- Email: [email protected]
First report of Ehrlichia infection in goats, China
Accepted Manuscript First report of Ehrlichia infection in goats, China Hui Zhang, Zhenyu Chang, Khalid Mehmood, Yajing Wang, Mujeeb Ur Rehman, Fazul Nabi, Ahamad Jawad Sabir, Xueting Liu, Xiaoxing Wu, Xinxin Tian, Donghai Zhou PII:
S0882-4010(17)30601-0
DOI:
10.1016/j.micpath.2017.07.012
Reference:
YMPAT 2350
To appear in:
Microbial Pathogenesis
Received Date: 24 May 2017 Revised Date:
7 July 2017
Accepted Date: 10 July 2017
Please cite this article as: Zhang H, Chang Z, Mehmood K, Wang Y, Rehman MU, Nabi F, Sabir AJ, Liu X, Wu X, Tian X, Zhou D, First report of Ehrlichia infection in goats, China, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.07.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT First report of Ehrlichia infection in goats, China
2
Hui Zhang a , §, Zhenyu Chang a , §, Khalid Mehmood a, b, Yajing Wang a, Mujeeb Ur
3
Rehman a, Fazul Nabi a, Ahamad Jawad Sabir c, Xueting Liu a, Xiaoxing Wu a, Xinxin
4
Tian a, Donghai Zhou a, *
5
a
6
People's Republic of China.
7
b
8
Bahawalpur, 63100, Pakistan
9
c
SC
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070,
M AN U
University College of Veterinary &Animal Sciences, Islamia University of
ICE&E, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan
* Corresponding author: [email protected]
11
§
14
15
16
17
18
EP
These authors made equal contributions to this article.
AC C
13
TE D
10
12
RI PT
1
ACCEPTED MANUSCRIPT ABSTRACT
20
Ehrlichiosis is an emerging infectious disease of domestic animals which is
21
transmitted by ticks. This disease has been reported earlier in most parts of China in
22
dogs, cattle and humans, but there is no published data regarding this disease in goats.
23
The present study provided the evidence of Ehrlichia infection in goats in Wuhan,
24
China on the basis of clinical signs, gross lesions, serum-biochemical,
25
histopathological and PCR. Twenty four goats were presented to the veterinary
26
hospital of Huazhong Agricultural University during July, 2016. The goats were
27
diagnosed for Ehrlichia in monocytic and granulocytic forms by blood smear
28
examination. Further confirmation was done by PCR examination, while
29
histopathological examination revealed degeneration and inflammation in different
30
tissues. The biochemical criterion and blood samples analysis showed significant
31
(P<0.05) changes. The present study reported that goats are naturally exposed to
32
Ehrlichia infection. To the best of our knowledge, this is the first clinical report of
33
Ehrlichia infection in goats infested with infected ticks.
34
Key words: Ehrlichiosis; infection; goats; China.
35
1. Introduction
36
Ehrlichiosis is recognized as rickettsial disease that affects various mammals
37
including cattle, sheep, goats, dogs and humans worldwide. Ehrlichiosis is one of the
38
most important public health issues across the world, especially in tropical and
39
subtropical regions. After infecting the host, Ehrlichia multiply in monocytes and
AC C
EP
TE D
M AN U
SC
RI PT
19
ACCEPTED MANUSCRIPT macrophages or in peripheral blood neutrophils, and spread to phagocyte cells of
41
different organs such as liver, spleen, lungs and lymph nodes [1]. Several Ehrlichiosis
42
forms have been identified however the disease is usually found in monocytic and
43
granulocytic forms [2,3]. Ehrlichiosis is maintained in an enzootic cycle between ticks
44
and vertebrate hosts through the bite of infected vectors [4]. Ehrlichiosis was first
45
reported in Algeria in 1935 [5]. In United States, it is recognized as pathogen of dogs
46
and human’s disease additionally causes serious illness in ruminants in Europe [6,7].
47
In China, the distribution of Ehrlichia bacteria is not clear across a massive land
48
having different climates and not currently recognize as a cause of death in goats in
49
the China. However, sporadic cases of Ehrlichiosis have been reported in dogs, mouse,
50
humans and ticks in China [8-10].
M AN U
SC
RI PT
40
Ehrlichia bacteria causes disease in ruminants from mild illness to fetal disease;
52
infected goats exhibited mild pyrexia, lethargy, inappetance, serous or mild nasal
53
discharge, elevated rectal temperature and decreased ALP activity [11]. The canine
54
Ehrlichiosisis characterized by a high fever, lymphadenomegaly, hemorrhagic
55
tendencies, depression, lethargy, anorexia and splenomegaly [12]. Generally, a
56
various lab tests, serological assay, clinical signs, history, and the detection of DNA
57
by PCR assay are primary research and clinical tools to achieve diagnosis of
58
Ehrlichiosis. However, diagnosis is still challenging due to its multiple phases and
59
various clinical manifestations of the disease. We evaluated the natural incidence of
60
Ehrlichiosis infection in goats with Ehrlichia organisms from Wuhan, China for the
AC C
EP
TE D
51
ACCEPTED MANUSCRIPT first time using clinical signs, serum-biochemical, histopathological and PCR
62
diagnosis.
63
2. Materials and methods
64
In our study, a total of 24 goats with same clinical symptoms form 5 flock were
65
presented to the veterinary hospital of Huazhong Agricultural University (Wuhan,
66
China) during July 2016. Initially, the diseased animals were diagnosed by clinical
67
examination and case history. The serological testing, histopathological and PCR
68
diagnosis were performed for further confirmation of Ehrlichiosis in goats.
SC
M AN U
69
RI PT
61
The concentration of GLU, BUN, CREA, P and Ca in the serum samples was measured
via
semi-automatic
biochemical
machine
(COULTER®LH
750,
71
Guangdong). The blood samples were tested through veterinary automatic auto blood
72
cell counter hematology analyzer for animal (Shenzhen Procan Electronics Inc.
73
China). The gross examinations were performed on internal organs to find out
74
appropriate lesions on different organs. Tissue samples from liver, lung, kidney and
75
spleen were fixed in 10% formalin for further analysis and subsequent use. After 72 h,
76
the samples were dehydrated, embedded in paraffin and cut into 5-µm thickness and
77
strained with hematoxylin & eosin (H&E). For molecular confirmation, the blood
78
samples were used to extract Ehrlichia organism using the TIANamp Virus
79
DNA/RNA Kit (TianGen, China) and specific primers for 23S gene as described
80
previously [5]. All the experiments were conducted after the approval of institutional
AC C
EP
TE D
70
ACCEPTED MANUSCRIPT Animal Welfare and Research, Ethics Committee guideline of Huazhong Agricultural
82
University, Wuhan, China.
83
3. Results
84
Briefly, the physical examination revealed a poor body condition with weakness,
85
emaciation, depression, odd colored yellow urine, and high rectal temperature in
86
infected goats. The clinical signs depicted dry skin with ticks bite symptom and
87
visible pale mucus membrane with yellow coating on the tongue. (Figure 1a-c). Blood
88
smear examination confirmed the presence of monocytic and granulocytic forms of
89
Ehrlichia bacteria in the blood (Figure 1d). Histopathological analysis of liver, spleen,
90
lung and kidney tissues revealed marked degeneration and presence of inflammation
91
in tissues (Figure 2). The biochemical criterion and blood samples analysis showed a
92
significant (P<0.05) elevated Retica, BUN, CREA and WBC count, while a
93
significantly decreased (P<0.05) values of PLT and RBC in infected goats as
94
compared with normal ones (Table 1). Molecular detection by PCR was performed
95
using specific primers which indicated that samples were positive for Ehrlichia
96
bacteria (Figure 3).
97
4. Discussion
98
Infectious diseases have been serious threat for animal health and productivity in
99
developing countries [13-15]. Ehrlichia organism is an obligatory and economically
100
important pathogens in ruminants, canine and humans. Phylogenetic and molecular
101
studies have concluded that Ehrlichiosis is becoming emerging and life-threatening
AC C
EP
TE D
M AN U
SC
RI PT
81
ACCEPTED MANUSCRIPT tick borne diseases in humans and animals, which is caused by genus Ehrlichia and
103
transmitted by ticks. Previous epidemiological and molecular detection demonstrated
104
that the goats and dogs were infected with tick-transmitted Ehrlichia organism in
105
United States and Malaysia respectively [11,16]. Domestic goats are parasitized by
106
the lone star tick, therefore, goats are naturally exposed to infection with Ehrlichia
107
species [17,18]. The presence of Ehrlichiosis in ticks, animals, and humans has been
108
earlier reported from China [2,8-10]. In recent years, it has been found that this
109
infection is present in humans and dogs [19-21]. However, scare information and
110
research is available regarding the detection of Ehrlichia in goats and sheep in China.
M AN U
SC
RI PT
102
Wuhan has hot summer weather around the year and climate is humid with
112
abundant rainfall, which could potentially increase the spread and growth of ticks.
113
Acute Ehrlichiosis should occur during peak season for the tick growth [22]. In our
114
study, the gross pathological examination, biochemical criterion, blood routine
115
analysis, histopathology and PCR analysis confirmed the Ehrlichia infection in goats
116
in Wuhan, China. Emerging infectious disease has a high economic burden in public
117
health and veterinary concerns. Pathogens usually cause mild to moderate infections
118
and complications resulted in severe outbreak of serious illness in livestock. Evidence
119
of caprine Ehrlichiosis is still limited in Wuhan China but it can be speculated from
120
diverse risk factors associated with free range and semi-intensive farming,
121
environment and dietary habitat of animals. The high levels of Ehrlichiosis emerging
122
may be due to possible wild animal contamination and free ranging farming sources.
123
Since, no study was conducted to evaluate the infection of Ehrlichiosis in domestic
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ACCEPTED MANUSCRIPT and wild animals in this region. Current study provides specific data on Ehrlichiosis in
125
goats in order to assure the safety of domestic animals through controlled farming,
126
effective surveillance systems and technical assistant. These outcomes could be useful
127
protecting food animals from infection and reduce spreading tick-born hazards in
128
Wuhan China.
129
Acknowledgments
SC
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124
This study was supported by Hubei Provincial Natural Science Foundation of
130
China (Grant No: 2014CFB244), supported by the Fundamental Research Funds for
132
the Central Universities (Program No: 2011PYO78).
133
Conflict of interest
None of the authors have any conflict of interest.
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M AN U
131
Author's contribution
DZ and HZ conceive and designed the experiments; ZC, MUR, YW, HZ, FN, XL,
EP
136
XW and XT performed the experiments and analyzed data; HZ, KM and AJS wrote
138
the manuscript and handled the revision.
139
References
140 141
AC C
137
[1]
J.S. Dumler, Anaplasma and ehrlichia infection, Annals of the New York Academy of Sciences. 1063 (2005) 361-373.
ACCEPTED MANUSCRIPT 142
[2]
Y. Ge, H. Yin, Y. Rikihisa, W. Pan, H. Yin, Molecular detection of tick-borne
143
Rickettsiales in goats and sheep from Southeastern China, Vector Borne
144
Zoonot. 16 (2016) 309. [3]
Ehrlichia infection in Italy, Emerg. Infect. Dis. 4 (1998), 663-665.
146 147
M. Nuti, D.A. Serafini, D. Bassetti, A. Ghionni, F. Russino, P. Rombolà, et al.,
RI PT
145
[4]
K. Mdladla, E.F. Dzomba, F.C. Muchadeyi, Seroprevalence of Ehrlichia ruminantium antibodies and its associated risk factors in indigenous goats of
149
South Africa, Prev. Vet. Med. 125 (2016) 99-105. [5]
M AN U
150
SC
148
M. Dahmani, A. Loudahi, O. Mediannikov, F. Fenollar, D. Raoult, B. Davoust,
151
Molecular detection of Anaplasma platys, and Ehrlichia Canis, in dogs from
152
Kabylie, Algeria, Ticks Tick-Borne Dis. 6 (2015) 198-203. [6]
R.S. Buller, M. Arens, S.P. Hmiel, C.D. Paddock, J.W. Sumner, Y. Rikihisa,
TE D
153
A. Unver, M. Gaudreault-Keener, F.A. Manian, A.M. Liddell, N.
155
Schmulewitz, G.A. Storch, Ehrlichia ewingii, a newly recognized agent of
156
human ehrlichiosis, N. Engl. J. Med. 341 (1999) 148–155.
158 159 160
[7]
H.I. Gokce, Z. Woldehiwet, The effects of Ehrlichia (Cytoecetes)
AC C
157
EP
154
phagocytophila on the clinical chemistry of sheep and goats. Zbl. Vet. Reihe B 46 (1999) 93–103.
[8]
T. Dong, Z.Y. Qu, L.J. Zhang, Detection of A. phagocytophilum and E.
161
chaffeensis in patient and mouse blood and ticks by a duplex real-time PCR
162
assay, PLoS One. 8 (2013) e74796.
ACCEPTED MANUSCRIPT 163
[9]
H. Pan, Y.H. Ma, S.D. Tong, Y. Sun, et al., Canine Ehrlichiosis caused
164
simultaneously by Ehrlichia canis and Ehrlichia platys, Microbiol. Immunol.
165
44 (2000) 737-739. [10]
D. Xu, J. Zhang, Z. Shi, C. Song, X. Zheng, Y. Zhang, et al., Molecular
RI PT
166 167
detection of vector-borne agents in dogs from ten provinces of China, Parasite
168
Vector. 8 (2015) 1-7.
illness in goats. Vet. Microbiol. 130 (2008) 398-402.
170 171
[12]
S. Harrus, T. Waner, Diagnosis of canine monocytotropic ehrlichiosis (ehrlichia canis): an overview. Vet. J. 187 (2011) 292-296.
172 173
A.D. Loftis, M.L. Levin, J.P. Spurlock, Two USA Ehrlichia spp. cause febrile
SC
[11]
M AN U
169
[13]
M.M. Elhaig, A. Selim, M.M. Mahmoud, E.K. El-Gayar, Molecular confirmation of Trypanosoma evansi and Babesia bigeminain cattle from
175
Lower Egypt, Pak. Vet. J. 36 (2016) 409-414.
176
[14]
TE D
174
X.B. Wen, H.T. Jiang, Y.L. Zhang, X.Y. Lang, J. Liu, H.B. Ni, Rapid and sensitive diagnosis of cattle anaplasmosis by loop-mediated isothermal
178
amplification (LAMP), Pak. Vet. J. 36 (2016) 174-178.
180 181 182
AC C
179
EP
177
[15]
R. Yilmaz, I.T. Cangul, K. Onat, A. Akkoc, M.O. Ozyigit, E. Akdesir, Histopathological, immunohistochemical and bacteriological characterization of Mycoplasma bovis pneumonia in cattle, Pak. Vet. J. 36 (2016) 316-321.
[16]
M. Nazari, S.Y. Lim, M. Watanabe, R.S.K. Sharma, N.A.B.Y. Cheng, M.
183
Watanabe, Molecular Detection of Ehrlichia canis in Dogs in Malaysia, PLoS
184
Neglected Trop. Dis. 7(2013) e1982.
ACCEPTED MANUSCRIPT 185
[17]
J.M. Lockhart, W.R. Davidson, D.E. Stallknecht, J.E. Dawson, E.W. Howerth, Isolation of Ehrlichia chaffeensis from white-tailed deer (Odocoileus
187
virginianus) confirms their role as natural reservoir hosts, J. Clin. Microbiol. 35
188
(1997) 1-5.
189
[18]
RI PT
186
V.G. Dugan, S.E. Little, D.E. Stallknecht, A.D. Beall, Natural infection of domestic goats with Ehrlichia chaffeensis, J. Clin. Microbiol. 38 (2000)
191
448-449. [19]
W. Li, L. Liu, X. Jiang, X. Guo, et al., Molecular identification of spotted
M AN U
192
SC
190
193
fever group Rickettsiae in ticks collected in central China, Clin. Microbiol,
194
Infect. 152 (2009) 279-280.
195
[20]
Y. Li, Z. Chen, Z. Liu, J. Liu, J. Yang, Q. Li, et al., Molecular survey of Anaplasma and Ehrlichia of red Deer and Sika deer in Gansu, China in
197
2013, Transbound. Emerg. Dis. 63 (2015) 228-236.
198
[21]
TE D
196
M. Lu, J.H. Tian, B. Yu, W.P. Guo, M.H. Li, C. Edward, et al., Extensive diversity of
200
Tick-borne Diseases. 8 (2017) 574-580.
202 203
204
205
AC C
201
rickettsiales bacteria in ticks from Wuhan, China. Ticks and
EP
199
[22]
T.M. Kollars Jr, J.H. Oliver Jr, L.A. Durden, P.G. Kollars, Host association and seasonal activity of Amblyomma americanum (Acari: Ixodidae) in Missouri, J. Parasitol. 86 (2000) 1156–1159.
M AN U
SC
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ACCEPTED MANUSCRIPT
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Figure 1. The physical examination of Ehrlichsiosis in goats. a. poor body condition
208
with weakness, emaciation, growth depression; b. ticks bite on the skin; c. pale oral
209
mucosa with yellow coating on the tongue; d. monocytic and granulocytic in blood.
212
213
214
215
216
EP
211
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ACCEPTED MANUSCRIPT
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Figure 2. The histopathological examination (H&E) of Ehrlichiosis in goats. a. liver;
219
b. spleen; c. lung; d. kidney
220
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221
Figure 3. Specific PCR amplification of 23S gene on 1.5% agarose gel. Marker: 5000
222
3000 2000 1000 750 500 250 100 bp DNA ladder
223
224
ACCEPTED MANUSCRIPT Table 1. Biochemical criterion and blood samples analysis in goats Items
Infected goats
1.21±0.36
1.44±0.53
BUN (mmol/L)
5.50±0.23
17.64±3.26*
CREA (mmol/L)
181.26±27.30
P (mmol/L)
2.05±0.05
Ca (mmol/L)
2.15±0.02
WBC (109/L)
13.39±1.60
RBC (1012/L)
2.49±0.18
1.76±0.57*
PLT (109/L)
259.23±37.91
108.42±26.73*
Retica (%)
0
0.23±0.02*
TE D
indicate significant difference (p<0.05)
EP
237.50±21.37* 2.13±0.12
M AN U
*
RI PT
GLU (mmol/L)
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226
Normal goats
2.19±0.15
SC
225
24.26±0.64*
ACCEPTED MANUSCRIPT Highlights 1. Ehrlichiosis is an emerging infectious disease in goats in China. 2. Our study find the evidence of Ehrlichiosis infection for the first time in goats, China.
AC C
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3. Ehrlichial organisms infections in goats through the bite of an infected ticks.
S0882-4010(17)30601-0
DOI:
10.1016/j.micpath.2017.07.012
Reference:
YMPAT 2350
To appear in:
Microbial Pathogenesis
Received Date: 24 May 2017 Revised Date:
7 July 2017
Accepted Date: 10 July 2017
Please cite this article as: Zhang H, Chang Z, Mehmood K, Wang Y, Rehman MU, Nabi F, Sabir AJ, Liu X, Wu X, Tian X, Zhou D, First report of Ehrlichia infection in goats, China, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.07.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT First report of Ehrlichia infection in goats, China
2
Hui Zhang a , §, Zhenyu Chang a , §, Khalid Mehmood a, b, Yajing Wang a, Mujeeb Ur
3
Rehman a, Fazul Nabi a, Ahamad Jawad Sabir c, Xueting Liu a, Xiaoxing Wu a, Xinxin
4
Tian a, Donghai Zhou a, *
5
a
6
People's Republic of China.
7
b
8
Bahawalpur, 63100, Pakistan
9
c
SC
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070,
M AN U
University College of Veterinary &Animal Sciences, Islamia University of
ICE&E, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan
* Corresponding author: [email protected]
11
§
14
15
16
17
18
EP
These authors made equal contributions to this article.
AC C
13
TE D
10
12
RI PT
1
ACCEPTED MANUSCRIPT ABSTRACT
20
Ehrlichiosis is an emerging infectious disease of domestic animals which is
21
transmitted by ticks. This disease has been reported earlier in most parts of China in
22
dogs, cattle and humans, but there is no published data regarding this disease in goats.
23
The present study provided the evidence of Ehrlichia infection in goats in Wuhan,
24
China on the basis of clinical signs, gross lesions, serum-biochemical,
25
histopathological and PCR. Twenty four goats were presented to the veterinary
26
hospital of Huazhong Agricultural University during July, 2016. The goats were
27
diagnosed for Ehrlichia in monocytic and granulocytic forms by blood smear
28
examination. Further confirmation was done by PCR examination, while
29
histopathological examination revealed degeneration and inflammation in different
30
tissues. The biochemical criterion and blood samples analysis showed significant
31
(P<0.05) changes. The present study reported that goats are naturally exposed to
32
Ehrlichia infection. To the best of our knowledge, this is the first clinical report of
33
Ehrlichia infection in goats infested with infected ticks.
34
Key words: Ehrlichiosis; infection; goats; China.
35
1. Introduction
36
Ehrlichiosis is recognized as rickettsial disease that affects various mammals
37
including cattle, sheep, goats, dogs and humans worldwide. Ehrlichiosis is one of the
38
most important public health issues across the world, especially in tropical and
39
subtropical regions. After infecting the host, Ehrlichia multiply in monocytes and
AC C
EP
TE D
M AN U
SC
RI PT
19
ACCEPTED MANUSCRIPT macrophages or in peripheral blood neutrophils, and spread to phagocyte cells of
41
different organs such as liver, spleen, lungs and lymph nodes [1]. Several Ehrlichiosis
42
forms have been identified however the disease is usually found in monocytic and
43
granulocytic forms [2,3]. Ehrlichiosis is maintained in an enzootic cycle between ticks
44
and vertebrate hosts through the bite of infected vectors [4]. Ehrlichiosis was first
45
reported in Algeria in 1935 [5]. In United States, it is recognized as pathogen of dogs
46
and human’s disease additionally causes serious illness in ruminants in Europe [6,7].
47
In China, the distribution of Ehrlichia bacteria is not clear across a massive land
48
having different climates and not currently recognize as a cause of death in goats in
49
the China. However, sporadic cases of Ehrlichiosis have been reported in dogs, mouse,
50
humans and ticks in China [8-10].
M AN U
SC
RI PT
40
Ehrlichia bacteria causes disease in ruminants from mild illness to fetal disease;
52
infected goats exhibited mild pyrexia, lethargy, inappetance, serous or mild nasal
53
discharge, elevated rectal temperature and decreased ALP activity [11]. The canine
54
Ehrlichiosisis characterized by a high fever, lymphadenomegaly, hemorrhagic
55
tendencies, depression, lethargy, anorexia and splenomegaly [12]. Generally, a
56
various lab tests, serological assay, clinical signs, history, and the detection of DNA
57
by PCR assay are primary research and clinical tools to achieve diagnosis of
58
Ehrlichiosis. However, diagnosis is still challenging due to its multiple phases and
59
various clinical manifestations of the disease. We evaluated the natural incidence of
60
Ehrlichiosis infection in goats with Ehrlichia organisms from Wuhan, China for the
AC C
EP
TE D
51
ACCEPTED MANUSCRIPT first time using clinical signs, serum-biochemical, histopathological and PCR
62
diagnosis.
63
2. Materials and methods
64
In our study, a total of 24 goats with same clinical symptoms form 5 flock were
65
presented to the veterinary hospital of Huazhong Agricultural University (Wuhan,
66
China) during July 2016. Initially, the diseased animals were diagnosed by clinical
67
examination and case history. The serological testing, histopathological and PCR
68
diagnosis were performed for further confirmation of Ehrlichiosis in goats.
SC
M AN U
69
RI PT
61
The concentration of GLU, BUN, CREA, P and Ca in the serum samples was measured
via
semi-automatic
biochemical
machine
(COULTER®LH
750,
71
Guangdong). The blood samples were tested through veterinary automatic auto blood
72
cell counter hematology analyzer for animal (Shenzhen Procan Electronics Inc.
73
China). The gross examinations were performed on internal organs to find out
74
appropriate lesions on different organs. Tissue samples from liver, lung, kidney and
75
spleen were fixed in 10% formalin for further analysis and subsequent use. After 72 h,
76
the samples were dehydrated, embedded in paraffin and cut into 5-µm thickness and
77
strained with hematoxylin & eosin (H&E). For molecular confirmation, the blood
78
samples were used to extract Ehrlichia organism using the TIANamp Virus
79
DNA/RNA Kit (TianGen, China) and specific primers for 23S gene as described
80
previously [5]. All the experiments were conducted after the approval of institutional
AC C
EP
TE D
70
ACCEPTED MANUSCRIPT Animal Welfare and Research, Ethics Committee guideline of Huazhong Agricultural
82
University, Wuhan, China.
83
3. Results
84
Briefly, the physical examination revealed a poor body condition with weakness,
85
emaciation, depression, odd colored yellow urine, and high rectal temperature in
86
infected goats. The clinical signs depicted dry skin with ticks bite symptom and
87
visible pale mucus membrane with yellow coating on the tongue. (Figure 1a-c). Blood
88
smear examination confirmed the presence of monocytic and granulocytic forms of
89
Ehrlichia bacteria in the blood (Figure 1d). Histopathological analysis of liver, spleen,
90
lung and kidney tissues revealed marked degeneration and presence of inflammation
91
in tissues (Figure 2). The biochemical criterion and blood samples analysis showed a
92
significant (P<0.05) elevated Retica, BUN, CREA and WBC count, while a
93
significantly decreased (P<0.05) values of PLT and RBC in infected goats as
94
compared with normal ones (Table 1). Molecular detection by PCR was performed
95
using specific primers which indicated that samples were positive for Ehrlichia
96
bacteria (Figure 3).
97
4. Discussion
98
Infectious diseases have been serious threat for animal health and productivity in
99
developing countries [13-15]. Ehrlichia organism is an obligatory and economically
100
important pathogens in ruminants, canine and humans. Phylogenetic and molecular
101
studies have concluded that Ehrlichiosis is becoming emerging and life-threatening
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ACCEPTED MANUSCRIPT tick borne diseases in humans and animals, which is caused by genus Ehrlichia and
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transmitted by ticks. Previous epidemiological and molecular detection demonstrated
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that the goats and dogs were infected with tick-transmitted Ehrlichia organism in
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United States and Malaysia respectively [11,16]. Domestic goats are parasitized by
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the lone star tick, therefore, goats are naturally exposed to infection with Ehrlichia
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species [17,18]. The presence of Ehrlichiosis in ticks, animals, and humans has been
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earlier reported from China [2,8-10]. In recent years, it has been found that this
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infection is present in humans and dogs [19-21]. However, scare information and
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research is available regarding the detection of Ehrlichia in goats and sheep in China.
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Wuhan has hot summer weather around the year and climate is humid with
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abundant rainfall, which could potentially increase the spread and growth of ticks.
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Acute Ehrlichiosis should occur during peak season for the tick growth [22]. In our
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study, the gross pathological examination, biochemical criterion, blood routine
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analysis, histopathology and PCR analysis confirmed the Ehrlichia infection in goats
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in Wuhan, China. Emerging infectious disease has a high economic burden in public
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health and veterinary concerns. Pathogens usually cause mild to moderate infections
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and complications resulted in severe outbreak of serious illness in livestock. Evidence
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of caprine Ehrlichiosis is still limited in Wuhan China but it can be speculated from
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diverse risk factors associated with free range and semi-intensive farming,
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environment and dietary habitat of animals. The high levels of Ehrlichiosis emerging
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may be due to possible wild animal contamination and free ranging farming sources.
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Since, no study was conducted to evaluate the infection of Ehrlichiosis in domestic
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ACCEPTED MANUSCRIPT and wild animals in this region. Current study provides specific data on Ehrlichiosis in
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goats in order to assure the safety of domestic animals through controlled farming,
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effective surveillance systems and technical assistant. These outcomes could be useful
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protecting food animals from infection and reduce spreading tick-born hazards in
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Wuhan China.
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Acknowledgments
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This study was supported by Hubei Provincial Natural Science Foundation of
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China (Grant No: 2014CFB244), supported by the Fundamental Research Funds for
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the Central Universities (Program No: 2011PYO78).
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Conflict of interest
None of the authors have any conflict of interest.
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Author's contribution
DZ and HZ conceive and designed the experiments; ZC, MUR, YW, HZ, FN, XL,
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XW and XT performed the experiments and analyzed data; HZ, KM and AJS wrote
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the manuscript and handled the revision.
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References
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[1]
J.S. Dumler, Anaplasma and ehrlichia infection, Annals of the New York Academy of Sciences. 1063 (2005) 361-373.
ACCEPTED MANUSCRIPT 142
[2]
Y. Ge, H. Yin, Y. Rikihisa, W. Pan, H. Yin, Molecular detection of tick-borne
143
Rickettsiales in goats and sheep from Southeastern China, Vector Borne
144
Zoonot. 16 (2016) 309. [3]
Ehrlichia infection in Italy, Emerg. Infect. Dis. 4 (1998), 663-665.
146 147
M. Nuti, D.A. Serafini, D. Bassetti, A. Ghionni, F. Russino, P. Rombolà, et al.,
RI PT
145
[4]
K. Mdladla, E.F. Dzomba, F.C. Muchadeyi, Seroprevalence of Ehrlichia ruminantium antibodies and its associated risk factors in indigenous goats of
149
South Africa, Prev. Vet. Med. 125 (2016) 99-105. [5]
M AN U
150
SC
148
M. Dahmani, A. Loudahi, O. Mediannikov, F. Fenollar, D. Raoult, B. Davoust,
151
Molecular detection of Anaplasma platys, and Ehrlichia Canis, in dogs from
152
Kabylie, Algeria, Ticks Tick-Borne Dis. 6 (2015) 198-203. [6]
R.S. Buller, M. Arens, S.P. Hmiel, C.D. Paddock, J.W. Sumner, Y. Rikihisa,
TE D
153
A. Unver, M. Gaudreault-Keener, F.A. Manian, A.M. Liddell, N.
155
Schmulewitz, G.A. Storch, Ehrlichia ewingii, a newly recognized agent of
156
human ehrlichiosis, N. Engl. J. Med. 341 (1999) 148–155.
158 159 160
[7]
H.I. Gokce, Z. Woldehiwet, The effects of Ehrlichia (Cytoecetes)
AC C
157
EP
154
phagocytophila on the clinical chemistry of sheep and goats. Zbl. Vet. Reihe B 46 (1999) 93–103.
[8]
T. Dong, Z.Y. Qu, L.J. Zhang, Detection of A. phagocytophilum and E.
161
chaffeensis in patient and mouse blood and ticks by a duplex real-time PCR
162
assay, PLoS One. 8 (2013) e74796.
ACCEPTED MANUSCRIPT 163
[9]
H. Pan, Y.H. Ma, S.D. Tong, Y. Sun, et al., Canine Ehrlichiosis caused
164
simultaneously by Ehrlichia canis and Ehrlichia platys, Microbiol. Immunol.
165
44 (2000) 737-739. [10]
D. Xu, J. Zhang, Z. Shi, C. Song, X. Zheng, Y. Zhang, et al., Molecular
RI PT
166 167
detection of vector-borne agents in dogs from ten provinces of China, Parasite
168
Vector. 8 (2015) 1-7.
illness in goats. Vet. Microbiol. 130 (2008) 398-402.
170 171
[12]
S. Harrus, T. Waner, Diagnosis of canine monocytotropic ehrlichiosis (ehrlichia canis): an overview. Vet. J. 187 (2011) 292-296.
172 173
A.D. Loftis, M.L. Levin, J.P. Spurlock, Two USA Ehrlichia spp. cause febrile
SC
[11]
M AN U
169
[13]
M.M. Elhaig, A. Selim, M.M. Mahmoud, E.K. El-Gayar, Molecular confirmation of Trypanosoma evansi and Babesia bigeminain cattle from
175
Lower Egypt, Pak. Vet. J. 36 (2016) 409-414.
176
[14]
TE D
174
X.B. Wen, H.T. Jiang, Y.L. Zhang, X.Y. Lang, J. Liu, H.B. Ni, Rapid and sensitive diagnosis of cattle anaplasmosis by loop-mediated isothermal
178
amplification (LAMP), Pak. Vet. J. 36 (2016) 174-178.
180 181 182
AC C
179
EP
177
[15]
R. Yilmaz, I.T. Cangul, K. Onat, A. Akkoc, M.O. Ozyigit, E. Akdesir, Histopathological, immunohistochemical and bacteriological characterization of Mycoplasma bovis pneumonia in cattle, Pak. Vet. J. 36 (2016) 316-321.
[16]
M. Nazari, S.Y. Lim, M. Watanabe, R.S.K. Sharma, N.A.B.Y. Cheng, M.
183
Watanabe, Molecular Detection of Ehrlichia canis in Dogs in Malaysia, PLoS
184
Neglected Trop. Dis. 7(2013) e1982.
ACCEPTED MANUSCRIPT 185
[17]
J.M. Lockhart, W.R. Davidson, D.E. Stallknecht, J.E. Dawson, E.W. Howerth, Isolation of Ehrlichia chaffeensis from white-tailed deer (Odocoileus
187
virginianus) confirms their role as natural reservoir hosts, J. Clin. Microbiol. 35
188
(1997) 1-5.
189
[18]
RI PT
186
V.G. Dugan, S.E. Little, D.E. Stallknecht, A.D. Beall, Natural infection of domestic goats with Ehrlichia chaffeensis, J. Clin. Microbiol. 38 (2000)
191
448-449. [19]
W. Li, L. Liu, X. Jiang, X. Guo, et al., Molecular identification of spotted
M AN U
192
SC
190
193
fever group Rickettsiae in ticks collected in central China, Clin. Microbiol,
194
Infect. 152 (2009) 279-280.
195
[20]
Y. Li, Z. Chen, Z. Liu, J. Liu, J. Yang, Q. Li, et al., Molecular survey of Anaplasma and Ehrlichia of red Deer and Sika deer in Gansu, China in
197
2013, Transbound. Emerg. Dis. 63 (2015) 228-236.
198
[21]
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196
M. Lu, J.H. Tian, B. Yu, W.P. Guo, M.H. Li, C. Edward, et al., Extensive diversity of
200
Tick-borne Diseases. 8 (2017) 574-580.
202 203
204
205
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rickettsiales bacteria in ticks from Wuhan, China. Ticks and
EP
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[22]
T.M. Kollars Jr, J.H. Oliver Jr, L.A. Durden, P.G. Kollars, Host association and seasonal activity of Amblyomma americanum (Acari: Ixodidae) in Missouri, J. Parasitol. 86 (2000) 1156–1159.
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Figure 1. The physical examination of Ehrlichsiosis in goats. a. poor body condition
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with weakness, emaciation, growth depression; b. ticks bite on the skin; c. pale oral
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mucosa with yellow coating on the tongue; d. monocytic and granulocytic in blood.
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Figure 2. The histopathological examination (H&E) of Ehrlichiosis in goats. a. liver;
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b. spleen; c. lung; d. kidney
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Figure 3. Specific PCR amplification of 23S gene on 1.5% agarose gel. Marker: 5000
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3000 2000 1000 750 500 250 100 bp DNA ladder
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ACCEPTED MANUSCRIPT Table 1. Biochemical criterion and blood samples analysis in goats Items
Infected goats
1.21±0.36
1.44±0.53
BUN (mmol/L)
5.50±0.23
17.64±3.26*
CREA (mmol/L)
181.26±27.30
P (mmol/L)
2.05±0.05
Ca (mmol/L)
2.15±0.02
WBC (109/L)
13.39±1.60
RBC (1012/L)
2.49±0.18
1.76±0.57*
PLT (109/L)
259.23±37.91
108.42±26.73*
Retica (%)
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0.23±0.02*
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indicate significant difference (p<0.05)
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237.50±21.37* 2.13±0.12
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GLU (mmol/L)
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Normal goats
2.19±0.15
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24.26±0.64*
ACCEPTED MANUSCRIPT Highlights 1. Ehrlichiosis is an emerging infectious disease in goats in China. 2. Our study find the evidence of Ehrlichiosis infection for the first time in goats, China.
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3. Ehrlichial organisms infections in goats through the bite of an infected ticks.