ABSTRACT Rabbit hemorrhagic disease is a contagious viral disease of rabbits controlled by vaccination. The present study was aimed to diagnose rabbit hemorrhagic disease from 11 infected farms from Qalubia governorate during 2019 and to prepare homologous vaccine against rabbit hemorrhagic disease virus 2. For this purpose, 11 liver samples were collected from suspected cases and subjected to detection and identification of circulating rabbit hemorrhagic disease virus. Ten samples were confirmed to be rabbit hemorrhagic disease virus using hemagglutination test, animal inoculation and reverse transcriptase polymerase chain reaction. Sequencing and phylogenetic analysis of two isolates (R5&R6) revealed the presence of rabbit hemorrhagic disease virus 2 (A/Qalubia/2019 and B/Qalubia/2019) under accession number MT07629 and MT067630 respectively. The inactivated rabbit hemorrhagic disease virus vaccines were prepared using Montanide ISA 206 oil or aluminum hydroxide gel adjuvants. Prepared vaccines were inoculated subcutaneously in susceptible rabbits and submitted to sterility, safety and potency tests. Obtained results showed that mean hemagglutination inhibition titer for aluminum hydroxide gel vaccine was 6,7.7,8.9 and 9.1 log2 while, Montanide vaccine reached to 6.7,8.7,9.2 and 9.5 log2 at 1st, 2nd, 3rd, and 4th weeks post vaccination, respectively. Immunized rabbits with Montanide vaccine showed better protection reach to 70%, 90%, 100% and 100% when compared to aluminum hydroxide gel vaccine 60%, 70%, 90% and 90% at 1st, 2nd, 3rd and 4th weeks post vaccination respectively. It was concluded that newly emerged rabbit hemorrhagic disease virus 2 was isolated from suspected cases. The two prepared vaccines were sterile, safe and potent. The oily adjuvanted rabbit hemorrhagic disease virus 2 vaccine stimulated an earlier and higher humoral immune response than the aluminum hydroxide gel adjuvanted vaccine. This humoral immune response achieved significant level of protection.

RESUMEN La enfermedad hemorrágica del conejo es una enfermedad viral contagiosa de los conejos que se controla mediante vacunación. El presente estudio tuvo como objetivo diagnosticar la enfermedad hemorrágica del conejo en 11 granjas infectadas de la provincia de Qalubia, durante 2019 y preparar una vacuna homóloga contra el virus de la enfermedad hemorrágica del conejo tipo 2. Para este propósito, se recolectaron 11 muestras de hígado de casos sospechosos y se sometieron a detección e identificación de virus circulante de la enfermedad hemorrágica del conejo. Se confirmó que diez muestras eran positivas al virus de la enfermedad hemorrágica del conejo, utilizando para ello la prueba de hemaglutinación, inoculación en animales y Reacción en cadena de la polimerasa con transcriptasa inversa. La secuenciación y el análisis filogenético de dos aislamientos (R5 y R6) revelaron la presencia del virus de la enfermedad hemorrágica del conejo tipo 2 (A/Qalubia/2019 y B/Qalubia/2019) con los números de acceso MT07629 y MT067630 respectivamente. Las vacunas inactivadas del virus de la enfermedad hemorrágica del conejo se prepararon usando adyuvantes de gel de hidróxido de aluminio o aceite Montanide ISA 206. Las vacunas preparadas se inocularon por vía subcutánea en conejos susceptibles y se sometieron a pruebas de esterilidad, seguridad y potencia. Los resultados obtenidos mostraron que el título medio de inhibición de la hemaglutinación para la vacuna en gel de hidróxido de aluminio fue de 6; 7,7; 8,9 y 9,1 log2, mientras que la vacuna de Montanide alcanzó 6,7; 8,7; 9,2 y 9,5 log2 en la 1ª, 2ª, 3ª y 4ª semanas después de la vacunación, respectivamente. Los conejos inmunizados con la vacuna Montanide tuvieron una mejor protección, alcanzándose niveles de 70%, 90%, 100% y 100% en comparación con la vacuna en gel de hidróxido de aluminio 60%, 70%, 90% y 90% en la 1ª, 2ª, 3ª y 4ª semanas después de la vacunación, respectivamente. Se concluyó que el virus de la enfermedad hemorrágica del conejo tipo 2 de reciente aparición se aisló de los casos sospechosos. Las dos vacunas preparadas fueron estériles, seguras y potentes. La vacuna contra el virus de la enfermedad hemorrágica del conejo tipo 2 con adyuvante oleoso estimuló una respuesta inmune humoral más temprana y mayor que la vacuna con adyuvante en gel de hidróxido de aluminio. Esta respuesta inmune humoral confirió un nivel significativo de protección.

The purpose of this work was to examine physiological differences between the yeast Geotrichum candidum and the mould Penicillium camembertii, organisms involved in the industrial process of cheese ripening. Three groups of amino acids had previously been characterized, based on their carbon assimilation and dissimilation by the two fungal species. For both of them, a diauxic growth phase had been shown for a group of amino acids, which however had not been examined in light of physiological differences between the two microorganisms. In this work, the higher level of enzymatic activities of P. camembertii if compared to G. candidum was confirmed since a continuous and sequential use of both carbon substrates, glucose and arginine, was recorded during P. camembertii culture; while after glucose depletion, a clear stationary phase was recorded before the assimilation of the considered amino acid as both carbon and nitrogen sources by G. candidum. This behaviour was confirmed for the three amino acids tested, i.e., arginine, proline and glutamic acid. Contrarily, during the two growth phases, on glucose and the test amino acid, respectively, higher growth rates were recorded for G. candidum compared to P. camembertii, showing higher substrate utilisation efficiency by G. candidum. Improving the knowledge regarding the metabolization of amino acids might be helpful in designing strategies aiming at improving processes such as cheese ripening. The work should be followed up by similar works using small peptides.