Leptospirosis is a zoonosis
of constant importance as causative
microbial agent, maintained by subclinical
infections in wild and domestic animals.
Currently, for active immunization in dogs
is used an inactivated vaccine, produced by
specialized companies in different countries.
The vast majority are polyvalent vaccines,
which include the two leptospire serovars
(Leptospira canicola and Leptospira
icterohaemorhagiae) and viral antigens for
prevention of infectious hepatitis Rubarht,
Carré's disease, coronavirus and
parainfluence. Specific immunity induced
by this inactivated vaccine, is active,
individual, installs in 10 to 21 days after
administration, lasting a variable time (4-6
months) and never causes a 100%
protection. This paper aims to investigate
how this immune response in carnivores and
its protection

Gheorghiță, Ileana

Pavli, Constantin

Perianu, Tudor

Tănase, Oana

Cercetari Agronomice in Moldova

Tanase, Irina-Oana

ALSE Repository

VICINAL IMMUNE RESPONSES IN CANINE LEPTOSPIROSIS   63  Cercetări Agronomice în Moldova Vol. XLIV , No. 4 (148) / 2011   RESEARCHES ON VICINAL IMMUNE RESPONSES IN CANINE LEPTOSPIROSIS  Oana TĂNASE1*, Ileana GHEORGHIŢĂ2, C. PAVLI1, T. PERIANU1  *E-mail: tanase_oana@yahoo.com  Received  July 6, 2011                                                       1 University of Agricultural Sciences and Veterinary Medicine Iaşi, Romania 2 State Laboratory for Animal Health Galaţi, Romania ABSTRACT - Leptospirosis is a zoonosis of constant importance as causative microbial agent, maintained by subclinical infections in wild and domestic animals. Currently, for active immunization in dogs is used an inactivated vaccine, produced by specialized companies in different countries. The vast majority are polyvalent vaccines, which include the two leptospire serovars (Leptospira canicola and Leptospira icterohaemorhagiae) and viral antigens for prevention of infectious hepatitis Rubarht, Carré's disease, coronavirus and parainfluence. Specific immunity induced by this inactivated vaccine, is active, individual, installs in 10 to 21 days after administration, lasting a variable time (4-6 months) and never causes a 100% protection. This paper aims to investigate how this immune response in carnivores and its protection.  Key words: Immune response; Leptospira icterohaemorhagiae; Leptospira canicola.  REZUMAT. Cercetări privind raspunsul imun post-vaccinal în leptospiroza canină. Leptospiroza este o zoonoză de permanentă actualitate, întrucât agentul microbian cauzativ este menţinut, prin infecţii subclinice, la animale sălbatice şi domestice. În prezent, pentru imunizarea activă a câinilor, sunt utilizate o gamǎ largǎ de vaccinuri inactivate, foarte eficace, produse de firme specializate din diverse ţǎri. În marea lor majoritate sunt vaccinuri polivalente, care includ, pe lângǎ cele douǎ serovariante de leptospire, întâlnite frecvent la canide (Leptospira canicola şi Leptospira icterohaemorhagiae), şi antigene virale pentru prevenirea hepatitei contagioase Rubarht, boala lui Carré, coronavirus şi parainfluenţa. Imunitatea specifică indusă de vaccin, inactivat este activă, individuală, se instalează în 10 - 21 de zile de la administrare, durează un timp variabil (4-6 luni) şi niciodată nu determină o protecţie de 100%. Prezenta lucrare are rolul de a investiga modul de instalare a răspunsului imun la carnivore, precum și protecţia acestuia.  Cuvinte cheie : răspuns imun; Leptospira icterohaemorhagiae; Leptospira canicola.  Oana TĂNASE,  Ileana GHEORGHIŢĂ, C. PAVLI, T. PERIANU   64 INTRODUCTION  Leptospira infections represent an important health issue, causing endemic infections being transmitted to humans. Mortality in humans isn’t high, but has very dramatic clinical manifestations (fever, toxic effect, headache, muscle pain, kidney damage, liver, nervous system, cardiovascular system) and is followed by a long convalescence (Blood et al.,1989; Vasiu, 2004; Velescu, 2002).  Leptospires are located at convoluted tubules level, and is excreted in the animal urine infecting the soil, water tanks, food, etc. (Perianu, 2003; Carter and Chenga, 1991; Constantinescu et al., 1997). People get sick during bathing in ponds infected during agricultural works on wetlands, etc. Enter the body through wounds or erosions of the skin and mucous membranes. Researches on the immune response after administration of different antileptospira vaccines, which included the two specific leptospire serovas (Leptospira canicola and Leptospira icterohaemorhagiae) were taken in 2009 on three groups of 10 dogs, of different races, close in size and maintenance over the age of one year, belonging to paddock "B" Galaţi Municipality.  MATERIALS AND METHODS  The research was undertaken in 2009 on three groups of 10 dogs, of different races, close in size and maintenance over the age of one year. For immunization were used commercial vaccines as: "Biocan L", "Nobivac LR", "Eurican DHPPI2LR". Before vaccines subcutaneously inoculation, the 30 dogs were subjected to blood collection and examined for the presence of specific antibodies to Leptospira canicola and Leptospira icterohaemorhagiae. To determine antileptospiric post-vaccination immune response from the three groups of dogs were collected blood samples 3 times every 10 days to 30 days and after 5 months after vaccine administration. Blood was harvested from saphenous vein, in sterile vacutainers and left at room temperature until the serum expression. Expressed serum was stored in a refrigerator until the execution of serological reactions. For determination of specific antibodies, anti-Leptospira canicola and anti-Leptopira icterohaemorhagiae, was used agglutination lyses reaction (RMAL), vs. eight leptospire serovas, maintained on Korthof environment. Antigens represented by live cultures of leptospire (Leptopira canicola and Leptopira icterohaemorhagiae), aged 6-12 days, incubated at 28 ° C in medium Korthof-looking uniform and a density of 100-600 micro-organisms on a microscope field (objective 20, ocular 15 Zeis) and not agglutinate spontaneously in the culture medium or in isotonic chloride solution. Cultures were checked before being used to a specific serum to see whether or not agglutinated. The technique consists in performing the reaction a glass slide with a thickness of 2-2.5 mm. To this end were made serum dilutions, starting with the dilution 1 / 50. On clean glass slide and defatted in advance to put one drop, with easy heads of each dilution over to add a drop as far as possible equal to, the antigen (culture leptospire). VICINAL IMMUNE RESPONSES IN CANINE LEPTOSPIROSIS   65 Reaction was read under a microscope with dark background, after contact for 20-30 minutes at room temperature. Sera positive at first dilution 1 / 50, were further diluted (1/100, 1/200, 1/400, 1/800, etc.) until exhaustion titer.  RESULTS AND DISCUSSION  Serological researches regarding immune response, after inoculation of the commercial vaccines on three test groups leaded to results depending leptospire serovar, vaccine structure and time since inoculation. Results analytical interpretation shows an immune response at ten days from inoculation, translated through presence of antibodies for both serovars (Table 1).   Table 1 - Results antileptospiric immune response after 10 days of vaccines  Specific antibody titer No. Vaccine Leptospire serovar Delivery number Samples 1/ 1001/ 2001/ 4001/ 8001/ 16001/ 32001/ 6400 Medium titer L. i.h. I 10 1 3 4 1 1 0 0 1/570 1. Biocan L L.canicola I 10 2 2 5 1 0 0 0 1/340 L. i.h. I 10 2 3 3 1 1 0 0 1/360 2. NobivacLRL. canicola I 10 2 3 4 1 0 0 0 1/320 L. i.h. I 10 3 3 2 2 0 0 0 1/330 3. Eurican DHPPI2LR L. canicola I 10 3 3 3 1 0 0 0 1/290    Figure 1 - Average titer values for the two leptospire  Oana TĂNASE,  Ileana GHEORGHIŢĂ, C. PAVLI, T. PERIANU   66 Considering results registered in Table 1 it can be observed the fact that immune response intensity differs considering serovars. The medium titer differ depending leptospire serovar, being estimated between 1/330 and 1/570 for Leptospira icterohaemoragiae and 1/290-1/340 in Leptospira canicola. From the point of view of the vaccine structure the highest titer value was obtained for Leptospira icterohaemorhagiae 1/570 respectively 1/340 in Leptospira canicola, in samples harvested from the dogs inocculated with vaccine Biocan L,  biological product that has only these two serovars in structure. Decreasing antibody titer was obtained in samples from dogs inoculated with the vaccine "NobivacLR" with three antigens (two bacterial and viral antigen), followed by a medium titer, of 1/330 in Leptospira icterohaemorhagiae and 1/290 in Leptospira canicola, in sera taken from dogs inoculated with "Eurican DHPPI2LR" vaccine which contains seven antigens (two bacterial and five viral). Monitoring post-vaccination immune response, after 30 days of administration of three vaccines we have obtained the results recorded in Table 2.  Table 2 - Immune response at 30 days from vaccine inoculation   Specific antibody titer No. Vaccine Leptospire serovar Delivery number Samples 1/ 100 1/ 2001/ 4001/ 8001/ 16001/ 32001/ 6400 Medium titer L. i.h. II 10 0 2 2 2 1 2 1 1/1720 1. Biocan L L.canicola II 10 0 1 3 2 1 1 1 1/1420 L. i.h. II 10 0 2 3 2 1 1 1 1/1440 2. NobivacLRL. canicola II 10 0 3 3 1 1 1 1 1/1220 L. i.h. II 10 1 1 2 3 1 1 1 1/1115 3. Eurican DHPPI2LR L. canicola II 10 0 3 3 2 1 1 0 1/820  From results analyze  is shown on the one hand an increase in the number of animals with high specific antibody value ranging from 1/800 and 1/6400 and on the other hand an increase in medium titer value  for both serovars of the three biological products. Thus the average antibody titer values were between 1/1115 and 1/1720 for Leptospira icterohaemorhagiae and 1/820 to 1/1420 for Leptospira canicola. Average titer values differ depending on vaccine used. Thus,   the highest value of average titer       of 1/1720 to Leptospira icterohaemorhagiae and 1 / 1420 to Leptospira canicola, were obtained for  vaccine "Biocan L", and the lowest average titer values were VICINAL IMMUNE RESPONSES IN CANINE LEPTOSPIROSIS   67 obtained in  Leptospira serovars icteroheamorhagae of 1 / 1115 and Leptospira canicola 1 / 820, contained in "Eurican DHPPI2LR" structure (Fig. 2).   Figure 2 - Average titer values after 30 days from inoculation  Table 3 - Immune response after 5 months from immunization   Antibody titer No. Vaccine Leptospire serovar Delivery  Samples 1/ 100 1/ 2001/ 4001/ 8001/ 16001/ 32001/ 6400 Average titer L. i.h. III 10 1 3 3 3 0 0 0 1/430 1. Biocan L L.canicola III 10 2 4 3 1 0 0 0 1/290 L. i.h. III 10 3 3 3 1 0 0 0 1/290 2. NobivacLRL. canicola III 10 3 4 2 1 0 0 0 1/270 L. i.h. III 10 4 3 2 1 0 0 0 1/260 3. Eurican DHPPI2LR L. canicola III 10 5 3 1 1 0 0 0 1/230  From results shown in Table 3, regarding immune response after 5 month, appears a rise in animal number with antibody titer values between 1/100-1/400, and on the other hand a decrease in medium titer value depending leptospire serovar. The highest values between 1/320 -1/430 were registered for Leptospira icterohaemorhagiae and 1/230 - 1/290 for Leptospira canicola. Average titer values are different depending on the antigenic structure of the vaccine inoculated. Thus the largest value of the average titre of 1/430 for Leptospira icterohaemorhagiae and 1/290 to Leptospira canicola has been obtained for the vaccine "Biocan L". Values less than 1/290 for Leptospira Oana TĂNASE,  Ileana GHEORGHIŢĂ, C. PAVLI, T. PERIANU   68 icterohaemorhagiae and 1/290 to Leptospira canicola have been obtained for the vaccine "NobivacLR" and the lowest values of average titer of 1/260 -1 / 230, were obtained using vaccine "Eurican DHPPI2LR" (Fig. 3).   Figure 3 - Average titer values after 5 months from immunisation  Analyzing the immune response dynamics at 10, 30 days and 5 months after administration of the  vaccines, there is, on the one hand, an increase in the average titer after 30 days for both leptospire serovars (Leptospira icterohaemorrhagiae and Leptospira canicola), included in the vaccines structure, compared with values obtained from the examination after 10 days and on the other hand, a decrease in the average titer after 150 days from immunization. Increased average titre value after 30 days after inoculation of the biological products, explains antigenicity and post-vaccination immunity. Lower average titer after 5 months post-vaccination explains the decrease of protection.  In fact, it is known that the immune response and therefore post-vaccination immunity is dependent as intensity and duration on nature of antigen (bacterial or viral, inactivated or attenuated). Average titer progressively drop after 5 months from imunisation, can be explained by the fact that the duration of immunity obtained using inactivated bacterial antigens, is shorter, being between 4-6 months.  CONCLUSIONS  Results obtained shows that after imunisation with three biological products, all animals exhibited  immune response, by the appearance of specific antibodies in blood serum. VICINAL IMMUNE RESPONSES IN CANINE LEPTOSPIROSIS   69 The intensity of immune response, and average titer values differ depending on the structure of Leptospira serovar present in the three types of vaccines, ranging from 1 / 420 - 1 / 1720 for  Leptospira icterohaemoragiae and Leptospira canicola 1/230-1/1420. Average titer values are different depending on the antigenic structure of the vaccine. The highest value of average titer of 1 / 430 for Leptospira icterohaemorhagiae and 1 / 290 to Leptospira canicola was obtained from the vaccine "Biocan L". Values of 1 / 290 for Leptospira icterohaemorhagiae and 1 / 290 for Leptospira canicola have been obtained for the vaccine "NobivacLR", and the lowest average titers of 1 / 260 and 1 / 230 were obtained for "Eurican DHPPI2LR ". Analyzing the immune response dynamic at 10, 30 days and 5 months, it appears, on the one hand, increased value of the average titer after 30 days in both leptospire serovars, compared with values obtained after 10 days, on the other hand, decreased antibody average titer after 5 months from immunisation. Average titre increased value after 30 days from inoculation of the biological products highlights antigenicity of the vaccine strains in the product structure, post-vaccination immunity. Lower average titer after 5 months shows post-vaccination protection decrease of immunity.  REFERENCES  Blood D.C., Radostits O.M., 1989 – Veterinary Medicine. Ed. Bailliere Tindall, London. Carter G.R., Chenga M.M., 1991 – Les zoonoses infectieuses, caires des maladies contagieuses. Ecole Nationales Veterinaires Froncaises, Sept. Symposium. Constantinescu Valerica, Petriceanu Gela, Rădulescu Alina Ruxandra, 1997 – Informaţii epidemiologice privind leptospiroza la animale în ultimii 10 ani (1985-1995) (Epidemiological information regarding leptospirosis in animals in past ten years (1985-1995). Al XXIII – lea simpozion „Actualităţi în Patologia Animalelor Domestice”, USAMV Cluj-Napoca. Gheorghiţă Ileana, Perianu T., 2009 – Observaţii privind leptospiroza canină în Municipiul Galaţi (Observations regarding canine leptospire in Galați country). Lucrări ştiinţifice, Vol.52(11), Seria Medicină veterinară. Partea II, 943-946.   Kmetty E., Dikken H., 1993 – Leptospira interrogans,classification and serovas history. University Bess Groningen, Nederlands.  Perianu T., 2003 – Boli infecţioase ale animalelor domestice. Bacterioze (Infectious diseases in animals. Bacteriosis).  Vol I, Edit. Fundaţiei Chemarea, Iaşi.  Vasiu C., 2004 – Boli infecţioase ale animalelor domestice. Bacterioze (Infectious diseases in animals. Bacteriosis). Edit.  MEGA, Cluj-Napoca.  Velescu Elena, 2002 – Patologia bolilor infecţioase la animale (Infectious diseases pathology in animals patologia). Edit. Terra Nostra, Iaşi.    

Researches on vicinal immune responses in canine leptospirosis

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Researches on vicinal immune responses in canine leptospirosis

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