Introduction
Colibacillosis, a disease caused by Escherichia coli (E. coli), is one of the most common infectious diseases in the poultry industry. Most E. coli serotypes are non-pathogenic and found forming part of the microflora of the gastro-intestinal tracts of birds, reptiles, and mammals. About 10%–15% of the intestinal coliforms belong to pathogenic serotypes [1,2]. Therefore, birds are continuously exposed to infection with these organisms through fecal contamination of feeds, water, and the environment [3]. Vertical transmission of the disease occasionally occurs [4]. However, Avian Pathogenic Escherichia coli (APEC) serotypes, as a group, are virulent for birds where they cause systemic diseases such as coligranuloma, colisepticemia, omphalitis, synovitis, swollen head syndrome, cellulitis, pericarditis, perihepatitis, airsacculitis, peritonitis, salpingitis, panophthalmitis, and so on. All types and age groups of birds are susceptible to the disease, ranging from chicks to adult layers, broilers, and breeders [5].
Whereas colibacillosis is primarily an enteric disease in mammals, in poultry it may be a localized or systemic disease, occurring mostly secondary to impairment of the host defense mechanisms [6]. The acute form of the disease is characterized by septicemia, which results in death, while its subacute form is also accompanied by pericarditis, airsacculitis and perihepatitis, reproductive tract infections like salpingitis and/or egg peritonitis, resulting in huge mortality [7].
Calcium is an essential macro-element that is primarily bound in the blood and its absorption and release is tightly controlled in the intestines, bones, and kidneys [8]. The homeostasis of calcium in chicken is mainly maintained by the following estrogens, hormones, calcitonin, parathormone (PTH), and thyroxin [9]. Some biological activities in the body such as egg laying, shell calcification, and embryonic development impose serious extra demands on calcium homeostasis in birds [10]. Estrogens increase the production of blood calcium-binding proteins by promoting the formation of vitellogenins from the liver which are lipoproteins that are incorporated into the egg yolk. The estrogen-controlled hypercalcemic effect is not noticed in other animals (mammals) and is thought to be due to the need to produce large, calcified eggs requiring a rapidly mobilized source of calcium [11]. The parathyroid gland produces PTH which is a protein hormone that plays a vital role in the control of calcium and phosphate levels [12] as well as maintaining calcium homeostasis by regulating the calcium liberation from bone and resorption from the kidneys [13]. PTH bioactivity is elevated during eggshell calcification, but plasma PTH decreases to a low level after the formation of the eggshell [14].
The search for laboratory diagnosis of the reproductive disorders reported in layers with colibacillosis requires establishing the specific endocrine and other haemato-biochemical alterations, consequent to the disease, which necessitated this study. Therefore, the aim of this study was to determine the reproductive endocrine and some electrolyte changes in layers experimentally infected with E. coli.
Materials and Methods
Ethical approval
Ethical approval was sought from the Ethical Committee on Animal Use and Care of Ahmadu Bello University, Zaria (ABUECAUC), with Approval number: ABUECAUC/2017/026.
Location of experimental study
This study was carried out in the Department of Veterinary Pathology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Kaduna State, which is located within the Northern Guinea Savannah Zone of Nigeria [15].
Experimental animals and design
A total of twenty laying (20 weeks old) chickens that were specifically raised for research purposes and vaccinated against endemic vaccinable diseases in the area, except E. coli infections, were purchased from a reputable farm in Kaduna State. The birds were housed and managed intensively in the poultry research pens of the Department. Prior to the arrival of the birds, the pens were thoroughly washed with detergent and sprayed with formalin at a concentration of 4 ml/liter of water. Throughout the experiment, the birds were fed standard commercial layer mash (Hybrid Feeds®) and water was provided to the birds ad libitum.
Source of bacterial organism
The E. coli used in this experiment, APEC serotype O1K1, was obtained from the bacteria bank of the National Veterinary Research Institute, Vom, Plateau State, Nigeria.
Grouping, subculture of bacterial organism, preparation of McFarland standards, and inoculation of birds with E. coli
The birds were kept for 4 weeks to acclimatize to the new environment and other handling conditions, after which they were divided at random into two groups (infected and control) of 10 layers each. The control birds were housed in a pen located far away from the pen in which the birds of the infected group were housed. The bacteria from a previously prepared slant were reactivated by sub-culturing on Eosin and methylene blue. The resulting colonies were then examined for their characteristic features, color, and morphology and tested for gram stain reaction. On the day of infection (Day 0), the bacterial inoculum was prepared using McFarland standards, which were prepared by adding barium chloride to sulphuric acid to obtain a barium precipitate of different turbidity standards. These were used to estimate the number of bacteria present in a liquid suspension [16]. In this test, the turbidity of the suspension of bacteria was compared with a turbidity of the appropriate standard. Each of the birds in the infected group was then challenged by inoculating each of the birds in the infected group with 0.5 ml of bacterial aliquot containing 109 colony forming units (CFUs) of the bacteria intratracheally [17]. After inoculation, the bacteria were recovered from infected birds by following conventional culture, isolation, and identification of bacteria by standard procedures as documented in Cowan and Steel [18,19].
Collection of blood samples for reproductive endocrine and biochemical analyses
Beginning from day 0 and, subsequently, on days 2, 4, 6, 14, 21, 28, 35, and 42 post-infection, blood samples (2 ml) were collected from the brachial vein of each of 5 birds selected at random from each group, at 08:00 to 09:00 hours of the day, using 23 G needles. Exactly 2 ml of the blood was dispensed into a heparinized vacutainer tube. The blood was centrifuged in the laboratory at 2,200 g for 10 minutes. The plasma was then dispensed into vials and stored at −20oC until used for determination of activities of PTH, estrogen, progesterone, calcium, and phosphate concentrations.
Statistical analysis
All the data obtained were subjected to statistical analysis including the calculation of the means and standard error of the means. Data between groups were evaluated using a student t-test and values of p < 0.05 were considered significant using Graph Pad Prism version 5.00 for Windows, Graph pad Software, San Diego California USA.
Determination of progesterone, estrogen, and PTH profiles using Elisa kits
Plasma progesterone, estrogen, and PTH profiles were measured using commercial ELISA kits (Monobind Inc., USA, Accubind progesterone, oestradiol, and PTH ELISA microwells), in accordance with the manufacturer instructions, while plasma calcium and phosphorus concentrations were determined using commercial test kits (Agappe, India) by means of an ultraviolet digital spectrophotometer (Perkin Elmer AAS 400).
Results
Plasma calcium concentration
The mean plasma calcium concentrations in the E. coli-infected and control layers are presented in Figure 1. Plasma level of calcium in the infected group remained relatively unchanged and comparable with that of the control group up to day 4 pi. A drop in the plasma calcium level was then observed beginning from day 6 (2.24 ± 0.05 mmol/l) to its lowest level (1.61 ± 0.11 mmol/l) on day 14 pi, which was significantly (p < 0.05) different from that of the control group. Plasma calcium concentration in the E. coli-infected group then showed a slight increase to a level that was still lower than that of the control group and remained so up to the termination of the experiment. The plasma calcium concentration in the control group was maintained at fairly the same level with only some non-significant fluctuations.
Figure 1.
Mean (± SEM) plasma calcium concentration in E. coli-infected and control groups of layers.
Plasma inorganic phosphate concentration
The mean plasma phosphate concentrations in the E. coli-infected and control layers are presented in Figure 2. The mean plasma phosphate concentrations in the infected and control groups were comparable up to day 6 pi, following which a sharp increase was observed in the E. coli-infected group, on day 14 pi, to a significantly (p < 0.05) higher level (1.64 ± 0.12) than in the control group. The mean plasma phosphate concentration in the infected group was, thereafter, maintained at higher levels compared to those of the control up to the end of the experiment.
Figure 2.
Mean (± SEM) plasma phosphate concentration in E. coli-infected and control groups of layers.
Plasma PTH profile
The mean plasma PTH profile in the E. coli-infected and control layers are presented in Figure 3. The mean plasma PTH profiles in the infected and control groups were at the same levels up to day 6 pi. A significant (p < 0.05) increase in the mean profile of this hormone was observed in the infected group, from day 6 value of 24.10 ± 0.56 pg/ml to 27.40 ± 0.79 pg/ml on day 14 pi and peaked (31.04 ± 0.80 pg/ml) on day 21 pi before it gradually dropped to a level comparable with that of the control group on day 42 pi. The mean plasma PTH profile in the control group remained unchanged throughout the experiment.
Figure 3.
Mean (± SEM) plasma PTH profile in E. coli-infected and control groups of layer.
Plasma estrogen profile
The mean plasma estrogen profile in the E. coli-infected and control layers are presented in Figure 4. The mean plasma estrogen profile in the infected group was comparable to that of the control birds up to day 14 pi following which a progressive decrease from 347 ± 2.55 to the lowest value (332.6 ± 5.41pg/ml) on day 28 pi was observed that differed significantly (p < 0.05) from the corresponding value in the control group. The mean plasma estrogen profile, thereafter, was maintained at significantly (p < 0.05) lower levels than the corresponding control values up to the end of the experiment.
Figure 4.
Mean (± SEM) estrogen profile of E. coli-infected and control groups of layers.
Plasma progesterone profile
The mean plasma progesterone profile in the E. coli-infected and control layers are presented in Figure 5. The mean progesterone profile in the infected group progressively decreased starting from day 4 pi (117.1 ± 2.98 pg/ml) to the lowest value (94.8 ± 1.98 pg/ml) on day 28 pi that differed significantly (p < 0.05) from that of the control group. The low levels of this hormone were maintained in the infected group up to day 42 pi when the experiment was terminated.
Figure 5.
Mean (± SEM) progesterone profile in E. coli-infected and control groups of layers.
Discussion
The significant decrease in the mean plasma concentration of calcium observed in the E. coli-infected layers, especially on days 14, 21, and 28, and with the level remaining significantly lower than in the control up to the termination of the experiment is a pointer to the fact that colibacillosis could be associated with some derangement in the metabolism of calcium. It was probable that the observed enteritis in the E. coli-infected layers in this study had interfered with either feed digestion or subsequent absorption of nutrients as earlier reported [20,21], including calcium, thus contributing to the lowered levels in the infected group. It is also probable that the observed decline in mean plasma estrogen activity had contributed to the lowering of plasma calcium concentration observed in this group of layers. This is because estrogen plays a role in the hepatic synthesis of vitellogenins, which are lipoproteins that bind calcium; a rise in their concentration is associated with a rise in plasma calcium concentration and vice versa [22].
The finding that the mean plasma phosphates concentration was significantly increased in the E. coli-infected layers from day 14 up to 42 pi in the face of lowered mean plasma calcium concentration was pathologic since the body physiology dictates that plasma calcium concentration is altered alongside that of phosphates [23]. This change suggests some impairment of renal excretion of phosphates, since in normal health, phosphorus is excreted in urine as part of an effort to maintain the calcium–phosphorus ratio [24].
The finding that the response to increase in the mean PTH profile, which started on day 14 pi, was only noticeable on day 35 pi suggests impairment at the hormone’s target cells. The observed renal tubular lesions might have been responsible for the delayed response to the PTH profile. This is because it has been reported that in renal failure, phosphate loading or hyperphosphataemia decreases the calcaemic response to PTH activity [8]. The significant increase in the mean plasma PTH profile is believed to be a physiological response to the significant decrease in plasma calcium [25,12].
The significant decrease in the mean plasma estrogen profile from day 14 up to 42 pi, observed in the E. coli-infected layers, suggests impairment of its secretion from the ovary. Indeed, the observed progressive decrease in mean plasma progesterone profile from days 4 to 42 pi, which coincided with the period of decline in estrogen profile, was supporting evidence that the infection with E. coli resulted in impairment of some ovarian functions, probably due to ovarian lesions. The observed congestion of the ovarian follicles in this study strongly supports this hypothesis. Escherichia coli infection-induced changes in ovarian follicles were similarly reported by Oh et al. [26] and Srinivasan et al. [27]. The significant decrease in the mean plasma progesterone profile in the E. coli-infected layers may also be due to lower feed intake which has an important correlation with circulating progesterone contents [28] and disappearance of pre-ovulatory follicles after a few days of feed withdrawal [8]. It could be concluded from this study that experimental infection with E. coli in layers caused a progressive significant decrease in mean plasma estrogen, progesterone, PTH, and calcium activities post-infection, which may partly be responsible for the reported reproductive disorders in layers infected with E. coli.
Acknowledgments
We thank Habib Paul Mamman and Hajiya Salamatu, from the Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, for their immense Laboratory support.
Conflict of interest
The authors declare that they have no conflict of interest.
Funding
This research work was not funded by any grant.
