Investigations into Increased Incidence of Severe Gizzard Erosions and Ulcerations in U.S. Commercial Broilers.

During a series of pathology surveys in four production complexes of a U.S. broiler integrator, the technical services veterinarians of an animal health company noted a high incidence of severe gizzard erosions and ulcerations (GEU), prompting further clinical investigation and a battery trial. No growth-promoting antibiotics or ionophore coccidiostats were used during the period of these surveys. All used tribasic copper chloride (TBCC) at ≤120 ppm added copper in broiler rations. Clostridium perfringens was isolated from 83% and 67% of gizzard lesions cultured in two complexes, and cecal C. perfringens most probable number determinations were higher in severely affected than in mildly affected or unaffected birds. Histopathology revealed both acellular koilin fusion defects characteristic of copper toxicity, as well as inflammatory cell infiltrates. Intralesional bacilli suggestive of C. perfringens were noted in 78% of affected flocks examined. Species E Aviadenovirus was isolated from one bird in one complex, and that bird had a single intranuclear inclusion body; no other flocks had Adenoviruses isolated or detected on PCR, nor any inclusion bodies. Other viruses detected were thought to be incidental. A pilot study using feed with supplemental copper from TBCC or copper sulfate and challenge with one of the isolated C. perfringens strains reproduced the lesions. A battery study was conducted with an unchallenged negative control group fed a diet with 16 ppm added copper, a group fed the control diet and orally challenged with 108 organisms of a field strain of C. perfringens at 21 and 22 days, and a group treated with the same diet containing 250 ppm added copper from TBCC and orally challenged with C. perfringens. Birds were necropsied at 23 and 28 days. All challenged groups developed lesions, with those receiving both TBCC and C. perfringens having significantly higher gross and histopathological lesion scores than the unchallenged negative controls. Lesions were qualitatively similar to those in the field and contained suspected C. perfringens bacilli. Because the levels of TBCC used in the commercial birds and in the battery trial generally have been considered safe, and because C. perfringens is usually regarded as a pathogen of the lower GI tract, the possible association of these two agents with GEU is a novel observation and warrants further investigation.

Investigaciones sobre el aumento de la incidencia de erosiones y ulceraciones severas en la molleja en pollos de engorde comerciales en los Estados Unidos. Durante una serie de estudios de patología en cuatro complejos de producción de un integrador de pollos de engorde de los Estados Unidos, veterinarios de servicio técnico de una empresa de salud animal observaron una alta incidencia de erosiones y ulceraciones severas de la molleja (GEU), lo que motivó una mayor investigación clínica y un estudio en batería. Durante el período de estas encuestas no se utilizaron antibióticos promotores del crecimiento ni coccidiostáticos ionóforos. Todos utilizaron cloruro de cobre tribásico (TBCC) con un nivel de ≤120 ppm de cobre agregado en raciones para pollos de engorde. Se aisló Clostridium perfringens del 83% y el 67% de las lesiones de molleja cultivadas en dos complejos, y las determinaciones del número más probable de C. perfringens en los sacos ciegos fueron mayores en aves severamente afectadas que en aves levemente afectadas o no afectadas. La histopatología reveló defectos de fusión de la capa córnea acelular característicos de la toxicidad por cobre, así como infiltrados de células inflamatorias. Se observaron bacilos intralesionales sugestivos de C. perfringens en el 78% de las parvadas afectadas examinadas. La especie Aviadenovirus E se aisló de un ave en un complejo, y esa ave tenía un único cuerpo de inclusión intranuclear; en ninguna otra parvada se aislaron o detectaron adenovirus mediante PCR, ni se observaron cuerpos de inclusión. Se pensó que otros virus detectados fueron incidentales. Un estudio piloto que utilizó alimento con cobre suplementario de cloruro de cobre tribásico o sulfato de cobre y con desafío con una de las cepas aisladas de C. perfringens reprodujo las lesiones. Se realizó un estudio de batería con un grupo de control negativo no desafiado alimentado con una dieta con 16 ppm de cobre agregado, un grupo alimentado con la dieta de control y desafiado por vía oral con 108 organismos de una cepa de campo de C. perfringens a los 21 y 22 días, y un grupo tratado con la misma dieta que contenía 250 ppm de cobre agregado de cloruro de cobre tribásico y desafiados por vía oral con C. perfringens. A las aves se les realizó la necropsia a los 23 y 28 días. Todos los grupos desafiados desarrollaron lesiones, y aquellos que recibieron cloruro de cobre tribásico y C. perfringens tuvieron puntuaciones de lesiones macroscópicas e histopatológicas significativamente más altas que los controles negativos no desafiados. Las lesiones eran cualitativamente similares a las del campo y contenían bacilos sospechosos de C. perfringens. Debido a que los niveles de cloruro de cobre tribásico utilizados en las aves comerciales y en el ensayo en batería generalmente se han considerado seguros, y debido a que C. perfringens generalmente se considera un patógeno del tracto gastrointestinal inferior, la posible asociación de estos dos agentes con erosiones y ulceraciones severas de la molleja es una observación reciente y justifica una mayor investigación.

Mycoplasma anserisalpingitidis sp. nov., isolated from European domestic geese (Anser anser domesticus) with reproductive pathology.

In 1983, Mycoplasma sp. strain 1220 was isolated in Hungary from the phallus lymph of a gander with phallus inflammation. Between 1983 and 2017, Mycoplasma sp. 1220 was also identified and isolated from the respiratory tract, liver, ovary, testis, peritoneum and cloaca of diseased geese in several countries. Seventeen studied strains produced acid from glucose and fructose but did not hydrolyse arginine or urea, and all grew under aerobic, microaerophilic and anaerobic conditions at 35 to 37 ˚C in either SP4 or pleuropneumonia-like organism medium supplemented with glucose and serum. Colonies on agar showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. Molecular characterization included analysis of the following genetic loci: 16S rRNA, 23S rRNA, 16S-23S rRNA ITS, rpoB, rpoC, rpoD, uvrA, parC, topA, dnaE, fusA and pyk. The genome was sequenced for type strain 1220T. The 16S rRNA gene sequences of studied strains of Mycoplasma sp. 1220 shared 99.02-99.19 % nucleotide similarity with M. anatis strains but demonstrated ≤95.00-96.70 % nucleotide similarity to the 16S rRNA genes of other species of the genus Mycoplasma. Phylogenetic, average nucleotide and amino acid identity analyses revealed that the novel species was most closely related to Mycoplasma anatis. Based on the genetic data, we propose a novel species of the genus Mycoplasma, for which the name Mycoplasma anserisalpingitidis sp. nov. is proposed with the type strain 1220T (=ATCC BAA-2147T=NCTC 13513T=DSM 23982T). The G+C content is 26.70 mol%, genome size is 959110 bp.

Focal duodenal necrosis in chickens: attempts to reproduce the disease experimentally and diagnostic considerations.

Focal duodenal necrosis (FDN) is an intestinal disease of egg-layer chickens characterized by multifocal necrosis of the duodenal loop and proximal jejunum. Affected flocks usually have decreased egg weights and drops in egg production. Previous studies have associated this condition with Clostridium perfringens infection. We tried to reproduce FDN by experimental infection of egg-laying chickens using different netB-positive and netB-negative C. perfringens strains, and duodenal homogenate obtained from FDN lesions. Chickens challenged with C. perfringens and/or duodenal homogenate developed duodenitis after challenge. Gross lesions included mucosal erosions, hyperemia, mucosal hemorrhages, and watery intestinal content. Microscopic lesions included mild enterocyte degeneration and necrosis, and mild-to-moderate hemorrhage and lymphoplasmacytic and heterophilic infiltration of the lamina propria. Two netB-positive C. perfringens strains closely related to necrotic enteritis pathogenic strains, by genomic composition, were re-isolated from lesions. Necrosis of intestinal crypts was observed in chickens challenged with duodenal homogenate with or without C. perfringens coinfection. Characteristic microscopic FDN lesions with significant necrosis and loss of villus enterocytes were not reproduced.

Evaluation of protective efficacy of inactivated Mycoplasma synoviae vaccine with different adjuvants.

Mycoplasma synoviae (MS) is a poultry pathogen with a reported distribution throughout the world. Vaccination is utilized as an important component of MS control programs for MS infection. The aim of this study was to evaluate protection efficacy of an inactivated MS vaccine (MS bacterin) with different adjuvants in broilers against a Chinese field isolate (CHN-BZJ2-2015). Vaccination with adjuvants ISA 71 VG and chitosan, respectively, enhanced specific lymphocyte proliferation responses and upregulated the expression of IL-1ß, IL-6, IL-2 and IFN-γ prior to challenge. Furthermore, vaccination with adjuvant ISA 71 VG elicited the highest antibody titers, exhibited significantly lower air sac, foot pad and tracheal lesions than the other groups (P < 0.05), and decreased MS colonization. These results demonstrated that inactivated MS vaccine with ISA 71 VG is able to induce both cellular and humoral immune response in broilers and confers a high level of protection upon challenge, demonstrating a potential application in the field.

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.

Identification of Strain-Specific Sequences That Distinguish a Mycoplasma gallisepticum Vaccine Strain from Field Isolates.

Despite attempts to control avian mycoplasmosis through management, vaccination, and surveillance, Mycoplasma gallisepticum continues to cause significant morbidity, mortality, and economic losses in poultry production. Live attenuated vaccines are commonly used in the poultry industry to control avian mycoplasmosis; unfortunately, some vaccines may revert to virulence and vaccine strains are generally difficult to distinguish from natural field isolates. In order to identify genome differences among vaccine revertants, vaccine strains, and field isolates, whole-genome sequencing of the M. gallisepticum vaccine strain ts-11 and several "ts-11-like" strains isolated from commercial flocks was performed using Illumina and 454 pyrosequencing and the sequenced genomes compared to the M. gallisepticum Rlow reference genome. The collective contigs for each strain were annotated using the fully annotated Mycoplasma reference genome. The analysis revealed genetic differences among vlhA alleles, as well as among genes annotated as coding for a cell wall surface anchor protein (mg0377) and a hypothetical protein gene, mg0359, unique to M. gallisepticum ts-11 vaccine strain. PCR protocols were designed to target 5 sequences unique to the M. gallisepticum ts-11 strain: vlhA3.04a, vlhA3.04b, vlhA3.05, mg0377, and mg0359 All ts-11 isolates were positive for the five gene alleles tested by PCR; however, 5 to 36% of field isolates were also positive for at least one of the alleles tested. A combination of PCR tests for vlhA3.04a, vlhA3.05, and mg0359 was able to distinguish the M. gallisepticum ts-11 vaccine strain from field isolates. This method will further supplement current approaches to quickly distinguish M. gallisepticum vaccine strains from field isolates.

Development, characterization and optimization of a new suspension chicken-induced pluripotent cell line for the production of Newcastle disease vaccine.

Traditionally, substrates for production of viral poultry vaccines have been embryonated eggs or adherent primary cell cultures. The difficulties and cost involved in scaling up these substrates in cases of increased demand have been a limitation for vaccine production. Here, we assess the ability of a newly developed chicken-induced pluripotent cell line, BA3, to support replication and growth of Newcastle disease virus (NDV) LaSota vaccine strain. The characteristics and growth profile of the cells were also investigated. BA3 cells could grow in suspension in different media to a high density of up to 7.0 × 10(6) cells/mL and showed rapid proliferation with doubling time of 21 h. Upon infection, a high virus titer of 1.02 × 10(8) EID50/mL was obtained at 24 h post infection using a multiplicity of infection (MOI) of 5. In addition, the cell line was shown to be free of endogenous and exogenous Avian Leukosis viruses, Reticuloendotheliosis virus, Fowl Adenovirus, Marek's disease virus, and several Mycoplasma species. In conclusion, BA3 cell line is potentially an excellent candidate for vaccine production due to its highly desirable industrially friendly characteristics of growing to high cell density and capability of growth in serum free medium.

Evaluation of the egg transmission and pathogenicity of Mycoplasma gallisepticum isolates genotyped as ts-11.

Live Mycoplasma gallisepticum vaccines are used for the control of respiratory disease, egg production losses and egg transmission associated with M. gallisepticum infection in long-lived poultry. The first field case of apparent increased virulence and vertical transmission of ts-11, a live M. gallisepticum vaccine, has been reported. In that study a M. gallisepticum isolate from the broiler progeny of ts-11-vaccinated breeders was genotyped as ts-11 by sequence analysis of four different genetic targets and Random Amplified Polymorphic DNA and found to be significantly more virulent than ts-11 vaccine. The objective of the current study was to evaluate the rate of egg transmission and pathogenicity of ts-11 vaccine and isolates recovered from ts-11-vaccinated breeders (K6222B) and their broiler progeny (K6216D) which had been genotyped as ts-11. Groups of 28-week-old specific pathogen-free chickens at 87% average weekly egg production were inoculated with sterile broth media (negative controls), ts-11 vaccine, K6222B, K6216D or R strain (positive controls) by eye-drop and aerosol. K6216D transmitted via the egg at an average rate of 4.0% in the third and fourth weeks post-infection, while egg transmission of K6222B and ts-11 vaccine was not detected. M. gallisepticum was isolated from the air sacs, ovaries and oviducts of hens infected with K6216D and K6222B, but not from those infected with ts-11 vaccine. K6216D and K6222B both induced respiratory signs and significantly more tracheal colonization and more severe tracheal and air sac lesions than ts-11 vaccine (P ≤ 0.05). There were no substantial differences in the egg production of ts-11, K6216D and K6222B infected groups. These results provide the first conclusive evidence of transovarian transmission of an isolate genotyped as ts-11 and indicate that isolates genotyed as ts-11 vary in their virulence and ability to transmit via the egg.

Comparison of multiple genes and 16S-23S rRNA intergenic space region for their capacity in high resolution melt curve analysis to differentiate Mycoplasma gallisepticum vaccine strain ts-11 from field strains.

Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses in the global poultry industry. In an attempt to compare and evaluate existing genotyping methods for differentiation of MG strains/isolates, high resolution melt (HRM) curve analysis was applied to 5 different PCR methods targeting vlhA, pvpA, gapA, mgc2 genes and 16S-23S rRNA intergenic space region (IGSR). To assess the discriminatory power of PCR-HRM of examined genes and IGSR, MG strains ts-11, F, 6/85 and S6, and, initially, 8 field isolates were tested. All MG strains/isolates were differentiated using PCR-HRM curve analysis and genotype confidence percentage (GCP) values of vlhA and pvpA genes, while only 0, 3 and 4 out of 12 MG strains/isolates were differentiated using gapA, mgc2 genes and IGSR, respectively. The HRM curve analysis of vlhA and pvpA genes was found to be highly correlated with the genetic diversity of the targeted genes confirmed by sequence analysis of amplicons generated from MG strains. The potential of the vlhA and pvpA genes was also demonstrated for genotyping of 12 additional MG strains from Europe and the USA. Results from this study provide a direct comparison between genes previously used in sequencing-based genotyping methods for MG strain identification and highlight the usefulness of vlhA and pvpA HRM curve analyses as rapid and reliable tools specially for diagnosis and differentiation of MG strains used here.

The development and application of a Mycoplasma gallisepticum sequence database.

Molecular analysis was conducted on 36 Mycoplasma gallisepticum DNA extracts from tracheal swab samples of commercial poultry in seven South African provinces between 2009 and 2012. Twelve unique M. gallisepticum genotypes were identified by polymerase chain reaction and sequence analysis of the 16S-23S rRNA intergenic spacer region (IGSR), M. gallisepticum cytadhesin 2 (mgc2), MGA_0319 and gapA genetic regions. The DNA sequences of these genotypes were distinct from those of M. gallisepticum isolates in a database composed of sequences from other countries, vaccine and reference strains. The most prevalent genotype (SA-WT#7) was detected in samples from commercial broilers, broiler breeders and layers in five provinces. South African M. gallisepticum sequences were more similar to those of the live vaccines commercially available in South Africa, but were distinct from that of F strain vaccine, which is not registered for use in South Africa. The IGSR, mgc2 or MGA_0319 sequences of three South African genotypes were identical to those of the ts-11 vaccine strain, necessitating a combination of mgc2 and IGSR targeted sequencing to differentiate South African wild-type genotypes from ts-11 vaccine. To identify and differentiate all 12 wild-types, mgc2, IGSR and MGA_0319 sequencing was required. Sequencing of gapA was least effective at strain differentiation. This research serves as a model for the development of an M. gallisepticum sequence database, and illustrates its application to characterize M. gallisepticum genotypes, select diagnostic tests and better understand the epidemiology of M. gallisepticum.

Influence of swab material on the detection of Mycoplasma gallisepticum and Mycoplasma synoviae by real-time PCR.

Recent reports have shown an increased recovery of cells from flocked nylon swabs which may improve the specimen quality and the real sensitivity of diagnostic tests in a clinical setting. In this study, the detection of Mycoplasma gallisepticum (MG) and M. synoviae (MS), using dry swabs of different materials (nylon flocked, cotton, and polyester), was investigated using real-time TaqMan PCR protocols. Different types of samples, including dilutions of pure broth cultures of MG and MS as well as swabs from tracheas of experimentally infected chickens and field cases of infection, were analyzed. There were no statistical differences in real-time PCR results among the different swab types (P < 0.05), indicating that this is not likely to be a significant factor in MG and MS detection by this method.

Ultrafast evolution and loss of CRISPRs following a host shift in a novel wildlife pathogen, Mycoplasma gallisepticum.

Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ∼2% of ancestral poultry strains and a nucleotide substitution rate of 0.8-1.2×10(-5) per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ∼50% of the CRISPR repertoire founding (1994-95) strains and have lost the CRISPR-associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs.

Detection and differentiation of avian mycoplasmas by surface-enhanced Raman spectroscopy based on a silver nanorod array.

Mycoplasma gallisepticum is a bacterial pathogen of poultry that is estimated to cause annual losses exceeding $780 million. The National Poultry Improvement Plan guidelines recommend regular surveillance and intervention strategies to contain M. gallisepticum infections and ensure mycoplasma-free avian stocks, but several factors make detection of M. gallisepticum and diagnosis of M. gallisepticum infection a major challenge. Current techniques are laborious, require special expertise, and are typically plagued by false results. In this study, we describe a novel detection strategy which uses silver nanorod array-surface-enhanced Raman spectroscopy (NA-SERS) for direct detection of avian mycoplasmas. As a proof of concept for use in avian diagnostics, we used NA-SERS to detect and differentiate multiple strains of avian mycoplasma species, including Acholeplasma laidlawii, Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma synoviae, and M. gallisepticum, including vaccine strains 6/85, F, and ts-11. Chemometric multivariate analysis of spectral data was used to classify these species rapidly and accurately, with >93% sensitivity and specificity. Furthermore, NA-SERS had a lower limit of detection that was 100-fold greater than that of standard PCR and comparable to that of real-time quantitative PCR. Detection of M. gallisepticum in choanal cleft swabs from experimentally infected birds yielded good sensitivity and specificity, suggesting that NA-SERS is applicable for clinical detection.

Molecular characterization of Mycoplasma gallisepticum isolates from Jordan.

Two groups of Mycoplasma gallisepticum (MG) isolates (n = 24) from Jordan were analyzed by molecular methods and compared with other Middle Eastern isolates, related international isolates, and reference strains. The first group (n = 19) was isolated from July 2004 to January 2005 (isolation period A), and the newer group (n = 5) from June 2007 to April 2008 (isolation period B). The groups of isolates are from chicken flocks from northern Jordan, but are not from the same farms. None of the flocks were vaccinated for MG. Random amplified polymorphic DNA analysis, targeted sequencing of the partial MG cytadhesin 2 (mgc2), and the MG 16S-23S rRNA intergenic spacer region (IGSR) divided the Jordanian isolates into two groups. All of the 19 isolates from time period A, in addition to two isolates from time period B, were indistinguishable from the F strain. Three of five isolates from time period B were characterized as wild types and were indistinguishable from each other. The wild-type field strain was readily distinguished from the F strain. It was 91% and 96.4% similar to the F strain based on Clustal-W alignments of sequences of mgc2 and IGSR, respectively. Sequence similarity of mgc2 gene of the Jordan wild-type strain to isolates from Israel and Egypt ranged from 96.5% to 100%, whereas for IGSR it was 99.4%-100%. We theorize that the F-strain live MG vaccine, commonly used in Jordan prior to 2007, was transmitted to nonvaccinated poultry in the region and was a predominant genotype during time period A.