Haematological and Serum Biochemistry Profile of Cockerels Experimentally Infected with Salmonella enterica Serovar Zega

Salmonellosis is a serious medical and veterinary problem worldwide and causes great concern in the food and livestock industries, especially the poultry industry which occupies a prominent position in the provision of animal protein and accounts for about 25% of local meat production in Nigeria particularly and is identified as a disease of major economic importance causing low performance in poultry production. The study was carried out at the experimental animal farm, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Nigeria. One hundred (100) five-week old chickens obtained from the Poultry division of National Veterinary Research Institute, Vom, were used for the experiment. The birds were randomly assigned to 4 groups of 25 birds per group (A, B, C, D). Each bird in all the groups received 0.5 ml of PBS containing 1 × 108 cfu/ml of Salmonella enterica serovar Zega as follows: Group A was infected with Salmonella Zega intra-nasally (IN). Group B was infected with Salmonella Zega intra-peritonealy (IP). Group C was infected with Salmonella Zega orally (OR). Group D was the Uninfected control (CT). There was a significant change (p < 0.05) in the mean corpuscular volume (MCV) post-infection in all infected groups, however no statistical significant changes (p > 0.05) in the mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in the infected groups compared across the different days post infection. There was significant increase (p < 0.05) in the mean aspartate transferase (AST) and mean alanine transferase (ALT) after infection in all the infected groups between How to cite this paper: Emennaa, P.E., Eze, D.C., Chah, F.K., Okoye, J.O.A., Ihejioha, J.I., Sati, M.N., Onyema, I., Okorie-Kanu, C., Dzikwi-Emennaa, A.A., Chidiebere, U., Olorufemi, K., Nwamo, A.C., Omeke, J.N. and Barde, I.J. (2019) Haematological and Serum Biochemistry Profile of Cockerels Experimentally Infected with Salmonella enterica Serovar Zega. Open Journal of Veterinary Medicine, 9, 171-184. https://doi.org/10.4236/ojvm.2019.912015 Received: November 13, 2019 Accepted: December 13, 2019 Published: December 16, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access P. E. Emennaa et al. DOI: 10.4236/ojvm.2019.912015 172 Open Journal of Veterinary Medicine day 7 and 10 post infection. There was also a marked increase (p < 0.05) in the mean serum total protein values in all infected groups following infection. The findings of this study showed that apart from Salmonella Pullorum and Salmonella Gallinarum that are known to cause pathology in birds Salmonella Zega which is none host specific for birds can also cause pathology in them. This is the first report in the study area to the best of our knowledge.


Introduction
In developing countries especially in Africa, poultry farming is a major source of livelihood [1]. Poultry occupies a prominent position in the provision of animal protein and accounts for about 25% of local meat production in Nigeria [2]. Nebiyu et al., [3] identified disease prevalence as one of the major factors that causes low performance in poultry production.
Salmonella infection is a serious medical and veterinary problem worldwide and causes great concern in the food industry [4]. In poultry, the infection is important both as a cause of clinical disease in poultry and as a source of food-borne contamination in humans [5]. Avian salmonellosis is one of the major disease challenges militating against poultry production in Nigeria.
Host-adapted salmonellae Salmonella Pullorum and Salmonella Gallinarum are responsible for avian salmonellosis, a severe systemic disease characterised by somnolence, inappetence, weight loss and death [6], in spite of efforts at vaccination and medication. There are however other salmonella serotypes that have the capacity to infect and replicate in poultry, usually resident in the gastrointestinal tracts causing negligible or very mild signs of illness [7]. Salmonella enterica serovar Zega, one of the over 3000 identified strains of salmonella were first isolated from dead ducklings in the Belgian Congo (now Democratic Republic of Congo) in 1952 [8]. Recently, it was listed amongst 37 Salmonella enterica strains isolated from commercial layer flocks in Southwest Nigeria [9] [10]. This suggests that the agent may be pathogenic for avian species. This strain has the potential of becoming a major factor in the epidemiologic picture of avian salmonellosis in Nigeria. There is therefore a need to formally ascertain the pathogenicity and pathology of the serovar in chickens.
Haematology and serum biochemistry indices of birds and mammals are useful in the evaluation of their physiological and pathological status following infection and assessing the outcome of pathological processes in vital organs [11].
This study was carried out to investigate the haematologic and serum biochemistry values that accompany an experimental infection of Salmonella Zega in chickens as a baseline for assessing its pathogenicity.

Experimental Site
The study was carried out at the experimental animal farm, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, South-eastern Nigeria. Nsukka is located between latitudes 5˚50' north and longitude 6˚52' and 7˚54' east [12]. It is an area of fairly high temperature ranging from 21.17˚C to 32.00˚C [12]. Rainfall months' span from March and October while the dry season months are between November and February with a relative humidity of 70% and 20% respectively.

Experimental Animals
A total of 100, five-week old chickens obtained from the Poultry division of National Veterinary Research Institute, Vom, were used for the experiment. These birds were housed in deep litter system in a concrete pen and uncontaminated quality commercial starter diet was provided for the chicks throughout the experiment which lasted for 21 days. Feed and water were provided ad-libitum. The 100 birds were randomly assigned to 4 groups of 25 birds per group (A, B, C and D) in different pens separated by wire mesh and ceiling board from bottom to top of the roof.

Bacteriological Monitoring before Infection
Before infection, cloacal swabs were collected from the birds on day 2 and 5 post arrival in Nsukka. This was in order to confirm if they were free from Salmonella organism. This was done by pre-enrichment of the swab samples in buffered peptone water, followed by plating on MacConkey agar (MCA) using standard laboratory methods [13] [14]. Only Salmonella free birds were used for the experiment.

Bacterial Isolate Used in the Study
Stocked culture of Salmonella enterica serovar Zega originally isolated from commercial layers in South-western Nigeria, identified at the Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, South Dakota, USA [9] and maintained at the bacterial bank, Bacterial Research Department, National Veterinary Research Institute, Vom, Nigeria was used in this study.

Culture and Determination of Bacterial Inoculum
The lyophilized bacterium from the culture bank was reactivated by culturing in peptone water, incubated overnight at 37˚C and sub-cultured on MacConkey agar (MCA). The resulting colonies were examined for their colony characteristics (colour and morphology) and tested for Gram-reaction (Gram-negative).
Five colonies were scooped and inoculated into 10 ml of Phosphate Buffered Saline (PBS) and this was incubated for 24 hours at 37˚C after which a ten-fold di-Open Journal of Veterinary Medicine lution was carried out in test tubes. The colony counts from the test tubes were determined. To obtain the number of organisms that was inoculated into the birds, the number of organisms was multiplied by volume by the dilution factor (CFU = No. of colony × Volume × Reciprocal of Dilution factor) [15].

Experimental Infection
The 100 birds were randomly assigned to 4 groups of 25 birds per group. (A, B, C, D). Each bird in all the groups received 0.5 ml of PBS containing 1 × 10 8 cfu/ml of Salmonella enterica serovar Zega as follows: (Group A) were infected with Salmonella Zega intra-nasally (IN).
(Group C) were infected with Salmonella Zega orally (OR).
(Group D) was the Uninfected control (CT).
Note: For group A, 0.25 ml was administered into each nostril of the bird.

Blood Sample Collection
On days 0, 3, 5, 7 and 10 post-infection (PI), two millilitres of blood were collected from 3 randomly selected birds in each group through the jugular vein following proper restraint using a sterile 2 ml syringe. The samples were collected within 1 minute of capture to ensure that the levels of the monitored parameters were not affected by any stress induced by handling [16] and delivered into sample bottle treated with ethylene diamine tetra-acetic acid for haematological study. The same procedure was employed to obtain serum samples with anti-coagulant free sample bottles [17] [18]. The blood samples were collected in the morning hours to avoid haemolysis of the RBC as a result of high temperature.

Haematological and Serum Biochemistry Procedures
The anti-coagulated blood was used to determine the packed cell volume (PCV),  [20].
The differential leukocyte count was done on Leishman stained blood films [21].
The stained slides were examined with immersion oil at x100 objective using a P. E. Emennaa et al. Open Journal of Veterinary Medicine light microscope. Two hundred cells were counted by the longitudinal counting method and each cell type was identified and scored using the differential cell counter. Results for each type of white blood cell were expressed as a percentage of the total count and converted to the absolute value per microliter of blood.
Sera were separated from the clotted blood following centrifugation from which serum metabolites (serum glucose, urea, cholesterol, albumin, globulin and creatinine) were determined by spectrophotometry. Values of all these parameters were analysed using the routine laboratory procedures of Dacie and Lewis [22].

Data Analysis
Data obtained were expressed as mean ± S.E.M (standard error of the mean).
They were subjected to Repeat Measure One Way Analysis of Variance

Discussion
An analysis of the haematologic parameters across days 0, 3, 5, 7 and 10 post-infection (PI), subsequently compared within the three different routes of infection showed a slight reduction in the mean packed cell volume (PCV), haemoglobin count and red blood cell (RBC) count (Figures 1-3). The reduction observed in those parameters at day 3 PI in the infected groups were similar to works done by Freitas-Neto et al., [23], Nwiyi and Omodamiro [24], Barde et al., [25]; they all reported an acute onset in experimental infections of salmonella gallinarum in chickens and quails. The anemia associated with acute fowl typhoid in chicken has been attributed to an increased ability of the reticuloendothelial cells to take up erythrocytes as reported by Assoku and Penhale [26].  (Figure 4 and Figure   5). As alluded to by Barde et al., [25] in his study with salmonella enterica serovar Gallinarum in quails, these may be as a result of haemorrhages and congestion of organs observed in the infected birds. The intra-peritoneal group (IP) showed a significant increase (p < 0.05) in the mean heterophil count as compared to the other infected groups and the control ( Figure 6) same with mean white blood cells count and mean lymphocytes count (Figure 7 and Figure 8).

Results
This increase according to Brar et al., [27] and Morgulis, [28] is common in general infections due to septicaemias caused by infectious agents such as sal-Open Journal of Veterinary Medicine

monella. Monocyte,, and basophil percentage values in chickens in this study
were varied and showed no relevant changes (Figure 9 and Figure 10), and were consistent with a previous report [29].
There was significant increase (p < 0.05) in the mean aspartate transferase (AST) and mean alanine transferase (ALT) after infection in all the infected groups between day 7 and 10 post infection (Figure 11 and Figure 12). There was also a marked increase (p < 0.05) in the mean serum total protein values in all infected groups following infection ( Figure 13) but no significant changes in albumin, gbobulin cholesterol and bilirubin values (Figures 14-17). This increase in mean aspartate transferase (AST) agrees with Brar et al., [27] and Freitas-Neto et al., [23] that interprete it as a consequence of an incapacity of the liver to synthesize protein due to the lesion intensity resulting from hepatic disease.

Conclusion
There was a significant change (p < 0.05) in the mean corpuscular volume (MCV) post-infection in all infected groups, however no statistical significant changes (p > 0.05) in the mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in the infected groups compared across the different days post infection. There was significant increase (p < 0.05) in the mean aspartate transferase (AST) and mean alanine transferase (ALT) after infection in all the infected groups between day 7 and 10 post infection. There was also a marked increase (p < 0.05) in the mean serum total protein values in all infected groups following infection.