Mycoplasma Gallisepticum and mycoplasma sinoviae in turkeys

Introduction

Mycoplasmosis is a disease associated with infections of the respiratory tracts of chickens and turkeys that cause sinusitis, conjunctivitis, ocular discharge, aerosaculitis and arthritis (Fatummb, 1984; Kang et al., 2002; Osorio et al., 2007).

The disease is primarily associated to a group of microorganisms named Mycoplasma gallicepticum (Mg.), Mycoplasma sinoviae (Ms), Mycoplasma meleagridis (Mm) and Mycoplama iowa, of which Mycoplasma iowa causes embryonic death in turkeys. While the Mg infection affects both chickens and turkeys, the latter develop a more severe picture called infectious sinusitis (Fatummb, 1984; Kleven, 2008; Kleven & Ferguson, 2008). Direct (horizontal) transmission from diseased birds to susceptible ones occurs by ocular and nasal secretions and is the most common form of infection. On the other hand, contamination of equipment, food and drinking water, including wild birds play an important role as sources of infection and Mg reservoir (Bradbury et al., 2000; Kang et al., 2002, Kleven, 2008; Kleven & Ferguson, 2008; Luttrell et al., 1992). Contaminated breeders play the role of reservoir and source of transmission of the disease to offspring via transovaric (vertical) transmission (Kleven, 2008; Kleven & Ferguson, 2008). In Argentina, Colusi & Menchaca (1965) made the first description of sinusitis in turkeys, with confirmation thereof in 1995 by Brandetti et al. (1995).

The objective of this study was the epizootiological, pathological and etiological description of a case of infectious sinusitis in turkeys produced by Mg and Ms.

Materials & Methods

In the month of April, 2010, for diagnostic purposes, 2 female turkeys were brought to the Laboratory for Diagnosis of Bird and Piliferous Diseases of the Faculty of Veterinary Sciences of the UNLP, by the owner of a family farm, located in the town of Berisso, Province of Buenos Aires, Argentina. The animals were 4 months old and were part of a batch of 30 turkeys that lived alongside 30 hens sharing water and food supplies with them.

Clinical manifestations began at the age of 3 months and were characterized by decay, serious respiratory disorders such as dyspnea, blockage, rales, ocular and nasal secretion and marked bilateral sinusitis. Morbidity and mortality was 25%. Since the beginning of the clinical picture, birds were medicated with tylosin at 2 g/l and enrofloxacyn 3 ml/l, without any beneficial results.

Blood was extracted from the axillary vein in order to obtain serum for rapid agglutination on plate serological tests for Mg and Ms (Fatummb, 1984). Autopsy was performed on the submitted turkeys. Samples for histopathological studies of nose, trachea, lung and visceral organs were obtained. The samples were fixed in buffered formaldehyde at 10%, and embedded in paraffin... Coloration of hematoxylin and eosin was used.

For the application of the PCR technique in real time, tracheae and joint samples were extracted. The DNA was extracted using the DNEASY BLOOD & TISSUE KIT Quiagen ® kit, following the steps recommended by the manufacturer. The primers used were MS Fw 5´- CTG TTA TAG CAA TTT CAT GTG GTG -3´ Rv 5´ - CTG TTG TAG TTG CTT CAA CTT GTC- 3´

And MG Fw 5´ - CTA GAG GGT TGG ACA GTT ATG- 3´ Rv 5´ - GCT GCA CTA AAT GAT ACG TCA AA -3´. The final volume of the PCR reaction was of 25 µl, composed of 12.5 of µl Biorad iQ ^TM SYBR GREEN Supermix®, 3 µl of sample, 1 µl of each of the primers and 7.5 µl of distilled water. As positive control known Mg and Ms DNA and as negative control sterile distilled water was used. The protocol applied was as follows: An initial cycle of 94 ° C for 30 seconds, a cycle of 45 repetitions at 94° C for 10 seconds, 58° C for 30 seconds, 72° C for 30 seconds; a third cycle of 72 ° C for 5 minutes, a final cycle of 121 repetitions with an initial temperature of 73° C and a final temperature of 85° C for 30 seconds (Callison et al., 2006).

Swab samples of trachea and joints were planted in tubes containing Frey broth and Frey agar plates (Frey et al., 1968), with 14% of pig serum supplemented with 1% of nicotinamide adenine dinucleotie (NAD); 1% of HCl cysteine was necessary for the growth of Ms. 1,000,000 UI/lt penicillin G and 5% of thallium acetate were added to prevent the growth of opportunistic microorganisms. Samples were incubated in microaerophile at 37 ° C, watching them on a daily basis for evidence of the growth of mycoplasm (Frey et al., 1968).

Results and Discussion

The autopsy showed that the infraorbitary sinus and the trachea contained sero-mucuos exudate and nasal turbinates cuts showed the presence of caseose exudate. The palmeral conjunctive showed serous exudate of sparkling kind. The lungs showed consolidated areas of wine red color and the air sacs were opaque. The histopathological study showed that the nasal turbinates were congested, bleeding and showed exudate to light, with a marked epithelial detachment and the presence of inflammatory exudate composed of mononuclear and polymorphonuclear cells. The trachea showed a moderate subepithelial lymphocyte infiltration in focal and diffuse form. The bronchi also revealed marked hemorrhage and subepithelial lymphocyte infiltration in focal and diffuse form. Large consolidation areas were observed in the lung, with lymphocyte accumulation in the parenchyma and the peribronchial area.

Sera were treated at 56 ° C for 60 minutes and faced to commercial antigens against Ms (MS RPA-Test. Solei SARL®) and Mg (MG RPA-Test. Solei SARL®), testing positive by rapid agglutination to both diseases. Bacteriological studies of tracheal swab and joint cultures permitted isolation of Mycoplasma synoviae colonies. The real-time PCR test procedure led to the following results: Respiratory tract swab positive to Mg and Ms; and joint swab positive to Mg. Only Ms was isolated from trachea and joint swabs.

The system of rearing the turkeys in conjunction with the hens, with access for wild birds involves a potential factor of infection for turkeys. (Ley, 2008; Kleven et al., 2004; Luttrell et al., 1992). It could be inferred that the sum of these factors can act as a means of transmission and maintenance of the Mycoplasms.

It has been shown that wild and backyard birds were positive to Mycoplasma gallisepticum and Mycoplasma sinoviae by rapid agglutination and the hemoagglutination inhibition test. Through bacteriological studies and the PCR technique it has been shown that hens laying eggs for consumption are most affected by mycoplasms (Bradbury et al., 2000; Bradbury, 2001; Kang et al., 2002; Kleven, 2008; Luttrell et al., 1992; Sanei et al., 2007)

Ms is identified as a producer of respiratory clinical signs, such as aerosaculitis, pneumonia, purulent arthritis and the fall in egg production in turkeys (Fatummb, 1984, Khehra et al., 1999). Mg is mentioned as the cause in most of the descriptions of infectious sinusitis in turkeys. However, through serological tests the presence of antibodies against Ms was determined, although it could not be pinpointed as potential pathogen (Fatummb, 1984; Kleven et al., 2004).

Luttrell et al. (1992) described a case in turkeys released after rearing in pens. One of the birds, showing clinical signs of infectious sinusitis was serologically positive to Ms and Mg and Ms was isolated from the infraorbitary sinus, leading to the conclusion that this was the agent causing the disease. The case is similar to the one described by Luttrell et al. (1992), in which both mycoplasms were isolated; we can infer an interaction between both in the development of the disease.

Law (2008) studying wild and backyard birds, demonstrated that they were positive to Mg and Ms. On the other hand, it has been demonstrated that hens laying eggs for consumption are most affected by mycoplasms (Bradbury, 2001).

Conclusions

On the basis of clinical signs, macroscopic lesions and histopathological, serological, bacteriological tests and PCR, final diagnosis of a case of infectious sinusitis caused by Mg and Ms was made.

On the other hand, the inconvenience of breeding birds of different species in the same place is demonstrated, since, although we do not know the origin of the infection, it can be attributed to hens, as chronic carriers, responsible for the transmission of infectious agents to the turkeys.

While the literature indicates that infectious sinusitis in turkeys is produced by Mg, it is necessary to perform supplementary studies for the purposes of determining if Ms acted as primary pathogen along with Mg or if it was only present in the described clinical picture as a secondary or enhancing agent of the Mg.

Bibliography

Bradbury JM, Dare CM, Yavari CA, Forrester A. 2000. Evidence of Mycoplasma gallisepticum in British wild birds. p. 253. In: Abstracts of the 13th Congress of the International Organization for Mycoplasmology, Fukuoka, Japan.

Bradbury JM. 2001. The mycoplasma a wolf in sheep's clothing- Department of Veterinary Pathology. University of Liverpool. p. 5. In: Proceedings of the 24th Technical Turkey Conference.

Brandetti E, Linzitto OR, Menendez NA, Radman NA, Sato H, Herrero Loyola MA. 1995. Descripción de un caso de sinusitis en el pavo. Therios 24:230-232.

Callison SA, Riblet SM, Sun S, Ikuta N, Hilt D, Leiting V, Kleven SH, Suarez Dl, García M. 2006. Development and validation of a real-time taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds. Avian Dis. 50:537-544-

Colusi AD & Menchaca ES. 1965. Sinusitis Infecciosa de los pavos, su comprobación en la República Argentina. Rev. Med. Vet. 1: 46.

Fatummb OO. 1984, Infectious Sinusitis in Turkeys at Ibadan, Nigeria. Avian Dis. 28 (3):734-736.

Frey MLL, Hanson LP, Anderson DP. 1968, A medium for the isolation of avian mycoplasmas. American 6- Frey MLL, Hanson. Vet Res 19:2163-2171.

Kang MS, Gazdzinski P, Kleven SH. 2002. Virulence of recent isolates of Mycoplasma synoviae in turkeys. Avian Dis. 46:102.110.

Khehra RS, Jones RC, Bradbury JM. 1999. Dual infection of turkey poults with avian pneumovirus and Mycoplasma synoviae. Avian Pathol. 28:401-404.

Kleven SH & Ferguson-Noel N. 2008. Mycoplasma synoviae infection. pp. 845-856. En Diseases of Poultry, 12va ed, Saif YM, Fadly AM, Glisson JR,McDougald LR, Nolan LK, Swayne DE (eds). Blackwell Publishing Ltd., Oxford, UK.

Kleven SH, Fulton RM, Garcia M, Ikuta VN, Leiting VA, Liu T, Ley DH, Opengart KN, Rowland GN, Wallner-Pendleton E. 2004. Molecular characterization of Mycoplasma gallisepticum isolates from turkeys. Avian Dis. 48:562-569.

Kleven SH. 2008. Mycoplasmosis. pp. 54-64. In: A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. Dufour-Zavala L, Swayne DE, Glisson JR, Pearson JE, Reed WM, Jackwood MW, Woolcock PR (eds.). American Association of Avian Pathologists, Athens, GA, USA.

Ley DH. 2008. Mycoplasma gallisepticum infection. pp. 807-845. In: Diseases of Poultry. Saif YM, Fadly AM, Glisson JR, McDougald LR, Nolan LK, Swayne DE (eds). Blackwell Publishing Ltd., Oxford, UK.

Luttrell MP, Kleven SH, Mahnke GM. 1992. Mycoplama synoviae in a Released Pen-Raised Wued Turkey. Avian Dis. 36:169-171.

Osorio C, Fletcher OJ, Abdul-Aziz T, Gonder E, Tilley B, Ley DH. 2007. Pneumonia of turkey breeder hens associated with Mycoplasma synoviae. Avian Dis. 51:791-796.

Sanei B, Barnes HJ, Vaillancourt JP, Ley DH. 2007. Experimental infections of chickens and turkeys with Mycoplasma gallisepticum reference strain S6 and North Carolina field isolate RAPD type B. Avian Dis. 51:106-111.