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Co-occurrence of Mycoplasma Species and Pigeon Herpesvirus-1 Infection in Racing Pigeons (Columba livia).

Abstract: Oropharyngeal swab samples were collected from 438 live racing pigeons (Columba livia), with and without signs of respiratory disease, that were housed in 220 lofts in 3 provinces in the western part of the Netherlands. Polymerase chain reaction (PCR) was used to identify Mycoplasma species and pigeon herpesvirus-1 (PHV-1) from the samples. In 8.6% of the pigeon lofts tested, signs of respiratory disease were present in pigeons at sampling, and in 30.9% of the sampled pigeon lofts, respiratory signs were observed in pigeons during the 6-month period immediately before sampling. A total of 39.8% of tested pigeons (54.5% of tested lofts) were positive for Mycoplasma species, and 30.6% of tested pigeons (48.6% of tested lofts) were positive for PHV-1. In 15.8% of the tested pigeons (26.8% of tested pigeon lofts), coinfection by Mycoplasma species and PHV-1 was identified. The number of pigeon lofts having pigeons coinfected by Mycoplasma species and PHV-1 was higher than that where only one of the infections was identified. Neither the presence of Mycoplasma species, PHV-1, nor the cooccurrence of both infections was significantly associated with signs of respiratory disease.

Key words: Mycoplasma, pigeon herpesvirus-1, the Netherlands, respiratory disease, avian, pigeon, Columba livia

Introduction

Various infectious agents have been associated with respiratory disease in racing pigeons (Columba livia) and may severely compromise racing performance. (1,2) However, evidence demonstrating an etiologic relationship between the detected agents and the observed clinical signs of respiratory disease often is lacking. (3) Moreover, the presence of facilitating factors and importance of concurrent infection favoring the onset of respiratory disease in pigeons often are left unrevealed. (2)

Mycoplasma columbinum, M columborale, and M columbinasale have been isolated from the oropharynx, sinuses, air sac, lung, trachea, and eye in healthy pigeons as well as pigeons with respiratory disease. (3-11) Signs that have been attributed to clinical mycoplasmosis in pigeons include dyspnea, abnormal respiratory sounds, sneezing, conjunctivitis, and rhinitis. (3,4,9) Mycoplasma columbinum has been isolated from a racing pigeon with severe arthritis of the shoulder joint, (12) and M columbinum and M columbinasale have been isolated from the brain in pigeons. (3,6) However, definitive evidence for a primary pathogenic role of Mycoplasma species in pigeons is lacking. (2,3,7,10,13,14) While some investigators claim that virtually all pigeons are infected with mycoplasmas (15) and consider the pathogenicity of all mycoplasmas in pigeons to be negligible, (16) coinfection of pigeons with Mycoplasma species and other bacterial as well as viral agents is presumed to promote the onset or worsen the clinical manifestation of respiratory disease in pigeons. (17,18)

Worldwide, more than 50% of racing pigeons are deemed to be infected persistently with pigeon herpesvirus-1 (PHV-1). (18) Although clinical infection does not occur readily, high morbidity and variable mortality associated with PHV-1 infection may be observed when infected racing pigeons are exposed to facilitating factors, such as inadequate hygiene and environmental conditions, overcrowding, transportation, and high-intensity training. (18,19) Clinical disease resulting from PHV-1 infection seems to be observed most frequently during summer and the breeding season when large numbers of chicks are present. (18,20,21) While acute mortality is the most common sign of PHV-1 infection in juvenile pigeons, coryza, conjunctivitis, diphtheroid stomatitis, and pharyngitis are the most prominent signs observed in clinically affected subadult to adult pigeons with PHV-1 infections. (18,20,21) Concurrent or secondary bacterial infection may worsen the clinical course of PHV-1 infection in racing pigeons. (20,21)

We determined the prevalence of Mycoplasma species and PHV-1 infection in racing pigeons in the western part of the Netherlands. In addition, the potential synergistic effect of PHV-1 towards the development of respiratory disease in racing pigeons was investigated.

Materials and Methods

Animals and sampling

From the end of July 2013 through the end of March 2014, oropharyngeal swab samples were collected from 438 racing pigeons housed at 220 lofts in 3 Dutch provinces comprising the western part of the Netherlands (North-Holland, South-Holland, and Utrecht). The total number of pigeons lofts in the 3 provinces was estimated at 2000, so approximately 11% of the lofts in those provinces were included in the study. On average, two subadult to adult pigeons were sampled per loft. Samples were collected from 80, 217, and 141 pigeons housed at 41, 109, and 70 lofts in Utrecht, North-Holland, and South-Holland, respectively. Before sample collection, a clinical examination was performed on each pigeon to identify signs suggestive of respiratory infection, such as abnormal breathing sounds, rhinitis, nasal discharge, dyspnea, coryza, or pharyngitis. A questionnaire was completed for each sampled loft to ascertain whether any signs of respiratory disease had occurred during the 6-month period immediately before sampling in pigeons that did not have clinical respiratory disease at sampling. In pigeon lofts where signs of respiratory disease were present at sample collection, oropharyngeal swab samples were collected exclusively from pigeons with clinical respiratory disease. Pigeon lofts were categorized as positive for PHV-1 and Mycoplasma species (Table 1; H+/M+) if co-occurrence of both infections was demonstrated in a single pigeon or if both infections were detected separately in 2 individual pigeons housed in the same loft.

Polymerase chain reaction (PCR)

Extraction of DNA from swab samples was performed as described previously by Kempf et al. (22) For identification of Mycoplasma species a 16S rRNA gene-specific PCR was performed as described previously. (23) For species identification, PCR products were sequenced and sequences were compared to those maintained in the GenBank database through the Basic Local Alignment Search Tool (BLAST). For identification of PHV-1 in samples, a nested PCR was performed based on the protocol described by VanDevanter et al. (24) The potential of using both PCR protocols was explored as a proxy for shedding of PHV-1 and mycoplasmas in sampled pigeons.

Statistical methods

Statistical analyses were performed to determine if the presence of Mycoplasma species was significantly associated with the presence of PHV-1 infection at the level of the entire study population of pigeons as well as at the level of pigeon lofts. In addition, we investigated whether infection with mycoplasmas, PHV-1, or the coinfection was associated with signs of respiratory disease. For this purpose, the infection status of the pigeons and pigeon lofts (4 categories based on the presence or absence of Mycoplasma species and/ or PHV-1) was related to the presence or absence of signs of respiratory disease (3 categories; no signs of respiratory disease at sample collection or during the 6-month period immediately before sampling, signs of respiratory disease within the 6-month period before sampling but not at sample collection, or signs of respiratory disease at sample collection; Table 1). These data were explored with simple descriptive and categorical statistical approaches. Categorical data analysis was performed using 2 X 2 contingency tables (%2 test) for which noninfected (H-/M-) pigeons or pigeon lofts with or without signs of respiratory disease were used as control groups. Furthermore, we investigated if the number of pigeons and pigeon lofts positive for Mycoplasma species and/or PHV-1 varied substantially among the 3 provinces. Due to the nonhomoscedasticity of the latter data, the nonparametric Friedman test was used. Levels of statistical significance were set at P < .05.

Results

Prevalence of Mycoplasma species and PHV-1 infections in racing pigeons and pigeon lofts

A total of 174 of 438 sampled pigeons (39.7%) representing 120 of 220 pigeon lofts (54.5%) tested positive for Mycoplasma species, whereas 134 of 438 sampled pigeons (30.6%) belonging to 107 of 220 pigeon lofts (48.6%) tested positive for PHV-1. In 69 of 438 pigeons (15.8%) belonging to 59 of 220 pigeon lofts (26.8%), co-occurrence of Mycoplasma species and PHV-1 shedding was demonstrated (Table 1). Based on these results, the PCR protocols used appeared suitable as proxies for shedding of PHV-1 and mycoplasmas.

The number of pigeons and pigeon lofts positive for Mycoplasma species and/or PHV-1 was not significantly different among the 3 provinces (Friedman test, P > .05; Table 2). Mycoplasma columbinum, M columborale, and M columbinasale were detected in pigeons from each of the 3 provinces. Based on 16S rRNA gene sequencing, 8% of the Mycoplasma-positive samples could not be assigned to any of the latter species or other known Mycoplasma species and were categorized as such. Prevalence of M columbinum infection was the highest for each province (52.3%) followed by M columbinasale (28.2%) and M columborale (10.9%).

Association of PHV-1 and Mycoplasma species infection in racing pigeons with respiratory disease

In 60.5% of the sampled pigeon lofts, no pigeons showed signs of respiratory disease at the sample collection or during the prior 6 months. Physical signs of respiratory disease were identified in 8.6% of the lofts at sampling and in 30.9% during the 6 months before sampling. Table 1 shows the number of individual pigeons and pigeon lofts that tested positive for PHV-1 and/or Mycoplasma species in each of these categories.

Among entire study population, the association between shedding of PHV-1 and Mycoplasma species was not significant (Friedman test; P = .06). However, when examined at the level of pigeon flocks, coinfection with Mycoplasma species and PHV-1 was identified in more flocks than one of the infections alone (Table 1).

Among the entire study population and among the pigeon lofts, there was no significant correlation between the presence of Mycoplasma, PHV-1, or mixed infection and signs of respiratory disease at sampling or within the 6 months before oropharyngeal sampling ([chi square] test, 2 x 2 contingency tables).

Discussion

Our results illustrated that Mycoplasma species and PHV-1 infection are commonly present in racing pigeons in the western part of the Netherlands. The relatively high number of Mycoplasmapositive pigeons was comparable to that reported for other countries. (3,4,8,9) As previously documented, the relative occurrence of different species of known mycoplasmas in pigeons seems to vary according to the investigated geographic region. (3,4) Mycoplasma columbinum was the most prevalent species in our study performed in the western part of the Netherlands.

Oropharyngeal sampling used to investigate the presence of Mycoplasma species and PHV-1 in our study was performed mainly during autumn and winter. Aside from climate variations, stressors, such as overcrowding, breeding, and participation in high-stakes pigeon races, may influence the number of PHV-1-positive pigeons detected by oropharyngeal sampling during certain seasonal periods. Especially for PHV-1, immunosuppression resulting from early coupling and breeding in the winter is considered an important facilitating factor promoting the onset of clinical herpesvirus infection in pigeons. (18) In addition to repetitive sampling of individual pigeons and pigeon lofts, continuous monitoring throughout the year to confirm seasonal variations and identify promoting factors is recommended.

When using oropharyngeal sampling, only pigeons that are excreting the tested agent(s) will be detected. Especially for PHV-1, latency and intermittent excretion are important characteristics. (18) Accordingly, repetitive oropharyngeal sampling of individual pigeons combined with serologic testing for PHV-1 and Mycoplasma species is necessary to estimate the overall prevalence of PHV-1 and Mycoplasma species in pigeon populations.

Although a trend toward significant co-occurrence of shedding of PHV-1 and mycoplasmas was demonstrated in the pigeon lofts, an etiologic relationship between the coinfection and the development of respiratory disease could not be demonstrated. Pathogenicity studies that focus on the 3 recognized Mycoplasma species in pigeons, the role of concurrent infections, and the presence of facilitating environmental factors may reveal the importance of mycoplasmas in inducing respiratory disease in racing pigeons.

References

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(12.) Hellebuyck T, Garmyn A, De Cooman L, et al. Mycoplasma columbinum isolated from a racing pigeon (Columba livia) with arthritis. J Avian Med Surg. 2014;28(3):240-241.

(13.) Pettersson B, Tully JG. Bolske G, Johansson K-E. Re-evaluation of the classical Mycoplasma lipopliilum cluster (Weisburg et al. 1989) and description of two new clusters in the hominis group based on 16S rDNA sequences. Int J Syst Evol Microbiol. 2001; 51(Pt 2):633-643.

(14.) Molokwu JU, Adegboye DS. Mycoplasma columborale and Mycoplasma columbinum from pigeons: a first report of their isolation in Nigeria. Rev Sci Tech Off Int Epizoot. 1988;7(3):635-638.

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(16.) Gerlach H. Ober das vorkommen von Mykoplasmen bei Tauben. Berl Munch Tierdrtzl Wochenschr 1977:90(7): 140-143.

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(18.) Vindevogel H, Debruyne H, Pastorett PP. Observation of pigeon herpesvirus 1 re-excretion during the reproduction period in conventionally reared homing pigeons. J Comp Pathol. 1985;95(1): 105-112.

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(23.) Kiss I, Matiz K, Kaszanyitzky E. et al. Detection and identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay. Vet Microbiol. 1997; 58(l):23-30.

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Tom Hellebuyck, DVM, PhD, Stephan Gobel, DVM, Frank Pasmans, DVM, PhD, Connie Adriaensen, PhD, and An Martel, DVM, PhD

From the Department of Pathology. Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.
Table 1. Prevalence of Mycoplasma species and-or PHV-1 infection, as
tested by PCR on oropharyngeal swab samples, in pigeon lofts and
racing pigeons with or without respiratory signs in 3 provinces in
the western part of the Netherlands.

                                N                  PHV1+/M-

                          Pigeon              Pigeon    Pigeons,
Presence of               lofts    Pigeons   lofts, %      %
respiratory signs

Absent at sampling/        133       266       12.7       13.2
  absent during
  6 mos before sampling
Absent at sampling/         68       135       17.6       17.7
  present during
  6 mos before sampling
Present at sampling         19       37        15.8       16.2
Total                      220       438       14.6       14.8

                                PHVI-/M+              PHV1+/M+

                           Pigeon    Pigeons    Pigeon    Pigeons,
Presence of               lofts, %      %       lofts, %      %
respiratory signs

Absent at sampling/         22.6       24.4       31.6       15.8
  absent during
  6 mos before sampling
Absent at sampling/         19.2       24.4       38.2       16.3
  present during
  6 mos before sampling
Present at sampling         15.8       18.9       31.6       13.5
Total                       20.3       24.0       26.8       15.8

                                PHV1-/M-

                           Pigeon    Pigeons,
Presence of               lofts. %      %
respiratory signs

Absent at sampling/         33.1       46.6
  absent during
  6 mos before sampling
Absent at sampling/         25.0       41.5
  present during
  6 mos before sampling
Present at sampling         36.8       51.4
Total                       38.3       45.4

Abbreviations: PVH-1 or PHV1, pigeon herpesvirus-1; M. Mycoplasma
species: PCR, polymerase chain reaction.

Table 2. Prevalence of Mycoplasma species and/or PHV-1 infection, as
tested by PCR on oropharyngeal swab samples, in pigeon lofts and
racing pigeons by province in the western part of the Netherlands.

                      N                  PHV1+/M-

                Pigeon              Pigeon
Province        lofts    Pigeons   lofts, %   Pigeons, %

Utrecht           41       80        56.2        42.5
North-Holland    109       217       42.8        26.2
South-Holland     70       141       48.6        29.0
Total            220       438       14.6        14.8

                      PHV1-/M+                PHV1+/M +

                 Pigeon                  Pigeon
Province        lofts, %   Pigeons, %   lofts, %   Pigeons, %

Utrecht           53.7        41.3        34.2        13.8
North-Holland     44.2        31.2        25.7        19.1
South-Holland     44.7        44.7        39.4        14.3
Total             20.3        24.0        26.8        15.8

Abbreviations: PVH-I or PHV1. pigeon herpesvirus-1; M, Mycoplasma
species; PCR, polymerase chain reaction.
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Title Annotation:Research Brief
Author:Hellebuyck, Tom; Gobel, Stephan; Pasmans, Frank; Adriaensen, Connie; Martel, An
Publication:Journal of Avian Medicine and Surgery
Date:Dec 1, 2017
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