Spectral technique for detection of changes in eggshells caused by Mycoplasma synoviae.

Mycoplasma synoviae (MS) is a major pathogen in chicken and turkeys, causing subclinical infection. MS infections are highly prevalent and may potentate and be involved in sinovitis, respiratory syndromes, as well as lead to eggshell apex abnormality (EAA). A deformed, inhomogeneous eggshell is susceptible to cracks and breaks through which microbes get in and additionally entails higher water loss in the egg during the entire incubation process. Not all eggs with eggshell apex abnormality possess characteristic deformation and that is why some eggs may be incorrectly classified during a visual inspection. To minimize the above risk, the spectral VIS technique and the analysis based on the classification tree method-CTM is proposed. The method makes use of specially defined parameters extracted from the shape of transmittance spectra of eggshells. Directional coefficients of the lines adjusted to the specific ranges of the transmittance spectrum are used in the process of classifying samples as those from MS-carrying hens and from healthy hens. Three CTM-based classifiers were created for a group of white, brown, and mixed shells. After comparing, it can be concluded that the best results were obtained for the group of brown shells (accuracy 88%, specificity 88%, and false negative rate 13%). The authors present a non-invasive spectral method that utilizes eggshells, i.e., the natural waste from chicken farms. The method enables entering data into the classifiers described in the article. The process provides an opportunity to correctly assign, the examined shell to the group of shells with increased risk-with approx. 86% accuracy. This means that, if a few of such results are registered, the herd is eligible more specific studies targeting MS bacteria. Regular spectral testing can support the detection of egg lesions in MS positive flocks.

Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics.

The temperature-sensitive (ts+) Mycoplasma synoviae vaccine strain MS-H harbors a non-synonymous mutation which results in Glycine to Arginine substitution at position 123 in the highly conserved glycine-rich motif of Obg-fold in the GTP-binding protein Obg. In-silico analysis of the wild-type and mutant Obgs of M. synoviae has indicated that this amino acid substitution affects structure of the protein, potentially leading to abrogation of Obg function in vivo. Present study was conducted to develop the first expression vector for M. synoviae and to investigate the potential effect(s) of complementation of MS-H vaccine with the wild-type obg from 86079/7NS, the parent strain of MS-H. An oriC vector, pKS-VOTL, harboring the 86079/7NS obg gene, downstream of the variable lipoprotein haemagglutinin (vlhA) gene promoter, also cloned from 86079/7NS, was used to transform MS-H. The plasmid was localised at the chromosomal oriC locus of MS-H without any detectable integration at the chromosomal obg locus. Analysis of the MS-H transformants revealed abundant obg transcripts as well as Obg protein, when compared to the MS-H transformed with a similar vector, pMAS-LoriC, lacking obg coding sequence. The MS-H transformants complemented with wild-type Obg maintained their original temperature-sensitivity phenotype (consistent with MS-H vaccine) but, when compared to the MS-H transformed with pMAS-LoriC, had significantly higher (p < 0.05) growth rate and viability at the permissive (33°C) and non-permissive temperature (39.5°C), respectively. Analysis of Obg expression in MS-H and its wild-type parent strain revealed comparatively lower levels of Obg in MS-H. These results indicate that not only the mutation in Obg, but also the level of Obg expression, can confer functional abnormalities in the bacterial host. Furthermore, with the construction of first expression vector for M. synoviae, this study has set foundation for the development of recombinant vaccine(s) based on MS-H.

RETRACTED: Mycoplasma synoviae vaccine modifies virus shedding and immune responses of avian influenza (H9N2) infection in commercial layers.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The authors retract the above paper due to: 1) conflict of interest among the authors; and 2) addition of co-author Dr. Muhammad Younus without his knowledge or permission. The authors apologize for these two grave mistakes.

Serological investigation of Mycoplasma synoviae infection in China from 2010 to 2015.

Mycoplasma synoviae can cause worldwide respiratory diseases, synovitis, or subclinical symptoms in poultry. M. synoviae infection occurs throughout the yr and causes significant economic losses, including increased mortality, condemnations, medication, and live production cost. In the present study, the seroprevalence of M. synoviae among 44,395 non-vaccinated chickens from 21 provinces in China from 2010 to 2015 was estimated using ELISA. The overall seroprevalence was 41.19%. Seropositive rates in different yr ranged between 24.70 and 57.20%; the highest seropositive rate was observed in 2010, and the lowest was observed in 2013. The prevalence rates varied greatly in different provinces from 5.10 to 100%. Of the 463 commercial flocks tested, 375 (80.99%) were positive for M. synoviae by ELISA. The seasonal distribution ranged between 26.83% (in October) and 53.98% (in July). An investigation of chickens according to age further showed that M. synoviae can infect chickens at any age. Our findings indicate that M. synoviae infection is very common in China and should prompt further research into its prevalence to develop effective control and prevention strategies.

Development of Multilocus Sequence Typing (MLST) for Mycoplasma synoviae.

Mycoplasma synoviae (MS) is a poultry pathogen that has had an increasing incidence and economic impact over the past few years. Strain identification is necessary for outbreak investigation, infection source identification, and facilitating prevention and control as well as eradication efforts. Currently, a segment of the variable lipoprotein hemagglutinin A (vlhA) gene (420 bp) is the only target that is used for MS strain identification. A major limitation of this assay is that colonality of typed samples can only be inferred if their vlhA sequences are identical; however, if their sequences are different, the degree of relatedness is uncertain. In this study we propose a multilocus sequence typing (MLST) assay to further refine MS strain identification. After initial screening of 24 housekeeping genes as potential targets, seven genes were selected for the MLST assay. An internal segment (450-711 bp) from each of the seven genes was successfully amplified and sequenced from 58 different MS strains and field isolates (n = 30) or positive clinical samples (n = 28). The collective sequence of all seven gene segments (3960 bp total) was used for MS sequence typing. The 58 tested MS samples were typed into 30 different sequence types using the MLST assay and, coincidentally, all the samples were typed into 30 sequence types using the vlhA assay. However, the phylogenetic tree generated using the MLST data was more congruent to the epidemiologic information than was the tree generated by the vlhA assay. We suggest that the newly developed MLST assay and the vlhA assay could be used in tandem for MS typing. The MLST assay will be a valuable and more reliable tool for MS sequence typing, providing better understanding of the epidemiology of MS infection. This in turn will aid disease prevention, control, and eradication efforts.

Quantification of the horizontal transmission of Mycoplasma synoviae in non-vaccinated and MS-H-vaccinated layers.

The number of newly infected birds attributable to one infectious bird per day (= transmission rate ß) was assessed in non-vaccinated and MS-H-vaccinated experimental specified pathogen-free White Leghorns after Mycoplasma synoviae challenge. Furthermore, the effect of vaccination on the shedding of the challenge strain was determined. The following groups were made: a negative control group (n = 5), a vaccinated (MS-H vaccine by eye drop (>105.7 colour changing units/bird)) non-challenged group (n = 5), two non-vaccinated challenged groups (n = 18 each) and two vaccinated challenged groups (n = 18 each). In the challenged groups, six seeder birds were intratracheally inoculated with 105.4 colony forming units (CFUs)/bird. Trachea swabs were taken at day (D)2, D3, D4, D5, D7, D9, D11, D14, D17, D21, D25, D28, D32, D35, D42 and D46 after contact with seeders and analyzed with a quantitative PCR able to detect the vaccine and field strain separately. The transmission rate and shedding were estimated using the susceptible exposed infectious transmission model and a linear mixed model, respectively. The mean shedding of the challenge strain was 106.4 CFU equivalents M. synoviae/g trachea mucus in vaccinates shedding MS-H, while in the birds not shedding the vaccine (non-vaccinates and vaccinates not shedding MS-H) it was 106.9 CFU equivalents M. synoviae/g trachea mucus. In vaccinates shedding MS-H, ß was 0.0012 (95% C.I.: 0.00048 - 0.0024), while in birds not shedding vaccine (non-vaccinates and vaccinates not shedding MS-H) a significantly higher ß of 0.022 (95% C.I.: 0.015 - 0.031) was found.

Phylogenetic and pathogenic analysis of Mycoplasma Synoviae isolated from native chicken breeds in China.

Mycoplasma synoviae (M. synoviae) infection leads to serious economic losses in the world every year. Between 2013 and 2014, the infectious synovitis, caused by M. synoviae infection, occurred in native chickens in China and resulted in the loss of millions of chickens in Chinese poultry farms. However, there has been no data about phylogenetic and pathogenic analysis of Chinese M. synoviae isolates. In this study, a total of 110 M. synoviae strains were isolated from M. synoviae infected chickens. The isolates identified in the present study were classified into a new distinct subgroup based on analysis of the 5'-end vlhA sequences, tentatively termed the K group. In addition, though the pathogenicity was significantly different among isolates, there was no close relationship between pathogenicity and genotype for Chinese M. synoviae based on a pathogenic analysis of the 5'-end of the vlhA gene.

Two strains of Mycoplasma synoviae from chicken flocks on the same layer farm differ in their ability to produce eggshell apex abnormality.

Mycoplasma synoviae (Ms) is considered to be an economically important poultry pathogen. Although the full economic costs of infection in layer chickens are still under debate, the prevalence of Ms is known to be high in some countries and earlier reports have shown a correlation between infection and Eggshell Apex Abnormality (EAA). This work is a continuation of an earlier study of a clinical case of EAA on a layer hen farm where the presence of two different strains of Ms, based on the sequence of the 5' end of the vlhA gene, was demonstrated. Both strains could be detected in the trachea but only one (designated strain PASC8) appeared able to colonize the oviduct, while the other (designated TRACH) was not found in the oviduct and has not been related to EAA. The PASC8 partial vlhA gene sequence differs from that of the TRACH in having a 39 nucleotide deletion in the proline rich region and three point mutations in the RIII region. Based on this information an experimental infection was performed in SPF chickens using groups infected with either the PASC8 or the TRACH strain and a non-infected control group. Both Ms strains were detected in the trachea of infected birds, but only the PASC8 strain was found in the oviduct. Furthermore, EAA developed only in the group infected with PASC8 strain. Compared to the control group, both strains produced an adverse impact on egg production: a decrease in the numbers laid and in their average weight (P<0.05) This work demonstrates a difference in oviduct tropism between two Ms strains and a possible relationship to the production of EAA in experimental conditions.

Laboratory investigations into the origin of Mycoplasma synoviae isolated from a lesser flamingo (Phoeniconaias minor).

BACKGROUND: The role of wild birds in the transmission and spread of mycoplasmas is not clear. Up to now different Mycoplasma species have been isolated from wild birds many of which are not considered pathogens sensu stricto for domestic flocks. This report describes the first isolation of Mycoplasma synoviae in a captive lesser flamingo (Phoeniconaias minor) held in a zoo in Italy and the laboratory investigations performed to elucidate its origin. Results showed that the strain was similar to the MS-H vaccine strain using the vlhA methods although no vaccination with this product was used in the zoo. CASE PRESENTATION: This paper describes investigations into a case in which 10 of 12 adult lesser flamingos (Phoeniconaias minor) died after having recently been moved from the Netherlands to a new zoo in Northern Italy. While most of the birds appeared to have died from the stress of movement and poor adaptation to their new environment, Mycoplasma synoviae, an important poultry pathogen in the layer and meat industry, was isolated for the first time from the trachea of one animal presenting catarrhal tracheitis and fibrinous airsacculitis. Genetic analysis of the conserved region of the vlhA was not able to differentiate the flamingo strain from the MS-H vaccine strain. However differences in the sequences of the obg gene of the flamingo and vaccine strain were detected. A test for temperature-sensitivity (ts) gave a ts (-) phenotype for the flamingo strain, in contrast to the ts (+) status of the MS-H strain. Based on this information and knowing that the flamingos were not vaccinated against M. synoviae, it is highly likely that the flamingo was infected with a genetically similar wild strain by contact with infected birds. CONCLUSIONS: This case provides evidence for the potential role of international trade of ornamental birds as a possible route of introduction of new mycoplasma strains between countries, and moreover highlight that vlhA gene sequencing was not sufficient to discriminate the wild strain isolated from the flamingo from the MS-H vaccine strain.

The effect of eggshell apex abnormalities on table egg quality during storage in 2 seasons of the year.

Mycoplasma synoviae infection of hens has been associated with problems of eggshell quality called eggshell apex abnormalities (EAA). Little is known about the quality of EAA eggs from a commercial point of view, especially during their storage. The study aimed to examine the differences between EAA and normal eggs during storage under controlled conditions in 2 seasons, summer and winter, by comparing internal and external quality parameters. In a conventional egg production farm with white laying hens of varying ages in the city of Bastos, state of São Paulo, Brazil, 232 eggs were used in the summer season and 400 eggs in the winter season. Half of the eggs had EAA, and the other half were considered normal eggs for each season. The eggs were analyzed at 2, 7, 14, 21, and 28 d after being laid and stored from 24.6 to 25.8°C in summer and from 24 to 25°C in winter. There was no difference (P > 0.05) in the average egg weight between EAA and normal eggs at any studied time point, but in both seasons, the weight loss in EAA eggs was higher than in normal eggs. The losses in Haugh unit scores from the first to the last measurements were approximately 40% regardless of egg type or season of production. In comparing eggshell thickness, only the apices of the EAA eggs were thinner (P < 0.0001) than normal eggs in the summer, but in the winter, the EAA egg apices (P < 0.0001) and sides (P = 0.03) were both thinner. The presence of EAA did not affect the eggshell weight (P > 0.05) or eggshell percentage (P > 0.05). The eggshell strength of the EAA eggs was lower (P < 0.0001) than normal eggs in both the summer (16.57%) and winter (19.86%). The presence of EAA did not affect the internal quality of the egg, but was related to a greater loss of external quality and weight during storage.

Eggshell apex abnormalities associated with Mycoplasma synoviae infection in layers.

Eggs exhibiting eggshell apex abnormalities (EAA) were evaluated for changes in shell characteristics such as strength, thickness, and ultrastructure. Mycoplasma synoviae (MS) infection was confirmed by serological assay along with isolation of MS from the trachea and oviduct. Changes in eggshell quality were shown to be statistically significant (p < 0.01). We also identified ultrastructural changes in the mammillary knob layer by Scanning Electron Microscopy. While eggs may seem to be structurally sound, ultrastructural evaluation showed that affected eggs do not regain their former quality. In our knowledge, this is the first report describing the occurrence of EAA in Korea.

Extended survival times of Mycoplasma gallisepticum and Mycoplasma synoviae on kanekalon synthetic hair fibres.

The survival times of Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) on washed and unwashed natural and synthetic kanekalon hair samples over a 5-d period were evaluated using the color changing unit method for comparison with results of previous studies conducted on natural hair. Regardless of whether synthetic or natural hair samples prewashed with a disinfectant shampoo were spiked with Mg or Ms, all viable organisms rapidly dropped below a count of 1 × 10(1)/mL of culture. Unwashed natural hair seeded with a titer of approximately 1 × 10(6)/mL of viable Mg or Ms decreased to 6 × 10(5)/mL and 6 × 10(3)/mL, respectively, by 4 h postseeding, but no viable Mg or Ms were detected on natural hair from 8 h onwards. By contrast, the titers of Mg and Ms on synthetic hair did not decline from the initial 1 × 10(6)/mL seed dose up to 96 h postseeding, and, in fact, viable Mg and Ms was still detectable at 9 d postinfection. Application of a real-time quantitative single-tube duplex PCR assay confirmed that no proliferation of Mg or Ms had occurred on the synthetic hair samples, the cells simply remained viable. The unexpected finding that Mg and Ms survive for extended periods on synthetic kanekalon hair fibers raises the question of whether attachment to a surface is a prerequisite for the survival and persistence of Mg and Ms in the extra-host environment. Future studies should be aimed at determining whether other synthetic hair types or indeed other types of plastics commonly found in the poultry house offer similar survival advantages to mycoplasmas.

Eggshell apex abnormalities associated with Mycoplasma synoviae infection in layers

Eggs exhibiting eggshell apex abnormalities (EAA) were evaluated for changes in shell characteristics such as strength, thickness, and ultrastructure. Mycoplasma synoviae (MS) infection was confirmed by serological assay along with isolation of MS from the trachea and oviduct. Changes in eggshell quality were shown to be statistically significant (p < 0.01). We also identified ultrastructural changes in the mammillary knob layer by Scanning Electron Microscopy. While eggs may seem to be structurally sound, ultrastructural evaluation showed that affected eggs do not regain their former quality. In our knowledge, this is the first report describing the occurrence of EAA in Korea.

Effect of Mycoplasma synoviae and lentogenic Newcastle disease virus coinfection on cytokine and chemokine gene expression in chicken embryos.

Mycoplasma synoviae and Newcastle disease virus (NDV) are 2 avian pathogens that cause modulation in expression of a variety of cytokine and chemokine genes in chickens. However, there is limited data about gene modulation after coinfection with these 2 pathogens and even less data about gene modulation after infection of chicken embryos. In this study, the effect of M. synoviae type strain WVU 1853 and lentogenic LaSota vaccine strain of NDV infection on cytokine and chemokine gene expression in chicken embryos was analyzed in the liver, spleen, bursa of Fabricius, and thymus by using quantitative real-time PCR. Three types of infection were performed; infection with M. synoviae on d 10, infection with NDV on d 17; and consecutive infection with both pathogens, where M. synoviae was inoculated on d 10 and NDV on d 17. Thus, simulation of consecutive infection that may occur after NDV infection of the M. synoviae-infected host was performed. Mycoplasma synoviae infection of embryos resulted in intensive upregulation of cytokine and chemokine genes, including interferon (IFN)-γ, IL-1ß, IL-6, IL-12p40, IL-16, IL-18, MIP-1ß (CCL4), inducible nitric oxide synthase (iNOS), XCL1, and lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF), with different expression profiles in the 4 organs. Inoculation of lentogenic NDV significantly upregulated IFN-γ, IL-6, and IL-16 genes in spleen and IFN-γ, IL-1ß, IL-2, IL-16, IL-21, XCL1, and MIP-1ß (CCL4) genes in the thymus, but to a lesser extent than M. synoviae. However, no genes were upregulated by NDV in the liver and bursa of Fabricius. Overall effect of NDV inoculation, regarding the number of modulated cytokine and chemokine genes and the extent of expression, was lower than M. synoviae. When NDV was introduced after on-going M. synoviae infection, most M. synoviae-induced cytokine and chemokine genes were significantly downregulated. This study provides the first evidence in chicken embryos that consecutive infection with NDV could suppress expression of cytokine and chemokine genes being significantly upregulated by the previous M. synoviae infection.

Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes.

The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans.

Mycoplasma synoviae cell invasion: elucidation of the Mycoplasma pathogenesis in chicken.

Fluorochrome-labelled cells of two field isolates and Mycoplasma synoviae (Ms) were inoculated onto monolayer cultures of fluorochrome-labelled HEp-2 cells and monitored by confocal laser scanning microscopy (CLSM). Ms was detected initially adhered to and subsequently inside the host cells. Between 24 and 48 h of infection, Ms was detected in the perinuclear region, and after 72 h of infection was confirmed by gentamicin invasion assay. High and low passage Ms strains showed no differences in adherence or invasion. The morphology and the actin filaments of the infected HEp-2 cells were preserved throughout the study period. The observed invasion by Ms is consistent with the biology of Mollicutes, and could explain the difficulties in recovering field isolates of the mycoplasma and in controlling the infection in birds even after long-term antibiotic treatment.

Induction of eggshell apex abnormalities in broiler breeder hens.

Recently, the causal relationship between eggshell apex abnormalities (EAA) and Mycoplasma synoviae was described. This eggshell pathology has only been documented in table egg layers both spontaneously and experimentally infected with M. synoviae, suggesting that meat-type layers are less prone to this condition. In this study the susceptibility of specified pathogen free (SPF) broiler breeder hens to produce eggs with EAA after M. synoviae infection was assessed. Five groups of 12 hens each were made: a negative control group, a group inoculated intratracheally (i.t.) with a M. synoviae EAA strain at 19 weeks of age, a group inoculated i.t. with this strain at 19 and 26 weeks of age, a group inoculated with M. synoviae i.t. at 19 weeks of age and infected 5 days earlier with infectious bronchitis virus D1466 (IBV), and a fifth group similar to the former but inoculated i.t. twice with an M. synoviae EAA strain at 19 and 26 weeks of age. Eggs with EAA were only produced after a single i.t. inoculation with the M. synoviae EAA strain if preceded by an infection with IBV. The production of eggs with EAA started 6 weeks after M. synoviae EAA inoculation and the proportion of eggs with EAA during the experiment was 9/449 (2%), which was much lower than that in SPF layer hens (14-22%). The present results suggest that broiler breeder hens are less susceptible to producing eggs with EAA after an infection with a M. synoviae EAA strain preceded by an IBV infection, compared with table egg layers. Similar to SPF egg layers, the mean daily egg production per hen was significantly reduced by the M. synoviae EAA strain and there was a general negative effect on eggshell strength by this strain, suggesting it could also have a detrimental effect on hatching egg quality.

Gene expression modulation in chicken macrophages exposed to Mycoplasma synoviae or Escherichia coli.

Mycoplasma synoviae and Escherichia coli are two avian pathogens that exhibit markedly different mechanisms for infection and pathogenicity and may be expected to manipulate the host innate immune response differently. The aim of this study was to determine the extent of modulated genes and make a comparison between the transcriptomes of chicken macrophages exposed to either M. synoviae type strain WVU 1853 (MS) or avian pathogenic E. coli strain V-G (APEC). To analyze temporal gene expression profile of monocyte-derived macrophages (MDM) and HD11 cell line macrophages after each exposure, two avian immunity microarrays were used: the avian macrophage microarray (AMM) and the avian innate immunity microarray (AIIM). The quantity of MS-modulated genes was estimated in three experiments, using both microarrays. A cross-section revealed 14 AMM/AIIM genetic elements that were modulated in both types of macrophages. Additionally, to compare immunomodulatory activity of MS and APEC, MDM were exposed to each pathogen and gene modulation was detected by AIIM microarray. This study revealed 157 elements uniquely modulated by MS and 1603 elements uniquely modulated by APEC. AIIM microarray analysis also revealed a core set of 146 elements modulated by both pathogens, with generally higher induction/repression levels after APEC exposure. Validation of selected gene expression was done by quantitative real time RT-PCR. The study shows higher transcription levels of IL-1beta, IL-6, iNOS, NCF1, peroxiredoxin 1 and cathepsin L genes after MDM exposure to APEC than after exposure to MS. Surprisingly, complement component C3 gene was repressed after MDM exposure to APEC, while being induced after exposure to MS.

Role of Mycoplasma synoviae in commercial layer Escherichia coli peritonitis syndrome.

Mycoplasma synoviae (MS) is an important pathogen of domestic poultry and is prevalent in commercial layers. During the last decade Escherichia coli peritonitis became a major cause of layer mortality. The possible role of MS in the E. coli peritonitis syndrome of laying hens was studied. Four groups of 64 mycoplasma-free commercial layers at the onset of lay (about 80% daily production) were challenged with a virulent MS strain or a virulent avian E. coli strain or both. The four experimental groups were identified as follows: negative control, E. coli, MS, and MS plus E. coli. A typical E. coli peritonitis mortality was reproduced and included one, three, zero, and five birds in the negative control, E. coli, MS, and MS plus E. coli groups, respectively. Only the increased mortality in the MS plus E. coli group had statistical significance. Four weeks postchallenge 10 clinically normal birds from each of the four experimental groups were necropsied. All of the examined birds in the two MS-challenged groups demonstrated severe tracheal lesions. Body cavity lesions were detected in two and four birds in the MS and MS plus E. coli groups, respectively. The results demonstrate a possible pathogenesis mechanism of respiratory origin with regard to the layer E. coli peritonitis syndrome, show the MS pathological effect in layers, and indicate that a virulent MS strain can act as a complicating factor in the layer E. coli peritonitis syndrome.

Serological responses of chickens to low challenge doses of Mycoplasma synoviae.

Groups of eight chickens were challenged with 10-fold dilutions of one of two strains of Mycoplasma synoviae (MS); each challenge group contained two noninfected sentinels. Both strains were highly efficient in colonizing the respiratory tract with challenge doses as low as 76 and 24 color-changing units/bird. Infection spread rapidly (within 7 days) to sentinels, while uninfected control chickens separated from infected chickens by two empty pens remained uninfected for the 56-day experimental period. Although sentinels and birds challenged with the lowest doses had weaker or slightly slower antibody responses in some cases as measured by serum plate agglutination, enzyme-linked immunosorbent assay (ELISA), and hemagglutination inhibition (HI), they generally exhibited a typical antibody response. Agglutination reactions tended to be weak, but a high percentage of tests (generally >30% from day 14 postchallenge) were positive. ELISA results were variable, and in some cases reactor rates were low (generally <20%), even though the chickens were colonized in the upper respiratory tract. The HI test was reliable in detecting infected groups; usually >50% were positive from 14 days postchallenge. Mean HI titers were higher when using hemagglutination antigens prepared from the homologous MS strain as compared with antigen prepared from the heterologous strain or with standard antigen prepared from WVU 1853.