https://doi.org/10.52973/rcfcv-e34380
Received: 17/01/2024 Accepted: 18/03/2024 Published: 26/06/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34380
ABSTRACT
Paratuberculosis, created by Mycobacterium avium subspecies
paratuberculosis (MAP), manifests as a chronic aiction marked
by persistent diarrhoea and granulomatous enteritis, pervasive
in both domestic and global wild ruminants. In this investigation,
DNA disruption in lesioned tissues of goat as natural infecte with
MAP was pathologically assessed. Accordingly, goats manifesting
symptoms suggestive to paratuberculosis, including pronounced
emaciation and continual episodic diarrhoea, were subjected to
an ELISA diagnostic procedure to ascertain the presence of MAP.
This diagnostic approach conrmed the presence of the infectious
agent in 20 patients. These patients were subsequently euthanized,
and tissue samples from intestinal and regional lenf nods. It were
subjected to Hematoxylin and Eosin (HE) staining for histopathological
investigatıon, Ziehl Neelsen (ZN) staining to identify acid–fast
mycobacteria, γ–H2AX to discern disruptions in double stranded
DNA, and 8–Ohdg to detect DNA oxidation by immunohistochemical
(IHC) method. Gross anatomical observation serous adipose atrophy,
augmented dimensions of mesenterial lymphatic nodes, mucosal
hypertrophy and non–retractable mucosal undulations. Histological
assessment highlighted epithelial cellular degeneration, an abundance
of epithelioid macrophages, lymphocytes, plasmocytes, inltrating in
mucosa. Acid–fast entities, discernible through ZN staining, appeared
as luminescent red conglomerates in intestinal and mesenterial
tissue. The immunohistochemical analyses evinced positive results
for both γ–H2AX and 8–Ohdg across all sampled tissues. Intriguingly,
this investigation presented the inaugural global evidence of γ–H2AX
and 8–Ohdg expression in a natural MAP infection, demonstrating that
this pathological agent precipitates DNA degradation and oxidation,
thereby augmenting comprehension of the disease’s pathogenesis.
Key words: Goat; MAP; paratuberculosis; γ–H2AX; 8–Ohdg
RESUMEN
La paratuberculosis, provocada por Mycobacterium avium subespecie
paratuberculosis (MAP), se maniesta como una afección crónica
marcada por diarrea persistente y enteritis granulomatosa,
generalizada en rumiantes domésticos y salvajes del mundo. En
esta investigación se evaluó patológicamente la alteración del DNA
en tejidos lesionados de cabra infectada naturalmente por MAP. En
consecuencia, las cabras que manifestaban síntomas sugestivos
de paratuberculosis, incluida emaciación pronunciada y diarrea
episódica continua, se sometieron a un procedimiento de diagnóstico
ELISA para determinar la presencia de MAP. Este procedimiento
de diagnóstico confirmó la presencia del agente infección en
20 pacientes. A continuación, se practicó la eutanasia a estos
pacientes y se tomaron muestras de tejido de los ganglios linfáticos
intestinales y regionales. Se sometieron a tinción de Hematoxilina
y Eosina (HE) para la investigación histopatológica, tinción de Ziehl
Neelsen (ZN) para identificar micobacterias acidorresistentes,
γ–H2AX para discernir alteraciones en el DNA de doble cadena
y 8–Ohdg para detectar la oxidación del DNA mediante el método
inmunohistoquímico (IHQ). Observación anatómica macroscópica
atroa adiposa serosa, aumento de las dimensiones de los ganglios
linfáticos mesenteriales, hipertroa mucosa y ondulaciones mucosas
no retráctiles. La evaluación histológica destacó degeneración
celular epitelial, abundancia de macrófagos epitelioides, linfocitos,
plasmocitos, inltrados en la mucosa. Las entidades ácido–alcohol
resistentes, distinguibles mediante tinción de ZN, aparecían
como conglomerados rojos luminiscentes en el tejido intestinal y
mesenterial. Los análisis inmunohistoquímicos mostraron resultados
positivos tanto para γ–H2AX como para 8–Ohdg en todos los tejidos
muestreados. Curiosamente, esta investigación presentó la evidencia
global inaugural de la expresión de γ–H2AX y 8–Ohdg en una infección
natural por MAP, demostrando que este agente patológico precipita la
degradación y oxidación del DNA, con lo que aumenta la comprensión
de la patogénesis de la enfermedad.
Palabras clave: Cabra; MAP; paratuberculosis; γ–H2AX; 8–Ohdg
Pathological Investigation of Double–Stranded DNA Breaks and DNA
Oxidation in Natural Infection with Mycobacterium avium subspecies
paratuberculosis in Goats
Investigación patológica de las roturas de DNA de doble cadena y la oxidación del DNA en
la infección natural por Mycobacterium avium subespecie paratuberculosis en cabras
Muhammet Bahaeddin Dörtbudak
1
* , Merve Öztürk
2
1
Harran University, Faculty of Veterinary Medicine, Department of Pathology. Sanliurfa, Türkiye.
2
Necmettin Erbakan University, Faculty of Veterinary Medicine, Department of Internal Medicine. Konya, Türkiye.
*Corresponding author: mbdortbudak@gmail.com
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INTRODUCTION
Paratuberculosis manifests as chronic diarrhea and cachexia
in ruminants. Globally prevalent, this disease inicts substantial
economic impacts through reductions in milk output, live weight, and
can even lead to the death of the animals. There’s evidence to suggest
a link between this disease in both wild and domestic ruminants and
Crohn’s disease in humans. However, it’s currently not identied as a
zoonosis. Furthermore, the discovery of the disease–causing agents
in raw milk from affected animals poses food safety concerns [1, 2, 3].
Mycobacterium avium subspecies paratuberculosis (MAP) is identied
as the etiological agent of paratuberculosis. This bacterium is acid–
resistant, gram–positive, devoid of spores, rod–shaped, and can persist
within macrophages. The disease exhibits a high prevalence but low
fatality rate. Transmission is commonly through water and food sources
contaminated with infectious secretions, including direct contact,
faeces, urine, and saliva. Reports also indicate possible intrauterine
transmission to the offspring. Although young ruminants are highly
vulnerable, manifestations aren’t evident in animals younger than 2
years, attributed to the disease’s extended incubation period [4, 5]. The
primary site of infection after oral intake is the gastrointestinal system,
predominantly affecting the terminal sections of the small intestine and
adjacent lymph nodes. The most evident clinical manifestation is chronic
diarrhea, frequently accompanied by dehydration, cachexia, depression,
submandibular swelling, and diminished milk production. In deceased
animals, granulomatous enterocolitis and localized lymphadenitis
are commonly present. On a histopathological level, infiltration of
lymphoplasmacytic cells, predominantly epithelioid histiocytes, is
noticeable. While no conclusive treatment exists and its zoonotic nature
remains unveried, a general recommendation is the culling of infected
animals, even though reporting the disease isn’t mandatory [3, 6, 7, 8].
Recent research underscores the impact of pathogenic agents on
cellular damage, with DNA damage being a fundamental mechanism of
such injury. Two pivotal biomarkers for DNA damage are γ–H2AX and
8–OhdG. The γ–H2AX protein, a member of the H2A family, is integral to
the DNA repair process. Phosphorylation of histone proteins initiates
the emergence of γ–H2AX, which facilitates the primary assembly of
DNA repair proteins at the site of double–strand breaks. Consequently,
γ–H2AX is currently recognized as a vital indicator of DNA breakages
in scientic investigations [9, 10]. On the other hand, 8–OHdG, another
DNA damage biomarker, is indicative of DNA oxidation. It emerges
from the oxidation of the 8th carbon atom of the guanine base in DNA
by free radicals and is acknowledged as a marker for DNA damage
attributed to oxidative stress [11, 12].
This research study aimed to probe the deleterious impact
of pathogens on DNA to shed light on the pathogenesis of
paratuberculosis, a signicant health concern for domestic ruminants
globally. To achieve this, the study assessed the presence of γ–H2AX
and 8–OhdG expressions, current biomarkers, in the affected tissues
of naturally infected animals, thereby ascertaining DNA damage.
MATERIALS AND METHODS
Animal Material
In this study, the materials of the study conducted with the ethical
approval of Bıngol University Animal Experiments Local Ethics Committee
(B.Ü. AELEC 2023/02–02/13) were used. The subjects of the research
were 20 female hair goats (Capra hircus), aged between 2 to 5 years.
Blood samples were collected from mature animals displaying symptoms
suggestive of paratuberculosis, such as consistent intermittent diarrhea,
pronounced emaciation, and diminished productivity. The presence of the
disease was diagnosed using the ELISA test. Subsequent to the testing,
all 20 animals suspected of having paratuberculosis were conrmed
to be infected with MAP. Given the lack of an effective treatment and
to thwart environmental contamination, the affected animals were
euthanized. Postmortem examinations of these animals displayed
signs of paratuberculosis, particularly in the intestines and mesenteric
lymph nodes. For further histopathological and immunopathological
evaluations, samples from the affected tissues were secured and
preserved in a 10% buffered formalin solution.
ELISA Test
Blood sera from suspected infected animals were analyzed
for MAP antibodies using a commercial ELISA kit (Paracheck 2,
no.63325, Prionics AG, Zurich, Switzerland) to detect the presence
of infection antibodies. The testing procedure adhered strictly to
the manufacturer’s prescribed protocol. Post–reaction, the optical
density (OD) of each well was measured using an ELISA reader (Rayto,
RT–2100C, China) tted with a 450 nm lter.
Tissue Processing
Tissues preserved in a 10% buffered formalin solution were rinsed
in running tap water before undergoing rutine tissue processing.
From the processed tissue samples, paran blocks were produced.
Sections measuring 5 µm in thickness were then cut from each
block onto both normal and polylysine–coated slides using a rotary
microtome (Leica, RM2135, Germany).
Histopathological Examination
Tissue sections placed on conventional slides were oven–dried for
an hour, followed by deparanization and rehydration. To observing in
tissue histopathological changes and acid–resistant microorganisms,
HE and ZN staining techniques were employed, respectively [13].
After staining, each tissue section was covered with a coverslip
using a drop of Entellan™ and subsequently observed under a light
microscope (Leica, DM2500, Germany).
Immunohistochemical Staining
Tissue sections placed on polylysine slides underwent oven drying
for an hour before deparanization and subsequent rehydration
through a xylol–alcohol series. Endogenous peroxidase activity was
quenched by immersing the tissues in 3% H
2
O
2
for 10 min. Following
a PBS wash, antigen retrieval was achieved by boiling the tissues
three times in a retrieval solution. Another PBS wash was followed
by outlining the tissue sections with a PAP pen and then blocking
non–specic binding sites using Ultra V Block for 20 minutes. The
primary antibodies, γ–H2AX (no. NB100–2280SS, NovusBio, USA) and
8–Ohdg (no. sc66036, Santa Cruz, USA), were diluted at a 1/100 ratio
and then incubated at +4°C overnight. After a PBS wash, biotinylated
secondary antibody was applied to the tissues, followed by a 20 min
incubation. Following another PBS wash, streptavidin–peroxidase
was added for an additional 20 min. Upon the nal PBS wash, 3,3′–
Diaminobenzidine (DAB) chromogen was introduced to the sections
to visualize the antigen–antibody interaction. After counterstaining
with Mayer Haematoxylin, the sections, now covered with coverslips,
were analyzed under a light microscope [13].
FIGURE 1. Gross pathology. A; Serous fat atrophy in the mesenterium (arrowhead)
and severe edema in the mesenterial lymph node (arrow). B; Thickening (arrow)
and transverse folds (arrowhead) of the intestinal mucosa
FIGURE 2. Histopatology. A; Atrophy and fusion of villi (arrowheads), inammatory
cell infiltrations in the lamina propria (thin arrows), epithelioid histiocyte
aggregations (white stars), lymphoid hyperplasia (black stars), inflammatory
cell infiltration and lymphangitis in the submucosa (thick arrows), HE, 4×. B;
Severe degeneration and desumation of enterocytes (arrowheads), edema and
inammatory cell inltration in villous lamina propria (thick arrow), cystic dilatation
in crypt glands and degeneration of epithelial cells (thin arrows), enlargement
of villous lacteals (star), HE, 10×. C; Degeneration in enterocytes (arrowheads),
epithelioid histiocyte aggregation in lamina propria (stars), mononuclear cell
inltration in lamina propria (thick arrow), cystic dilation in crypt glands (thin
arrows), HE, 10×. D; Epithelioid cell granulomas (stars), edema and inammatory
cell inltrates (thick arrow), lymphoid hyperplasia (arrowheads), HE, 10× in the
mesenterial lymph node. E; Areas showing acid–fast mycobacteria suggestive of
Mycobacterium avium subspecies paratuberculosis (arrows) in the lamina propria,
ZN, 10×. F; Areas showing acid–fast mycobacteria suggestive of Mycobacterium
avium subspecies paratuberculosis (arrows) in mesenterial lymph node ZN, 10×
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RESULTS AND DISCUSSIONS
ELISA Test
ELISA test was performed on the blood sera of 20 goats with
suspected MAP infection in clinical findings. According to the
arithmetic mean (S/P (+) ≥ 0.50) calculated from the optical densities
of the tested serum samples, 20 goat sera with suspected infection
in the study were positive for MAP infection.
Macroscopic Findings
Upon postmortem examination of infected goats that were
euthanized and sent to slaughter, notable lesions were detected in
the intestine and its adjacent lymph nodes. In the abdominal cavities,
there was a presence of about 2–3 L of either serous or serobrinous
exudate. The mesentery, omentum, and subperitoneal fatty tissue
exhibited signs of atrophy and were substituted by a widespread
yellowish gelatinous edema. The serosa of the intestine appeared
cloudy due to pervasive edema. Furthermore, mucosal thickening
and undulating folds, which remained rigid even when tugged, were
evident. The intestinal chambers contained watery content. While
the lesions permeated almost all segments of the intestine, they were
predominantly located in the ileum, jejunum, and to some extent in
the proximal section of the cecum. The lymphatic vessels appeared
cord–like due to thickening, and the mesenteric lymph nodes were
markedly enlarged. Cross–sections of the edematous lymph nodes
revealed them to be engorged, making it challenging to discern
between the cortex and medulla. (FIG.1. A–B)
macrophages, lymphocytes, and plasmocytes, complemented by
a presence of neutrophils and eosinophils. Notably, collections of
focal epithelioid macrophages were commonly identied, while the
presence of giant cells was infrequent. The described epithelioid
cells typically exhibited a round to oval morphology with peripherally
positioned euchromatic nuclei and cytoplasm that was either
eosinophilic or foamy. The villous lacteals and Lieberkühn crypts
displayed notable cystic dilatation and desquamation (FIG. 2. A–B).
In the submucosa, inammatory manifestations mirrored those in
the lamina propria. The lymphoid structures within the submucosa
displayed hyperplasia and perilymphangitis. Despite an absence
of detectable lesions in the muscularis layer, the serosa exhibited
edema, subtle leukocyte inltrations, and lymphangitis (FIG. 2. C).
In examining the mesenteric lymph nodes, a notable enlargement
and uid retention were observed in nearly all sinuses. The cortex
demonstrated generalized lymphoid hyperplasia, and microgranulomas
consisting of epithelioid macrophages were evident within the
paracortical region and subcapsular sinuses (FIG. 2. D). ZN staining
Histopathological Findings
Histopathological evaluations indicated pervasive proliferative
inflammation encompassing nearly all intestinal layers, with
pronounced effects in the jejunum and ileum. Noteworthy
morphological alterations included villi atrophy, fusion, and attening,
coupled with substantial degeneration and shedding of enterocytes.
The mucosal lamina propria displayed edema, intensive inammatory
cell inltrations, and marked expansion, partly attributed to increased
brous connective tissue. Predominantly, the inammatory cell
population was composed of mononuclear leukocytes, including
FIGURE 3. Immunopathology; A; Expression of γ–H2AX in epithelioid histiocytes,
inammatory cells and enterocytes (arrowheads), B; Expression of γ–H2AX in
villus epithelium and inammatory cells (arrowheads), IHC, 20×. C; Expression
of γ–H2AX (arrowheads) in inammatory cells in the mesenterial lymph node,
IHC, 20×. D; 8–Ohdg expression in epithelioid histiocytes, inammatory cells and
enterocytes (arrowheads), IHC, 20×. E; 8–Ohdg expression (arrowheads) in villi
epithelium and inammatory cells, IHC, 20×. F; 8–Ohdg expression (arrowheads)
in inammatory cells in the mesenterial lymph node, IHC, 20×
DNA damage in goat paratuberculosis / Dörtbudak and Öztürk _____________________________________________________________________
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showcased a plethora of vivid red acid–fast entities, primarily
internalized by epithelioid cells within the villous lamina propria or in
external clusters. Epithelioid macrophages, and extracelluler areas
in mediastinal lenf nodes, acid–fast mycobacteria suggestive of
Mycobacterium avium subspecies paratuberculosis as conrmed,
albeit in reduced numbers (FIG. 2. E–F).
Immunohistochemical Findings
Immunohistochemical analysis using γ–H2AX revealed
immunoreactivity within the degenerated mucosal epithelium,
lamina propria–inltrating leukocytes, crypt epithelial cells, and in
the nuclei of most epithelioid macrophages. Additionally, intranuclear
staining of epithelioid macrophages was evident in the mediastinal
lymph nodes, particularly in areas with lymphoid hyperplasia (FIG 3.
A–C). When employing 8–Ohdg for immunohistochemical staining,
intranuclear staining was identied in leukocytes and epithelioid cells
within the lamina propria, submucosa, and mediastinal lymph nodes.
Furthermore, degenerated enterocytes displayed immunoreactivity
for 8–Ohdg (FIG. 3. D–F).
Paratuberculosis, stemming from MAP, represents a signicant
health concern for domestic ruminants, inflicting substantial
economic repercussions. This ailment, prevalent across various
global regions for an extended period, persists in its widespread
nature, with aicted animals typically exhibiting chronic diarrhea and
deteriorating physical conditions [3, 14]. The present study’s subjects,
based on their clinical histories and symptoms, predominantly
displayed intermittent, persistent diarrhea coupled with pronounced
emaciation, mirroring ndings from prior natural and experimental
infection research.
The macroscopic alterations discerned during necropsies of MAP–
aicted animals provide valuable diagnostic insights, distinguishing
it from other gastrointestinal maladies. Notably, while most intestinal
infections in ruminants typically manifest as erosive–ulcerative or
exudative, MAP infections are hallmarked by proliferative inammation
within the tissues [15, 16]. Such inammation predominantly exhibits
macroscopic tissue thickening, a feature also observed in this study,
especially in the mucosal thickening and folding of the intestines.
Concurrently, the disease impacts the mesenterial lymph nodes and
lymphatic vessels. While certain studies have highlighted frequent
occurrences of caseous necrosis within mesenterial lymph nodes,
others reported its rarity [17, 18].
In the made research, such necrotic clusters were infrequently
identied. Lymphatic vessel thickening stands out as a recurrent
macroscopic observation in MAP infections. It’s theorized that
obstructions arising from lymphangitis may play a pivotal role in
the accumulation of uid within the abdominal cavity. Furthermore,
the pronounced oedema witnessed in both the abdominal region and
beneath the chin can primarily be attributed to acute hypoproteinemia,
a consequence of malabsorption due to extensive intestinal mucosal
damage. Interestingly, despite the glaring hypoproteinemia, the
incidence of dehydration–induced mortality remains relatively
low, potentially owing to the duodenum’s minimal damage, which
is responsible for absorbing a signicant portion of the stomach’s
uid content. Consequently, fatalities due to acute dehydration are
infrequent. Although affected animals maintain regular appetite levels,
they exhibit cachexia and oedema stemming from hypoproteinemia.
malabsorption, in turn, culminates in serous fat atrophy resulting
from energy decits [15, 16, 17]. Corroborating prior research, this
study identied lymphadenitis, lymphangitis, ascites, cachexia, and
serous fat atrophy in affected subjects.
During chronic infections, there is typically an influx of
lymphoplasmacytic cells and broblasts at the inammation site.
In the case of chronic MAP infection, the predominant inammatory
cell inltration is primarily composed of mononuclear leukocytes. The
hallmark microscopic manifestation of MAP infection is the aggregation
of epithelioid macrophages. These inflammatory responses are
primarily located within the villous lamina propria and submucosa,
accompanied by vascular alterations. Concurrently, along with
proliferative characteristics, lesions indicative of tissue destruction
is also evident. It’s recognized that inammatory modications are
predominantly found in the small intestine’s distal portion [8, 19, 20].
This localized prevalence is believed to arise because the causative
agents predominantly are captured by M cells in this region to
breach the submucosa. When examining MAP infection through
numerous studies, its histopathological presentation can generally
be categorized into two distinct forms. The rst is the paucibacillary
form, characterized by a lymphocyte predominance and associated
with a Th–1 cell–mediated immune response. In contrast, the
multibacillary form is dominated by epithelioid macrophages and
correlates with a Th–2 humoral immune response. Both experimental
and naturally occurring infection studies have consistently reported
the multibacillary form as the more prevalent [2, 21, 22].
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Similar to the inltration patterns seen in the intestinal submucosa,
mesenterial lymph nodes also exhibit lymphoplasmacytic and
macrophage inltrations. However, the inammation’s intensity in
the lymph nodes has been described variably across studies, with
some reporting severe manifestations and others indicating milder
presentations [18, 23]. In the present investigation, the observed
patterns aligned with those from prior research. Additionally, it was
ascertained that the multibacillary form was present in almost all
studied animals, with lesions in the lymph nodes appearing less severe.
Cellular damage underpins the majority of diseases, and there’s a
prevailing theory that pathogens may directly or indirectly inict DNA
damage, especially in the case of infections [24, 25]. While various
studies have delved into the pathogenesis of MAP infection, the
complete understanding of its pathogenesis remains elusive. In an
attempt to elucidate this, past research on MAP infections in cattle,
sheep, goats, and camels primarily focused on proinammatory
cytokines, acute phase proteins, and oxidative stress parameters
[23, 26, 27]. Yet, there seems to be a gap in the literature regarding
DNA damage assessment in MAP infection.
In the present study, showcased the expression of γ–H2AX, a
biomarker indicative of double–stranded DNA breaks, in the case
of MAP infection. There is limited research utilizing γ–H2AX as a
biomarker in animals. For instance, Nakamura et al. [28] applied this
biomarker to identify DNA breaks in bovine lymphocytes post the
Fukushima disaster. Toyoda et al. [29] highlighted the DNA damage
in genotoxic urinary bladder cancers using γ–H2AX expression. Both
Fradet–Turcotte et al. [30] and Sakakibara et al. [31] postulated
that papilloma viruses induce DNA damage as evidenced by γ–
H2AX expression. Drawing parallels with previous studies, in the
this research underscores that MAP indeed induces DNA breaks in
affected tissues, as evidenced by γ–H2AX expression.
Infection–induced tissue damage triggers inflammation,
subsequently activating phagocytic cells. This activation gives rise to
the production of free radicals. These reactive oxygen species amplify
the extent of tissue damage. In the case of MAP infection, oxidative
stress markers such as Superoxide Dismutase (SOD), Malondialdehyde
(MDA), Glutathione (GSH), Nitric Oxide (NO), and Thiobarbituric Acid
Reactive Substances (TBARS) have been examined across various
animal species [23, 27, 32]. Yet, research pinpointing DNA oxidation
in MAP infection remains absent. The modern biomarker, 8–Ohdg, has
been employed to elucidate DNA oxidation in certain animal species
and varied diseases. For example, Karakurt et al. highlighted DNA
damage in ovine pulmonary adenocarcinoma using 8–Ohdg [33].
In a separate study, Karakurt underscored DNA oxidation in bovine
papilloma and bropapilloma via 8–Ohdg expression [34]. In the made
study, the occurrence of DNA oxidation in the case of MAP infection
was depicted through 8–Ohdg expression.
CONCLUSION
To effectively combat any disease and establish an impactful
treatment regimen, a thorough understanding of its pathogenesis
is imperative. Given the signicance of MAP infection, perceived as
a global threat affecting both animal and human health and resulting
in economic loss, this study illuminates that the causative agent
induces DNA breaks and oxidation in infected tissues. In made been
this research has pioneered the elucidation of the DNA damage
mechanism in MAP infection, marking a signicant contribution to
the global scientic community. In addition, this is the rst study
reported in the world to show the expression of γ–H2AX, a current
DNA damage marker, and 8–Ohdg, an important DNA oxidation
biomarker, in natural infection of Mycobacterium avium subspecies
paratuberculosis in goats.
Conict of interests
No conicts of interest for all authors are declared.
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