Водные патогены присутствуют в сточных водах крупного молочного завода летом и зимой

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Элмуродов, Б., Пурди, К., & Ралеих, Р. (2006). Водные патогены присутствуют в сточных водах крупного молочного завода летом и зимой. in Library, 1(1), 20–23. извлечено от https://inlibrary.uz/index.php/archive/article/view/31585
Бозорбой Элмуродов, Ветеринарный научно-исследовательский институт

Директор Научно-исследовательского ветеринарного института, профессор, доктор ветеринарных наук

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Аннотация

Сточные воды молочного производства являются ценным ресурсом в полузасушливых регионах, который может быть переработан, поэтому потенциальное содержание в них эпизоотических возбудителей, эндотоксинов и других опасных веществ очень актуально и важно. Бактериальные и грибковые эпизоотические патогены были изучены в сточных водах молочной фермы на 4500 коров в Южных Хай-Плейнс, США. Сточные воды собирались в трех экземплярах из 13 различных точек сбора вдоль дренажной системы сточных вод, начиная с доильного зала и заканчивая лагуной; и из ирригационной системы Centei, которая использовала воду из лагуны, смешанную с пресной водой, для кормовых культур, которые позже использовались в качестве корма для дойных коров. Возбудители были обнаружены во всех пунктах сбора, кроме двух пунктов, где применялось хлорирование. Было идентифицировано восемь сероваров Sa/monella; большинство проб сточных вод содержали Escherichia coli 0157'.H7; и Listeria monocytogenes обнаруживалась в средних концентрациях от 1 х 104 до 1 х 105 КОЕмл. Средние концентрации Staphytococcus spp (1; t0,1" i; lozCFu/мл) и Enterococcc,rspp (от 1x104 до 10x105 CfUlmiy) также были высокими. Средняя концентрация мезофильных грибов колебалась от 1x1A2 до 1. x 10s КОЕ/мл Th; сточные воды центрального водохранилища содержали меньшее количество каждого изученного патогена, и на этом участке не были выявлены виды Enterococcus. Одним из наиболее биологически активных веществ, обнаруженных в сточных водах лагуны, был эндотоксин, средняя концентрация которого составляла 37 712 ЕЭ. /мл Эти данные показывают, что сточные воды могут быть значительным источником эпизоотических патогенов и эндотоксинов.

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20

WATER PATHOGENS PRESENT IN THE WASTEWATER FLOW OF A LARGE DAIRY IN

THE SUMMER AND WINTER

C.W. Purdy, R.H. Raleigh, D.C. Straus, B.A Elmurodov

rom the Conservation and Production Research Laboratory, Renewable Energy, and Manure

Management Research Unit, Agricultural Research Service, United States Department of Agriculture,

P.O. Drawer 10, Bushland, Texas 79012 (Purdy); Veterinary Diagnostic Laboratory, Texas A&M

University, Amarillo Blvd West, Amarillo, Texas 79109 (Raleigh); Department of Microbiology and

Immunology, Texas Tech University Health Science Center, Lubbock, Texas 79430 (Straus); Uzbek

Veterinary Research Institute, Taylak, Samarkand, Republic of Uzbekistan 704453 (Elmurodov).

The authors thank Will Willis for technical assistance.

Abstract

Dairy wastewater is a valuable resource in semi-

arid regions that can be recycled, therefore it’s potential

content of epizootic pathogens, endotoxins and other hazardous substances is very timely and important. Bacterial
and fungal epizootic pathogens were studied in wastewater on a 4,500 cow dairy in the Southern High Plains, U.S.A..
The wastewater was collected in triplicate from 13 different collection points along the wastewater drainage system,
starting in the

milking

parlor and ending in a lagoon; and from an irrigation center pivot that applied the lagoon

wastewater mixed with fresh water to forage crops, later used as feed for dairy cows. Pathogens were found at all
collections points, except two points where chlorination was used. Eight

Salmonella

serovars were identified; most

wastewater samples contained

Escherichia coll

O157:H7; and

Listeria monocytogenes

was found in mean

concentrations from 1 x 10

4

to 1 x 10

5

CFU/ml. The mean concentrations of

Staphylococcus spp,

(1 x 10

4

to 1 x

10

7

CFU/ml) and

Enterococcus spp

(1 x 10

4

to 10 x10

5

CFU/ml) were also high. The mean concentration of mesophilic

fungi ranged from 1 x 10

2

to 1 x 10

5

CFU/ml. The center pivot wastewater contained fewer of each pathogen studied,

and no

Enterococcus spp

were identified from this site. One of the most biologically active substances found in the

lagoon wastewater was endotoxin which had a mean concentration of 37,712 EU/ml. These data show dairy
wastewater can be a significant source of epizootic pathogens and endotoxin.

Introduction

Water is very precious in a semi-arid region and dairy wastewater is used as a valuable resource. The 30

year average precipitation in this region was 470 mm/year, and 30 year normal temperature in degrees centigrade
was: maximum, 22, mean, 14, and minimum, 7. If this water contains dangerous pathogens there is the potential for
contaminating larger areas of crops and pasture land. This then becomes a reservoir that can infect mammals and
birds (Purdy, et al., 2001). Dairy wastewater that is used for dust control or for forage crops soon after irrigation,
become problematic as a potential source of enteric pathogens.

Modem dairies in the U.S.A, have an ingenious way of using water to carry away much of the solid waste

from the holding areas, milking parlors, and feed alleys located outside. The wastewater flows through ditches, and
settling basins, before entering waste holding ponds (lagoons). The lagoon wastewater is often pumped through pipes
to the irrigation center pivot irrigation systems, where it is usually blended with fresh well water prior to use.

The source of chlorinated water used to flush the fresh manure from the milking parlor floor was previously

used to cool the large milk reservoir tanks. This same source of water is frequently used in sprinklers directed up to
partially wash the cows udders of organic material while the cows wait in the holding area before they enter the
milking parlor. The water used to flush manure from the long feeding alleys is recycled lagoon water.

The objective of this study was to determine the concentration of bacteria, and fungi, and to identify specific

pathogen concentrations at 13 points along the flow of wastewater from the milking parlor to the lagoon and after
pumping to the center pivot used for irrigation. In addition, the endotoxin concentration was determined at four lagoon
sites.

Materials & Methods
Climate-

The 30 year average precipitation in the Southern High Plains was 120.4 mm in the winter and 349.5 mm

in the summer. The 30 year normal temperature in degrees centigrade for


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21

the winter months (October through March) was: maximum, 15.1, mean, 7.5, and minimum, - 0.1; summer (April
through September) was: maximum, 28.8, mean, 21.0, and minimum, 13.2.

Dairy-The

dairy was located in the

Southern High Plains, where 4,500 head of cows were milked three times a day.

Sample collection for wastewater

and

dilution

technique-Three-two-liter wastewater samples were collected in

sterile plastic bottles from each of 13 points (milk parlor flush, holding area sprinklers, milk parlor effluent, beginning
feeding alley flush, end feeding alley flush, beginning sand ditch, end sand ditch, settling basin, North lagoon, South
lagoon, East lagoon, West lagoon, and center pivot). Each water sample was assayed for bacterial and fungal
concentration. Briefly, 10-fold serial dilutions (W

1

to 10'

7

) were prepared from each sample by inoculating 10 ml of

wastewater into glass bottles containing 90 ml of sterile water. Aliquots of 0.1 ml/per dilution were spread over the
surface of the Petri plates used in the dilution series for each medium used.

Culture medium

- Specialized media were used to determine the concentration of bacteria and fungi, and to identify

the pathogens (Brain-Heart-Infusion agar identified mesophilic and thermophilic bacteria[BHI], Difco; MacConkey
agar was used for identifing all coliforms [MAC], Difco; Enterococcosel agar for identifing

Enterococcus spp.

[EGA],

BBL;

Listeria

selective agar for identifing

Listeria monocytogenes.

[LSA], Oxoid; Bard Parker agar for identifing Gram-

positive cocci; inducted were

Staphylococcus aureus, S. epidermidis, S. saprophyticus, and Micrococcus spp

[BPA],Oxoid; Malt Extract agar for identifing thermal fungi [ME], Difco; Littman oxgall agar for identifing mesophilic
fungi [LOA], Difco. The fungi were further identified by colony formation, color and by spore identification (Crisan, EV,
1959; Barnett, HL., 1965; Larone, DH, 1995). The following inhibitory mediums (EC broth, Difco) were used for
differentiating and enumerating

Escherichia coli

O157:H7, and a selenite-brilliant green- sulfapyridine enrichment

broth medium (SBG plus Sulfa, Difco) was used in part for the isolation of

Salmonella spp.

Additional specific Difco media (blood agar base supported with 5% bovine blood [BA] was used to help in

the identification of hemolytic

Staphylococcus spp, Streptococcus spp, Corynebacterium spp,

and to determine that

Salmonella spp

were not hemolytic. In addition, Xylose-lysine-desoxycholate agar [XLD], Difco; Brilliant Green agar

[BG], Difco; Lysine Iron agar [LIA], Difco; and Triple Sugar Iron agar [TSI], Difco were used to differentiate

Salmonella

spp.

The

Salmonella spp

were serotyped by the National Veterinary Services Laboratory located at Ames, Iowa.

Culture incubation

temperatures-

Thermophilic bacteria (BHI medium) were incubated at 55°C, and thermophilic

fungi (LOA medium) were incubated at 50°C. Pathogens were grown at 37°C, and mesophilic bacteria and fungi were
incubated at 28°C.

Kinetic

Limulus

lysate

assay-Endotoxin was measured by use of the kinetic chromogenic quantitative

Limulus

ameobocyte lysate assay (Williams and Halsey, 1997). Methods for the assay are fully described by Purdy et al.
(2001).

Statistical analyses-Data

were analyzed by use of ANOVA, using the general linear models procedure

a

(SAS user’s

guide, 1988). Significant differences between wastewater assay positions were further evaluated by use of the
Bonferroni adjusted paired t-test. Differences were considered significant at P < 0.05.

Results

Eight

Salmonella

serotypes were identified in the winter and summer

(Table 1).

A total of 86

Salmonella

isolates

were identified, 6

Salmonella

were untypeable, and 4 were multiple

Salmonella

serotypes. Sixty

Salmonella

suspected isolates were not

Salmonella,

which gave a 63% recovery rate.

Salmonella agona

was only found in the

winter.

Salmonella spp

was found at 11 of the 13 collection sites, except in the milk barn flush water and the water

from the milk barn holding area floor sprinklers.

Endotoxin mean concentration for the lagoon wastewater differed significantly with the seasons (P > 0.007),

49,800 (std error 3006) EU/ml in the winter and 25,624 (3992) EU/ml in the summer.

Escherichia coli

O157:H7 was recovered from all EC enriched water samples, except those collected from

the center pivot in the winter. The water from the origin of milk barn flush


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22

and from the floor sprinklers were considered sterile, however occasional microbe contaminate were encountered
due to the collection process.

The most numerous mesophylic fungal pathogens identified from the wastewater collection sites

were

Mucor spp,

and

Aspergillus spp,

and the most numerous thermophilic fungi were

Mucor pusillus

and

Aspergillus fumigatus.

The overall mean concentrations of bacteria and fungi found in the dairy wastewater collection

areas are reported as colony forming units (CFU)/ml for selective media

(Table 2).

There were significant

differences (P < 0.045) between the summer and winter and among the collection sites (P < 0.004) for
mesophilic bacterial concentrations. There were no significant differences between mean concentration of
thermophilic bacteria between seasons or among collection sites. There were significant differences
between seasons and collection sites for the mean concentration of mesophilic fungi

(Table 2).

The mean

concentrations of theromphilic fungi were determined only in the winter. At that time the mean
concentrations were significantly different among collection sites (P < 0.0001).

Discussion

Dairy wastewater appears to be a significant source of enteric pathogens,

Salmonella spp,

and

Escherichia coli

O157:H7. In addition numerous Gram-positive pathogens such as S.

aureus,

Streptococcus spp,

and

Corynebacterium spp

were isolated from the wastewater.

Listeria monocytogenes

was recovered most frequently. The endotoxin concentration of the dairy wastewater was high 37,712
(5121) EU/ml, but lower than that found in feedyard playas (8,321 ng/ml or 83,210 EU/ml) (Purdy, CW, et
al., 2001).

Thermal fungi and thermal bacteria in wastewater were 100 times less frequent than mesophilic

fungi and mesophilic bacteria at each collection site. (Data not shown). It is interesting that one to two log
differences were seen at different sides of the lagoon using the selective media. This indicates that the
lagoon microflora are not thoroughly mixed in the lagoon even under continuous multiple aerators.

The variety of bacterial pathogens and high levels of endotoxin should be considered before using

this water for aerial spraying to abate dust. This practice would certainly increase environmental
contamination, and increase the aerosol risk to animals and humans. The use of this untreated wastewater
in the irrigation of plants that will be fed as forage without sustaining some drying (curing) period should be
discouraged.

The spread of epizootic pathogens by dairy wastewater to other animals should be avoided. For

example, in this study, eight

Salmonella

serovars were identified and many more serovars were probably

present. We recommend that dairy wastewater used to settle dust be chlorinated to kill the pathogens
present. It should be realized that the less organic material that the wastewater contains the more efficient
chlorination will be. Additional animal research is needed to determine if the aerosolized endotoxin level
induced by high pressure application of wastewater to settle dust is sufficient to lower milk production and
induce fever in exposed livestock.

It appears that the wildlife most vulnerable to these wastewater pathogens would be migratory

water fowl, which can gain access to the lagoon, regardless of fencing.

References

Barnett, H.L. 1965. Illustrated Genera of Imprefect Fungi, 2

nd

Ed., Burgess Publishing Co., Minneapolis,

Minnesota.

Crisan, E.V. August 1959. The isolation and identification of Thermophilic Fungi. A Master of Science

Thesis Submitted to the Faculty of Purdue University, Department, of Microbiology, Purdue Press,

Larone, D.H. 1995. Medically important fungi: A guide to identification, 3

rd

Ed., ASM Press, Washington, D

C.

Purdy, C.W., Straus, D C., Parker, D.B., and Williams, B.P. 2001. Water quality in cattle feedyards playas

in winter and summer. American Journal of Veterinary Research. 62:1402- 1407.
SAS

Users’ Guide:Version 6.03 Edition.

1988. Cary, NC: SAS Institute Inc.


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23

Williams P.B. and Halsey, J.E. 1997. Endotoxin as a factor in adverse reactions to latex gloves.
Annals of Allergy, Asthma, and Immunology 79:303-310. ______________________________

Table 1.

Salmonella

serovars identified from dairy wastewater and the number of -each serovar

isolated.

Salmonella

serovars

Number isolated

agona

12

give

6

minnesota

14

montevideo

6

muenster

1

newport

10

typhimurium

36

Worthington

1

Untypeable Salmonella

4

Multiple Salmonella
serotypes

6

Table 2. Mean microbial

colorfy.

forming units (CFU)/ml based on the dairy wastewater collection site and the

selective media on which the wastewater dilutions were grown.
Collection
site

Mean
CFU/ml BHI

Mean
CFU/ml LOA

Mean CFU/ml
MAC

Mean
CFU/ml BPA

Mean
CFU/ml LSA

Mean
CFU/ml ECA

Milk Parlor
Effluent

1.35E+08
(8.96E+07)

1.62E+05
(9.10E+04)

1.65E+05
(6.92E+04)

4.08E+06
(1.82E+06)

5.97E+05
(1.60E+05)

6.37E+05
(4.73E+05)

Alley Flush
Top

4.28E+07
(1.03E+07)

8.10E+05
(3.33E+05)

4.26E+04
(1.25E+04)

2.55E+06
(9.35E+05)

1.34E+05
(5.55E+04)

5.23E+04
(7.80E+03)

Alley Flush
Bottom

2.66E+07 (9.51
E+06)

4.30E+05
(6.63E+04)

9.25E+04
(1.79E+04)

1.12E+07 (6.41
E+06)

1.19E+05
(8.73E+04)

7.26E+04
(3.09E+04)

Sand Ditch
Top

5.53E+07
(1.94E+07)

2.06E+05
(9.30E+04)

2.38E+05
(7.12E+04)

6.33E+08
(2.45E+06)

2.34E+05
(7.14E+04)

9.38E+04
(1.45E+04)

Sand Ditch
Bottom

3.33E+07
(4.32E+06)

3.80E+05
(1.71E+05)

1.51E+05
(5.10E+04)

6.69E+06
(2.47E+06)

2.26E+05 (6.31
E+04)

1.07E+05
(3.22E+04)

Collection
site

Mean
CFU/ml BHI

1

Mean CFU/ml
LOA

2

Mean CFU/ml
MAC

3

Mean CFU/ml
BPA

4

Mean CFU/ml
LSA

5

Mean CFU/ml
ECA

6

Settling Basin

7.95E+07 (2.51
E+07)

3.76E+05
(1.74E+05)

3.61E+04
(1.38E+04)

2.04E+07
(1.09E+07)

2.14E+05
(5.57E+04)

1.26E+05
(5.62E+04)

Lagoon North

6.23E+06
(2.40E+06)

1.00E+05
(8.16E+04)

3.62E+04
(1.54E+04)

2.88E+06 (1.41
E+06)

1.70E+05
(1.23E+04)

1.84E+04
(7.52E+03)

Lagoon
South

5.62E+06
(2.05E+06)

7.17E+02
(3.00E+02)

6.05E+04 (2.91
E+04)

8.47E+05
(2.92E+05)

1.33E+05
(1.52E+04)

7.97E+03
(3.56E+03)

Lagoon East

1.84E+07
(7.38E+06)

5.02E+04
(5.00E+04)

1.83E+05
(9.34E+04)

3.86E+06
(1.78E+06)

1.77E+05
(2.90E+04)

1.78E+04
(7.23E+03)

Lagoon West

1.19E+06
(3.05E+05)

4.37E+04
(2.29E+04)

7.06E+04
(2.17E+04)

5.80E+05
(7.29E+04)

1.26E+05
(1.82E+04)

6.72E+03
(1.97E+03)

Collection
site

Mean
CFU/ml BHI

Mean
CFU/ml LOA

Mean CFU/ml
MAC

Mean
CFU/ml BPA

Mean
CFU/ml LSA

Mean
CFU/ml ECA

Center Pivot

3.68E+05
(3.39E+05)

2.54E+03
(1.83E+03)

6.33E+02

'

(3.31

E+02)

4.57E+02
(2.38E+02)

1.67E+00
(1.67E+00)

0.00E+00
(0.00E+00)

1. Brain heart infusion agar is a rich all purpose bacterial medium; 2. Littman oxgall agar specific for fungal
isolation; 3. MacConkey agar specific for Gram-negative bacteria and followed EC enrichment broth which limited
the growth to

Escherichia coli

and

E. coli

O157:H7; 4. Bard Parker selective media for

Staphylococcus spp

and

Micrococcus spp;

5.

Listeria

selective agar for

Listeria monocytogenes;

Enterococcosel agar selective medium for

Enterococcus spp.

Библиографические ссылки

Barnett, H.L. 1965. Illustrated Genera of Imprefect Fungi, 2 nd Ed., Burgess Publishing Co., Minneapolis, Minnesota.

Crisan, E.V. August 1959. The isolation and identification of Thermophilic Fungi. A Master of Science Thesis Submitted to the Faculty of Purdue University, Department, of Microbiology, Purdue Press,

Larone, D.H. 1995. Medically important fungi: A guide to identification, 3 rd Ed., ASM Press, Washington, D C.

Purdy, C.W., Straus, D C., Parker, D.B., and Williams, B.P. 2001. Water quality in cattle feedyards playas in winter and summer. American Journal of Veterinary Research. 62:1402- 1407. SAS Users’ Guide:Version 6.03 Edition. 1988. Cary, NC: SAS Institute Inc.

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