# Molecular Detection ofAnaplasma spp. andEhrlichia spp. in Ruminants from Twelve Provinces of China

Authors: Haixiang Qiu; Patrick John Kelly; Jilei Zhang; Qinghua Luo; Yi Yang; Yongjiang Mao; Zhangping Yang; Jing Li; Hongzhuan Wu; Chengming Wang
Journal: Canadian Journal of Infectious Diseases and Medical Microbiology
 (2016)

---

## Abstract

Anaplasma spp. andEhrlichia spp. are tick-transmitted bacteria that are of significant economic importance as they can infect large and small ruminants and also people. There is little information on anaplasmosis and ehrlichiosis in ruminants in China. 16S rRNA FRET-qPCRs were used to screen convenience whole blood samples from 2,240 domestic ruminants in 12 provinces of China forAnaplasma spp. andEhrlichia spp. Positive samples were further analyzed with a standard PCR for thegltA.Anaplasma spp. DNA was detected in the sheep (11.7%; 13/111), goats (81.8%; 219/270), cattle (13.2%; 241/1,830), and water buffaloes (6.9%; 2/29).Ehrlichia spp. DNA was detected in sheep (1.8%; 2/111), goats (1.1%; 3/270), and cattle (3.6%; 65/1830) but not in water buffaloes (0/29). Sequencing ofgltA PCR products showed thatA. marginale,A. ovis,Ehrlichia canis, andEhrlichia sp. (JX629807) were present in ruminants from China, while the 16S rRNA FRET-qPCR sequence data indicated that there might also beA. platys,A. phagocytophilum,Anaplasma sp. BL126-13 (KJ410243), andAnaplasma sp. JC3-6 (KM227012). Our study shows that domestic ruminants from China are not uncommonly infected with a variety ofAnaplasma spp. andEhrlichia spp.


---

## Body

## 1. Background

Anaplasma spp. andEhrlichiaspp. are tick-transmitted, intracellular Gram-negative bacteria that are important animal and human pathogens. The majorAnaplasmaspecies that impact animal and human health areAnaplasma marginale,A. ovis,A. centrale,A. bovis,A. phagocytophilum, andA. platys[1]. The most important of these in ruminants isA. marginale which causes bovine anaplasmosis (formerly gall-sickness) which is associated with fever, anemia, icterus, and often death. The major pathogenicEhrlichia species areE. canis,E. chaffeensis,E. ewingii,E. muris, andE. ruminantium[2] with the latter causing heartwater in domestic ruminants. This disease is prevalent in Africa, where it causes high mortality (up to 90%) and extensive economic losses [3].In China, there is little information on theAnaplasmaandEhrlichiaspecies in domestic ruminants.Anaplasma bovis, A.marginale, andA. ovis have been described in various Provinces, including Xinjiang, Gansu, Henan, Yunnan, Hubei, Guizhou, and Zhejiang [1, 4–7]. In addition,A. phagocytophilumhas been reported in ruminants (sheep and cattle) in Henan and Xinjiang as well as in dogs and ticks [1, 6–14], andA. platys has been detected in red deer and sika deer from Gansu province [4]. In the case ofEhrlichiaspp. in ruminants in China, an unclassified species has been reported in cattle in Tibet [15] andE. canis, a species that infects dogs worldwide, has been reported in sika deer from Gansu [4].To provide further information onAnaplasma spp. andEhrlichia spp. infections in domestic ruminants of China, we carried out a PCR survey for the organisms in blood samples from ruminants in 12 provinces of China. Our findings are reported below.


## 2. Materials and Methods

### 2.1. Blood Samples

This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Between 2007 and 2013, convenience whole blood samples (around 6 mL) from cattle (n=1,830), water buffaloes (n=29), goats (n=270), and sheep (n=111) were collected in 12 provinces/municipalities of China as described before [16, 17] (Table 1). DNA was extracted from the whole blood samples using a standard phenol-chloroform method as described previously [16] and stored at −80°C until PCRs were performed.Table 1
Molecular detection ofAnaplasma spp. and Ehrlichia spp. in Chinese ruminants.
Animal species Subspecies/breed Province City Anaplasma positivity Ehrlichia positivity Positive/total, % Positive/total, % Cattle (n=1,830) Bos p. taurus Holsteins Anhui Bengbu 3/109, 2.8% 1/109, 0.9% Wannan Anhui Wuhu 17/17, 100.0% 14/17, 82.4% Holsteins Beijing Sanyuan 0/107, 0.0% 0/107, 0.0% Holsteins Jiangsu Yancheng 1/395, 0.3% 2/395, 0.5% Holsteins Jiangsu Yangzhou 0/269, 0.0% 1/269, 0.4% Holsteins Heilongjiang Qiqihar 1/111, 0.9% 0/111, 0.0% Simmentals Inner Mongolia Chifeng 0/132, 0.0% 0/132, 0.0% Luxi Shandong Jining 3/42, 7.1% 0/42, 0.0% Bohaiblack Shandong Binzhou 1/33, 3.0% 0/33, 0.0% Holsteins Shanghai Shanghai 0/255, 0.0% 0/255, 0.0% Holsteins Tianjin Tianjin 0/94, 0.0% 0/94, 0.0%  Cattle (n=1,830) Bos p. indicus Minnan Fujian Putian 17/24, 70.8% 2/24, 8.3% Leiqiong Hainan Haikou 74/74, 100.0% 15/74, 20.3% Yunling Yunnan Kunming 124/168, 73.8% 30/168, 17.9%  Water buffalo (n=29) Haizi Jiangsu Yancheng 2/29, 6.9% 0/29, 0.0%  Goats (n=270) Yangtze River Delta White Jiangsu Yangzhou 119/172, 69.2% 3/172, 1.7% Xinjiang Xinjiang Urumqi 10/98, 10.2% 0/98, 0.0%  Sheep (n=111) Sishui Fur Inner Mongolia Xilingol 0/72, 0.0% 1/72, 1.4% Wuranke Shandong Jining 13/39, 33.3% 1/39, 2.6%


### 2.2. FRET-qPCR

As described previously, FRET-qPCRs for the 16S rRNA ofAnaplasma spp. [18] andEhrlichia spp. [19], and the mammalian HMBS gene [20] as an endogenous internal control, were performed on a Roche Light-Cycler 480-II PCR Instrument. Positive PCR products were verified by gel electrophoresis and sequenced using forward and antisense primers (BGI, Shanghai, China). Negative controls consisting of sterile molecular grade water were used to detect cross-contamination during DNA extraction and PCR processing.


### 2.3. Standard PCR for the Citrate Synthase Gene (gltA)

To further characterize theAnaplasmaandEhrlichiaspecies detected above, we carried out standard PCRs for the citrate synthase gene (gltA) ofAnaplasma spp. as described previously [21] and forEhrlichia spp. with primers designed for the study (forward primer: GGTTTATGGTGCTTTTCCTAGTGTTGA; reverse primer: TTACAGATTTCTCAGGAGTATATGCCTCC). The PCR products we obtained were verified by gel electrophoresis and sequenced (BGI, Shanghai, China).


### 2.4. DNA Sequence Data Analysis

Compilation and assembly of the multiple sequences generated from each template were performed using the Vector NTI. Sequence alignment was performed with Align (Vector NTI).


## 2.1. Blood Samples

This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Between 2007 and 2013, convenience whole blood samples (around 6 mL) from cattle (n=1,830), water buffaloes (n=29), goats (n=270), and sheep (n=111) were collected in 12 provinces/municipalities of China as described before [16, 17] (Table 1). DNA was extracted from the whole blood samples using a standard phenol-chloroform method as described previously [16] and stored at −80°C until PCRs were performed.Table 1
Molecular detection ofAnaplasma spp. and Ehrlichia spp. in Chinese ruminants.
Animal species Subspecies/breed Province City Anaplasma positivity Ehrlichia positivity Positive/total, % Positive/total, % Cattle (n=1,830) Bos p. taurus Holsteins Anhui Bengbu 3/109, 2.8% 1/109, 0.9% Wannan Anhui Wuhu 17/17, 100.0% 14/17, 82.4% Holsteins Beijing Sanyuan 0/107, 0.0% 0/107, 0.0% Holsteins Jiangsu Yancheng 1/395, 0.3% 2/395, 0.5% Holsteins Jiangsu Yangzhou 0/269, 0.0% 1/269, 0.4% Holsteins Heilongjiang Qiqihar 1/111, 0.9% 0/111, 0.0% Simmentals Inner Mongolia Chifeng 0/132, 0.0% 0/132, 0.0% Luxi Shandong Jining 3/42, 7.1% 0/42, 0.0% Bohaiblack Shandong Binzhou 1/33, 3.0% 0/33, 0.0% Holsteins Shanghai Shanghai 0/255, 0.0% 0/255, 0.0% Holsteins Tianjin Tianjin 0/94, 0.0% 0/94, 0.0%  Cattle (n=1,830) Bos p. indicus Minnan Fujian Putian 17/24, 70.8% 2/24, 8.3% Leiqiong Hainan Haikou 74/74, 100.0% 15/74, 20.3% Yunling Yunnan Kunming 124/168, 73.8% 30/168, 17.9%  Water buffalo (n=29) Haizi Jiangsu Yancheng 2/29, 6.9% 0/29, 0.0%  Goats (n=270) Yangtze River Delta White Jiangsu Yangzhou 119/172, 69.2% 3/172, 1.7% Xinjiang Xinjiang Urumqi 10/98, 10.2% 0/98, 0.0%  Sheep (n=111) Sishui Fur Inner Mongolia Xilingol 0/72, 0.0% 1/72, 1.4% Wuranke Shandong Jining 13/39, 33.3% 1/39, 2.6%


## 2.2. FRET-qPCR

As described previously, FRET-qPCRs for the 16S rRNA ofAnaplasma spp. [18] andEhrlichia spp. [19], and the mammalian HMBS gene [20] as an endogenous internal control, were performed on a Roche Light-Cycler 480-II PCR Instrument. Positive PCR products were verified by gel electrophoresis and sequenced using forward and antisense primers (BGI, Shanghai, China). Negative controls consisting of sterile molecular grade water were used to detect cross-contamination during DNA extraction and PCR processing.


## 2.3. Standard PCR for the Citrate Synthase Gene (gltA)

To further characterize theAnaplasmaandEhrlichiaspecies detected above, we carried out standard PCRs for the citrate synthase gene (gltA) ofAnaplasma spp. as described previously [21] and forEhrlichia spp. with primers designed for the study (forward primer: GGTTTATGGTGCTTTTCCTAGTGTTGA; reverse primer: TTACAGATTTCTCAGGAGTATATGCCTCC). The PCR products we obtained were verified by gel electrophoresis and sequenced (BGI, Shanghai, China).


## 2.4. DNA Sequence Data Analysis

Compilation and assembly of the multiple sequences generated from each template were performed using the Vector NTI. Sequence alignment was performed with Align (Vector NTI).


## 3. Results

The mammalian HMBS gene endogenous internal control was positive for all samples, indicating that DNA extraction had been successful.


### 3.1.Anaplasma spp. DNA in Ruminants

Overall, 17.2% (385/2240) of the ruminants from 8 of the 12 provinces studied (Table1 and Figure 1) were positive for the 16S rRNA ofAnaplasma spp. with copy numbers ranging from 50 to 52,000/mL blood (median 1,720 copies/mL blood). Goats were most frequently positive (81.1%; 219/270), followed by cattle (13.2%; 241/1,830), sheep (11.7%; 13/111), and water buffaloes (6.9%; 2/29).Figure 1
Detection ofAnaplasma spp. andEhrlichia spp. in ruminants from 12 provinces of China. Blood samples of ruminants (2,240) were collected from twelve provinces (in bisque) of China. The prevalence is shown forAnaplasmaspp. (red) andEhrlichiaspp. (blue).When we sequenced the positive 16S rRNA FRET-qPCRs, we obtained clean sequencing data for 38 of the samples from cattle (23) and goats (15) (Table2). The sequences in the cattle were most commonly identical to those ofA. phagocytophilum(12/23; 52%) andA. marginale(10/23; 44%) with positive animals in 4 and 2 of the 9 provinces studied, respectively. Representative sequences were deposited in GenBank forA. phagocytophilum(KX279691) andA. marginale(KX279690). The one otherAnaplasmasp. found was in a Wannan cow from Anhui which had a 16S RNA sequence identical to that of a poorly characterizedAnaplasmasp. BL126-13 (KJ410243). We also found evidence for this organism in three goats from Jiangsu. OtherAnaplasmaspp. we detected in goats wereA. ovis (KX279688) in Xinjiang (2),A. platys(KX279689) in Jiangsu (2), and a poorly characterizedAnaplasmasp. (KM227012) in Jiangsu (5). The 16S rRNA sequences we deposited in GenBank (KX279683; KX279685) are identical to those ofAnaplasmasp. (KM227012) andAnaplasmaBL126-13 (KJ410243), respectively.Table 2
Source ofAnaplasma and Ehrlichiaspp. identified in this study based on 16S rRNA and gltA gene sequences.
Organism Species 16S RNA Species gltA GenBank # Numbers Animal City, province GenBank # Numbers Animal City, province Anaplasmaspp. A. marginale KX279690 10 Cattle Binzhou, Shandong (1) A. marginale KX506005 1 Cattle Kunming, Yunnan Kunming, Yunnan (9) A. platys KX279689 2 Goat Yangzhou, Jiangsu A. phagocytophilum KX279691 15 Cattle Bengbu, Anhui (2) Haikou, Hainan (5) Kunming, Yunnan (4) Wuhu, Anhui (1) Goat Yangzhou, Jiangsu (3) A. ovis KX279688 2 Goat Urumqi, Xinjiang (2) A. ovis KX506006 1 Goat Urumqi, Xinjiang Anaplasmasp. KX279685 4 Cattle Wuhu, Anhui (1) Goat Yangzhou, Jiangsu (3) Anaplasmasp. KX279683 5 Goat Yangzhou, Jiangsu (5)  Ehrlichiaspp. Ehrlichiasp. KX279682 14 Sheep Inner Mongolia (1) Cattle Yunnan, Kunming (10) Ehrlichiasp. KX506007 5 Cattle Yunnan, Kunming Goat Yangzhou, Jiangsu (3) E. canis KX506008 1 Goat Yangzhou, JiangsuSince there is limited polymorphism in the 16S rRNA FRET-qPCR sequences of different ruminantAnaplasmaspp. (Figure 2), to enable more definitive species differentiation we carried out a PCR and sequencing of the more polymorphicgltA on 20% of the 16S rRNA positive samples with the highest copy numbers (range of 610 to 52,000/mL blood; median 2,300). Only two samples provided clean sequencing data with one (16S rRNA FRET-qPCR copy number 52,000/mL blood) from a bovine in Yunnan having 100% identity withA. marginale (0/620 mismatches with CP006847.1) and the other (16S rRNA FRET-qPCR copy number 47,700 copies/mL blood) from a goat in Xinjiang having 99.7% identity withA. ovis (1/438 mismatches with KJ410284.1). ThegltA sequence ofA. marginale we identified was deposited in GenBank under the accession number KX506005 and that ofA. ovis as KX506006. The 16S rRNA sequences forAnaplasmaspp. from ruminants in this study are compared with those of other representingAnaplasma spp. (Figure 3).Figure 2
Alignment of the sequences obtained with the 16S rRNA FRET-qPCR we used in our study and those ofAnaplasma spp. in GenBank. “.” denotes the identical nucleotide sequence to that ofA. bovis. Organisms with GenBank accession numbers identified in the study are in red.Figure 3
Phylogenetic comparison ofAnaplasmaspp. from ruminants in this study. The 16S rRNA sequences (in red font and accession number) are compared with those of other representingAnaplasma spp. (in black font and accession number). Branch lengths are measured in nucleotide substitutions and numbers show branching percentages in bootstrap replicate. Scale bar represents the percent sequence diversity.


### 3.2.Ehrlichia spp. DNA in Ruminants

A total of 70 animals (70/2,240, 3.1%) were positive for DNA ofEhrlichia spp. in our 16S rRNA FRET-qPCR with copy numbers varying from 50 to 42,900/mL blood (median 5,100). Cattle were most frequently positive (3.6%, 65/1,830) followed by goats (1.1%, 3/270) and sheep (1.8%, 2/111). None of the water buffaloes (29) were positive. We foundEhrlichia sp. positive animals in over half (7/12) the provinces we studied with the highest prevalence in Wuhu (82.4%, 14/17) of Anhui province and lower prevalence in the tropical provinces in the south of China, mainly Hainan (20.3%, 15/74), Yunnan (17.9%, 30/168), and Fujian (8.3%, 2/24). No positive animals were detected from the more northern provinces of Beijing, Shanghai, Heilongjiang, and Tianjin (Table 1; Figure 1).Standard PCR and sequencing of thegltA were performed on samples positive in the FRET-qPCR for 16S rRNA with higher copy numbers, namely, those between 14,400 and 42,900/mL blood (median 16,700). UseablegltA sequences were obtained for six of the animals studied (copy numbers 33,100 to 42,900/mL blood, mean 37,600) showing five, all cattle from Yunnan, to be infected with anEhrlichia sp. having an identical sequence (563/563; 100%) to that of a new species closely related toE. canis and found inRhipicephalus microplus in the Czech republic (JX629807) [22]. The remaining animal, a goat from Jiangsu, was found to be infected withE. canis having a sequence almost identical (549/563; 98%) to that ofE. canis (Oklahoma strain; AF304143) [23] and a strain found in a dog in Thailand (KJ459920) [24].ThegltA sequence ofE. canis we identified was deposited in GenBank under the accession number KX506008 and that of a representative of theEhrlichia sp. (JX629807) as KX506006. ThegltA sequences forEhrlichiaspp. from ruminants in this study are compared with those of other representingEhrlichia spp. (Figure 4).Figure 4
Phylogenetic comparison ofEhrlichiaspp. from ruminants in this study. ThegltA sequences (in red font and accession number) are compared with those of other representingEhrlichia spp. (in black font and accession number). Branch lengths are measured in nucleotide substitutions and numbers show branching percentages in bootstrap replicate. Scale bar represents the percent sequence diversity.


## 3.1.Anaplasma spp. DNA in Ruminants

Overall, 17.2% (385/2240) of the ruminants from 8 of the 12 provinces studied (Table1 and Figure 1) were positive for the 16S rRNA ofAnaplasma spp. with copy numbers ranging from 50 to 52,000/mL blood (median 1,720 copies/mL blood). Goats were most frequently positive (81.1%; 219/270), followed by cattle (13.2%; 241/1,830), sheep (11.7%; 13/111), and water buffaloes (6.9%; 2/29).Figure 1
Detection ofAnaplasma spp. andEhrlichia spp. in ruminants from 12 provinces of China. Blood samples of ruminants (2,240) were collected from twelve provinces (in bisque) of China. The prevalence is shown forAnaplasmaspp. (red) andEhrlichiaspp. (blue).When we sequenced the positive 16S rRNA FRET-qPCRs, we obtained clean sequencing data for 38 of the samples from cattle (23) and goats (15) (Table2). The sequences in the cattle were most commonly identical to those ofA. phagocytophilum(12/23; 52%) andA. marginale(10/23; 44%) with positive animals in 4 and 2 of the 9 provinces studied, respectively. Representative sequences were deposited in GenBank forA. phagocytophilum(KX279691) andA. marginale(KX279690). The one otherAnaplasmasp. found was in a Wannan cow from Anhui which had a 16S RNA sequence identical to that of a poorly characterizedAnaplasmasp. BL126-13 (KJ410243). We also found evidence for this organism in three goats from Jiangsu. OtherAnaplasmaspp. we detected in goats wereA. ovis (KX279688) in Xinjiang (2),A. platys(KX279689) in Jiangsu (2), and a poorly characterizedAnaplasmasp. (KM227012) in Jiangsu (5). The 16S rRNA sequences we deposited in GenBank (KX279683; KX279685) are identical to those ofAnaplasmasp. (KM227012) andAnaplasmaBL126-13 (KJ410243), respectively.Table 2
Source ofAnaplasma and Ehrlichiaspp. identified in this study based on 16S rRNA and gltA gene sequences.
Organism Species 16S RNA Species gltA GenBank # Numbers Animal City, province GenBank # Numbers Animal City, province Anaplasmaspp. A. marginale KX279690 10 Cattle Binzhou, Shandong (1) A. marginale KX506005 1 Cattle Kunming, Yunnan Kunming, Yunnan (9) A. platys KX279689 2 Goat Yangzhou, Jiangsu A. phagocytophilum KX279691 15 Cattle Bengbu, Anhui (2) Haikou, Hainan (5) Kunming, Yunnan (4) Wuhu, Anhui (1) Goat Yangzhou, Jiangsu (3) A. ovis KX279688 2 Goat Urumqi, Xinjiang (2) A. ovis KX506006 1 Goat Urumqi, Xinjiang Anaplasmasp. KX279685 4 Cattle Wuhu, Anhui (1) Goat Yangzhou, Jiangsu (3) Anaplasmasp. KX279683 5 Goat Yangzhou, Jiangsu (5)  Ehrlichiaspp. Ehrlichiasp. KX279682 14 Sheep Inner Mongolia (1) Cattle Yunnan, Kunming (10) Ehrlichiasp. KX506007 5 Cattle Yunnan, Kunming Goat Yangzhou, Jiangsu (3) E. canis KX506008 1 Goat Yangzhou, JiangsuSince there is limited polymorphism in the 16S rRNA FRET-qPCR sequences of different ruminantAnaplasmaspp. (Figure 2), to enable more definitive species differentiation we carried out a PCR and sequencing of the more polymorphicgltA on 20% of the 16S rRNA positive samples with the highest copy numbers (range of 610 to 52,000/mL blood; median 2,300). Only two samples provided clean sequencing data with one (16S rRNA FRET-qPCR copy number 52,000/mL blood) from a bovine in Yunnan having 100% identity withA. marginale (0/620 mismatches with CP006847.1) and the other (16S rRNA FRET-qPCR copy number 47,700 copies/mL blood) from a goat in Xinjiang having 99.7% identity withA. ovis (1/438 mismatches with KJ410284.1). ThegltA sequence ofA. marginale we identified was deposited in GenBank under the accession number KX506005 and that ofA. ovis as KX506006. The 16S rRNA sequences forAnaplasmaspp. from ruminants in this study are compared with those of other representingAnaplasma spp. (Figure 3).Figure 2
Alignment of the sequences obtained with the 16S rRNA FRET-qPCR we used in our study and those ofAnaplasma spp. in GenBank. “.” denotes the identical nucleotide sequence to that ofA. bovis. Organisms with GenBank accession numbers identified in the study are in red.Figure 3
Phylogenetic comparison ofAnaplasmaspp. from ruminants in this study. The 16S rRNA sequences (in red font and accession number) are compared with those of other representingAnaplasma spp. (in black font and accession number). Branch lengths are measured in nucleotide substitutions and numbers show branching percentages in bootstrap replicate. Scale bar represents the percent sequence diversity.


## 3.2.Ehrlichia spp. DNA in Ruminants

A total of 70 animals (70/2,240, 3.1%) were positive for DNA ofEhrlichia spp. in our 16S rRNA FRET-qPCR with copy numbers varying from 50 to 42,900/mL blood (median 5,100). Cattle were most frequently positive (3.6%, 65/1,830) followed by goats (1.1%, 3/270) and sheep (1.8%, 2/111). None of the water buffaloes (29) were positive. We foundEhrlichia sp. positive animals in over half (7/12) the provinces we studied with the highest prevalence in Wuhu (82.4%, 14/17) of Anhui province and lower prevalence in the tropical provinces in the south of China, mainly Hainan (20.3%, 15/74), Yunnan (17.9%, 30/168), and Fujian (8.3%, 2/24). No positive animals were detected from the more northern provinces of Beijing, Shanghai, Heilongjiang, and Tianjin (Table 1; Figure 1).Standard PCR and sequencing of thegltA were performed on samples positive in the FRET-qPCR for 16S rRNA with higher copy numbers, namely, those between 14,400 and 42,900/mL blood (median 16,700). UseablegltA sequences were obtained for six of the animals studied (copy numbers 33,100 to 42,900/mL blood, mean 37,600) showing five, all cattle from Yunnan, to be infected with anEhrlichia sp. having an identical sequence (563/563; 100%) to that of a new species closely related toE. canis and found inRhipicephalus microplus in the Czech republic (JX629807) [22]. The remaining animal, a goat from Jiangsu, was found to be infected withE. canis having a sequence almost identical (549/563; 98%) to that ofE. canis (Oklahoma strain; AF304143) [23] and a strain found in a dog in Thailand (KJ459920) [24].ThegltA sequence ofE. canis we identified was deposited in GenBank under the accession number KX506008 and that of a representative of theEhrlichia sp. (JX629807) as KX506006. ThegltA sequences forEhrlichiaspp. from ruminants in this study are compared with those of other representingEhrlichia spp. (Figure 4).Figure 4
Phylogenetic comparison ofEhrlichiaspp. from ruminants in this study. ThegltA sequences (in red font and accession number) are compared with those of other representingEhrlichia spp. (in black font and accession number). Branch lengths are measured in nucleotide substitutions and numbers show branching percentages in bootstrap replicate. Scale bar represents the percent sequence diversity.


## 4. Discussion

Our results are consistent with other PCR studies from China [1, 6–10, 12, 13] showing that domestic ruminants from the country are infected with a range ofAnaplasmaandEhrlichiaspp. Differences in the prevalence of animals on the farms we studied were most likely due to differing husbandry practices and tick exposure, with dairy animals and water buffaloes raised intensively having the lowest levels of positivity. Also, although our sample numbers were small, our results indicate that ruminants are generally more commonly infected withAnaplasmaandEhrlichiaspp. in the more southern provinces (Yunnan and Hainan) and along the seaboard (Fujian, Anhui, and Jiangsu) where there are more tropical conditions and tick vectors are expected to be more prevalent. Lower prevalence was found in the cooler northern provinces (Heilongjiang, Beijing, Inner Mongolia, and Xinjiang).Although we obtained relatively large numbers of animals positive by FRET-qPCR for the 16S rRNA ofAnaplasmaspp., we were only able to amplify a small number of these with thegltAprimers. We presume this was because of low parasitemia in affected animals and different numbers of target sequences for the PCRs [25], since we could only amplify thegltA from animals with high copy numbers in the 16S rRNA FRET-qPCR. It might also, however, have been because of different sensitivities of the PCRs we used as has been described before with molecular detection of different genes inAnaplasmaspp. [10, 25, 26].The oneAnaplasmawe definitively identified with thegltAPCR wasA. marginalewhich is the agent of bovine anaplasmosis, a very common disease of cattle around the world in tropical and subtropical countries [27]. Infections are mainly transmitted byRhipicephalus microplus and although most infections are subclinical, there can be fever and severe anemia resulting in production losses from decreased milk production and abortion. Studies in China have shown that the organism can be found inRhipicephalus(Boophilus)microplus[28] and also that the organism might be transmitted byHyalomma asiaticum[5]. The organism appears to be widespread in domestic ruminants in China and it has been reported to be a relatively common infection of cattle in southern and northern China [5, 15, 29, 30].The otherAnaplasmawe definitively identified,A. ovis, has also been reported previously in China in goats (15%) from central and southern China [1] and in sheep and goats (41%) in Henan and Xinjiang [6, 7]. This organism is the agent of ovine anaplasmosis which can be transmitted byDermacentor nuttalli,Hyalomma asiaticum kozlovi, andRhipicephalus pumilio in China where infections mostly result in subclinical anemia in indigenous animals [31]. Recently, the organism has been shown to infect humans [32] as has a closely related organism in China, putatively named “Anaplasma capra” [33].Although we were not able to usegltAsequencing to definitively identify most of theAnaplasmaspp. we detected with our16S rRNAFRET-qPCR, we could confirm the accuracy of the qPCR in detectingA. marginaleandA. ovis.Anaplasma marginalewas one of the most commonAnaplasmaspecies we detected with the 16S rRNA FRET-qPCR along withA. phagocytophilumwhich has also already been described in China where it appears to be common in ruminants [7, 11, 12, 34].Anaplasma phagocytophilumis the agent of tick-borne fever of ruminants and is transmitted byDermacentor silvarum,Haemaphysalis concinna, H. longicornis, andIxodes persulcatusin China [35, 36]. The organism is now known to infect a wide variety of domestic and wild animals and is the agent of human granulocytic anaplasmosis [27].OtherAnaplasmaspp. we appear to have identified based on their 16S rRNA sequences includeA. platys, the agent of infectious canine cyclic thrombocytopenia [3], which has been described in dogs in Asia [24, 37] and in sika deer, goats, and cattle in China [4, 19]. The remaining twoAnaplasmaspp. we appear to have found are as yet only poorly characterized withAnaplasmasp. (KM227012) first reported inProcapra gutturosa,the Mongolian gazelle, in China and appear to be most closely related toA. ovisandA. centrale[38].Anaplasmasp. BL126-13 (KJ410243) has only been identified in aHyalomma asiaticumfrom Xinjiang [39]. As we found that these poorly characterized organisms seem to occur relatively commonly, especially in goats, it would seem appropriate that they should be studied further as they could be important pathogens.A number ofEhrlichiaspp. have been described in China,E. chaffeensis,E. canis,E. platys,E. ewingii(granulocytic ehrlichial agent), and also a novel species closely related toE. chaffeensisandA. marginale [14, 15, 33, 40]. The most importantEhrlichiaspecies infecting ruminants,E. ruminantium, is restricted to Africa and some Caribbean islands [19] and has not been reported in China. Of the otherEhrlichiaknown to infect ruminants (summarized in Zhang et al., 2015 [19]), we only found evidence of infection withE. canisin the domestic ruminants we studied in China. AlthoughE. canis is best known as a very common dog pathogen around the world, infections have also been described in people [41] and in cats [42], and there is thus growing evidence thatE. canis has a wider host range than previously thought [2, 19, 43]. Our finding ofE. canisand closely related organisms in a goat and cattle in China further supports this evidence and is consistent with the findings of a study showing thatE. canis or very closely related organisms are present in domestic ruminants in the Caribbean [19] and also a study showing thatE. canisoccurs in sika deer in China [4]. Further studies are underway in our laboratory to determine the pathogenicity ofE. canisin domestic ruminants.In summary, we found DNA ofAnaplasmaspp. andEhrlichiaspp. relatively common in the blood of the goats (81.1% and 1.1%, resp.), cattle (13.7% and 3.6%, resp.), sheep (11.7% and 1.8%, resp.), and water buffaloes (6.9% and 0%, resp.) we studied from China. Further, our data from 12 provinces show that a wide range ofAnaplasmaspp. andEhrlichiaspp. occur in ruminants in China and further larger scale studies are indicated to determine more accurate prevalence data for these agents and their impact on health and production. The low copy numbers we commonly found indicate that chronic infections are common and this did not enable us to obtain reliable multigene sequence data from most samples. It would appear best, then, for future studies on the presence ofAnaplasmaandEhrlichiaspp. to rather be conducted on organisms cultured from infected animals. Ticks should also be considered for such studies as they generally contain relatively high numbers ofAnaplasmaandEhrlichia spp. (2,530 to 970,000/positive tick) [25].



---

*Source: 9183861-2016-12-20.xml*