Introduction
Mycoplasma are relatively small with a preference for mucosal surfaces. They rely on the hosts to supply their nutritional requirements (Bradbury, Reference BRADBURY2005). The relationship between the host and the colonising pathogen is very important when considering both diagnosis and control measures. The most commonly encountered mycoplasma in poultry are M. gallisepticum (MG), M. synoviae (MS), M. meleagridis (MM), and lastly M. iowae (MI). Mycoplasma iowae is relatively uncommon compared to those previously mentioned. They tend to be host specific but cross species isolation can occur. Avian species are the only ones known to be infected with MI (Bradbury and Kleven, Reference BRADBURY and KLEVEN2003).
Mycoplasma iowae was first reported by Yoder and Hofstad (Reference YODER and HOFSTAD1962) as a new mycoplasma serotype isolated from chickens and turkeys. This became known as the Iowa 695 strain of avian mycoplasma. Classification of MI had been hindered by the marked antigenic variation and this created difficulty in designating types (Rhoades, Reference RHOADES1984). This has mostly been alleviated with the use of polymerase chain reaction (PCR) assays that have allowed the detection of species specific DNA segments. It can also be used to identify some MI strains (Zhao and Yamamoto, Reference ZHAO and YAMAMOTO1990; Boyle et al., Reference BOYLE, GOOD and MORROW1995; Cai et al., Reference CAI, BELL-ROGERS, PARKER, FERENCZ and POZDER2008).
Mycoplasma iowae is economically significant as certain strains are pathogenic to the turkey embryo. Although the turkey is the natural host for MI, it has also been occasionally reported in chickens, as well as wild birds (Bozeman et al., Reference BOZEMAN, KLEVEN and DAVIS1984; Bradbury and McCarthy, Reference BRADBURY and MCCARTHY1984). It can be difficult to detect as there are usually no clinically visible signs in the adult turkey. Natural infections are usually characterized by a reduction in hatchability due to embryonic deaths. This reduction in hatchability is variable with a range of estimates from 2% to 10% (Jordan, Reference JORDAN1996; Bradbury and Kleven, Reference BRADBURY and KLEVEN2003). A result of this variation is that the economic impact is difficult to quantify (Alankari and Bradbury, Reference ALANKARI and BRADBURY1996). The unpredictable variation in losses is related to differences in turkey strain susceptibility and also variations in pathogenicity between different strains of MI, making it a very complex pathogen.
The spread of Mycoplasma iowae
vertical transmission
Infection of eggs in the oviduct and subsequent vertical transmission is the most important mode of spread. In closed populations, it is the means by which permanent infections are maintained. Infected embryos that survive through to hatch remain in the population to complete the cycle and infect the following generation. An infected hen may develop an immune response and with it the level of egg transmission decreases. The immune response is often slow, so the reduction in egg transmission often occurs late in lay. The development of a response can be anywhere from a few weeks to three months into lay (Jordan, Reference JORDAN1996). The number of birds shedding MI at any one time can be quite variable and identifying those birds is difficult (Cai et al., Reference CAI, BELL-ROGERS, PARKER, FERENCZ and POZDER2008). It is our opinion that intermittent or variable shedding is more a function of the ability of the diagnostic test, such as bacterial culture, to identify the presence of MI than the variance in MI shedding by the carrier. We have repeatedly shown, irrespective of laboratory used, that when birds of known positive status are sampled bacterial culture will identify only a small percentage (10-15%) as positively infected compared with those identified positive with PCR. This we believe would lead previous authors who relied upon bacterial culture alone to infer that the organism was intermittently shed.
lateral transmission
Lateral spread within the hatchery and during brooding is possible but is considered a less significant route of infection (Burch and Valks, 2003). This may occur if susceptible birds are hatched on the same day or if cleaning between hatches is not thorough. In older birds housed together, the density is likely to have a role in the degree of lateral transmission via infected faeces (Alankari and Bradbury, Reference ALANKARI and BRADBURY1996). The potential for spread of disease between flocks in separate housing by direct or indirect contact is difficult to quantify. Mycoplasma iowae appears to have no aerosol transmission compared with other important mycoplasma such as M. gallisepticum, and as such, it should be easier to control.
Venereal transmission with infected tom semen and artificial insemination (AI) is by far the most significant mode of lateral transmission and initiates oviduct infection within a breeder hen (Shah-Majid and Rosendal, 1986). The rate of infection is probably increased by semen pooling that is normally used commercially. Poor hygiene during insemination, leading to cloacal cross-contamination, may contribute to the spread in a breeder flock. We have shown that infection between birds occurs within a flock and cross contamination can be stopped with strict biosecurity and the elimination of faecal transfer between infected and non-infected birds. The technique used was similar to a previously documented mycoplasma and salmonella eradication program (Wood et al., Reference WOOD, BUDDIGER, VAN DE HOEF, KOSTAL and FERENCZ2008).
Mechanisms by which Mycoplasma iowae causes disease
Although mycoplasmas are successful pathogens, the mechanisms by which they enter the host, multiply, and evade defences is not well understood (Jordan et al., Reference JORDAN, HORROCKS, JONES and CLEE1992; Bradbury, Reference BRADBURY2005). The heterogeneity between MI strains, antigenic variation, and pathogenic variation means there are differences in the host's response to each strain. The virulence of a particular strain may also have an impact on the ability of it to establish infection within the bird and this means certain strains are more commonly found in field infections (Mirsalimi et al., Reference MIRSALIMI, ROSENDAL and JULIAN1989). When there is a pathological response resulting from an MI, it is usually in the embryo or young bird and much of the pathology occurs as a response to inflammation. This response can result in congestion, necrosis, exudation, and cellular infiltration of the infected tissues (Jordan, Reference JORDAN1996). Lastly, there are strains that have no apparent clinical effect (Cai et al., Reference CAI, BELL-ROGERS, PARKER, FERENCZ and POZDER2008).
In poults, MI prefers the gastrointestinal tract, especially the caeca and large intestine. Other avian mycoplasma species such as MG and MS typically target the respiratory system and this is also true in the newly hatched, where MI has been found in the upper respiratory tract (Bradbury et al., Reference BRADBURY, IDERIS and OO1988; Mirsalimi et al., Reference MIRSALIMI, ROSENDAL and JULIAN1989). It can also be isolated from the extra-embryonic fluid and meconium of pipped and dead-in-shell embryos. In mature hens, it is harboured in the ovaries, oviduct, cloacae, and the mucosal folds of the vagina. In the tom, the phallus and semen are primary harbouring sites (Shah-Majid and Rosendal, Reference SHAH-MAJID and ROSENDAL1986; Trampel and Goll, Reference TRAMPEL and GOLL1994). These infected organs appear grossly free of clinical disease as MI rarely causes any damage to the tissue in the adult bird. The ability of MI to sequester quietly in the tissue and then move into the egg when required allows transmission to the following generation to occur.
Signs of infection and effects on the immune system
Increased embryo mortality is the most common sign of an MI infection. Variation in strain virulence has an impact on the severity of the effects on the embryos that survive, and as a consequence, there is a range of clinical signs. Mycoplasma iowae can kill between 2-10% of infected embryos but values are usually in the lower end of this range with natural field infections (Jordan, Reference JORDAN1996; Bradbury and Kleven, 2003). With both experimental and natural infection, embryonic death is generally between 18 and 24 days of incubation, although later occurrences have been reported (Yoder, Reference YODER1970; Bradbury et al., Reference BRADBURY, IDERIS and OO1988; Burch and Valks, Reference BURCH and VALKS2003).
In experimentally infected poults, clinical signs have included airsacculitis, stunting, poor feathering, and leg abnormalities including chondrodystrophy (Bradbury et al., Reference BRADBURY, IDERIS and OO1988; Mirsalimi et al., Reference MIRSALIMI, ROSENDAL and JULIAN1989). The stunting and poor feathering results from metabolic changes in both the embryo and poult. Leg abnormalities are not pathognomonic for MI, as other causes such as overheating during incubation and some nutritional deficiencies may present similar lesions. Other mycoplasmas such as M. meleagridis should be on a list of differentials when airsacculitis is identified. Unexpectedly, there are few clinical reports of MI outbreaks under field conditions presenting these clinical findings, although there is a recent report of a natural infection that displayed chondrodystrophy with leg and vertebral deformities (Trampel and Goll, Reference TRAMPEL and GOLL1994; Ley et al., Reference LEY, MARUSAK, VIVAS, BARNES and FLETCHER2010). This would demonstrate that the route of infection appears to be important in the severity of the clinical disease that develops and that experimental infection may not replicate the route of natural infection in the field and resulting pathology.
It has been suggested that Mycoplasma iowae may be slightly immunosuppressive for adult turkeys and has transient immunosuppressive effects in young birds. The slight delay in immune responses and the temporary reduction in the size of the bursa of Fabricius support this suggestion (Bradbury and McCarthy, Reference BRADBURY and MCCARTHY1984). Inflammatory cellular infiltrates in the bursa may have an effect on the production of B-lymphocytes: however, the mechanism of immunosuppression by MI is still largely unknown (Bradbury, Reference BRADBURY2005). Histologically, inflammatory responses can be seen in the spleen, bursa of Fabricius, and the lamina propria of some gastrointestinal tissues.
Confirmation of a Mycoplasma iowae infection
culture
Culture remains the most common diagnostic test for MI; however, issues arise because of its slow growth rate when plated. It can sometimes take up to 2-3 weeks before a colony is detected (Boyle et al., Reference BOYLE, GOOD and MORROW1995). Positive cultures may also require follow up immunofluorescence testing to determine the species specific strain. This uses species specific anti-sera which increases costs and further delays results. Other culturing issues can include competition from other non-pathogenic mycoplasmas in the growth medium and in contaminated culture can lead to problems with bacterial overgrowth (Cai et al., Reference CAI, BELL-ROGERS, PARKER, FERENCZ and POZDER2008). The use of cloacal samples in particular causes contamination, so both broth and agar media are used to maximise the chance of success (Alankari and Bradbury, Reference ALANKARI and BRADBURY1996). Mycoplasma iowae (and particularly field strains) do not tolerate some culture media and it has been shown that direct plating methods on agar can be more favoured than broth inoculation (Amin and Jordan, Reference AMIN and JORDAN1978). Culture had been used as the primary diagnostic tool until the development of a number of field-strain validated PCR assays (Garcia et al., Reference GARCIA, GERCHMAN, MEIR, JACKWOOD, KLEVEN and LEVISOHN1997; Cai et al., Reference CAI, BELL-ROGERS, PARKER, FERENCZ and POZDER2008). Culture remains a viable and important diagnostic method and is significantly less expensive than PCR. It has been our experience that the sensitivity of MI culture is particularly low when compared with PCR.
Polymerase Chain Reaction (PCR)
There are early descriptions of the experimental use of PCR for detecting MI (Zhao and Yamamoto, Reference ZHAO and YAMAMOTO1990; Kempf et al., Reference KEMPF, BLANCHARD, GERSBERT, GUITTET and BENNEJEAN1994; Boyle et al., Reference BOYLE, GOOD and MORROW1995). It is a rapid, sensitive, and simple diagnostic method with Garcia et al. (Reference GARCIA, JACKWOOD, HEAD, LEVISOHN and KLEVEN1996) describing the successful development and experimental use of PCR as part of a multi-mycoplasma species test that included MI. The PCR DNA amplification process was designed to identify and amplify a particular section of RNA sequence (in this case 16S rRNA) common to the mycoplasmas. Detection limits have been improved from 100 colony forming units (CFU) per PCR reaction (equivalent to 1000 CFU obtained per tracheal swab) using improved amplification products to 32 CFU per PCR reaction (Garcia et al., Reference GARCIA, GERCHMAN, MEIR, JACKWOOD, KLEVEN and LEVISOHN1997). At this level, it is a suitable method for the detection of MI in dead-in-shell where the concentration of mycoplasma is particularly low. The recent development and field validation of PCR was able to achieve a published diagnostic sensitivity and specificity of 97.6% and 95.5% respectively, with a detection limit of 80 CFU per swab, or 32 CFU per reaction, making PCR a viable diagnostic alternative to culture (Cai et al., Reference CAI, BELL-ROGERS, PARKER, FERENCZ and POZDER2008). In practice this sensitivity would appear to be much higher. This has also been documented on field samples with recent work focussed on improving the specificity (Ramirez et al., Reference RAMIREZ, DARE, YAVARI and BRADBURY2012).
Although PCR is rapid and is becoming the diagnostic gold standard in terms of sensitivity and specificity, it has some disadvantages. It is significantly more expensive in terms of both the assay and equipment required. DNA extraction can be affected by the faecal and bacterial loads on the swab and contamination can still potentially hinder testing (Cai et al., Reference CAI, BELL-ROGERS, PARKER, FERENCZ and POZDER2008). PCR will only identify MI to the species level without differentiating between serotypes and, as a consequence, culture may be necessary if isolation and serotype identification is required. Both culture and PCR can be affected by sample contamination and require further tests for serotype determination; however, by using PCR the major advantage is the speed with which a result is produced. To decrease the expense of PCR testing we have shown that up to 10 samples can be tested per reaction without loss of sensitivity or specificity.
serological tests
There are no reliable diagnostic serological tests available for detection of MI due to the weak serum antibody response as discussed previously (Garcia et al., Reference GARCIA, JACKWOOD, HEAD, LEVISOHN and KLEVEN1996; Bradbury, Reference BRADBURY1999).
Treatment and eradication
Treatment primarily describes the methods used to eradicate or reduce the incidence of MI within a population. The success of an eradication scheme largely hinges upon the use of a sensitive antimicrobial in the adult bird or egg (dipping and injection) or the use of treatments such pre-incubation heating. Unlike other mycoplasma species such as MG and MS, prophylactic vaccination for MI is not available.
Mycoplasmas do not have a cell wall and consequently, antimicrobials that affect cell wall synthesis, such as penicillin and its derivatives, are ineffective. Furthermore, MI is generally more resistant to antimicrobials than other avian pathogenic mycoplasmas and the availability of effective treatment options is often limited (Gautier-Bouchardon et al., Reference GAUTIER-BOUCHARDON, REINHARDT, KOBISCH and KEMPF2002). This is probably due, in part, to the long standing use of antimicrobials to control other mycoplasma infections and as a result resistance has developed (Kleven, Reference KLEVEN2008). The use of antimicrobials to eradicate other more pathogenic mycoplasma such as MM without accessing the concurrent eradication of MI has probably led to selection for resistance within these populations. Enrofloxacin (now unavailable for use in food producing animals in North America) and tiamulin, appear to be the most commonly used antibiotics due to high level resistance identified to other antimicrobials such as tylosin, oxytetracycline and lincomycin (Jordan et al., Reference JORDAN, HORROCKS and JONES1991; Reference JORDAN, HORROCKS and FROYMAN1993; Burch and Valks, Reference BURCH and VALKS2003).
antimicrobials in adult birds
Administration by feed, water or injection have all been used as delivery routes and have been shown to experimentally reduce infection (Jordan et al., Reference JORDAN, HORROCKS and FROYMAN1993). No antimicrobial can entirely eliminate an MI infection in a flock, or eggs produced by that flock, but it does appear to reduce the losses via embryonic mortality (Garcia et al., Reference GARCIA, GERCHMAN, MEIR, JACKWOOD, KLEVEN and LEVISOHN1997). Depending on the antibiotic used, treatment will decrease the shedding by the hen and can place measurable antibiotic residues in the egg for up to 12 days (Valks and Burch, Reference VALKS and BURCH2002). This treatment approach would appear to be effective only when attempting to reduce the effect of a pathogenic MI that is showing severe clinical signs and the benefit must be weighed against the potentially substantial treatment cost.
semen treatment with antimicrobials
In addition to parenteral treatments, Tomczyk and Minta (Reference TOMCZYK and MINTA2002) used tylosin and enrofloxacin as a turkey semen additive before artificial insemination and completely eliminated mycoplasma. The use of antimicrobials in the semen did not appear to have a negative effect on fertility. This treatment would only be effective in stopping the spread in hens that were already negative.
egg treatment – dipping
The objective of egg dipping is to reduce the presence of the organism on the shell membrane as the eggshell can be treated with traditional egg disinfection methods. This procedure is mostly used in combination with egg injection as a dual procedure (see following) (Ghazikhanian et al., Reference GHAZIKHANIAN, YAMAMOTO, MCCAPES, DUNGAN, LARSON and ORTMAYER1980). Increased uptake can be achieved with the presence of either a temperature or pressure gradient. When dipping, there can be irregularities in uptake due to handler variation, contaminated dipping solution, and pre-treatment washes. Uptake can be determined by measuring the weight of the egg pre- and post-dip, and this should be combined with monitoring the antimicrobial concentration through regular checks and renewal. Bacteriological monitoring is needed to prevent the over growth of micro-organisms such as pseudomonas (Hafez, Reference HAFEZ2006).
egg treatment – egg injection
Egg injection is another delivery method and is considered more accurate than dipping; however, but it is still not 100% effective. Mycoplasma iowae and meleagridis have been successfully eliminated using a combination of egg dipping and injection in turkeys, but this resulted in up to a 45% loss in hatchability in an eradication program (Wood et al., Reference WOOD, BUDDIGER, VAN DE HOEF, KOSTAL and FERENCZ2008). The mortality level was highly correlated to the concentration and amount injected. Increased mortalities may result from antimicrobial injection into the air sac and, for this reason, injection into the pointed end of the egg is recommended (Nascimento et al., Reference NASCIMENTO, NASCIMENTO, SANTOS, DIAS, RESENDE and SILVA2005).
egg treatment – heat treatment
Pre-incubation heat treatment using an incubator modified with additional heating elements has been found to be effective against other mycoplasma species (Yoder, Reference YODER1970). Despite the effectiveness of pre-incubation temperatures of over 47°C on other mycoplasmas, MI was still recovered from poults after heat treatment (Jordan and Amin, Reference JORDAN and AMIN1978). A negative effect of heat treatment is the reduction in hatchability, which have been reported to vary between 8%-65% depending on the protocol employed (Yoder, Reference YODER1970; Jordan and Amin, Reference JORDAN and AMIN1978). A number of different temperature profiles, maximum temperatures, and time at maximum temperatures have been presented with mixed results.
None of the above mentioned treatments are 100% effective in eliminating an infection from the adult bird or egg population. These methods would typically need to be used in conjunction with small group rearing and the elimination of positive groups (Wood et al., Reference WOOD, BUDDIGER, VAN DE HOEF, KOSTAL and FERENCZ2008). This methodology acknowledges the imperfect response to treatment and chance that some birds or groups will in fact still be infected and not have had the pathogen eliminated. Maintaining negative groups that are clear of infection after positive groups can be removed depends on the biology of the particular MI involved (i.e. either aerosol or close contact spread) and the maintenance of biosecurity between the groups. Removal of the usually limited number of positive small groups, then allows the remaining majority of MI free birds to be housed together without fear of horizontal or later vertical infection.
Environmental persistence and biosecurity
The viability of mycoplasmas outside the host is short due to both their structure and reliance on the host. Mycoplasma iowae is able to survive and remain viable for up to five days on feathers under experimental conditions whilst a field strain remained viable for six days (Christensen et al., Reference CHRISTENSEN, YAVARI, MCBAIN and BRADBURY1994). The same field strain was also shown to survive and remain viable on human hair, cotton, rubber, feathers, and straw for six days. This resilience has implications for mechanisms of spread from neighbouring poultry. As a consequence biosecurity and appropriate disinfectant use play a significant role in keeping MI out of uninfected flocks and premises.
Conclusions
Mycoplasma iowae has sporadically caused problems in the turkey industry as specific pathogenic strains cause embryonic mortality and lead to reductions in hatchability. Management of the risk associated with the introduction and spread of MI involves the development and implementation of a range of practices such as rapid diagnosis, treatment, hygiene, biosecurity, and risk assessment. The control of pathogenic MI at the commercial level is primarily through the sourcing of pathogenic MI free stock and the maintenance of appropriate biosecurity levels.
