Apparent effect of chronic Plasmodium infections on disease severity caused by experimental infections with Mycoplasma gallisepticum in house finches.

An epidemic caused by a successful host jump of the bacterial pathogen Mycoplasma gallisepticum from poultry to house finches in the 1990s has by now spread across most of North America. M. gallisepticum causes severe conjunctivitis in house finches. We experimentally show that M. gallisepticum transmission to birds with or without chronic Plasmodium infection does not differ. However, once infected with M. gallisepticum house finches chronically infected with Plasmodium develop more severe clinical disease than birds without such infection. We speculate as to possible effects of coinfection.

Introduction

Chronic infections with Plasmodium may adversely impact birds as experimentally shown by effects on reproduction (Merino et al., 2000, Knowles et al., 2010), on feather growth and quality (Marzal et al., 2013) or on telomere-length degradation (Asghar et al., 2015, Asghar et al., 2016). Mycoplasma gallisepticum is a bacterium that jumped from poultry to house finches (Haemorhous mexicanus) in the early 1990s in which it causes severe conjunctivitis (Ley et al., 1996, Hochachka et al., 2013) and can have severe impact on population size (Hochachka and Dhondt, 2000, Dhondt et al., 2006). As it spread across the continent M. gallisepticum rapidly evolved (Delaney et al., 2012, Tulman et al., 2012, Hochachka et al., 2013) and increased in virulence once endemic in a region (Hawley et al., 2013). Because haemosporidian parasites are frequently detected in house finches (Kimura et al., 2006, Davis et al., 2013), and given the increasing interest of possible effects of coinfection (Cressler et al., 2016) we tested to what extent chronic Plasmodium infections would impact house finches experimentally exposed to M. gallisepticum.

The objective of the experiment was to determine if (1) birds naturally infected with Plasmodium would become more rapidly infected with M. gallisepticum through horizontal transmission compared to individuals in which no Plasmodium was detected; (2) if birds infected with both M. gallisepticum and Plasmodium would develop more severe M. gallisepticum-induced clinical disease than birds infected only with M. gallisepticum; and (3) to what extent the response would vary with the virulence of the M. gallisepticum strain used.

Material and methods

Juvenile house finches were captured August–October 2015 in Ithaca, Tompkins County, New York (42°46′ N, 76° 45′ W) under permit (New York State Fish and Wildlife License 39, Albany, NY; and United States Geological Survey, Department of the Interior, Laurel, MD, permit 22669). Experiments were approved by Cornell University's IACUC protocol 2009-034.

Only birds negative for M. gallisepticum were used. Infection status was determined by visual inspection for eye lesions (Kollias et al., 2004), by Realtime Polymerase Chain Reaction (qPCR) designed to test for the presence of M. gallisepticum DNA from conjunctival swabs (Grodio et al., 2008), and by Rapid Plate Agglutination (RPA) to test for the presence of M. gallisepticum-specific antibodies in blood (Sydenstricker et al., 2006). We identified haemosporidian infection by lineage using DNA extracted from blood and using the nested polymerase chain (PCR) reactions described by Hellgren et al. (2004) and Waldenstrom et al. (2004) targeting the mitochondrial cytochrome b gene for Plasmodium and Haemoproteus. We repeated the PCR test three times for each sample. All PCR products positive for any haemosporidian infection were sequenced and the product compared to the Malavi database (Bensch et al., 2009).

Captive house finches were placed in six groups of 12 individuals housed in large semi-outdoor octagonal aviaries with a ground surface area of 6.87 m2 and a volume of 17.87 m3 (Dhondt et al., 2013). Each aviary contained water and a multiperch tube feeder that dispensed ad libitum a pelleted diet (Roudybush, Inc. Cameron Park, CA) (2/3) mixed with sunflower seeds (1/3). One artificial tree and multiple acrylic perches were distributed in identical fashion inside each aviary, as well as ceramic heat lamps. Five of the six groups consisted of six birds with and six without evidence of haemosporidian parasites and one group contained seven birds with and five without haemosporidian parasites.

To introduce M. gallisepticum in a group of birds we selected at random one group member with and one without Plasmodium and instilled 0.05 ml of M. gallisepticum inoculum in each conjunctival sac. We used three M. gallisepticum strains that differed in virulence (Hawley et al., 2013): CA2008 a weakly virulent strain, and NC2008 and CA2015 two more virulent ones. Because the number of colony forming units (CFU) varied between inocula, and as the CFU/ml was lowest for CA2008 at 6.20 × 106 CFU/ml, we diluted the other inocula with Frey's medium to equalize the CFU/ml in all three inocula. The precise identifiers for the inocula used are: CA2008- 2008.028-2-3P; NC2008 2008.031-4-3P; CA2015–2015.002-3-2P. Each M. gallisepticum strain was introduced in two of the groups. The single group in which 5 birds were negative and 7 positive for Plasmodium was assigned to CA2008.

We examined the birds for eye lesions, and collected conjunctival swabs for qPCR on day 0, 4, 10, 17, 31, 38, 45, 52, and 59 following the introduction of M. gallisepticum in the group on 30 November 2015 (Day 0); we took a blood sample for antibody testing by RPA on Day PI (post introduction) 0, 17, 31, 45, and 59. Eye lesions were scored on a scale of 0 (no lesions visible) to 3 (severe lesions) for each eye (Sydenstricker et al., 2005). Eye scores used here are the sum of the score in each eye, with a combined maximum of 6. The experiment was terminated on day 59 PI.

Sampling for presence of M. gallisepticum DNA was done by swabbing the conjunctiva of both eyes of a bird using a separate sterile cotton tipped 3 inch wood handle swab for each eye (Fisher Scientific) that was then placed in 200 μl tryptose phosphate broth (TPB) and stored at −25 °C. DNA extraction from conjunctival swab samples was carried out using a Qiagen DNeasy blood and tissue kit (Qiagen, Valencia, California, USA), following the manufacturer's recommended protocol for the purification of total DNA from animal tissues. Conjunctival swabs were tested for the presence of M. gallisepticum DNA using qPCR as described by Grodio et al. (2008). For antibody testing blood samples taken from a bird's left brachial vein were collected into micro-capillary tubes. Serum was tested for M. gallisepticum antibodies by Rapid Plate Agglutination using commercially available M. gallisepticum antigen produced by Charles River Laboratories, Inc using the A5969 M. gallisepticum poultry strain.

Statistical methods

To determine the rate of horizontal transmission between the inoculated index birds and the naïve group members we used survival analysis in the Statistix10 software package, (Tallahassee, FL). For each naïve individual we determined the time to first evidence of infection as the Day PI that an individual developed any sign of infection: eye score (score >0), M. gallisepticum-DNA in the eye swab, or evidence of presence of antibodies if neither other sign of infection was observed. We used the Logrank test which allows for censored data, and report the χ 2 and P-values of the Logrank tests.

To test for effects of the M. gallisepticum-strain and of Plasmodium on M. gallisepticum-load and on eye scores we summed the 8 values of M. gallisepticum-load and eye lesions from day 4 to day 59 PI and used these values in a two-way Analysis of Variance (Statistix10 software package). M. gallisepticum-load values were log (value + 1)-transformed. We first calculated the model with an interaction term between Plasmodium and M. gallisepticum-strain. If the interaction term was not significant, we recalculated the ANOVA without it.

Results

Presence of haemosporidians in juvenile house finches

During our initial screening we detected three different Plasmodium haplotypes, but no Leucocytozoon nor Haemoproteus. BLASTN results of the sequences identified in the MALAVI database (Bensch et al., 2009) showed that out of 46 haplotypes PADOM11 (Plasmodium sp.) was found 35 times (76.1%), WW3 (Plasmodium sp.) was found 9 times (19.6%) and SEIAUR01 (Plasmodium cathemerium) was found twice (4.5%). We distributed the birds among the six groups to equalize the number of birds with each haplotype. Thus in 5 groups five birds were infected with PADOM11 and one bird with WW3; the sixth group, in which we introduced the M. gallisepticum CA2008 strain, had five birds with PADOM11, one with WW3 and one l bird with SEIAUR01.

Response of index birds to M. gallisepticum inoculation

The index birds are the initial source of the horizontal transmission in each group, although as other birds become infected with M. gallisepticum, these can also contribute to further transmission. To provide an idea about differences between the M. gallisepticum strains used in this experiment, and between birds with and without Plasmodium we summarize the mean summed M. gallisepticum-load across the 8 samples taken on days 4–59 PI for each index bird as shown in Table 1. In all aviaries there was a source of M. gallisepticum infection, although in one of the aviaries in which NC2008 was introduced only one index birds developed disease. Given the very small sample sizes we did not perform statistical tests to compare M. gallisepticum-load between isolates or birds with/without Plasmodium, although the data suggest that the three M. gallisepticum isolates differ in virulence as expected (CA2008 < NC2008 ≤ CA2015) and that birds with Plasmodium developed infections with a higher M. gallisepticum -load and tended to have more severe lesions.

Table 1

Mean and standard errors of M. gallisepticum-load summed to day 59 PI in index birds.

	CA2008	NC2008	CA2015
No Plasmodium (n = 2)	17.62 ± 1.17	19.26 ± 1.52	15.99 ± 2.05
Plasmodium (n = 2)	15.74 ± 9.80	21.65 ± 21.65a	33.08 ± 5.04

One of the index birds, although it seroconverted, did not develop disease and hence would not have transmitted M. gallisepticum. The summed M. gallisepticum-load of the 2nd bird in the same aviary was 43.29.

Horizontal transmission and Plasmodium infection

Time to first infection

To determine the time to first infection through horizontal transmission of each naïve individual we determined what day post introduction (PI) of M. gallisepticum in the aviary each individual developed any sign of infection: eye lesions (eye score >0), M. gallisepticum-DNA present in the eye swab, or evidence of presence of antibodies if neither other sign of infection was observed.

Survival analysis showed that the three M. gallisepticum strains differed significantly in transmission rates (χ 2 = 13.32, df = 2, P = 0.0013). Transmission was significantly slower in the group with CA2008 than in both other groups (NC2008 versus CA2008: χ 2 = 8.39, df = 1, P = 0.004; CA2015 versus CA2008: χ 2 = 8.20, df = 1, P = 0.004), but there was no significant difference between CA2015 and NC2008 (χ 2 = 0.28, df = 1, P = 0.60). The lower transmission in the groups exposed to CA2008 was primarily caused by the fact that by day 59 PI, when the experiment was terminated, half of the naïve birds still showed no signs of having become infected, while in the other groups more birds showed evidence of having been infected by M. gallisepticum.

While the time to infection did differ between groups exposed to different M. gallisepticum isolates, there was no effect of Plasmodium: birds with and without Plasmodium did not differ in time to infection in any of the groups (all P > 0.13).

Severity of disease if infected

Some birds did not develop any signs of infection. Because we wanted to address the question how birds responded if infected by horizontal transmission we used only those birds that either developed eye lesions or in which M. gallisepticum-load in the conjunctiva was non-zero. This reduced the sample sizes to 10/20 for CA2008, 16/20 for NC2008 and 18/20 for CA2015.

In order to answer questions 2 (effect of Plasmodium?) and 3 (effect of M. gallisepticum strain used?) we performed a two-way analysis of variance with interaction to determine if either M. gallisepticum-strain or the presence of Plasmodium impacted M. gallisepticum-load and/or eye lesions. Given that the interaction term was not significant for M. gallisepticum-load (P = 0.80) nor for eye lesions (P = 0.12) we only report the results without interaction (Table 2) and illustrate the change of M. gallisepticum-load and eye lesions for one M. gallisepticum-strain over time in Fig. 1. The results show that the response to infection through horizontal transmission in a group varied significantly between M. gallisepticum-strains to which the birds were exposed (P < 0.0001) but also that birds with a chronic Plasmodium infection developed more severe clinical disease (P < 0.03).

Table 2

Mean and standard errors of eye scores (eye score) and M. gallisepticum-loads (MG load) summed to day 59 PI of house finches exposed to different M. gallisepticum isolates that had (Yes) or did not (No) have chronic Plasmodium infections before the start of the experiment. Groups of 12 birds were held in large aviaries into which M. gallisepticum was introduced. Sample sizes vary between groups because only birds that developed an infection are included in the analysis.

MG isolate	Plasmodium present	n	Eye score	MG load
CA2008	No	6	2.83 ± 1.25	1.53 ± 1.48
CA2008	Yes	4	1.00 ± 0.58	5.14 ± 5.10
NC2008	No	9	7.11 ± 2.34	7.11 ± 2.03
NC2008	Yes	7	11.43 ± 4.46	14.47 ± 4.92
CA2015	No	9	12.11 ± 3.54	18.73 ± 2.24
CA2015	Yes	9	24.89 ± 3.62	27.63 ± 2.57

Fig. 1

Mean severity of eye lesions ± SE (circles), and M. gallisepticum-load (log (qPCR+1)) (triangles) of birds infected through horizontal transmission with the CA2015 strain of M. gallisepticum. Birds in which Plasmodium was detected by PCR (grey symbols) developed more severe disease than birds in which Plasmodium was not found (white symbol). As there is no significant interaction between M. gallisepticum –strain and Plasmodium infection (Table 2) the results for the other M. gallisepticum strains are qualitatively similar and therefore not shown.

Discussion

House finches chronically infected with Plasmodium spp. and exposed to M. gallisepticum are equally likely to become infected with M. gallisepticum through horizontal transmission than group members without a chronic Plasmodium infection. A chronic Plasmodium infection thus does not make a bird more sensitive to infection. In this experiment one of the two birds that were the source of M. gallisepticum in each group had and one did not have a chronic Plasmodium infection. In order to determine the role of chronic Plasmodium infections on M. gallisepticum transmission one would need to compare groups in which both birds used to introduce M. gallisepticum in the aviary did not or did have chronic Plasmodium infections. The latter would deposit more M. gallisepticum on the feeder, which would have an effect on M. gallisepticum transmission rates (Adelman et al., 2013).

Although the Plasmodium-positive birds developed more severe clinical disease in response to the M. gallisepticum exposure, this could be attributed to other traits that correlate with both maintenance of a chronic Plasmodium infection and a stronger response to M. gallisepticum infection, and therefore this experiment, although suggestive, does not conclusively prove cause and effect. For example, social status or multilocus heterozygosity by influencing immunocompetence (Hawley et al., 2005, Hawley et al., 2007) might alter the response to both infections. Experiments in which birds, naïve to both pathogens, are experimentally infected with Plasmodium and with M. gallisepticum are needed to confirm that the relationship found is causal.

Dhondt and Dobson (2017) speculated that in birds infected both with the widespread Plasmodium parasite and the emerging M. gallisepticum bacteria coinfection might facilitate transmission of both (Dhondt and Dobson, in press). Thus, if we can confirm that birds coinfected with Plasmodium carry higher M. gallisepticum loads for a longer period of time this could lead to higher transmission rates (Hawley et al., 2013, Williams et al., 2014) resulting in a higher proportion of the population being infected with M. gallisepticum. The observation that prevalence of mycoplasmal conjunctivitis increases from North to South among house finch population in eastern USA (Altizer et al., 2004) might in part be explained by higher Plasmodium parasitaemia in those regions (Davis et al., 2013). Reciprocally because house finches infected with M. gallisepticum transiently increase corticosterone levels (Love et al., 2016), they are more attractive to mosquitoes (Gervasi et al., 2016). The increased mosquito feeding rate could facilitate Plasmodium transmission. This remains to be tested experimentally.