Avian Orthoavulavirus 1 Vaccination Failure in Minnesota Turkeys.

Minnesota is the leading state in number of turkeys produced in the United States. Turkey flocks in the field are usually vaccinated several times with live avian orthoavulavirus 1 (AOAV-1) vaccines starting as early as 2 wk of age (WOA). During the years 2018-2019, many turkey flocks were diagnosed with low-virulence AOAV-1 infection around 9 WOA that led to respiratory disease, although they were previously vaccinated. This study was designed to investigate the immunity against AOAV-1 in Minnesota turkey flocks in the field and experimentally after vaccination. We reviewed antibody titers against AOAV-1 from turkey flocks tested by ELISA at Minnesota Poultry Testing Laboratory (n = 1292). Up to 9 WOA, more than 85% of the field flocks tested had unprotective antibody titers against AOAV-1. However, commercial poults at 3 WOA experimentally vaccinated by eye-drop method had an ELISA geometric mean titer of 6011 at 7 WOA. Oropharyngeal virus shedding after vaccination was 10%, 70%, 80%, and 40% at 1, 3, 5, and 7 days postvaccination, respectively. This study demonstrates that experimentally vaccinated turkeys respond very well to AOAV-1 vaccine when properly administered. However, there is clear vaccination failure in the field, where vaccine is commonly administered in drinking water, a method that is more susceptible to failure because of many variables in this procedure. We recommend choosing the most effective method of vaccine administration. Given the high incidence of inadequate immunity induced in commercial turkeys on mass application of live AOAV-1 vaccines in water, alternative application methods and subsequent monitoring of the serologic antibody response must be undertaken to ensure a proper immune response.

Artículo regular­Fracaso de la vacunación contra el Orthoavulavirus aviar 1 en pavos de Minnesota. Minnesota es el estado líder en número de pavos producidos en los Estados Unidos. Las parvadas de pavos en el campo generalmente se vacunan varias veces con vacunas vivas con Orthoavulavirus Aviar 1 (AOAV-1) comenzando desde las 2 semanas de edad (WOA). Durante los años 2018­2019, muchas parvadas de pavos fueron diagnosticadas con infección por Orthoavulavirus Aviar 1 de baja virulencia alrededor de las nueve semanas de edad que condujeron a una enfermedad respiratoria, aunque las aves fueron vacunadas previamente. Este estudio fue diseñado para investigar la inmunidad contra Orthoavulavirus Aviar 1 en parvadas de pavos de Minnesota en el campo y experimentalmente después de la vacunación. Se revisaron los títulos de anticuerpos contra Orthoavulavirus Aviar 1 de parvadas de pavos analizados por ELISA en el Laboratorio de Diagnóstico Avícola de Minnesota (n = 1292). Hasta las nueve semanas de edad, más del 85% de las parvadas de campo analizadas tenían títulos de anticuerpos no protectores contra Orthoavulavirus Aviar 1. Sin embargo, los pavipollos comerciales a las tres semanas de edad vacunados experimentalmente por el método de gota ocular tenían un título medio geométrico de ELISA de 6011 a las siete semanas de edad. La diseminación del virus orofaríngeo después de la vacunación fue del 10%, 70%, 80% y 40% a los 1, 3, 5 y 7 días después de la vacunación, respectivamente. Este estudio demuestra que los pavos vacunados experimentalmente respondieron muy bien a la vacuna con Orthoavulavirus Aviar 1 cuando se administra correctamente. Sin embargo, existe un claro fracaso de la vacunación en el campo, donde la vacuna se administra comúnmente en el agua potable, un método que es más susceptible al fracaso debido a muchas variables en este procedimiento. Se recomienda elegir el método de administración de vacunas más eficaz. Considerando la alta incidencia de inmunidad inadecuada inducida en pavos comerciales con la aplicación masiva de vacunas vivas con Orthoavulavirus Aviar 1 en agua, se deben llevar a cabo métodos de aplicación alternativos y monitoreo posterior de la respuesta de anticuerpos serológicos para asegurar una respuesta inmune adecuada.

Characterization of H9N2 avian influenza viruses from the Middle East demonstrates heterogeneity at amino acid position 226 in the hemagglutinin and potential for transmission to mammals.

Next-generation sequencing (NGS) technologies are a valuable tool to monitor changes in viral genomes and determine the genetic heterogeneity of viruses. In this study, NGS was applied to clinical poultry samples from Jordan to detect eleven H9N2 low pathogenic avian influenza viruses (LPAIV). All of the viruses tested belonged to Middle East A genetic group of G1 lineage. Deep sequencing demonstrated a high degree of heterogeneity of glutamine and leucine residues at position 226 in the hemagglutinin (HA) gene, which increases specificity to either avian or mammalian-type receptors. Moreover, additional amino acid changes in PB1, PA, M1, M2, and NS1 were identified among the viruses tested. Compared to single gene amplification, application of NGS for surveillance and characterization of H9N2 LPAIV provides a complete genetic profile of emerging isolates and better understanding of the potential of zoonotic transmissions to mammals.

Vaccine Efficacy Against a New Avian Influenza (H9N2) Field Isolate from the Middle East (Serology and Challenge Studies).

Avian influenza subtype H9N2 is endemic in many countries in the Middle East. The reported prevalence of infection was variable between countries and ranged from 28.7% in Tunisia to 71% in Jordan. Several commercial killed whole-virus vaccine products are used as monovalent or bivalent mixed with Newcastle disease virus. Recently, we have noticed that many of the vaccinated broiler flocks did not show a production advantage over nonvaccinated flocks in the field. A new avian influenza field virus (H9N2) was isolated from these vaccinated and infected broiler flocks in 2013. This virus had 89.1% similarity of its hemagglutinin (HA) gene to the classical virus used for manufacturing the classical vaccine. Inactivated autogenous vaccine was manufactured from this new field isolate to investigate its serological response and protection in specific-pathogen-free (SPF) and breeder-male chickens compared to the classical vaccine. Oropharyngeal virus shedding of vaccinated breeder-male chickens was evaluated at 3, 9, 10, and 14 days postchallenge (DPC). Percentage of chickens shedding the virus at 3 DPC was 64%, 50%, and 64% in the classical vaccine group, autogenous vaccine group, and the control challenged group, respectively. At 7 DPC percentage of virus shedding was 42%, 7%, and 64% in the classical vaccine group, autogenous vaccine group, and the control challenged group, respectively. At 10 DPC only 9% of classical vaccine group was shedding the virus and there was no virus shedding in any of the groups at 14 DPC. There was statistical significance difference (P < 0.05) in shedding only at 7 DPC between the autogenous vaccine group and the other two groups. At 42 days of age (14 DPC), average body weight was 2.720, 2.745, 2.290, and 2.760 kg for the classical vaccine group, autogenous vaccine group, control challenged group, and control unchallenged group, respectively. Only the control challenged group had significantly (P < 0.05) lower average body weight. In another experiment, vaccinated SPF chicks had hemagglutination inhibition (HI) geometric mean titers (GMTs), with classical antigen, of 8.7 and 3.1 log 2 for classical and autogenous vaccine groups, respectively. When the autogenous antigen was used for HI, GMTs were 6.0 and 8.1 log 2, respectively. Both vaccines protected against body weight suppression after challenge. However, autogenous vaccine elicited significantly higher HI titer and reduced viral shedding at 7 DPC. In conclusion, it is important to revise the vaccine virus strains used in each region to protect against and control infection from new field strains. Further field experiments are needed to demonstrate the efficacy of new vaccines under field conditions.

Decay of maternal antibodies in broiler chickens.

The objective of this study was to determine the decay rate of maternal antibodies against major broiler chicken pathogens. A total of 30 one-day-old broiler chicks were obtained from a commercial hatchery and reared in isolation. These chicks were retrieved from a parent flock that received a routine vaccination program. Chicks were bled at hatch and sequentially thereafter every 5 d through 30 d of age. Maternal antibody titers were measured by ELISA for avian encephalomyelitis (AEV), avian influenza virus (AIV), chicken anemia virus (CAV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and reovirus (Reo). Maternal antibody titers for Newcastle disease virus (NDV) were measured using a hemagglutination inhibition test. Half-life estimates of maternal antibody titers were 5.3, 4.2, 7, 5.1, 3.9, 3.8, 4.9, 4.1, 6.3, and 4.7 d for AEV, AIV, CAV, IBDV, IBV, ILTV, MG, MS, NDV, and Reo, respectively. The statistical analysis revealed significant differences among half-lives of maternal antibody titers against certain pathogens. Furthermore, all maternal antibody titers were depleted by 10 d of age except for IBDV.

Mycoplasma gallisepticum experimental infection and tissue distribution in chickens, sparrows and pigeons.

The most effective approaches to control the spread of Mycoplasma gallisepticum include strict biosecurity measures, continuous surveillance and eradication of infected flocks. The rapid expansion of the poultry industry worldwide in restricted geographical areas and severe economic losses due to M. gallisepticum outbreaks make it crucial to identify and better control the vectors responsible for the transmission of the disease. In the present study we evaluated the susceptibility of sparrows and pigeons to M. gallisepticum and the tissue distribution of M. gallisepticum in these species as compared with chickens. This information will further define the role of these common avian species in M. gallisepticum transmission. Twenty-six chickens, pigeons, and sparrows were experimentally inoculated with a field strain of M. gallisepticum and were monitored for the development of clinical signs, seroconversion, productive infection by culture, and M. gallisepticum distribution in their tissues by immunohistochemistry. All M. gallisepticum-inoculated chickens showed mild respiratory signs, seroconverted (haemagglutination inhibition geometric mean titre = 494) and shed M. gallisepticum in their tracheas. M. gallisepticum antigens were observed at high levels by immunohistochemistry in their tracheas, conjunctivas, nasal turbinates, and air sacs. The pigeons and sparrows did not show clinical signs or seroconvert but M. gallisepticum was reisolated up to 7 days post inoculation from pigeons and intermittently from sparrows. M. gallisepticum antigens were observed at low level in the conjunctiva of some pigeons and sparrows, as well as in the trachea of some sparrows. We conclude that pigeons and sparrows are partially susceptible to M. gallisepticum infection but do not seroconvert or maintain a steady carrier state similar to chickens and that these species may play a role in M. gallisepticum transmission between poultry farms as mechanical vectors.

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.

Change in antimicrobial susceptibility of Mycoplasma gallisepticum field isolates.

This study compares the antimicrobial susceptibility over time between two groups of Mycoplasma gallisepticum (MG) isolates from the same geographical area. Minimum inhibitory concentration of 13 antimicrobials was determined against two groups of MG isolates from chickens. Group 1 strains (n=22) were isolated in 2004-2005 while group 2 strains (n=7) were isolated in 2007-2008. Minimum inhibitory concentration 50 for group 1 versus group 2 was 4/4, 0.5/0.5, ≤ 0.031/≥ 64, ≤ 0.031/2, ≤ 0.031/0.125, 1/0.5, 1/1, ≤ 0.031/≤ 0.031, ≤ 0.031/2, ≤ 0.031/2, 1/4, ≤ 0.031/0.062, and 0.062/2 µg/ml against gentamicin, spectinomycin, erythromycin, tilmicosin, tylosin, florfenicol, thiamphenicol, tiamulin, ciprofloxacin, enrofloxacin, chlortetracycline, doxycycline, and oxytetracycline, respectively. There was a statistically significant increase in resistance of group 2 to erythromycin, tilmicosin, tylosin, ciprofloxacin, enrofloxacin, chlortetracycline, doxycycline, and oxytetracycline. This dramatic increase in resistance against 8 antimicrobials belonging to three different families of antimicrobials in a relatively short period of time appears to be rare and of concern. The cause of this increased resistance observed in group 2 of MG isolates was not determined and should be further investigated. Monitoring of MG field strain susceptibility is highly recommended to implement successful treatment and prophylaxis programs in endemic areas.

Molecular typing and antimicrobial susceptibility of Clostridium perfringens from broiler chickens.

Clostridium perfringens (Cp) causes necrotic enteritis disease in commercial poultry. Antimicrobials are used to control and treat this disease and sometimes clinical outbreaks do not respond well to certain treatments. This study was designed to isolate Cp from clinical cases, type these isolates by multiplex PCR, and determine their antimicrobial susceptibility by micro-dilution method. A total of 67 Cp isolates were obtained from 155 broiler chicken flocks. All isolates were classified as type A and non-enterotoxin producers. Lincomycin, erythromycins, and tilmicosin showed very high minimal inhibitory concentration (MIC) 50 of ≥256 µg/ml. However, tylosin, amoxicillin, ampicillin, penicillin, florfenicol, danofloxacin, enrofloxacin, chlortetracycline, doxycycline, and oxytetracycline had variable MIC50 of 64, 0.5, 1, 1, 8, 4, 8, 4, 8, 0.5 µg/ml, respectively. It is recommended that Cp infections in Jordan be treated with either penicillins or tetracyclines especially amoxicillin and oxytetracycline.

Pathogenicity of an avian influenza virus serotype H9N2 in chickens.

A low pathogenic avian influenza virus (AIV) serotype H9N2 affected many commercial flocks in the Middle East in late 1990s and early 2000s. Due to the varying pathogenicity ofAIV H9N2 reported in previous studies, this study was carried out to determine the pathogenicity of a Jordanian isolate of H9N2 in broiler and specific-pathogen-free (SPF) chickens. Mild tracheal rales were observed in the broilers but not in the SPF birds starting 3 days postinfection (DPI) and until the end of the experiment at 16 DPI. Infected chickens had gross and histologic changes limited to the respiratory system (sinuses, trachea, lungs, and air sacs) characterized by congestion and lymphoplasmacytic inflammation. However, the lesions in the broiler chickens were more severe than those in the SPF chicks. Furthermore, the virus caused significant (P = 0.004) reduction (230 g) in average body weight of the infected broiler group compared with the uninfected broiler group. Both broiler and SPF-infected groups seroconverted, and they had a geometric mean titer of 2(8.2) and 2(9.3), respectively, on the hemagglutination inhibition test at 16 DPI. Cloacal virus shedding was not detected by 9 DPI and 15 DPI in broiler and SPF-infected groups, respectively. This study demonstrated the pathogenic nature of the local Jordanian H9N2 isolate and the variation from what it has been reported in other countries of the region. Regional effort should be directed to start an eradication program of this disease because of its pathogenicity for chickens, wide distribution, and possible interference with surveillance for H5N1 serotype.

Infectious bronchitis virus serotypes in poultry flocks in Jordan.

Infectious bronchitis virus (IBV) causes respiratory disease in chickens all over the world. IBV has many serotypes that do not confer cross protection against each other. Hemagglutination inhibition (HI) test has been used to determine the serotypes of IBV as a substitute to the more laborious virus neutralization test and the more sophisticated restriction endonuclease digestion or sequencing of the S1 gene. In Jordan, no previous studies have been carried out to determine the involvement of IBV in respiratory disease in chickens, or the serotypes of IBV that possibly exist. In this study, serum from different chicken flocks (n=20) that suffered from respiratory disease were tested for IBV antibodies using commercial IBV antibody ELISA at time of the initial signs of the respiratory disease and repeated on serum samples from the same flocks 10-14 days later. ELISA titer for IBV increased in 14 out of 20 flocks (70%) after 10-14 days of the initial signs of the respiratory disease and this indicates a recent exposure to IBV. The second serum samples from these 14 flocks were further examined against a panel of five IBV antigens (Ark, Conn, DE-072, JMK, and Mass) by HI test to determine the serotype(s) of IBV they have been exposed to. The HI test results indicated that the exposure of some of these flocks were to Ark, DE-072, and Mass like serotypes. However, the HI titers against the antigens used in this study were relatively similar in 10 out of the 14 flocks (71%) and the serotype of IBV that these flocks were exposed to could not be determined and the possible causes of this are discussed.

Comparison and verification of quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) and real time RT-PCR for avian leukosis virus subgroup J.

Avian leukosis virus subgroup J (ALV-J) infections cause significant economic losses because of increased mortality, tumor production, decreased production, and cost for eradication. Current quantification methods for ALV-J expressed by TCID(50) are difficult to determine because of the lack of cytopathic effect in cell cultures and non-specificity of currently available antigen-capture ELISA tests. In this study, a one-tube fluorescent probe based real time RT-PCR method was developed for quantification of ALV-J and compared with available quantification methods. Cell lysates with different TCID(50)s determined by cell culture and antigen capture ELISA (ag-ELISA) were used for one-tube real time RT-PCR using fluorogenic probe and quantitative competitive RT-PCR (QC-RT-PCR). The results of QC-RT-PCR and real time RT-PCR were highly correlated to the TCID(50)s determined by conventional culture methods. They were also very specific, sensitive, easy to perform, reproducible, and rapid compared with conventional methods. These RT-PCR based quantification methods of ALV-J viral RNA will be useful for virological and pathogenesis studies.