Efficacy of Tylosin Tartrate on Mycoplasma Infections and Eggshell Apex Abnormalities in Layer Hens Under Field Conditions

The aim of this report was to describe a clinical case of concurrent infection with Mycoplasma Gallisepticum (MG) and Mycoplasma Synoviae (MS) in layers, complicated with Escherichia coli and Pasteurella multocida, as well as to evaluate the efficacy of tylosin tartrate treatment (Pharmasin ® 100% WSG, Huvepharma) on Mycoplasma under field conditions. A flock of 18,500 ISA Brown pullets, 21 weeks of age and MS positive, showed an increase in weekly mortality, with no influence on egg production. At 24 weeks of age, post mortem was carried out and blood samples were collected, as well as samples for microbiological cultures from the lesions. In necropsy, several findings of pericarditis, airsacculitis of posterior thoracic sacs, prolapse and egg yolk peritonitis were found. Sporadic neurological signs were also apparent and post mortem examination revealed yellow caseous exudate accumulations in cranial bones. Although egg production was not influenced, Eggshell Apex Abnormalities (EAA) were increased with a significant number of both broken and misshapen eggs together with discoloration. Elisa serological examinations for Infectious Bronchitis Virus (IBV) and Newcastle Disease Virus (NDV) were within vaccination program limits, but MS, MG and Avian Rhinotracheitis (ART) results showed a significant seroconversion. Microbiology also revealed the presence of Escherichia coli and Pasteurella multocida. On week 25, a drug treatment with tylosin tartrate was applied at a dose of 105 mg kg −1 body weight in drinking water for five consecutive days. Three days after treatment, misshapen, broken and discolored eggs were decreased. The mortality was also significantly decreased within a week since the beginning of tylosin administration and at 30 weeks of age was within acceptable levels. Post mortem evaluation confirmed the absence of previously gross lesions in primary organs. In summary, tylosin treatment in complicated Mycoplasma spp. infections under field conditions improved egg shell quality and decreased mortality, thus the economic losses linked to decreased egg production and labor costs were reduced.


INTRODUCTION
Mycoplasmas remain one of the most important diseases in modern intensive poultry production, causing great economic losses and affecting performance of the birds. Economic losses in chickens are attributable not only to overt respiratory disease but also to poor weight gain, downgrading of broilers at slaughter due to Science Publications AJAVS airsacculitis and loss of egg production in layers (Bradbury and Morrow, 2008). Mycoplasma gallisepticum and Mycoplasma synoviae are the most relevant for commercial chickens from a clinical and an economical point of view. In the last decade MG infections are rare, except for multi-age egg production sites, whereas MS infections became very frequent in poultry flocks in European countries (Bradbury et al., 2005).
Mycoplasma synoviae is considered the second most important mycoplasma affecting chickens (Kleven and Ferguson-Noel, 2008). It causes respiratory disease and subsequent condemnations due to airsacculitis, although most respiratory tract infections seem to be subclinical. M. synoviae also causes synovitis in chickens and turkeys (Landman and Feberwee, 2004). The effect of M. synoviae infections on egg production by layer chickens remains unclear. In one report a cumulative egg production loss of 10% was seen in flocks infected with M. synoviae (Morrow et al., 1990), but in another study no association was seen between M. synoviae infection and egg production (Mohammed et al., 1987). However, from the year 2000 onwards also M. synoviae strains with oviduct tropism able to induce or associated with Eggshell Apex Abnormalities (EAA) have been increasingly found (Catania et al., 2010;Feberwee et al., 2009;Ranck et al., 2010;Strugnell et al., 2011). EAA are characterized by altered shell surface, shell thinning, increased translucency and the occurrence of cracks and breaks. EAA were first described in white layers housed in cages, but were later also seen in brown layers housed in cages and in both types of birds kept in other housing systems.
Different antimicrobials, especially antibiotics have been used to treat flocks infected with MG and MS in order to reduce severity of clinical signs, lesions and production losses (Bebear and Kempf, 2005). Such treatment can reduce severity of disease, economic losses, populations of Mycoplasmas, but does not eliminate MG and MS from the infected poultry flock.
One of the most widely used molecules in medication programs is tylosin, a macrolide antibiotic. Catania et al. (2009) reported that tylosin treatment improved egg shell quality and this reduced the economic losses linked to labor costs and moreover the increased average weight of eggs from treated birds contributed to improve production performances.
We recently observed a clinical case of concurrent infection with Mycoplasma gallisepticum and Mycoplasma synoviae in layers, complicated with Escherichia coli and Pasteurella multocida. The purpose of this report was to evaluate the efficacy of tylosin tartrate treatment (Pharmasin ® 100% WSG, Huvepharma) on Mycoplasma infections and EAA in layers under field conditions.

Flock Details
The reported case occurred in a layers farm located in Nicosia, Cyprus. A flock of 18,500 ISA Brown pullets, 15 weeks of age, imported from Greece, was introduced in one house under intensive and controlled environment system. No major disease problems were previously recorded in the farm. The farm was completely isolated, with high standards of biosecurity and held complete records of feed and water consumption, daily production data and veterinary instructions.

Clinical History
During the rearing period, the flock was fully vaccinated against Infectious Bronchitis Virus (IBV) and Newcastle Disease Virus (NDV), whereas vaccination against MG, MS and ART was not applied.
The first day of arrival (15 weeks of age), layers already laid the first eggs reaching 2% at the age of 16 weeks. The flock was also found serologically positive in MS and negative in MG at the time of arrival. Production data, feed consumption and mortality were accordingly to the breed specifications, with the exception of layers body weight which was 100 g heavier than the breed standard. No signs of any disease manifestation were shown and no production problems were observed up to 21 weeks of age, even though the flock was tested MS positive. At the onset of 21st week of age, the average weekly mortality was raised gradually up to 0.98% in a period of five weeks (26 weeks of age), the egg production was not influenced, but the percentage of broken and misshapen eggs rapidly increased (Fig. 1). Fig. 1. Production data before and after treatment with tylosin tartrate at 25 weeks of age

Clinical Examination
At 21st week of age there was a slight increase in mortality which was first attributed to prolapse ( Fig.  2) and peritonitis ( Fig. 3) due to early production. Due to an outbreak of ND in broilers at that time and to strict biosecurity measures, no post mortem examination was carried out.
At week 24, clinical inspection and post mortem examination was carried out. During the clinical inspection of the flock, the main symptoms observed were unilateral or bilateral swelling of the eyelids and facial skin, with no other major symptoms. Primary lesion findings were pericarditis-airsacculitis of posterior thoracic air sacs (Fig. 4) and at a later stage, post mortem examination revealed yellow caseous exudate accumulations in cranial bones (Fig. 5) with apparent, but very sporadic, neurological signs (Fig. 6). Egg quality parameters were also compromised with increased number of misshapen and broken eggs, together with discoloration of brown eggs (Fig. 7).

Sample Collection and Laboratory Examination
At week 24, blood samples were also collected, as well as samples from the lesions for microbiological analysis. The results revealed E. coli isolation from infected air sacs, heart and egg yolk, as well as Pasteurella multocida in the case of purulent inflammation of cranial bones. Elisa analysis (Biochek, Holland) showed high positive titres for MG, MS and ART, indicating infection with a field strain, whereas NDV and IBV seroconversion was indicative of a vaccination (Mean Titer and CV according to Biochek baselines) well implemented ( Table 1).

Treatment
On week 25, a treatment with tylosin tartrate (Pharmasin ® 100% WSG, Huvepharma) was applied at a dose of 105 mg kg −1 (3.5 bags of 1.1 kg each per day with bird average body weight of 1.8 kg) in drinking water for five consecutive days.

Outcome
Three days after start of treatment, the numbers of misshapen, broken and discolored eggs were decreased. The mortality was also significantly decreased at the time that administration of tylosin was finished and at 30 weeks of age was within acceptable levels. Post mortem evaluation confirmed the absence of previously gross lesions in primary organs. After the treatment, the flock recovered normally with a major improvement in health and production data and with no evidence of gross lesions.

DISCUSSION
A clinical case of Mycoplasma gallisepticum and Mycoplasma synoviae in layers under field conditions was studied. Mycoplasmas remain one of the most important diseases in modern intensive poultry production causing great economic and health impact worldwide. The most economic effects of MG infections are well known and measures for the control and eradication are included in European Union legislation (EEC, 1990;CEU, 2009;EC, 2011) which prohibits intra-community trade in live poultry or hatching eggs derived from infected flocks. The economic impact of MS infection is less well understood but the organism is becoming more and more widespread in commercial birds, especially layers and broiler-breeders.
It is well known that the effects of Mycoplasma in uncomplicated infection may be minimal but are often enhanced in the presence of other respiratory pathogens such as pathogenic serotypes of E. coli and the viruses of ND and IBV, even vaccine strains (Ley, 2008). In this study, MG, Escherichia coli, Pasteurella multocida and ART were identified as factors that further complicated the infection with MS found at the time of arrival, whereas IBV and NDV seroconvertion was a clear indication of proper vaccine implementation. The role of MS infection in the layer chicken industry is still unclear and under debate, but it is also well known that the effects of MS in uncomplicated infection may be minimal (Ley, 2008). This report is in agreement with the clinical findings of the present study up to 21 weeks of age, when obviously the increase of mortality coincided with the infection with MG and the other pathogens. Up to that time, no clinical findings were observed due to the presence of MS. The microbiological and serological results at 24 weeks of age revealed the co-infection and the severity of the disease was enhanced in the presence of the identified respiratory pathogens such as MG, ART and the presence of pathogenic serotypes of E. coli and Pasteurella multocida. Drual et al. (1992) attributed significant respiratory disease, synovitis and high mortality in commercial broilers to a dual infection of MS and an atypical strain of Pasteurella gallinarum. In addition, the higher MS titers compared to MG titers at 24 weeks of age indicated that the flock was infected with MG a few weeks later than MS. Unfortunately, due to NDV biosecurity measures was not able to identify the exact time of infection. In all such mixed infections the timing of the infections with respect to each other and to the age of the bird may well influence the outcome and the disease severity. Furthermore it is apparent that there are more complex interactions between some pathogens whereby a mixture of three agents could cause more severe respiratory disease than any two of them combined. MG infection in chickens with E. coli and respiratory vaccine viruses is one such example (Nakamura et al., 1994).
The clinical inspection and necropsy revealed symptoms and gross lesions in agreement to those found in previous studies (Stipkovits and Kempf, 1996;Kleven and Ferguson-Noel, 2008). In addition, some of the infected laying hens appeared to exhibit neurological signs such as torticollis, due to purulent inflammation of cranial bones. Such conditions have been described in turkeys (Chin et al., 1991), but description of such neurological sings is scarce in chickens.. Although the mycoplasma infections, specially complicated with other pathogens, are associated with loss in egg production (Stipkovits and Kempf, 1996;Landman, 2013), the egg production in the present study was not influenced by the severity of the infection. Our data are in agreement with another study which found no association between MS infection and egg production (Mohammed et al., 1987). A decrease in egg production was noted at 27 week of age which was attributed to a 2% drop of crude protein in layers feed, due to a shortage of soya and maize and subsequent replacement in the ratio with wheat and soya concentrated proteins. Nevertheless, the possibility of tylosin efficacy on retaining the high level of egg production by controlling mycoplasma infections should not be excluded.
The last few years, a novel eggshell pathology, characterized by an altered shell surface, thinning, increased translucency and cracks and breaks in the eggshell apex, has become increasingly common in layer flocks of various breeds in Netherlands (Feberwee et al., 2009), in Italy (Catania et al., 2010) and other countries (Ranck et al., 2010;Strugnell et al., 2011). Eggshell Apex Abnormalities (EAA) are characterized by altered shell surface, shell thinning, increased translucency and the occurrence of cracks and breaks and has been induced or associated with M. synoviae strains with oviduct tropism (Landman, 2013). In the present study, the infection with MS and complicated pathogens showed an increase in broken and misshapen eggs together with discoloration. The time of EAA increase coincided with the increase in egg production and concurrent infection of MG and other pathogens. Landman (2013) reported that the proportion of affected eggs varied between flocks, from a few percent up to 25% and after onset, abnormal eggs were produced throughout Science Publications AJAVS the remainder of the laying period. Catania et al. (2010) found that approximately 1.3% to 1.8% of eggs with EAA resulted in great deal of breakage and spoilage of healthy eggs. In the present study, broken and misshapen eggs reached 3.7% per week and decreased to 1.98% per week in a period of 5 weeks after the tylosin treatment.
Different antimicrobials, especially antibiotics have been used to treat flocks infected with MG and MS in order to reduce severity of clinical signs, lesions and production losses (Bencina, 2005). One of the most widely used molecules in medication programs is tylosin, a macrolide antibiotic. There are several very recent studies that provide evidence of tylosin efficacy under field or experimental conditions. Treatment with tylosin, administered in the drinking water for 5 days, resulted in an immediate improvement of eggshell quality and egg weight in flocks of multi-age layer hens chickens housed on farms in Northern Italy with eggshell abnormalities in the apex of eggs under field conditions, (Catania et al., 2010; concluded that in a challenge study, using a particular MG strain, tylosin proved to be efficacious in protecting against the detrimental consequences of the challenge and there were no significant differences in efficacy of the 35 mg kg −1 BW group compared to the 100 mg kg −1 BW group for most parameters, e.g., clinical respiratory score. Vereecken et al. (2013) investigated the sensitivity for tylosin of 10 European Mycoplasma synoviae strains, isolated out of clinical diseased poultry. The researchers calculated the MIC 90 and MIC 50 values (0.12 and <0.015 µg mL −1 respectively) and found that tylosin can be used effectively under field conditions as a therapeutic for Mycoplasma synoviae infections in poultry. In the presented study and under severe field conditions, a dosage of 105 mg kg −1 BW tylosin tartrate (Pharmasin ® 100% WSG, Huvepharma) was efficient enough to reduce the severity of Mycoplasma infection. The choice to select the maximum dosage was imperative due to increased mortality and the risk of deterioration of the flock's health status. De  stated that "there are important differences in official registered dosage across countries and even more important are the inconsistencies in actual given dosages that can be found all over the world. These differences in actually used dosages cannot solely be attributed to regional differences in susceptibility of MG to tylosin. This means that either under dosing or overdosing, or both are a real problem". Therefore, more research is needed under field conditions in order to establish a dosage that would be efficient and, at the same time, cost effective.

CONCLUSION
In conclusion, Mycoplasma spp causes severe health and production problems in layers. Tylosin tartrate treatment in right time and dosage under field conditions can be used to treat the disease, to improve egg shell quality, to increase average weight of eggs from treated birds and to improve production performances by reducing the economic losses linked to labor costs.