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Molecular detection of -like organisms in cattle drinking
water
Nick Wheelhouse, Michelle Sait, Jo Gidlow, Rita Deuchande, Nicole Borel,
James Baily, George Caldow, David Longbottom
To cite this version:
Accepted Manuscript
Title: Molecular detection of Chlamydia-like organisms in cattle drinking water
Authors: Nick Wheelhouse, Michelle Sait, Jo Gidlow, Rita Deuchande, Nicole Borel, James Baily, George Caldow, David Longbottom
PII: S0378-1135(11)00217-3
DOI: doi:10.1016/j.vetmic.2011.03.040
Reference: VETMIC 5266
To appear in: VETMIC
Received date: 11-2-2011 Revised date: 29-3-2011 Accepted date: 31-3-2011
Please cite this article as: Wheelhouse, N., Sait, M., Gidlow, J., Deuchande, R., Borel, N., Baily, J., Caldow, G., Longbottom, D., Molecular detection of
Chlamydia-like organisms in cattle drinking water, Veterinary Microbiology (2010),
doi:10.1016/j.vetmic.2011.03.040
Accepted Manuscript
1 Short Communication 2 3 4Molecular detection of Chlamydia-like organisms in cattle drinking water 5
6
Nick Wheelhousea,1,*, Michelle Saita,1, Jo Gidlowb, Rita Deuchandec, Nicole Boreld, 7
James Bailye, George Caldowb, David Longbottoma 8
a
Moredun Research Institute, Pentlands Science Park, Bush Loan, Edinburgh EH26 9
0PZ, UK 10
b
SAC Consulting: Veterinary Services, Disease Surveillance Centre, Greycook, St 11
Boswells, Roxburghshire, TD6 0EU, UK 12
c
Veterinary Laboratories Agency, Sutton Bonington, The Elms, College Road, Sutton 13
Bonington, Loughborough, Leicestershire, LE12 5RB, UK 14
d
Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, 15
Winterthurerstrasse 268, 8057 Zurich, Switzerland 16
e
SAC Edinburgh, Allan Watt Building, Bush Estate, Penicuik, Midlothian, EH260QE 17
18
*Corresponding author. Tel +44 (0)1314455111; fax +44 (0)1314556735 19
Accepted Manuscript
Abstract
24
A substantial proportion of the causes of infectious bovine abortion remain largely 25
undiagnosed, potentially due to the presence of previously unrecognised infectious 26
agents. Recently, several reports have demonstrated the presence of Parachlamydia 27
sp. in placental and foetal tissues derived from bovine abortions of unknown 28
aetiology but the route of transmission remains undefined. The drinking water from 29
one such recent case study was analysed for the presence of Parachlamydia sp. as a 30
potential source of infection. Chlamydiales sp. 16S rRNA genes were PCR-31
amplified from the drinking water and a 16S rRNA gene clone library constructed. 32
DNA sequencing of thirty-one clones indicated the presence of organisms belonging 33
to the Parachlamydiaceae, specifically the genera Parachlamydia and 34
Neochlamydia. Seven 16S rRNA gene sequences were identical to a Parachlamydia 35
sp. sequence obtained from placental tissue from an abortion case originating from 36
the same farm. These results raise the possibility that the drinking water is a source 37
of Parachlamydia, which may play a role in infectious bovine abortion. 38
39
Keywords 40
Parachlamydiaceae; cattle; abortion; water 41
Accepted Manuscript
431. Introduction
44
Bovine abortion is a significant animal welfare issue and a cause of significant 45
economic loss, yet despite this, investigations leading to no definitive diagnosis remain 46
high (Cabell, 2007). In Great Britain, figures from the 2002-09 Veterinary Investigation 47
Surveillance (VIDA) Report show that 77% (6,560 of 8,549 samples) of diagnosed 48
bovine fetopathies resulted from an infectious cause, whereas no diagnosis was reached 49
for 80% (34,462 of 43,011 samples) of all cases of bovine fetopathy 50
(http://www.defra.gov.uk/vla/reports/docs/rep_vida_cattle_2009.pdf). This could in part 51
be explained by the presence of unidentified infectious abortifacient agents. While 52
Chlamydophila abortus is recognised as a known aetiological agent of ruminant 53
abortion (Longbottom and Coulter, 2003), several novel species of Chlamydia-like 54
organisms have also emerged as putative ruminant abortifacients. These species include 55
Waddlia chondrophila, isolated originally from an aborted bovine fetus in the USA 56
(Dilbeck et al., 1990) and subsequently identified in a stillborn calf in Germany 57
(Henning et al., 2002). Recent studies carried out on aborted bovine placentas in 58
Switzerland, using both molecular and immunohistochemical techniques, identified the 59
presence of other non-characterised chlamydia-like organisms and Parachlamydia sp. 60
(Borel et al., 2007; Ruhl et al., 2009). These findings were supported by the 61
identification of Parachlamydia, Rhabdochlamydia and Neochlamydia species in foetal 62
and placental tissue samples from the UK (Deuchande et al., 2010; Wheelhouse et al., 63
2010). However, although several reports have demonstrated the presence of these 64
organisms in aborted bovine tissues, no studies have investigated possible sources of 65
infection. Many of the identified strains of Parachlamydia were discovered as 66
Accepted Manuscript
including water supplies (Greub and Raoult, 2002). This study was carried out to 68examine whether contaminated drinking water could be a potential source of 69
transmission of Parachlamydia on a farm that experienced an abortion storm in which 70
parachlamydial involvement was confirmed (Deuchande et al., 2010). 71
72
2. Materials and Methods
73
2.1 Isolation of DNA 74
Samples were obtained from a Scottish farm on which suspected 75
parachlamydial involvement in cases of bovine abortion were previously reported 76
(Deuchande et al., 2010). Water samples were taken from each of the cattle drinking 77
troughs in the calving sheds and also directly from the bore hole supplying them. Prior 78
to DNA extraction 35ml of each sample was centrifuged at 20,000 rpm for 60 min using 79
an SW32Ti rotor in a Beckman Coulter Optima™ L-90K ultracentrifuge (Beckman 80
Coulter (UK) Ltd., High Wycombe, Buckinghamshire, UK). DNA was extracted from 81
the subsequent pellet using the QIAamp DNA stool mini kit (Qiagen, Crawley, UK). 82
83
2.2 PCR, cloning and sequencing 84
A pan-Chlamydiales PCR targeting the 16S rRNA gene was performed using 85
forward primer 16S FOR2 (5’-CGTGGATGAGGCATGCAAGTCGA-3’) and reverse 86
primer 16S REV2 (5’-CAATCTCTCAATCCGCCTAGACGTCTTAG-3’) to generate 87
amplicons of approximately 260bp (Ossewaarde and Meijer, 1999). Negative-control 88
reactions contained DNA-free water instead of extracted DNA. PCR products were 89
purified (Qiaquick®PCR purification kit, Qiagen) prior to cloning into the pGEM-T® 90
vector (Promega, Hampshire UK). Plasmids were transformed into Escherichia coli 91
Accepted Manuscript
ampicillin resistance and blue-white colony selection, according to standard procedures. 93The insertion of PCR products was confirmed by colony PCR using the 16S rRNA gene 94
pan-Chlamydiales primers. Positive colonies resulting from each water trough were 95
grown in overnight broths, the plasmid DNA extracted using a Qiagen Miniprep kit 96
(Qiagen), and sequenced. Sequencing was achieved using primers directed against the 97
T7-promoter sequence of the plasmid by dideoxy chain termination / cycle sequencing 98
on an ABI 3730XLDNA sequencer (Eurofins MWG Operon, Ebersberg, Germany). 99
100
2.3 Sequence analysis 101
102
16S rRNA gene sequences were aligned in ClustalW2 (Larkin et al., 2007) and 103
the Phylip program DNAdist (Felsenstein, 1989) was used to calculate a distance matrix 104
using a Jukes Cantor model of nucleotide substitution (Jukes and Cantor, 1969). The 105
software package MOTHUR was used to assign sequences to operational taxonomic 106
units (OTUs) and generate rarefaction curves to assess diversity and sampling intensity 107
(Schloss et al., 2009). The phylogenetic classification of a representative sequence from 108
each OTU was determined using the Ribosomal Database Project (RDP) Classifier 109
program with a bootstrap cut-off of 50% (Wang et al., 2007) and by comparison with 110
sequences in the NCBI DNA databases. All sequences were submitted to GenBank. 111
112
3. Results 113
PCR analysis revealed the presence of chlamydial DNA in each of the drinking 114
troughs but not in the original bore-hole water source. The 31 16S rRNA gene 115
sequences (GenBank accession HQ702405-HQ702435) were grouped into 12 OTUs 116
using a cut-off of ≥99% sequence identity (Table 1). Rarefaction curves appeared to be 117
Accepted Manuscript
diversity present in the water sample and that further sampling was unlikely to reveal 119additional diversity (Figure 1). 120
Fifteen and sixteen of the cloned 16S rRNA gene sequences each representing 6 121
OTUs originated from organisms belonging to the genera Neochlamydia and 122
Parachlamydia respectively (Table 1). BLAST analysis revealed that representative 123
sequences from each OTU were closely related to Parachlamydiaceae sequences 124
obtained from environmental samples and clinical samples taken from bovine abortion, 125
ovine ocular infections and fish epitheliocystis. Seven (22.5%) of the 16S rRNA gene 126
sequences obtained from the water troughs and allocated to OTU 1 were identical to a 127
16S rRNA gene recovered from a placental sample originating from a case of bovine 128
abortion from the same farm (Deuchande et al., 2010). 129
130
4. Discussion
131
Parachlamydiaceae have now emerged as potential causes of bovine abortion 132
both in the UK (Deuchande et al., 2010; Wheelhouse et al., 2010) and in Switzerland 133
(Borel et al., 2007; Ruhl et al., 2009). Despite this there have been no studies 134
investigating the possible sources of infection. This study was carried out to determine 135
whether drinking water could be a possible medium for parachlamydial transmission in 136
cattle in which Parachlamydia had been detected in aborted placental tissue 137
(Deuchande et al, 2010). Given that the water at source was negative, yet the water in 138
the drinking troughs was positive by PCR for the presence of parachlamydial DNA, 139
suggests that contamination of the trough water occurred after extraction from the 140
borehole in this group of animals. Moreover, the similarity in sequence of many of the 141
samples with one obtained from a placenta on the same farm suggests that the water is a 142
Accepted Manuscript
although the source of this contamination is unclear at present, P. acanthamoebae has 144been demonstrated to exist as an endosymbiont of Acanthamoeba sp. that commonly 145
inhabit water samples. Furthermore, while P. acanthameoba exists as an endosymbiont 146
at environmental temperatures it is lytic to its protozoan host at physiological 147
temperatures, exposing the animal to the organism and promoting infection (Greub et 148
al., 2003). This could suggest that thorough cleaning and disinfection of the troughs 149
before the re-introduction of animals into housing should be considered to reduce the 150
risk of infection. 151
In addition to bovine abortion, Chlamydia-like organisms have also been 152
reported to play possible roles in other pathogenic conditions, such as ocular infections 153
in sheep (Polkinghorne et al., 2009). Indeed, for 16 of the 31 submitted sequences the 154
top DNA database matches were sequences obtained from ocular swabs from sheep 155
with ocular disease, possibly indicating that the same organisms may cause a range of 156
conditions in different host species. As well as their potential roles in animal disease 157
there is a possible threat to human health, with evidence supporting a role for 158
Parachlamydia sp. in human pneumonia and ocular disease (Greub, 2009). There is also 159
evidence that P. acanthamoebae can cross the human placenta to the unborn fetus (Baud 160
et al., 2009) and be a cause of neonatal morbidity and hospitalisation (Lamoth et al., 161
2009). Its isolation from human nasal mucosa (Amann et al., 1997), the presence of it 162
within guinea-pig (Lutz-Wohlgroth et al., 2006) nasal secretions and its detection in 163
ocular secretions of both sheep (Polkinghorne et al., 2009) and domesticated cats 164
(Richter et al., 2010) suggests that contamination of the drinking water by ocular or 165
nasal secretions may act as a potential source for transmission to livestock, which 166
Accepted Manuscript
In summary, this report demonstrates the presence in the environment of168
Parachlamydia sp. that have been linked to cases of bovine abortion and the possible 169
role of drinking water as a source of infection. These findings support the need for 170
further investigations to determine the role of Parachlamydiaceae sp. in bovine abortion 171
and their potential environmental reservoirs. 172
173
Acknowledgments
174
This work was funded by the Scottish Government Rural and Environment 175
Research and Analysis Directorate (RERAD) and by the Biological and Biotechnology 176
Sciences Research Council (BBSRC). 177
178
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Table 1. OTU grouping and phylogenetic assignment of Parachlamydiaceae 16S rRNA gene sequences retrieved from water trough samples.
Operational taxonomic unit (OTU)
Clones assigned to OTU Genus assignment by RDP Classifier (% confidence)
Top BLAST match, (% similarity), source
1 WT16, WT20, WT15, WT45,
WT48, WT19, WT17
Parachlamydia (70%) FJ160739. Uncultured Chlamydiales bacterium clone USC7 (99%) Sheep with mild ocular disease
2 WT2, WT10, WT9, WT8 Neochlamydia (73%) FJ160741. Uncultured Chlamydiales bacterium clone USC9 (90%) Sheep with severe ocular disease
3 WT30, WT23, WT27, WT24 Parachlamydia (68%) FJ160733. Uncultured Chlamydiales bacterium clone USC1 (93%)
Sheep with mild ocular disease
4 WT34, WT36, WT31 Neochlamydia (52%) AF097198. Uncultured Chlamydiales bacterium clone CRG15 (94%)
Human respiratory tract infection
5 WT4, WT21, WT3 Neochlamydia (64%) AY013469. Uncultured Chlamydiales bacterium clone CRG93 (99%)
Human respiratory tract infection
Accepted Manuscript
6 WT44, WT47, WT46 Neochlamydia (84%) AY326518. Uncultured soil bacterium clone 4-1 (94%)
Soil
7 WT41, WT13 Parachlamydia (70%) AY013438. Uncultured Chlamydiales bacterium clone CRG62 (99%)
Human respiratory tract infection
8 WT42 Parachlamydia (74%) FJ160739. Uncultured Chlamydiales bacterium clone USC7 (97%)
Sheep with mild ocular disease
9 WT22 Parachlamydia (83%) AM408789. Endosymbiont of Acanthameoba sp. EI2 (88%)
Acanthamoeba sp.
10 WT14 Parachlamydia (54%) AY013438. Uncultured Chlamydiales bacterium clone CRG62 (95%)
Human respiratory tract infection
11 WT6 Neochlamydia (69%) FJ376381. Uncultured Chlamydiales bacterium clone UFC3 (91%)
Fish with epitheliocystis
12 WT7 Neochlamydia (55%) FJ532292. Parachlamydiaceae bacterium clone CRIB39 (93%)
