Mycoplasma gallisepticum, a pathogen of worldwide economic importance in poultry, is recovered in chickens, especially from the respiratory tract. Some strains, however, are specialized to other tissues and because it jumps from poultry to wild birds, the new strains also cause severe conjunctivitis in new hosts. Nevertheless, most studies of M. gallisepticum in wild birds use choanal swabs or combine choanal and conjunctival swabs to quantify bacterial load. Because the clinical signs associated with M. gallisepticum infection differ markedly between poultry and House Finches (Haemorhous mexicanus), we compared the bacterial load in choanal and conjunctival samples following experimental inoculation of House Finches with M. gallisepticum isolates originating from poultry or from House Finches. This allowed us to test two hypotheses: M. gallisepticum changed tissue tropism, or M. gallisepticum simply expanded its within-host niche. By comparing bacterial loads from choanal and conjunctival swabs in birds inoculated with one of a suite of M. gallisepticum isolates, we found support for hypothesis 2. The choanal loads in House Finches did not differ between isolates, while the conjunctival loads of birds inoculated with poultry isolates were lower than in birds inoculated with House Finch isolates. When measuring the bacterial load of M. gallisepticum in birds, it is important to sample and analyze separately choanal and conjunctival swabs, as quantifying bacterial loads in pooled samples may not provide reliable information on differences in virulence.

Mycoplasma gallisepticum is a pathogen of worldwide economic importance, particularly for the poultry industry. In chickens, the bacterium is recovered, especially from the respiratory tract (Levisohn and Kleven 2000), where it typically causes chronic respiratory disease, including inflammation of the air sacs, lungs, and trachea (Pflaum et al. 2020). In turkeys, it frequently causes infraorbital sinusitis (Ferguson 2013; Raviv and Ley 2013). Extensive variability in tissue tropism has been reported (Levisohn and Kleven 2000), including isolation of encephalitic forms in turkeys (Thomas et al. 1966; Chin et al. 1991), strains with a proclivity for the cloacal tissue (Macowan et al. 1983), strains causing unilateral enlargement of the eyeball (Power and Jordan 1976), and rare cases of keratoconjunctivitis in layer chickens (Nunoya et al. 1995). Around 1993–94, an epidemic of mycoplasmal conjunctivitis in wild birds, especially House Finches (Haemorhous mexicanus), emerged in the Baltimore, Maryland, area (Doster 1994), caused by a novel strain of M. gallisepticum (Ley et al. 1997; Hochachka et al. 2013). The epidemic spread rapidly across eastern North America (Fischer et al. 1997; Dhondt et al. 1998) and later also spread westward causing disease, especially in House Finches (Ley et al. 2006). In finches, clinical signs and gross lesions ranged from mild to severe unilateral or bilateral conjunctival swelling with serous to mucopurulent drainage and nasal exudate. Microscopic lesions consisted of chronic lymphoplasmacytic conjunctivitis, rhinitis, and sinusitis (Ley et al. 1996).

Given that M. gallisepticum disease in poultry is primarily associated with the upper respiratory tract, the traditional location for sampling in most poultry studies is the trachea or choanal cleft (e.g., Feberwee, Mekkes, de Wit et al. 2005; Feberwee, Mekkes, Klinkenberg 2005; García et al. 2005; Feberwee et al. 2006; Gharaibeh and Hailat 2011; Pflaum et al. 2017). Similarly, older studies in wild birds sampled and detected M. gallisepticum in the trachea, for example, in House Sparrows, Passer domesticus (Jain et al. 1971) and Tree Sparrows, Passer montanus (Shimizu et al. 1979). After the emergence of M. gallisepticum in wild birds in North America, most studies continued to use choanal swabs (Farmer et al. 2002, 2005; Ganapathy et al. 2007) or a pooled sample of choanal and conjunctival swabs (e.g., Luttrell et al. 1996, 1998, 2001; Fischer et al. 1997; Staley et al. 2018; Tardy et al. 2019; Bonneaud et al. 2020). Few studies have sampled and analyzed conjunctival and choanal samples separately (e.g., Michiels et al. 2016). In our finch studies, we historically sampled the conjunctiva rather than the choana (Hartup and Kollias 1999; Hartup et al. 2000, 2001; Altizer et al. 2004; Dhondt et al. 2005, 2007, 2008, 2013, 2015, 2017; Sydenstricker et al. 2006; Hawley et al. 2010; Fleming-Davies et al. 2018). In the one study in which we took both choanal and conjunctival swabs from House Finches, M. gallisepticum was detected separately at both locations up to 20 wk after inoculation (Kollias et al. 2004) by PCR following the Lauerman (1998) protocol.

The lack of attention to a possible effect of the sampling location on the detection and bacterial load of M. gallisepticum is illustrated in a recent meta-analysis identifying wild bird species in which M. gallisepticum has been detected (Sawicka et al. 2020). The results compiled in that study were grouped using multiple criteria that did not, however, include the sampling location in the host.

It is evident that the clinical signs following a M. gallisepticum infection differ markedly between poultry and House Finches; the M. gallisepticum strain that emerged in the early 1990s in House Finches clearly forms a separate phylogenetic clade (HOFI_MG) from the POULTRY_MG clade causing disease in poultry (Ley et al. 1997; Hochachka et al. 2013); and gene expression in the same tissues using the same M. gallisepticum strain differed between poultry and House Finches (Pflaum et al. 2020). Additionally, we had previously recovered POULTRY_MG from wild asymptomatic finches (Hochachka et al. 2013), while Ferguson et al. (2003) had found HOFI_MG in turkeys. For these reasons, we decided to compare the bacterial load in choanal and conjunctival samples following experimental inoculation of House Finches with diverse POULTRY_MG and HOFI_MG strains.

Our experiments tested the following hypotheses. Hypothesis 1: M. gallisepticum changed tissue tropism following its evolutionary change to HOFI_MG. If that is correct, choanal swabs should have lower bacterial loads in birds inoculated with HOFI_MG than in birds inoculated with POULTRY_MG and higher bacterial loads in the conjunctiva of the birds inoculated with HOFI_MG. Hypothesis 2: M. gallisepticum expanded its within-host niche. This hypothesis predicts a similar bacterial load in the choana of birds infected with POULTRY_MG and HOFI_MG; the conjunctival load, however, should be higher in birds inoculated with HOFI_MG than in birds inoculated with POULTRY_MG.

Capture and housing of the birds

In February and March 2019, and again in the fall of 2020, juvenile House Finches were trapped in and around Ithaca, New York (42°46′N, 76°45′W). Trapping activities were conducted under New York State Fish and Wildlife license 39 (Albany, New York) and permit 22669 from the United States Geological Survey, Department of the Interior (Laurel, Maryland). To test if the birds had been previously exposed to M. gallisepticum, we used a blood sample taken from each bird's brachial vein in a rapid plate agglutination (RPA) assay to test for the presence of M. gallisepticum–specific antibodies as described (Sydenstricker et al. 2006). Birds negative for antibodies were transferred to aviaries in a large barn (Dhondt et al. 2012), housed individually in wire bar cages (45 × 45 × 75 cm), and consumed food and water ad libitum. The food consisted of two-thirds pelleted diet (Daily Maintenance Diet, Roudybush Inc., Woodland, California, USA) and one-third black sunflower seeds (Wild Birds Unlimited, Indianapolis, Indiana, USA). Ceramic heat lamps were used as needed to prevent the water dishes from freezing. The lights were on an automatic timer and provided the finches 12 h of daylight each day. The experimental activities were approved by Cornell University's Institutional Animal Care and Use Committee (protocol 2009-0034).

After a minimum of 1 wk of quarantine, birds were inspected for the presence of eye lesions and retested for antibodies and for the presence of M. gallisepticum DNA. Only birds that remained negative were included in the experiment.

Experiments

On 4 April 2019 (day 0), three groups of juvenile House Finches (n=6 or 7) were inoculated with 50 µL of inoculum in each eye. The strains used had been grown in the Geary Lab (University of Connecticut, Storrs, Connecticut, USA) and were used at the concentration provided. The inocula used are listed in Table 1. The F and the Rlow strain are standard laboratory strains; the NY2001 strain was obtained from an asymptomatic House Finch (Ithaca, New York, USA) in 2001 (see Hochachka et al. 2013; Ley et al. 2016) but phylogenetically belongs to the POULTRY_MG group.

Table 1

Identities and concentrations of poultry (POULTRY_MG) and House Finch, Haemorhous mexicanus (HOFI_MG) Mycoplasma gallisepticum isolates inoculated into the eyes of juvenile House Finches (Haemorhous mexicanus) from Ithaca, New York, USA, and number of birds inoculated in each group.

Identities and concentrations of poultry (POULTRY_MG) and House Finch, Haemorhous mexicanus (HOFI_MG) Mycoplasma gallisepticum isolates inoculated into the eyes of juvenile House Finches (Haemorhous mexicanus) from Ithaca, New York, USA, and number of birds inoculated in each group.
Identities and concentrations of poultry (POULTRY_MG) and House Finch, Haemorhous mexicanus (HOFI_MG) Mycoplasma gallisepticum isolates inoculated into the eyes of juvenile House Finches (Haemorhous mexicanus) from Ithaca, New York, USA, and number of birds inoculated in each group.

On 26 October 2020 (day 0), three groups were inoculated as in the previous experiment. The HOFI_MG strains used were CA2006 (n=5), CA2015 (n=4), and NC2006 (n= 5; Table 1). A negative control group (n=6) was sham inoculated with Frey medium provided by Edan Tulman (University of Connecticut, Storrs, Connecticut, USA). As all control birds remained negative throughout the experiment, we will not further report the results from these birds.

Sampling

To determine whether inoculation led to infection, we examined the House Finches for conjunctival lesions, recorded the eye score (Sydenstricker et al. 2005), measured bacterial load via swab samples from the conjunctival sac and choanal cleft, and assessed the presence of M. gallisepticum–specific antibodies from blood samples. Before inoculation, baseline data on antibodies (from blood samples) and mycoplasmal bacterial load (from conjunctival and choanal swabs) for all the birds in both experiments were collected. Following inoculation, eye lesions were recorded, and choanal and conjunctival swabs for quantitative PCR (qPCR) and blood samples for antibody testing were taken on days postinoculation (DPI), 5, 10, 15, 21, 28, in 2019 and on DPI, 4, 7, 10, 14, 21, 28, in 2020. Eye lesions were recorded on a scale from 0 (no lesions) to 3 (severe lesions) following Sydenstricker et al. (2006). The choanal and conjunctival samples were placed separately each in a tube with 300 µL of Frey medium and stored at –80 C. Blood samples for RPA assays were taken from the brachial vein and placed in microcapillary tubes. After centrifugation, the plasma was separated to be used in the antibody test (Kleven 2008). Upon termination of the study, the birds were humanely euthanized by means of carbon dioxide gas inhalation.

Laboratory procedures

To assess M. gallisepticum loads, DNA was extracted from the conjunctival and choanal swab samples using a DNeasy 96 Blood & Tissue Kit (Qiagen, Valencia, California, USA) following the manufacturer's recommended protocol. The total DNA was used in quantifying the mycoplasmal bacterial load by means of qPCR, as described by Grodio et al. (2008) and modified by Hawley et al. (2013). The target gene of interest was mgc2, and the number of mgc2 copies per sample reflects the bacterial load (Hawley et al. 2013). Given that both House Finch and poultry isolates of M. gallisepticum were used in this experiment, two different probes were used in qPCR reactions to assess bacterial load. Samples from birds in groups inoculated with a POULTRY_MG isolate or sham inoculated with Frey medium were tested with a qPCR using the poultry MG probe (IDEXX, Westbrook, Maine, USA) specific for poultry M. gallisepticum. All birds inoculated with a HOFI_MG isolate were tested using the House Finch–specific M. gallisepticum qPCR probe described by Grodio et al. (2008), as modified by Hawley et al. (2013) and Fleming-Davies et al. (2018).

Statistical analyses

Bacterial loads were log10 transformed for statistical analyses. To compare the results of the bacterial loads between the two experiments, we calculated the area under the curve (AUC) between DPI 4 and 28. Because in 2019 the first sampling day was DPI 5, we calculated the AUC between DPI 5 and 28, calculated the mean bacterial load value per day, and added that to the DPI 5-28 AUC value to have comparable values. For each individual, we summed the eye scores for both eyes recorded on DPI 4 (5), 10, 14 (15), 21, and 28 (the DPIs on which eye scores were done in the 2020 experiment only are in brackets). We used analysis of variance (ANOVA) to compare the bacterial loads and eye scores between groups of birds inoculated with different isolates. If the ANOVA yielded a statistically significant result, we performed a Tukey honestly significant difference (HSD) post hoc test to make all pairwise comparisons. To compare how choanal and conjunctival loads differed from one another, we performed a one-sample t-test. All statistical tests were performed using Statistix 10 (Analytical Software 2021).

Antibodies and eye lesions

All House Finches were successfully infected, as shown by all developing M. gallisepticum–specific antibodies. Antibodies were first detected in 14 birds inoculated with HOFI_MG by DPI 4, while in 18 of 19 birds inoculated with POULTRY_MG, antibodies were first detected only by DPI 10 (χ2=29.2; df=1; P<0.0001), indicating a significantly more rapid immunologic response to infection when exposed to HOFI_MG.

None of the birds inoculated with the POULTRY_MG isolates developed eye lesions, while all birds inoculated with HOFI_MG had eye lesions (χ2=33.0; df=1; P<0.0001). The severity of the eye lesions summed to DPI 28 was significantly lower in the birds inoculated with CA2006 (mean 11.80±SE 1.202; A) compared with CA2015 (mean 27.67±1.552; B), and NC2006 (mean 29.80±1.202; B), as shown by an ANOVA (F2,12=58.43; P<0.0001) and a Tukey HSD post hoc test showing two homogenous groups: CA2006 A; and CA2015 B and NC2006 B.

Choanal and conjunctival MG loads

Choanal M. gallisepticum loads did not differ significantly between isolates, as shown by an ANOVA (F5,32=1.05; P=0.40). Conjunctival MG-loads, however, did differ between the isolates (ANOVA: F5,31=55.98; P<0.0001; see Supplementary Material Table S1). The Tukey HSD post hoc test (at the α=0.05 level) indicated how the conjunctival bacterial loads differed between isolates: the three POULTRY_MG isolates formed one homogenous group with very low bacterial loads in the conjunctiva; and birds inoculated with HOFI_MG isolates had much higher bacterial loads. Among those, NC2006 demonstrated the highest load, and CA2006 had a significantly lower load. The CA2015 strain demonstrated statistically intermediate conjunctival loads (Table 2 and Fig. 1). When exploring the trajectories of the M. gallisepticum loads (Fig. 2), it can be seen that conjunctival loads of POULTRY_MG isolates remained very low throughout the experiment, while the loads of birds infected with HOFI_MG isolates remained high. The loads in birds infected with the low virulence CA2006 isolate decreased much more rapidly compared with those in birds infected with the more virulent CA2015 and NC2006 isolates. In contrast, the choanal loads of all isolates remain at a similar level throughout the experiment, although all declined by DPI 28.

Table 2

The AUC values for the choanal and conjunctival loads per isolate of Mycoplasma gallisepticum isolates inoculated into the eyes of juvenile House Finches (Haemorhous mexicanus) from Ithaca, New York, USA.a

The AUC values for the choanal and conjunctival loads per isolate of Mycoplasma gallisepticum isolates inoculated into the eyes of juvenile House Finches (Haemorhous mexicanus) from Ithaca, New York, USA.a
The AUC values for the choanal and conjunctival loads per isolate of Mycoplasma gallisepticum isolates inoculated into the eyes of juvenile House Finches (Haemorhous mexicanus) from Ithaca, New York, USA.a
Figure 1

Comparison of mean±SE area under the curve values of conjunctival (above) and choanal (below) swab samples between days postinoculation 4 and 28 of House Finches (Haemorhous mexicanus) inoculated with three poultry (NY2001, Rlow, and F) and three House Finch isolates (CA2005, CA2015, and NC2006) of Mycoplasma gallisepticum. Bacterial loads of conjunctival swabs differed between isolates (F5,31=55.98; P<0.0001), while the bacterial loads did not differ between choanal samples). The letters above the columns indicate groups that were significantly different by a Tukey honestly significant difference post hoc test at α=0.05.

Figure 1

Comparison of mean±SE area under the curve values of conjunctival (above) and choanal (below) swab samples between days postinoculation 4 and 28 of House Finches (Haemorhous mexicanus) inoculated with three poultry (NY2001, Rlow, and F) and three House Finch isolates (CA2005, CA2015, and NC2006) of Mycoplasma gallisepticum. Bacterial loads of conjunctival swabs differed between isolates (F5,31=55.98; P<0.0001), while the bacterial loads did not differ between choanal samples). The letters above the columns indicate groups that were significantly different by a Tukey honestly significant difference post hoc test at α=0.05.

Close modal
Figure 2

Comparison of mean±SE bacterial loads per days postinoculation (DPI) of conjunctival (above) and choanal (below) swab samples on different DPI of House Finches (Haemorhous mexicanus) inoculated with three poultry isolates (circles; NY2001, Rlow, and F) and three House Finch isolates (triangles; CA2006, CA2015, and NC2006) of Mycoplasma gallisepticum.

Figure 2

Comparison of mean±SE bacterial loads per days postinoculation (DPI) of conjunctival (above) and choanal (below) swab samples on different DPI of House Finches (Haemorhous mexicanus) inoculated with three poultry isolates (circles; NY2001, Rlow, and F) and three House Finch isolates (triangles; CA2006, CA2015, and NC2006) of Mycoplasma gallisepticum.

Close modal

The one-sample t-test showed that birds inoculated with the POULTRY_MG isolates had a much lower bacterial load in the conjunctiva than in the choana, while the opposite was true in birds inoculated with the virulent HOFI_MG strains (Table 2). In birds inoculated with CA2006, an isolate of low virulence, the bacterial loads did not differ between choanal and conjunctival swabs.

Our experiments clearly show that the conjunctival M. gallisepticum load in House Finches inoculated with a POULTRY_MG isolate was very low both compared with the choanal load in the same individual and compared with the conjunctival loads of birds inoculated with a HOFI_MG isolate. Choanal M. gallisepticum loads, on the other hand, did not differ between M. gallisepticum isolates, indicating that HOFI_MG has simply expanded its within-host niche to the eye as compared with its POULTRY_MG–like ancestors, in support of our hypothesis 2 and contrary to the predictions of hypothesis 1. Isolates of HOFI_MG cause a more rapid immunologic response in House Finches than POULTRY_MG and more severe lesions. Among the House Finch M. gallisepticum isolates, those that cause the most severe eye lesions also had the highest conjunctival load, as expected from Hawley et al. (2013), but no difference in the choanal load.

Motility is the capability of bacteria to reach and remain within host niches for nutrients or shelter from the host's defense mechanisms. Mycoplasma gallisepticum is known to have gliding motility, which appears to be essential for its parasitic lifestyle and for spreading in the respiratory tract once it has infected a host (Indikova et al. 2014). After the mycoplasmal conjunctivitis outbreak in wild birds, it was determined that strains of M. gallisepticum infecting House Finches form a separate phylogenetic clade (Hochachka et al. 2013). Tulman et al. (2012) reported a few coding changes of genes related to cytoadherence, such as hlp3, plpA, mgc2, gapA, crmA, and crmB-like. Other studies with different strains of M. gallisepticum have reported a high variation in cell adherence among HOFI_MG isolates (Perez et al. 2020), and compared with POULTRY_MG, HOFI_MG strains were shown to be qualitatively more adhesive and invasive than the poultry counterpart (Dowling et al. 2020), which seems to be related to coding changes, in particular, of three proteins MGC2, GapA and CrmA, essential not only for cytoadherence but also for gliding motility (Indikova et al. 2014). These reported observations align with hypothesis 2, because HOFI_MG isolates with a better adherence might be able to better spread within the host. Additionally, although Perez et al. (2020) showed a variation in adherence among HOFI_MG isolates, they did not find any sign of changes in glycan linkage specificity and binding avidity that are associated with changes in tissue tropism.

Sampling is a critical stage for diagnosis in live animals, especially for cases with low bacterial loads. This study has confirmed that when testing birds for infection with M. gallisepticum, it is important to sample and test the choana and the conjunctiva separately. Measuring bacterial loads in samples pooled from the two sites may be misleading.

We thank Adrianne Chissus for capturing the birds and helping with the experiments. We thank Avery August and Toshi Kawate, College of Veterinary Medicine at Cornell University, for use of their facilities. This work was supported by the Cornell University multistate grant 1020824, awarded through the NC1180 program Control of Emerging and Re-emerging Poultry Respiratory Diseases in the United States funded through the National Institute of Food and Agriculture – USDA.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-21-000187.

Analytical Software.
2021
.
Statistix 10.
Analytical Software
,
Tallahassee, Florida
.
Altizer
S,
Hochachka
WM,
Dhondt
AA.
2004
.
Seasonal dynamics of mycoplasmal conjunctivitis in eastern North American house finches.
J Anim Ecol
73
:
309
322
.
Bonneaud
C,
Tardy
L,
Hill
GE,
McGraw
KJ,
Wilson
AJ,
Giraudeau
M.
2020
.
Experimental evidence for stabilizing selection on virulence in a bacterial pathogen.
Evol Lett
4
:
491
501
.
Chin
RP,
Daft
BM,
Meteyer
CU,
Yamamoto
R.
1991
.
Meningoencephalitis in commercial meat turkeys associated with Mycoplasma gallisepticum.
Avian Dis
35
:
986
993
.
Dhondt
AA,
Altizer
S,
Cooch
EG,
Davis
AK,
Dobson
A,
Driscoll
MJL,
Hartup
BK,
Hawley
DM,
Hochachka
WM,
Hosseini
PR.
2005
.
Dynamics of a novel pathogen in an avian host: Mycoplasmal conjunctivitis in house finches.
Acta Trop
94
:
77
93
.
Dhondt
AA,
Dhondt
KV,
Hawley
DM,
Jennelle
CS.
2007
.
Experimental evidence for transmission of Mycoplasma gallisepticum in house finches by fomites.
Avian Pathol
36
:
205
208
.
Dhondt
AA,
Dhondt
KV,
Hochachka
WM.
2015
.
Response of black-capped chickadees to house finch Mycoplasma gallisepticum.
PLoS One
10
:
e0124820
.
Dhondt
AA,
Dhondt
KV,
Hochachka
WM,
Ley
DH,
Hawley
DM.
2017
.
Response of house finches recovered from Mycoplasma gallisepticum to reinfection with a heterologous strain.
Avian Dis
61
:
437
441
.
Dhondt
AA,
Dhondt
KV,
Hochachka
WM,
Schat
KA.
2013
.
Can American goldfinches function as reservoirs for Mycoplasma gallisepticum?
J Wildl Dis
49
:
49
54
.
Dhondt
AA,
Dhondt
KV,
McCleery
BV.
2008
.
Comparative infectiousness of three passerine bird species after experimental inoculation with Mycoplasma gallisepticum.
Avian Pathol
37
:
635
640
.
Dhondt
AA,
States
SL,
Dhondt
KV,
Schat
KA.
2012
.
Understanding the origin of seasonal epidemics of mycoplasmal conjunctivitis.
J Anim Ecol
81
:
996
1003
.
Dhondt
AA,
Tessaglia
DL,
Slothower
RL.
1998
.
Epidemic mycoplasmal conjunctivitis in house finches from eastern North America.
J Wildl Dis
34
:
265
280
.
Doster
G.
1994
.
Eye infections in house finches.
Southeast Coop Wildl Dis Study Newsl
10
:
1
2
.
Dowling
AJ,
Hill
GE,
Bonneaud
C.
2020
.
Multiple differences in pathogen-host cell interactions following a bacterial host shift.
Sci Rep
10
:
6779
.
Farmer
KL,
Hill
GE,
Roberts
SR.
2002
.
Susceptibility of a naïve population of house finches to Mycoplasma gallisepticum.
J Wildl Dis
38
:
282
286
.
Farmer
KL,
Hill
GE,
Roberts
SR.
2005
.
Susceptibility of wild songbirds to the house finch strain of Mycoplasma gallisepticum.
J Wildl Dis
41
:
317
325
.
Feberwee
A,
Landman
WJ,
von Banniseht-Wysmuller
T,
Klinkenberg
D,
Vernooij
JC,
Gielkens
AL,
Stegeman
JA.
2006
.
The effect of a live vaccine on the horizontal transmission of Mycoplasma gallisepticum.
Avian Pathol
35
:
359
366
.
Feberwee
A,
Mekkes
DR,
de Wit
JJ,
Hartman
EG,
Pijpers
A.
2005
.
Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections.
Avian Dis
49
:
260
268
.
Feberwee
A,
Mekkes
DR,
Klinkenberg
D,
Vernooij
JC,
Gielkens
AL,
Stegeman
JA.
2005
.
An experimental model to quantify horizontal transmission of Mycoplasma gallisepticum.
Avian Pathol
34
:
355
361
.
Ferguson
NM.
2013
.
Mycoplasmosis.
In:
Diseases of poultry
, 13th Ed.,
Swayne
DE,
Glisson
JR,
McDougald
LR,
Nolan
LK,
Suarez
DL,
Nair
VL,
editors.
Wiley-Blackwell
,
Ames, Iowa
, p.
877
.
Ferguson
NM,
Hermes
D,
Leiting
VA,
Kleven
SH.
2003
.
Characterization of a naturally occurring infection of a Mycoplasma gallisepticum house finch-like strain in turkey breeders.
Avian Dis
47
:
523
530
.
Fischer
JR,
Stallknecht
DE,
Luttrell
P,
Dhondt
AA,
Converse
KA.
1997
.
Mycoplasmal conjunctivitis in wild songbirds: The spread of a new contagious disease in a mobile host population.
J Emerg Infect Dis
3
:
69
72
.
Fleming-Davies
AE,
Williams
PD,
Dhondt
AA,
Dobson
AP,
Hochachka
WM,
Leon
AE,
Ley
DH,
Osnas
EE,
Hawley
DM.
2018
.
Incomplete host immunity favors the evolution of virulence in an emergent pathogen.
Science
359
:
1030
1033
.
Ganapathy
K,
Saleha
AA,
Jaganathan
M,
Tan
CG,
Chong
CT,
Tang
SC,
Ideris
A,
Dare
CM,
Bradbury
JM.
2007
.
Survey of Campylobacter, Salmonella and mycoplasmas in house crows (Corvus splendens) in Malaysia.
Vet Rec
160
:
622
624
.
García
M,
Ikuta
N,
Levisohn
S,
Kleven
S.
2005
.
Evaluation and comparison of various PCR methods for detection of Mycoplasma gallisepticum infection in chickens.
Avian Dis
49
:
125
132
.
Gharaibeh
S,
Hailat
A.
2011
.
Mycoplasma gallisepticum experimental infection and tissue distribution in chickens, sparrows and pigeons.
Avian Pathol
40
:
349
354
.
Grodio
JL,
Dhondt
KV,
O'Connell
PH,
Schat
KA.
2008
.
Detection and quantification of Mycoplasma gallisepticum genome load in conjunctival samples of experimentally infected house finches (Carpodacus mexicanus) using real-time polymerase chain reaction.
Avian Pathol
37
:
385
391
.
Hartup
BK,
Bickal
JM,
Dhondt
AA,
Ley
DH,
Kollias
GV.
2001
.
Dynamics of conjunctivitis and Mycoplasma gallisepticum infections in house finches.
Auk
118
:
327
333
.
Hartup
BK,
Kollias
GV.
1999
.
Field investigation of Mycoplasma gallisepticum infections in house finch (Carpodacus mexicanus) eggs and nestlings.
Avian Dis
43
:
572
576
.
Hartup
BK,
Kollias
GV,
Ley
DH.
2000
.
Mycoplasmal conjunctivitis in songbirds from New York.
J Wildl Dis
36
:
257
264
.
Hawley
DM,
Dhondt
KV,
Dobson
AP,
Grodio
JL,
Hochachka
WM,
Ley
DH,
Osnas
EE,
Schat
K,
Dhondt
AA.
2010
.
Common garden experiment reveals pathogen isolate but no host genetic diversity effect on the dynamics of an emerging wildlife disease.
J Evol Biol
23
:
1680
1688
.
Hawley
DM,
Osnas
EE,
Dobson
AP,
Hochachka
WM,
Ley
DH,
Dhondt
AA.
2013
.
Parallel patterns of increased virulence in a recently emerged wildlife pathogen.
PLoS Biol
11
:
e1001570
.
Hochachka
WM,
Dhondt
AA,
Dobson
A,
Hawley
DM,
Ley
DH,
Lovette
IJ.
2013
.
Multiple host transfers, but only one successful lineage in a continent-spanning emergent pathogen.
Proc Biol Sci
280
:
20131068
.
Indikova
I,
Vronka
M,
Szostak
MP.
2014
.
First identification of proteins involved in motility of Mycoplasma gallisepticum.
Vet Res
45
:
1
14
.
Jain
NC,
Chandiramani
NK,
Singh
IP.
1971
.
Studies on avian pleuro-pneumonia-like organisms. 2. Occurrence of Mycoplasma in wild birds.
Indian J Anim Sci
41
:
301
305
.
Kleven
SH.
2008
.
Mycoplasmosis.
In:
A laboratory manual for the isolation, identification and characterization of avian pathogens
, 5th Ed.,
Dufour-Zavala
L,
editor.
American Association of Avian Pathologists
,
Athens, Georgia
, pp.
59
64
.
Kollias
GV,
Sydenstricker
KV,
Kollias
HW,
Ley
DH,
Hosseini
PR,
Connolly
V,
Dhondt
AA.
2004
.
Experimental infection of house finches with Mycoplasma gallisepticum.
J Wildl Dis
40
:
79
86
.
Lauerman
LH.
1998
.
Nucleic acid amplification assays for diagnosis of animal diseases.
American Association of Veterinary Laboratory Diagnosticians
,
Madison, Wisconsin
,
152
pp.
Levisohn
S,
Kleven
S.
2000
.
Avian mycoplasmosis (Mycoplasma gallisepticum).
Rev Sci Tech
19
:
425
442
.
Ley
DH,
Berkhoff
JE,
Levisohn
S.
1997
.
Molecular epidemiologic investigations of Mycoplasma gallisepticum conjunctivitis in songbirds by random amplified polymorphic DNA analyses.
Emerg Infect Dis
3
:
375
380
.
Ley
DH,
Berkhoff
JE,
McLaren
JM.
1996
.
Mycoplasma gallisepticum isolated from house finches (Carpodacus mexicanus) with conjunctivitis.
Avian Dis
40
:
480
483
.
Ley
DH,
Hawley
DM,
Geary
SJ,
Dhondt
AA.
2016
.
House finch (Haemorhous mexicanus) conjunctivitis, and Mycoplasma spp. isolated from North American wild birds, 1994–2015.
J Wildl Dis
52
:
669
673
.
Ley
DH,
Sheaffer
DS,
Dhondt
AA.
2006
.
Further western spread of Mycoplasma gallisepticum infection of house finches.
J Wildl Dis
42
:
429
431
.
Luttrell
MP,
Fischer
JR,
Stallknecht
DE,
Kleven
SH.
1996
.
Field investigation of Mycoplasma gallisepticum infections in house finches (Carpodacus mexicanus) from Maryland and Georgia.
Avian Dis
40
:
335
341
.
Luttrell
MP,
Stallknecht
DE,
Fischer
JE,
Sewell
CT,
Kleven
SH.
1998
.
Natural Mycoplasma gallisepticum infection in a captive flock of house finches.
J Wildl Dis
34
:
289
296
.
Luttrell
MP,
Stallknecht
DE,
Kleven
SH,
Kavanaugh
DM,
Corn
JL,
Fischer
JR.
2001
.
Mycoplasma gallisepticum in house finches (Carpodacus mexicanus) and other wild birds associated with poultry production facilities.
Avian Dis
45
:
321
329
.
Macowan
KJ,
Randall
CJ,
Brand
TF.
1983
.
Cloacal infection with Mycoplasma gallisepticum and the effect of inoculation with H120 infectious bronchitis vaccine virus.
Avian Pathol
12
:
497
503
.
Michiels
T,
Welby
S,
Vanrobaeys
M,
Quinet
C,
Rouffaer
L,
Lens
L,
Martel
A,
Butaye
P.
2016
.
Prevalence of Mycoplasma gallisepticum and Mycoplasma synoviae in commercial poultry, racing pigeons and wild birds in Belgium.
Avian Pathol
45
:
244
252
.
Nunoya
T,
Yagihashi
T,
Tajima
M,
Nagasawa
Y.
1995
.
Occurrence of keratoconjunctivitis apparently caused by Mycoplasma gallisepticum in layer chickens.
Vet Pathol
32
:
11
18
.
Perez
K,
Mullen
N,
Canter
JA,
Ley
DH,
May
M.
2020
.
Phenotypic diversity in an emerging mycoplasmal disease.
Microb Pathog
138
:
103798
.
Pflaum
K,
Tulman
ER,
Beaudet
J,
Liao
X,
Dhondt
KV,
Dhondt
AA,
Hawley
DM,
Ley
DH,
Kerr
KM,
Geary
SJ.
2017
.
Attenuated phenotype of a recent house finch-associated Mycoplasma gallisepticum isolate in domestic poultry.
Infect Immun
85
:
e00185
17
.
Pflaum
K,
Tulman
ER,
Canter
J,
Dhondt
KV,
Reinoso-Perez
MT,
Dhondt
AA,
Geary
SJ.
2020
.
The influence of host tissue on M. gallisepticum vlhA gene expression.
Vet Microbiol
251
:
108891
.
Power
J,
Jordan
FT.
1976
.
Unilateral enlargement of the eye in chicks infected with a strain of M. gallisepticum.
Vet Rec
99
:
102
103
.
Raviv
Z,
Ley
DH.
2013
.
Mycoplasma gallisepticum infection.
In:
Diseases of poultry
, 13th Ed.,
Swayne
DE,
Glisson
JR,
McDougald
LR,
Nolan
LK,
Suarez
DL,
Nair
VL,
editors.
Wiley-Blackwell
,
Ames, Iowa
, pp.
877
893
.
Sawicka
A,
Durkalec
M,
Tomczyk
G,
Kursa
O.
2020
.
Occurrence of Mycoplasma gallisepticum in wild birds: A systematic review and meta-analysis.
PLoS One
15
:
e0231545
.
Shimizu
T,
Numano
K,
Uchida
K.
1979
.
Isolation and identification of mycoplasmas from various birds: An ecological study.
J Vet Med Sci
41
:
273
282
.
Staley
M,
Bonneaud
C,
McGraw
KJ,
Vleck
CM,
Hill
GE.
2018
.
Detection of Mycoplasma gallisepticum in house finches (Haemorhous mexicanus) from Arizona.
Avian Dis
62
:
14
17
.
Sydenstricker
KV,
Dhondt
AA,
Hawley
DM,
Jennelle
CS,
Kollias
HW,
Kollias
GV.
2006
.
Characterization of experimental Mycoplasma gallisepticum infection in captive house finch flocks.
Avian Dis
50
:
39
44
.
Sydenstricker
KV,
Dhondt
AA,
Ley
DH,
Kollias
GV.
2005
.
Re-exposure of captive house finches that recovered from Mycoplasma gallisepticum infection.
J Wildl Dis
41
:
326
333
.
Tardy
L,
Giraudeau
M,
Hill
GE,
McGraw
KJ,
Bonneaud
C.
2019
.
Contrasting evolution of virulence and replication rate in an emerging bacterial pathogen.
Proc Natl Acad Sci U S A
116
:
16927
16932
.
Thomas
L,
Davidson
M,
McCluskey
RT.
1966
.
Studies of PPLO infection. I. The production of cerebral polyarteritis by Mycoplasma gallisepticum in turkeys; the neurotoxic property of the Mycoplasma.
J Exp Med
123
:
897
912
.
Tulman
ER,
Liao
X,
Szczepanek
SM,
Ley
DH,
Kutish
GF,
Geary
SJ.
2012
.
Extensive variation in surface lipoprotein gene content and genomic changes associated with virulence during evolution of a novel North American house finch epizootic strain of Mycoplasma gallisepticum.
Microbiology
158
:
2073
2088
.

Supplementary data