Infection affects embryonic development – Mycoplasma iowae in turkeys: Part 2

Mycoplasma iowae in turkeys leads to decreased hatchability, late embryo mortality and leg and skeletal abnormalities in growing birds. The history and occurrence of this disease were discussed in the first part of this article in the previous edition of WP. How M. iowae can be recognised and diagnosed and which measures can be taken to prevent flocks from being infected, is discussed in this second part.

By Dr. Tahseen Aziz, Rollins Animal Disease Diagnostic Laboratory, Raleigh, NC, USA and Dr. H. John Barnes, College of Veterinary Medicine, NC State University, Raleigh, NC, USA

Embryonic mortality and inferior poult quality are the primary manifestations of vertical infection of turkeys with Mycoplasma iowae. Embryos die during late incubation, typically between days 18 and 24. Decreased hatchability in affected turkey flocks averages 2 – 5%. Infected poults that hatch may be of second quality. Embryonic mortality and decreased hatchability due to M. iowae infection can vary widely from minimal to significant and of extended duration.

The extent of hatchability loss depends on the virulence of the M. iowae strain, rate of egg transmission (i.e., number of hens transmitting mycoplasmas into eggs), and dose of the organism. Other factors related to egg-incubation conditions may also influence the rate of embryonic mortality in hatches of M. iowae-positive flocks. There is evidence that high temperatures during incubation can exacerbate mortality in M. iowae-infected embryos. It is important to realise that considerable variation exists among M. iowae strains in their pathogenicity for turkey embryos. Some isolates are more embryo lethal, causing death of the embryo with marked lesions, particularly stunting and edema, while others have little to no affect on embryo viability.

Lesions in natural and experimental M. iowae infected embryos include stunting, edema (particularly around the head area), hemorrhage, excess urates in ureters, poor feathering, abnormal feathers (clubbing of down), hepatomegaly, splenomegaly, and occasional hemorrhage of the chorioallantoic membrane. Although airsacculitis has been described in cull day-old poults naturally infected with M. iowae, the air sac lesions could not be attributed convincingly to M. iowae, as infection with other mycoplasmas, especially M. meleagridis, was not ruled out. However, turkey poults inoculated directly into their air sacs with M. iowae developed lesions of varying severity. Histologically, there was exudative and cellular airsacculitis characterised by fibrinous exudate and infiltrates of heterophils and mononuclear inflammatory cells.

Deformities of legs and bones
Different leg deformities have been described in poults naturally or experimentally infected with M. iowae. Chondrodystrophy (shortening and thickening of bones) is the most obvious lesion. Mycoplasma iowae was isolated from cloacae, air sacs, and joints of 17-day-old commercial poults with leg problems that included chondrodystrophy, valgus deformity, excessive clear fluid in joint cavities, curled toes, and splayed legs. More recently, M. iowae was associated with chondrodystrophy in a flock of meat-type turkeys at 3 to 7 weeks of age. Birds with chondrodystrophic lesions constituted 17.3% of the total culling and mortality in the flock. Poor growth and lameness were noted in the affected birds.

Distended feather tips result in “Clubbed Down,” a lesion often seen late in development or at hatching in Mycoplasma iowae infected embryos. Clubbed down is not specific for M. iowae infection. Other causes include high temperatures during incubation and B-vitamin deficiencies. (Courtesy of Dr. Colin Baxter-Jones)

Gross lesions of chondrodystrophy were characterised by deformities in bones of the legs and vertebral column including: shortening, thickening, and bowing of tarsometatarsi (shank bones); enlarged hock joints; slight widening of the growth plate in proximal tibiotarsi; shortening of the vertebral column; marked deformity of the posterior segment of the thoracic vertebrae (due to deformity of the articulating thoracic vertebra and caudal articulation of the notarium); uneven spacing between ribs; and twisting (rotation) of the neck. Histopathologic lesions in the free, articulating thoracic vertebrae of birds with vertebral column deformity were described in detail.

Weak and poor performance
Experimentally, when day-old turkey poults were inoculated via air sacs and foot pads with M. iowae, poor feathering and poor and uneven growth were apparent in some poults by the second week of age. From the second to third weeks of age, most of the inoculated poults showed one or more of the following skeletal abnormalities: chondrodystrophy, rotated tibia, splayed leg, deviated toes, excessive fluid in joint cavity, pitted articular cartilage, and wrinkled, sigmoid folding, and/or ruptured digital flexor tendons; excessive joint fluid was the most common abnormality.

Turkey, 18 days of age, chondrodystrophy. In chondrodystrophy, appositional growth exceeds longitudinal growth. Legs of chondrodystrophic poults are short, thick, and have prominent joints. Poults infected with Mycoplasma iowae typically show chondrodystrophy, but not all poults with chondrodystrophy are infected with M. iowae. Other causes of this lesion include genetics, high incubator temperatures, and malnutrition. Bowing of legs or other angular deformities are often seen in chondrodystrophic limbs.

Histologically, there was evidence of mild lymphocytic tenosynovitis in digital flexor tendons. The day-old poults that were inoculated orally with a strain of M. iowae showed only a mild clinical disease (poor feathering, slight growth depression) compared with poults inoculated via the air sacs and foot pads with the same strain. In the same study, most of the 60 turkey embryos inoculated via yolk sac with M. iowae at 21 days of age failed to hatch.

Those that did hatch exhibited weakness, poor growth, abnormal feathering and poor appetite. Fourteen of the 18 poults that hatched died within the first week after hatching and two more poults died by 16 days of age. The two poults that survived into the third week developed chondrodystrophy. Chickens inoculated with strains of M. iowae into air sacs and foot pad at one day of age developed marked gross and histologic lesions in the digital flexor tendons above and below the hock joints.

Turkey, 28 days of age, chondrodystrophy. Proximal tibiotarsus from a chondrodystrophic poult (left) is markedly thickened, has a “U” shape, and the growth plate lacks a uniform thickness. In contrast, the proximal tibiotarsus from an unaffected bird (right) has a normal thickness, a “V” shape, and uniform growth plate.

Findings in naturally and experimentally infected poults indicate that M. iowae is most pathogenic for turkey embryos, and it appears that skeletal deformities (particularly chondrodystrophy) occur only in poults infected in-ovo or during the first few days after hatching. At a commercial level, M. iowae is not recognised as an important cause of tenosynovitis and leg deformities in turkeys (or chickens). There is no report of clinical disease, adverse effect on production, or lesions in mature hens and toms due to infection with M. iowae.

Samples for diagnosis
Low grade reduction in hatchability with increased late embryonic mortality should arouse suspicion of M. iowae infection. In-shell-dead embryos (especially those that die during late incubation) and piped, unhatched embryos should be examined for gross lesions. If infection with M. iowae is suspected, samples may be collected for mycoplasma culture and isolation or for molecular detection by Polymerase Chain Reaction (PCR) assay. Although M. iowae is present in several tissues of infected embryos, extraembryonic fluid, swabs of oropharynges, and cloacal swabs are reliable and easy to collect.

A recommended sampling technique is inserting a sterile swab between the embryo and shell followed by swabbing the oropharynx (throat) of the embryo. Using fluorescent-antibody microscopy and electron microscopy, M. iowae was identified on or adhering to intestinal mucosa of chick embryos inoculated following yolk sac inoculation. While intestine and air sacs are excellent sites for isolation or detection of M. iowae in infected embryos, these sites are more difficult to sample. Isolation or detection of M. iowae may be attempted from very young cull, second-quality poults; cloacal swabs, intestine, air sacs, and oropharynx are recommended sites of sampling from young poults. It is important to collect samples within the first few days after hatching, as the isolation rate from vertically infected poults decreases with time.

Turkey, 30 days of age, wry neck. Occasionally turkeys with wry neck occur in flocks with Mycoplasma iowae infected birds. The neck is rotated and permanently held to one side. M. iowae has been isolated from intervertebral spaces in the affected part of the neck.

Although M. iowae is not believed to be an important cause of tenosynovitis of digital flexor tendons in commercial turkey flocks (as well as chicken flocks), it should be considered a possibility in the absence of other infectious agents. Experimentally infected poults and chickens developed joint/tendon lesions from which M. iowae was isolated. However, the organism was also isolated from swabs of the hock joint and digital flexor tendons that did not have lesions. When poults have chondrodystrophy M. iowae can be isolated from swabs of affected bones, adjacent joint spaces, and associated tendons. Isolation rates of 25-30% from hock joints and flexor tendons and approximately 50% from spinal lesions have been obtained in turkeys up to six weeks of age.

It has been suggested that swabbing might not be the best way to collect samples and that it might be preferable to macerate pieces of tendons or articular cartilage to release mycoplasmas located within the tissues. It is also possible that the organism does not persist very long in affected joints and tendons.

Frequent sampling and testing
In breeder hens and toms, isolation or detection of M. iowae should be attempted from the cloaca of hens, and the cloaca, phallus, or semen of toms. Cloaca yields the organism most consistently. Frequent sampling is essential as shedding of organisms from infected oviducts is intermittent; hens and toms may be infected but not shedding the organism. Even hens with a very low rate of M. iowae isolation from oviducts can still transmit the organism vertically to their progeny at a significant rate, and toms that are culture-negative can still introduce infection to M. iowae-free hens.

Turkey, 28 days of age, spinal chondrodystrophy. Chondrodystrophy of the spine is characterised by thickening and deformity of the free thoracic vertebra, which lies between the notarium cranially and synsacrum caudally. M. iowae is isolated from the intervertebral joint spaces of a high percentage of these lesions. In contrast to chondrodystrophy of the legs, spinal chondrodystrophy is specific for M. iowae infection and is not known to have other causes.

It is recommended that sampling and inoculation of swabs be done on the same day. If this is not possible, swabs should be stored at 4oC and plated as soon as possible. Swabs should not be stored for longer than 24 hours at room temperature as this may compromise survival of any mycoplasma. Moistening swabs with mycoplasma broth before they are used for sampling may aid survival of the mycoplasma. Mycoplasma iowae is fastidious in its requirement for yeast extract. When new batches of yeast extract are obtained for incorporation into solid or liquid media, they need to be tested to be sure they support growth of the organism. Recovering M. iowae from tissues is increased if the swab is directly plated onto agar media rather than inoculated into broth.

Turkey, 45 days of age, spinal chondrodystrophy. Lesions of spinal chondrodystrophy likely persist for the life of the turkey. Recently we found typical lesions in 18 week-old toms. How long Mycoplasma iowae remains in the lesion is unknown. Severe deformity of the back can result in scoliosis as seen here.

Mycoplasma iowae does not elicit a strong humoral immune response (antibody production) that can be detected by conventional serologic tests, such as the plate agglutination test. It is believed that the lack of immune response results from limited invasion of M. iowae in adult birds, but it could also be due to weak immunogenicity. Antibodies to M. iowae could not be detected by the plate agglutination test at six weeks of age in poults inoculated orally or via air sacs and foot pad at one day of age. Additionally, the marked antigenic variation among strains potentially complicates the effectiveness of serologic diagnosis. In one study, none of the 21 antigens prepared from 21 different strains of M. iowae gave positive plate-agglutination reaction with antisera against five reference strains of M. iowae.

Turkey, 26 days of age, spinal chondrodystrophy, (x2, left is cranial). In the spinal chondrodystrophic lesion (bracket), the Free Thoracic Vertebra (FTV) and cranial vertebra of the synsacrum are markedly distorted and compressed. Masses of proliferating cartilage extend along the ventral vertebral body and fill the caudal articulation between the FTV and synsacrum. Cartilage forming the cranial articulation is mildly thickened. There is moderate kyphosis (arching of the spine) due to loss of bone in the vertebral bodies and vertebral deformity has resulted in compression of the spinal cord (arrow).

Elisa was also found unreliable because of increased false-positive reactions with negative turkey sera. The hemagglutination– inhibition test cannot be used because the hemagglutinin of M. iowae is weak and unstable. Hemagglutination activity of 4 strains of M. iowae became undetectable 24 hours after antigens were stored at 4oC, -20oC, or -70oC.

Start with great grandparent flocks
As M. iowae is vertically transmitted, prevention must start with eradication of the organism from great grandparent flocks, a task for which primary turkey breeding companies are responsible. Any eradication strategy requires a thorough knowledge of the biology and transmission (especially egg transmission patterns) of M. iowae , as well as considerable effort and expense. No study in the USA about prevalence, extent, and economic significance of M. iowae infection in turkeys has been done, so the need for M. iowae eradication remains controversial.