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Alterations in host biomarkers in Cryptosporidium infected goats

N.U. Khan, M.H. Saleem, M.S. Sarwar, I.U. Khan, M.A. Khan, A. Ali,
H. Ali, G. Rashid, M. Shafee, M. Ali, A.U. Khan, K. Prince,
Shakirullah, A. Ullah, I. Khan, W. Ahmad, M.L. Sohail

PII: S0921-4488(20)30204-2
DOI: https://doi.org/10.1016/j.smallrumres.2020.106255
Reference: RUMIN 106255

To appear in: Small Ruminant Research

Received Date: 13 January 2020

Revised Date: 27 September 2020
Accepted Date: 1 October 2020

Please cite this article as: Khan NU, Saleem MH, Sarwar MS, Khan IU, Khan MA, Ali A, Ali H,
Rashid G, Shafee M, Ali M, Khan AU, Prince K, Shakirullah, Ullah A, Khan I, Ahmad W, Sohail
ML, Alterations in host biomarkers in Cryptosporidium infected goats, Small Ruminant
Research (2020), doi: https://doi.org/10.1016/j.smallrumres.2020.106255

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Alterations in host biomarkers in Cryptosporidium infected goats

Khan,N.U.a, Saleem, M.H.b, Sarwar, M.S.a, Khan, I.U.c,Khan, M.A.b, Ali, A.d, Ali, H.1, Rashid,
G.b, Shafee, M.e, Ali, M.b,Khan, A.U.f,Prince, K.g, Shakirullahc,Ullah, A.a,Khan, Ia, Ahmad, W.h,
Sohail, M.L.g* mls@cuvas.edu.pk

a
College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University Mardan,

Pakistan
b
Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences,

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Lahore, Pakistan
c
Faculty of Veterinary and Animal Sciences, Gomal University, Dera Ismail Khan , Pakistan

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d
Department of Sociology, Abdul Wali Khan University Mardan, Pakistan

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e
Center of Excellence in Vaccinology and biotechnology, Quetta, Pakistan
f
Department of Animal Health, The University of Agriculture Peshawar
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g
Department of Medicine, Cholistan University of Veterinary and Animal Sciences, Bahawalpur,
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Pakistan
h
Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore,

Pakistan
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*CORRESPONDING AUTHOR
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Dr. Muhammad Luqman Sohail

Department of Medicine, Cholistan University of Veterinary and Animal Sciences,
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Bahawalpur, Pakistan.
+92-334-958-3248
Highlights

 A total of 384 fecal and blood samples were randomly collected and diagnosed using

two-step nested PCR, after initial screening. Blood samples were processed for

measurement of hematological and biochemical parameters.

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  Diseased goats showed significant increase (P<0.05) in lymphocytes, eosinophils and

packed cell volume. Biochemical analysis showed significant decrease (P<0.05) in total

proteins, albumin, ALP, sodium and zinc, while significant increase (P<0.05) was

recorded in copper, urea and creatinine.

Abstract

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Cryptosporidiosis is the second major cause of diarrheal illness in humans and animals. Despite

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being a major cause of digestive anomalies in goats of Khyber Pakhtunkhwa (Pakistan), there is

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a serious dearth of data regarding the infection induced alterations in physiological biomarkers.

The present study was conducted to record changes in vital body parameters caused by the
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cryptosporidiosis. A total of 384 fecal and blood samples were randomly collected from adult

goats showing clinical signs of the disease. Fecal samples were examined using the modified
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Ziehl Neelsen acid fast staining, and were confirmed for the infection using two-step nested PCR

assay. A total of 66 animals were included in the diseased group, while 57 were included in
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healthy animals group. Blood samples were processed for measurement of hematological and

biochemical parameters using automatic analyzer. Diseased goats showed significant increase in
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lymphocytes, eosinophils and packed cell volume (P<0.05). Biochemical analysis showed
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significant decrease in total proteins, albumin, ALP, sodium and zinc (P<0.05), while significant

increase (P<0.05) was recorded in copper, urea and creatinine in diseased goats.

Key words: Goat, Cryptosporidium, hematology, Biochemical profile

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INTRODUCTION

Enteric protozoa are a major menace for the productivity potential of livestock, exerting

negative effects on economy (Squire et al., 2017). Moreover, a number of such parasites are of

public health concern as zoonotic transmission may occur to humans exposed to infected animals

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(Khan et al., 2017). Cryptosporidium is one of such important enteric protozoa which infect a

wide range of vertebrates, including reptiles, fish and mammals (Liet al., 2016). The

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Cryptosporidium infection is highly endemic in small and large ruminants in Pakistan and its

prevalence is reported over 30% in small ruminants (Khan et al., 2017).

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Cryptosporidiosis in small and large ruminants is considered as a major cause of infection
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in humans, due to the dissemination of oocysts in environment (Squire et al., 2017).

Conventional microscopic fecal examination remains the primary diagnostic technique for
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detection of parasite. It is cheap, but requires skillful laboratory personnel and results often

depend on proper fecal collection (Plutzer et al., 2018). Due to limited ability of this traditional
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diagnostic tool to differentiate the source of parasite, various aspects of fecal-oral and other

transmission routes (person to person, contact with animals, contaminated food and municipal
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drinking water) are still unclear (Fall et al., 2003). Recently, development of different molecular

techniques for detection and differentiation of Cryptosporidium spp. are now used in
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epidemiological studies and have improved our understanding of the interspecies transmission

(Xiao, 2010).

Parasitic infection brings about several alterations in host biomarkers and changes the

blood picture of infected animal (El Manyaweet al., 2010). There is a serious dearth of data

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regarding the changes in hematological and biochemical parameters induced by Cryptosporidium

in small ruminants in region. So this study is aimed to record such changes in goats suffering

from cryptosporidiosis.

MATERIALS AND METHODS

The current study was carried out in three districts of province Khyber Pakhtunkhwa i.e; Lakki

Marwat, Kohat and Bannu.

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Lakki Marwat lies at 32° 36' 27" north, 70° 54' 45" east, at an altitude of 262 meters.

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Kohat is located between north latitude 32° 47' and 33° 53' and east longitude 70° 34' and 72° 17

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and is at the elevation of 489 meters from sea level. Bannu is situated at 32.99° north latitude,

70.61° east longitude and is 371 meters above the sea level.
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All the fecal samples (n=384) were collected directly from the rectum of adult goats (12 to 18
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months old). Sampling was carried out using a cross-sectional study design and animals showing

clinical signs of the disease (mild mental depression, diarrhea of different severity) were

selected. Cryptosporidium oocysts were confirmed by simple microscopic examination after

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Modified Ziehl- Neelsen (MZN) staining technique (Casemore et al., 1985). All the slides were

observed through calibrated light microscope at 100X magnification as described by Bakiret et

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al., 2003. A fecal sample was considered positive if at least one, clearly identifiable oocyst was
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identified. Furthermore, the positive samples were subjected to nested PCR for confirmatory

diagnosis and only the PCR positive goats were included in diseased group. Animals included in

the healthy animals group were asymptomatic and were tested negative for the Cryptosporidium

and other common infections prevalent in the area of study. All the healthy animals were tested

negative for commonly occurring blood and fecal parasites (theileria, babesia, anaplasma, liver

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fluke, hemonchus contortus, coccidiosis and other cestodes and enteric parasites). All the healthy

animals were following complete vaccination schedule recommended by livestock department. .

Haematobiochemical Profile

Blood sample (3ml) was collected from jugular veinin plain (for biochemical analysis) and

anticoagulant added (for hematological analysis) vacutainers, each, from PCR positive

symptomatic goats. Serum was harvested and kept at -20°C till further analysis. Changes in

blood profile, liver and renal biomarkers and serum biochemical profile were carried out in

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healthy and diseased animals. Hematological parameters, hematocrit (PCV), hemoglobin (Hb),

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RBC’s count, and differential leukocytic count were recorded using automatic hematological

analyzer (Abacus junior vet, Diatron, Austria). Liver enzymes, aspartate aminotransferase

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(AST), alanine transaminase (ALT), alkaline phosphatase (ALP),renal function parameters,
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creatinine and blood urea nitrogen (BUN)were recorded by Pictus 700 chemistry analyzer

(Diatron, Hungary). Mineral concentration (Calcium, Magnesium, Zinc, sodium, copper and
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potassium)of healthy and diseased animals were also determined. Serum levels of zinc and

copper were recorded (Ali et al., 2013). Briefly, each of serum and nitric acid (0.5 ml) were kept
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at 90°Cfor 15 minutes. After cooling, 2.5 ml perchloric acid was mixed with the mixture and

heated to bring the volume down to 2 ml. These samples were further processed for the
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measurement of copper level by spectrophotometer (Perkins Elmer Analyst 800, USA). Zinc

level was measured by commercially available kits and chemistry analyzer. Serum level of
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calcium, magnesium, sodium and potassium was measured by automated blood chemistry

analyser (Hitachi, Japan), and chemical reagents (Thermo Fisher Scientific Inc. USA). Total

serum protein was measured by biuret method, while albumin was estimated through

commercially available reagent kits.

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Statistical analysis

The statistical analysis was done using SPSS version 20.0. Blood and serum biochemical

markers of healthy and diseased animals (n=57, n= 67, respectively) were analyzed through

student t test, keeping confidence interval 95%, with alpha value of <0.05. Data set for individual

variables was checked for normal distribution.

RESULTS

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Table1 shows the alterations recorded in 66 adult diseased goats in comparison to healthy goats

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(n=57). Hematology revealed significant increase in number of lymphocytes and eosinophils

(P<0.05). Packed cell volume (PCV) also experienced significant increase during the infection

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(P<0.05). No significant differences were found in hemoglobin concentration, monocytes and

basophil counts (P>0.05).

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Results showed significant changes in renal and hepatic biomarkers during cryptosporidiosis.
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Liver enzyme ALP significantly decreased (P<0.05). Blood urea nitrogen (BUN) and creatinine

increased significantly during cryptosporidiosis (P<0.05). Results showed significant decrease in

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total protein and albumin in diseased goats (P<0.05). Results revealed significant increase in

potassium and copper, whereas sodium and zinc were decreased significantly (P<0.05).
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DISCUSSION

Clinicopathologic data helps in differentiating gastrointestinal diseases, developing a prognosis

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and plan for treatment (Navarre and Pugh, 2002). Keeping this in consideration, this study

addresses the alterations in biomarkers of adult goats having cryptosporidiosis, a major digestive

anomaly in small ruminants.

Results showed significant increase in circulating lymphocytes count in diseased goats. During

the disease lymphocytes, macrophages and polymorphonuclear cells migrate to lamina propria as

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a part of the host defense mechanism against this parasite (Waters et al., 1996). Circulating

eosinophils were also increased for animals categorized as diseased. PCV increased significantly

in the diseased animals. This increase may be attributed to excessive loss of body fluid and lesser

fluid intake due to diarrhea during the disease (Osman et al., 2008). Thakre et al., 2016 also

reported increase in PCV because of dehydration and increased Hb. Tajik and Nazifi, 2013 also

correlated higher PCV with prolonged dehydration in case of diarrheic illnesses.

Various significant biochemical alterations were recorded during cryptosporidiosis. Similar to

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previous studies, total protein and albumin concentrations were significantly reduced in the

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diseased animals (El Manyawe et al., 2010). This decrease may be due to the intestinal protein

loss during diarrhea, a prominent feature of cryptosporidiosis. Albumin is a negative acute phase

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protein and its concentration decreases during inflammation as induced by cryptosporidiosis
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(Hashemnia et al., 2014). Excessive protein break down, loss of albumin and poor absorption of

nutrients during cryptosporidial infection also causes a decrease in albumin concentration

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(Bollam et al., 2005). El Manyawe et al., 2010 also reported similar alterations in renal indicators

along with liver enzymes. These findings indicate altered renal and hepatic function in infected
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animals. Carmena, 2010 stated deleterious effects of cryptosporidiosis on liver in human

subjects. Creatinine and urea were recorded to be increased significantly in diseased animals as
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compared to healthy animals. This increase may be attributed to decreased blood flow to kidneys

and lesser urine formation to maintain body hydration status and to avoid dehydration during
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diarrhea. Increase in creatinine and BUN could be related to reduced glomerular filtration rate

(GFR) as a pre renal event (e.g. dehydration) given PCV was higher in this cohort. Wang et al.,

2016 also reported elevated creatinine level during diarrheic illness indicating altered renal

function.

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In this study, serum electrolytes and trace elements (sodium, zinc, potassium and copper)

experienced significant alterations in diseased animals. This may be due to fecal losses of

electrolytes and possible translocation of intracellular and extracellular compartments (Tajik and

Nazifi, 2013). Mageed et al., 2015 also reported significant decrease in sodium level during

Cryptosporidiosis, which is attributed to loss of sodium ions due to intestinal secretions and

diarrhea. El Manyawe et al., 2010 also described low level of sodium due to diarrhea and

malabsorption in the infected tissue of intestine during the disease. In contrast, potassium ions

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increased significantly, likely due to the acidosis associated with enteritis, in which extracellular

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potassium level increases due to K/H exchange mechanism, in response to metabolic acidosis

(Radostits, 2007). A significant decrease in level of zinc and increase in copper level was

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recorded in diseased animals. Our results are in agreement with Yones et al., 2015, who reported
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similar findings in human subjects with parasitic infections, which may be due to inverse relation

of zinc and copper concentrations. A decrease in zinc concentration increases copper absorption
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by gastrointestinal tract. It is linked to synthesis of metallothionein, which has higher affinity for

copper than zinc.

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This study reports significant alterations in hematological and biochemical parameters of goats

suffering from cryptosporidiosis, along with changes in level of electrolytes and trace elements,
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which must be taken into consideration when addressing the disease in field conditions.

Financial Disclosure
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This research did not receive any specific grant from funding agencies in the public, commercial,

or not-for-profit sectors..

Declaration of interest

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Authors declare there is no conflict of interest

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Table 1: Various parameters of healthy and cryptosporidium infected goats
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Healthy animals
Parameters Infected animals (n=66)
(n=57)
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Hematology

TEC (×1012 /l) 13.67 ±0.04a 13.21 ±0.03a

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Hb (g/L) 105.30 ± 1.01a 107.40± 0.45a

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PCV (L/L) 0.36± 0.07a 0.45±0.05b

TLC (109/L) 10.42 ± 1.56a 12.33 ± 1.21a

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Lymphocyte (109/l) 4.85 ±0.93a 8.11 ±0.44b

Monocyte (109/l) 0.57 ±0.09a 0.59 ±0.21a

Eosinophil (109/l) 0.39 ±0.07a 0. 53 ±0.04b

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 Basophil (109/l) 0.09±0.03a 0.13 ±0.05a

Neutrophil (109/l) 4.52 ±0.51a 5.54±0.45b

Serum Biochemistry

Total protein (g/l) 79.11 ± 2.42a 61.07 ± 1.59b

Albumin (g/l) 33.83 ± 1.23a 19.20 ± 0.95b

ALT(µKat/L) 0.283 ± 0.09a 0.44 ± 0.14a

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AST(µKat/L) 0.69 ± 0.18a 0.51 ± 0.12a

ALP(µKat/L) 3.29 ± 0.23a 1.69 ± 0.31b

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Calcium (mmol/l) 2.28 ± 0.15a 2.02 ± 0.22a

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Magnesium
0.723 ± 0.09a 0.594 ± 0.14a
(mmol/L)
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Sodium(mmol/L) 135.35 ± 0.83a 107.12 ± 1.19b

3.41 ± 0.17a 4.61 ± 0.11b

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Potassium(mmol/l)

Zinc (µmol/l) 13.76 ± 0.67a 8.52 ± 0.42b

Copper (µmol/l) 16.52 ± 0.67a 23.76 ± 0.81b

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Urea (mmol/l) 8.39 ± 0.77a 12.46 ± 0.56b

52.9 ± 2.45a 137.3 ± 3.64b

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Creatinine (mmol/l)

Values (Mean ± Standard error mean) having different superscripts (a, b) in rows are
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significantly different in each parameter (P<0.05).

TLC= Total Leukocyte Count, Hb= Haemoglobin, PCV= Packed Cell Volume, TEC= Total
Erythrocyte Count, ALT= Alanine Aminotransferase, AST= Aspartate Aminotransferase, ALP=
Alkaline Phophatase

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