The First Occurrence of Hepatitis-Hydropericardium Syndrome in Iran and Effective Applied Control Measures in the Affected Commercial Broiler Flock.

Fowl adenoviruses cause three economically important diseases in broiler chicken flocks: hepatitis-hydropericardium syndrome (HHS), inclusion body hepatitis (IBH), and adenoviral gizzard erosion. IBH has not been considered a serious threat in northeast Iran since the last decade, because no major effect on flock performance has been noticed along with a low mortality rate. During this period, all the sporadic IBH outbreaks have also been investigated for HHS without finding any confirmed case. In March 2021, a 15-day-old commercial broiler flock in northeast Iran showed a 50% mortality rate, and birds underwent postmortem examination, histopathology, molecular testing, and phylogenetic analysis for possible disease agents. Typical gross lesions of HHS were observed postmortem that included hydropericardium with an unusual accumulation of jelly-like and straw-colored fluid in the pericardial sac (without right ventricular failure); petechial or ecchymotic hemorrhages on the myocardium, myocardial valves, and endocardium; and discolored and mottled liver along with small white foci and petechial or ecchymotic hemorrhages. Histopathologic analysis showed necrosis of hepatocytes and basophilic inclusion bodies in the livers. The molecular tests performed for detection of fowl adenovirus (FAdV), H5 avian influenza virus, Newcastle disease virus, avian infectious bronchitis virus (IBV), H9N2, chicken infectious anemia virus (CIAV), infectious bursal disease (IBD) virus, Marek's disease virus, Ornithobacterium rhinotracheale, Mycoplasma gallisepticum, and Mycoplasma synoviae turned out positive for FAdV, CIAV, IBD vaccine virus, and IBV serotypes 793B and variant I. The phylogenetic tree based on the hexon gene loop 1 demonstrated a FAdV serotype 4 (FAdV-4) that was identical to Pakistani isolate PARC-1/98. Because it was the first detection of a FAdV-4 in Iran, the stamping out program was applied immediately on the basis of HHS gross lesions and positive PCR reaction on pericardial jelly-like fluid. It seems that this eradication strategy was successful because no outbreaks were noticed for 2 mo after the initial outbreak. It was concluded that the use of gross pathologic baselines, quick diagnosis of disease, and close collaboration between governmental and private sectors were the critical factors that helped locally control the first occurrence of HHS in Iran.

Reporte de caso- Primera presentación del síndrome de hepatitis-hidropericardio (HHS) en Irán y medidas efectivas de control aplicadas en la parvada comercial de pollos de engorde afectada. Los adenovirus del pollo causan tres enfermedades económicamente importantes en las parvadas de pollos de engorde: síndrome de hepatitis-hidropericardio (HHS), hepatitis con cuerpos de inclusión (IBH) y erosión de la molleja por adenovirus. La hepatitis con cuerpos de inclusión no se ha considerado una amenaza grave en el noreste de Irán desde la última década, porque no se ha observado un efecto importante en el rendimiento de la parvada junto con una baja tasa de mortalidad. Durante este período, todos los brotes esporádicos de hepatitis con cuerpos de inclusión también han sido analizados para el síndrome de hepatitis-hidropericardio sin encontrar ningún caso confirmado. En marzo del 2021, una parvada comercial de pollos de engorde de 15 días de edad en el noreste de Irán mostró una tasa de mortalidad del 50 % y las aves se sometieron a un examen post mortem, histopatología, pruebas moleculares y análisis filogenético para detectar posibles agentes patógenos. Se observaron lesiones macroscópicas post mortem típicas del síndrome de hepatitis-hidropericardio que incluían hidropericardio con una acumulación inusual de líquido gelatinoso y de color pajizo en el saco pericárdico (sin insuficiencia ventricular derecha); hemorragias petequiales o equimóticas en el miocardio, válvulas miocárdicas y endocardio; e hígado decolorado y moteado junto con pequeños focos blancos y hemorragias petequiales o equimóticas. El análisis histopatológico mostró necrosis de hepatocitos y cuerpos de inclusión basófilos en los hígados. Se realizaron pruebas moleculares para la detección de adenovirus del pollo (FAdV), virus de la influenza aviar H5, virus de la enfermedad de Newcastle, virus de la bronquitis infecciosa aviar, virus de influenza tipo H9N2, virus de la anemia infecciosa de los pollos, virus de la enfermedad infecciosa de la bolsa, virus de la enfermedad de Marek, Ornithobacterium rhinotracheale, Mycoplasma gallisepticum y Mycoplasma synoviae con resultados positivos para adenovirus del pollo, virus de la anemia infecciosa, virus vacunal de Gumboro y virus de la bronquitis infecciosa serotipos 793B y variante I. El árbol filogenético basado en la asa 1 del gene del hexon demostró la presencia del serotipo 4 del adenovirus del pollo (FAdV-4) que era idéntico al aislamiento pakistaní PARC-1/98. Debido a que fue la primera detección de un adenovirus de pollo serotipo 4 en Irán, el programa de despoblación sanitario se aplicó de inmediato sobre la base de lesiones macroscópicas del síndrome de hepatitis-hidropericardio y por reacción de PCR positiva en el líquido pericárdico gelatinoso. Parece que esta estrategia de erradicación tuvo éxito porque no se notaron brotes durante dos meses después del brote inicial. Se concluyó que el uso de líneas base de patología macroscópica, el diagnóstico rápido de la enfermedad y la estrecha colaboración entre los sectores gubernamental y privado fueron los factores críticos que ayudaron a controlar localmente la primera presentación del síndrome de hepatitis-hidropericardio en Irán.Key words: hepatitis-hydropericardium syndrome, FAdV-4, control measures, Iran.

Cytotoxic Effect of Biebersteinia multifida Alcoholic Extracts on MCF-7, HeLa, and A2780 Cell Lines.

Conventional cancer treatments are costly and have different serious side effects for patients. Natural herbal treatments are widely accepted among people because of their minimal side effects, although there is little scientific knowledge about them. One of these remedies utilizes the root of Biebersteinia multifidi that has been used for years in Iran to treat different chronic genital diseases. The current study examined the effects of methanolic and ethanolic extracts of B. multifida (induction of necrosis and apoptosis) on breast cancer (MCF-7), ovarian cancer (A2780), and human cervix cancer (HeLa) cell lines in comparison with normal breast cells. These effects were determined to be morphological alterations in cell light microscopy, by flow cytometry (staining with annexin V and propidium iodide), and by measuring live cells and inhibition concentrations by MTT assay. IC50 of B. multifida on the MCF-7 cell line (methanolic extract) was 400 µg/ml and for A2780 was 250 µg/ml. The IC50 amount of B. multifida on the MCF-7 cell line (ethanolic extract) was 750 µg/ml and 1500 for A2780. Results demonstrated that apoptosis and necrosis occurred in MCF-7 and A2780 following the addition of ethanolic and methanolic extracts of B. multifida to the medium. These findings confirmed the anti-cancer effects of mehthanolic extracts of Biebersteinia multifida root and its safety for normal cells; thus, it can be applied in cancer therapy as a novel medication.

Attenuation and amino acid changes in infectious bronchitis virus Iranian 793/B serotype during serial passaging in embryonated chicken eggs.

The use of live attenuated vaccine (LAV) is the main method for controlling infectious bronchitis (IB). It is advisable to develop a LAV using a dominant serotype in the region in the case of vaccine failure. Since 793/B serotype is one of the most predominant circulating IB viruses in Iran, attenuation of three Iranian 793/B isolates (IR/773/2001, IR/794/2002 and IR/520/2002) was done by serial passaging in specific pathogen free (SPF) embryonated chicken eggs up to 90 passages to assess the degree of their attenuation to achieve a native LAV in the future. Virulence and pathogenicity of passage levels 15 and 90 of isolates 773 and 794 were compared using histopathology, ciliostasis and potency tests. The results showed a decrease in the virulence and pathogenicity of the isolates at passage 90 compared to passage 15, although this decrease in pathogenicity was very mild and viruses after passage 90 were not adequately attenuated. Each isolate underwent some amino acid changes at passage 90. In case of isolate 773 it was 5 aa changes, while in isolate 794 it was 19 aa changes. Some amino acid changes resulted in change into amino acid with different hydrophobicity characteristics. No amino acid change was found at passage level 15 compared to wild type viruses. Interestingly, we did not find previously reported change in amino acid 95 in passage levels 15 and 90. Keywords: infectious bronchitis; live attenuated vaccine; 793/B serotype; pathogenicity; attenuation; nucleotide sequencing.

Nucleotide Sequence Analysis of S1 Gene among Iranian Avian Infectious Bronchitis Viruses Isolated during 2001-2002.

Infectious bronchitis (IB) virus genome codes for four structural proteins, among which the S1 subunit of spike glycoprotein comprises the major epitopes to induce neutralizing antibodies. This study involved the comparison of the full S1 sequences of five IB viruses, namely two Massachusetts and three 793/B serotypes, isolated from IB outbreaks during 2001-2002, with all other Iranian and foreign 793/B isolates and 10 known serotypes. Analysis of S1 subunit showed three unique amino acid changes at positions 349 (V to L), 392 (T to N), and 393 (Q or R to T or K or S) for the Iranian 793/B isolates, compared to those of the foreign 793/B isolates reported before 2006 (onset of vaccination with 793/B vaccine in Iran). They were used as amino acid markers for the differentiation of Iranian 793/B isolates for years. Sequence alignment of the Iranian isolates with those of the foreign ones reported after 2006 demonstrated that amino acids 392 and 393 were no longer considered as amino acid markers, and only the change in amino acid 349 still remained specific to the Iranian 793/B isolates. Phylogenetic tree sequence analysis revealed that the Iranian 793/B isolates were closely related indicating that they came from a single source, more probably from France. There was a very close correlation between the first detection of 793/B serotype and the time of French chicken meat importation. Moreover, it was shown that one of the Massachusetts isolates was completely identical with the H120 vaccine strain. Furthermore, the other Massachusetts isolate with two amino acid changes at positions 64 (G to E) and 95 (S to R) was very similar to this vaccine strain. It seems that the latter isolate is a passaged chicken H120 vaccine strain.

Detection of 793/B serotype of infectious bronchitis virus in tissue sample by indirect immunoperoxidase assay.

The indirect immunoperoxidase (IIP) assay was compared with the reverse transcription-polymerase chain reaction (RT-PCR) for detection of 793/B serotype of infectious bronchitis virus in tissues samples collected from experimentally infected chickens. This technique was optimized in specific pathogen-free (SPF)-embryonated chicken eggs and broiler chickens inoculated with the Iranian IR/773/2001 strain of 793/B serotype The trachea, lung, kidney, and cecal tonsil tissue samples from experimentally infected chicken embryos and chickens were collected in order to prepare tissue sections in IIP assay and to detect in RT-PCR. The sensitivity and specificity values of IIP assay were, respectively, 83 and 84 %, and the positive and negative prediction values were 71 and 91 % when compared with RT-PCR.

Characterization of three infectious bursal disease virus isolates obtained from layer chickens in Iran.

Three infectious bursal disease viruses (IBDVs) were isolated from field outbreaks in IBDV-vaccinated and non-vaccinated layer chicken flocks. Agar gel precipitation test (AGPT), immunoperoxidase staining, transmission electron microscopy (TEM), inoculation into embryonated eggs, and chicken embryo fibroblasts (CEFs) confirmed that the isolates were IBDVs. RT-PCR, restriction fragment length polymorphism (RFLP), and phylogenetic analysis demonstrated that the isolates were very virulent IBDV (vvIBDV) and showed a nucleotide sequence similarity of 96.3 to 99.8% in comparison with other vvIBDV strains. It was concluded that the Iranian isolates represented vvIBDV of serotype 1 originating from Europe, Japan, and China.

Biological and molecular characterization of Avian influenza virus (H9N2) isolates from Iran.

Three Influenza A virus (H9N2) isolates obtained from three separate broiler flocks with variable mortality rates were cloned twice in embryonated SPF chicken eggs by limiting dilution. Biological properties of these isolates were examined in 4-week-old SPF chickens and chick embryo fibroblast (CEF) cultures. The isolates neither caused mortality in the inoculated chickens nor produced CPE in cell cultures, indicating low pathogenicity. PCR products of 486 bp containing the sequences for hemagglutinin (HA) cleavage site, which were generated from the isolates, were subjected to nucleotide sequencing. Sequence analysis of the HA region containing the cleavage site of the isolates showed a similar sequence motif (PARSSRG) but different flanking regions. Phylogenetic analysis of deduced amino acid sequences revealed that the isolates were closely related to those isolated earlier, indicating a common source. Moreover. the amino acid sequences of the recent isolates were very similar to those from Saudi Arabia, Germany and Pakistan. It is postulated that, except for some Chinese isolates, the pathogenicity of Iranian isolates seems to be similar to that of other Eurasian isolates. It is possible that an elevation in mortality rate under field condition could be caused by co-infection of recent isolates with the bacteria such as mycoplasma, Escherichia coli, and Ornithobacterium rhinotracheale rather than by an emerging a pathogenic H9N2 subtype of the virus.

Detection of egg drop syndrome 1976 virus by polymerase chain reaction and study of its persistence in experimentally infected layer birds.

Polymerase chain reaction (PCR) assay was developed for the detection of Egg drop syndrome 1976 (EDS-76) virus in tissues, namely in the uterus, spleen and buffy coat. It was also used to study the persistence of the virus in tissues of experimentally infected layer birds. The PCR assay could detect as little as 10 fg of purified EDS-76 viral DNA. It also amplified the DNA of Fowl adenovirus serotypes 4 (FAV-4) and 8 (FAV-8). The virus persisted in the uterus up to day 21 post infection (p.i.). Detection of EDS-76 viral DNA in the buffy coat could be useful for studying the occurrence of the respective disease in layer bird flocks.

Differentiation of classical and very virulent strains/isolates of Infectious bursal disease virus by reverse transcription-polymerase chain reaction.

Reverse transcription-polymerase chain reaction (RT-PCR) is a rapid method for identification and differentiation of viruses. It was used to differentiate very virulent from classical (field/vaccine) strains/isolates of Infectious bursal disease virus (IBDV). RT-PCR products of 552 bp were generated by amplification of variable region of VP2 gene in three field classical isolates, two vaccine strains and two very virulent isolates of IBDV. The PCR products were digested with SacI, HhaI, SspI and StuI. Digestion of the PCR products with SacI and HhaI revealed the presence of a single restriction site in all the field classical isolates and vaccine strains, but no such a restriction site in very virulent strains. On the other hand digestion of these products with SspI and StuI showed the presence of a single restriction site in very virulent strains but no such a restriction site in classical field isolates and vaccine strains. Although the restriction profiles of classical field Indian isolates and vaccine strains were identical, all of these enzymes could differentiate very virulent Indian strains from classical field isolates and vaccine strains.

Development of sandwich elisa for the detection of fowl adenovirus 4 associated with hydropericardium syndrome in experimentally infected chicken.

A sandwich ELISA was standardized to detect fowl adenovirus (FAV) group I antigen in various tissues, namely liver, spleen, bursa, thymus and kidneys of chicks experimentally infected with fowl adenovirus 4 (FAV-4) isolated from cases of inclusion body hepatitis-hydropericardium syndrome (IBH-HPS). The assay was found to be more sensitive and more specific in comparison to an agar gel immunodiffusion (AGID) test, as it could detect FAV antigen below the titer of 20,000 TCID50/ml and below 1.14 microg in 5% (w/v) suspensions of liver tissue. In 2-week-old experimentally infected chicks, the antigens were detectable by ELISA in liver from 3 to 15 days, in thymus from 3 to 7 days, and in kidneys, bursa and spleen from 3 to 10 days post infection (p.i.). Maximum antigen concentration in terms of ELISA absorbance values was detected in liver and kidneys, which could be used as tissues of choice for virus isolation or detection of viral antigens from IBH-HPS cases.

Amino acid changes in the variable region of VP2 in three infectious bursal disease viruses with different virulence, originating from a common ancestor.

An infectious bursal disease virus (IBDV), Hyd(C), from an outbreak was isolated and plaque-purified in BGM-70 cells. From the moderately virulent plaque-purified virus, two IBDVs with relatively high and low virulence were obtained by passaging the virus in specific pathogen free chickens and BGM-70 cells 10 and seven times, respectively. Comparison of amino acid sequences of the VP2 variable region of these viruses revealed that three amino acids at positions 279, 284 and 300 (Asp, Thr and Glu, respectively, in the plaque-purified virus) were changed. In in vitro- passaged virus, amino acid residues 279, 284 and 300 were Asn, Thr and Glu, whereas these were Asp, Ala and Gln in the in vivo -passaged virus. Change of residue 284 (Thr -->Ala) had a critical role in cell culture infectivity, whereas the change in residue 279 (Asp -->Asn) was associated with attenuation of the virus. No correlation could be observed between amino acid changes at position 300 and virulence or cell culture infectivity. Moreover, residue 330 (Arg) in heptapeptide motif SWSAR 330 GS was not found to be associated with the cell culture infectivity or virulence.