House Finches Research Articles

The house finch ocular microbiome is altered by infection, but does not predict disease transmission

Recent studies have highlighted the importance of host-associated microbial communities in inhibiting growth or colonization of pathogenic species and mitigating disease severity. Much research has been conducted on how the microbiome of individuals may impact their disease susceptibility; however, less attention has been given to the role of microbiomes in population-level disease processes. In this study, we examined the effects of the ocular microbiome on pathogen load, disease severity, and transmission success of the conjunctival bacterial pathogen Mycoplasma gallisepticum (MG) in house finches (Haemorhous mexicanus). Fifteen male house finches were exposed to MG and housed pairwise with uninfected females for 32 days. Swabs for ocular microbiome characterization via 16 S rRNA gene amplicon sequencing were collected from male and female finches before and nine days after MG inoculation. MG load and disease severity were quantified throughout the experiment for all individuals. We examined whether ocular microbiome composition prior to MG infection was associated with pathogen load, disease severity, or transmission efficacy. Although the presence of certain taxa in the ocular microbiome is associated with disease protection in this species, we did not find any evidence that ocular microbiome composition was associated with any metric used to quantify disease transmission. MG infection did alter the resident bacterial communities, which aligns with results from previous studies. However, this was only true for the indicator birds that were directly inoculated with MG. Our results suggest the resident ocular microbiome of house finches shifts in responses to direct contact with pathogens, but may be more resilient, at least at the timescales examined, when host exposure is via an infected conspecific. This suggests that outcomes of interactions between pathogens and resident microbial communities may depend on the mode or dose of pathogen exposure.

Links between Innate and Adaptive Immunity Can Favor Evolutionary Persistence of Immunopathology.

Immunopathology, or the harm caused to an organism's own tissues during the activation of its immune system, carries substantial costs. Moreover, avoiding this self-harm may be an important mechanism underlying tolerance of infection, helping to reducing fitness costs without necessarily clearing parasites. Despite the apparent benefits of minimizing immunopathology, such damage persists across a range of host species. Prior work has explored a trade-off with resistance during a single infection as a potential driver of this persistence, with some collateral damage being unavoidable when killing parasites. Here, we present an additional trade-off that could favor the continued presence of immunopathology: robust immune responses during initial infection (e.g., innate immunity in vertebrates) can induce stronger memory (adaptive immunity), offering protection from future infections. We explore this possibility in an adaptive dynamics framework, using theoretical models parameterized from an ecologically relevant host-parasite system, house finches (Haemorhous mexicanus) infected with the bacterial pathogen, Mycoplasma gallisepticum. We find that some degree of immunopathology is often favored when immunopathology during first infection either reduces susceptibility to or enhances recovery from second infection. Further, interactions among factors like transmission rate, recovery rate, background mortality, and pathogen virulence also shape these evolutionary dynamics. Most notably, the evolutionary stability of investment in immunopathology is highly dependent upon the mechanism by which hosts achieve secondary protection (susceptibility vs. recovery), with the potential for abrupt evolutionary shifts between high and low investment under certain conditions. These results highlight the potential for immune memory to play an important role in the evolutionary persistence of immunopathology and the need for future empirical research to reveal the links between immunopathology during initial infections and longer-term immune protection.

Correlates of Co-Infection with Coccidiosis and Avian Malaria in House Finches (Haemorhous mexicanus).

Pathogens have traditionally been studied in isolation within host systems; yet in natural settings they frequently coexist. This raises questions about the dynamics of co-infections and how host life-history traits might predict co-infection versus single infection. To address these questions, we investigated the presence of two parasites, a gut parasite (Isospora coccidians) and a blood parasite (Plasmodium spp.), in House Finches (Haemorhous mexicanus), a common passerine bird in North America. We then correlated these parasitic infections with various health and condition metrics, including hematological parameters, plasma carotenoids, lipid-soluble vitamins, blood glucose concentration, body condition, and prior disease history. Our study, based on 48 birds captured in Tempe, Arizona, US, in October 2021, revealed that co-infected birds exhibited elevated circulating lutein levels and a higher heterophil:lymphocyte ratio (H/L ratio) compared to those solely infected with coccidia Isospora spp. This suggests that co-infected birds experience heightened stress and may use lutein to bolster immunity against both pathogens, and that there are potentially toxic effects of lutein in co-infected birds compared to those infected solely with coccidia Isospora sp. Our findings underscore the synergistic impact of coparasitism, emphasizing the need for more co-infection studies to enhance our understanding of disease dynamics in nature, as well as its implications for wildlife health and conservation efforts.

Urban house finches are more resistant to the effects of artificial light at night

Rapid urbanization of habitats alters the physical, chemical, auditory, and photic environments of human and wild animal inhabitants. One of the most widespread transformations is caused by artificial light at night (ALAN), but it is not clear the extent to which individuals acclimate to such rapid environmental change. Here, we tested the hypothesis that urban birds show increased resistance to harmful behavioral, parasitological, and physiological effects of ALAN. We captured house finches (Haemorhous mexicanus), a bird that commonly inhabits cities and their natural surroundings, from two urban and two rural sites in Phoenix, Arizona, USA, which differ by both degree of urbanization and by multiple orders of magnitude in ALAN intensity, and placed them in a common garden laboratory setting. We exposed half of the birds from each habitat type to ecologically relevant levels of night lighting during the subjective night and found that, while ALAN exposure reduced sleep in both urban and rural birds, ALAN-exposed urban birds were able to sleep longer than ALAN-exposed rural birds. We also found that ALAN exposure increased the proliferation rate of an intestinal coccidian parasite (Isospora spp.) in both urban and rural birds, but that the rate of proliferation was lower in urban relative to rural birds. We found that night lighting suppressed titers of feather corticosterone in rural but not urban birds, suggesting that light impairs HPA function through chronic stress or suppression of its circadian rhythmicity, and that urban birds were again resistant to this effect. Mediation analyses show that the effect of ALAN exposure in rural birds was significantly sleep-mediated for feather corticosterone but not coccidiosis, suggesting a diversity of mechanisms by which ALAN alters physiology. We contribute further evidence that animals from night-lit habitats can develop resistance to ALAN and its detrimental effects.

Slow and steady: auditory features for discriminating animal vocalizations.

We propose that listeners can use temporal regularities - spectro-temporal correlations that change smoothly over time - to discriminate animal vocalizations within and between species. To test this idea, we used Slow Feature Analysis (SFA) to find the most temporally regular components of vocalizations from birds (blue jay, house finch, American yellow warbler, and great blue heron), humans (English speakers), and rhesus macaques. We projected vocalizations into the learned feature space and tested intra-class (same speaker/species) and inter-class (different speakers/species) auditory discrimination by a trained classifier. We found that: 1) Vocalization discrimination was excellent (> 95%) in all cases; 2) Performance depended primarily on the ~10 most temporally regular features; 3) Most vocalizations are dominated by ~10 features with high temporal regularity; and 4) These regular features are highly correlated with the most predictable components of animal sounds.

House finch leg color changes with infection

In numerous animals, dramatic coloration (e.g. bright red or yellow) often indicates potential fitness, but it is less clear whether subtle coloration in the same individuals (e.g. grey or brown) could also encode such signals. To determine if subtle coloration could indicate health in a bird species with colorful feathers, house finches (Haemorhous mexicanus), we used spectrometry to objectively quantify leg brightness and saturation before, six days after, and 13 days after captive individuals were inoculated with a naturally‐occurring bacterial pathogen, Mycoplasma gallisepticum. We found that legs became less bright six days after infection (near the typical peak of infection), then more bright and less saturated at 13 days (entering the typical recovery period). These results suggest that subtle colors could reliably indicate current infection status, and therefore survival potential.

ECR Spotlight – Rebecca Koch

ECR Spotlight is a series of interviews with early-career authors from a selection of papers published in Journal of Experimental Biology and aims to promote not only the diversity of early-career researchers (ECRs) working in experimental biology but also the huge variety of animals and physiological systems that are essential for the ‘comparative’ approach. Rebecca Koch is an author on ‘ Captivity affects mitochondrial aerobic respiration and carotenoid metabolism in the house finch (Haemorhous mexicanus)’, published in JEB. Rebecca is a postdoctoral researcher in the lab of Matthew Toomey, Department of Biological Sciences, University of Tulsa, investigating how birds communicate information during courtship, particularly through the use of coloration.

Captivity affects mitochondrial aerobic respiration and carotenoid metabolism in the house finch (Haemorhous mexicanus).

In many species of animals, red carotenoid-based coloration is produced by metabolizing yellow dietary pigments, and this red ornamentation can be an honest signal of individual quality. However, the physiological basis for associations between organism function and the metabolism of red ornamental carotenoids from yellow dietary carotenoids remains uncertain. A recent hypothesis posits that carotenoid metabolism depends on mitochondrial performance, with diminished red coloration resulting from altered mitochondrial aerobic respiration. To test for an association between mitochondrial respiration and red carotenoids, we held wild-caught, molting male house finches in either small bird cages or large flight cages to create environmental challenges during the period when red ornamental coloration is produced. We predicted that small cages would present a less favorable environment than large flight cages and that captivity itself would decrease both mitochondrial performance and the abundance of red carotenoids compared with free-living birds. We found that captive-held birds circulated fewer red carotenoids, showed increased mitochondrial respiratory rates, and had lower complex II respiratory control ratios - a metric associated with mitochondrial efficiency - compared with free-living birds, though we did not detect a difference in the effects of small cages versus large cages. Among captive individuals, the birds that circulated the highest concentrations of red carotenoids had the highest mitochondrial respiratory control ratio for complex II substrate. These data support the hypothesis that the metabolism of red carotenoid pigments is linked to mitochondrial aerobic respiration in the house finch, but the mechanisms for this association remain to be established.

Urbanization alters the song propagation of two human-commensal songbird species.

Urban expansion has increased pollution, including both physical (e.g., exhaust, litter) and sensory (e.g., anthropogenic noise) components. Urban avian species tend to increase the frequency and/or amplitude of songs to reduce masking by low-frequency noise. Nevertheless, song propagation to the receiver can also be constrained by the environment. We know relatively little about how this propagation may be altered across species that (1) vary in song complexity and (2) inhabit areas along an urbanization gradient. We investigated differences in song amplitude, attenuation, and active space, or the maximum distance a receiver can detect a signal, in two human-commensal species: the house sparrow (Passer domesticus) and house finch (Haemorhous mexicanus). We described urbanization both discretely and quantitatively to investigate the habitat characteristics most responsible for propagation changes. We found mixed support for our hypothesis of urban-specific degradation of songs. Urban songs propagated with higher amplitude; however, urban song fidelity was species-specific and showed lowered active space for urban house finch songs. Taken together, our results suggest that urban environments may constrain the propagation of vocal signals in species-specific manners. Ultimately, this has implications for the ability of urban birds to communicate with potential mates or kin.

Using point-of-care devices to examine covariation among blood nutritional-physiological parameters and their relationships with poxvirus infection, habitat urbanization, and male plumage coloration in house finches (Haemorhous mexicanus).

The development of inexpensive and portable point-of-care devices for measuring nutritional physiological parameters from blood (e.g., glucose, ketones) has accelerated our understanding and assessment of real-time variation in human health, but these have infrequently been tested or implemented in wild animals, especially in relation to other key biological or fitness-related traits. Here we used point-of-care devices to measure blood levels of glucose, ketones, uric acid, and triglycerides in free-ranging house finches (Haemorhous mexicanus)-a common songbird in North America that has been well-studied in the context of urbanization, nutrition, health, and sexual selection-during winter and examined (1) repeatability of these methods for evaluating blood levels in these wild passerines, (2) intercorrelations among these measurements within individuals, (3) how blood nutritional-physiology metrics related to a bird's body condition, habitat of origin (urban vs. suburban), poxvirus infection, and sex; and (4) if the expression of male sexually selected plumage coloration was linked to any of the nutritional-physiological metrics. All blood-nutritional parameters were repeatable. Also, there was significant positive covariation between concentrations of circulating triglycerides and glucose and triglycerides and uric acid. Urban finches had higher blood glucose concentrations than suburban finches, and pox-infected individuals had lower blood triglyceride concentrations than uninfected ones. Last, redder males had higher blood glucose, but lower uric acid levels. These results demonstrate that point-of-care devices can be useful, inexpensive ways of measuring real-time variation in the nutritional physiology of wild birds.

Varying conjunctival immune response adaptations of house finch populations to a rapidly evolving bacterial pathogen.

Pathogen adaptations during host-pathogen co-evolution can cause the host balance between immunity and immunopathology to rapidly shift. However, little is known in natural disease systems about the immunological pathways optimised through the trade-off between immunity and self-damage. The evolutionary interaction between the conjunctival bacterial infection Mycoplasma gallisepticum (MG) and its avian host, the house finch (Haemorhous mexicanus), can provide insights into such adaptations in immune regulation. Here we use experimental infections to reveal immune variation in conjunctival tissue for house finches captured from four distinct populations differing in the length of their co-evolutionary histories with MG and their disease tolerance (defined as disease severity per pathogen load) in controlled infection studies. To differentiate contributions of host versus pathogen evolution, we compared house finch responses to one of two MG isolates: the original VA1994 isolate and a more evolutionarily derived one, VA2013. To identify differential gene expression involved in initiation of the immune response to MG, we performed 3'-end transcriptomic sequencing (QuantSeq) of samples from the infection site, conjunctiva, collected 3-days post-infection. In response to MG, we observed an increase in general pro-inflammatory signalling, as well as T-cell activation and IL17 pathway differentiation, associated with a decrease in the IL12/IL23 pathway signalling. The immune response was stronger in response to the evolutionarily derived MG isolate compared to the original one, consistent with known increases in MG virulence over time. The host populations differed namely in pre-activation immune gene expression, suggesting population-specific adaptations. Compared to other populations, finches from Virginia, which have the longest co-evolutionary history with MG, showed significantly higher expression of anti-inflammatory genes and Th1 mediators. This may explain the evolution of disease tolerance to MG infection in VA birds. We also show a potential modulating role of BCL10, a positive B- and T-cell regulator activating the NFKB signalling. Our results illuminate potential mechanisms of house finch adaptation to MG-induced immunopathology, contributing to understanding of the host evolutionary responses to pathogen-driven shifts in immunity-immunopathology trade-offs.

Characterizing living ocular bacterial communities and the effects of antibiotic perturbation in house finches

AbstractDNA‐based methods to measure the abundance and relative abundance of bacterial taxa can be skewed by the presence of dead or transient bacteria. Consequently, the active, functional members of the community may be a small subset of the detected bacterial community. This mismatch can make inferences about the roles of communities in host health difficult and can be particularly problematic for low‐abundance microbiomes, such as those on conjunctival surfaces. In this study, we manipulated bacterial communities on bird conjunctiva with a bacteriostatic antibiotic, reducing bacterial activity while preserving viability, to identify the living and active conjunctival communities using comparisons of 16S ribosomal DNA and RNA in paired samples. DNA amplicons included many more sequence variants than RNA amplicons from the same communities, with consequent differences in diversity. While we found that changes in communities in DNA samples broadly represent shifts in the living (RNA‐amplicon) communities, assessments of community function may be better described by RNA samples, reducing background noise from dead cells. We further used these data to test RNA:DNA ratios, used in other microbiological contexts, to detect shifts in bacterial activity after antibiotic disruption but were unable to detect changes in bacterial activity with this method.

Male pathology regardless of behaviour drives transmission in an avian host-pathogen system.

Host sex is an important source of heterogeneity in the severity of epidemics. Pinpointing the mechanisms causing this heterogeneity can be difficult because differences in behaviour among sexes (e.g. greater territorial aggression in males) can bias exposure risk, obfuscating the role of immune function, which can lead to differences in pathology, in driving differential susceptibility between sexes. Thus, sex-biased transmission driven by differences in immune function independent of behaviour is poorly understood, especially in non-mammalian systems. Here we examine the previously unexplored potential for male-biased pathology to affect transmission using an avian host-pathogen system. We employ a sex-dependent multistate transmission model parameterized with isolated, individual-based experimental exposures of domestic canaries and experimental transmission data of house finches. The experiment revealed that male birds have shorter incubation periods, longer recovery periods, higher pathogen burdens and greater disease pathology than females. Our model revealed that male-biased pathology led to epidemic size rapidly increasing with the proportion of male birds, with a nearly 10-fold increase in total epidemic size from an all-female to an all-male simulation. Our results demonstrate that female-biased resistance, independent of male behaviour, can drive sex-dependent transmission in wildlife, indicating that sex-based differences in immune function, not just differences in exposure risk, can shape epidemic dynamics.

Let's stick together: Infection enhances preferences for social grouping in a songbird species.

Acute infections can alter foraging and movement behaviors relevant to sociality and pathogen spread. However, few studies have directly examined how acute infections caused by directly transmitted pathogens influence host social preferences. While infected hosts often express sickness behaviors (e.g., lethargy) that can reduce social associations with conspecifics, enhanced sociality during infection might be favored in some systems if social grouping improves host survival of infection. Directly assaying social preferences of infected hosts is needed to elucidate potential changes in social preferences that may act as a form of behavioral tolerance (defined as using behavior to minimize fitness costs of infection). We tested how infection alters sociality in juvenile house finches (Haemorhous mexicanus), which are both highly gregarious and particularly susceptible to infection by the bacterial pathogen Mycoplasma gallisepticum (MG). We inoculated 33 wild-caught but captive-held juvenile house finches with MG or media (sham control). At peak infection, birds were given a choice assay to assess preference for associating near a flock versus an empty cage. We then repeated this assay after all birds had recovered from infection. Infected birds were significantly more likely than controls to spend time associating with, and specifically foraging near, the flock. However, after infected birds had recovered from MG infection, there were no significant differences in the amount of time birds in each treatment spent with the flock. These results indicate augmented social preferences during active infection, potentially as a form of behavioral tolerance. Notably, infected birds showed strong social preferences regardless of variation in disease severity or pathogen loads, with 14/19 harboring high loads (5-6 log10 copies of MG) at the time of the assay. Overall, our results show that infection with a directly transmitted pathogen can augment social preferences, with important implications for MG spread in natural populations.

Rapid adaptation to a novel pathogen through disease tolerance in a wild songbird.

Animal hosts can adapt to emerging infectious disease through both disease resistance, which decreases pathogen numbers, and disease tolerance, which limits damage during infection without limiting pathogen replication. Both resistance and tolerance mechanisms can drive pathogen transmission dynamics. However, it is not well understood how quickly host tolerance evolves in response to novel pathogens or what physiological mechanisms underlie this defense. Using natural populations of house finches (Haemorhous mexicanus) across the temporal invasion gradient of a recently emerged bacterial pathogen (Mycoplasma gallisepticum), we find rapid evolution of tolerance (<25 years). In particular, populations with a longer history of MG endemism have less pathology but similar pathogen loads compared with populations with a shorter history of MG endemism. Further, gene expression data reveal that more-targeted immune responses early in infection are associated with tolerance. These results suggest an important role for tolerance in host adaptation to emerging infectious diseases, a phenomenon with broad implications for pathogen spread and evolution.

EFFECTS OF MYCOPLASMA GALLISEPTICUM INFECTION ON PREENING BEHAVIORS AND FEATHER QUALITY IN HOUSE FINCHES (HAEMORHOUS MEXICANUS).

Infections can have far-reaching sublethal effects on wildlife, including reduced maintenance of external structures. For many wildlife taxa, daily maintenance of external structures (termed preening in birds) is critical to fitness, but few studies have examined how infections alter such maintenance. Mycoplasma gallisepticum is a common pathogen in free-living House Finches (Haemorhous mexicanus), where it causes mycoplasmal conjunctivitis. Despite documented behavioral changes associated with M. gallisepticum infections in finches, no studies have examined how preening behavior may change with infection and how potential differences in preening may affect feather quality. To test this, we experimentally inoculated captive House Finches with M. gallisepticum or a control treatment, and we collected behavioral and feather quality data to detect potential changes in feather maintenance due to infection. We found that finches infected with M. gallisepticum preened significantly less often, and within the infected treatment, birds with the highest conjunctivitis severity preened the least often. However, there was no difference in the quality scores for secondary flight feathers collected from control versus infected birds. We also assayed feather water retention and found that the degree of water retention correlated with our feather quality scores, such that feathers with poor scores retained more water. However, as with quality scores, feather water retention did not differ with infection; this may be due to the controlled environment that the birds experienced while in captivity. Our data suggest that, in addition to sickness behaviors previously observed in finches, M. gallisepticum infection decreases other behaviors critical to survival, such as preening. While the consequences of reduced preening on feather maintenance were not apparent in captive conditions, further work is needed to determine whether House Finches in the wild that are infected with M. gallisepticum experience a fitness cost, such as increases in ectoparasite loads, due to this reduced feather maintenance.

Variation in plasma protein levels in House Finches (Haemorhous mexicanus): effects of season, disease state, and urbanization

Variation in plasma protein levels in House Finches (Haemorhous mexicanus): effects of season, disease state, and urbanization

Are Purple Finches (Haemorhous purpureus) the Next Host for a Mycoplasmal Conjunctivitis Epidemic?

Ever since 1994, when the bacterial pathogen Mycoplasma gallisepticum jumped from poultry to wild birds, it has been assumed that the primary host species of this pathogen in wild North American birds was the house finch (Haemorhous mexicanus), in which disease prevalence was higher than in any other bird species. Here we tested two hypotheses to explain a recent increase in disease prevalence in purple finches (Haemorhous purpureus) around Ithaca, New York. Hypothesis 1 is that, as M. gallisepticum evolved and became more virulent, it has also become better adapted to other finches. If this is correct, early isolates of M. gallisepticum should cause less-severe eye lesions in purple finches than in house finches, while more-recent isolates should cause eye lesions of similar severity in the two species. Hypothesis 2 is that, as house finch abundance declined following the M. gallisepticum epidemic, purple finches around Ithaca increased in abundance relative to house finches and purple finches are thus more frequently exposed to M. gallisepticum-infected house finches. This would then lead to an increase in M. gallisepticum prevalence in purple finches. Following an experimental infection with an early and a more-recent M. gallisepticum isolate, eye lesions in purple finches were more severe than in house finches. This did not a support Hypothesis 1; similarly, an analysis of Project Feeder Watch data collected around Ithaca did not show differences in changes in purple and house finches' abundance since 2006, a result which does not support Hypothesis 2. We conclude that purple finch populations will, unlike those of house finches, not suffer a severe decline because of a M. gallisepticum epidemic.

High virulence is associated with pathogen spreadability in a songbird-bacterial system.

How directly transmitted pathogens benefit from harming hosts is key to understanding virulence evolution. It is recognized that pathogens benefit from high within-host loads, often associated with virulence. However, high virulence may also directly augment spread of a given amount of pathogen, here termed 'spreadability'. We used house finches and the conjunctival pathogen Mycoplasma gallisepticum to test whether two components of virulence-the severity of conjunctival inflammation and behavioural morbidity produced-predict pathogen spreadability. We applied ultraviolet powder around the conjunctiva of finches that were inoculated with pathogen treatments of distinct virulence and measured within-flock powder spread, our proxy for 'spreadability'. When compared to uninfected controls, birds infected with a high-virulence, but not low-virulence, pathogen strain, spread significantly more powder to flockmates. Relative to controls, high-virulence treatment birds both had more severe conjunctival inflammation-which potentially facilitated powder shedding-and longer bouts on feeders, which serve as fomites. However, food peck rates and displacements with flockmates were lowest in high-virulence treatment birds relative to controls, suggesting inflammatory rather than behavioural mechanisms likely drive augmented spreadability at high virulence. Our results suggest that inflammation associated with virulence can facilitate pathogen spread to conspecifics, potentially favouring virulence evolution in this system and others.

CHANGES IN TISSUE TROPISM OF MYCOPLASMA GALLISEPTICUM FOLLOWING HOST JUMP.

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.