Assessment of blood changes post-challenge with Corynebacterium pseudotuberculosis and its exotoxin (phospholipase D): A comprehensive study in goat

Aim: There is very little information regarding blood changes during the challenge of phospholipase D (PLD) in goats. Therefore, this experiment was conducted to study the changes in blood after the challenge with Corynebacterium pseudotuberculosis and its exotoxin, PLD to fill in the gap of caseous lymphadenitis (CLA) research. Materials and Methods: Twenty-six crossbred Boer goats aged 12-14 months were divided into 3 groups; the first group n=6 was inoculated with 1 ml phosphate buffered solution s.c. as the control. The second group n=10 was inoculated with C. pseudotuberculosis 1 × 109 cfu s.c. The third group n=10 was intravenous injected with PLD 1 ml/20 kg body weight. Serial blood collections were done at 1 h, 3 h, 5 h, 8 h, and 12 h then every 24 h post-inoculation for the first 30 days of the experiment. Subsequently, the blood collection continued twice a week till the end of the experiment (90 days post-challenge). Results: Both C. pseudotuberculosis and PLD treated groups showed significant changes (p<0.05) in red blood cell count, hemoglobin (Hb), packed cell volume, mean corpuscular volume, mean corpuscular Hb concentration, white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, globulin, and total plasma proteins. Similarly, both treated groups showed significant changes (p<0.05) in alanine transaminase, alkaline phosphatase, aspartate transaminase, total bilirubin, calcium concentration, creatine phosphokinase, creatinine, gamma-glutamyl transpeptidase, urea concentration, lactate dehydrogenase, prothrombin time, and activated partial thromboplastin time. Conclusion: It concluded that C. pseudotuberculosis and PLD have a negative impact on the goat’s health in general reflected by all those changes recorded in the hemogram, leukogram, and the blood chemistry.


Introduction
The blood changes caused by infectious diseases are commonly first discovered during routine blood work. Hemogram, leukogram, and blood biochemistry results give an overall assessment of any pathologically related blood changes. Most of these changes are indirectly caused by the pathogen or by the host response to that pathogen. Nonetheless, the host defense response varies according to many types of the infective bacteria, course of the disease and the type of the inflammatory reaction. Acute bacterial infections commonly lead to neutrophilia whilst chronic bacterial infection may lead to lymphocytosis and monocytosis [1].
Too often, caseous lymphadenitis (CLA) in small ruminants is a subclinical disease that has no clear clinical signs until the lesions become visible in the superficial lymph nodes and are discovered on clinical inspection, yet the infected animals are apparently healthy. Indeed, animals with no obvious CLA lesions do not dismiss the fact that internal abscesses could have developed especially if there is CLA infection in a flock or herd; such deep lesions are only discovered at necropsy [2,3].
CLA decreases production and productivity via lowering the milk and meat production and reduces the value of the hide, and in severe cases fatality may arise of the infected animal itself resulting in financial loss and even rendering the sheep and goats rearing unprofitable [4,5]. Extraordinarily, CLA has a long incubation period ranging between 3 and 20 weeks. However, shorter incubation periods have been reported [6][7][8] during which only a few animals may develop distinct clinical signs such as fever with some other changes in their vitals such as heart and respiratory rates, inappetence and decreased food consumption and alteration in the general health condition. Oddly, CLA has no significant changes on hemogram in goats challenged with C. pseudotuberculosis, but toll reflected significantly Available at www.veterinaryworld.org/Vol.8/September-2015/13.pdf on the leukogram between challenged groups at different sampling time [2,5]. Sheep experimentally challenged with C. pseudotuberculosis showed changes in the plasma proteins and the hemogram [9].
Phospholipase D (PLD) hydrolyzes the sphingomyelin in mammalian cell membranes increasing the vascular permeability especially the endothelial layer leading to plasma proteins leakage from the blood into the surrounding tissue space and from there into the lymphatic system. This contributes to the dissemination of C. pseudotuberculosis from the primary infection site to other parts of the animal's body [3].
The blood changes during the course of C. pseudotuberculosis infection and PLD treatment were reported in mice model of CLA [10]. However, there is scanty information regarding blood changes during the PLD challenge in goats. Therefore, this study was designed to further understand the blood changes during infection with C. pseudotuberculosis and the challenge with PLD to fill the gap of CLA research in goats.

Ethical approval
The experiment was performed according to the guidelines of the care and use of experimental animals provided by institutional animal care and use committee (IACUC). The experimental procedures were approved by Universiti Putra Malaysia, animal care committee (UPM/FPV/PS/3.2.1.551/AUP-R119).

Isolation and identification of C. pseudotuberculosis
Bacteria were isolated from clinical cases of CLA in goats. Isolates were sent to the Veterinary Laboratory Service Unit, Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia for identification and confirmation of the bacteria according to principles and methods described in the microbiological diagnostic laboratory (University of California, Davis, Revised Edition 2008).

PLD extraction
C. pseudotuberculosis exotoxin was extracted following the method described by Zaki [11]. Briefly, 2 or 3 loops of a 48 h culture of C. pseudotuberculosis were inoculated in a flask of freshly prepared bovine heart-liver medium. The flask was incubated anaerobically for 7 days at 37°C in slanting position of 15-20°. The culture that developed a pellicle was used. PLD separation started with centrifugation of the culture medium at 8000 rpm/15 min in a refrigerated centrifuge. The supernatant was collected and passed via sterile cellulose membrane filter (0.2 μm) and stored at 4°C then used in the experiment.

Experimental inoculations
Twenty-six crossbred Boer goats (13 bucks and 13 does) aged between 12 and 14 months with no history of vaccination against CLA were screened twice (3 months apart) for CLA using agar gel immunodiffusion test prior to the experiment.
The goats were divided randomly into 3 groups; the 1 st group consisted of 6 goats (3 males and 3 females) housed separately and inoculated with 1 ml phosphate buffered solution subcutaneously as a control. The 2 nd group consisted of 10 goats (5 males and 5 females housed separately) was inoculated with C. pseudotuberculosis 1 × 10 9 cfu subcutaneously; the 3 rd group also consisted of 10 goats (5 males and 5 females housed separately) injected with PLD 1 ml/20 kg body weight intravenously. Serial blood collections were done at 1 h, 3 h, 5 h, 8 h, and 12 h then every 24 h post-inoculation for the first 30 days of the experiment. Subsequently, the blood collection continued twice a week till the end of the experiment (12 weeks post-inoculation).

Complete blood count (CBC) and blood biochemistry
The blood samples were collected from the treated groups and the control animals for hematological and blood biochemical analysis. The blood samples were analyzed using Animal Blood Counter special for veterinary use (abc ™ ), and the serum samples were analyzed using 902 Automatic Analyzer (Hitachi ® , Japan). The examined parameters were red blood cell count (RBC), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular Hb concentration (MCHC), white blood cell count (WBC), neutrophils, lymphocytes, monocytes, eosinophils, basophils, globulin, and total plasma proteins.

Statistical analysis
Statistical analysis was performed using SPSS version 19.0. Repeated measure Analysis of Variance was used to analyze the hemogram, leukogram, and blood biochemistry. All values were reported as mean ± standard error at 95% confidence level.

Results
CBC is the most common performed blood tests for clinical or research purposes that provide an overview of patient's general health status and can also indicate the presence of any kind of disease. In any research, monitoring blood biochemical parameters is crucial to detect any changes in an early period and it can be of greater help to anticipate preliminary results, even though there are no obvious symptoms that manifest on clinical inspection. Blood analysis was conducted throughout the experimental period (12 weeks) to ensure complete understanding of CLA pathogenesis via blood profiling.

RBC count
C. pseudotuberculosis infected group showed a significant decrease (p<0.05) in RBC count toward Available at www.veterinaryworld.org/Vol.8/September-2015/13.pdf the end of the experiment, distinctively in week 10, week 11, and week 12 compared to the control. PLD challenged group showed no changes in their RBC count (Table-1).

Hb concentration
Hb concentration was significantly decreased (p<0.05), in C. pseudotuberculosis infected group in week 10, week 11, and week 12 compared to PLD challenged group and to the control (Table-2). PCV C. pseudotuberculosis infected group showed a significant (p<0.05) increase in packed cell volume in week 2, week 3, and week 5 compared to the control, whilst PLD challenged group showed a significant (p<0.05) increase in week 10 compared to the control ( Table-3).

MCV
Infection with C. pseudotuberculosis showed a significant increase (p<0.05) in week 8 and a significant decrease (p<0.05) in week 11, and week 12 in MCV compared to the control. In PLD challenged group, animals showed a significant increase (p<0.05) in week 8 and week 10 in MCV compared to the control (Table-4).

MCHC
The MCHC showed a significant decrease (p<0.05) in week 4, week 10, week 11, and week 12 in C. pseudotuberculosis infected group compared to the control, and a significant decrease (p<0.05) in week 5 in PLD challenged animals compared to the control ( Table-5).

WBCs count
The group infected with C. pseudotuberculosis showed a significant increase (p<0.05) in WBC, 2 times higher than the normal level in week 2 only,    and it's maintained significantly higher (p<0.05) than the control in week 4, week 5, week 8, and week 10. PLD challenged animals showed a significant increase (p<0.05) in WBC in week 8, week 10, week 11, and week 12 compared to the control (Table-6).

Neutrophil count
Infection with C. pseudotuberculosis showed a significant increase (p<0.05) in week 2, week 3, week 4, week 5, and week 8 compared to the control, whilst in PLD challenged group, neutrophil count was significantly decreased (p<0.05) in week 1 compared to the control, and significantly increased (p<0.05) toward the end of the experiment, specifically week 10, week 11, and week 12 compared to the control (Table-7).

Lymphocyte count
Both, C. pseudotuberculosis and PLD challenged animals showed a significant decrease (p<0.05) in week 6 compared to the control. There was significant increase (p<0.05) in week 1, week 5, week 7, and week 10 in C. pseudotuberculosis infected group as well as significant increase (p<0.05) in PLD challenged group in week 1, week 5, week 7, week 9, week 10, week 11, and week 12 compared to the control (Table-8).

Monocyte count
C. pseudotuberculosis infection showed a significant increase (p<0.05) in monocyte count in week 1, week 2, week 4, week 5, and week 10 compared to the control, whereas challenging with PLD showed a significant increase (p<0.05) in week 4, week 5, week 10, and week 12 with a significant decrease (p<0.05) in week 2, week 6, and week 9 compared to the control (Table-9).

Eosinophil count
Eosinophil count showed a significant increase (p<0.05) in C. pseudotuberculosis infected group in week 5 and week 9, with a significant decrease (p<0.05) in week 2 compared to the control. Challenging with PLD showed a significant increase (p<0.05) in week 1, week 2, and week 4, with a significant decrease (p<0.05) in week 9, week 10, week 11, and week 12 compared to the control (Table-10).

Basophil count
Infection with C. pseudotuberculosis showed a significant increase (p<0.05) in basophil count in week 4, week 5, week 6, week 8, week 9, week 10, week 11, and week 12 compared to the control. Moreover, PLD challenged group showed a significant increase (p<0.05) in basophil count between week 1 and week 5 and between week 8 and week 12 compared to the control (Table-11). ALT C. pseudotuberculosis infection showed a significant decrease (p<0.05) in ALT concentration in week, week 3, week 4, week 5, week 10, and week 11 compared to the control, while PLD challenged group showed a significant increase (p<0.05) in ALT concentration in week 6 only, with a significant decrease (p<0.05) in week 1, week 4, week 5, week 7, week 9, and week 11 compared to the control (Table-12).

ALP
Challenging with both C. pseudotuberculosis and PLD showed a significant decrease (p<0.05) in ALP concentration 3 times lower than the control in week 1, week 2, week 3, and week 4. However, week 6 showed 5 times lower in C. pseudotuberculosis infected group and 2 times lower in PLD challenged group compared to the control. Moreover, ALP showed 3 times lower than the control in week 7, week 8, week 9, and week 12 with 2 times lower in week 11 compared to the control (Table-13).

AST
The concentration of AST increased significantly (p<0.05) in week 1, week 3, week 4, week 5, and week 7, and decreased significantly (p<0.05) in week 11 post-infection with C. pseudotuberculosis compared to the control. In PLD challenged group, AST concentration showed a significant increase (p<0.05) in week 1, week 7, and week 10. Strikingly, AST peaked in week 6 compared to the control (Table-14).

Total bilirubin concentration
Total bilirubin concentration showed a significant increase (p<0.05) in week 2 and week 9, with        (Table-15).

Calcium concentration
Infection with C. pseudotuberculosis showed a significant increase (p<0.05) in calcium concentration in week 7, and a significant decrease (p<0.05) in week 6, week 9, week 10, week 11, and week 12 compared to the control. Calcium concentration showed a significant increase (p<0.05) in week 10 post-challenge with PLD compared to the control (Table-16).

Creatinine concentration
Concentration of the creatinine in C. pseudotuberculosis infected animals showed a significant increase (p<0.05) in week 2, week 4l and week 7. In week 3, there was a significant decrease (p<0.05), week 5, week 6, week 9, week 10, week 11, and week 12 compared to the control. PLD challenged group showed a significant increase (p<0.05) in creatinine concentration in week 4 and week 7, and a significant decrease (p<0.05) in week 5, week 8, and week 10 compared to the control (Table-17).   one only and a significant decrease (p<0.05) in week 6, week 9 and week 10 compared to the control, whilst PLD challenged group showed a significant increase (p<0.05) in week 1, week 4, and peaked in week 12, and a significant decrease (p<0.05) in week 6, week 7, week 9, and week 10 compared to the control (Table-18).

GGT concentration
Infection with C. pseudotuberculosis showed 4 times significant increase (p<0.05) in GGT concentration in week 5, and a significant decrease (p<0.05) in week 11 and week 12 compared to the control. Concentration of GGT concentration was significantly increased (p<0.05) during week 6 to week 10, with a significant decrease (p<0.05) in week 1 and week 2 compared to the control (Table-19).

Urea concentration
Urea concentration showed a significant increase (p<0.05) post-infection with C. pseudotuberculosis in week 2, week 3, and week 7, and a significant decrease (p<0.05) in week 9 and week 10 compared to the control. Challenged with PLD group showed a significant increase (p<0.05) in urea concentration in week 1, week 2, week 3, week 5, week 7, week 8, week 9, and week 10, and a significant decrease (p<0.05) in week 4 compared to the control (Table-20).

Total protein concentration
Infection with C. pseudotuberculosis showed a significant increase (p<0.05) in total protein concentration in week 5 and week 7, and a significant decrease (p<0.05) in week 2, week 6, week 9, and week 10 compared to the control. Moreover, challenged with PLD group showed a significant increase (p<0.05) in total protein concentration in week 5, and a significant decrease (p<0.05) in week 2 and week 10 compared to the control (Table-21).

LDH concentration
Infection with C. pseudotuberculosis showed a significant increase (p<0.05) in LDH concentration in week 1, week 2, week 3, and week 7, and a significant decrease (p<0.05) in week 4, week 6, week 8, week 10, week 11, and week 12 compared to the control. PLD challenged group, LDH concentration showed a significant increase (p<0.05) in week 1, week 2, week 6, week 7, and week 12, and a significant decrease (p<0.05) in week 8, week 10, and week 11 compared to the control (Table-23).

PT
PT was significantly increased (p<0.05) post-infection with C. pseudotuberculosis in week 2, week 5, week 8, and week 9, and a significant decrease (p<0.05) in week 1 and week 10 compared to the control. Challenge with PLD also showed a significant increase (p<0.05) in PT in week 1 and week 8, and a significant decrease (p<0.05) in week 10 compared with the control (Table-24).
APTT Infection with C. pseudotuberculosis showed a significant increase (p<0.05) in APTT in week 2, week 3,   and a significant decrease (p<0.05) in week 2, week 9, and week 10 compared to the control. PLD challenged group showed a significant increase (p<0.05) in globulin concentration in week 1 and week 5, and a week 8, and week 9, and a significant decrease (p<0.05) in week 6 compared to the control, whilst in PLD challenged group, APTT was significantly increased (p<0.05) in week 1, week 8, week 9, and week 10, and a significant decrease (p<0.05) in week 4, week 5, week 6, and week 11 compared to the control (Table-25).

Discussion
Laboratory analysis of bodily fluids such as blood or urine via different means, hematology and blood biochemistry is of great value to validate predictive disease diagnosis and/or prognosis; this applies to infectious diseases, cancer and immunopathy. This study reported a wide range of hematological and blood biochemical changes in induced CLA in goats and compared it with PLD challenged goats. Hematological evaluation reached a tipping point in the current study exposing many significant changes post-challenged with C. pseudotuberculosis and PLD. Previous studies [5,10,[12][13][14] reported similar findings of our study where there were significant changes in Hb concentration, RBC count, MCV, and MCHC concentrations. These results could be inferred as harmful effects of C. pseudotuberculosis exotoxin, PLD, on endothelial cells of the vascular system disrupting the normal physiology of the hemopoietic system. Moreover, it was hypothesized that thrombocytopenia is one of many reasons for anemia or failure of megakaryocytes response in bone marrow [10,13]. A striking difference was revealed in the current study by PLD inoculated animals where the experimental goats showed no significant changes in RBC count and Hb concentrations. This odd outcome may be indicative of an insufficient single-dose of PLD that was inoculated into the animals. Moreover, it may be due to the absence of the pathogen, named, C. pseudotuberculosis and lack of its pathological role in the process of the disease to affect or change the studied parameters.
Although, C. pseudotuberculosis infection in goats resulted in mastitis and classical CLA cases, but it did not affect RBC count nor PCV [5]. The result from this study disagreed with Junior et al. [5] where PCV showed a significant increase in both C. pseudotuberculosis and PLD challenged groups. This can be explained as direct effects of the PLD and/or indirect effects of the C. pseudotuberculosis causing substantial degeneration of RBCs membrane leading reticuloendothelial system to remove it from the circulation [12,14]. All the findings from this study indicate the different means by which the PLD and the C. pseudotuberculosis affecting the body in general and the blood parameters in particular. In addition, other factors such as sex, age, physiological status of the animal, and the individual variations may also impose critical effects on the overall health status of the goats which in turn reflected in the blood work.
Naturally CLA infected sheep showed a significant increase in WBC count due to the increased neutrophil, monocyte and lymphocyte counts [5,12]. Similarly, in this study WBC, neutrophil, monocyte and lymphocyte counts were significantly high post-inoculation with the C. pseudotuberculosis and the PLD. These findings are  in accord with previous studies findings [10,12,15] who stated that C. pseudotuberculosis resulted in significant increase in the parameters under the study in sheep and mice model of CLA. Theoretically, and from a pathophysiological point of view, inoculation of PLD should not lead to any significant changes in the WBC, neutrophil, monocyte and lymphocyte counts. However, we hypothesize, that PLD inoculation resulted in transient immunosuppression which in turns led to an activation of some of the opportunistic pathogens contributing to the significant increase in WBC, neutrophil, monocyte and lymphocyte counts.
In the current study, eosinophil and basophil counts were significantly changed in both challenged    [10] both studies have reported no significant changes in basophils count during the course of CLA in sheep or in mice. However, histologically, CLA abscessation in the lymph nodes of sheep and goats showed immense infiltration with neutrophil and to a lesser extent with eosinophils; these eosinophils give the push its greenish shade [16]. A recent study in mice suggests that basophil may involve in cellular immunity as a T-cell regulator to mediate the magnitude of the secondary immune response [17]. This study hypothesized that the significant increase in basophil count is due to the cellular immunity response. Hence, that C. pseudotuberculosis is a facultative intracellular pathogen and has the ability to live inside the macrophages. Blood biochemistry assessment orchestrated with the hemogram and the leukogram revealing various significant changes post-infection with C. pseudotuberculosis and PLD challenge in the current study. ALT and AST are the most elevated enzymes in liver diseases. Both enzymes could be elevated before any clinical disease is apparent such as in acute hepatic necrosis and their levels may reach as high as 100 times the normal level with peak activity between 7 and 12 days [18]. In this study, liver enzymes such as ATL, ALP, AST, and GGT concentrations were significantly changed in both the PLD and the C. pseudotuberculosis inoculated groups. These findings are agreed with those described by some authors [10,12,[18][19][20][21] who stated that liver damage, bacterial invasion induced liver damage, hepatotoxicity, obstruction of the biliary tree, disease processes that involved hepatocytes integrity and hemolytic diseases can significantly change ATL, ALP, AST, and GGT serum concentrations. We hypothesize that the high fluctuation in liver enzymes can reflect the severity and/or the chronic nature of the disease affecting the liver. Hence, that C. pseudotuberculosis inoculation was led to abscess formation in the liver. In addition, the toxic nature of the PLD had the same role in evoking these liver enzymes (ATL, ALP, AST, and GGT) which indicates rather similar pathophysiological mechanism of the C. pseudotuberculosis, even though PLD did not lead to any abscess formation in the liver or any other organs.
The bilirubin metabolic defects resulted in jaundice which is in most cases inherited or due to hemolysis. The bilirubin is the end product of Hb breakdown which excreted in the bile. Whilst urea is the end product of protein catabolism process in the body and it is cleared by the kidneys. Measurement of plasma urea concentration reflects the kidney function. Additionally, creatinine is the end product of creatine phosphate breakdown in the muscles. Both, creatinine and urea are cleared from the body by means of the kidneys through the process of glomerular filtration [18]. In our study, total bilirubin, creatinine, urea, and CPK concentrations were significantly changed in both the C. pseudotuberculosis and the PLD challenged goats. This result in contrast with Osman et al. [10] who stated that there were no significant changes in direct or total bilirubin post-inoculation with C. pseudotuberculosis and PLD in mice model of CLA. However, the findings accords with Ibtisam [12] who reported the significant high level of serum creatinine and blood urea concentrations in sheep naturally infected with CLA. This could be attributed to the degenerative effect of C. pseudotuberculosis toxin leading to impairment of renal function. The authors suggested that decreased blood flow into the kidneys could increase the serum creatinine and urea concentrations. CPK was significantly high up to 1000 fold post-inoculation of C. pseudotuberculosis and PLD in mice. CPK is an indicator for muscle damage, and its elevation could be attributed to the damage imposed by the bacteria and/or its exotoxin on cardiac or skeletal muscles [10,22,23].
Serum LDH is commonly found in the liver, skeletal muscles, heart, and kidneys. Hence, its level pattern changes due to tissue damage and it can be considered a marker of the instability of cellular integrity or cell death caused by pathological condition [24][25][26]. In this study, LDH was significantly changed post-inoculation with the C. pseudotuberculosis and the PLD. It suggested that LDH is a precise indicator that has a positive association with liver diseases, heart problems, uric acid elevation, and hematocrit, and it is proposed to be a marker for cardiac dysfunction [27]. Elevation in LDH serum concentration may raise from tissue damage by the toxic materials or pathological lesions in the liver, lymph nodes, lung, bone marrow, and spleen and it's hypothesized that LDH source in such cases may be the inflammatory cells [24]. Since, in this study, the pathophysiological mechanism of C. pseudotuberculosis and PLD inoculation involved inflammation, tissue damage, high blood urea, high PCV, and toxicity which reflected as high serum LDH concentration. Moreover, estimation of serum LDH level is a useful tool for detection of acute myocardial infarction and if combined with CPK it makes a good diagnostic method for delayed detection [28]. Therefore, we believe that LDH can be used in CLA diagnosis scheme; since, it's very sensitive biomarker for tissue damage especially in the lung, liver, and lymph nodes, a major organs that affected by CLA in small ruminant.
Cations, particularly, calcium is the most common element in the body, and 99% of it is found in the skeleton. Calcium disorder is presented into two main metabolic forms, hypocalcemia and hypercalcemia. It is within the realm of science that hypocalcemia is due to fall in free calcium, albumin-bound calcium or both whilst hypercalcemia is due to the influx of the calcium from the calcium pool into the extracellular fluids is more than its efflux [18]. In the current study, calcium concentration showed significant changes in both the C. pseudotuberculosis and the PLD challenged goats. These findings disagree with those reported by Osman et al. [10] who stated there was no significant change in serum calcium concentration post-inoculation with