Brucellosis is still remaining a serious problem in farm animals. Previous studies threw the light upon the marked role of sheep and goat especially mature ones, in the epidemiological pattern of brucellosis (Nayel et al., 2020; Rabah et al., 2022). In this study, the seroprevalence of sheep brucellosis was (17.50%, 22.50%) by RBPT and cELISA respectively, while the seroprevalence of goat brucellosis was (30%, 31.25%) by RBPT and cELISA respectively. Lower disease percentages were recorded by different authors in the northern coast area of Egypt (Nayel et al., 2020; Rabah et al., 2022). This differences may be attributed to the time and place of sampling and people habits in reporting cases as well. The high prevalence ratio of brucellosis observed in this study (22.50 % in sheep and 31.25 % in goat by cELISA) may be because of the wrong management practices followed by animals’ owners as large-scale animal grazing and mixed breeding which facilitate the spread of infection. Several factors such as lack of research, illegal animal movement, communal clashes, unreported outbreaks, poor vaccination coverage, and absence of brucellosis control programs in this region, may also participate in the increase of brucellosis prevalence, establishment, and maintenance (Nayel et al., 2020; Rabah et al., 2022).
In parallel, the erythrogram of the brucella-infected ewes and does characterized by macrocytic hypochromic anemia. This anemia has an immunological origin as the activated pro-inflammatory cytokines due to the bacterial infection suppress the erythropoiesis process by interfering with erythropoietin formation. It also enhances ferritin and hepcidin synthesis, leading to inadequate intestinal iron absorption and decreased macrophage iron recycling. This action is mainly to prevent iron accessibility to the invading bacteria to prevent its growth and multiplication (Kushwaha et al., 2014; Hashem et al., 2020). By the time, the iron bioavailability to the host bone marrow decreased too, causing the noted reduction of RBCs, Hb, PCV, and MCHC in the infected animals of both species. Similar results were obtained before in brucella-infected camels (El-Boshy et al., 2009), cattle (Kushwaha et al., 2014), and cows (Hashem et al., 2020). On the other hand, the increased MCV values pointed to a regenerative bone marrow response and an increased reticulocytes release in the peripheral blood. These data are compatible with Sikder et al. (2012) findings in cattle infected with Brucella abortus.
The activated pro-inflammatory cytokines stimulate the formation of different leukocytes, differentiation, maturation, and migration from bone marrow and lymph nodes (lymphocytes) to the circulation for infection fight (Gul et al, 2013; Hashem et al., 2020). This explains the pronounced neutrophilia, lymphocytosis, monocytosis, and eosinophilia and the subsequent leukocytosis which observed in current study in both infected species. The leukocytosis is an evident in Brucella infection reported before in brucella-infected cow, ewe and horse (Sikder et al., 2012; Gul et al, 2013; Hashem et al., 2020). This leukocytosis is an important reaction from the host as neutrophils are the first line of defense against bacterial infection and monocytes act as uterine tissue debris scavengers (present due to retained placenta and delayed natural uterine cleaning in case of Brucella infection).
Regarding the biochemical parameters, the current data depicted a marked hyperglobulinemia resulted in total hyperproteinemia and decreased A/G ratio in the infected ewes and does. As the presence of the Brucella organism in the host body stimulates its immune system to produce more innate non-specific immune proteins and humoral-specific proteins. Innate proteins such as cytokines, acute phase proteins, and matrix metalloproteinases are responsible for limiting the spread of the pathogen and multiplication till the specific immunity development and they result in the elevation of α and β globulin fractions (Kumar et al., 2015; Mahboub et al., 2015). While, the humoral-specific proteins (immunoglobulins) are responsible for pathogen destruction and removal, resulting in γ globulin elevation (Kumar et al., 2015; Mahboub et al., 2015). Hyperglobulinemia was determined before in brucella-infected ewes (Kumar et al., 2015; Mahboub et al., 2015) and cattle (Kushwaha et al., 2014). Likewise, the hypoalbuminemia in the brucella-infected sheep and goat detected in this study was assigned to the immune response and the strong acute phase response usually observed with Brucella infection (Sharifiyazdia et al., 2012; Hamdy et al., 2019). Albumin is a negative acute phase reactant, sensitized in the liver like the above-mentioned α and β globulins. During the infection, the liver gives absolute priority to α and β globulins synthesis at the expense of albumin synthesis causing the significant hypoalbuminemia recorded in both diseased groups. Similar results were obtained by Uluğ et al. (2010), Kumar et al. (2015), Mahboub et al. (2015), and Hashem et al. (2020) in brucella-infected humans, ewes, and cows respectively.
Additionally, the immune response was involved in the hyperthermia and anorexia commonly noted in diseased animals because of prostaglandin E2 secretion and bradykinin formation (de Goeij et al., 2013). The hyperthermia and anorexia had a great contribution to the prior hypoalbuminemia, hypoglycemia and T/HDL/LDL-hypocholesterolemia detected in the infected animals in this research, because of the dietary amino acids, carbohydrates, and fatty acids deficiency. While, hypertriglyceridemia referred to increased adipose tissue lysis and usage of body fat stores to get the energy to overcome the hypoglycemia related to the disease. Similar findings were recorded in brucella-infected different species by Mahboub et al. (2015), Singh et al. (2016), Merhan et al. (2017), Kumar et al. (2015), and Hashem et al. (2020). Anorexia was also implicated in the spotted hyponatremia, hypokalemia, hypochloremia, hypocalcemia, hypomagnesemia, hypophosphatemia, hypozincemia and hypocupremia in the infected animals (Radostits et al., 2002).
The oxidative stress noticed in brucella-infected ewes and does in the current work (represented by the decreased TAC levels in both diseased groups) is another outcome for the above-described cell-mediated immune response. The immune cells, mainly neutrophils, and macrophages generate free radicals under the effect of the invigorated pro-inflammatory cytokines. Free radicals react with the pathogen cellular components and absorb their electrons to achieve their stability (Merhan et al., 2017; Shalby et al., 2020). Physiologically, free radicals are controlled by the anti-oxidants (enzymes and vitamins) but in this research as well as previous researches free radicals exceed the anti-oxidants neutralizing ability and oxidative stress strongly appeared (Kataria et al. 2010; Bozukluhan et al., 2017; Merhan et al., 2017; Shalby et al., 2020). Anorexia partially participated in this process, whereas Zn and Cu are necessary elements for antioxidants synthesis and their decreased concentrations in the infected animals means lower levels of antioxidants and lead to oxidative stress augmentation (Kataria et al., 2010; Bozukluhan et al., 2017; Merhan et al., 2017; Shalby et al., 2020).
Oxidative stress is a reasonable cause for liver and kidney damage usually determined in brucella-infected species. This was indicated in the current data and previous data by the elevated hepatic and renal function tests in the infected animals (Gul et al., 2013; Mahboub et al., 2013; Kumar et al., 2015; Singh et al., 2016; Bozukluhan et al., 2017; Maruf et al., 2019; Hashem et al., 2020). The liver is the concerned organ with albumin and cholesterol formation and glucose blood levels regulation. Thus, its damage took a part in the hypoalbuminemia, hypoglycemia and T/HDL/LDL-hypocholesterolemia obtained in the infected ewes and goat here (Bozukluhan et al., 2017; Maruf et al., 2019; Hashem et al., 2020). In the same way, the decreased minerals and electrolytes levels in the diseased animals may be attributed to renal insufficiency and its inability to restore minerals and electrolytes from urine due to its damage (Gul et al., 2013; Mahboub et al., 2013; Kumar et al., 2015; Singh et al., 2016; Bozukluhan et al., 2017; Maruf et al., 2019; Hashem et al., 2020). Therefore, oxidative stress had an indirect role in the reported hypoalbuminemia, hypoglycemia, and T/HDL/LDL-hypocholesterolemia and decreased minerals and electrolytes levels in the infected ewes and goat. Interestingly, oxidative stress involved in the hypoalbuminemia noticed in the affected species through another way. As the accumulated free radicals consume the albumin because of its antioxidant characters. These free radicals also attack the circulating erythrocytes causing their lysis and subsequent anemia magnification (Kataria et al., 2010; Bozukluhan et al., 2017; Merhan et al., 2017; Shalby et al., 2020). Consequently, oxidative stress which begins as apart from the host innate immunity, has a major role in the disease pathogenesis exacerbation.
Matrix metalloproteinases are a group of proteolytic zinc-containing Ca-dependent enzymes. They are responsible for extracellular matrix remolding through its degradation. They are upregulated in different physiological and pathological conditions under the effect of the pro-inflammatory cytokines (Scian et al., 2011). Miraglia et al. (2013) and Šiširak and Hukić (2014) referred to their elevated activities in brucella-infected patients especially those with osteoarticular complications and neurobruollsis. They attributed this increase to the elevated pro-inflammatory cytokines activity usually accompanied Brucella infection (Demirdag et al., 2003; Scian et al., 2011; Hashem et al., 2020). The current results were compatible with these authors` opinions, as MMP-2 and MMP-9 showed high concentrations in the brucella-infected ewes and does.
The comparison between the two species demonstrated that most of the clinicopathological alterations in goat were more considerable than in sheep. Many researchers found that brucellosis is more prevalent in goat than in sheep (Nayel et al., 2020; Rabah et al., 2022). So, goat has a stronger immune response than sheep against Brucella (represented by the higher percentages of hyperglobulinemia, hypoalbuminemia, decreased A/G and increased MMPs activity). This response may cause a higher degree of anemia, hepatic damage, anorexia and hyperthermia and a lower degree of bonne marrow regenerative response and leukocytosis (total and differential) in the affected does than the affected ewes (Tizard, 2013). The hepatic damage and anorexia explained the higher percentages of T/HDL/LDL-hypocholesteremia, hypertriglyceridemia, decreased minerals, and electrolytes levels and trace elements recorded in does with brucellosis than in ewes with brucellosis (Radostits et al., 2002). While, oxidative stress and the subsequent renal damage were more prominent in sheep than in goat because of the higher leukocytosis and neutrophilia degree (are responsible for free radicals production) recorded in sheep than in goat (Malech et al., 2014).
Regarding the importance of the studied markers in the Brucella diagnosis, the present work suggested TAC and Zn (especially if combined together) as excellent biomarkers for caprine and ovine brucellosis and excluded Cu, MMP-2, and MMP-9 as markers for the disease. These results completely disagreed with Demirdag et al. (2003) and Scian et al. (2011) opinions who nominated MMPs as sensitive biomarkers for human brucellosis. Shalby et al. (2020) also disagreed with this result, as they excluded Zn and oxidative stress indicators as markers for ovine brucellosis. This disagreement may be due to the difference in the host species, Brucella species, and the disease stage.
