Healthy Cattle Microbiome and Dysbiosis in Diseased Phenotypes

Abstract

: Cattle farming is an ancient practice, with roots in the early Neolithic era that has retained its status in the food industry today, with global beef market revenue amounting to $385.7B, as of 2018. Hence, cattle maintenance is naturally essential to cater to nutritional requirements of modern civilization. This extensive review aims to provide a holistic overview of cattle microbiome, analysing the native microbial composition within respiratory tract, gastrointestinal tract, reproductive tract, and skin. The dysbiosis associated with various diseases such as bovine respiratory disease, bovine digital dermatitis, mastitis, Johne's disease, uterine diseases (metritis and endometritis) and metabolic disorders (ruminal acidosis and ketosis) has been discussed. Moreover, various non-antibiotic microbial therapies including phage therapy, prebiotics and probiotics have been examined as potential means to reduce disease-associated dysbiosis. In general, this review highlights the importance of the microbiome in maintenance of health in cattle and its potential in alleviating bovine diseases, with an aim to enhance cattle health and production.

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Citation: Khalil, A.; Batool, A.; Arif,
S. Healthy Cattle Microbiome and
Dysbiosis in Diseased Phenotypes.
Ruminants 2022,2, 134–156. https://
doi.org/10.3390/ruminants2010009
Academic Editors: Ceferino M.
López Sández and Silvia Preziuso
Received: 28 September 2021
Accepted: 10 February 2022
Published: 23 February 2022
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Review
Healthy Cattle Microbiome and Dysbiosis in Diseased Phenotypes
Alaa Khalil 1, * , Aamina Batool 2and Sania Arif 3
1Department of Animal Science, Georg-August-University, Burckhardtweg 2, 37077 Göttingen, Germany
2Faculty of Life Sciences, University of Central Punjab, Punjab 54000, Pakistan; aaminabatool.9@gmail.com
3Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University,
Grisebachstraße 8, 37077 Göttingen, Germany; sarif@gwdg.de
*Correspondence: akhalil2@gwdg.de
Abstract:
Cattle farming is an ancient practice, with roots in the early Neolithic era that has retained a
major status in the food industry, with global beef market revenue amounting to $385.7B (
as of 2018
).
Hence, cattle maintenance is essential for catering to the nutritional requirements of modern civiliza-
tion. This extensive review aims to provide a holistic overview of the cattle microbiome, analyzing
the native microbial composition within the respiratory tract, gastrointestinal tract, reproductive tract,
and skin of cattle. The dysbiosis associated with various diseases such as bovine respiratory disease,
bovine digital dermatitis, mastitis, Johne’s disease, uterine diseases (metritis and endometritis), and
metabolic disorders (ruminal acidosis and ketosis) have been discussed. Moreover, various non-
antibiotic microbial therapies including phage therapy, prebiotics, and probiotics have been examined
as potential means to reduce disease-associated dysbiosis. In general, this review highlights the
importance of the microbiome in the maintenance of health in cattle and its potential in alleviating
bovine diseases, with an aim to enhance cattle health and production.
Keywords: microbiome; cattle; Johne’s disease; dysbiosis; mastitis
1. Introduction
Microbiome encompasses an interactive and dynamic micro-ecosystem, established
by the genetic elements, structures, and metabolites of characteristic microbiota. The
microbiota inhabits diverse ecological niches, including eukaryotic hosts. In eukaryotic
host environments, the importance of the microbiome in the maintenance of physiolog-
ical functionality has been indicated as a neglected niche by many researchers [
1
]. The
potential influence of the microbiome on cattle growth and the immune system has been
thoroughly investigated [
2
]. The commensal microbiota confers beneficial effects to animal
health through various mechanisms; such as helping to digest the otherwise indigestible
plant fiber [
3
]. Moreover, it provides the host with nutrients and energy sources (volatile
fatty acids) [
4
], building units (carbohydrates, peptides, lipids) [
1
,
5
], and modulates the
immune system via cytokines and antibodies [
6
]. Microbiota creates a physical barrier
between pathogens and immune cells, competing with the pathogens for adhesion sites
and nutrients [
7
,
8
]. Moreover, these microbes inhibit pathogenic growth by producing
antimicrobial compounds, such as organic acids, hydrogen peroxide, bacteriocins, and
biosurfactants [9,10].
The holistic view of the cattle microbiome indicates the colonization of skin, body
cavities, and mucosal surfaces, of respiratory, gastrointestinal, and urogenital tracts, by
microbial communities [
11
–
14
]. The distribution varies throughout these organs, depend-
ing on the host factors and distinct physiochemical properties of the colonization site [15].
Although cattle microbiome co-evolves with its host (influenced by inter-microbial interac-
tions, host attributes, and environmental factors) [
16
], the core functional microbiome in
any organism is more conservative [
17
]. However, during disease conditions, the delicate
growth balance of microbial consortium is prone to intrusions, followed by either loss or
Ruminants 2022,2, 134–156. https://doi.org/10.3390/ruminants2010009 https://www.mdpi.com/journal/ruminants

Ruminants 2022,2135
gain of different microbial species, leading to dysbiosis [
18
]. Dysbiosis may cause or aggra-
vate multiple disease phenotypes in cattle such as Johne’s disease, uterine and metabolic
diseases (ruminant acidosis) [
19
,
20
]. To explore the therapeutic and prophylactic role of
the cattle microbiome, understanding the microbial composition (particularly associated
with health and disease phenotypes) is paramount. This understanding could very well
aid in laying out a well-defined microbiome manipulation strategy for better health and
production outcomes [
21
]. Therefore, this review aims at summarizing the natural bacterial
microflora associated with a healthy cattle microbiome and how the composition shifts
during disease conditions. Alternative therapeutics such as probiotics, prebiotics, and
bacteriophages that have proven efficacy in combating dysbiosis have been discussed [
22
].
2. Development and Succession of Cattle Microbiome with Age
Since the microbiota represents a dynamic ecosystem where the host factors play
a crucial role in the selection, adaptation, and stabilization of microbial communities, it
would be prudent to observe the developmental changes in the microbial consortium
throughout the cattle lifespan [
23
]. Previous studies report significant fluctuations in the
microbiome throughout the cattle’s lifetime [
17
,
24
]. These changes could very well be
influenced by infant transitions (weaning age, weaning strategy, mode of delivery, and type
of milk feeding), as well as environmental factors and host genetics [
25
,
26
]. Soon after birth,
the infant microflora starts to establish and is derived from mother’s vaginal, skin and
environmental microbiota [
27
]. It implies that the mode of delivery, either through natural
(vaginal) or cesarean section (C-section), could be a deciding factor regarding the initial in-
fant microbiome [
26
]. Recent studies observed a significant microbiome difference between
these two modes of delivery, as vaginal delivery led to higher species richness, evenness,
and diversity in human and cattle infants as compared to C-section delivery [
17
,
28
]. Fol-
lowing vaginal delivery, the species belonging to the phylum Proteobacteria were most
abundant in calves’ rumen, in addition to the increased relative abundance of gastrointesti-
nal tract-related genera Bifidobacteria and Bacteroides [
29
] and the vaginal tract-associated
genera Prevotella and Butyrivibrio spp. [
17
] In contrast, the rumen of calves, delivered by
C-section, are more likely to be colonized by species from the phylum Firmicutes, and the
genera Peptostreptococcus and Dorea. The reduced colonization and low maternal microbial
persistence over time in calves after C-section make them more prone to infections [17].
The newborn calves lack a fully functional rumen, as the milk bypasses the rumen,
reticulum, and omasum. The milk passes through the oesophageal groove to the abomasum
where it is enzymatically digested [
30
]. In young ruminants shortly after birth, aerobic and
facultative anaerobes gradually consume oxygen which contributes to the predominance
of the anaerobes in the rumen microbiome [
24
]. As shown in Figure 1, during the first three
days, rumen microbiota is dominated by Proteobacteria,Firmicutes (whereas Streptococcus
represents 75% of the Firmicutes reads), and Bacteroidetes with varying minor contributions
of other phyla including Actinobacteria, Fusobacteria, Tenericutes, and Cyanobacteria [
24
,
31
].
The microbiome abundance trend shifts from the phylum Proteobacteria (that dominate
for the first three days) to Bacteroidetes (that dominate during 3–12 days of age
[24,32,33]
).
Another prominent microbial transition during early stages to adult development is char-
acterized by the Verrucomicrobiaceae genus Akkermansia decline (from 23–40% during the
early days to 2–4% at first month) [
17
,
24
]. Among the minor phyla, Actinobacteria mainly
Actinomycetales and Fusobacteria are prominent in the newborns [
24
]. Taxonomical diver-
sity increases with age, especially upon the introduction of a new fermentation substrate,
i.e., solid
food [
34
]. Prevotellaceae family is observed to be the most abundant family in the
phylum Bacteroidetes from day 6 to 83 with 31 to 72% abundance, respectively [17,24,32].

Ruminants 2022,2136
Ruminants 2022, 2, FOR PEER REVIEW 3
Figure 1. Dynamics of bacterial communities across different ages [24,32].
3. Healthy Cattle Microbiome Composition
3.1. Respiratory Tract Microbiome
The respiratory tract harbors a distinct microbial make-up within nostrils, nasophar-
ynx, hard plate, oropharynx, tonsils, trachea, and lungs [35]. Overall, a healthy cattle res-
piratory tract microbiome could be attributed to six phyla, namely Proteobacteria, Firmicu-
tes, Tenericutes, Actinobacteria, Fusobacteria, and Bacteroidetes [36]; although, the relative
abundance of each phylum is varied across individual organs. Tenericutes dominated the
distal trachea to the lungs, the exception being the secondary bronchi of the left and right
caudal lobes, which were colonized mainly by Actinobacteria [35]. Proteobacteria prevailed
in the nostrils, nasopharynx, and oropharynx, whereas Firmicutes were extensively pre-
sent on the floor and hard palate of the mouth and Fusobacteria colonized the tonsils. At
the genus level, Mycoplasma, Moraxella, Streptococcus, Fusobacterium, and Streptomyces were
the most abundant along the respiratory tract with uneven distribution as following: My-
coplasma predominated the trachea, lung, nostril, and nasopharynx; Streptococcus predom-
inated the floor and hard plate of the mouth; Bibersteinia were localized in oropharynx;
and Fusobacterium dominated the tonsils [35].
3.2. Gastrointestinal Microbiome
Given the significance of oral health in organisms, extensive studies have been de-
voted to investigating the human, cat, sheep, and dog oral microbiome [37–39]. Most stud-
ies on cattle oral health are limited to dental formulas and diseases, and not much data
are available regarding the oral microbiome. Recently, it was reported that the most prev-
alent genera associated with healthy cattle oral cavity are Arcobacter, Gastranaerophilales,
Planifilum, Escherichia-Shigella, Actinobacteria, Burkholderia, and Pseudomonas (Figure 2). In-
terestingly, studies pertinent to periodontitis reported that healthy samples demonstrate
low intra-sample variability and are clustered separately from disease-prevalent samples
[40].
3.3. Rumen Microbiome
The ruminal stomach is made of four compartments, the reticulum, rumen, omasum
(pre-stomach), and abomasum (true stomach) [41]. Being the largest compartment of the
stomach, the complexity of the microbial ecosystem allows rumen to utilize fiber-rich diets
in addition to complex carbohydrates [42], resulting in the digestion of 60–70% of the in-
gested cellulose [1]. Bacteria are the most abundant prokaryotes, making up more than
Figure 1. Dynamics of bacterial communities across different ages [24,32].
3. Healthy Cattle Microbiome Composition
3.1. Respiratory Tract Microbiome
The respiratory tract harbors a distinct microbial make-up within nostrils, nasophar-
ynx, hard plate, oropharynx, tonsils, trachea, and lungs [
35
]. Overall, a healthy cattle respi-
ratory tract microbiome could be attributed to six phyla, namely Proteobacteria,
Firmicutes
,
Tenericutes, Actinobacteria, Fusobacteria, and Bacteroidetes [
36
]; although, the relative abun-
dance of each phylum is varied across individual organs. Tenericutes dominated the distal
trachea to the lungs, the exception being the secondary bronchi of the left and right caudal
lobes, which were colonized mainly by Actinobacteria [
35
]. Proteobacteria prevailed in the
nostrils, nasopharynx, and oropharynx, whereas Firmicutes were extensively present on
the floor and hard palate of the mouth and Fusobacteria colonized the tonsils. At the genus
level, Mycoplasma,Moraxella,Streptococcus,Fusobacterium, and Streptomyces were the most
abundant along the respiratory tract with uneven distribution as following: Mycoplasma
predominated the trachea, lung, nostril, and nasopharynx; Streptococcus predominated
the floor and hard plate of the mouth; Bibersteinia were localized in oropharynx; and
Fusobacterium dominated the tonsils [35].
3.2. Gastrointestinal Microbiome
Given the significance of oral health in organisms, extensive studies have been de-
voted to investigating the human, cat, sheep, and dog oral microbiome [
37
–
39
]. Most
studies on cattle oral health are limited to dental formulas and diseases, and not much
data are available regarding the oral microbiome. Recently, it was reported that the most
prevalent genera associated with healthy cattle oral cavity are Arcobacter,Gastranaerophilales,
Planifilum,Escherichia-Shigella,Actinobacteria, Burkholderia, and Pseudomonas (Figure 2). Inter-
estingly, studies pertinent to periodontitis reported that healthy samples demonstrate low
intra-sample variability and are clustered separately from disease-prevalent samples [40].
3.3. Rumen Microbiome
The ruminal stomach is made of four compartments, the reticulum, rumen, omasum
(pre-stomach), and abomasum (true stomach) [
41
]. Being the largest compartment of the
stomach, the complexity of the microbial ecosystem allows rumen to utilize fiber-rich
diets in addition to complex carbohydrates [
42
], resulting in the digestion of 60–70% of
the ingested cellulose [
1
]. Bacteria are the most abundant prokaryotes, making up more
than 95% of the ruminal microbiota at a cellular density of 1010–1011 cells/g [
2
]. The
most abundant phylum in the rumen are Bacteroidetes (with genus Prevotella comprising

Ruminants 2022,2137
45–57% of total 16S rRNA sequences and 90% of Bacteroidetes population), followed by
28% of phylum Firmicutes. Furthermore, genera Dialister, Succiniclasticum,Ruminococcus,
Butyrivibrio, and Mitsuokella represented more than 1% of the total bacterial genera in the
rumen [43].
3.4. Small Intestine
The main function of the small intestine is the absorption of protein and carbohy-
drates [
1
] and its compartments (duodenum, jejunum, and the ileum) vary in their function
and microbial communities. Interestingly, a study reported that phylum Firmicutes were
dominant within all sections of cattle gastrointestinal tract except jejunum (Proteobacteria
were dominant here). Enterobacteriaceae were highly abundant within the small intestine,
while jejunum was enriched by Ruminococcus, Acetitomaculum, and Lachnospiraceae [
44
].
Compared to the rumen, the relative abundance of the phylum Bacteroidetes significantly de-
creased (0.4–1.1%) while that of phylum Firmicutes elevated drastically, reaching up
to 80%
of relative abundance. Studies based on low abundance phyla Actinobacteria (6–13%),
Proteobacteria (0.8–5.8%), and Tenericutes (0.4–4%) have also been reported. Furthermore,
other significant genera pertinent to the small intestine include Ruminococcus, Butyrivibrio,
Lactobacillus, Bulleidia, Mogibacterium, Mitsuokella, and Propionibacterium [43].
3.5. Large Intestine
Bacterial abundance in the cecum, colon, and rectum is about 1012 to 1014 cells/mL [
1
].
The large intestine plays an important role in water absorption and digestion as 30% of the
cellulose digestion takes place in the large intestine [
1
,
45
]. Different regions of the large in-
testine represent distinct microbial intensity and diversity in the corresponding microbiota.
In the cecum, Firmicutes have been the predominant phylum reaching up
to 81%
of the total
phyla while Bacteroidetes contribute to 18–26%. Spirochetes, Tenericutes, and Actinobacteria
have also been reported in the cecum. Furthermore, Prevotella, Turicibacter, Coprococcus,
Ruminococcus, Dorea, Blautia, Clostridium, and Oscillospira have been the most abundant
genera in the cecum [
43
]. Similar to the cecum, the colon contains phylum Firmicutes
as 81% of microbial relative abundance, followed by Bacteroidetes at 21–33%. Moreover,
Spirochetes, Tenericutes, Proteobacteria, Actinobacteria, and Fibrobacteres have been in the great-
est abundance among the rest of 23 phyla. The most abundant genera were Prevotella,
Ruminococcus, Coprococcus, Dorea, Turicibacter, Blautia, Oscillospira, and Parabacteroides [
43
,
46
].
Similarly, the rectum has also been dominated by the phylum Firmicutes. Moreover, the
genera that dominated the rectum were Clostridium, Roseburia, Osillospira, Succinivibrio,
Ruminococcus, Bacteroides, Prevotella, Blautia, Coprococcus, and Turicibacter [47].
3.6. Reproductive Tract Microbiome
Reproductive efficiency greatly influences the overall productivity of cattle as well as
the regulation of health and homeostasis. From that perspective, the need for understanding
the reproductive tract microbiome becomes significant [
48
]. Despite the availability of
advanced next-generation sequencing techniques (NGS), the healthy cattle reproductive-
tract microbiota are not fully understood due to a lack of definitive research. Insights in
the healthy reproductive tract microbiome would greatly improve and influence cattle
reproduction, maternal and neonatal therapeutic practices [49].
3.7. Female Reproductive Tract
Significant differences in the bovine reproductive-tract microbiome are linked with
the type of breed [
50
], anatomical regions in the tract [
13
], and physiological status [
10
].
Four major phyla have been reported by several studies, namely Firmicutes, Bacteroidetes,
Proteobacteria [
10
,
13
,
50
,
51
], and Actinobacteria [
13
,
51
]. The predominant genera reported
were Aeribacillus,Bacillus, Clostridium, Ruminococcus, Rikenella, Alistipes, Eubacterium, and
Prevotella [
10
,
51
]. The cattle uterus contains the uterine body and two uterine horns; and
each horn has its oviduct [
52
]. The concept of a sterile uterine system has been challenged

Ruminants 2022,2138
by several studies [
7
]. The uterine microbiome exists before the occurrence of pregnancy
as the female reaches reproductive maturity, and it is maintained throughout pregnancy.
The major phyla colonizing the uterus reported for five pregnant and 10 virgin cows were
Firmicutes, Bacteroidetes, and Proteobacteria [
8
]. Another study by
Clemmons et al., 2017
compared the healthy microbiome communities in the bovine vagina and uterus for non-
pregnant cows after insemination. They reported that Firmicutes were the most dominant
phyla in both uterus and vagina. The next phyla, in order of abundance, were Proteobacteria,
followed by Actinobacteria, and Bacteroidetes. At the genus level, the vagina showed the high-
est abundance of an undetermined genus pertinent to order Bacteroidales. The next in order
of abundance were genus 5-7N15 pertinent to family Bacteroidaceae, followed by Oscillospira,
Butyrivibrio, Ureaplasma, Campylobacter, Dorea, CF231 (pertinent to family Paraprevotellaceae),
Clostridium, Helcococcus, and Corynebacterium. Alternatively, the uterus contained a dominat-
ing abundance of Corynebacterium, Ureaplasma, Staphylococcus, Microbacterium, Butyrivibrio,
and Helcococcus [13].
3.8. Bull Reproductive Tract
Selection of the bulls is a vital practice for the cattle industry [
53
]. Despite the microbial
significance in the maintenance of fertility and reproductive health, the research found on
this topic remains nominal. A study published in 2019 investigated the microbiome of the
penis and prepuce [
54
]. According to the authors, the most abundant phyla were Firmicutes,
Fusobacteria, Bacteroidetes, Proteobacteria, and Actinobacteria. Community composition was
consistent with clustering patterns and low and high diversity clusters were identified at
the genera level. For low diversity samples, Bradyrhizobium had constantly been detected,
whilst the high diversity samples exhibited multiple genera. The dominant colonizers in the
prepuce were Bacteroides, unclassified Ruminococcaceae,Histophilus, and Streptobacillus [
55
].
3.9. Skin Microbiome
Skin is a potent physical barrier against pathogenic invasion, environmental factors,
and physical trauma [
48
]. Interestingly, studies that sought to analyze changes in human
and cattle skin microbial communities caused by close environmental contact reported
a weak correlation in the characterization of the cattle skin microbiome. Much focus
has been reserved for the teat skin microbiome, particularly as it related to raw milk
microbial diversity. Some of the major taxa found to be associated with teat skin were
Corynebacteriales, Clostridium, Atopobium, Bifidobacteriales, Lachnospiraceae, and Coriobacteriia
as well as Pediococcus, Aerococcus, Staphylococcus, Pantoea, Enterobacter, Enterococcus, and
Proteobacteria [
56
,
57
]. Several commensal microbial species associated with teat skin are
not common in milk, indicating a breakdown of microbial flow from teat skin to milk [
57
].
Ample research has been devoted to the characterization of the microbiome of digits as
well [
58
]. Healthy digits have been characterized by elevated levels of phyla Proteobacteria,
Bacteroidetes, and Firmicutes [
59
–
61
]. Furthermore, Actinobacteria and Spirochetes have also
been detected in healthy digits [
59
]. A study by Mamuad et al., 2020 indicated two
more phyla that preceded the aforementioned phyla in abundance. The study reported
Tenericutes and Bacteroidetes, with 14.1% and 11.8% mean abundance respectively [
61
].
The families commonly found in healthy skin belong to Ruminococaceae, Aerococcaceae,
Corynebacteriaceae, and Moraxellaceae [60].

Ruminants 2022,2139
Ruminants 2022, 2, FOR PEER REVIEW 6
Figure 2. Variation in healthy cattle microbiome at different organs. Predominant bacteria in skin,
reproductive organs, oral cavity, gastrointestinal tract, and respiratory tract are shown
[10,13,35,40,51,55–57,60,62].
4. Cattle Diseases Associated with Microbiome Dysbiosis
4.1. Bovine Respiratory Disease BRD
Bovine respiratory disease (BRD) is a complex multifactorial disease associated with
host susceptibility, pathogenic load, and environmental stimuli [63]. BRD is a major con-
cern for producers worldwide due to the high treatment costs as well as high morbidity
Figure 2.
Variation in healthy cattle microbiome at different organs. Predominant bacte-
ria in skin, reproductive organs, oral cavity, gastrointestinal tract, and respiratory tract are
shown [10,13,35,40,51,55–57,60,62].
4. Cattle Diseases Associated with Microbiome Dysbiosis
4.1. Bovine Respiratory Disease BRD
Bovine respiratory disease (BRD) is a complex multifactorial disease associated with
host susceptibility, pathogenic load, and environmental stimuli [
63
]. BRD is a major con-
cern for producers worldwide due to the high treatment costs as well as high morbidity
and mortality rate [
64
]. Among the environmental factors, transportation stress along
with temperature fluctuations and ventilation play a crucial role in aggravating the dis-
ease [
65
]. Dysbiosis has been reported to be one of the hallmarks of disease [
11
]. Disease

Ruminants 2022,2140
conditions are exacerbated through infections and immune system suppression caused
bybovine pestivirus A, bovine orthopneumovirus, and bovine respirovirus 3 [
66
]. Ac-
cording to previous studies, the most commonly described bacterial communities in BRD
comprised of Mycoplasma spp., particularly Mycoplasma bovis,Mannheimia haemolytica, and
Pasteurella multocida
[
67
,
68
]. Many studies have also reported the natural abundance of
these bacteria in the healthy respiratory tract microbiome. Nevertheless, these species are
classified as opportunistic [
69
] and their relative abundances are higher in diseased animals
as compared to their healthy counterparts. For instance, the abundance of Mycoplasma and
Mannheimia has increased substantially over time in infected calves, though some studies
also report a rise in Moraxella spp., indicating its role in the pathogenesis of BRD [
66
,
67
].
Pseudomonas fluorescens abundance has also been significantly higher in BRD-infected calves;
however, its contributing role in respiratory disease remains unclear [
67
]. Recent studies
in healthy calves reported the abundance and probiotic effect of Lactobacillus lactis and
Lactobacillus casei colonization against M. haemolytica growth, which has also been proved
in vitro [68] (Table 1).
4.2. Bovine Digital Dermatitis
Bovine Digital Dermatitis (DD) is characterized by painful ulcerative lesions that
might persist in chronic disease [
70
] and is identified as a major cause of cow lameness.
Being a highly prevalent infectious disease, DD causes major economic losses in dairy
herds worldwide and affects animal health, productivity, and welfare [
71
]. It is a ge-
netic, multifactorial disease, influenced by both environmental as well as pathogenic
involvement [
72
]. Disease pathogenesis is still not fully understood, though several re-
search papers have been published since its first description in 1974 [
60
]. Several stud-
ies have frequently identified spirochetes from Treponema genus as a major pathogen
in DD lesions
[59,60,72–74]
. A distinct six phylogroups were highly associated with
the disease and classified as
Treponema phagedenis
, Treponema medium,Treponema putidum,
Treponema denticola
,
Treponema matophilum
, and
Treponema paraluiscuniculi
groups [
75
]. De-
spite the continual presence of Treponema spp. In DD lesions, the DD disease phenotype
could not be reproduced by using Treponema pure culture [
76
]. Other identified microbes in-
cluded
Mycoplasma spp.
,Fusobacterium necrophorum, Bacteroides
spp., Porphyromonas levii [60,73]
,
Prevotella spp., Corynebacterium spp., and Tissierella spp. [
59
] These pieces of evidence sug-
gested the potential association of these bacteria with the disease pathogenesis as secondary
opportunistic pathogens [
77
]. A metagenomic study by Zinicola et al., 2015, could not
detect any viral or fungal DNA in the DD lesions and thus presently, it cannot be linked
to the disease [
74
,
77
]. The positive response against antibiotic treatment also indicated
the bacterial nature of the disease [
76
]. A correlation has been proposed between the
main pathogen Dichelobacter nodosus involved in bovine foot rot and DD lesions by several
studies [
78
,
79
]. Nevertheless, this correlation was negated by another study that reported
a lack of presence of D. nodosus in Swiss cattle affected by DD [
80
]. Another study by
Krull et al., 2014
stated the presence of D. nodosus in DD lesions but no statistical signifi-
cance was reported [
60
]. The Treponema spp. were also observed within the rumen and
fecal microbiome of infected cattle [
74
]. Another study suggested that foremilk and udder
cleft skin might be an important reservoir for Trepenoma spp. [81] (Table 1).
4.3. Mastitis
Mastitis is characterized by an inflammatory response to the intra-mammary in-
fection that disturbs the physical barrier of the mammary quarters [
82
]. This disease
is a major economic burden on the dairy industry worldwide, affecting animal health
and productivity [
83
]. Mastitis has been classified into two subgroups based on the
severity of the disease (clinical and subclinical). Observable physiological changes in
the milk and udder are a consequence of clinical mastitis while in subclinical mastitis
no symptoms are observed, despite the presence of pathogens [
84
]. Mastitis’s aetiolog-
ical agent usually has a microbial origin that involves various bacterial species. The

Ruminants 2022,2141
major pathogens included
Staphylococcus aureus
,
Streptococcus agalactiae
,Escherichia coli,
Streptococcus dysgalactia
,
Mycoplasma spp.
, whilst the minor pathogens were represented by
coagulase-negative staphylococci and
Corynebacterium spp.
[
85
] Environmental pathogens
including Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aeorogenes,
Streptococcus uberis Pseudomonas spp., and Prototheca have also been involved in mastitis
pathogenesis [
83
,
86
]. NGS has made a transition from identifying a single organism to char-
acterizing the aetiological microbiome associated with mastitis infection [
87
]. Recent studies
reported that alteration in the mammary gland microbiome (dysbiosis) is related to mastitis
(whether the dysbiosis is a sequence or consequence of mastitis is still a debate)
[88–90]
. An
increased microbial load and relative reduction in bacterial diversity have been reported in
mastitis milk [
91
]. This can be attributed to the increased amount of pathogenic colonization
with the progression of infection, accompanied by a reduction in the healthy commensal
bacteria. Altering the intra-mammary commensal bacteria has a deleterious effect on mam-
mary gland homeostasis [
92
]. Shifts in the milk microbiome have been observed in healthy
cows and those infected with mastitis. Phylum Firmicutes have dominated the healthy
milk and the usual most abundant phyla were Firmicutes,Bacteroidetes,Actinobacteria, and
Proteobacteria [
88
,
93
]. In contrast, a higher representation of Proteobacteria was over observed
in mastitis milk [
88
]. Hoque et al., 2019 identified 26 unreported strains associated with
mastitis, namely the strains belonging to genera
Acinetobacter
,Pseudomonas, Streptococcus,
Corynebacterium Staphylococcus, Enterococcus, Bacillus, and Escherichia [88] (Table 1).
Recent studies suggested that gut microbiome dysbiosis played an important aetiolog-
ical role in intra-mammary infections (IMI) [
94
]. In a study by Ma Chen et al., 2018, it was
proposed that the intestinal microbiota can induce mastitis. To examine this suggestion,
the researchers induced IMI by fecal transplantation from a cow suffering from mastitis to
the germ-free mice, which led to the development of mastitis symptoms [
95
]. Interestingly,
a promising result was obtained when probiotics were introduced in mice, parallel with
inoculation of the fecal microbiota of infected cattle. A relatively great relief from mastitis
symptoms was observed in the probiotics-treated mice group. In this group, a functional
shift in the intestinal microbiota was also observed (to a state different from healthy and
diseased microbiota), which approved the author’s supposition regarding the correlation of
intestinal microbiota in mastitis induction [
95
]. Another supportive investigation revealed
that gut dysbiosis could aggravate S. aureus mastitis severity and the blood–milk barrier
permeability. These effects were reversed when fecal microbiota transplant from healthy
mice was applied [
96
]. Nevertheless, the correlation between the gut microbiome and
mastitis may also be influenced by other factors. One study found out that the overpro-
duced lipopolysaccharides (LPS) by the rumen, moved into the blood and disturbed the
blood–milk barrier by accumulating in the mammary glands. Consequently, inducing IMI
in subacute rumen acidosis (SARA) infected cows, and increases the severity of mastitis
infection [
94
]. Ruminal short-chain fatty acids (SCFAs) have a protective effect on the
blood–milk barrier, and therefore against IMI [97] (Table 1).
4.4. Johne’s Disease
Johne’s disease is characterized by persistent diarrhea that leads to malnutrition,
emaciation, and significant economic losses in the livestock industry worldwide [
98
,
99
].
Johne’s disease is caused by slow-growing non-motile mycobacteria—Mycobacterium avium
subsp. Paratuberculosis (MAP) [
100
]. MAP is also being investigated in correlation with
human Crohn’s disease [
99
,
101
–
103
]. The pathological similarities between Johne’s and
Crohn’s disease affirmed these speculations [
104
]. Recent studies suggested that gastroin-
testinal microbiome dysbiosis facilitated MAP infection and aggravated disease sever-
ity [
19
,
105
]. However, a very limited number of investigations on cattle have been reported
so far(Table 1).
A study by Fecteau et al., in 2016 compared the fecal microbial communities between
MAP infected, exposed, and negative controls. A distinct bacterial community has been
observed in the MAP infected group compared to negative and exposed samples (which

Ruminants 2022,2142
showed similarity to each other). A relative increase in the phylum Actinobacteria and
Proteobacteria along with a significant decrease in the phylum Bacteroidetes and Firmicutes
abundance was observed. Interestingly, the genus Arthrobacter has dominated the phylum
Actinobacteria in all positive samples [105].
Another study investigated the fecal and ileal microbiota during early infection with
MAP. The fecal microbiota showed a relative decrease in the genera Verrucomicrobia and
Akkermansia.Planococcaceae and Paraprevotellaceae showed higher abundance in the infected
samples. Analysis of ileal microbiota showed less abundance of phylum Proteobacteria in
MAP infected calves. Moreover, changes in the microbiome induced functional changes in
its metabolites, thus an increased level of metabolism was observed in MAP negative calves.
However, increased lysine and histidine metabolism pathways have been associated with
MAP infection [19] (Table 1).
4.5. Uterine Diseases
Metritis and endometritis are the most important postpartum uterine diseases, affect-
ing up to 40% of dairy cattle [
106
,
107
]. Uterine diseases are a major economic concern
directly affecting animal health, milk production, and reproductive performance [
108
].
Metritis is an inflammation of the uterine wall, which is characterized by systematic signs of
illness. The symptoms, such as fever and toxemia usually occur during the first 21 days after
parturition [
109
]. Endometritis is an inflammation in the inner lining of the uterus without
signs of systematic illness, it can occur during or after 21 days post-partum. Endometritis
is classified into clinical and subclinical types [
109
,
110
]. Uterine diseases are multifactorial
where environmental factors affect the occurrence of the disease and host defense plays an
important role against bacterial pathogenesis [
111
]. Cow uterus is exposed to bacterial con-
tamination during calving and up to two weeks postpartum. A healthy cow immune system
can resist pathogenic colonization. However, failure to resist a pathogenic colonization leads
to disease progression [
112
]. Several risk factors, including parity, dystocia, retinal placenta,
abortion, twins, and calving season, play a role in triggering uterine diseases [
113
]. Culture-
dependent studies have identified pathogenic bacteria associated with uterine infections
from diseased cows.
Trueperella pyogenes
and
Escherichia coli
, in addition to gram-negative
pathogens
Porphyromonas levii
,Fusobacterium necrophorum,
Prevotella melaninogenica,
and
Bacteroides spp.
were frequently isolated
[114–117]
.
Trueperella pyogenes
is usually diag-
nosed in clinical endometritis [
117
,
118
]. Escherichia coli is also reported as one of the major
pathogens causing uterine infection [
119
]. Piersanti et al., 2019 reported that these two pathogens
can induce Endometritis in the Holstein Friesian model [
118
].
Porphyromona, Bacteroides
, and
Fusobacteria have been associated with metritis [
111
]. A dysbiosis has been reported in the
infected uterus, characterized by a decrease in microbial diversity and enhanced loss of
heterogeneity [
111
]. Increased abundance of Bacteroidetes and Fusobacteria and decrease
in Proteobacteria were observed in metritis cow [
20
,
115
]. The ecological interaction be-
tween bacteria seems to be important in the development of metritis, such as increased
abundance of the genes of Fusobacterium of the Bacteroides spp. is an essential hallmark.
Furthermore, Helcococcus,Porphyromonas, and Filifactor were also found to be associated
with metritis [
115
]. Moreover, the uterine microbiota of clinical endometritis (CE) cows
exhibited an increased abundance of Fusobacterium,Trueperella, and Peptoniphilus, whilst
subclinical endometritis (SE) cows almost lacked the aforementioned phyla [
117
] (Table 1).
Proteobacteria, Bacteroidetes, and Fusobacteria have been identified as uterine disease
risk factors. Cows with retained placenta and healthy cows exhibit the same total bacterial
count in the first days postpartum. However, Bacteroidetes and Fusobacteria tend to be in
higher abundance after day-7 in the cows with retained placenta, postpartum fever assisted
parturition, and twin delivery. Proteobacteria were found to be more abundant in metritis.
The author concluded that the total bacterial load and the microbiome composition are
associated with specific risk factors of uterine disease [114].

Ruminants 2022,2143
4.6. Metabolic Disorders
4.6.1. Ruminal Acidosis
Ruminal acidosis is a gastrointestinal metabolic disease of dairy cattle. The prognosis
predicts intensive feeding of a high carbohydrate diet by cattle, leading to accumulation
of short-chain fatty acids, lactic acid, and LPS in the rumen [
120
]. This results in an
extreme drop of rumen pH reaching <5.8 for at least 3 h in a day [
121
]. There are two
forms of ruminal acidosis; clinical (acute) and subclinical ruminal acidosis (SARA) [
122
].
Disease sequelae include lameness, liver abscesses, paint brush hemorrhage, weight loss,
scouring, and epistaxis apart from deleterious effects on the reproductive system [
27
,
123
].
Ruminal acidosis is the most important disorder of the dairy industry as it causes low milk
production, low milk protein, and fat leading to huge economic losses [123].
Rumen microbial community alterations are observed in association with rumen
acidosis [
124
]. Severe reduction or elimination is observed in protozoan communities [
27
].
Rumen bacterial diversity and richness reduction have also been associated with the
disease [
125
]. Changes in Firmicutes to Bacteroidetes ratio have also been reported, resulting
in a decrease in Bacteroidetes and cellulolytic bacteria as well as an increase in starch
fermenting bacteria [
25
,
126
]. Plaizier et al., 2017 reported an equal relative abundance
in Firmicutes and Bacteroidetes in the rumen, which indicated a reduction in Bacteroidetes
phylum [126].
Several studies evaluated the composition and functional activity of ruminal mi-
crobiota challenged with SARA. Microbiome dysbiosis was indicated by a decrease in
cellulolytic bacteria including Fibrobacter succinogenes,Ruminococcus albus,
Streptococcus bovis
,
Butyrivibrio fibrisolvens
Prevotella bryantii, Selenomonas ruminantium, and
Ruminococcus bicirculans
in association with the acidotic challenge [
127
,
128
]. Streptococcus,
Lactobacillus Succiniclasticum
,
and Clostridium levels were increased during ruminal acidosis [
31
,
128
]. Enrichment in
carbohydrate-, amino acids-, energy-, vitamin co-factor- metabolism pathways in addi-
tion to biofilm formation pathways [
127
] and high concentration of LPS, have also been
reported to be associated with SARA. Inflammation induced by ruminal LPS over-activated
nuclear factor kappa-B and mitogen-activated protein kinase inflammatory pathways and
significantly increased proinflammatory cytokine synthesis [120] (Table 1).
4.6.2. Ketosis
Acetonemia is a metabolic disorder of dairy cattle and affects cattle health, fertility, and
milk production during the early lactation period. This disorder contributes to a negative
energy balance when energy demands exceed energy intake [129,130].
Ketosis often occurs when high glucose demand is fulfilled through adipose utilization
as an energy source. However, fat mobilization may increase non-esterified fatty acid
concentration in blood [
129
]. Prolonged periods of high blood concentrations of non-
esterified fatty acids can impair liver function, which leads to partial oxidation of non-
esterified fatty acids. Consequently, overproduction and accumulation of ketone bodies,
predominantly Beta-hydroxybutyrate (BHB), cause ketosis [
131
]. BHB levels can be used as
a biomarker for ketosis diagnosis. Cows experiencing BHB levels higher than 1.4 mmol/L
are usually diagnosed with ketosis [121].
Ketosis can result in less dry matter intake, reduction in milk production, and weight
loss. Some cows also develop neurological disorders, excitable uncoordinated, and aggres-
sive behavior. Furthermore, ketosis may be a risk factor for mastitis [132].
Shifts in the microbiome in cows with ketosis have been detected by qPCR, T-RFLP,
and 16S rRNA sequencing. An increase in phyla Firmicutes, and Proteobacteria was detected
in cows with ketosis. A significant decrease in Euryarchaeota was also reported with
elevated levels of A. lipolytica, P. bryantii, M. elsdenii, and Lachnospiraceae [
133
]. Furthermore,
the genus Ruminococcaceae,Methanobrevibacter,Erysipelotrichaceae, and Atopobium were
rarefied [27,133] (Table 1).

Ruminants 2022,2144
Table 1. Significant transitions in the microbiome associated with cattle disease.
Disease Significant Changes in the Microbiome Reference
Bovine respiratory disease
↑Mycoplama bovis
↑Mannheimia haemolytica
↑Pasteurella multocida
↑Mycoplasma
[67,68]
Mastitis
↑Staphylococcus aureus
↑Streptococcus agalactiae
↑Escherichia coli
↑Klebsiella
↑Streptococcus dysgalactia,
↑Corynebacterium bovis
[85,86,88]
Johnes Disease
↑Arthrobacter
↑Bacillus
↑Enterococcus
↑Camobacterium
↑Desemzia
↑Trichococcus
↑Planococcaceae
↓Paraprevotellaceae
↓Faecalibacterium
↓Akkermansia
[19,105]
Metritis and endometritis
↑Trueperella pyogenes
↑Escherichia coli
↑Porphyromonas levii
↑Fusobacterium necrophorum
↑Prevotella melaninogenica
↑Bacteroides spp
[20,111,114,116,117]
Bovine digital dermatitis
↑Treponema spp.
↑Fusobacterium spp.
↑Mycoplasma spp.
↑Porphyromonas spp.
↑Prevotella spp.
↑Corynebacterium spp.
↑Tissierella spp.
[59]
Rumen Acidosis
↓Bacteroidetes
↓Fibrobacter succinogenes
↓Ruminococcus albus
↓Ruminococcus bicirculans
↓Butyrivibrio fibrisolvens
↑Prevotella bryantii
↑Selenomonas ruminantium
↓Streptococcus
↑Lactobacillus
↑Succiniclasticum
↑Clostridium
[31,127,128]
Ketosis
↑Lachnosparaceae
↑A. lipolytica,
↑P. bryantii
↑M. elsdenii
↓Ruminococcaceae
↓Methanobrevibacter
↓Erysipelotrichaceae
↓Atopobium
↓F. succinogenes
↓Butyrivibrio proteoclasticus
↓Euryarchaeota
[27,133]
The direction of the arrows shows either an increase or decrease in the microbial abundance.

Ruminants 2022,2145
5. Non-Antibiotics Microbial Therapy
5.1. Probiotics
Probiotics are live microorganisms associated with human and animal health that con-
fer a beneficial effect on health when administrated in sufficient quantities [
134
]. Probiotics
can exert their beneficial effects through various mechanisms [
9
]. They can modulate the
host-microbiota by competing with pathogens over the adhesion sites and nutrients [135].
Moreover, probiotics maintain intestinal homeostasis, improving barrier function [
136
].
Furthermore, they can produce antimicrobial metabolites, including lactic acid, diacetyl,
and antimicrobial peptides, such as bacteriocins that inhibit competing bacteria [
137
]. In
other cases, probiotics can directly interact with the host cells to modulate the immune
system [
138
,
139
]. The most common microorganisms in probiotics are safe, food-grade
bacteria related to genera lactic acid bacteria (LAB), Lactobacillus, and Bifidobacterium [
140
].
Apart from them, enterococcus and streptococcus form part of probiotics as well [141].
Due to the significance of rumen acidosis in dairy cattle, studies infusing probiotics
to treat rumen acidosis were a reasonable choice. Goto et al., 2016 reported that intro-
ducing a probiotic cocktail (Miyarisan Pharmaceutical Co., Ltd., Tokyo, Japan) including
L. plantarum
,
E. faecium
, and
C. butyricum
for seven days could improve the pH and lactic
acid level [
142
]. Yeast has also been incorporated within probiotics in cattle as various stud-
ies provide sufficient evidence of its beneficial impact against ruminal acidosis [
124
,
127
].
Mohammed et al., 2017
reported that Saccharomyces cerevisiae supplementation could re-
duce subacute rumen acidosis, but it did not reduce acute acidosis [124].
In line with the global efforts to reduce antibiotics usage, researchers have applied pro-
biotics as a potential alternative approach for preventing and controlling mastitis
[143,144]
.
LAB-based probiotics have successfully stimulated the host immune response that may
get approved as non-antibiotic mastitis therapy [
9
]. A study by Pellegrino et al., 2017
investigated the immunomodulatory effect of L. perolens CRL1724 and L. lactis subsp. lactis
CRL1655. When inoculated in healthy cows during the dry-off period, the study demon-
strated an increase in immunoglobulin (Ig) G in milk and blood samples. They were able
to recognize probiotics against S. aureus isotopes which could prevent mastitis infection
during the dry-off period [
145
]. Another study conducted by Souza et al., 2018 showed
that against S. aureus infection, L. casei BL23 exhibited anti-inflammatory properties on
infected bovine mammary epithelial cells and did not obstruct the induction of host cell
defensins [
146
]. A study by Wallis et al., 2018 assessed 13 LAB strains for their ability to
form biofilms, producing a barrier against the pathogens and adhere to bovine glandular
mammary epithelium. The biofilm formation and adherence were observed in all the
strains, showing strain dependency [
9
,
147
]. Another study by the same group investigated
the ability of five LAB strains to remove and replace the biofilm formed by pathogenic
staphylococci. All five strains were able to remove the staphylococcal biofilm [
148
]. To
assess the potential probiotic potential of Lactococcus lactis LMG 7930, Armas et al., 2017
examined its
in vitro
potential against ten mastitis-causing pathogens. The strain showed
antagonistic properties against many pathogens such as S. agalactiae and S. aureus strains.
Interestingly, the probiotic strain was adhesive to bovine mammary epithelial cells. Al-
though the probiotic strain tends to decrease the internalization of some pathogenic strains,
it did not significantly affect pathogen invasion. The author suggested further studies
to assess strain safety and efficiency in the field [
149
]. Another probiotic combination by
Pellegrino et al., 2019 to bovine teat canal epithelial cells (BTCEC) selected two strains,
lactis subsp. lactis CRL 1655 and Lactobacillus perolens CRL 1724 based on their adhesion
patterns and their ability to co-aggregate and inhibit the pathogenesis. The characteristics
of these two strains suggested that they may be good candidates for mastitis prevention
during the dry-off period [
150
]. Yu et al., 2017 compared a commercial disinfectant against
a probiotic disinfectant of L. plantarum IMAU80065 and IMAU10155 combination. The
study showed a gradual decrease in the SCC (somatic cell count; an indicator of milk
quality) following the cleaning protocol. Moreover, it was lower in the LAB group than the
commercial disinfectant. The complete 16S rRNA sequencing of raw milk samples revealed

Ruminants 2022,2146
substantial diversity in microbial make-up within samples. The authors suggested that
probiotic disinfectants can replace chemical disinfectants [151].
Additionally, LAB has been evaluated as a potential preventive method against up-
per respiratory tract infection (bovine respiratory disease) caused by the M. haemolytica
associated pathogen cluster [
59
,
80
]. In a study by Amat et al., 2020 inter-nasal inoculations
with L. amylovorus,L. buchneri,L. curvatus, and L. paracasei, isolated from healthy calves,
had been done to investigate their longitudinal effect on the nasopharyngeal microbiome.
A significant decrease in M. haemolytica, because of the colonization resistance had been
reported [
36
,
152
]. Another promising study introduced Dietzia spp. as a potential pro-
biotic to inhibit Mycobacterium avium subsp. Paratuberculosis (MAP) and Johne’s disease
in vitro
[
153
]. The susceptible calf, following MAP infection, had been administered probi-
otics to impede the development of Johne’s disease. For this study, Dietzia subsp. C79793-74
was added to antibiotic-free milk feed for 60 days. None of the ten treated calves developed
the symptoms as they aged, in contrast to the untreated group in which seven out of eight
developed MAP symptoms. Moreover, a group was treated with a combination of Dietzia
and tetracycline, as Dietzia has proven sensitivity to tetracycline contamination. This group
also exhibited a high infection rate (six out of eight were infected) which signified the need
for good practices along with probiotic administration [154].
Metritis affects up to 40% of the dairy herd and is usually treated with antibiotics [
155
].
However, recently, the intravaginal introduction of several LAB strains has been used
to prevent or reduce the incidence of postpartum uterine infections [
156
]. As reported
by Deng et al., 2015, the intravaginal administration of a cocktail of LAB composed of
Lactobacillus sakei
FUA 3089, Pediococcus acidilactici FUA 3138, and FUA 3140 resulted
in a lower uterine infection and an overall improvement in local and systematic im-
mune response [
157
]. Another study suggested that the LAB combination consisting
of
Lactobacillus rhamnosus
,Pediococcus acidilactici,Lactobacillus reuteri, and Lactobacillus sakei
showed reduced inflammation in the uterus against endometrium inflammation and E. coli
infection [158,159].
Calf diarrhea has a high incidence, especially in the first four weeks of a calves’ life,
resulting in a high mortality and morbidity rate [
160
]. Long-term, broad-spectrum an-
tibiotics have conventionally been applied as therapy for the disease [
161
]. However,
probiotic research has been implemented recently for preventing and controlling the
disease.
Renaud et al., 2019
reported a reduction in the duration of diarrhea in calves
treated with multispecies probiotic bolus (MSP). MSP contained
Pediococcus acidilactici
,
Lactobacillus acidophilus
,Lactobacillus casei, Enterococcus faecium, Bifidobacterium bifidum, pep-
tide extract, enzyme blend, killed yeast extract, dried whey, and natural flavors. The authors
recommended further investigations to assess clinical and economic relevance [162].
Another study administrated fermented milk with Lactobacillus murinus CRL 1695,
Lactobacillus mucosae CRL 1696, Lactobacillus johnsonii CRL1693, and Lactobacillus salivarius
CRL 1702 to treat calves. Significantly low mortality and morbidity rates were reported in
calves treated with fermented milk and LAB as compared to the control group. However,
the viable bacterial number showed no difference between the two groups [
163
]. The
study by Fukuda et al., 2019 examined a commercial probiotic product (Bio Three for
animal, Toa Pharmaceutical Co., Ltd., Tokyo, Japan) comprised of Bacillus mesentericus,
Clostridium butyricum
, and
Enterococcus faecalis
, orally administrated for eight days. The
antibiotic-treated group was medicated with ampicillin (Kyritsuseiyaku, Tokyo, Jaban)
for the first five days, followed by kanamycin sulfate (Meiji Seika Co., Tokyo, Japan)
administration till the eighth day. The results showed no significant differences in fecal
score between the two treatment regimes, leading to the confirmation that probiotics can
be an alternative therapy to antibiotic treatment [
161
], the main points are summarized
in Table 2.

Ruminants 2022,2147
Table 2. Important probiotic combinations against disease treatment.
Disease Probiotic Combination Reference
Rumen acidosis L. plantarum,E. faecium, and C. butyricum [142]
Saccharomyces cerevisiae [124]
Mastitis L. perolens CRL1724 and L. lactis subsp. lactis CRL1655 [145]
L. casei BL23 [146]
L. rhamnosus ATCC 7469, L. plantarum 2/37, L. Brevis 104/37, L. plantarum 118/37,
and L. plantarum 6E [148]
Lactococcus lactis LMG 7930 [149]
L. plantarum IMAU80065 and IMAU10155 [151]
Bovine respiratory disease L. amylovorus, L. buchneri, L. curvatus, and L. paracasei, [36,152]
Dietzia spp. [153]
Dietzia subsp. C79793-74 [154]
Metritis Lactobacillus sakei FUA 3089, Pediococcus acidilactici FUA 3138, and FUA 3140 [157]
Lactobacillus rhamnosus, Pediococcus acidilactici, Lactobacillus reuteri,
and Lactobacillus sakei [158]
Calf diarrhoea Pediococcus acidilactici, Lactobacillus acidophilus, Lactobacillus casei, Enterococcus
faecium, and Bifidobacterium bifidum [162]
Lactobacillus murinus CRL 1695, Lactobacillus mucosae CRL 1696, Lactobacillus
johnsonii CRL1693, and Lactobacillus salivarius CRL 1702 [163]
Bacillus mesentericus, Clostridium butyricum and Enterococcus faecalis [161]
5.2. Phage Therapy
Bacteriophages are viruses acting as intracellular parasites that infect bacteria to
replicate within the bacterial cell [
164
]. The phages can replicate through either the lytic
(virulent) or lysogenic (temperate) cycle. These mechanisms play a paramount role in
phage therapeutic potential. In the lytic cycle, the virus infects the bacteria and kills the
cell [
165
], whereas, in the lysogenic cycle, the virus can either lysogenize the host cell by
integrating its genome into the host genetic material or move to the lytic cycle [
166
]. The
phages are extremely diverse in nature [
167
], and are thus highly specific in infecting their
bacterial hosts [168].
In cattle, bacteriophages have been an attractive alternative to antibiotic usage, as
investigated by many researchers [
169
]. Many
in vitro
and mouse models show promising
results for bacteriophage therapy potential. However, a few clinical studies have been
conducted [
170
]. The phages have been widely introduced against mastitis pathogens such
as staphylococcus, E. coli, and Streptococci [
144
]. Studies on utilizing bacteriophages for
treating mastitis caused by S. aureus were reported on the lytic phage SA isolated from
purified sewage water to be active against six S. aureus strains out of 13. The highest
lytic activity was recorded at pH 7 at 37
◦
C [
171
]. In another study, SA phage exhibited a
relatively narrow host range against 10 S. aureus strains compared to SA2 and SNAF phages.
Remarkably, phage SNAF exhibited a significant growth reduction in S. aureus compared
to SA and SA2 phages [
172
]. The lytic activity of the SAJK-IND phage reached 100%
against
S. aureus
isolated from mastitis milk, as reported by Ganaie et al., 2018. However,
using only MSP exhibited just 40% lytic activity against the isolates [
173
]. Another study
investigated a mixture of three phages, STA1.ST29, EB1.ST11, and EB1.ST27 against S.
aureus from pasteurized as well as raw bovine mastitis milk. The results reported that the
bacteriophage mixture significantly reduced S. aureus in the pasteurized milk [174].
Various laboratory models have been used to evaluate the effectiveness of bacterio-
phages as a treatment in many studies. Iwano et al., 2018 reported that the lytic phage
Φ
SA012 and
Φ
SA039 were effective against 93 S. aureus strains and six MRSA strains.
Furthermore, studies investigated the effectiveness of phage
Φ
SA012 and
Φ
SA039
in vivo
using a mouse model.
Φ
SA012 showed higher effectiveness in reducing S. aureus prolifera-
tion and consequently mammary gland inflammation [
175
]. The cocktail bacteriophages are

Ruminants 2022,2148
more effective than single phage, as reported by a study by Geng et al., 2020, investigating
the efficiency of lytic phage cocktail vGSM-A1 and vBSP-A2 in the mouse model [176].
Other studies used bacteriophages against E. coli. Porter et al., 2016 demonstrated the
effectiveness of a cocktail of four bacteriophages against E. coli through several
in vitro
tests
and reported a significant reduction in E. coli adhesion and intracellular survival. Moreover,
the phage inhibited E. coli growth when challenged by 1.6
×
10
3
CFU/mL [
177
]. A more
recent study by Da Silva et al., 2018, evaluated the effect of UVF13 phage against E. coli in
induced mastitis in a murine model. The result indicated a 10-fold reduction in bacterial
load in the phage-treated group [178].
Escherichia coli and Trueperella pyogenes are among the pathogens driving metritis [
111
].
Despite the effectiveness of bacteriophage therapy against E. coli [
178
], the intrauterine
administration of bacteriophages did not affect the uterine pathogens [
170
]. However,
the administration of UFV13 can reduce the T. pyogenes adhesion and disturb the biofilm
formation [179].
The phage-derived endolysins have also been proposed as a potential antimicrobial
agent. In a study by Zhou et al., 2017, the lytic enzyme LysK
∆
amidase exhibited a broad
lytic activity against 137 methicillin-resistant and susceptible staphylococci isolated from
bovine mastitis milk samples and human patients [
180
]. Another study by
Fan et al., 2016
investigated trx-SA1 endolysin isolated from S. aureus bacteriophage IME-SA1 as a possible
treatment for bovine mastitis. The results showed that trx-SA1 could control mild infec-
tion of clinical mastitis [
181
]. An interesting result had been obtained from one study by
Scholte et al., 2018
, which used PlyC, peptidoglycan hydrolyzed derived from streptococ-
cus C1 bacteriophage against streptococcus uberis cell wall. The study reported that a low
dose of PlyC 1.0 µg/mL can induce lytic activity [182].
Bacteriophages have also been used against M. haemolytica, a bacterium that is im-
plicated in bovine respiratory disease pathogenesis. However, none of the studies have
reported a promising result against M. haemolytica [36].
5.3. Prebiotics
Prebiotics are defined as organic nutrients that are indigestible by an animal’s up-
per gastrointestinal tract enzymes but can be digested by one or a limited cohort of gut
bacteria and consequently increase their growth and activity. The cumulative effect is an
improvement of the host’s health [
183
,
184
]. The most used prebiotics in animals are manno-
oligosaccharides, fructo-oligosaccharides, and trans-galacto-oligosaccharides. Prebiotics
can reduce the attachment of pathogenic bacteria and improve the immune response in
cattle [
185
]. In a study by Grispoldi et al., 2017, the introduction of prebiotics in the Holstein
Friesian diet resulted in the reduction of E. coli prevalence [186].
6. Future Perspectives
In this review, we endeavour to summarize the microbiota composition and diver-
sity related to cattle niches, as well as its contribution to animal health and production.
The commensal microbiota composition is directly affected by various environmental
and host factors, for instance, the initial infant microflora is derived from the mother’s
vaginal, skin, and environmental microbiota [
27
] while the rumen of calves, delivered by
C-section, are more likely to be colonized by species from the phylum Firmicutes instead
of
Proteobacteria
[
17
,
28
]. It implies that characterization of native colonized microbiome
should be analyzed in relation to the potential microbial sources such as water, feed, soil,
air, animal handling conditions, etc., and the host characters to be considered are cattle age,
breed, immunity, etc. The association studies considering more variables would sort out a
similar set of phylogenetic and functional biomarkers among consensus microbiome for a
specific niche or organ. For analysis of complete microbiome, microbiome-wide association
studies (MWAS) can be undertaken with reference to select the host and environmental
parameters [1].

Ruminants 2022,2149
Moreover, the lack of consistency within the reported predominance of bacterial phyla
within the healthy cattle microbiome results in ambiguity. This uncertainty points to an
inevitable need to establish improved standardized methods for characterizing whole cattle
metagenome at the omics level. This characterization will lead to a deeper understanding of
conserved functional pathways within varying cattle species around the world. Therefore,
substantiating the concept of a standardized probiotics therapy that can be administered
alongside traditional therapies for effective treatment. The knowledge based on microbial
shifts within diseased and healthy microbiota can be utilized to understand and exploit
commensal relationships within various microbes. The healthy microbiota can be selected
for their prognostic effect, as candidates for preventive probiotics [6].
In light of the commensal relations within microbiota that generate synergistic benefits
for cattle, the researchers must focus on selecting and optimizing the most effective mi-
crobial combination. These combinations will relieve dysbiosis during disease conditions.
Once these combination therapies are established, their efficacy within clinical trials can be
initiated to standardize the dosage, time, and other parameters [187–189].
7. Conclusions
The review has aimed to build a case for a combination of prebiotic and probiotic
therapies alongside traditional antibiotic medicines. The diseases characterized by dysbiosis
are promising targets for such an approach. The bioceutical or nutraceutical therapies
have shown physiological benefits in humans already. With a deeper understanding of
cattle microbiome prevalence and function, these therapies can be extended to all cattle
management systems for improved cattle health and lifespan.
Understanding the cattle microbiome is critical for the maintenance of cattle health
and productivity. Holistic knowledge of the cattle microbiome has the potential to generate
a myriad of benefits, not limited to economic, therapeutic, industrial setups. Despite the
advancements in sequencing technologies, our understanding of the complexities of mi-
crobiome diversity and interactions with the host remains superficial. Understanding the
native microbial composition within could help in designing preventive and therapeutic
approaches against the dysbiosis associated with various diseases. Although the research
gaps are filling up rapidly and promising results have been elucidated in several studies
pertinent to microbiome manipulation, more research is needed before the microbiome
can be exploited for effective disease prevention and treatment strategies. Hence, pro-
longed, horizontal studies with a large sample size are recommended to provide more
precise explanations of the mechanisms influencing microbiome composition, diversity,
and function.
Author Contributions: A.K. conceptualized and wrote the original draft. A.B. and S.A. contributed
to figures drawing and editing the manuscript. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors would like to acknowledge Jens Tetens and Michael Hoppert for
their constructive suggestions. We also thank Ahmed Khalil, Osman Abdalmonem and Christoph
Ammer-Herrmenau for evaluating the review article.
Conflicts of Interest: The authors declare no conflict of interest.

Ruminants 2022,2150
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