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 Chlamydia-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 Bacteria (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 Chlamydiales, which currently include six family-level lineage, i.e.
43 the Chlamydiaceae, Parachlamydiaceae, Criblamydiaceae, Simkaniaceae,
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 Chlamydophila 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, Waddlia 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 chlamydiae. 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