Keila V. Dhondt

Experimental evidence for transmission of Mycoplasma gallisepticum in house finches by fomites.

Ever since Mycoplasma gallisepticum emerged among house finches in North America, it has been suggested that bird aggregations at feeders are an important cause of the epidemic of mycoplasmal conjunctivitis because diseased birds could deposit droplets of pathogen onto the feeders and thereby promote indirect transmission by fomites. In this paper we bring the first experimental evidence that such transmission (bird-to-feeder-to-bird) does actually take place. House finches infected via this route, however, developed only mild disease and recovered much more rapidly than birds infected from the same source birds but directly into the conjunctiva. While it is certainly probable that house finch aggregations at artificial feeders enhance pathogen transmission, to some degree transmission of M. gallisepticum by fomites may serve to immunize birds against developing more severe infections. Some such birds develop M. gallisepticum antibodies, providing indication of an immune response, although no direct evidence of protection.

Detection and quantification of Mycoplasma gallisepticum genome load in conjunctival samples of experimentally infected house finches (Carpodacus mexicanus) using real-time polymerase chain reaction.

A TaqMan®-based real-time, quantitative polymerase chain reaction (qPCR) assay utilizing the mgc2 gene was developed to detect Mycoplasma gallisepticum in conjunctival swabs of experimentally infected house finches. The assay was demonstrated to be quantitative by the standard curve method with reproducible results within runs and between runs. The detection limit of the mgc2 assay was examined using two standards. The test had a detection limit of less than 14 copies per reaction when tested with a plasmid standard and less than 10 copies per reaction when tested with M. gallisepticum genomic DNA. All M. gallisepticum-negative birds (10 specific pathogen free chickens and 10 house finches) were negative by mgc2 qPCR assay. Existing evidence suggests that an important part of M. gallisepticum pathogenesis includes both its attachment to and invasion of host cells. Thus, our test also made use of rag-1 as an internal control gene. The rag-1 qPCR results showed that host cell quantity varied greatly between conjunctival samples. After inoculation, M. gallisepticum levels in the house finch conjunctiva increased over the 7-day period post infection. The bird with the most pronounced clinical conjunctivitis harboured the highest level of M. gallisepticum and the bird that did not develop conjunctivitis had very low numbers of M. gallisepticum. Thus, it appears that development of conjunctivitis may correlate with M. gallisepticum load.

Common garden experiment reveals pathogen isolate but no host genetic diversity effect on the dynamics of an emerging wildlife disease

Host genetic diversity can mediate pathogen resistance within and among populations. Here we test whether the lower prevalence of Mycoplasmal conjunctivitis in native North American house finch populations results from greater resistance to the causative agent, Mycoplasma gallisepticum (MG), than introduced, recently‐bottlenecked populations that lack genetic diversity. In a common garden experiment, we challenged wild‐caught western (native) and eastern (introduced) North American finches with a representative eastern or western MG isolate. Although introduced finches in our study had lower neutral genetic diversity than native finches, we found no support for a population‐level genetic diversity effect on host resistance. Instead we detected strong support for isolate differences: the MG isolate circulating in western house finch populations produced lower virulence, but higher pathogen loads, in both native and introduced hosts. Our results indicate that contemporary differences in host genetic diversity likely do not explain the lower conjunctivitis prevalence in native house finches, but isolate‐level differences in virulence may play an important role.

Comparative infectiousness of three passerine bird species after experimental inoculation with Mycoplasma gallisepticum

Mycoplasma gallisepticum has been isolated from various species of free-living birds, and we therefore tested the hypothesis that bird species other than the main host, the house finch (Carpodacus mexicanus), could play a role in the epidemiology of the infection. We compared the disease course in the house finch, American goldfinch (Carduelis tristis) and house sparrow (Passer domesticus) after inoculation into the conjunctival sac with M. gallisepticum, and also the degree to which the three species were infectious to other naïve house finches. Severity of clinical signs was least in house sparrows, intermediate in American goldfinch and the highest in house finch. House sparrows were only mildly infectious to naïve house finches for a short time, whereas American goldfinches remained infectious for up to 49 days post inoculation, although by then there were no physical signs of disease. We conclude that since American goldfinches can be infectious without showing any conjunctivitis, and since they often make long-distance movements, they might play an as yet unsuspected but important role in M. gallisepticum dynamics in house finches.

Evidence of trade‐offs shaping virulence evolution in an emerging wildlife pathogen

In the mid‐1990s, the common poultry pathogen Mycoplasma gallisepticum (MG) made a successful species jump to the eastern North American house finch Haemorhous mexicanus (HM). Subsequent strain diversification allows us to directly quantify, in an experimental setting, the transmission dynamics of three sequentially emergent geographic isolates of MG, which differ in the levels of pathogen load they induce. We find significant among‐strain variation in rates of transmission as well as recovery. Pathogen strains also differ in their induction of host morbidity, measured as the severity of eye lesions due to infection. Relationships between pathogen traits are also investigated, with transmission and recovery rates being significantly negatively correlated, whereas transmission and virulence, measured as average eye lesion score over the course of infection, are positively correlated. By quantifying these disease‐relevant parameters and their relationships, we provide the first analysis of the trade‐offs that shape the evolution of this important emerging pathogen.

Pathogenicity and immunogenicity of three Mycoplasma gallisepticum isolates in house finches (Carpodacus mexicanus)

Mycoplasma gallisepticum (MG) has become a common cause of conjunctivitis in free-living house finches (Carpodacus mexicanus) since its emergence in the early 1990s. To date, temporal and spatial genotypic variation in MG has been documented, but phenotypic variation in pathogenicity and immunogenicity has not been examined. House finches were inoculated with MG isolates Virginia (VA)1994, California (CA)2006, or North Carolina (NC)2006, which were cultured from free-living house finches with conjunctivitis in 1994, 2006, and 2006, respectively. Infection with NC2006 resulted in the most severe eye lesions, highest pathogen loads, and highest levels of pathogen-specific lachrymal and serum antibodies. Infection with CA2006 caused the least severe eye lesions, lowest pathogen load, and lowest levels of antibodies. A small number of birds in each group developed protracted, severe disease in spite of robust antibody responses, suggesting that immunopathology may contribute to the lesions. Immunoblot analyses indicated that isolates are antigenically similar; thus, there may be partial cross-protection if a house finch encounters two or more strains of MG throughout the course of its lifetime. This study provides evidence that MG strains or strain variants circulating in house finch populations vary in their ability to cause disease, induce antibody responses, and persist in the host.

Understanding the origin of seasonal epidemics of mycoplasmal conjunctivitis.

1. Many host-pathogen systems show regular seasonal oscillations. 2. Seasonal variation in mycoplasmal conjunctivitis prevalence in house finches is an example of such oscillations. 3. An annual pulse of Mycoplasma gallisepticum-naïve juveniles increasing the number of susceptibles, seasonal changes in flocking behaviour increasing transmission rate and a gradual loss of resistance to reinfection with time are sufficient to model the observed seasonal variation in disease prevalence. Nevertheless, experiments are needed to test the underlying mechanisms. 4. We carried out an 18-month experiment with small groups of birds in large aviaries to test two hypotheses. 5. To test the first hypothesis that an influx of naïve juveniles in a group of recovered adults is sufficient to cause an outbreak, we added eight juveniles to a group of 11 adults that had recovered from an earlier infection. In all, three replicates juveniles became infected, but only after some of the adults relapsed. 6. To test the second hypothesis that reintroduction of M. gallisepticum into a multiage group of previously exposed but fully recovered house finches causes a new outbreak, we inoculated two birds in each group in March of the 2nd year. Contrary to what happens in the wild at that time disease prevalence increased rapidly after reintroduction of M. gallisepticum. 7. We conclude that asymptomatic, recovered adults can initiate an epidemic and transmit M. gallisepticum to naïve house finches and that the reintroduction of M. gallisepticum is sufficient to cause a new outbreak, even at a time of the year when mycoplasmal conjunctivitis is low in free-living birds. Date, as such, seems to be less important to explain seasonal variation in conjunctivitis than the presence of naïve juveniles or the introduction on M. gallisepticum. 8. Seasonality in outbreaks is most likely tightly linked to seasonal variation in bird movements and behaviour.

Effects of route of inoculation on Mycoplasma gallisepticum infection in captive house finches

The routes by which Mycoplasma gallisepticum initiates infection during outbreaks of conjunctivitis in house finches remain uncertain. As M. gallisepticum recovered from the cloaca of chickens remains viable for up to 3 days in chicken faeces, the possibility of spread via faecal contamination has been suggested. To test the hypothesis that food or water contaminated with M. gallisepticum may initiate infection, 20 house finches were experimentally inoculated by the oral or the conjunctival route. Clinical and immunological responses were compared. All inoculated birds seroconverted, thus demonstrating infection. Only two of the birds inoculated via the oral route developed very mild unilateral conjunctivitis while all 10 of those infected by eye-drop inoculation developed severe bilateral conjunctivitis. The orally inoculated birds had reduced levels of activity for only a few days, while those infected by conjunctival inoculation had reduced activity for several weeks. M. gallisepticum DNA was detected in conjunctival swabs by polymerase chain reaction in only three orally inoculated birds but in all birds in the conjunctivally inoculated group. Antibodies developed more slowly after oral inoculation than after conjunctival inoculation. We showed that oral exposure to M. gallisepticum can initiate infection, disease, and a serological response, which suggests that food or water contaminated with secretions or excretions may be a route of transmission between house finches.

CAN AMERICAN GOLDFINCHES FUNCTION AS RESERVOIRS FOR MYCOPLASMA GALLISEPTICUM

We performed experiments to test if American Goldfinches (Spinus tristis) could be a competent reservoir for Mycoplasma gallisepticum and play a role in the epidemic spread of mycoplasmal conjunctivitis among House Finches (Carpodacus mexicanus) in North America. We infected one of two individuals housed together in a cage and determined if transmission occurred to the second bird. Probability of transmission between an American Goldfinch and a House Finch (in either direction) was similar to that between two House Finches. In a second experiment small groups of birds (6–8) were housed in large aviaries. Two source birds were inoculated with M. gallisepticum, and transmission to the naive birds in the aviary was recorded. Transmission occurred among House Finches, among American Goldfinches, and from House Finches to American Goldfinches. Transmission was more likely between House Finches than among American Goldfinches, and between House Finches and American Goldfinches. We conclude that American Goldfinches are a competent reservoir for Mycoplasma gallisepticum and could have played a role in the spread of the epidemic as they are more migratory than House Finches.

Mycoplasma sturni from a California House Finch with Conjunctivitis Did Not Cause Disease in Experimentally Infected House Finches

Mycoplasma gallisepticum conjunctivitis emerged in 1994 as a disease of free-ranging House Finches (Carpodacus mexicanus) in North America and has also been isolated from other songbirds with conjunctivitis. A key feature for the successful study of natural and experimental disease has been the apparent, very-high correlation between characteristic eye lesions and M. gallisepticum. Mycoplasma sturni was originally isolated from an adult European Starling (Sturnus vulgaris) with bilateral conjunctivitis and has since been reported in a relatively small number of other avian species, but not in House Finches. We identified as M. sturni a mycoplasma isolate from a California House Finch with conjunctivitis. However, experimental infection of House Finches with the M. sturni isolate failed to reproduce the disease. Therefore, M. gallisepticum remains the primary known cause of conjunctivitis in House Finches.