmo-
A
Salmonella serotypes and phage types found in mne ammal feedmills
Table 4
S. berm
S. qiobo
S. typhimertam
I
DT IOJL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2.1
Sampling period
spillage
1 (S. 0 0 1 (S. 0 0 1 LS. 0 0 1 (S. 1 (S.
0
goldcoast)
goldcoast)
goldcoast)
goldcoast)
8,2O:i: - )
2 Ls. indiana) 0 1 (S. 16:-:-j 0 0 2 (S. goldcoast)
0
0
Intake pit spillage
sub-samples
sampling
Number of positive
Table 5 Mill J-fortnightly
from various
1 (S. 0 0 1 (S. 1 (S. 1 (S. 0 0 1 (S. 0 0 0 0 0 1 (S. 0 0 0 2 (S. 1 (S. 0
kentucky/S. mbandaka)
infantis)
CiKh0W)
16:-:-j
newbrunswick)
indiana)
tentlessee)
Mixer/weigher blending bin area spillage
cubana)
sampling
0
0 0 0
0 0 0
0 0 0 0
0 0 I (S.
0 0 0
2 ts. agonlz) 1 (S. goldconst) 0 0
1 (S. kentuck?;) 0 0
tees)
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0 0 0 0 0 1 (S. uirchmv)
0
Cooler aggregate
0 0 0 1 (S. mbandaka) 1 (S. oranienberg) 0 0 0
0
Press spillage
serotype)
1 (S. mbnndaka) 2 (S. idikan) 0 0 1 (S. h/k) 0 0 0
0
Grinder spillage
sites (Salmonella
0 2 tS. ~irchow, S. lic~ingstonr) 0 0 2 tS. agona. S. goldcoast) 0 0 0 0 0 0 0 0 0 0 0 0
0
0
0
Bagger spillage
l/l2 3/12 3/12 2/12 6/12 o/12 o/12 6/12 o/12 l/l2 o/12 o/12 l/l2 3/12 l/l2 l/l2 o/12 3/12 2/12 2/12
-//I2--
I
Totals
40 41 42
25 26 27 28 29 30 31 32 33 34 35 36 31 38 39
22 23 24
mbandoka) typhimurium ~i~ingstone)
goldcoasr)
DT104)
DT104L,
agama)
0
0 II/84 (13.1)
2 (S. Ohio/S. 26/84 (30.9)
0
1 (S. ~phimuritm 0 1 (S. rennessee) 0 0 0 0 0 0 0
0
0
0
0
0
0
0
0
0
cubana)
2 (S. typhimurium S. idikan)
I (S. indiana) 0 2 (S. goldcoast/S. 0 0
1 (S. enteritidis PT4) 2 (S. tennessee)
2 (S. 0 0 I (S. 1 (S. 1 (S. 0 DT99)
0
(4.8)
6/84 (7.1)
14,‘84 (16.7)
(7.1)
6/84
0
0
0 4/84
0
I (S. morrte~idro) 0 0
1 (S. t~irchnw) 0 0 0 0 0 0 0 0 0 _
0
0
0
0
0 0 0
2 (S. tees/S. derby) I (S. liuingstone) 0 0 0 0 0 0 0 _
0
0
0
0
0 0
0
0
0
mbandaka)
derby)
li~~ingstone) mbandaka)
1 (S. L,irchow) 0 0 0 0 0 0 0 0 0 0 0 0 0 I (S. melengridis) 1 (S. fetmssee) 0 0
0
0
0
0 0 0 0 2 (S. 1 (S. 0 1 (S. 0 0 0 0 0 0 0 2 (S. 0 0
o/12 2/12 65/504 (12.9%)
O/12
2/12
4/12 o/12
2/12 l/12
2 2 s 2
2 a% 3
z % _,
3 2 ‘L.
<
s
0
t. _?
2/12 l/12 l/12 o/12
b
2/12
3/12 o/12 o/12 l/12 4/12 4/12 l/12 l/12
166
R.H. Davies, C. Wray/
Veterinary Microbiology
51 (1997) 159-169
No salmonella was found in mouse or rat droppings collected from the mills. A very low overall prevalence of salmonella contamination was found in two of the mills (B and I). A wide range of salmonella serotypes was found in some of the mills, particularly mill H where 18 different serotypes were found (Table 4). This was also the mill with the highest level of contamination apart from mills D, E and F (in which cooler contamination was present), which generally presented with a more restricted range of serotypes, especially in finished product areas. S. typhimurium was found in six of the nine mills but the multiresistant strain of S. typhimurium DT104L was only found in three of these. S. enteritidis was found in three of the nine mills and only one of these isolates was phage type 4. The results of fortnightly monitoring of a limited number of sites in one feed mill (J) for 84 weeks are shown in Table 5. Twenty-six different serotypes (including those with incomplete antigenic structures) were isolated. Mill J was notable for frequent isolation of S. goldcoast and S. virchow. S. enteritidis was isolated on one occasion and S. typhimurium on three occasions. Salmonella isolates were occasionally made from inside the coolers but there was no persistent contamination by specific serotypes.
4. Discussion Salmonella infection in chicks via contaminated feed can be established with as little as one organism (Milner and Shaffer, 1952) and other factors such as age, production, nutritional and environmental stress, intercurrent disease and the fat content of feed may also be involved (Jones et al., 1982; Lax et al., 1995). There is also a marked serotype and strain variation in infectivity with birds acting as biological filters for the more virulent strains in feed, which may not necessarily be the most prevalent strains isolated during routine monitoring (Jones and Richardson, 1996). Acid treatment of feed has been shown to reduce flock infection in chickens (Shapcott, 1984; Humphrey and Lanning, 1988) but the predominant serotypes found in acidified feed from mills A, D and J have also been found in poultry flocks supplied with their feed (Davies, unpublished observation). Monitoring individual rations for salmonella is an inefficient process because of the need to sample large volumes of material with a heterogenous distribution of contamination (McChesney, 1995), therefore, a means of assessing contamination of the milling process is required (Malmqvist et al., 1995). This is particularly true of cereal ingredients which form the largest part of all rations and are considered a low risk ingredient as whole grains are not a natural reservoir for salmonella compared with finely processed ingredients such as oil seed residue meals (McChesney, 1995). Cross-contamination of grains with faeces of rodents, wild birds and cats containing S. typhimurium DT104L has been found frequently on cattle and pig farms where grain stores are close to infected livestock housing (unpublished data). An improved method of indirectly sampling grains as well as a risk assessment approach to grain storage is, therefore, required. As contaminated dust tends to settle locally (Schmidt and Hoy, 1996), sampling spillage samples from specific areas in the mill can provide an indication of the nature
R.H. Da&s.
C. Wray/
Veterinary Microbiology
51 (1997) 159-169
16’1
and extent of contamination problems. A high contamination rate with a wide range of salmonella serotypes in ingredient areas suggests poor ingredient selection, whereas, a high contamination rate of ingredient spillage collected after storage, but not before, suggests persistent contamination of storage bins. Contamination in spillages around the coolers suggests intrinsic contamination of the fatty aggregate which builds up at the entry point of the cooler. This can be confirmed by taking samples from inside the empty cooler, which requires the sampler to climb up inside the cooler to reach the feed entry point where the most contaminated aggregate develops. Samples of rations taken before and after cooling during the first five minutes production after an overnight shutdown period can also be used to demonstrate cooler contamination. Collection and culture of wild bird droppings by feed companies can also be used to identify the dangers of contamination of intake pits, outloading gantries and warehouses by birds and prompt more decisive action on spilled feed clearance, bird-proofing and deterrents. As well as identifying specific contamination hazards isolation of salmonella from spillage, samples collected from defined sites in the mill is a convenient way of comparing the relative contamination risks associated with a particular mill. The isolation rate may vary with time according to the level of contamination of ingredients which have recently been processed or seasonally with increases in contamination due to higher atmospheric humidity in spring and early winter (Kiilher. 1993). To take account of this, repeat sampling (as in mill J where the frequent isolation of S. uirchow and S. goldcoast, which are otherwise uncommon in feed, was noteworthy) using limited numbers of samples is desirable for ongoing monitoring of a mill. A single independent investigation in which large numbers of samples are taken should also give a good indication of the risks associated with a particular mill. This latter approach may be useful for a customer which wishes to independently evaluate the salmonella status of a potential feed supplier. Although few isolates of S. enteritidis and S. typhimurium were found during this study, their presence did indicate that ingredients contaminated by them had been used. Many mills use heat treatment for broiler rations. It is suggested that temperatures of 80°C for at least one minute are likely to be successful but there is no clear guidance of effective conditioning temperatures and times in current mill equipment where very short conditioning times are usual (McIlroy et al., 1989; Voeten and van de Leest, 1989). S. typhimurium habituated to highly dehydrated conditions has been shown to survive 60 min at 100°C (Kirby and Davies, 19901. Even when feeds are effectively heat-treated, there is still a risk of recontamination in coolers. This is a particular problem in mills where ruminant rations conditioned at relatively low temperatures share the same lines as poultry feeds, although specialist poultry mills may also be affected if salmonella survives conditioning in the first half hour before full conditioning temperatures are reached following a shutdown period and becomes established in the cooling system. Many poultry breeder and layer rations are treated with organic acid compounds in an attempt to reduce salmonella contamination and this may be helpful (Humphrey and Lanning, 1988) but these may be less effective under humid storage conditions than formaldehyde, which has recently been licensed for use in finished feeds in the USA
168
R.H. Dauies, C. Wray/
Veterinary Microbiology
51 (1997) 159-169
(Smyser and Snoeyenbos, 1979; Brown, 1996). There is a danger, however, that blanket application of anti-salmonella treatments may encourage poor ingredient selection and less stringent application of other salmonella control measures. If the level of contamination rises because of this, it may be too much for the activity of the treatment or batches of highly contaminated feed may escape treatment when application of the antibacterial fails because of blockages or faulty metering devises.
Acknowledgements This work was funded by the Ministry of Agriculture, Fisheries and Food. The authors are grateful to Mrs. S. Bedford and Mrs. K. McIntosh for technical assistance, to colleagues in the Animal Health and Welfare Group and the State Veterinary Service for help with arranging study sites and to the various feed companies involved for permission to take the samples.
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Schmidt, R., Hoy, S.E., 1996. Investigations on dust emission from chicken and layer houses. Berlin Mllnch. Tiertiztl. Wschr. 109, 95-100. Shapcott, R-C., 1984. Practical aspects of salmonella control. Progress report on a programme in a large broiler integration. In: Snocyenbos, G.H. (Ed.), Proceedings of the International Symposium on salmonella. American Association of Avian Pathology, Kennett Square, PA, USA, pp. 109-l 14. Smyser, CF., Snoeyenbos, G.H., 1979. Evaluation of organic acids and other compounds as salmonella antagonists in meat and bone meal. Poultry Sci. 58, 50-54. Veldman. A., Vahl, H.A., Borggreve, G.J., Fuller, D.C., 1995. A survey of the incidence of salmonella species and enterobacteriaceae in poultry feeds and feed components. Vet. Rec. 136, 169-172. Voeten, A.C., van de Leest, L., 1989. Influence of the pelleting temperature used for feed on salmonella infection in broilers. Arch. Gefhigelk. 53, 225-230.