Journal of Parasitology

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Jan 21, 2014 - Veterinary Parasitology 161: 178-186. Boal, C. W., R. W. Mannan, and K. S. Hudelson. 1998. Trichomoniasis in Cooper's hawks from Arizona.

Journal of Parasitology Racing Pigeons: A Reservoir for Nitro-Imidazole-Resistant Trichomonas gallinae --Manuscript Draft-Manuscript Number:

13-359R1

Full Title:

Racing Pigeons: A Reservoir for Nitro-Imidazole-Resistant Trichomonas gallinae

Short Title:

Pigeons and Nitro-Imidazole-Resistant Trichomonas gallinae

Article Type:

Research Notes

Corresponding Author:

Lieze Oscar Rouffaer, DVM University of Ghent Merelbeke, Belgium BELGIUM

Corresponding Author Secondary Information: Corresponding Author's Institution:

University of Ghent

Corresponding Author's Secondary Institution: First Author:

Lieze Oscar Rouffaer, DVM

First Author Secondary Information: Order of Authors:

Lieze Oscar Rouffaer, DVM Edwin Claerebout, Prof. Dr. Connie Adriaensen Cindy De Boeck An Martel, DVM, MSc, PhD, Dip ECZM

Order of Authors Secondary Information: Abstract:

Trichomonas gallinae, the cause of avian trichomonosis, is most commonly found in the order Columbiformes. Racing pigeons are often treated preventively with nitroimidazoles which could result in the emergence of resistant isolates. These isolates can be a threat to wildlife when exchange of isolates would occur. The sequence type of 16 T. gallinae isolates obtained from racing pigeons and 15 isolates from wild pigeons was determined based on the ITS1/5.8S rRNA/ITS2 region sequence. The resistance profiles of these isolates against five different nitro-imidazoles (metronidazole, dimetridazole, ronidazole, tinidazole and carnidazole) were determined. Two different Trichomonas sequence types were isolated. Sequence type A isolates were recovered from racing and wild pigeons, in contrast to sequence type B which was only isolated from wild pigeons. Isolates with sequence type B were all susceptible to the tested nitro-imidazoles, except for tinidazole-resistance in 3 isolates. Resistance to the nitro-imidazoles was observed more frequently in isolates obtained from racing pigeons than from wild pigeons, with most isolates belonging to sequence type A. A higher percentage of the sequence types A isolated from racing pigeons, in comparison with those isolated from the wild pigeons, were resistant to the nitroimidazoles and displayed higher MLC values. Two isolates belonging to sequence type A, 1 recovered from a racing pigeon and 1 from a wild pigeon, displayed a similar resistance pattern, suggesting a potential exchange of resistant isolates between racing pigeons and wild pigeons.

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RH: RESEARCH NOTES Racing Pigeons: A Reservoir for Nitro-Imidazole-Resistant Trichomonas gallinae L. O. Rouffaer, C. Adriaensen, C. De Boeck, E. Claerebout*, and A. Martel, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; *Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; Correspondence should be sent to: [email protected] ABSTRACT: Trichomonas gallinae, the cause of avian trichomonosis, is most commonly found in the order Columbiformes. Racing pigeons are often treated preventively with nitroimidazoles which could result in the emergence of resistant isolates, and these isolates can be a threat to wildlife when exchanges occur. The sequence type of 16 T. gallinae isolates obtained from racing pigeons and 15 isolates from wild pigeons was determined based on the ITS1/5.8S rRNA/ITS2 region sequence. In addition, the resistance profiles of these isolates against 5 different nitro-imidazoles (metronidazole, dimetridazole, ronidazole, tinidazole and carnidazole) were determined. Two different Trichomonas sequence types were isolated. Sequence type A isolates were recovered from racing and wild pigeons, in contrast to sequence type B which was only isolated from wild pigeons. Isolates with sequence type B were all susceptible to the tested nitro-imidazoles, except for tinidazole-resistance in 3 isolates. Resistance to the nitro-imidazoles was observed more frequently in isolates obtained from racing pigeons than from wild pigeons, with most isolates belonging to sequence type A. A higher percentage of the sequence type A isolated from racing pigeons, in comparison with those isolated from the wild pigeons, were resistant to the nitro-imidazoles and displayed higher MLC values. Two isolates belonging to sequence type A, 1 recovered from a racing

pigeon and 1 from a wild pigeon, displayed a similar resistance pattern, suggesting a potential exchange of resistant isolates between racing pigeons and wild pigeons. Trichomonas gallinae, a flagellate belonging to the order Trichomonadida, is the cause of avian trichomonosis. The parasite is located in the upper digestive and sometimes in the respiratory tract of a large variety of birds, although mostly in the order Columbiformes (rock dove as a primary host) and birds of prey (Stabler, 1954; Boal et al., 1998; Krone et al., 2005; Forrester and Foster, 2008). Recently, outbreaks of trichomonosis have been reported in wild finches in Great Britain (Robinson et al., 2010). The lesions caused by T. gallinae range from mild, often subclinical infections, to serious inflammation of the upper digestive tract with caseous lesions in the pharynx which can cause death by starvation due to the blockage of the lumen of the oesophagus. Some strains are more virulent than others and can cause systemic disease with migration to the liver and other organs with a potential fatal outcome (Stabler, 1954; Höfle et al., 2004; Forrester and Foster, 2008). The drugs of choice for the treatment of trichomonosis are nitro-imidazoles. Since 1990 resistance to these drugs has been observed, presumably due to the use of subtherapeutic doses and the preventive use of these drugs against trichomonosis (Lumeij and Zwijnenberg, 1990). Based on the 5.8S rRNA and the surrounding Internal Transcribed Spacers (ITS)1 and ITS2, 15 different sequence types of Trichomonas spp. have been described (Gerhold et al., 2008; Sansano-Maestre et al., 2009; Grabensteiner et al., 2010). It has been suggested that a correlation may exist between certain sequence groups, the resistance profile and virulence profile of T. gallinae (Sansano-Maestre et al., 2009; Zimre-Grabensteiner et al., 2011). By direct or indirect contact between racing pigeons and wild birds at shared drinking facilities, the exchange of resistant Trichomonas isolates could be facilitated, possibly giving rise to problems in treating trichomonosis in wild birds in bird rescue centres.

In this study, the resistance profiles of T. gallinae isolates acquired from racing pigeons and wild pigeons, using metronidazole, dimetridazole, ronidazole, carnidazole and tinidazole were compared. In addition, sequence typing of the ITS1-5.8S-ITS2 region was performed for each T. gallinae isolate. Trichomonas gallinae isolates were recovered from crop swabs from 16 racing pigeons and 15 wild pigeons. The racing pigeons originated from 16 different lofts and sampling was performed at the veterinary clinic of Ghent University during routine clinical examination. The group of wild pigeons consisted of 6 rock pigeons (Columba livia forma domestica), 6 wood pigeons (Columba palumbus) and 3 collared doves (Streptopelia decaocto). These animals were sampled at their arrival in a bird rescue centre, before receiving any treatment. None of the pigeons showed macroscopical signs of trichomonosis during routine examination. The crop swabs were incubated at 37 C in lnPouchTMTF medium (BioMed Diagnostics, Santa Clara, California). When the samples were positive, the Trichomonas parasites were transported to TYM medium (Clark and Diamond, 2002). Trichomonas sequence typing was done by amplifying and sequencing the 5.8S rRNA and surrounding Internal Transcribed Sequences (ITS1 and ITS2). DNA of the parasite was extracted from the swabs using the DNeasy Tissue extraction kit according to the manufacturers’ instructions (Qiagen Benelux B.V., Venlo, Nederland). To amplify the 5.8S rRNA gene and Internal Transcribed Sequences (ITS1 and ITS2), PCR was done as described by Grabensteiner et al. (2010). All PCR products were sequenced. Direct fluorescence based sequencing, with PCR primers (Trichomonad specific: TFR1 and TFR2), BigdyeTerminator RR 3.1 and BigdyeSequence Buffer (Applied Biosystems, Halle, Belgium) was used in the MJ Research PTC-200 Peltier Thermal Cycler (Group BIOzym, Landgraaf, Netherlands). The sequenced products were analysed using a Gene analyser (Applied Biosystems), and compared with existing sequences in the genbank (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Susceptibility tests for nitro-imidazoles (metronidazole, dimetridazole, ronidazole, carnidazole and tinidazole) were performed in anaerobic conditions without additional antibiotics. A serial dilution for each anti-protozoal agent was made in duplicate in a range from 500 µg/ml to 0.49 µg/ml. Each dilution contained 10,000 Trichomonas trophozoites confirmed by the Bürkerse counting chamber. After 24 hr incubation at 37 C the plate was visualised beneath the microscope (100x) to detect motile T. gallinae. The Mean Lethal Concentration was determined as the concentration after 24 hr at which no motile trophozoites were detected (Zimre-Grabensteiner et al., 2011). The cut-off to determine resistance to the nitro-imidazoles was set on 15.6 µg/ml (Narcisi and Secor, 1996). The Yates-corrected chisquare test was used for the statistical analyses (http://statpages.org/ctab2x2.html) Two different sequence types could be distinguished. Twenty-three T. gallinae isolates showed 100% identity with T. gallinae genbank accession number EU881912 (SansanoMaestre et al., 2009) and belonged to sequence type A. Eight isolates showed 100% identity with T. gallinae genbank accession number EU881911 (Sansano-Maestre et al., 2009) and belonged to sequence type B. One hundred percent of the isolates obtained from racing pigeons belonged to sequence type A, in comparison with only 47% of the isolates from the wild pigeons. Fifty-three percent of the isolates isolated from the wild birds belonged to sequence type B. The MLC values for all anti-protozoal agents used in this study are presented in Table I. Resistance to metronidazole, dimetridazole, ronidazole, carnidazole and tinidazole was more frequently observed in isolates isolated from racing pigeons (75%, 50%, 75%, 88% and 56% of the isolates were resistant to the nitro-imidazoles respectively) than in the ones from wild pigeons (13%, 20%, 13%, 7% and 40% were resistant to the nitro-imidazoles respectively). Statistical significant results were obtained for metronidazole (p-value: 0.002),

ronidazole (p-value: 0.002), carnidazole (p-value: 500

Total number of isolates

µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml

(percentage of resistance)

15,6

31,25

62,5

125

250

500

Metronidazole Wild pigeons

A

1

2

2

3

2

1

A

2

2

A

1

1

2

4

1

1

1

2

5

1

1

B Racing pigeons

2

1

7

1

15 (13%)

8 4

2

3

2

16 (75%)

1

Dimetridazole Wild pigeons

B Racing pigeons

2

A

1

1

7

1

15 (20%)

8 1

3

2

16 (50%)

1

Ronidazole Wild pigeons

Racing pigeons Carnidazole

A

1

1

2

1

B

2

3

1

2

1

2

1

A

1

7

15 (13%)

8 1

2

3

4

1

1

16 (75%)

Wild pigeons

Racing pigeons

A

3

1

2

B

1

5

2

A

2

7

1

15 (7%)

8 2

4

2

2

2

16 (88%)

2

Tinidazole Wild pigeons

Racing pigeons

A

2

2

B

1

2

2

1

4

A

2

7

1

8

3 2

15 (40%)

1

4

4

16 (56%)

Table I: Trichomonas gallinae: susceptibility tests: Susceptibilities of the T. gallinae strains recovered from wild pigeons and racing pigeons for each anti-protozoal agent. Trichomonas spp. with MLC ≥ 15.6 µg/ml for metronidazole, dimetridazole, ronidazole, carnidazole and tinidazole were considered resistant (resistance is indicated in bold). A = sequence type A, B = sequence type B

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