oxics
A icle
A No el Mul i-App oach P o ocol o he
Cha ac e iza ion o Occupa ional Exposu e o O ganic
Dus —Swine P oduc ion Case S udy
Ca la Viegas 1,2,*ID , Tiago Fa ia 1,3, Ana Mon ei o 1, Liliana A anha Cae ano 1,4 ID ,
Elisabe e Ca olino 1, Ani a Quin al Gomes 1,5 and Susana Viegas 1,2 ID
1GIAS, ESTeSL—Escola Supe io de Tecnologia da Saúde de Lisboa, Ins i u o Poli écnico de Lisboa,
1990-096 Lisbon, Po ugal; [email p o ec ed] (T.F.); [email p o ec ed] (A.M.);
[email p o ec ed] (L.A.C.); [email p o ec ed] (E.C.); [email p o ec ed] (A.Q.G.);
[email p o ec ed] (S.V.)
2Cen o de In es igação em Saúde Pública, Escola Nacional de Saúde Pública, Uni e sidade NOVA
de Lisboa, 1600-560 Lisbon, Po ugal
3Cen o de Ciências e Tecnologias Nuclea es, Ins i u o Supe io Técnico, Uni e sidade de Lisboa,
E.N. 10 ao km 139,7, 2695-066 Bobadela LRS, Po ugal
4Resea ch Ins i u e o Medicines (iMed.ULisboa), Facul y o Pha macy, Uni e si y o Lisbon,
1649-004 Lisbon, Po ugal
5Facul y o Medicine, Uni e si y o Lisbon Ins i u e o Molecula Medicine, 1649-028 Lisbon, Po ugal
*Co espondence: [email p o ec ed]
Recei ed: 13 No embe 2017; Accep ed: 25 Decembe 2017; Published: 27 Decembe 2017
Abs ac :
Swine p oduc ion has been associa ed wi h heal h isks and wo ke s’ symp oms.
In Po ugal, as in o he coun ies, la ge-scale swine p oduc ion in ol es se e al ac i i ies in he
swine en i onmen ha equi e di ec in e en ion, inc easing wo ke s’ exposu e o o ganic dus .
This s udy desc ibes an upda ed p o ocol o he assessmen o occupa ional exposu e o o ganic
dus , o un eil an accu a e scena io ega ding occupa ional and en i onmen al isks o wo ke s’
heal h. The pa icle size dis ibu ion was cha ac e ized ega ding mass concen a ion in i e di e en
size anges (PM0.5, PM1, PM2.5, PM5, PM10). Biobu den was assessed, by bo h ac i e and passi e
sampling me hods, in ai , on su aces, loo co e ing and eed samples, and analyzed h ough cul u e
based-me hods and qPCR. Smalle size ange pa icles exhibi ed he highes coun s, wi h indoo
pa icles showing highe pa icle coun s and mass concen a ion han ou doo pa icles. The limi
alues sugges ed o o al bac e ia load we e su passed in 35.7% (10 ou o 28) o samples and o
ungi in 65.5% (19 ou o 29) o samples. Among Aspe gillus gene a, sec ion Ci cumda i was he mos
p e alen (55%) on mal ex ac aga (MEA) and Ve sicolo es he mos iden i ied (50%) on dichlo an
glyce ol (DG18). The esul s documen a wide cha ac e iza ion o occupa ional exposu e o o ganic
dus on swine a ms, being use ul o policies and s akeholde s o ac o imp o e wo ke s’ sa e y.
The me hods o sampling and analysis employed we e he mos sui able conside ing he pu pose o
he s udy and should be adop ed as a p o ocol o be ollowed in u u e exposu e assessmen s in his
occupa ional en i onmen .
Keywo ds: o ganic dus ; occupa ional exposu e; swine; cul u e-based me hods; molecula ools
1. In oduc ion
Du ing he pas ew yea s, mos animal husband y p ac ices in Eu ope and he Uni ed S a es
ha e been indus ialized, esul ing in li es ock ope a ions aising housands o animals in a single
acili y [
1
,
2
]. The con ined p oduc ion o swine p omo es was e by-p oduc s and eed concen a ion,
Toxics 2018,6, 5; doi:10.3390/ oxics6010005 www.mdpi.com/jou nal/ oxics
Toxics 2018,6, 5 2 o 14
wi h he subsequen gene a ion o high le els o o ganic dus , including dus s, gases, mic oo ganisms,
mic obial me aboli es and o he po en ial ai bo ne heal h haza ds [1].
The biobu den, comp ising ungi and bac e ia, should be conside ed among o ganic dus as an
impo an ai bo ne pollu an in swine p oduc ion acili ies [
3
]. O ganic dus in swine p oduc ion
may be gene a ed by a ious mic obial g ow h subs a es, such as eeding ma e ials [
4
,
5
], moldy hay,
eces [
6
–
9
], manu e and bedding ma e ial [
10
], en ila ion, il a ion and mis ing applica ions [
11
].
This di e si y o con amina ion sou ces wo sens he occupa ional p oblem and hinde s he e ec i eness
o con ol measu es [
8
]. O no e, his occupa ional en i onmen may be associa ed wi h high
ae osoliza ion o pa icula e ma e , boos ing exposu e o mic oo ganisms such as ungi [
8
,
9
] and o
he myco oxins p oduced by hem [8,10].
Swine wo ke s ha e an inc eased p e alence o se e al espi a o y symp oms and diseases, such
as ch onic b onchi is, ch onic obs uc i e pulmona y disease and o ganic dus oxic synd ome [
9
,
12
–
16
].
In addi ion, wo k p ac ices such as he ypes and me hods o swine eeding, he use o wood sha ings
o animal bedding and he use o some speci ic disin ec an s ha e also been ela ed o wo ke s’
symp oms [
8
,
10
,
17
–
19
]. When eeding occu s, abundan o ganic dus pa icles om eed become
ae osolized o a long ime o deposi ed on he loo [
10
]. High dispe sion o mic oo ganisms and hei
me aboli es also occu s as hey a e esuspended du ing he eeding ask [20,21].
Al hough he need o manual wo k in swine p oduc ion is dec easing, mos ly due o he
con inemen o swine p oduc ion o acili ies wi h au oma ion, in Po ugal, as in o he coun ies,
some ac i i ies s ill equi e a me s’ di ec in e en ion, such as pigle ail cu and accina ion,
among o he ac i i ies ela ed o swine b eeding [
8
]. In hese ac i i ies, inc eased occupa ional exposu e
o o ganic dus occu s [
5
,
8
,
9
], as p e iously epo ed o ungal con amina ion [
5
], myco oxins [
8
] and
pa icula e ma e [
9
] in Po uguese swine a ms. In his s udy, a di e en p o ocol o he assessmen
o exposu e o o ganic dus is desc ibed, implemen ed o un eil a mo e accu a e occupa ional
exposu e scena io ega ding he o ganic dus isk ac o . In ac , besides he assessmen o pa icles,
bac e ia and ungi in ai and su ace samples, molecula ools we e ex ensi ely applied o a ge
ha m ul ungal species, and eed samples we e also sc eened.
2. Ma e ials and Me hods
2.1. Swine Fa ms’ Cha ac e is ics and Collec ion o En i onmen al Samples
Fi e Po uguese swine a ms we e p ospec ed in he Lisbon dis ic be ween June and July
o 2017. Swine a ms we e selec ed acco ding o he ollowing c i e ia: loca ion wi hin Lisbon
dis ic , highes numbe o animals (Table 1) and numbe o wo ke s. The i e a ms had been
e alua ed, among o he s, in an ea lie s udy om ou g oup [
5
], wi h speci ic ecommenda ions
on he need o implemen sa e y measu es in he con ex o en i onmen al and occupa ional heal h.
Howe e , no modi ica ions in wo king ac i i ies o sa e y p ocedu es we e obse ed.
In he swine a m A, ma e ni y was he main ac i i y, despi e ha ing o he a eas wi h on-going
ac i i ies. The loo in he swine ma e ni ies was co e ed wi h jou nal pape . Manu e emo al sys ems
we e p esen in all a m acili ies, wi h comple e emo al om he building se e al imes a day.
The en ila ion sys ems in he s udied a m buildings consis ed o mechanical en ila ion by wall
exhaus ans coupled wi h na u al en ila ion h ough he ope a ion o a winch-cu ain. Fa m wo ke s
did no use espi a o y p o ec ion de ices in any o he a ms.
Twen y o wen y- i e ai samples om i e dis inc a eas (pig ges a ion si e, ma e ni y, s alls,
pig a ening a ea and qua an ine con inemen ) (Table 1) and one ou doo ai sample ( o be used as
a e e ence) we e aken in each swine a m. Samples om su aces (wall swab), loo co e age om
ma e ni ies (jou nal pape ) and eed om pig ges a ion si e and/o pig a ening we e also collec ed a
each a m (Table 1).
Toxics 2018,6, 5 3 o 14
Table 1. Numbe o samples collec ed and animal quan i y in each a m.
Swine
Fa ms
No. o Ai
Samples
Impac ion *
No. o Ai
Samples
Impinge
No. o Su aces
Samples (Walls)
No. o Feed
Samples
No. o Floo
Co e Samples
Animal
Quan i y
A 20 5 5 2 1 1768
B 20 # 5 5 2 1 8000
C 20 4 # 5 2 1 3300
D 20 5 5 2 1 6000
E 16 + 4 4 2 1 7000
* A each wo king si e, 4 ai samples we e aken o each media (mal ex ac aga (MEA), dichlo an glyce ol (DG18),
yp ic soy aga (TSA), Viole Red bile aga (VRBA)); +: a m wi hou pig a ening; #: one sample los .
2.2. Pa icula e Ma e Assessmen
Pa icle measu emen was pe o med wi h di ec - eading equipmen (Handheld Pa icle Coun e
om Ligh house Wo ldwide Solu ions (Model 3016/5016)). This measu emen equipmen gi es
in o ma ion ega ding pa icle mass concen a ion (mg
×
m
−3
) (PMC) a i e di e en sizes
(PM0.5, PM1, PM2.5, PM5, PM10). Pa icle coun s (PNC) by pa icle diame e size we e also ob ained
wi h he same equipmen o six di e en diame e s (0.3
µ
m, 0.5
µ
m, 1
µ
m, 2.5
µ
m, 5
µ
m and 10
µ
m).
Measu emen s we e pe o med nea he nose o each wo ke unde ypical en ila ion condi ions and
du ing he de elopmen o asks pe o med in each o he i e a eas o in e es .
2.3. Biobu den Sampling and Analysis by Cul u e-Based Me hods
Ai samples (50 L) we e collec ed a a 1-m heigh on o ou media: 2% mal ex ac aga (MEA)
wi h 0.05 g/L chlo amphenicol media; dichlo an glyce ol (DG18) aga -based media; yp ic soy aga
(TSA) wi h 0.2% nys a in; Viole Red bile aga (VRBA), wi h a low a e o 140 L o ai pe minu e.
Fo su ace samples, he walls o he conside ed indoo loca ions we e swabbed using a 10 by 10 cm
squa e s encil, disin ec ed wi h 70% alcohol solu ion be ween samples, acco ding o he In e na ional
S anda d ISO 18593-2004, and swabs we e pla ed on o he selec ed media.
Fo loo co e age and eed samples, 4.4 g o each (no o en-d ied p io o p ocessing,
hus e aining na u al wa e con en ) we e washed in 40 mL o s e ilized dis illed wa e o 20 min
a 200 pm, and 0.15 mL o his suspension we e sp ead on o he ou media. A e incuba ion o
MEA and DG18 a 27
◦
C o 5 o 7 days o ungi and TSA and VRBA a 30
◦
C and 35
◦
C o 7 days
o mesophilic bac e ia and coli o ms (G am-nega i e bac e ia), espec i ely, biobu den densi ies
(colony- o ming uni s, CFU
·
m
−3
, CFU
·
m
−2
, CFU
·
g
−1
) we e calcula ed. Fungal species we e iden i ied
mic oscopically using ease moun o Sco ch ape moun and lac ophenol co on blue moun p ocedu es.
Mo phological iden i ica ion was achie ed h ough mac o- and mic oscopic cha ac e is ics [22].
2.4. Fungal Sampling and Molecula De ec ion by Real-Time PCR
Ai samples (300 L) we e collec ed using he impinge Co iolis
µ
ai sample (Be in Technologies)
wi h a low a e o 300 L o ai pe minu e. Samples we e collec ed on o 10-mL s e ile
phospha e-bu e ed saline (PBS) wi h 0.05% T i on X-100, and an aliquo was subsequen ly used
o DNA ex ac ion using he ZR Fungal/Bac e ial DNA MiniP ep Ki (Zymo Resea ch, I ine, CA,
USA) acco ding o he manu ac u e ’s ins uc ions. Aliquo s o su ace samples, loo co e age
(jou nal pape ) and eed samples we e also used o DNA ex ac ion ollowing he same s eps as he
impinge samples (Table 1).
A Viia7 qPCR De ec ion Sys em (The mo Fishe Scien i ic, Wal ham, MA, USA) was used o
pe o m eal- ime PCR (RT-PCR) o he molecula de ec ion o Aspe gillus species/s ains (Table 2).
Reac ions included 1
×
iQ Supe mix (Bio-Rad, Amado a, Po ugal), 0.5
µ
M o each p ime and 0.375
µ
M
o TaqMan p obe in a o al olume o 20
µ
L. Ampli ica ion ollowed a h ee-s ep PCR: 40 cycles wi h
dena u a ion a 95
◦
C o 30 s, annealing a 52
◦
C o 30 s and ex ension a 72
◦
C o 30 s. A non- empla e
Toxics 2018,6, 5 4 o 14
con ol was used in e e y PCR eac ion. As posi i e con ols o he species, DNA samples we e
ob ained om e e ence s ains om he Mycology Labo a o y om he Na ional Ins i u e o Heal h
Dou o Rica do Jo ge (INSA).
Table 2. Sequence o p ime s and TaqMan p obes used o eal- ime PCR.
Aspe gillus
Sec ions Ta ge ed Sequences Re e ence
Fumiga i
Fo wa d P ime 50-CGCGTCCGGTCCTCG-30
Re e se P ime 50-TTAGAAAAATAAAGTTGGGTGTCGG-30C uz-Pe ez e al. 2001 [23]
P obe 50-TGTCACCTGCTCTGTAGGCCCG-30
Ve sicolo es
Fo wa d P ime 50-CGGCGGGGAGCCCT-30
Re e se P ime 50-CCATTGTTGAAAGTTTTGACTGATcTTA-30
P obe
5
0
-AGACTGCATCACTCTCAGGCATGAAGTTCAG-3
0EPA 2017 [24]
2.5. S a is ical Analysis
The so wa e SPSS, Ve sion 24.0 o Windows, IBM, Lisbon, Po ugal, 2016 was used o s a is ical
analysis. The esul s we e conside ed signi ican a he 5% signi icance le el. The Shapi o–Wilk
es was used o es da a no mali y. F equency analysis (n, %) was used o he quali a i e da a.
Minimum, maximum, median and in e qua ile ange we e de e mined in he quan i a i e da a,
since no mali y was no e i ied. The F iedman es was used o compa e he pa icle concen a ion
o di e en dimensions (ei he he coun s o he mass), since he no mali y assump ion was no
e i ied. The K uskal–Wallis es was used o compa e he concen a ion o pa icles o di e en sizes
(ei he coun s o mass) be ween he i e swine a ms s udied and be ween wo kplaces, since he
no mali y assump ion was no e i ied. The K uskal–Wallis es was used o compa e ungi and
bac e ia concen a ion, bo h in ai and on su aces, be ween he i e swine a ms s udied and be ween
wo kplaces, since he no mali y assump ion was no e i ied. Spea man’s co ela ion coe icien was
used o s udy he ela ionship be ween ungi and bac e ia concen a ion (ai and su ace) and pa icle
concen a ion (coun s and mass).
3. Resul s
3.1. Pa icula e Ma e
S a is ically-signi ican di e ences we e de ec ed o pa icle coun s (PNC) o di e en dimensions
and o pa icle mass concen a ions (PMC) o a ious dimensions (
χ2
F
(4) = 120.000, p= 0.000)
(F iedman’s mul iple compa isons). Smalle pa icles (0.3
µ
m) we e he ones wi h signi ican ly highe
coun s and lowe mass concen a ions.
Smalle pa icle (0.3
µ
m) coun s we e signi ican ly di e en among swine a ms (
χ2
K−W
(4) = 27.832,
p= 0.000), pa icula ly among Swine Fa m D and Swine Fa ms A (p= 0.002) and E (p= 0.000), and among
Swine Fa ms C and E (p= 0.005). Rega ding he coun s o 0.5
µ
m-sized pa icles, signi ican di e ences
we e also ound be ween swine a ms (
χ2
K−W
(4) = 25.353, p= 0.000), namely among Swine Fa m D and
Swine Fa ms A (p= 0.000) and E (p= 0.000). Th ough mean anks analysis, i was ound ha 0.3
µ
m-
and 0.5
µ
m-sized pa icle coun s we e signi ican ly highe in Swine Fa m E, ollowed by Swine Fa m A.
No s a is ically-signi ican di e ences (p> 0.05) we e de ec ed o pa icle coun s o he emaining sizes.
Rega ding pa icle mass esul s, he PM0.5 concen a ion was signi ican ly di e en be ween
swine a ms (
χ2
K−W
(4) = 27.832, p= 0.000), namely among Swine Fa ms D and Swine Fa ms A (
p= 0.002
)
and E (p= 0.000) and among Swine Fa ms C and E (p= 0.005). Highe mass concen a ions o PM0.5
pa icles we e obse ed in Swine Fa ms E and A. The di e ences ound o PM1.0 concen a ions
be ween swine a ms (
χ2
K−W
(4) = 27.389, p= 0.000) ollowed he same end, i.e., Swine Fa m D di e ed
Toxics 2018,6, 5 5 o 14
signi ican ly om Swine Fa ms A (p= 0.001) and E (p= 0.000) and C di e ed om E (
p= 0.008
).
Swine Fa ms E and A also exhibi ed he highes mass concen a ions o PM1.0 pa icles. As o he
mass concen a ion o he pa icles PM2.5, s a is ically-signi ican di e ences we e also de ec ed among
he swine a ms (
χ2
K−W
(4) = 13.159, p= 0.011), namely among Swine Fa ms D and A (p= 0.007), wi h he
highes concen a ions obse ed in Swine Fa m A. No s a is ically-signi ican di e ences (p> 0.05)
we e de ec ed o mass concen a ion o he emaining pa icles.
Highe alues o pa icle coun s and pa icle mass concen a ion we e ound indoo , in compa ison
o ou doo ai samples. No s a is ically-signi ican di e ences we e ound among he wo k a eas
(ma e ni y, ges a ion, ba e ies, a ening and qua an ine), nei he in coun s no on mass concen a ions
o he di e en pa icle sizes (p> 0.05).
3.2. Biobu den: Bac e ial Con amina ion
Resul s o o al bac e ial load in indoo ai anged om 1800 o 54,840 CFU
·
m
−3
, wi h Swine Fa m
E p esen ing he highes median alue (28,210 CFU
·
m
−3
) Swine Fa ms A, B, C and D wi h mean alues
o 18,688, 13,660, 11,944 and 14,720 CFU
·
m
−3
, espec i ely. In 35.7% (10 ou o 28) o he sampling
si es, he o al bac e ial load exceeded he limi alues al eady sugges ed [
25
] (10,000 CFU
·
m
−3
o o al
bac e ia o eigh hou s o wo k o ag icul u al en i onmen s). G am-nega i e bac e ia load in he ai
anged be ween 0 and 220 CFU
·
m
−3
wi h he highes median alue (72 CFU
·
m
−3
) ound in Swine Fa ms
A and B, ollowed by Swine Fa ms C, D and E, wi h mean alues o 4, 24 and 60 CFU
·
m
−3
, espec i ely.
None o he sampled si es exceeded he limi alues sugges ed by Goye [
25
] (1000 CFU
·
m
−3
o
G am-nega i e bac e ia o eigh hou s o wo k o ag icul u al en i onmen s) (Figu e 1). The bac e ial
load in ou doo ai was lowe han in indoo ai in each swine a m, excep o he “ a ening” a ea in
Fa m C, p esen ing a sligh ly lowe alue indoo s han he o al bac e ial load ou doo s.
Toxics2018,6,5 5o 14

(p=0.008).SwineFa msEandAalsoexhibi ed hehighes massconcen a ionso PM1.0pa icles.
As o  hemassconcen a iono  hepa iclesPM2.5,s a is ically‐signi ican di e enceswe ealso
de ec edamong heswine a ms(߯௄ିௐ
ଶ(4)=13.159,p=0.011),namelyamongSwineFa msDandA
(p=0.007),wi h hehighes concen a ionsobse edinSwineFa mA.Nos a is ically‐signi ican 
di e ences(p>0.05)we ede ec ed o massconcen a iono  he emainingpa icles.
Highe  alueso pa iclecoun sandpa iclemassconcen a ionwe e oundindoo ,in
compa ison oou doo ai samples.Nos a is ically‐signi ican di e enceswe e oundamong he
wo ka eas(ma e ni y,ges a ion,ba e ies, a eningandqua an ine),nei he incoun sno onmass
concen a ionso  hedi e en pa iclesizes(p>0.05).
3.2.Biobu den:Bac e ialCon amina ion
Resul s o  o albac e ialloadinindoo ai  anged om1800 o54,840CFU∙m
−3
,wi hSwine
Fa mEp esen ing hehighes median alue(28,210CFU∙m
−3
)SwineFa msA,B,CandDwi hmean
alueso 18,688,13,660,11,944and14,720CFU∙m
−3
, espec i ely.In35.7%(10ou o 28)o  he
samplingsi es, he o albac e ialloadexceeded helimi  aluesal eadysugges ed[25]
(10,000CFU∙m
−3
 o  o albac e ia o eigh hou so wo k o ag icul u alen i onmen s).
G am‐nega i ebac e ialoadin heai  angedbe ween0and220CFU∙m
−3
wi h hehighes median
alue(72CFU∙m
−3
) oundinSwineFa msAandB, ollowedbySwineFa msC,DandE,wi hmean
alueso 4,24and60CFU∙m
−3
, espec i ely.Noneo  hesampledsi esexceeded helimi  alues
sugges edbyGoye [25](1000CFU∙m
−3
 o G am‐nega i ebac e ia o eigh hou so wo k o 
ag icul u alen i onmen s)(Figu e1).Thebac e ialloadinou doo ai waslowe  haninindoo ai 
ineachswine a m,excep  o  he“ a ening”a eainFa mC,p esen ingasligh lylowe  alue
indoo s han he o albac e ialloadou doo s.

Figu e1.Bac e ialloadob ained o ai andsu acesamples.
Rega dingsu acesamples,mesophilicbac e ialpopula ion anged om3×10
4
 o
516×10
4
CFU∙m
−2
,wi h hehighes median alue oundinSwineFa mC(192×10
4
CFU∙m
−2
),
ollowedbySwineFa msA,B,DandEwi hmean alueso 93.4×10
4
,182.4×10
4
,128.4×10
4
and
162×10
4
CFU∙m
−2
, espec i ely.G am‐nega i ebac e ia angedbe ween0and104×10
4
CFU∙m
−2
,
Figu e 1. Bac e ial load ob ained o ai and su ace samples.
Rega ding su ace samples, mesophilic bac e ial popula ion anged om 3
×
10
4
o
516
×
10
4
CFU
·
m
−2
, wi h he highes median alue ound in Swine Fa m C (192
×
10
4
CFU
·
m
−2
),
ollowed by Swine Fa ms A, B, D and E wi h mean alues o 93.4
×
10
4
, 182.4
×
10
4
, 128.4
×
10
4
and
162
×
10
4
CFU
·
m
−2
, espec i ely. G am-nega i e bac e ia anged be ween 0 and 104
×
10
4
CFU
·
m
−2
,

Toxics 2018,6, 5 6 o 14
wi h only one sampling si e in each swine a m p esen ing coli o m g ow h (G am-nega i e bac e ia),
mainly in he “qua an ine” and in he “ma e ni y” a ea in Swine Fa m B.
Feed bac e ial con amina ion anged be ween 81 and 1237 CFU
·
g
−1
, and he “ma e ni y” om
Swine Fa m E showed coun less mesophilic bac e ia. G am-nega i e bac e ia concen a ions anged
om 0 o 363 CFU
·
g
−1
, and he same sampling si e showed coun less colonies, whe eas in he wo o he
sampling si es, isola es we e no obse ed (“s alls” in Swine Fa m A and “qua an ine” in Swine Fa m C).
Conce ning bedding esul s, coun less mesophilic bac e ia we e p esen in all swine a ms, excep
in Fa m D wi h a alue o 1529 CFU
·
g
−1
. Rega ding G am-nega i e bac e ia, swine Fa m E p esen ed
coun less coli o ms, and he o he swine a ms anged be ween 489 and 1552 CFU·g−1.
These esul s sugges a highe con ibu ion o G am-posi i e han G am-nega i e bac e ia in he
bac e io a load.
3.3. Biobu den: Fungal Con amina ion
Fungal load in indoo ai anged om 40 o 3120 CFU
·
m
−3
on MEA, wi h Swine Fa m E p esen ing
he highes median alue (2500 CFU
·
m
−3
), ollowed by Swine Fa ms A, B, C and D wi h mean alues
o 124, 140, 604 and 104 CFU
·
m
−3
, espec i ely. No ewo hy, 65.5% (19 ou o 29) o he sampling
si es showed highe ungal load han he limi s imposed by he Wo ld Heal h O ganiza ion (WHO)
(maximum alue o 150 CFU
·
m
−3
) (2). Two ou o he 29 (6.9%) ai samples collec ed in he i e swine
a ms p esen ed highe ungal load when compa ed o he ou doo sampling. Swine Fa m A was he
excep ion, wi h highe ungal load indoo s ( wo ou o i e collec ed samples).
Simila esul s we e ound on DG18, wi h ungal load anging om 80 o 3400 CFU
·
m
−3
and wi h
Swine Fa m E also p esen ing he highes median alue (2680 CFU
·
m
−3
), ollowed by Swine Fa ms
A, B, C and D, wi h mean alues o 160, 400, 604 and 356 CFU
·
m
−3
, espec i ely. DG18 e ealed an
inc eased amoun o sampling si es (82.8%; 24 ou o 29) wi h ungal load exceeding he WHO limi s
(maximum alue o 150 CFU
·
m
−3
) (Figu e 2). Nine ou o he 29 (31%) ai samples p esen ed highe
indoo ungal load when compa ed o he ou doo sampling. Swine Fa m B was he one wi h he
highes numbe o ai samples p esen ing highe load indoo s ( ou ou o i e collec ed samples).
Toxics2018,6,5 6o 14

wi honlyonesamplingsi eineachswine a mp esen ingcoli o mg ow h(G am‐nega i e
bac e ia),mainlyin he“qua an ine”andin he“ma e ni y”a eainSwineFa mB.
Feedbac e ialcon amina ion angedbe ween81and1237CFU∙g
−1
,and he“ma e ni y” om
SwineFa mEshowedcoun lessmesophilicbac e ia.G am‐nega i ebac e iaconcen a ions anged
om0 o363CFU∙g
−1
,and hesamesamplingsi eshowedcoun lesscolonies,whe easin he wo
o he samplingsi es,isola eswe eno obse ed(“s alls”inSwineFa mAand“qua an ine”in
SwineFa mC).
Conce ningbedding esul s,coun lessmesophilicbac e iawe ep esen inallswine a ms,
excep inFa mDwi ha alueo 1529CFU∙g
−1
.Rega dingG am‐nega i ebac e ia,swineFa mE
p esen edcoun lesscoli o ms,and heo he swine a ms angedbe ween489and1552CFU∙g
−1
.
These esul ssugges ahighe con ibu iono G am‐posi i e hanG am‐nega i ebac e iain
hebac e io aload.
3.3.Biobu den:FungalCon amina ion
Fungalloadinindoo ai  anged om40 o3120CFU∙m
−3
onMEA,wi hSwineFa mE
p esen ing hehighes median alue(2500CFU∙m
−3
), ollowedbySwineFa msA,B,CandDwi h
mean alueso 124,140,604and104CFU∙m
−3
, espec i ely.No ewo hy,65.5%(19ou o 29)o  he
samplingsi esshowedhighe  ungalload han helimi simposedby heWo ldHeal h
O ganiza ion(WHO)(maximum alueo 150CFU∙m
−3
)(2).Twoou o  he29(6.9%)ai samples
collec edin he i eswine a msp esen edhighe  ungalloadwhencompa ed o heou doo 
sampling.Swine
Fa mAwas heexcep ion,wi hhighe  ungalloadindoo s( woou o  i ecollec edsamples).
Simila  esul swe e oundonDG18,wi h ungalload anging om80 o3400CFU∙m
−3
and
wi hSwineFa mEalsop esen ing hehighes median alue(2680CFU∙m
−3
), ollowedbySwine
Fa msA,B,CandD,wi hmean alueso 160,400,604and356CFU∙m
−3
, espec i ely.DG18
e ealedaninc easedamoun o samplingsi es(82.8%;24ou o 29)wi h ungalloadexceeding he
WHOlimi s(maximum alueo 150CFU∙m
−3
)(Figu e2).Nineou o  he29(31%)ai samples
p esen edhighe indoo  ungalloadwhencompa ed o heou doo sampling.SwineFa mBwas
heonewi h hehighes numbe o ai samplesp esen inghighe loadindoo s( ou ou o  i e
collec edsamples).

Figu e2.Fungalloaddis ibu ionin he i eassessedswine a ms.Thedashedline ep esen s he
e e encelimi ssugges edby heWo ldHeal hO ganiza ion(WHO).
Twen y i edi e en  ungalspecieswe e oundinai samplesonMEAand18onDG18.
Cladospo iumsp.was hemos p e alen inindoo ai samplesinbo hmedia(59.4%MEA;66.5%
DG18), ollowedbyFusa iumg aminea umspeciescomplex(13.2%)onMEAandUlocladiumsp.
(14.6%)onDG18(Table3).
Figu e 2.
Fungal load dis ibu ion in he i e assessed swine a ms. The dashed line ep esen s he
e e ence limi s sugges ed by he Wo ld Heal h O ganiza ion (WHO).
Twen y i e di e en ungal species we e ound in ai samples on MEA and 18 on DG18.
Cladospo ium sp. was he mos p e alen in indoo ai samples in bo h media (59.4% MEA; 66.5% DG18),
ollowed by Fusa ium g aminea um species complex (13.2%) on MEA and Ulocladium sp. (14.6%) on
DG18 (Table 3).
Toxics 2018,6, 5 7 o 14
Fungal load in subs a e ( eed and loo co e age) and su ace samples was dis ibu ed as
ollows: 0 o 4 CFU
·
g
−1
(MEA) and 0 o 39 CFU
·
g
−1
(DG18) in eed; 0 o 1 CFU
·
g
−1
(MEA)
and no isola es (DG18) in loo co e ing; 0 o 59
×
10
4
CFU
·
m
−2
(MEA) (maximum alues o
14
×
10
4
CFU
·
m
−2
in Swine Fa m D, ollowed by 6
×
10
4
CFU
·
m
−2
in Swine Fa m E) and 0 o 370,500
(DG18) (maximum alues o 10 ×104CFU·m−2in Swine Fa m D) on wall su aces.
Th ee di e en ungal species we e ound in eed samples on bo h media, he Cladospo ium
genus being he mos p e alen (71.4% MEA; 82.2% DG18). Fungi isola es om loo co e ing
samples we e only obse ed on MEA, wi h p edominan Penicillium genus (50.0%) ollowed by
Al e na ia sp. (37.5%). Ten ungal species we e ound in su ace samples on MEA and ou on DG18.
Cladospo ium sp. (53.8%) and Scopula iopsis b e icaulis (33.3%) we e he mos de ec ed species on
MEA, whe eas Scopula iopsis candida (50.3%) and Aspe gillus sec ion Ci cumda i (19.9%) we e he mos
obse ed on DG18. T icho hecium oseum was ound only on su aces (Table 3).
Table 3.
Fungal dis ibu ion in en i onmen al and subs a e ma ices a e inocula ion on o MEA and
DG18 media.
MEA DG18
Ai (CFU·m−3) (%; n)Ai (CFU·m−3) (%; n)
Cladospo ium sp. 59.4; 12,100 Cladospo ium sp. 66.5; 14,120
Fusa ium g aminea um 13.2; 2700 Ulocladium sp. 14.6; 3100
Al e na ia sp. 5.7; 1160 Ch ysonilia si ophila 4.7; 1000
O he s 21.7; 4420 O he s 14.2; 3020
Su aces (CFU·m−2) (%; n)Su aces (CFU·m−2) (%; n)
Cladospo ium sp. 53.8; 210,000 Scopula iopsis candida 50.3; 580,000
Scopula iopsis
b e icaulis 33.3; 130,000 Aspe gillus sec ion
Ci cumda i 19.9; 230,000
Penicillium sp. 12.8; 50,000 Cladospo ium sp. 13; 150,000
O he s 0.1; 500 O he s 16.7; 193,000
Feed (CFU·g−1) (%; n)Feed (CFU·g−1) (%; n)
Cladospo ium sp. 71.4; 10 Cladospo ium sp. 82.2; 37
Penicillium sp. 21.4; 3 Penicillium sp. 8.9; 4
Fusa ium culmo um 7.1; 1 Fusa ium culmo um 8.9; 4
Floo co e ing (CFU·g−1) (%; n)Floo co e ing (CFU·g−1) (%; n)
Penicillium sp. 50; 4 --
Al e na ia sp. 37.5; 3 --
Cladospo ium sp. 12.5; 1 --
Aspe gillus gene a we e obse ed on MEA and DG18 (2.26%). Aspe gillus sec ion Ci cumda i was
he mos p e alen (55%) on MEA ollowed by Aspe gilli (25%). Di e en Aspe gillus sec ions we e
mo e p e alen on DG18, Ve sicolo es being he mos iden i ied (50%) ollowed by Us i (20.8%) (Table 4).
Table 4. Aspe gillus sec ions’ dis ibu ion in ai samples.
MEA DG18
Ai (CFU·m−3) (%; n)Ai (CFU·m−3) (%; n)
Ci cumda i 55; 220 Ve sicolo es 50; 240
Aspe gilli 25; 100 Us i 20.8; 100
Nig i 10; 40 Aspe gilli 12.5; 60
Ve sicolo es 5; 20 Candidi 12.5; 60
Fla i 5; 20 Nidulan es 4.2; 20
No Aspe gillus sec ion Fumiga i no Aspe gillus sec ion Ve sicolo es we e de ec ed by qPCR.
Toxics 2018,6, 5 8 o 14
3.4. Co ela ion and Compa ison Resul s
No s a is ically-signi ican di e ences we e de ec ed be ween he swine a ms in ei he su ace o ai
samples, ega ding o al bac e ial load (
χ2
K−W
(4) = 1.936, p= 0.748,
χ2
K−W
(4) = 3.676, p= 0.452, espec i ely)
o G am-nega i e bac e ia (χ2
K−W(4) = 0.081, p= 0.999, χ2
K−W(4) = 7.132, p= 0.129, espec i ely).
S a is ically-signi ican di e ences we e ound be ween he ungal load on MEA in he swine a ms
in su ace (
χ2
K−W
(4) = 13.699, p= 0.008) and in ai (
χ2
K−W
(4) = 14.602, p= 0.001) samples. Fungal load
was signi ican ly di e en among Swine Fa ms C and D (p= 0.036) in su ace samples and among
Swine Fa m D and Swine Fa ms A (p= 0.007) and B (p= 0.025) in ai samples. Swine Fa m D p esen ed
he highes mean ank alues o ungal load in bo h su ace and ai samples. S a is ically-signi ican
di e ences we e also obse ed o ungal load on DG18 be ween he ai samples collec ed in he swine
a ms (
χ2
K−W
(4) = 12.621, p= 0.013), namely, be ween Swine Fa ms B and D (p= 0.005), wi h Fa ms D
and B exhibi ing he highes and he lowes alues, espec i ely (Table 5).
Table 5.
Resul s o he K uskal–Wallis es o he compa ison o ungi and bac e ia concen a ion,
bo h on su aces and in ai , be ween he i e swine a ms (n= 24).
Bac e ia/Fungus Swine
Fa ming nRanks Tes S a is ics aK uskal–Wallis Mul iple
Compa isons
Mean Rank Chi-Squa e d p
To al Bac e ia Su ace
(CFU·m−2)
A 5 12.00
1.936 40.748
B 5 10.00
C 5 14.10
D 4 10.75
E 5 15.30
G am Nega i e
Bac e ia-Su ace
(CFU·m−2)
A 5 12.40
0.081 40.999
B 5 12.60
C 5 12.00
D 4 12.75
E 5 12.80
Fungi (MEA)-Su ace
(CFU·m−2)
A 5 17.90
13.699 40.008 *
C6=D (p= 0.036)
B 5 12.50
C 5 6.00
D 4 19.50
E 5 8.00
Fungi (DG18)-Su ace
(CFU·m−2)
A 5 18.60
8.430 40.077
B 5 13.60
C 5 10.30
D 4 8.50
E 5 10.70
To al bac e ia-Ai
(CFU·m−3)
A 5 12.10
3.676 40.452
B 5 14.10
C 5 10.40
D 4 17.50
E 5 9.40
G am Nega i e
Bac e ia-Ai
(CFU·m−3)
A 5 10.20
7.132 40.129
B 5 15.00
C 5 15.00
D 4 16.50
E 5 6.60
Fungi (MEA)-Ai
(CFU·m−3)
A 5 6.40
17.602 40.001 *
A6=D (p= 0.007)
B 5 8.20 B 6=D (p= 0.025)
C 5 9.40
D 4 22.50
E 5 18.00
Fungi (DG18)-Ai
(CFU·m−3)
A 5 11.60
12.621 40.013 *
B6=D (p= 0.005)
B 5 6.10
C 5 10.80
D 4 22.50
E 5 13.50
aK uskal–Wallis es ; * s a is ically-signi ican di e ences a he 5% signi icance le el.
Toxics 2018,6, 5 9 o 14
Among he sampling si es (ma e ni y, ges a ion, ba e y, a ening and qua an ine),
no s a is ically-signi ican di e ences we e de ec ed in ungi no in bac e ia load o ei he su ace o ai
samples (p’s > 0.05).
The ela ionship be ween ungi, bac e ia (su ace and ai ) and pa icles’ concen a ion
(coun ing and mass) was pe o med sepa a ely o each swine a m. In Swine Fa m A, a signi ican
nega i e co ela ion, wi h s ong in ensi y, was ound be ween he ungal concen a ion on DG18 in
su ace samples and he ungal concen a ion on MEA in he ai (
s
=
−
0.975, p= 0.005). This esul
indica es ha highe ungal concen a ions on DG18 in he su ace a e ela ed o lowe ungal
concen a ions on MEA in he ai . In Swine Fa m B, highe o al bac e ia loads in su ace samples
we e ound o be signi ican ly co ela ed wi h lowe coun s o 0.5 mic on (
s
=
−
0.9, p= 0.037),
1.0 mic on (
s
=
−
0.9, p= 0.037), 2.5 mic on (
s
=
−
0.9, p= 0.037), 5.0 mic on (
s
=
−
0.9, p= 0.037) and
10.0 mic on (
s
=
−
0.9, p= 0.037) pa icles. Addi ionally, highe ungal loads on DG18 in su ace samples
we e ound o be posi i ely co ela ed wi h highe ungal loads on MEA in su ace samples (
s
= 0.918,
p= 0.028
) and wi h highe o al bac e ia loads in he ai (
s
= 0.894, p= 0.041). A s a is ically-signi ican
co ela ion was also ound in Swine Fa m B be ween highe PM0.5 mass concen a ion and highe
ungal loads on MEA in he ai (
s
= 0.9, p= 0.037). In Swine Fa m C, o al bac e ia load in he ai was
ound o be nega i ely co ela ed wi h ungal load on DG18 in he ai (
s
=
−
0.9, p= 0.037), sugges ing
ha highe concen a ions o o al bac e ia in he ai a e ela ed o lowe concen a ions o ungi in he
ai . No signi ican co ela ions we e ound o Swine Fa ms D and E.
4. Discussion
O ganic dus has been he ocus o se e al epidemiological s udies, as exposu e o o ganic
dus is desc ibed as causing mucous memb ane i i a ion in he eyes and uppe and lowe ai ways,
in lamma ion by alle gic and non-alle gic mechanisms and ai way obs uc ion [
26
]. O ganic dus in
swine p oduc ion can be gene a ed om se e al sou ces such as eed, skin cells, hai and d ied eces.
Acu e exposu es o high le els o dus may p o oke inc eased phlegm p oduc ion and pulmona y
in lamma ion 4
−
10 h a e exposu e. In addi ion, ch onic exposu es may esul in b onchi is and
as hma [27].
I has al eady been epo ed ha ae osols p oduced in swine p oduc ion acili ies may ha e
pa icle size dis ibu ions conside ably smalle han 1.0
µ
m in diame e [
28
,
29
], comp ising an
impo an size ac ion o he espi able ae osols ha should be assessed in swine p oduc ion [
28
,
29
].
Fu he mo e, pa icula e ma e can be he ehicle o he biobu den p esen in swine a ms, eaching
wo ke s espi a o y sys ems and, consequen ly, enhancing he occupa ional exposu e o swine wo ke s
o o ganic dus [
9
]. The e o e, mic oo ganisms adhe ing o pa icula e ma e , along wi h coexis ing
gases and oxins a e a gene al cause o conce n ega ding co-exposu e o se e al isk ac o s and
possible addi i e and syne gis ic heal h e ec s [
20
,
30
]. This scena io ein o ces he heo y ha
occupa ional exposu e is a ely associa ed wi h a single ac o , since i is commonly a combina ion o
se e al isk ac o s [31].
I has been sugges ed ha s a iona y sample s can be adequa ely used in he assessmen o
pe sonal exposu e o ai bo ne ungi in con ined ag icul u al en i onmen s [
7
] as is he case o
animal p oduc ion. Howe e , we should combine no only mo e han one ac i e me hod o ai
sampling [32,33]
, bu also couple i wi h passi e me hods [
34
,
35
], such as su ace swabs, loo co e ing
and eed o ob ain a mo e accu a e isk cha ac e iza ion [
33
]. As such, his s udy was pe o med
using a mul i-app oach p o ocol o he cha ac e iza ion o occupa ional exposu e o o ganic dus by
applying he impac ion me hod o ob ain a biobu den iable ac ion om o ganic dus , he impinge
me hod o he de ec ion o ha m ul ungal species and he su ace swabbing o complemen ai
samples’ esul s ega ding he di e si y o he biobu den [
31
,
35
]. In addi ion, he collec ion o loo
co e ing and li e (besides su aces swabs) samples allowed de e mining he con amina ion le els
om a la ge pe iod o ime (weeks o se e al mon hs), whe eas ai samples can only e lec he load
om a sho e pe iod o ime (mos ly minu es) [33,34,36].