UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE

CLUJ-NAPOCA

PhD SCHOOL

FACULTY OF VETERINARY MEDICINE

IANCU FLORINA-ALEXANDRA (KRUPACI)

THE EFFECT OF THE PHYLOGENETIC POSITION AND HABITAT

ARTIFICIALIZATION ON THE PORTING MICROFLORA AND ZOONOTIC

RISK REPRESENTED BY PHEASANTS AND RABBITS

(SUMMARY OF Ph.D. THESIS)

Scientific coordinator:

Prof. Dr. BRUDAȘCĂ GHEORGHE FLORINEL

CLUJ-NAPOCA

2013

PHD THESIS SUMMARY

XV

SUMMARY

Domestic and wild animals was always considered important as the reservoir of

infectious agents to humans.

The pheasant (Phasianus colchicus) and the hare (Lepus europaeus) represent game

species of major importance in our country, being hunted for their meat wich possesed

dietary indisputable qualities.

The hare can be reservoir or carrier of zoonotic disease. Besides disease like

stafilococosis, pasteurelosis, pseudotuberculosis and brucelosis, the hare can be carrier

for other zoonotic agents, among those, tularemia, listeriosis, toxoplasmosis, leptospirosis

and borreliosis are the most dangerous if the carrier animals are handled without caution

(Bourque et al., 1984; Pikula, 2007).

For the important role that it has in the living ecosystem and also for the fact that the hare

do not go far from the place that he live, makes from the hare un important bioindicator

of the environment factors.

Because of the direct or indirect contact that the hare has with different species, including

human, is very important from epizootic and veterinary point of view the knowledge of

the bacterial porting microflora and especially of the bacterial species.

Also, rabbit breeding for meat and fur gained increasing importance in the world

(Combe et al., 2005; Xiccato, 1999).

People, in many parts of the world have used rabbits as source of food and fur

(http://en.wikipedia.org/wiki/Rabbit).

An comparison between hares and rabbits, respectively game pheasants and household

pheasants, regarding microbial porting flora, is needed because is important to know the

existent differences and especially is important to analyze the implication of the

environmental factors upon the microbial profile evolution.

Because of the differences between the sylvatic and domestic life environment,

differences can be shaped also regarding the immune response of the two animals

category, translated thru a higher or lower resistance at disease that affect those animals.

Also, because of the ‟‟articial‟‟ raising conditions, the animals from this enviroments are

more protected from certain pathogens compared to wild animals.

Microbiological monitoring of the game species from huntig fields is an action that

should continuous conductind because the climatic changes and the seasonal conditions

can affect the disease evolution regarding the spreed and dissemination, emergence and

persistence in new habitats. Pathogen invasion can lead to new complex diseases,

presenting majore challenges for sustainable future of the animals and agriculture at

global level (de La Rocque et al., 2008).

Also, a study regarding the influence of the phylogenetic position (Aves and Mammalia)

upon poting microflora and a correlation with the studied species immune system had not

been performed so far.

The studies included in this paper were performed between 2009-2013, and had as main

objectives the verification of following hypotheses:

Microbial porting of farmed animals whether we are talking about pheasant

or rabbit, is limited compared to that of the wild animals;

PHD THESIS SUMMARY

XVI

Microbial flora present in a relatively small living environment, as is the case of animals

kept by man in closed systems (farms, households) is controlled by human intervention

(disinfection, surveillance of livestock movement, livestock closed circuit, etc) so we

assumed that in such environment, the bacterial load will be less, both in total and and

variety of species. On the other hand, the sylvatic environment where game species

studied are found, the circulation, therefore, the risk of contamination, are more

restricted, is present in the self-cleaning abilities of segments (for example, streams).

The comparative evaluation of microbial contaminants from the two habitats correlated

with the porting flora will allow to confirm or refute the issued hypothesis.

Differences in structure and function of the immune system in the two studied

classes could cause differences between the porting microflora and its

pathogenicity for the individuals concerned and for contacts. Class Aves, respectively, class Mammalia, by time evolution have acquired structures and

immunological competence conditioned by the living environment, the food search

behavior, the reproductive behavior, etc. In this way have emerged structures with

sophisticated roles, able to respond to multiple environmental aggressions but also at

thoso coming from inside.

According to some authors, the immune system in birds is even more complex than in

mammals, this being motivated by the presence of well defined structures, capable of

providing one, for example, training of B lymphocytes (bursa of Fabricius) or complex

protection by an organ (Harder gland). Nevertheless, is considered that, in the case of

mammals the antiagresional response pathways are more developed, the response being

more prompt, regardless the aggressor.

Antibiotic resistance is greater for the bacterial species of medical interest

from domestic animals compared with those of wild animals.

Animals kept for economic purpose are exploited under "unnatural" conditions, the

living environment being artificialized and controlled. In the attempt to avoid harmful

external influences and also the microbial aggression, has become established the

practice of use antimicrobials, among those, antibiotics, frequently without

discrimination. In such a context, is easy to understand that resistance at the antibiotics of

the porting flora can be installed, especially at the usual ones. It was assumed that

„domestic” studied animals, frequently being exposed to antibiotic therapy, will be

carriers of resistant microbial species or even or multiresistant to antibiotics.

This paper is divided into two parts, first part entitled " Literature Review " containing 50

pages and the second part containing 135 pages is entitled “Own Research”.

This study contained 35 figures photo, 17 charts and 30 tables. The first part of the thesis,

"Literature Review", highlights the current state of knowing and extends itself over 6

chapters.

In the Introduction are described the importance of the studied species and an brief

historical of the most frequent diseases encountered in these species, respectively the

evaluation of the losses due to those and the risks to man.

In the second chapter are described briefly pheasants and hare species, also being

conducted scientific classifications, described the spread, habitat and their behavior.

In chapter three of the first part, is described the species habitat, game pheasant habitat

and household pheasant, respectively, hare habitat and rabbit habitat.

PHD THESIS SUMMARY

XVII

The fourth chapter describe the porting microflora in pheasants and hare/rabbit, this flora

being classified upon animal category, specific to wildlife/domestic, respectively,

common to the both animal species and depending on the germs importance for animal

and human health.

In chapter five is described the bacterial flora commonly isolated from living

environments of the studied species, respectively from sylvatic and domestic life

environment. Chapter six covers aspects of the immune system in mammals and birds,

the ways that this develops and the way of „intervention” in case of infection outbreak.

The secon part of the thesis is intended to own research and comprises seven chapters. In

this part are described the objectives of the thesis, materials and methods that was used to

obtain results that will confirm or refute the issued hypothesis.

The presentation of experimental part is followed by an assertion of general conclusions,

recommendations and the literature cited.

In this study were cited 291 references.

The chapter II.I. is intended to describ the habitat for studied species so here are

described the hunting funds from where the samples were taken.

The hunting funds were described using public data site of Ministry of Environment and

Forests, paying attention to the limits of the funds, to feed available for hunting

population and to game effectives of these funds.

Were described the hunting funds on 4 counties, for a better understanding of the location

and to their contents, were attached figures respresenting technical drawings of the funds.

Were described the hunting funds: VIȘTEA (Cluj), CHIEJD (Sălaj), SÂNMIHAIU

ALMAŞ (Sălaj), SÂNTIMREU (Bihor), ŞIMIAN (Bihor), GRINDENI (Mureș).

After de description of the hunting funds were briefly described the rabbits and pheasants

households from wich the sample were collected. It was intended to describe the animals

maintenance conditions, making dietary specifications and treatments that animals have

undergone, it was described the manufacturing of the boxes, respectively the lofts, in

order to have a clear view of the way that artificial life environmental conditions of the

studied animals category could influence the porting microflora and their immunity.

In the chapter II.II. were described materials and methods applied in order to identify the

porting microbial flora from pheasants and hares/rabbits.

Were processed a total of 246 samples (pharyngeal and cloacal swabs) from game

pheasants and 46 samples from household pheasants. The samples were processed after

the classical microbiological techniques and the identification of the bacterial species was

made using API tests.

Upon performing the API tests for the bacterial species isolated from game

pheasants have been identified these bacterial strains: Enterobacter aerogenes (5,22%),

Staphylococcus spp. (12,95%), Pseudomonas aeruginosa (3,63%), E. coli (28,86%),

Enterobacter cloacae (15,22%), Serratia marcescens (5,22%), Corynebacterium spp.

(2,27%), Streptococcus spp. (2,5%), Micrococcus spp. (1,59%), Clostridium spp.

(3,63%), Enterobacter sakazakii (4,09%), Rahnella aquatillis (1,59%), Serratia

liquefaciens (2,04%), Chryseomonas luteola (0,22%), Pantoea spp. (1,59%), Serratia

rubidaea (0,68%), Proteus spp. (2,04%), Citrobacter braakii (0,22%), Aerococcus

viridans (2,27%), Ewingella americana (1,13%), Enterobacter amnigenus (1,81%),

Citrobacter freundii (0,45%), Enterobacter cancerogenus (0,22%), Salmonella arizonae

(0,45%).

PHD THESIS SUMMARY

XVIII

By comparing the bacterial strains isolated from game pheasants pharynx and

cloaca it have been seen that there is a statistically significant difference regarding the

prevalence of Enterobacter aerogenes, isolated with a significantly higher frequency

from pharynx (p = 0.008).

From household pheasants were identified these bacterial strains: Enterobacter

cloacae (38,8%), E. coli (9,25%), Klebsiella terrigena (5,55%), Enterobacter aerogenes

(7,40%), Staphylocccus spp. (9,25%), Clostridium spp. (18,51%), Escherichia fergusonii

(1,85%), Butiauxella agrestis (3,7%), Streptococcus spp. (3,70%), Serratia fonticola

(1,85%).

Differences regarding the isolation of the bacterial strains from pheasants pharynx

and cloaca are not statistically significant.

Regarding the prevalence of different bacterial strains in game and household pheasants,

have been observed statistically significant differences only for Enterobacter cloacae (p

= 0.020), Klebsiella terrigena (p = 0.021), Clostridium spp (p = 0.002) and Butiauxella

agrestis (p = 0.021).

From hares have been isolated from 162 samples, 280 bacterial strains, 157 of

these from pharynx and 123 from rectum.

The strains isolated from hares were identified thru API tests beeing identified

these bacterial strains: E. coli (28,57%), Enterobacter sakazakii (12,5%), Enterobacter

cloacae (9,64%), Clostridium spp. (6,42%), Corynebacterium spp. (5%), Micrococcus

spp. (3,21%), Serratia liquefaciens (6,07%), Serratia marcescens (1,78), Staphylocccus

spp. (9,28%), Pseudomonas aeruginosa (3,92%), Rahnella aquatillis (4,28%),

Streptococcus spp. (2,85%), Citrobacter freundii (2,14%), Enterobacter aerogenes

(2,85%), Pantoea spp. (1,42%).

Regarding the bacterial strains isolated from the pharynx and rectum of the hares

have been noted that are statistically significant differences for the prevalence of the

folowing strains: E. coli (p = 0.014), Staphylocccus spp. (p = 0.024), Streptococcus

spp.(p = 0.012), Enterobacter aerogenes (p = 0.007) și Citrobacter freundii (p = 0.039);

for the other strains were not statistically significant differences.

From rabbits were isolated from 140 samples, from pharynx 98 strains and from

rectum 109 bacterial strains.

The bacterial strains isolated from rabbits were tested thru API tests beeing

identified these bacterial species: E. coli (16,90%), Enterobacter cloacae (14%),

Staphylocccus spp. (6,28%), Micrococcus spp. (7,72%), Aerococcus viridans (3,38%),

Lactococcus lactis (2,41%), Klebsiella ornithinolitica (3,38%), Klebsiella pneumoniae

(1,44%), Enterobacter aerogenes (6,76%), Proteus mirabilis (3,38%), Serratia odorifera

(2,41%), Klebsiella planticola (4,34%), Rahnella aquatillis (1,93%), Pantoea spp.

(3,86%), Celullomonas spp. (6,76%), Clostridium spp. (11,11%), Citrobacter freundii

(3,86%).

Regarding the distribution of the bacterial strains isolated from rabbits pharynx

and rectum, statistically significant results were observed for the bacterial strains as

Enterobacter cloacae (p = 0.001), Staphylocccus spp. (p = 0.000), Aerococcus viridans

(p = 0.021), Klebsiella ornithinolytica (p = 0.021), Enterobacter aerogenes (p < 0.001),

Proteus mirabilis (p = 0.021), Klebsiella planticola (p = 0.006), Pantoea spp. (p =

0.012), Citrobacter freundii (p = 0.007), Celullomonas spp. (p < 0.001).

PHD THESIS SUMMARY

XIX

Statistically analyzing differences between bacterial strains isolated from hares and

rabbits can be seen statistically significant differences for the strains of Corynebacterium

spp (p = 0.001), Serratia liquefaciens (p < 0.001), Pseudomonas aeruginosa (p = 0.004),

Streptococcus spp. (p = 0.021), Celullomonas spp. (p = 0.000), Aerococcus viridans (p =

0.012), Lactococcus lactis (p = 0.048), Klebsiella ornithinolytica (p = 0.012), Proteus

mirabilis (p = 0.012), Serratia odorifera (p = 0.048), Klebsiella planticola (p = 0.003).

The predominant bacterial specie isolated from studied animals was E. coli whose

increased pathogenic potential is known and frequently reported in animals causing major

economic losses (Owen, 1992; Davies, 2006).

Although some of the identified bacterial species (Celullomonas spp., Aerococcus

viridans, Serratia spp., Enterobacter spp., Erwinia spp., Klebsiella terrigena) are

conditioned pathogenic, the isolation of those from pheasants and hares/rabbits posse risk

of disease, beeing known that physiological bacterial flora, under certain conditions, can

become pathogenic (Krupaci et al., 2011).

The bacterial species isolated from pheasants and hares/rabbits has the potential

pathogen, without any exception, existing data in the literature about infections they can

cause in animals or humans.

In conclusion from the studied wildlife were isolated more germs with zoonotic

risk than from the species from households, wich shows that in a sylvatic, unprotected

environment, pathogens are in a highr number compared to a controlled environment.

In the chapter II.III. was identified the microbial flora from the living environment of the

studied species, respectively, from the game pheasants and hares environment were

identified the following bacterial agents: Micrococcus spp., Staphylococcus spp.,

Corynebacterium spp., E. coli, Serratia rubidaea, Enterobacter sakazakii.

It have been seen that the waters from the hunting grounds are more contaminated

than soil, given the fact that from water were isolated several bacterial species, whose

pathogen potential should not be neglected.

From the living environment of household pheasants were identified the following

bacterial species: Corynebacterium spp., Erwinia spp., Serratia rubidaea, Vibrio

fluvialis, Enterobacter cloacae, Staphylococcus spp., Clostridium spp..

From the living environment of the rabbits were identified the following bacterial

species: Micrococcus spp., Erwinia spp., Corynebacterium spp., Enterobacter cloacae,

Clostridium spp., Klebsiella terrigena, Klebsiella pneumoniae, Streptococcus spp..

Most of the bacterial species isolated from the life environment of pheasants and

rabbits were isolated also from the samples that came from those animal species.

Nevertheless, there are bacterial species isolated only from the life environment.

Those bacterial species are represented by Vibrio fluvialis, bacterium with

pathogenic role for humans and animals, and Erwinia spp. that has no potential pathogen.

In conclusion, although in both types of living environments, sylvatic and

domestic were identified pathogenic and zoonotic bacteria, risk of disease is higher in the

domestic environment, because at this level have been isolated pathogen bacterial species

like Vibrio fluvialis.

The chapter II.IV. was designed to evaluate the antibiotic resistance level of the

bacterial strains isolated from hares/rabbits and pheasants, so were tested for sensitivity

to antibiotics strains of E. coli and Staphylococcus spp., bacterial species often cited in

literatura for their resistance to antibiotics.

PHD THESIS SUMMARY

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For the testing sensitivity germs to antibiotics was used Kirby-Bauer disc diffusion

method. The antibiograms were performed according to standard CLSI (Clinical and

Laboratory Standards Institute). It were tested 10 strains of E. coli and 10 strains of

Staphylococcus spp. from game pheasants, and 4 from household pheasants were tested 5

strains of E. coli and 4 strains of Staphylococcus spp.

After measuring the diameter of inhibition zones around each microdisk, the tested

bacterial species were interpreted as sensitive, resistant or intermediate sensitive to

antibiotics after CLSI 2011 standard.

After E. coli strains was isolated from game pheasants, susceptibility was tested

and has been noticed that the most effective antibiotic was chloramphenicol, 90% of the

tested strains were sensitive to its action. Efficient to the same strains was the gentamicin,

that 50% of the strains tested were susceptible at his action and the other 50% were

intermediate sensitive. The following efficient antibiotic for the tested strains was

ciprofloxacin, in this case were noticed the existence of two resistant strains. 50% from

the tested strains were sensitive to polymyxin B and the other 50% were resistent.

Trimethoprim sulfametoxazole was ineffective for the tested strains from this study, all of

those beeing resistant at the antibiotic action. To tetracycline, none of the tested bacterial

strains was sensitive.

For the E. coli strains isolated from household pheasants, it have been seen that

the only antibiotics in vitro corresponding effective were gentamicin (sensitivity 100%)

and ciprofloxacin (sensitivity 80%). At the action of nalidixic acid and tetracycline all the

strains were resistant.

In conclusion, the E. coli strains isolated from wild bird shows higher sensitivity to

antibiotics compared to those isolated from household birds.

In recent years the focus was on the occurrence of MRSA (Methicillin-resistant

Staphylococcus aureus) and MRCNS (Coagulase-negative staphylococci methicillin-

resistant) (Mártonová et al., 2008) so it is understandable the susceptibility testing for

some strains of staphylococci isolated from pheasants destinated to human consumption.

The testing antibiotics susceptibility for the Staphylococcus spp. strains isolated from

game pheasants shows that all strains of Staphylococcus spp were sensitive at the

enrofloxacin, amoxicillin + clavulanic acid, tetracycline action, and the rest of the

antibiotics were ineffective for all the tested strains (100% antibacterial resistance).

For antibiotic susceptibility testing of Staphylococcus spp. isolated from

household pheasants, it was found that at the action of the three antibiotics

(sulfametoxazole trimethoprim, ampicillin and penicillin) those strains showed 100%

resistance.

The tetracycline was the most effective antibiotic against all the tested strains from

this animal specie ( 100% sensitivity). At the action of enrofloxacin, the tested strains

were sensitive 50% and 50% intermediate sensitive and at the amoxicillin + clavulanic

acid, 75% of the strains were sensitive, rest of them being resistant.

From hares were tested 15 strains of E. coli and 10 strains of Staphylococcus spp.

and from rabbits, 15 strains of E. coli and 10 strains of Staphylococcus spp.

For the E. coli strains isolated from hares it was noticed that the most effective

antibiotics were ciprofloxacin and gentamicin, more of the half of the strains were

resistant at trimethoprim sulfametoxazole and the most ineffective antibiotic was the

tetracycline.

PHD THESIS SUMMARY

XXI

The most effective antibiotic for the E. coli strains isolated from rabbits was the

gentamicin, followed by ciprofloxacin and chloramphenicol. The most ineffective

antibiotic was the tetracycline.

It was seen that there are differences between the bacterial strains isolated from

hares and rabbits regarding the antibiotics sensitivity, such differences were describes by

Pissoni et al., 2004 who also noted that a biotype of E. coli isolated twice from the same

rabbit farm usually has the same behavior at the antibiotic, but the antibiotic

susceptibility is easily changing when the same biotype is isolated from other rabbit farm.

Such differences were found between strains of E. coli from wildlife and those from

domestic animals, the antimicrobial profile of the strains was for some of them different.

Differences regarding the resistance to antibiotics of the E. coli strains isolated

from the digestive tract of the hares and rabbits were noticed also by Krupaci et al., 2012,

in the sense of increased sensitivity to antibiotics for the wildlife strains compared to

those domestic.

Some bacteria have developed resistance at all antibiotics groups discovered untill

now, and the most common type of resistance is acquired and transmitted horizontally

through mobile genetic elements, like plasmids, integrons and transposomi (Tiemersma

et al., 2004). Staphylococcal infections are often treated with antibiotics and

consequently apear the acquired antibiotic resistance phenomenon (Normand et al.,

2000).

When testing the antibiotic susceptibility of the staphylococci strains isolated

from hares it was seen that the most effective antibiotics against all strains were the

amoxicillin + clavulanic acid and the enrofloxacin. The use of these two antibiotics was

not detected any resistant strain from those tested. The most ineffective antibiotics used

were the ampicillin and penicillin G, for those 80%, respectively 90% of the tested strains

presented resistance.

The most effective antibiotic for the staphylococci strains isolated from rabbits

was the enrofloxacin (90% of strains were sensitive) at the amoxicillin + clavulanic acid

and 70% from strains were sensitive. The most resistant strains were found at the

ampicillin action.

It was assumed that „domestic” animals studied, being exposed frequently to

antibiotic therapy will be carriers of antibiotic-resistant or multiresistant microbial

species. This assumption proved valid, being observed commonly the antibiotic

resistance phenomenon for the E. coli strains of domestic origin compared to those of

wild origin. Also, even with antibiotics that have antimicrobial activity against strains

from both origins, it can be seen that the diameters of the bacterial inhibition zones are

smaller for the strains of domestic rabbits.

The chapter II.V. entitled OVERALL ASSESSMENT OF THE BACTERICIDAL

ACTIVITY OF THE SERUM DEPENDING OF THE PORTING MICROFLORA

contains information about this parameter of innate immunity.

The serum capacity to inhibit bacterial growth is assessed by the presence of complement

components that can modulate the concentration of natural antibodies against other

ubiquitary agents in the environment, particularly enterobacteria (Moscati et al., 2008).

In addition to testing the immunity of studied organisms, by evaluating serum

bactericidal capacity can be assessed also the the pathogenicity of the bacteria tested so

PHD THESIS SUMMARY

XXII

serum the resistance property of invasive bacteria to serum is an important aspect of their

pathogenicity (Moll et al., 1980).

In the study were included 36 serum samples from rabbits that came from 4

different households and 15 serum samples from household pheasants. It was tested the

bactericidal capacity of the rabbit serum against two E. coli strains, reference strain (E.

coli ATCC 10536) and the other came from rabbits. Rabbit sera were tested only for the

reference strain.

The bactericidal potential of serum was evaluated in vitro by the microdilution plate

technique. For this purpose serial dilutions of the sera were made in broth by performing

5 dilutions (1/10, 1/20, 1/40, 1/80 şi 1/160). Consecutive the obtaining of serial dilutions

for each serum sample, every well was inoculated with 3 ml inoculum of reference strain

E. coli ATCC 10536, respectively E. coli strain from rabbits.

Rabbit sera had bactericidal activity for E. coli ATCC 10536 reference strain

accordind the dilution.

After conducting unimodal ANOVA and post hoc Tukey test were seen

statistically significant differences (p = 0,036) between rabbit group 1 and 2 regarding the

bactericidal activity of the sera at 1/80 dilution against the E. coli reference strain.

Between the two groups, at none dilution were seen statistically significant

differences.

Rabbit sera from group 1 had maximum efficiency for E. coli strain ATCC 10536

at the second dilution (1/20), for the rabbits from groups 2 and 3 was observed that at

higher dilutions of the sera (1/40, 1/80) the bactericidal activity increases. For the rabbits

from group 4, at the maximun serum concentration (1/10) is observed minimal effect

translated thru major bacterial turbidity (Chart 1).

PHD THESIS SUMMARY

XXIII

Chart 1. Rabbit serum bactericidal potential against reference strain E. coli

ATCC 10536

Statistically analyzing the obtained results for testing the bactericidal effect of

pheasant sera compared to the effect of the rabbit sera were found significant differences

in 4 of the 5 dilutions tested, so: at dilution 1/10 of the serum were found statistically

significant differences between pheasants and group 2 of rabbits(F(4,46) = 5,332, p =

0,001), at dilution 1/20 were found statistically significant differences between the

pheasants and group 1 of rabbits 1/40 (F(4,46) = 4,516, p = 0,001), at dilution 1/80

(F(4,46) = 5,871) and dilution 1/160 were found statistically significant differences

between group 1 of rabbits and pheasants F(4,46) = 4.289, p = 0.002).

Pheasant sera had maximum efficiency for the growth inhibition of the bacterial

reference strain E. coli at dilution 1/20, as seen in the chart 16 that at higher serum

dilutions bacterial growth is more intense.

PHD THESIS SUMMARY

XXIV

Chart 2. Pheasant serum bactericidal potential against reference strain E.

coli 10536. Error bars represent one standard deviation

Rabbit sera from group 1 had negative effect against E. coli isolated from rabbits

at dilution 1/10; as dilutions rate increased it had seen an incressed of the turbidity in the

wells wich corresponds to a more intense bacterial growth in the absence of serum

factors.

Also, from comparative testing activity of rabbit sera against E. coli reference

strain and the strain isolated from rabbits it was seen that the sera was more effective

against the strain isolated from rabbits, and it was observing the uniformity of the results

(Chart 2).

Analyzing the bactericidal capacity of sera from pheasant against reference E. coli

strain it can be seen that at the dilution of 1/20 (1/20 = 0,249 UDO) the serum has the

maximum effect, as the dilutions rate increases the turbidity increases to (1/160 = 0,256

UDO).

Chart 17. Rabbit serum bactericidal potential against E. coli strain isolated from

domestic rabbits. Error bars represent one standard deviation

PHD THESIS SUMMARY

XXV

In conclusion, the satisfactory bactericidal activity of the serum for the tested strains

even at high dilutions, in rabbit and pheasant,

shows enough concentration of potentially

bactericidal serum factors.

In the chapter II.VI. was performed The Determination of Total Gammaglobulins, very

important test to assess the operational status of the immune system for the studied species.

Determination of circulating antibodies thru laboratory methods creates the possibility of

estimating the anti-infective humoral protective potential, respectively allow diagnosis of

some diseases.

As a result, the determination of circulating immunoglobulin concentration, may provide

clues on the stage or level of protection.

Following the results was found that total immunoglobulins showed variations between

groups of studied rabbits.

In order to determinate differences between the 6 groups of rabbits was used

unimodal ANOVA statistical test using the program R for Windows.

It was found that between different groups are statistically significant differences, F

(5,63) = 2,38, p = 0,048.

Using post-hoc Tukey test was found that the average values of gammaglobulins at rabbits

from group 6 is significantly higher compared with group 4 (p = 0,035).

Table 1. Statistical analysis for total immunoglobulins

Specie

n

Average±SD

Rabbits

69

0,0338±0,0891 ODU

3,38±8,91 Vernes degree

Pheasants

15

0,0226±0,0132 ODU

2,26±1,32 Vernes degree

3,25±2,30 grade Vernes

The significantly higher values of immunoglobulin for the rabbits from group 4

compared to other groups may indicate a more efficient response from the immune

system at different pathogens.

The significantly higher values of immunoglobulin for the rabbits from group 4

compared to other groups may indicate a more efficient response from the immune

system at different pathogens.

Correlation between the existence of potentially pathogenic bacterial species in the

studied animals and their health status indicates a high potential protection from

PHD THESIS SUMMARY

XXVI

infections and allow meanwhile the classification of the determination of

immunoglobulins results in the category of physiological values for these species.

In the last chapter of this thesis, chapter II.VII. were assayed the circulating

immune complexes, wich are formed after coupling of specific immunoglobulin antigen

molecules aggression.

These circulating elements can provide us data on various aspects, such as the degree of

invasion or viral assault of the organism or the responsiveness of the attacked body thru the

antibodies formation degree. Due to the large size of the circulating imune complexes (CIC) can

be precipitated by polymers with high molecular weight as polyethyleneglycol (PEG), even at

lower concentration of the complexes.

For the determination of circulating immune complexes in pheasants and rabbits

the precipitation method with PEG was used (Haskova).

The results were expressed in optical density units (ODU). The work test applied

was the the one precipitating with PEG 4,2%, to estimate levels of circulating immune

complexes (CIC). This technique was applied by a modification of the original version at

the micromethod.

The hemolyzed samples were eliminated from testing, being considered

unsuitable. The samples were kept at room temperature for 60 minutes, then the was read

in spectrophotometer SUMAL PE2 (Carl Zeiss) compared to distilled water at a

wavelength of 450 nm in 96-well plates.

The results were expressed în units:

(U) CIC = (the sample extinction - witness extinction) x 1000

Are considered positive the values exceeding 100 U and negative those under this limit.

Individual values of circulating immune complexes (results expressed in U = units) in

rabbits varied in range from 0 to 253 with a mean of 16,34 and in pheasants were in range

from 2-40, with a mean of 12,6.

In order to determine the differences between the 6 groups of rabbits the unimodal

ANOVA statistical test was used with the R for Windows program. It was found that

between the different groups are statistically significant differences F (5,63) = 3,38, p =

0,009.

Using post-hoc Tukey test was found that the average for the values of the circulating

immune complexes in group 4 of rabbits is significantly higher compared to the group 2

(p = 0,002), group 5 (p = 0,03) and group 6 (p = 0,006).

It is considered that a wide variety of chronic debilitating diseases are caused by

circulating immune complexes.

Antigen-antibody complexes are deposited in the blood vessels of the affected organs and

lesions are initiated by activating the complement system, causing an inflammatory

response (Levinsky, 1978).

Except for two values of circulating immune complexes identified in group 4 of rabbits

that excess the 100 conventional units, all values of circulating immune complexes in

rabbits and pheasants are considered to be normal.

PHD THESIS SUMMARY

XXVII

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