VETERINARSKI ARHIV 68 (1), 33-38, 1998

Changes in the white blood cells and specific phagocytosis in chicken with experimental
acute fowl typhoid

Todor Kokosharov*

Laboratory of Bacteriology, Regional Veterinary Station, Haskovo, Bulgaria

* Contact address:
Prof. Dr. Todor Kokosharov,
Regional Veterinary Station, 6300 Haskovo, Bulgaria,
Phone: 359 38 200 32; Fax: 359 38 200 55

ISSN 0372-5480
Printed in Croatia

KOKOSHAROV, T.: Changes in the white blood cells and specific phagocytosis in chicken with experimental acute fowl typhoid. Vet. arhiv 68, 33-38, 1998.

ABSTRACT

The study was performed on Salmonella-free, five-month-old Plymouth Rock chickens infected orally with a strain of Salmonella gallinarum. The dynamics of the number and structure of white blood cells and phagocytosis of the leukocytes in healthy and infected chickens were tested. The experimental typhoid infection caused a leukocytosis on the 3^rd day after infection, followed by leukopenia from the 4^th day until the 14^th day inclusive, and reversed as late as the 21^st day. The results demonstrated that chickens with S. gallinarum infection produced an initial rise followed by fall in the percentage of circulating mature leukocytes in the recovery period and a percentage rise in immature myelocytes and bandnuclear cells. Heterophilia was associated with a relative lymphopenia. This study is the first report of enhancement of the specific phagocytic function (phagocytic activity and phagocyte number) of the leukocytes during 3 - 7 days post infection. All of these changes were found to precede the onset of the first clinical signs of the disease and they may improve early diagnosis of acute fowl typhoid in a clinical situation.

Key words: chicken, acute fowl typhoid, Salmonella gallinarum, white blood cells, phagocytosis

Introduction

Leukocytes play a central role in the inflammatory response and host defense to infection. They participate in the effective cleaning of gram-positive and gram-negative bacteria (AINSWORTH et al., 1996; HOLST et al., 1996; LAICHALK et al., 1996). Monocytes and granulocytes possess a variety of receptor and enzymatic mechanisms, enabling them to engulf and kill microbes (MARRA et al., 1990; LAWRENCE, 1992). Natural killer cells, or spontaneous or naturally cytotoxic lymphocytes, comprise a population of lymphocytes with an ever increasing biological role by mediating non-specific host resistance against bacterial disease (DUFFUS, 1989).

The discovery of the phagocytosis by Metchnikoff in 1882 led to approximately 100 years of fruitful research into the functions of phagocytic cells. For the phagocyte cell biologist, however, the most exciting research era is beginning right now (KRAUSE et al., 1997). Effective host defense against bacterial invasion is dependent upon the vigorous recruitment and activation of inflammatory cells. In the setting of bacterial infection the myeloid cells are instrumental in both the phagocytosis and killing of bacterial organisms (DUFFUS, 1989; KRAUSE et al., 1997).

Little information is available on the changes of white blood cells (WILSON, 1946; ASSOKU et al., 1970; ALLAN and DUFFUS, 1971) and nothing is known about phagocytosis in poultry with acute fowl typhoid. The present study shows results from both the dynamics of the number and structure of white blood cells (WBCs) and phagocytic activity (PA), phagocytic number (PN) and phagocytic index (PI) of the leukocytes (Lc) in domestic poultry with experimental acute Salmonella gallinarum infection.

Materials and methods

Healthy Salmonella-free five-month-old Plymouth Rock chickens were inoculated orally with 18 h bacterial Salmonella gallinarum culture into the crop. Each group (control and infected) comprised seven birds. The strain S. gallinarum was isolated from a dead hen and was preliminarily tested. Total white blood cells (WBCs) were measured in Bolotnikov stained solution in Bürker camera. Leukocytes were stained violet, erythrocytes remained colorless. White blood cell differential counts were performed on Giemsa stained blood smears. Because a dynamic relationship exists between host and pathogen it is often necessary to use live virulent bacteria to obtain accurate experimental results (ALPUCHE et al., 1992), we used the same strain S. gallinarum for phagocytosis. Phagocytosis was examined by methods described by (BOLOTNIKOV, 1982). Briefly: 0.5 ml blood (1:4 with 3.8% natrium citratum as an anticoagulant) was incubated with 0.25 ml bacterial culture for 30 min at 40.5 °C, shaking every 10 min. The samples were centrifuged at 1500 × g 10 min. Slides were air dried, fixed with methanol and stained with Giemsa dye. The number of leucocytes (Lc) associated bacteria was determined by counting at least 100-200 cells from each experiment in a light microscope under oil immersion 1000 ×. Phagocytosis was represented as:

Leucocyte controls (without bacteria) and bacterial controls (without leukocytes) were also included.

The experiments were repeated at least twice. All results were statistically analyzed using the Student-Fisher test. Values are reported as mean ± standard error (SE).

Results

Leukocytosis was observed on the 3^rd day after infection 5.6 ± 0.31 109/l). Total WBC count fell gradually from the 4^th day until the 14^th day, inclusive (1.6 - 1.2 109/l) and reversed as late as on the 21^st day. Changes were significant (Table 1).

The percentage of the myelocytes increased from the 4^th day (9.4%) and the percentages of the band and segmentnuclear cells-from 2^nd day (8.9 resp. 18.9%). Total heterophils count raised on 2^nd day (41.3%). This continoued till the 21^st day following infection. This granulocytosis was characterized with only myelocytosis and enhanced rate of the bandnuclear granulocytes on the 21^st day. Monocytes remained in normal range. Lymphocytes dropped slowly as early as the 1^st day (Table 1). Basophilic and eosinophilic cells didn't change their values (data not shown).

Table 1. Dynamics (mean±SE) of total and differential white blood cells for chickens with experimental acute typhoid (N=7 in each group)

a = in each group WBCs were determinated on 7 birds
^x = significantly different from control (P<0.05)

Table 2. The changes (mean±SE) in the values of phagocytosis in chickens with experimental acute typhoid (N=7 in each group)

PA=phagocytic activity; PN=phagocytic number; PI=phagocytic index
^x = significanly different from control (P<0.05)

Phagocytosis by the circulating leukocytes is shown in Table 2. It is evident that phagocytic activity and phagocytic number were elevated in the period between the 3^rd day - 7^th day. The difference was significant. The values of this indices reestablished during the recovery period. No significant difference was observed of phagocytic index during the infection (Table 2).

Discussion

Total WBCs count drastically enhanced to two times original values of the 3^rd day in the acute infection with S. gallinarum, but declined very rapidly as early as the 4^th to 14^th day. This study confirmed some of the observations made elsewhere (ASSOKU et al., 1970; ALLAN and DUFFUS, 1971). According PECHKOVSKY et al. (1996) leukocytosis is induced by IL-I and TNF-a, which accelerate release of mature and immature heterophils from the bone marrow postmitotic reverse pool (AINSWORTH et al., 1996). At the same time total heterophils increased rapidly from the very early stage reaching a peak at 4^th day and remained high the thereafter (from 2^nd to 21^st day). This is in agreement with experimental model where peak levels of the myelocytes are enhanced 4 days after the infection, band-and segmentnuclear cells-on 2^nd day. The results demonstrated that chickens with S. gallinarum infection produced an initial arise followed by fall in the circulating mature leukocytes percentage in the recovery period and the rose of the percentage of immature myelocytes and bandnuclear cells. This showed that the bone marrow was still able to produce young granulocytes and is good prognostic sign. AINSWORTH et al. (1996) and TERASHIMA et al. (1997) found that inflammatory stimuli increase the rate of Lc production from the precursors, shorten their maturation time, decrease the Lc reside in the bone marrow and cause both mature and immature cells to enter the circulation. Differential Lc counts showed that the pronounced leukopenia was the result of the decrease in the percentage of circulating lymphocytes. Heterophilia was associated with a relative lymphopenia. Heterophilia was redistributed but lymphopenia due to the migration to primer inflammatory locuss (JANLUZOV, 1977). Other authors (ASSOKU et al., 1970; ALLAN and DUFFUS, 1971) didn't discover lymphopenia in poultry with experimental typhoid. This difference is probably based on the different virulence of experimental S. gallinarum strains. WILSON (1946) and RUSOV and DUKIC (1980) established that the appearance of letal symptoms is accompanied with leukocytosis, heterophilia, lymphopenia, immature granulocytosis and erythrocytopenia. The percentage of monocytes didn't change. CHANSORIYA et al. (1993) discoverd that several monocytes from 12 h onward after infection appeared transformed into macrophages and probably this was the reason for the monocytes change.

On the other hand in this article we report for the first time enhancement specific phagocytic function of the Lc in infected with S. gallinarum chickens. Independently that the bacterial virulence determinants from an antiphagocytic arsenal (CHEN et al., 1996; COTTER and MILLER, 1996) the host generate defence mechanisms. The cellular response to S. gallinarum infection was marked. During 3-7 days post infection we observed an abrupt growth of the percentages of active phagocytes and the number of engulfed bacteria from one active Lc. No significant change of the engulfed function of the Lc (PI) was observed. Phagocytic reaction manifestes itself typpically as a highly perfected protective mechanism primarily under condition of bacterial invasion (LAWRENCE, 1992). The increase in the phagocytic potential as demonstrated in this study is very interesting and biologically significant. It was established that Lc need to engulf foreign antigens such as bacteria soon after their entry in the host. Any increase in Lc phagocytic potentiality will increase the change of early antigen uptake and quicker elimination. This enhancement in phagocytic function will be desirable as a part of the "first line of immunological defense". It was established that with the recovery the phagocytosis activity of the Lc subsided and was normalized. The observed results could be explained by pathogenetic action of S. gallinarum on the chickens. All of these changes were found to precede the onset of the first clinical signs of the disease (KOKOSHAROV et al., 1997) and they are early indicators and may improve the early diagnosis of acute fowl typhoid in the clinical situation.

References

AINSWORTH, T. M., E. B. LYNAM, L. A. SKLAR (1996): Neutrophil function in inflammation and infection. In: Cellular and molecular pathogenesis. (Sirica, A. E., Ed.). pp. 37-55.  Lippincott-Raven Publ. Philadelphia.

ALLAN, D., W. P. DUFFUS (1971): The immunopathology in fowls (Gallus domesticus) of acute and subacute Salmonella gallinarum infection. Res. Vet. Sci. 12, 140-151.

ALPUCHE, A. C. M., J. A. SWANSON, W. P. LOMMIS, S. I. MILLER (1992): Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosome. Proc. Natl. Acad. Sci. USA 89, 10079-82.

ASSOKU, R. K. G., W. J. PENHALE, A. BUXTON (1970): Haematological changes in acute experimental Salmonella gallinarum infection in chickens. J. Comp. Pathol. 80, 473-485.

BOLOTNIKOV, I. A. (1982): Immunoprophylaxis of the poultry infectious diseases. Roselhozizdat Press. Moskva.

CHANSORIYA, M., R. P. AWADHIYA, J. L. VEGAS, A. K. KATIYAR (1993): Studies on the cellular response in avian inflammation using a simple subcutaneous pouch model. Avian Pathol. 22, 591-603.

CHEN, L. M., K. KANIGA, J. E. GALAN (1996): Salmonella spp. are cytotoxic for cultured macrofage. Mol. Microb. 21, 1101-1115.

COTTER, P. A., J. F. MILLER (1996): Triggering bacterial virulence. Science 273, 1183-1184.

DUFFUS, W. P. H. (1989): Immunity to infection. In: Veterinary clinical immunology (Haliwell, R. E. W., N. T. Gorman, Eds.). pp. 135-164. W. B. Saunders Co. Philadelphia.

HOLST, O., A. J. ULMER, H. BRADE, H. D. FLAD, E. T. RIETSCHEL (1996): Biochemistry and cell biology of bacterial endotoxins. FEMS Immun. Med. Microb. 16, 83-104.

JANLUZOV, P. (1977): Changes in the cell composition of the lymph organs in the course of the infectious and immunizatory processes of chicken cholera. Gen. Comp. Pathol. 4, 29-35.

KOKOSHAROV, T., H. HRISTOV, L. BELCHEV (1997): Clinical, bacteriological and pathological studies on experimental fowl typhoid. Indian Vet. J. 74, 547-549.

KRAUSE, K. H., R. A. CLARK, M. P. WYMANN (1997): European workshop on the cell biology of phagocytes. J. Leuc. Biol. 61, 1-5.

LAICHALK, L. L., J. M. DAUFORTH, T. J. STANDIFORD (1996): Interleukin-IO inhibits neutrophil phagocytic and bactericidal activity. FEMS Immun. Med. Microb. 15, 181-187.

LAWRENCE, G. G. (1992): New pathways of phagocyte activation: the coupling of receptor-linked phospholipase D and the role of tyrosine in primer neutrophils. FEMS Microb. Immunol. 105, 229-238.

MARRA, M. N., C. G. WILDE, J. E. GRIFFITH, J. L. SNABLE, R.W. SCOTT (1990): Bactericidal/permeability-increasing protein has endotoxin, neutralizing activity. J. Immunol. 144, 662-666.

PECHKOVSKY, D. V., M. P. POTAPNEV, O. M. ZALUTSKAYA (1996): Different patterns of cytokine regulation of phagocytosis and bacterial killing by human neutrophils. Intern. J. Antimicr. Agents. 7, 33-41.

RUSOV, G., B. DUKIC (1980): Dijagnosticki znacaj citomorfoloske dijagnostike akutne salmoneloze zivine. Veterinaria (Sarajevo) 29, 289-92.

TERASHIMA, T., B. WIGGS, D. ENGLISH, J. C. HOGG, S. F. V. EEDEN (1997): Phagocytosis of small carbon particles (PM10) by alveolar macrophages stimulates the release of polymorphonuclear leukocytes from bone marrow. Am. J. Resp. Crit. Care Med. 155, 1441-1447.

WILSON, J. E. (1946): Fowl typhoid. Certain aspects of experimentally produced disease. Vet. Rec. 58, 269-272.

Received: 31 October 1997
Accepted: 12 January 1998

KOKOSHAROV, T.: Promjene u bijeloj krvnoj slici i fagocitoznoj aktivnosti u pilica s pokusno izazvanim akutnim kokosjim tifusom. Vet. arhiv 68, 33-38, 1998.

SAZETAK

Istrazivanje je provedeno na petomjesecnim pilicima pasmine Plymouth Rock, slobodnima od salmonela, oralno inficiranima vrstom Salmonella gallinarum. Istrazena je promjenljivost broja i grade bijelih krvnih stanica te fagocitoza leukocita u zdravih i inficiranih pilica. Pokusno izazvani kokosji tifus ocitovao se leukocitozom treceg dana nakon infekcije, a potom leukopenijom od cetvrtog do 14. i oporavkom od 21. dana. Rezultati su pokazali da se u pilica pokusno inficiranih bakterijom S. gallinarum pocetno povecao, a zatim u fazi oporavka smanjio postotak cirkulirajucih zrelih leukocita, a povecao se postotak promijelocita i nesegmentiranih neutrofilnih granulocita. Heterofilija je bila povezana s relativnom limfopenijom. Ovo je prvo izvjesce o pojacanju specificne fagocitne funkcije (aktivnosti i broja fagocita) leukocita u razdoblju od treceg do sedmog dana nakon infekcije. Sve navedene promjene prethodile su pojavi prvih klinickih znakova bolesti i mogu posluziti za poboljsanje rane dijagnostike akutnog kokosjeg tifusa.

Kljucne rijeci: pilici, akutni kokosji tifus, Salmonella gallinarum, bijele krvne stanice, fagocitoza