Borel, N; Pospischil, A; Greub, G (2010). Parachlamydia acanthamoebae and Its Zoonotic Risk. Clinical Microbiology Newsletter, 32(24):185-191. Postprint available at: http://www.zora.uzh.ch University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch Originally published at: Borel, N; Pospischil, A; Greub, G (2010). Parachlamydia acanthamoebae and Its Zoonotic Risk. Clinical Microbiology Newsletter, 32(24):185-191. Winterthurerstr. 190 CH-8057 Zurich http://www.zora.uzh.ch

Year: 2010

Parachlamydia acanthamoebae and Its Zoonotic Risk

Borel, N; Pospischil, A; Greub, G

Borel, N; Pospischil, A; Greub, G (2010). Parachlamydia acanthamoebae and Its Zoonotic Risk. Clinical Microbiology Newsletter, 32(24):185-191. Postprint available at: http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Borel, N; Pospischil, A; Greub, G (2010). Parachlamydia acanthamoebae and Its Zoonotic Risk. Clinical Microbiology Newsletter, 32(24):185-191. Parachlamydia acanthamoebae and Its Zoonotic Risk

Abstract

Parachlamydia acanthamoebae has been implicated as an emerging agent of pneumonia in humans. Recently, it has been linked to miscarriage and neonatal infections. In contrast, its role in atherosclerosis remains controversial. In animals, there is strong evidence for Parachlamydia as a new abortigenic in cattle, and further data on this agent in other animal species is accumulating. New diagnostic methods, such as specific real-time PCR and immunohistochemistry with specific anti-Parachlamydia antibodies to detect the organism within lesions, are now available to facilitate the diagnosis of parachlamydial infections in humans and animals. This review discusses parachlamydial infections in clinical settings in humans and animals, as well as their zoonotic potential and possible modes of transmission. Current diagnostic tools are presented, and finally, the antibiotic susceptibility of Parachlamydia is described for potential future preventive and therapeutic options. Nevertheless, more data on Parachlamydia and -like organisms are needed to elucidate their host range and pathogenic potential in humans and animals. 1 Parachlamydia acanthamoebae and its zoonotic risk

2

1 1 2, 3 3 NICOLE BOREL , ANDREAS POSPISCHIL , GILBERT GREUB

4

5 1Institute of Veterinary Pathology, University of Zurich, Vetsuisse Faculty, Switzerland

6 2Center for Research on Intracellular (CRIB), Institute of Microbiology,

7 University Hospital Center and University of Lausanne, Lausanne, Switzerland, and

8 3Infectious Diseases Service, University Hospital Center, Lausanne, Switzerland

9

10 Nicole Borel, DVM, FHV

11 Andreas Pospischil, DVM, ECVP, FHV

12 Gilbert Greub, MD, PhD

13

14 Corresponding author:

15 Nicole Borel, DVM, FVH

16 Institute of Veterinary Pathology, Vetsuisse Faculty

17 University of Zurich, Winterthurerstrasse 268

18 CH-8057

19 Zurich, Switzerland

20 Tel.: +41-44-635-8576

21 Fax.: +41-44-635-8934

22 Email: [email protected]

23

1 24 Abstract

25 Parachlamydia acanthamoebae has been implicated as an emerging agent of

26 pneumonia in humans. Recently, it has been linked to miscarriage and neonatal

27 infections. In contrast, its role in atherosclerosis remains controversial. In animals,

28 there is strong evidence for Parachlamydia as a new abortigenic in cattle and further

29 data on this agent in other animal species is accumulating. New diagnostic methods

30 such as specific real-time PCR and immunohistochemistry with specific anti-

31 Parachlamydia antibodies to detect the organism within lesions are now available to

32 facilitate the diagnosis of parachlamydial infections in humans and animals. This review

33 discusses parachlamydial infections in clinical settings in humans and animals as well

34 as their zoonotic potential and possible modes of transmission. Current diagnostic tools

35 are presented and finally, the antibiotic susceptibility of Parachlamydia is described for

36 potential future preventive and therapeutic options. Nevertheless, more data on

37 Parachlamydia and Chlamydia-like organisms are needed to elucidate their host range

38 and pathogenic potential in humans and animals.

39

40 Introduction

41 Parachlamydia acanthamoebae is a gram-negative obligate intracellular bacterium

42 belonging to the order , which currently include six family-level lineage, i.e.

43 the , , Criblamydiaceae, ,

44 Rhabdochlamydiaceae and Waddliaceae (1-5). Bacteria of the Chlamydiaceae family

45 include well established animal and human pathogens of worldwide distribution and of

46 high clinical importance. Chlamydiaceae are characterized by their particular

2 47 developmental cycle including elementary, intermediate and reticulate bodies taking

48 place in eukaryotic hosts. Parachlamydiaceae developmental cycle is characterized by

49 elementary and reticulate bodies comparable to the one of Chlamydiaceae and a third

50 stage, the crescent body (Figure 1D) (6). P. acanthamoebae is naturally infecting free-

51 living amoebae (Figure 1A) and may also enter and marginally replicate within

52 pneumocytes and lung fibroblasts (7). The Parachlamydiaceae family includes other

53 genus, species and species-level lineages (Table 1). However, this review will mainly

54 focus on P. acanthamoebae, which has been more studied and for which there are

55 some evidence for a pathogenic role towards animals and humans and for a zoonotic

56 risk.

57

58 Parachlamydia in ruminants

59 Chlamydia causes abortion in large and small ruminants leading to significant economic

60 losses worldwide (8). The typical chlamydial abortion in ruminants is caused by

61 abortus and around 20% of the sheep population in Switzerland is

62 infected with C. abortus (9). Chlamydial abortion in cattle seems to be much less

63 frequent compared to the situation in sheep and goat indicating species-specific

64 differences (10). Despite comprehensive laboratory investigations, up to 70% of cattle

65 abortion cases remain without an etiological diagnosis (11) suggesting the possible

66 presence of new yet undescribed abortigenic agents. In 1986, chondrophila

67 type strain WSU 86-1044 was implicacted as a new abortigenic agent in cattle, when it

68 was isolated in lung, liver and other tissues of an aborted fetus in the USA (12,13). A

69 second report described the same agent in a bovine abortion co-infected with Neospora

3 70 caninum (14), but could not clarify if abortion was caused by the former or the latter

71 agent. Further reports of W. chondrophila associated to ruminant abortion are still

72 lacking. Studies ongoing in the authors’s laboratory focusing on ruminant abortion did

73 not show evidence of Waddlia by specific real-time PCR or immunohistochemistry.

74 A previous study focusing on bovine chlamydial abortion (due to C. abortus) in the

75 eastern part of Switzerland where chlamydial abortion in small ruminants is endemic (9),

76 found C. abortus in only 4% of late-term abortion (10). In contrast, 18.3% of cases were

77 positive for Chlamydia-like organisms by broad-range 16S rRNA PCR. Sequencing of

78 PCR products surprisingly revealed no similarities to W. chondrophila but to P.

79 acanthamoebae resulting in the first PCR-based detection of this organism in a bovine

80 abortion. To confirm these preliminary findings, a follow-up study was initiated and

81 demonstrated the presence of Parachlamydia in bovine placenta by

82 immunhistochemical protocols using a specific anti-Parachlamydia antibody and by

83 transmission electron microscopy (15). In 70% of abortion cases with amplification of

84 Chlamydia-like organisms DNA, the presence of Parachlamydia could be further

85 confirmed by positive immunohistochemistry demonstrating the infectious agent within

86 the placental lesions (15). The development of more specific new molecular methods

87 such as a Parachlamydia-specific real-time PCR (16) and further improvement of the

88 Parachlamydia-specific immunohistochemistry (15) using mouse polyclonal antibodies

89 (17) enabled a comprehensive investigation on retrospectively collected bovine abortion

90 cases (18). Placenta samples from late-term abortion cases in cattle were investigated

91 by real-time PCR diagnostic for Chlamydiaceae and Parachlamydia spp., respectively

92 and histological sections of cases positive by real-time PCR were further examined by

4 93 immunohistochemistry to demonstrate the agent within the placental lesion.

94 Parachlamydial DNA was detected in 18.3% of cases and of these 81.4% was

95 confirmed by immunohistochemistry. By immunohistochemistry, parachlamydial antigen

96 was localized mainly intracytoplasmic within trophoblastic epithelium of the placenta

97 (Figure 2D). The main histopathological feature in these parachlamydial abortion cases

98 was purulent to necrotizing placentitis (Figure 1B). Severe placentitis will result in

99 placental insufficiency and consecutive abortion. Presence of parachlamydial antigen

100 within necrotic and inflamed placental tissue as demonstrated by immunohistochemistry

101 could link placental damage to the bacteria. Although final proof is lacking by

102 experimental infection in pregnant animals, there is strong evidence that Parachlamydia

103 might represent a new abortigenic agent in cattle.

104 Similar data were obtained in small ruminant abortion when placenta and fetal tissue

105 from 211 abortion cases of goat and sheep were investigated (19). Thus, 26.1% of

106 abortion cases were positive for C. abortus whereas only 2 cases (0.9%) were positive

107 for Parachlamydia by real-time PCR immunohistochemistry. In both cases, necrotizing

108 placentitis was present and interestingly, in one case fetal organs were available

109 showing interstitial pneumonia and mixed cellular periportal hepatitis. Parachlamydia

110 could be further detected in the fetal lung by PCR and immunohistochemistry. Mixed

111 infection with C. abortus and Parachlamydia was present in this case and simultaneous

112 presence of both agents could be demonstrated in the necrotic trophoblastic epithelium

113 neutrophilic exsudate of the placenta (19).

114 By 16S rRNA PCR, DNA of Chlamydia-like organisms was detected in semen samples

115 of bulls and rams (20). The presence of these organims suggests the possibility of

5 116 venereal transmission of Parachlamydia and of other members of this highly diverse

117 group of emerging pathogens.

118

119 Parachlamydia in other animals

120 Reptiles

121 A retrospective study on reptile tissues with granulomatous inflammation focused on the

122 presence of mycobacteria and . By 16S rRNA PCR, in 54.4% of cases DNA

123 of Chlamydia-like organisms with variable sequence similarity (88-97%) to P.

124 acanthamoebae and Simkania negevensis was detected in chelonians, lizards and

125 snakes from zoo, shops and privat owners (21). At that time, specific antibodies for

126 Parachlamydia were not available and samples are no more available. Thus,

127 demonstration of the infectious agent within the granulomatous lesions was not possible

128 and it remains unknown if Chlamydia-like organisms are involved in granuloma

129 formation in reptiles.

130

131 Swine

132 Cervical swabs and genital tracts of sows were examined in a recent study on the

133 diagnostic investigation into the role of chlamydiae in cases of increased rates of return

134 to estrus in pigs (22). By broad-range 16S rRNA PCR amplifying short-length

135 fragments, DNA of Chlamydia-like organisms were detected in 28.2% on the swabs

136 from sows with and 22.0% of sows without reproductive problems, respectively

137 (p>0.05). Although there was no significant difference between the two groups, the high

6 138 prevalence of Chlamydia-like organisms in cervical swabs, uteri, oviducts and tissues of

139 aborted fetuses was unexpected.

140 By the same method, DNA of Chlamydia-like organisms was detected in semen of

141 boars (23). However, the significance of this finding related to reproductive disorders in

142 boars and sows remains to be elucidated.

143

144 Other mammals

145 An association of ocular diseases in cats, koalas, guinea pigs, pigs and sheep with

146 Chlamydia-like organisms was reported as DNA of these bacteria was amplified by PCR

147 (24-29). However, the pathogenicity of these organisms remains unclear and need

148 further investigation.

149

150 Parachlamydia in humans

151 Respiratory Infections

152 P. acanthamoebae is considered as an emerging agent of pneumonia in humans

153 (recently reviewed by Greub, 2009) (5). The first association was provided by recovery

154 of P. acanthamoebae strain Hall’s coccus from the water of a humidifier associated with

155 an outbreak of fever (30). Then, serological and/or molecular evidence further

156 supported the role of Parachlamydia in bronchitis (31), community-acquired pneumonia

157 (32), aspiration pneumonia (33) and bronchiolitis (16). The source of infection (i.e

158 aerosolizied water, animals, or interhuman transmission) remained unidentified in all

159 these subsequent reports. Noteworthy, some cases were identified among two

7 160 immunocompromised patients, i.e. an HIV-infected individual with low CD4 count (34)

161 and a grafted patient (32).

162 The possible role of Parachlamydia was further supported by its ability to remain viable

163 for more than 2 weeks in pneumocytes (7) and by its ability to resist to destruction by

164 human macrophages (35,36), which are in the lower respiratory tract the first line of

165 defense of innate immunity against invading pathogens. Finally, a murine model of

166 pneumonia caused by P. acanthamoebae was developed: intratracheal inoculation of

167 P. acanthamoebae induced a pneumonia in 100% of infected mice and led to a

168 pneumonia-related mortality in 50% of them 5 days post-inoculation (37).

169 Histopathology of the lungs was characterized by purulent pneumonia in the acute

170 stage (2-4 das post-infection) and interstitial pneumonia in the subacute phase (7-10

171 days post-infection) (Figure 1C). Parachlamydia could be demonstrated within the lung

172 lesions by PCR, immunohistochemistry and electron microscopy (Figure 1D, 2C).

173 Morever, living Parachlamydia could be recovered by amoebal co-culture fulfilling the

174 Koch’s postulates.

175

176 Miscarriage and neonatal infections

177 P. acanthamoebae has been linked to human miscarriage and has been detected in

178 cervical smears (38). Direct maternal-fetal transmission was documented by

179 demonstrating the presence of parachlamydial DNA in an amniotic fluid taken from a

180 female patient with cough and flu-like symptoms (39). A very recent study investigated

181 the presence of Parachlamydia in premature neonates by specific real-time PCR (40).

182 DNA of Parachlamydia was detected in 31% of respiratory samples from 29 neonates.

8 183 Most of these patients had severe respiratory distress syndrome and the authors

184 hypothesized that superinfection with Chlamydia-related bacteria during mechanical

185 ventilation could have led to more severe respiratory disease. The mode of transmission

186 of Parachlamydia could have been either (i) through a systemic infection during

187 pregnancy, (ii) a chorio-amnionitis, or (iii) an infection at delivery by Parachlamydia

188 colonizing the vaginal mucosa. Alternatively, infection of the newborns with

189 Parachlamydia could have taken place during mechanical ventilation, free-living

190 amoebae being used as carrier by these strict intracellular bacteria (41).

191

192 Atherosclerosis

193 Some reports identified a possible role of Parachlamydia in atherosclerosis. Thus,

194 Parachlamydia DNA was amplified from abdominal aortic aneurysms and from 18.5% of

195 aortic and carotid atherosclerotic lesions (42). A very recent study did not confirm the

196 former results as there was neither parachlamydial DNA amplification from peripheral

197 blood mononuclear cells nor serological evidence of anti-Parachlamydia IgG in 354

198 patients with past history of atherosclerosis (43). Although it is difficult to definitively

199 conclude based on these few studies, an association between atherosclerosis and

200 Parachlamydia infection seems unlikely (43). In the past, atherosclerosis has been

201 linked to various infectious agents but significant amount of data is mainly available for

202 Chlamydia pneumoniae (44,45,46), which role in this setting remains nevertheless

203 controversial.

204

205 Zoonotic potential of Parachlamydia and mode of transmission

9 206 Coxiella burnettii and C. abortus are well-established infectious causes of adverse

207 pregnancy outcomes after contact with infected ruminants (48-50). The real case

208 numbers are probably underestimated as these agents are obligate intracellular and

209 need special diagnostic tools. Pregnant women should thus avoid contact to sheep and

210 goats flocks during the lambing season (48). Parachlamydia has been related

211 repeatedly to ruminant abortion (10,15,48,49), but is also an emerging respiratory

212 pathogen in humans (5) and has been linked to human miscarriage (38). Thus, either

213 aerosol (comparable to transmission of C. burnettii) or oral transmission (comparable to

214 transmission of C. abortus) of Parachlamydia from ruminant abortion material is

215 possible and should be considered as a potential zoonotic risk. This zoonotic risk is

216 further supported by the fact that maternal-fetal transmission of P. acanthamoebae was

217 documented in a young woman working as a butcher in a rural area known for cattle

218 breeding near from a small village named Bercher (Figure 3) (39). Moreover, contact to

219 animals as infection source was clearly observed in a serological study where 3 out of

220 482 healthy Swiss men were found positive for Parachlamydia sp., all coming from the

221 same rural region near Lausanne (see figure 3) (51). Alternative routes of transmission

222 are through contaminated water where free-living amoebae contain Parachlamydia or

223 ingestion of uncooked meat or raw milk from infected ruminants.

224 Pet animals such as dogs, cats and guinea pigs are additional sources of potential

225 zoonotic agents. Indeed, Chlamydia-like organisms have been reported in cats (24,29)

226 and guinea pigs with conjunctivitis (26). In the latter study, Parachlamydia-DNA was

227 found in six symptomatic and two asymptomatic guinea pigs. Interestingly, the owner

10 228 contact lenses also harbored DNA of P. acanthamoebae suggesting a zoonotic

229 transmission.

230

231 Diagnosis of parachlamydial infections

232 Diagnosis of parachlamydial infections is hampered by its obligate intracellular growth,

233 by its relatively recent discovery and the very recent availability of specific diagnostic

234 tools. Nucleic acid amplification of parachlamydial DNA was initially performed by

235 broad-range 16S rRNA PCR (1), and by a variation of it (3) followed by sequencing.

236 Diagnosis of well-known chlamydial infections is mostly either based on species-specific

237 PCR tests for detection of Chlamydia trachomatis in humans or Chlamydiaceae-specific

238 PCR methods followed by sequencing or Microarray in animals. Such PCR assays are

239 not able to detect new Chlamydia-like organisms such as Parachlamydia. Thus, specific

240 real-time PCR have then been developed allowing the detection of as few as 10 DNA

241 copies of Parachlamydia in clinical samples (16,34). One of these PCRs is targeting the

242 ADP/ATP translocase, an enzyme only present in Chlamydiae, Rickettsiae and plant

243 plastids and absent from all other bacterial species (including those colonizing the oral

244 cavity and potentially contaminating the lower respiratory tract samples), thus explaining

245 the excellent specifity of the test (34). The other PCR is targeting the 16S rRNA and this

246 will likely amplified also some other yet undiscovered species within the Parachlamydia

247 genus (16). These PCRs have been applied to various human and animal samples.

248 To define the possible pathogenic potential of these novel organisms, new diagnostic

249 tool were needed to demonstrate the agent within tissue lesions. In clinical settings,

250 samples could have been taken from a contaminated environment in the first place, i.e.

11 251 deep respiratory specimens contaminated by bacteria present in the upper airway.

252 Thus, it is important to ensure that the infectious agent diagnosed is related to the

253 lesion. Immunofluorescence and immunohistochemistry techniques are then the

254 methods of choice and can be used as second confirmatory tests in addition to PCR.

255 Mouse sera against various Chlamydia-like organisms were raised in the senior authors’

256 lab and the serological cross-reactivity between different Chlamydia-related organisms

257 was determined in a preliminary study by immunofluorescence and Western Blotting

258 (17). Subsequently, the specificity of these antisera was tested by Tissue Microarray

259 (TMA) technology associated with immunohistochemistry on formalin-fixed and paraffin-

260 embedded pellets (52) (Figure 2A & 2B). As expected, mice antisera against P.

261 acanthamoebae strain Hall’s coccus exhibited strong cross-reactivity to P.

262 acanthamoebae strain BN9, but not with distantly related species (52). Antisera against

263 P. acanthamoebae strain Hall’s coccus were then already successfully applied to

264 formalin-fixed and paraffin-embedded bovine placenta specimens and resulted in the

265 first report of Parachlamydia in bovine abortion (15) and follow-up studies on ruminant

266 abortion (18,19) (Figure 2D). Rabbit anti-Parachlamydia antibodies have been applied

267 to lung samples from mice experimentally infected with P. acanthamoebae showing

268 good correlation with histopathological lesions and real-time PCR (27) (Figure 2C).

269

270 Parachlamydia and antimicrobial susceptibility

271 When tested in an amoebal co-culture system, Parachlamydia acanthamoebae

272 appeared to be susceptible to macrolides and doxycycline and to be resistant to

273 quinolones (53), exhibiting a similar susceptibility pattern than that of Waddlia

12 274 chondrophila (54). This resistance to quinolones is likely due to mutations in the

275 quinolone-rersistance determining region of the gyrA and parC encoding genes of

276 Parachlamydia acanthamoebae (55). Although, no animal models have confirmed these

277 in vitro data, we thus currently recommend to use macrolides and/or doxycycline to treat

278 infections due to any Parachlamydiaceae.

279

280 Conclusion

281 The role of P. acanthamoebae as an emerging agent of pneumonia in humans and as

282 an abortigenic agent in ruminants has led to further study the pathogenesis of this strict

283 intracellular bacteria and its biology, and has triggered the development of

284 Parachlamydia-specific diagnostic tools to diagnose parachlamydial infections in

285 humans and animals. The parallel detection of this new bacterial agent suggested a

286 possible zoonotic risk. However, zoonotic transmission of Parachlamydia between

287 animals and humans remains to be proven.

288 Persons in contact with aborted material from ruminants such as owner, animal

289 caretakers, veterinarians should be aware of the potential zoonotic risk of well-known

290 abortigenic agents such as C. burnettii and C. abortus, and of more recently described

291 agents with yet poorly defined impact on human health such as P. acanthamoebae.

292

293 Acknowledgements

294 This work was supported by COST Action 855, Animal Chlamydiosis and Zoonotic

295 Implications, Switzerland (SBF Nr.: C05.0141). Gilbert Greub (Lausanne, Switzerland)

13 296 is supported by the Leenards Foundation through a career award entitled “Bourse

297 Leenards pour la relève académique en médecine clinique à Lausanne”.

298

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434

435 Figure legends

436 Figure 1. Histopathology (Hematoxylin and eosin staining, H & E) and transmission

437 electron microscopy in infections with Parachlamydia acanthamoebae. A.

438 Histopathology of Acanthamoeba castellanii infected with P. acanthamoebae strain

439 Hall’s coccus (52). Parachlamydial infection within amoebae is visible as finely

440 basophilic stippeling. H & E staining. B. Histopathology of a bovine placenta. Purulent to

441 necrotizing placentitis in a case of parachlamydial abortion positive by real-time PCR

442 and immunohistochemistry for P. acanthamoebae (18). H & E staining. C. Lung

443 histopathology of a mouse experimentally infected with living P. acanthamoebae at day

444 7 post-inoculation (37). Interstitial pneumonia is present. H & E staining. D.

445 Transmission electron microscopy of the lung of a mouse experimentally infected with

446 living P. acanthamoebae and sacrificed 4 days post-infection (37). Presence of several

447 Parachlamydia in an apoptotic cell including the crescent stage.

448

20 449 Figure 2. Immunohistochemistry (IHC) with mouse antiserum against Parachlamydia

450 acanthamoebae strain Hall’s coccus. A. Uninfected Acanthamoeba castellanii. IHC with

451 mouse antiserum against P. acanthamoebae strain Hall’s coccus (negative control). B.

452 A. castellanii infected with P. acanthamoebae strain Hall’s coccus. IHC with mouse

453 antiserum against Parachlamydia acanthamoebae strain Hall’s coccus (52). C. Positive

454 IHC of the lung of a mouse infected with living Parachlamydia (37). D. IHC of a bovine

455 placenta with the anti-Parachlamydia antibody. Presence of positive granular reaction

456 within trophoblastic epithelium (18). All sections were stained with the AEC/peroxidase

457 method, hematoxylin counterstain.

458

459 Figure 3. Map of Switzerland showing the location where the major outbreak of bovine

460 abortion due to Parachlamydia acanthamoebae occurred (15) and the rural area near

461 Bercher where a cluster of human infection due to Parachlamydia has been observed

462 during a seroepidemiological study in young Swiss men (n=3) and during an

463 investigation of amniotic fluids (n=1) (39,51).

464

21 Table 1. Pathogenicity of Parachlamydiaceae in humans and animals

Species Host Pathogenicity References

Parachlamydia acanthamoebae Ruminants Abortion 10,15,18,19

Guinea pigs Conjunctivitis 26

Reptiles Granulomatous inflammation 21

Mouse Pneumonia (experimental) 37

Cats Corneal disease 29

Humans Community-acquired pneumonia 30,32,34

Humans Aspiration pneumonia 33

Humans Bronchitis 31

Humans Bronchiolitis 16

Humans Miscarriage, premature labor 38,39

Humans Neonatal pneumonia 40

Humans Arteriosclerosis 33,42,43

Humans Ocular discharge 26

Neochlamydia hartmanellae Cats Conjunctivitis, keratitis 24

Neochlamydia sp. Fish Gill infection 31

Protochlamydia naegleriophila Humans Community-acquired pneumonia 56

Protochlamydia amoebophila Humans Community-aquired pneumonia 57

A B

C D D A B

C D BASEL ZURICH

LUZERN NEUCHATEL BERN

LUZERN DAVOS GRAUBUNDEN RURAL AREA NEAR BERCHER LAUSANNE

LOCARNO MATERNO-FETAL TRANSMISSION GENEVA & 3 SEROPOSITIVE PATIENTS DOCUMENTED CASES OF ABORTION IN BOVINES