TIERÄRZTLICHE HOCHSCHULE HANNOVER - TIHO ELIB
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Tierärztliche Hochschule Hannover
Assoziationen zwischen der Keimdichte im Haltungsumfeld und
an der Zitzenspitze – ein Beitrag zur Bekämpfung von
Mastitiden mit umweltassoziierten Mastitiserregern in
Milchviehbetrieben
INAUGURAL-DISSERTATION
zur Erlangung des Grades einer Doktorin
der Veterinärmedizin
- Doctor medicinae veterinariae -
(Dr. med. vet.)
vorgelegt von
Maria - Franziska Hohmann
Forst/Lausitz
Hannover 2020Wissenschaftliche Betreuung: Prof. Dr. Volker Krömker
Section for Production, Nutrition and Health
Department of Veterinary and Animal Science
Faculty of Health and Medical Science
University of Copenhagen
1. Gutachter: Prof. Dr. Volker Krömker
2. Gutachter: Prof. Dr. Nicole Kemper
Tag der mündlichen Prüfung: 10.11.2020Unsere größte Schwäche liegt im Aufgeben. Der sicherste Weg zum Erfolg ist immer, es doch noch einmal zu versuchen. Thomas Alva Edison (Amerikanischer Erfinder, 1847 - 1931)
Meinen Eltern in tiefster Dankbarkeit
Teile der vorliegenden Arbeit wurden bereits auf den folgenden Tagungen vorgestellt: Hohmann, M.-F., Wente, N., Zhang, Y., Krömker; V. Bestimmung der Keimdichte auf der Haut der Zitzenspitze Mastitisnachmittag: Forschung für die Praxis Hannover, 01.03.2019 Hohmann, M.-F., Wente, N., Zhang, Y., Krömker; V. Ausgewählte Risikofaktoren für Mastitiden in der Haltungsumwelt 44. Leipziger Fortbildungsveranstaltung: Labordiagnostik in der Bestandsbetreuung Leipzig, 21.06.2019 Hohmann, M.-F., Wente, N., Zhang, Y., Krömker; V. Mastitiserreger auf der Zitzenspitze - was beeinflusst die Erregerdichte auf der Haut Mastitisnachmittag: Forschung für die Praxis Hannover, 06.03.2020 Teile der vorliegenden Arbeit wurden bereits publiziert: Hohmann, M.-F., Wente, N., Zhang, Y., Klocke, D., Krömker, V. Comparison of two teat skin sampling methods to quantify teat contamination (Vergleich zweier Methoden zur Quantifizierung der Zitzenkontamination) Milk Science International, 73, 2-6 Veröffentlicht im Februar 2020 Hohmann, M.-F., Wente, N., Zhang, Y., Krömker; V. Bacterial load of the teat apex skin and associated factors at herd level (Bakteriendichte auf der Zitzenspitze und assoziierte Faktoren auf Herdenebene) Animals, 10, 1647, DOI: 10.3390/ani10091647 Veröffentlicht: 14.09.2020
Inhaltsverzeichnis
Inhaltsverzeichnis
1. Einleitung……….……………………………………………………………………..9
2. Publikation I ………………………………………………………...………………14
2.1. Abstract………………………………………………………………………15
2.2. Introduction………………………………………………………………….16
2.3. Material and methods………………………………………………………17
2.4. Results……………………………………………………………………….19
2.5. Discussion……………………………………………….…………………..21
2.6. Conclusion…………………………….………………………………….…24
2.7. References…………………………………………………………………..25
3. Publikation II……………………………………………………………….………..28
3.1. Simple Summary…………………………………..……………………….29
3.2. Abstract…………………….………………………………………………..29
3.3. Introduction………………………………………………………………….30
3.4. Material and methods………………………………………….…………..31
3.5. Results……………………………………………….………………………36
3.6. Discussion………………….………………………………………………..44
3.7. Conclusion………………….……………………………………………….51
3.8. Appendix…………………………………………………………………….52
3.9. References………………………………………………………….……….56
4. Diskussion……………………..…………………………………………………….61
5. Zusammenfassung……………..…………………………………………………..71
6. Summary……………...……………………………………………………………..73
7. Literaturverzeichnis……………………………..………………………………….75
8. Danksagung ………………...……………………………………………………...81Einleitung
1. Einleitung
Mastitis, die Entzündung der Milchdrüse, ist eine der wirtschaftlich bedeutendsten
Erkrankungen der Milchkuh im 21. Jahrhundert. Aufgrund verminderter
Syntheseleistung, veränderter Inhaltsstoffe, erhöhter somatischer Zellgehalte und
Behandlungskosten ist sie von enormer Bedeutung für das einzelne Tier sowie für den
ökonomischen Erfolg des Milchviehbetriebes (Seegers et. al., 2003). Es handelt sich
um eine Entzündung, die in den meisten Fällen in Folge verminderter
Abwehrreaktionen des Wirtsorganismus mit nachfolgender Invasion pathogener
Mikroorganismen entsteht (Krömker, 2007). Abgesehen von speziellen Mastitiden, für
die eine endogene Invasion auf hämatogenem, lymphogenem bzw. perkutanem Weg
diskutiert wird, gelangen die meisten Mastitiserreger galaktogen über den Zitzenkanal
an der Zitzenspitze in die Milchdrüse (DVG, 2002). Dort vermehren sie sich und führen
zu einer Entzündung der auskleidenden Schleimhäute und des Drüsengewebes mit
den oben beschriebenen Folgen. Mikroorgansimen, die für die Entstehung einer
Mastitis verantwortlich sind, werden epidemiologisch kategorisiert in „kuhassoziiert“
und „umweltassoziiert“. Neuere Untersuchungen weisen darauf hin, dass für manche
Erreger, zum Beispiel Streptococcus (Sc.) agalactiae und Klebsiella pneumoniae,
gewisse „Grauzonen“ existieren (Zadoks et al., 2011). Kuhassoziierte Mastitiserreger
stammen aus dem erkrankten Euterviertel und werden in den meisten Fällen beim
Melken über die Melktechnik oder über die Milch von Tier zu Tier übertragen. Zu dieser
Gruppe werden Staphylococcus (Staph.) aureus, Sc. agalactiae, Sc. dysgalactiae
sowie Streptokokken der serologischen Gruppe G und L gezählt (Krömker, 2007).
Jørgensen et al. (2016) vermuten ein Reservoir von Sc. agalactiae im bovinen
Gastrointestinaltrakt sowie im Haltungsumfeld der Milchkuh. Sie diskutieren einen
kontagiösen und einen oro-fäkalen Infektionsweg (Jørgensen et al., 2016). Durch
strategische Bekämpfungsmaßnahmen und gezielten Antibiotikaeinsatz in den letzten
Jahren konnte die Inzidenz von Infektionen mit kuhassoziierten Erregern immer mehr
zurückgedrängt werden, wodurch umweltassoziierte Mastitiserreger an Bedeutung
gewonnen haben (Makovec und Ruegg, 2003). Diese stammen aus dem
Haltungsumfeld der Tiere und ihre Übertragung findet überwiegend zwischen den
Melkzeiten im Stallbereich oder auf der Weide statt. Zu dieser Gruppe zählen Sc.
uberis und Enterococcus spp., vornehmlich Enterococcus faecium und Enterococcus
faecalis (zusammengefasst als äskulinspaltende Streptokokken) sowie coliforme
Keime, wie Escherichia (E.) coli, Klebsiella spp. und Enterobacter spp. (Krömker,
9Einleitung
2007). Laut aktuellen Untersuchungen sind die am häufigsten aus Milchproben
klinischer Mastitiden isolierten Umwelterreger Enterokokken, Sc. uberis und coliforme
Keime (Pinzón-Sánchez & Ruegg, 2011; Wente et al., 2016). Die Entstehung von
„Umweltmastitiden“ wird durch zwei Faktoren maßgeblich beeinflusst: 1. Die Dichte an
pathogenen Mastitiserregern an der Zitzenspitze respektive im Haltungsumfeld und 2.
Durch die Immunkompetenz des Tieres. Letztere wird unter anderem bestimmt durch
das Laktationsstadium (Burvenich et al., 2003), die Energiebalance (Suriyasathaporn
et al., 2000), Vitamindefizite (Smith et al., 1997) und den Impfstatus (Bradley et al.,
2015). Die anfälligste Zeit an einer Mastitis, verursacht durch umweltassoziierte
Erreger, zu erkranken ist die Zeit zu Beginn der Trockenstehphase und peripartal um
die Kalbung. Hier kommt es aufgrund vieler Stoffwechselumstellungen zu einer starken
Beeinträchtigung der immunologischen Abwehr der Kuh (Krömker, 2007; Bradley et
al., 2015). Weiterhin stellt der Zitzenkanal an der Zitzenspitze für die meisten
Mastitiserreger die Eintrittspforte zur Milchdrüse dar, weshalb die Erregerdichte vor der
Zitzenspitze die Entstehung von „Umweltmastitiden“ maßgeblich beeinflusst. Zur
Quantifizierung von Erregerdichten an der Zitzenkuppe sind mehrere Verfahren in der
Literatur beschrieben: Die meisten Autoren verwenden einen einzelnen Baumwoll-
oder Mulltupfer, welcher trocken (De Visscher et al., 2014) oder leicht angefeuchtet
(Baumberger et al., 2016; Verdier-Metz, 2012; Monsallier et al., 2012) verwendet wird.
Dabei werden die Zitzen beprobt, indem die Tupfer entweder um die Zitzenspitze
geführt werden (De Visscher et al., 2014) oder indem die Zitzen beginnend an der
Zitzenbasis, absteigend über die Zitzenspitze und wieder aufsteigend bis zur
Zitzenbasis abgefahren werden (Baumerberger et al., 2016). Pfannenschmidt
modifizierte in seiner Promotion 2003 die Nass-Trockentupfer-Technik (DIN 10113-1;
1997-07), zur Bestimmung von Keimdichten in Melkanlagen. Paduch & Krömker
(2011) modifizierten diese Technik, sodass Keimdichten an der Zitzenspitze
zuverlässiger bestimmt werden können. Diese optimierte Methode wurde in weiteren
Studien verwendet, um die mikrobielle Belastung an der Zitzenspitze quantifizieren zu
können (Paduch et al., 2012 & 2013; Svenneson et al., 2019).
Mittels unterschiedlicher Techniken konnte schon früh belegt werden, dass die
Rate neuer intramammärer Infektionen mit zunehmender bakterieller Belastung an den
Zitzenenden steigt (Neave et al., 1969; Pankey, 1989). Physikalische Eigenschaften
der Zitzenspitzen selbst können dabei Einfluss auf die Eutergesundheit nehmen:
Paduch et al. (2012) konnten zeigen, dass Zitzen mit ausgeprägten Hyperkeratosen,
10Einleitung
die durch eine unzureichende Anpassung der Melktechnik an die Tiere verursacht
werden, eine höhere mikrobielle Belastung mit E. coli und Sc. uberis aufwiesen als
Zitzen mit weniger rauhen Zitzenspitzen. Hyperkeratosen fördern durch Rhagaden die
Keimakkumulation und erschweren die Reinigung der Zitzenspitze. Ein
Zusammenhang zwischen Hyperkeratosen und einer erhöhten Inzidenz von Mastitiden
wird immer wieder kontrovers in der Literatur diskutiert (Neijenhuis et al., 2001; Zoche-
Golob et al., 2015; Guarin et al., 2017; Svenneson et al., 2019). Assoziationen
zwischen Zitzendimensionierungen (Länge, Zitzen- & Zitzenspitzendurchmesser) und
der Belastung mit mastitisrelevanten Mikroorganismen konnte bislang nicht festgestellt
werden. In derselben Studie wurden jedoch höhere Erregerdichten auf Zitzen der
Hinterviertel als auf Zitzenhautabstrichen der vorderen Zitzenpaare gefunden. Dies
folgt vermutlich aus einer höheren Spritzkontamination beim Laufen sowie durch
Anhaften von Schmutz und Kot der hinteren Gliedmaßen beim Liegen (Guarin et al.,
2017). Eine erhöhte mikrobielle Belastung sowie ein erhöhtes Mastitisrisiko durch stark
verschmutzte Euter wurde mehrfach beschrieben (Schreiner & Ruegg, 2003; Munoz
et al., 2008; Firth et al., 2019). Hauptreservoir von Umweltkeimen sind benutzte und
unbenutzte Einstreu sowie Kot. Mehrfach wurde beschrieben, dass die Erregerdichte
an der Zitzenspitze mit den Erregerdichten in der Einstreu positiv korrelieren (Lowe et
al., 2015; Rowbotham & Ruegg, 2016). Generell scheint organische Einstreu höhere
Keimdichten an coliformen Keimen und Streptokokken aufzuweisen, als anorganische
Einstreu, wie beispielsweise Sand. Für Sägespäne wurden höhere Keimdichten an
Klebsiella spp. beschrieben, während in Stroh höhere Werte an Streptococcus spp.
ermittelt wurden (Hogan et al., 1989). Ob die unterschiedlichen Einstreumaterialien
allerdings als Ursprungsquelle oder eher als geeignetes Medium zur Vermehrung für
die jeweiligen Pathogene dienen, ist nicht genau bekannt. Neben dem direkten
Einfluss der Erregerdichten in der Umwelt beeinflussen auch Managementpraktiken
die mikrobielle Flora an der Zitzenspitze: Wissenschaftler aus den USA und den
Niederlanden stellten heraus, dass die Keimdichten an E.coli und Streptococcus spp.
in der Einstreu und an der Zitzenspitze sinken, je häufiger die Laufwege im Stall
gesäubert werden (Lowe et al., 2015; Santmann-Berends et al., 2016). Paduch et al.
(2013) wiesen weiterhin deutlich geringere Keimdichten von Sc. uberis, E. coli und
anderen coliformen Keimen nach, wenn die Einstreu mit alkalisierenden Substanzen,
in diesem Fall einem Mischprodukt mit Löschkalk, behandelt wurde, als in
unbehandelter Einstreu. Höhere Gehalte an Lactobacillus spp. und Enterococcus spp.
11Einleitung
wurden mit einer auf Silage basierenden Ernährung, Freilaufställen mit Stroheinstreu
und mäßiger Melkhygiene, aber auch mit multiparen Kühen in Verbindung gebracht
(Monsallier et al., 2011). Während Fliegen als Überträger für Staph. aureus und Sc.
agalactiae beschrieben wurden, wird die gemeine Stechfliege oder Stallfliege,
Stomoxys calcitrans, als Überträger für E. coli vermutet (Castro et al., 2016).
Zur Verbesserung des Tierwohls und zur ökonomischen Stabilisierung von
Milchviehbetrieben erscheint es sinnvoll, Konzepte zur Reduktion oben beschriebener
Mikroorganismen auf der Haut der Zitzenspitze entwickeln zu können. In der
konventionellen Milchviehhaltung wie in der Nutztierhaltung insgesamt steht der
Einsatz von Antibiotika durch befürchtete Resistenzbildungen zunehmend in der Kritik
(Oliver et al., 2011). Jeder Einsatz antibakterieller Wirkstoffe in der Veterinär- und in
der Humanmedizin fördert die Selektion von Resistenzen in Mikroorgansimen (Kaspar
et al., 2015). Infektionen, welche durch antibiotikaresistente Bakterien verursacht
werden, nehmen zu, sind schwieriger zu therapieren und führen folglich häufiger zu
komplizierteren Krankheitsverläufen. Die WHO kategorisierte Antibiotika anhand ihrer
humanmedizinischen, kritischen Bedeutung (Critically Important Antimicrobials; CIA),
um für einen bedachten Einsatz und eine weiterhin erhaltene Wirksamkeit zu sorgen.
Viele als „kritisch“ angesehen Antibiotika werden auch in der Tierhaltung und in der
Mastitistherapie eingesetzt, zum Beispiel Cephalosporine der 3. und 4. Generation und
Fluorchinolone (WHO, 2019). Gemäß der 2015 vom Bundeskabinett verabschiedeten
„Deutschen Antibiotika-Resistenzstrategie 2020“(DART 2020) gilt die Vermeidung von
Infektionen als eine der Hauptmaßnahmen, die zur Verringerung des
Antibiotikaeinsatzes nötig ist, um im Rahmen des „One health“ - Ansatzes die
Entstehung und Ausbreitung von Antibiotikaresistenzen sektorübergreifend
eindämmen zu können. Der Fokus im Umgang mit Mastitiden liegt folglich auf deren
Prävention. Daraus lässt sich schließen, dass dies eine verringerte Exposition der
Zitzenspitze gegenüber umweltassoziierten Mastitiserregern sowie eine Verbesserung
der immunologischen Reaktion des Wirts auf bakterielle Herausforderungen
voraussetzt.
Oben beschriebene Arbeiten, welche sich mit den Einflussfaktoren auf die
Erregerdichte an der Zitzenspitze beschäftigt haben, beziehen sich in den meisten
Fällen auf das einzelne Tier oder wurden unter vorher bestimmten
Versuchsbedingungen durchgeführt. Im Fokus des modernen Herdenmanagements
12Einleitung
bzw. der heutigen tierärztlichen Bestandsbetreuung steht zunehmend die Herde als
Gesamtheit. Deshalb war es das Ziel dieser Arbeit, praxisnah mögliche Risikofaktoren
für die Dichte umweltassoziierter Mastitiserreger an der Zitzenspitze auf Herdenebene
ableiten und benennen zu können. In einem ersten Versuch wurde dazu die
analytische Übereinstimmung zweier Methoden zur Quantifizierung von
Mikroorganismen auf der Zitzenspitze verglichen. Die in der Literatur häufig
verwendete modifizierte Nass-Trockentupfer-Technik (NTT) galt hierfür als
Referenzmethode und wurde mit einer neuen Technik verglichen, bei der die
Zitzenspitze für einen definierten Zeitabschnitt in einen Behälter mit steriler
Ringerlösung getaucht wurde. Grund für die Etablierung neuer Methoden ist der nicht
gänzlich auszuschließende Untersucherbias bei der Entnahme von Nass-
Trockentupfern (Druck, der auf die Tupfer ausgeübt wird und Geschwindigkeit, mit der
die Tupfer um die Zitzenspitze geführt werden) sowie der enorme Zeitaufwand,
welcher in die Vor- und Nachbereitung der Tupfer investiert werden muss. In einem
zweiten, größer angelegten Versuch wurden auf zufällig ausgewählten
Milchviehbetrieben unterschiedlichster Größen Assoziationen zwischen der
Zitzenhautbesiedlung mit äskulinspaltenden Streptokokken und coliformen Bakterien
und Betriebsgegebenheiten (mikrobielle Belastung der Umwelt, Hygienemanagement,
Melkmanagement) untersucht. Hierfür wurden in regelmäßigen Abständen
Betriebsbesuche durchgeführt. Neben mikrobiologischen Untersuchungen von Nass-
Trockentupfern der Zitzenspitze, Einstreuproben und Luftkeimproben, wurden
individuelle Betriebsfaktoren berücksichtigt. Die Betriebe wurden hierfür nicht
vorselektiert, lediglich Betriebe, die ein Automatisches Melksystem benutzen, wurden
ausgeschlossen. Um die sich verändernden Risikofaktoren auf die Erregerdichten der
Zitzenspitze berücksichtigen zu können, wurden die Daten je Besuchstermin erfasst
und statistisch analysiert.
13Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
2. Publikation I: Comparison of two teat skin sampling methods to quantify
teat contamination
(Vergleich zweier Methoden zur Quantifizierung der Kontamination auf der
Zitzenhaut)
Maria-F. Hohmann1, Nicole Wente1, Yanchao Zhang1, Doris Klocke1, Volker
Krömker1,2
1Fakultät II, Abteilung für Bioverfahrenstechnik – Mikrobiologie, Hochschule
Hannover
2 Faculty
of Health and Medical Sciences, Department of Veterinary and Animal
Sciences, Section for Production, Nutrition and Health, University of Copenhagen,
Gronnegardsvej 2, 1870, Frederiksberg C, Denmark
Milk Science International 2020, 73: 2-6.
Eingereicht: 17.12.2019
Akzeptiert: 28.01.2020
14Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
2.1. Abstract
The aim of this research was to compare two sampling methods quantifying
microbial load on teat ends, especially mastitis pathogens originating from the cows’
surroundings. Methods were compared using a split udder design, including 132 teat
pairs in the study. For the first method, the wet/dry swab technique, a moistened swab
was rotated 360° around the teat end, followed by a dry swab in the same manner. For
the second and new method, the dipping technique, teat ends were immersed in a cup
filled with Ringer’s solution and were removed after five seconds. Microbial load per
milliliter as well as per teat end was calculated by determining the number of total
aerobic mesophilic bacteria as well as environmental pathogenic bacteria, including
coliform bacteria and esculin-positive streptococci. The concordance correlation
coefficient (CCC) was used to quantify the agreement between two series of
measurements and revealed the following coefficients: 0.112 for total aerobic
mesophilic bacteria; 0.008 for coliform bacteria and 0.001 for esculin positive
streptococci. The results of this study point out that under field conditions, the new
method does not provide similar results when compared with the wet/dry swab
technique for determining teat end microbial load.
Keywords
Teat end colonization, mastitis pathogens, wet/dry swab technique, dipping
technique, microbial load
15Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
2.2. Introduction
Bovine mastitis, the inflammation of the mammary gland, is a complex disease
considering its etiology, pathogenesis and therapy. Due to its enormous importance
for the individual cow as well as for the economic losses caused by the disease, it is
necessary to further characterize causative pathogens in order to develop control
strategies [1,2].
A wide variety of microorganisms are discussed as being responsible for the
development of mastitis and these can be epidemiologically categorized into
contagious, originating from infected quarters, or environmental, located in the
surroundings of dairy cows [3,4,5,6]. While the prevalence of contagious mastitis has
been reduced by control programs in recent years, environmental pathogens are
becoming increasingly important [4]. Most prevalent environmental microorganisms
isolated in milk samples of clinical mastitis cases are esculin-positive streptococci,
Escherichia coli and Klebsiella spp. [7].
In recent years, many authors have shown that teat end bacterial load can affect
udder health [8,9,10]. Microorganisms found on the teat surface, especially coliforms
and streptococci, are chemotrophic organisms requiring organic material to use for
their metabolism. If these bacteria are present in large populations on the teat skin,
this reflects a transient rather than a resident flora [11]. Furthermore, Paduch et al.,
2012 [12] pointed out that environmental bacterial load of the teat canal increases with
highly calloused teat ends. Furthermore, some studies revealed a lower microbial load
on teat skin in primiparous cows, which might be caused by less contact with litter due
to a lower udder depth or smoother surface [6]. Thus, it can be assumed that the teat
skin serves as a reservoir for pathogens, posing a risk for udder health.
As can be seen, it is necessary to gain more information concerning the variation
in the bacterial load on teat epithelia. For this purpose, some researchers described
methods quantifying teat end bacterial load. Most authors used only one cotton or
gauze swab, either dry [13] or moistened [14,15]. Teats were sampled by rotating
[13,16] or by wiping one side of the teat barrel from top to bottom, passing over the
teat end and wiping the other side of the teat barrel from top to bottom [14]. Paduch &
Krömker [17] modified the wet/dry swab technique (DIN 10113-1; 1997-07) used in a
previous study by Pfannenschmidt, 2003 [18] for determining the bacterial content in
milking equipment. Furthermore, the technique was used in other investigations
16Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
dealing with teat end bacterial load [10,12,19]. Nevertheless, personal influences,
particularly the pressure exerted on the swab or the speed of swabbing, together with
the amount of work invested in sample collection and preparation (two swabs) need to
be reduced to provide reliable results [18]. To make the procedure faster and more
objective, we evaluated a new method where teats were dipped in a sample vessel
filled with Ringer’s solution for a constant period of time. The aim of the study at hand
was to compare the analytical performance of two methods, the wet/dry swab
technique and a new dipping technique, for quantifying the microbial load of
environmental mastitis pathogens on dairy cows’ teat skin.
2.3. Material and Methods
The study took place from June to July 2018 and was conducted on two
commercial dairy farms in Lower Saxony, northwestern Germany. Two herds
participated: One herd of 35 cows mainly included German Holstein Red cows, with
an average milk yield of 8,095 kg (Dairy Herd Improvement Association, DHIA) and
mean bulk milk somatic cell count of 269,000 cells/mL; the second herd of 65 animals
mainly consisted of German Holstein Black cows, with an average milk yield of 10,856
kg (DHIA) and a mean bulk milk somatic cell count of 218,000 cells/mL. All cows were
housed in free-stall barns, either sawdust-bedded or bedded in dried horse manure
mixed with shredded straw and alkalized with lime. Healthy lactating primiparous and
multiparous cows were selected as described by Paduch & Krömker [17], according to
the following criteria: Four functional quarters without udder infections or signs of
clinical mastitis (i.e., no clotting or discoloration of milk, no swelling or udder redness
and no heat upon udder palpation), apparently clean udders (teat skin without
splashing or plaques of manure), no visible udder lesions or trauma, teat tissue and
skin that appeared normal.
The study included the results of 66 cows, so that a total of 132 teat pairs were
sampled. A split-udder design was used to compare the methods at udder half level.
A teat was matched with its contralateral teat to eliminate individual influences. In the
following, each method was used once per udder half and could be compared on the
basis of this. Two teat pairs were tested per cow.
Two contralateral teats (e.g., left front, right rear or right front, left rear) were
sampled with the modified wet/dry swab technique after the pre-cleaning and pre-
milking routine before milking as described by Paduch and Krömker [17]. The first swab
17Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
(ultrafine, Dry Swab, Check Diagnostics GmbH, Westerau, Germany) was moistened
with ¼ Ringer’s solution (Merck, Darmstadt, Germany) and rotated 360° around the
teat canal orifice at a distance of 1 cm from the teat apex. The same procedure was
carried out with the dry swab. Immediately after sampling, the tips of both swabs were
transferred to one tube containing 2 mL of sterile Ringer’s solution.
The remaining contralateral teats were prepared in the same way. These were
dipped in a cup filled with 40 mL of ¼ Ringer’s solution (Merck, Darmstadt, Germany)
until the lower 1.5 cm of the teat had been moistened. After five seconds, the teat was
removed from the dip solution. All samples were taken during the morning milking by
one researcher and then transported at 5 °C to the microbiology laboratory at the
University of Applied Sciences and Arts Hannover (Germany) within 8 h. Samples were
discarded directly at the time of sampling when any obvious contamination took place
or liquid was spilled. Therefore, a new pair of teats was sampled on another cow in a
similar manner. In the laboratory, both swab samples and dipping samples were
vortexed (Vortex Genie2, Scientific Industries Inc., Bohemia, NY, USA) each for 20
seconds and swabs were then removed from the swab samples with sterile tweezers.
Serial 1:10 dilutions were prepared with ¼ Ringer’s solution and a volume of 0.1 mL
was spread in duplicate over the whole of a pre-dried 9 cm diameter agar plate with a
Drigalski spatula. The total number of aerobic mesophilic bacteria was determined with
Plate Count agar (Merck, Darmstadt, Germany) and incubated at 30 °C for 72 h.
ChromoCult Coliform agar (Merck, Darmstadt, Germany) was used for detecting
coliform bacteria, while esculin positive streptococci (e.g., Streptococcus (S.) uberis,
Lactococcus lactis, Enterococcus spp.) were determined with Kanamycin Esculin
Azide agar (Merck, Darmstadt, Germany). The latter two were incubated at 37 °C for
24 h. Plates with 1-300 colonies were used to calculate bacterial counts in swab and
dipping solution [17]. The weighted arithmetic mean was calculated for each pathogen
group and results were indicated in colony-forming units per milliliter (cfu/mL) as well
as colony-forming units per teat end. A teat end represents the lower 1 cm (wet/dry
swab technique) or 1.5 cm (dipping technique) of the teat and is assumed to have a
diameter of 19.6 mm [20], giving an estimated teat skin area of around 9.2 cm² or 12.3
cm², respectively.
As bacterial counts were not normally distributed, results were log10-transformed
prior to further analysis. To calculate the reliability between bivariate pairs of
18Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
observations, we used the Lin’s Concordance Correlation Coefficient, as suggested by
Watson & Petrie and Koch & Spoerl [21,22,23]. The CCC provides a measure of
reliability based on correspondence. For measuring the agreement between two
continuous variables, values from -1 to +1 occur, whereby 1 indicates strong
concordance [24]. The CCC was computed by SPSS 25.0 (IBM Inc., Chicago, IL,
USA).
As there was no normal distribution of bacterial counts, we used the Kruskal-Wallis test
to determine whether the teat end bacterial load showed a tendency towards a specific
lactation number, related to the results of Rowbotham & Ruegg [6]. In accordance with
Rowbotham & Ruegg [6] and because the wet/dry swab technique represents our
reference method, results thereof were used for this purpose.
2.4. Results
The calculations included bacterial loads of 132 teat pairs (Table 1). In teat skin
swabs, the median total mesophilic count was 5.008 log10 cfu/mL Ringer’s solution or
rather 5.309 log10 cfu/teat end. In dipping solution, the median was 3.374 log 10 cfu/mL
and 4.975 log10 cfu/teat end for total mesophilic counts. Calculation of concordance
yielded the following results for total aerobic mesophilic counts: CCC = 0.112 (CI: 0.057
- 0.165). A closer evaluation of the group of environmental pathogens revealed the
following for coliform bacteria: Teat swabs: median = 1.301 log10 cfu/mL or in relation
to the sampled area: median = 1.477 log10 cfu/teat end. In the dipping solution, the
median was 1.000 log10 cfu/mL and 1.000 log10 cfu/teat end. The CCC result was 0.008
(CI: 0.005 - 0.01) for log10-transformed counts. The other main group of environmental
bacteria, esculin-positive streptococci, were found in wet/dry swabs with a median of
2.249 log10 cfu/mL or rather 2.538 log10 cfu/teat end. For the new dipping technique
method, a median of 1.000 log10 cfu/mL was found. Extrapolating the results to the teat
apex provided a median of 1.000 log10 cfu/teat end. Nevertheless, the CCC was 0.001
(CI: - 0.001 - 0.003).
We could not detect any differences in the bacterial load of the teat ends between
the lactation numbers. Results of the Kruskal-Wallis test are presented in Table 2.
19Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
Table 1: Calculations of log10 cfu/mL and log10 cfu/teat end of 132 teat skin pairs classified by
wet/dry swab technique and dipping technique. Concordance correlations coefficient (CCC) is given to
evaluate the inter-rater reliability.
Pathogen group Wet/dry swab technique Dipping technique CCC1
Median Median
Median Median
[log10 cfu/teat [log10 cfu/teat
[log10 cfu/mL] [log10 cfu/mL]
end] end]
(Minimum - (Minimum -
(Minimum - (Minimum -
Maximum) Maximum)
Maximum) Maximum)
0.112
Total aerobic 5.008 5.309 3.374 4.975
(CI: 0.057 -
mesophilic bacteria (3.107 - 6.477) (3.407 - 6.778) (1.653 - 6.233) (3.149 - 7.835)
0.165)
0.008
1.301 1.477 1.000 1.000
Coliform bacteria (CI: 0.005 -
(1.000 - 3.138) (1.000 - 3.438) (1.000 - 2.952) (1.000 - 4.549)
0.010)
0.001
Esculin positive 2.249 2.538 1.000 1.000
(CI: -0.001 -
streptococci (1.000 - 4.477) (1.000 - 4.778) (1.000 - 2.975) (1.000 - 4.573)
0.003)
1Strength-of-agreement criteria for Lin’s concordance correlation coefficient by McBride [24]
> 0.99 almost perfect agreement
0.95 - 0.99 substantial agreement
0.90 - 0.95 moderate agreement
< 0.90 poor agreement
Table 2: Ranking means of teat end microbial loads of 132 teats examined by wet/dry swab
technique, classified by lactation number and pathogen group. The Kruskal-Wallis test was performed
to test for lactation-dependent differences in the microbial load of teat skin.
Aerobic mesophilic Esculin-positive
Coliform bacteria, cfu/mL,
No. Lactation N bacteria, cfu/mL, streptococci, cfu/mL,
ranking means
ranking means ranking means
1 54 63.558 60.417 64.278
2 22 56.750 83.614 73.955
3 28 71.036 63.446 65.786
4 28 70.446 67.839 65.643
p-value1 132 0.491 0.089 0.791
1 p < 0.05, results of the Kruskal-Wallis test
20Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
2.5. Discussion
Swabbing surfaces to determine their bacterial load is one of the oldest methods
for this purpose. This sampling procedure is easy to handle for researchers and can
be applied in places which are difficult to access as well as requiring low expenditure
in terms of equipment and time. Referring to the above-mentioned points, the swabbing
method has become a widely used method in many studies [11,17,18,26]. On the other
hand, the high work-load involved in preparing and processing the swabs and the
deficiencies of a standardized procedure are regarded as disadvantages. Above all,
the inconsistency of the pressure applied on the swabs for removing bacteria from
surfaces is to be mentioned [18]. In order to standardize the method for determining
the microbial load on the teat, we tried to verify a comparative method, which could
lead to similar results as the more complicated method described above.
With regard to the microbial load on teat skin, Paduch & Krömker [17] reported
similar results for wet/dry skin swabs as we did. They found that the largest populations
on teat skin were S. uberis (maximum: 6.48 log10 cfu/mL) and coliforms (maximum:
6.48 log10 cfu/mL). These results were comparable with those of our study, whereby
the largest populations of environmental pathogens were esculin-positive streptococci
(maximum: 4.477 log10 cfu/mL) found in wet/dry teat skin swabs. It can therefore be
suspected that esculin-positive streptococci mainly originated from the environment
which the cows’ teats were exposed to for most of the day or that esculin-positive
streptococci are a part of the teat skin flora [6,14]. The latter disagrees with findings of
other researchers examining the microbial teat skin flora, by showing that esculin-
positive streptococci can be influenced by bedding and milking hygiene [19,25]. On the
contrary, the median amount of these streptococci in our study (median: 2.249 log10
cfu/mL) was above those values published by Paduch & Krömker (median: 1.71 log 10
cfu/mL) [17] as we considered all streptococci, hydrolyzing esculin (S. uberis,
Lactococcus spp., Enterococcus spp.), while Paduch & Krömker only referred to S.
uberis, subcultivated on modified Rambach agar. The median values for coliforms
differed slightly in both studies. The different results for esculin-positive streptococci
and coliforms can be explained by the larger sample size examined by Paduch &
Krömker [17] (n = 839 teat skin swabs from 32 herds), therefore leading to a wider
variation in teat skin bacterial load. These differences reveal that the udder skin is not
a uniform system and that the bacterial population differs from udder to udder [27].
21Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
Reasons for this might be physiological or environmental selective processes, possible
preferences of microorganisms for particular udder sites or any bacterial antagonism
mechanisms [26]. Furthermore, the above listed individual influence of the sampling
itself might be responsible for differing results. Cullen & Herbert [27] observed
fluctuations in bacterial load of staphylococci on teat skin throughout the year,
considering seasonal changes and changing stages of lactation. Furthermore,
antagonistic organisms on teat skin, particularly Staphylococcus chromogenes, are
suspected to affect the presence of other organisms [28].
Another source of differences to the above-named study of Paduch & Krömker [17]
could be that cows from different numbers of lactation were sampled in different
proportions in the studies. It is not mentioned in which ratio the primiparous and
multiparous cows were sampled. Rowbotham & Ruegg, 2016 [6] concluded that
primiparous cows have less bacterial teat load than multiparous cows, which, in turn,
leads to different study results depending on whether more primiparous or multiparous
cows are tested. Although the amount of cows per number of lactation is low in the
present study, the number of lactations is not decisive because both methods were
tested on the same cow in a split-udder design. To determine the extent of influence
of parity on teat skin bacterial load, we used the findings of the wet/dry swab technique.
However, remarkable differences could not be found for all pathogen groups, which
means that teat end bacterial load does not depend on the number of lactations in the
current study, as reflected in our results for wet/dry swabs. With regard to teat skin
bacterial load, varying correlations clarify that it is affected by several factors.
Referring to the CCC described by Lin, 1989 [21], comparing data of the new
dipping technique with the wet/dry swab technique for investigating teat end bacterial
load showed large differences between the two methods for esculin-positive
streptococci as well as for coliform counts. Highest concordance between the results
of the two measurement series existed with the total aerobic mesophilic bacteria,
where the CCC was 0.112, which, however, describes a poor agreement [24] .
In addition, the bacterial loads per teat end determined by the dip technique were
basically lower than those values determined with the wet/dry swab technique. This
probably results from the fact that the dipping solution cannot remove a sufficient
number of bacteria from the teat skin in the short duration of time. Prolonging the
dipping time could cause the results of the samplings to converge. In a pilot study,
22Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
different immersion times were tested, wherein five seconds turned out to be the
longest span accepted by the cows. However, the dipping method actually sampled a
greater teat area, as the swabs only dabbed the side walls of the lower 1 cm of the
teat, whereas the dipping solution also covered the teat apex with the teat canal orifice.
On the other hand, the dipping technique does not involve the mechanical action of
wiping, as with the wet/dry swab technique, so that a lower yield could be expected for
the dipping technique. Thus, the reasons for the poor concordance of the methods
could be the differences in location, surface sizes and the mechanical impacts.
Determining bacterial load is a destructive method, one sampling excluding a
subsequent one, since the bacterial load after the first sampling would of course be
reduced regardless of which sampling method is carried out first [29]. Therefore, we
used the split udder design, although we presumed that different teats would also differ
in their teat skin bacterial load. Furthermore, lower bacterial counts produced by the
dipping method were suspected to be a result of the higher volume and the higher
dilution of Ringer’s solution. While establishing the method, we tried to keep the volume
as low as possible but the limiting factor was the sampling vessel, which had to be
large enough for the teats to fit in and to dip them in the solution.
For both techniques, time and effort in the laboratory was the same. Without having
measured the time for sampling, the time required for the dipping technique seems to
be less, as the two swabs have to be taken out of their sterile packages one by one.
However, after some repetitions, the wet/dry swab technique with two swabs is a
practicable method as well. It also has to be noted that the Ringer’s solution for the
dipping technique should be at room temperature. Otherwise, there was a massive
defense reaction by the cows.
Neither for esculin-positive streptococci nor for coliforms was the CCC greater than
0.112, which was calculated for total mesophilic counts. This means that the dipping
method only produces results compared to those of the wet/dry swab technique with
poor concordance. The median values of each method for each pathogenic group differ
with inconsistently wide ranges, which might be unacceptable for standardized
measurements.
23Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
2.6. Conclusion
In general, teat skin bacterial load is affected by many factors and alongside many
other influences it can affect udder health. Our investigations showed that determining
teat skin bacterial load with the tested dipping technique does not produce similar
results to those when using the wet/dry swab technique. Dipping the teats in the test
medium does not produce equivalent results or results that correlate sufficiently well
with those gained from swabbing the teats. In addition, the procedure appears neither
faster nor easier to handle.
24Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
2.7. References
1. Seegers H, Fourichon C, Beaudeau F. Production effects related to mastitis and
mastitis economics in dairy cattle herds. Vet Res. 2003;34(9):475-491.
2. Hogeveen H, Huijps K, Lam TJGM. Economic aspects of mastitis: New
developments. NZ Vet J. 2011;59(1):16-23.
3. Watts JL. Etiological agents of bovine mastitis. Vet Microbiol. 1988;16(1):41-66.
4. Makovec JA, Ruegg PL. Results of Milk Samples Submitted for Microbiological
Examination in Wisconsin from 1994 to 2001. J Dairy Sci. 2003;86(11):3466-
3472.
5. Haveri M, Hovinen M, Roslöf A, Pyörälä S. Molecular types and genetic profiles
of Staphylococcus aureus strains isolated from bovine intramammary infections
and extramammary sites. J Clin Microbiol. 2008;46(11):3728-3735.
6. Rowbotham RF, Ruegg PL. Bacterial counts on teat skin and in new sand,
recycled sand, and recycled manure solids used as bedding in freestalls. J Dairy
Sci. 2016;99(8):6594-6608.
7. Pinzón-Sánchez C, Ruegg PL. Risk factors associated with short-term post-
treatment outcomes of clinical mastitis. J Dairy Sci. 2011;94(7):3397-3410.
8. Pankey JW. Premilking Udder Hygiene. J Dairy Sci. 1989;72(5):1308-1312.
9. Guarín JF, Baumberger C, Ruegg PL. Anatomical characteristics of teats and
premilking bacterial counts of teat skin swabs of primiparous cows exposed to
different types of bedding. J Dairy Sci. 2017;100(2):1436-1444.
10. Svennesen L, Nielsen S, Mahmmod Y, Krömker V, Pedersen K, Klaas I.
Association between teat skin colonization and intramammary infection with
Staphylococcus aureus and Streptococcus agalactiae in herds with automatic
milking systems. J Dairy Sci. 2019;102(1):1-11.
11. Rendos JJ, Eberhart RJ, Kesler EM. Microbial Populations of Teat Ends of Dairy
Cows, and Bedding Materials. J Dairy Sci. 1975;58(10):1492-1500.
12. Paduch JH, Mohr E, Krömker V. The association between teat end
hyperkeratosis and teat canal microbial load in lactating dairy cattle. Vet
Microbiol. 2012;158(3-4):353-359.
13. De Visscher A, Supre K, Haesebrouck F, Zadoks RN, Piessens V, Van CoillieE
et al. Further evidence for the existence of environmental and host-associated
species of coagulase-negative staphylococci in dairy cattle. Vet Microbiol.
2014;172:466-474.
14. Baumberger C, Guarín JF, Ruegg PL. Effect of 2 different premilking teat
sanitation routines on reduction of bacterial counts on teat skin of cows on
commercial dairy farms. J Dairy Sci. 2016;99(4):2915-2929.
25Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
15. Verdier-Metz I, Gagne G, Bornes S, Monsallier F, Veisseire P, Delbes-Paus,
C, et al. Cow teat skin, a potential source of diverse microbial populations for
cheese production. Appl Environ Microbiol. 2012;78(2):326-333.
16. De Vliegher S, Laevens H, Devriese LA, Opsomer G, Leroy JLM, Barkema HW
et al. Prepartum teat apex colonization with Staphylococcus chromogenes in
dairy heifers is associated with low somatic cell count in early lactation. Vet
Microbiol. 2003;92(3):245-252.
17. Paduch JH, Krömker V. Besiedlung von Zitzenhaut und Zitzenkanal laktierender
Milchrinder durch euterpathogene Mikroorganismen. [Colonization of the teat
skin and the teat canal by mastitis pathogens in dairy cattle.] Tierarztl Prax.
Ausgabe Grosstiere - Nutztiere. 2011;39(2):71-76.
18. Pfannenschmidt F. Eignung des Nass-Trockentupfer Verfahrens (NTT) DIN
10113; 1997-07 zur Bestimmung des Hygienestatus in Melkanlagen
[dissertation]. Tierärztliche Hochschule Hannover, 2003.
19. Paduch JH, Mohr E, Krömker V. The association between bedding material and
the bacterial counts of Staphylococcus aureus, Streptococcus uberis and
coliform bacteria on teat skin and in teat canals in lactating dairy cattle. J Dairy
Res. 2013;80(2):159-164.
20. Guarín JF, Ruegg PL. Short communication: Pre- and postmilking anatomical
characteristics of teats and their associations with risk of clinical mastitis in dairy
cows. J Dairy Sci. 2016;99(10):8323-8329.
21. Lin LI-K. A Concordance Correlation Coefficient to Evaluate Reproducibility.
Biometrics. 1989;45:255-268.
22. Watson PF, Petrie A. Method agreement analysis : A review of correct
methodology. Theriogenology. 2010;73(9):1167-1179.
23. Koch R, Spoerl E. Statistische Verfahren zum Vergleich zweier Messmethoden
und zur Kalibrierung : Konkordanz-, Korrelations- und Regressionsanalyse am
Beispiel der Augeninnendruckmessung. [Statistical Methods for Comparison of
Two Measuring Procedures and for Calibration: Analysis of Concordance,
Correlation and Regression in the Case of Measuring Intraocular Pressure.] Klin
Monatsbl Augenheilk. 2007;224:52–57.
24. McBride G. A proposal for strength-of-agreement criteria for Lin’s Concordance
Correlation Coefficient. NIWA Client Rep. 2005;45(1):307-310.
25. Monsallier F, Verdier-Metz I, Agabriel C, Martin B, Montel MC. Variability of
microbial teat skin flora in relation to farming practices and individual dairy cow
characteristics. Dairy Sci Technol. 2012;92(3):265-278.
26. Guarín JF, Baumberger C, Ruegg PL. Anatomical characteristics of teats and
premilking bacterial counts of teat skin swabs of primiparous cows exposed to
different types of bedding. J Dairy Sci. 2017;100(2):1436-1444.
26Publikation I: Comparison of two teat skin sampling methods to quantify teat contamination
27. Cullen GA, Hebert CN. Some ecological Observations on Microorganisms
inhabiting Bovine Skin, Teat Canals and Milk. Br Vet. J. 1967;123(1):14-25
28. De Vliegher S, Opsomer G, Vanrolleghem A, Devriese LA, Sampimonc OC, Sol
J, et al. In vitro growth inhibition of major mastitis pathogens by Staphylococcus
chromogenes originating from teat apices of dairy heifers. Vet Microbol.
2004;101(3):215-221.
29. Merli D, Amadasi A, Mazzarelli D, Cappella A, Castoldi E, Ripa S, et al.
Comparison of Different Swabs for Sampling Inorganic Gunshot Residue from
Gunshot Wounds: Applicability and Reliability for the Determination of Firing
Distance. J. Forensic Sci. 2018;24:1-7.
27Publikation II: Bacterial load of the teat apex skin and associated factors at herd level
3. Publikation II: Bacterial load of the teat apex skin and associated factors at
herd level
(Bakterielle Belastung der Zitzenspitzenhaut und damit verbundene Faktoren auf
Herdenebene)
Maria-F. Hohmann1, Nicole Wente1, Yanchao Zhang1, Volker Krömker1,2
1Fakultät II, Abteilung für Bioverfahrenstechnik – Mikrobiologie, Hochschule
Hannover
2 Faculty
of Health and Medical Sciences, Department of Veterinary and Animal
Sciences, Section for Production, Nutrition and Health, University of Copenhagen,
Gronnegardsvej 2, 1870, Frederiksberg C, Denmark
Animals
Eingereicht: 20.08.2020
Akzeptiert: 10.09.2020
28Publikation II: Bacterial load of the teat apex skin and associated factors at herd level
3.1. Simple Summary
The bacterial load on the teat apex of dairy cows, causing intramammary infections,
is to a large extent due to environmental impact. The aim of our study was to describe
factors at herd level that are associated with bacterial load of environmental mastitis
pathogens on the teat end’s skin. On visits to 31 dairy farms over a one-year period,
farm conditions were documented and environmental bacterial loads were examined.
We found seasonal fluctuations and direct correlations between the temperature-
humidity index (THI) in the barn and the bacterial load at the teat end. Significantly
more environmental mastitis pathogens were found in herds with a high percentage of
normal and slightly rough teat ends. The time since the last fresh bedding was added
to the cubicles as well as the frequency in which cubicles were cleaned also effect the
pathogen load on the teat skin. Pre-cleaning teats before milking as well as post-
dipping after milking showed a decreasing effect of teat skin bacterial load at herd level.
3.2. Abstract
In order to reduce antimicrobial treatment and prevent environmental mastitis, the
aim of the present study was to investigate associations between herd level factors
and microbial load on teat ends with environmental mastitis pathogens. Quarterly farm
visits of 31 dairy farms over a one-year period were used for statistical analysis. During
each farm visit, teat skin swabs, bedding and air samples were taken, and
management practices and herd parameters were documented. Total mesophilic
bacteria, esculin-positive streptococci and coliform bacteria were examined in the
laboratory procedures from teat skin and environmental samples. Esculin-positive
streptococci and coliform bacteria on teat ends increased with high THIs in the barn
and during the spring and summer. Significantly more coliform bacteria on teat ends
were found in herds with an increased percentage of normal or slightly rough teat ends.
Cleaning cubicles more frequently, pre-cleaning teats before milking as well as post-
dipping them after milking had a decreasing effect of teat skin load with total mesophilic
and coliform bacteria at herd level. To conclude, teat skin bacterial load with
environmental pathogens is subjected to fluctuations and can be influenced by
hygienic farm aspects.
Keywords
Teat end colonization, mastitis pathogens, wet-dry swab technique, bedding, season
29Publikation II: Bacterial load of the teat apex skin and associated factors at herd level
3.3. Introduction
Bovine mastitis, inflammation of the mammary gland, is a complex disease
considering its etiology and pathogenesis. As reducing antimicrobial usage is a social
concern as well as mastitis causes economic losses (reduced milk yield, discarded
milk, culls, therapy costs), it is necessary to further characterize causative pathogens
in order to develop control strategies [1,2]. A wide variety of microorganisms are
discussed as being responsible for the development of mastitis. These can be
epidemiologically categorized into contagious, originating from infected quarters, or
environmental, located in the surroundings of dairy cows [3-6]. While the prevalence
of contagious mastitis has been reduced by control programs in recent years,
environmental pathogens are becoming increasingly important [4]. Most prevalent
environmental microorganisms isolated in milk samples of clinical mastitis cases
occurring on German dairy farms are esculin-positive streptococci, Escherichia coli
and Klebsiella spp. [7].
The teat skin seems to act as a reservoir of microorganisms, especially Gram-
positive catalase-positive bacteria including coagulase-negative staphylococci [8].
Pathogenic bacteria can enter the udder through the teat canal and might cause
intramammary infection (IMI). In recent years, many authors have shown that teat end
bacterial load can affect udder health [9-11]. To gain more information concerning the
variation in the bacterial load on teat epithelia, some researchers described methods
quantifying teat end bacterial load. The wet-dry swab technique, described by Paduch
and Krömker [12], enables a semi-quantitative investigation of the teat end
colonization. Some genera of physiological teat skin flora are stated to inhibit some
isolates of mastitis pathogens [13,14]. Nonetheless, generally, the microbial
community of the teat surface depends on the respective farm environment. Monsallier
et al. [8] showed that farming practices could interact with microbial flora on teat skin.
Early on, it was recommended to reduce the environmental pathogen contamination of
the teat end as a method for controlling environmental mastitis [15]. Cows spend most
of the day lying down, making bedding a primary source for environmental pathogens
to stick onto the teat end skin. It has been published that numeric differences in the
distribution of Streptococcus spp., Staphylococcus spp., and Gram-negative bacteria
on teat skin are linked to different kinds of bedding materials [6,16,17]. Furthermore,
some researchers observed a reduction in teat skin bacterial load of environmental
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