Attenuated ehrlichiosis vaccine

Abstract

The present invention relates to an attenuated strain of Ehrlichia canis and a vaccine comprising said attenuated strain for protection of mammals against ehrlichiosis. The invention further relates to methods of preventing ehrlichiosis and of attenuating the pathogenicity Ehrlichia canis.

What is claimed is: 
     
         1 . A method for preparing an attenuated strain of Ehrlichia, wherein the attenuated strain of Ehrlichia is protective against clinical manifestation of ehrlichiosis, the method comprising the steps of:
 (i) selecting a pathogenic Ehrlichia strain capable of causing clinical manifestation of ehrlichiosis in a susceptible mammalian host species;   (ii) infecting a first cell line derived from the susceptible mammalian host species with the pathogenic Ehrlichia strain, and thereafter serially passaging the infected first cell line;   (iii) infecting a cell line derived from a mammalian species other than said susceptible mammalian host species with the Ehrlichia strain obtained from step (ii), and thereafter serially passaging the infected cell line, and   (iv) infecting a second cell line derived from said susceptible mammalian host species with the Ehrlichia strain obtained from step (iii), and thereafter serially passaging the infected second cell line, wherein the passaging in steps (ii) to (iv) is sufficient to attenuate the pathogenicity of said Ehrlichia strain so as to obtain an attenuated Ehrlichia strain protective against clinical manifestation of ehrlichiosis upon administration to the susceptible mammalian host species.   
     
     
         2 . The method of  claim 1 , wherein the pathogenic Ehrlichia strain is selected from the group consisting of:  E. canis, E. chaffeensis, E. ewingii, E. muris , and  E. ruminantium.    
     
     
         3 . The method of  claim 2 , wherein the pathogenic Ehrlichia strain is an  E. canis  strain. 
     
     
         4 . The method of  claim 3 , wherein the  E. canis  strain is selected from the group consisting of: Israel 611, Ebony, Broadfoot, Florida, Kogashima 1, Louisiana, Oklahoma, Venezuela, Sao Paulo, Jake, Demon, DJ, and Fuzzy. 
     
     
         5 . The method of  claim 3 , wherein the cell line of steps (ii) and (iv) is a canine cell line and wherein the cell line of step (iii) is a non-canine cell line. 
     
     
         6 . The method of  claim 5 , wherein the canine cell line of steps (ii) and (iv) is the same cell line or a different cell line. 
     
     
         7 . The method of  claim 5 , wherein the canine cell line is independently selected from the group consisting of: DH82, DLC 02, MDH and a primary cell culture of canine monocytes, or wherein the non-canine cell line is derived from a mammal selected from the group consisting of: murine, bovine, ovine, porcine, equine, feline, primate and human, or wherein the non-canine cell line is selected from the group consisting of: J774.A1, P388D1, RAW264.7, Mm1, BDM-1 and IC-21. 
     
     
         8 . The method of  claim 7 , wherein the canine cell line is DH82, and the non-canine cell line is J774.A1. 
     
     
         9 . The method of  claim 1 , wherein the attenuated strain of Ehrlichia confers resistance to one or more pathogenic strains of Ehrlichia. 
     
     
         10 . The method of  claim 1 , wherein the susceptible mammalian host is selected from the group consisting of: dog, wolf, fox, jackal, deer and human. 
     
     
         11 . The method of  claim 1 , further comprising a step of preparing a suspension of the Ehrlichia strain obtained from step (iv); or further comprising a step of administering a sample of the Ehrlichia strain obtained from step (iv) to test animals of the susceptible mammalian host species and assessing said animals for Ehrlichia infection and clinical manifestation of ehrlichiosis, wherein occurrence of infection concurrent with the substantial absence of clinical manifestation of ehrlichiosis is indicative of attenuation. 
     
     
         12 . The method of  claim 1 , further comprising one or more additional cycles of steps (ii)-(iv), or any combination thereof. 
     
     
         13 . The method of  claim 1 , further comprising at least one step of freezing, wherein the at least one step of freezing is carried out during or between any one of steps (ii)-(iv). 
     
     
         14 . The method of  claim 1 , wherein the number of passages in each of steps (ii) to (iv) is about 10 to about 100. 
     
     
         15 . The method of  claim 1 , wherein the administration is via a route selected from the group consisting of: parenteral, oral and intranasal administration. 
     
     
         16 . A vaccine, comprising an attenuated strain of Ehrlichia obtained by the method of  claim 1 . 
     
     
         17 . The vaccine of  claim 16 , comprising (i) an effective immunizing amount of the attenuated strain of Ehrlichia and (ii) a pharmaceutically acceptable carrier. 
     
     
         18 . The vaccine of  claim 16 , formulated for parenteral, oral, or intranasal administration. 
     
     
         19 . The vaccine of  claim 16 , wherein the Ehrlichia is an  E. canis.    
     
     
         20 . The vaccine of  claim 16 , further comprising an additional active immunizing agent, wherein the additional active immunizing agent is an attenuated or inactivated form of a pathogen selected from the group consisting of: rabies virus,  Borrelia burgdorferi , canine distemper virus, canine parvovirus, canine adenovirus, canine corona virus,  Giardia, Leptospira interrogans  and combinations thereof.