Use of Esienia fetida Worms to Produce Peptone for Clostridium perfringens Vaccine Production.

Concurrent with an increase in the human population on the earth, more than ever, the creation of energy and maintenance of health is necessary, and nowadays, various sources of energy supply are being developed. The general global view in this regard is to provide protein and energy from available and cheap sources. Iran is no exception to this general rule, only in the field of ensuring the health of livestock resources every year, about 10 tons of peptone is needed for producing clostridial vaccines. Vermicomposting worms (Esienia fetida) with high protein percentages and rapid reproductions are a suitable source for peptone production. Based on this, the vaccine strain of Clostridium perfringens type D cultivated in two different media contain peptone produced from worms and meat peptone. The growth rate, epsilon toxin (ETX), and alpha toxin (CPA) of Cl. perfringens have been compared in two media. The results showed that the growth rate of bacteria in the worm peptone medium in 48 h was 22% higher than that of the meat peptone. Additionally, the activity of alpha toxin (phospholipase C) was in worm peptone 15% higher than meat peptone during 80 min of measurement. Regarding epsilon toxin lethality, all three mice of the N-worm peptone group died, while all three mice of the meat peptone group survived even 72 h after injection. The average survival time of mice in the N-worm peptone group was 1700 min. Therefore, we suggest the worms' protein is more suitable than industrial meat in peptone production for vicinal propose. To eliminate the need for hydrolyzed protein in the production of vaccines in the future, we suggest an increase in the fields of employment and the development of fertilizer and worm farms in Iran.

The role of the major viral pathogens in a respiratory disease outbreak of broiler flocks in Eastern Iran.

Background: Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and avian influenza virus (AIV) H9N2 are major viral pathogens in broiler respiratory disease. Aims: Following a respiratory disease outbreak and economic losses in eastern Iran 2020-2021, we investigated the role of major viral pathogens and the implemented vaccination programs. Methods: Thirty-six respiratory disease affected broiler flocks in South Khorasan province were sampled, molecularly tested, and coinfections were investigated. The vaccination programs were obtained and the detected IBV were genotyped. Results: IBV, virulent NDV, and AIV H9N2 were detected in twenty-five, seven, and seven flocks, respectively. IBV+AIV, IBV+NDV, and NDV+AIV coinfections were respectively detected in six, five, and one flocks. Most IBV infected flocks (84%) had been immunized with a live IBV-Mass vaccine. All NDV infected flocks and 14.2% of AIV infected flocks had been vaccinated. IBV genotyping showed a high prevalence of variant 2 (83.3%), followed by Mass-type (12.5%), and Q1-type (4.2%). Variant 2 IB viruses were widely distributed in the province and half of them were mostly similar to the ones that had been detected in northern neighboring province, Khorasan Razavi. Conclusion: Single infection with variant 2 IBV was a major cause of the respiratory disease outbreak in which use of the Mass vaccine was probably not effective. The high coverage and multiple doses of vaccination against Newcastle disease possibly had reduced the prevalence of NDV. Considering the regional origin of IBV strains, strong biosecurity measures should be implemented and vaccination programs using appropriate vaccine strains should be used.

Evaluation of Cholera Toxin Adjuvanticity Effect on the Production of Specific Antibodies Induced by Avian Infectious Bronchitis Vaccine in Chickens.

Cholera toxin (CT) is one of the most well-known immunostimulants. Mammalian studies have shown that CT can generate immune responses against antigen. However, it has not exhibited a definite effect on poultry yet. In this study, focusing on a cost-effective method, the effect of different concentrations of CT obtained from Vibrio cholerae biotype El Tor and serotype Inaba was investigated on the immunogenicity of infectious bronchitis vaccine. After culturing and concentrating CT, different concentrations of CT (0.1, 1, 2, and 5 micrograms) were combined with avian infectious bronchitis vaccine strain H120 produced by Razi Vaccine and Serum Research Institute (RVSRI) and, at 7 days of age, inoculated via the eye drop administration in 42 specific-pathogen-free chickens (seven groups of six chicks that included four experimental groups, two negative control groups (PBS and toxin), and one positive control group). Blood samples were taken weekly from the wing veins of the chickens, and the immunoglobulin G (IgG) titer was checked by enzyme-linked immunosorbent assay. The results showed that 2 µg of CT in comparison with other concentrations caused a significant increase in the antibody titer against avian infectious bronchitis in the blood serums of the chickens. One-way ANOVA test showed that all the results of this study were significant at P<0.05 level. Our data show that CT has the potential to further stimulate the immune system of chickens and may increase the immunogenicity of the infectious bronchitis vaccine. However, more research is needed to examine all aspects of the use of this toxin in animal vaccines.

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.