Introduction

Corynebacterium pseudotuberculosis is the etiological agent for caseous lymphadenitis (CLA), a chronic contagious disease mainly affecting sheep and goat, and occasionally deer, cattle, horse, water buffalo, camelids, primates, wild ruminants, fowl, and pigs. The disease also infects humans, particularly farm workers and meat inspectors, hence, it is being considered as a potential zoonotic disease (Jesse et al. 2013). C. pseudotuberculosis is a gram-positive, intracellular bacterium, non-sporing, non-encapsulated, fimbriated, and facultative anaerobic bacterium that grows well on blood agar characterized by forming small whitish opaque colonies (Levinson & Jawetz, 1994). Eradication of this organism is almost impossible because it transmits fast in the herd and can survive up to 8 months at various temperatures and in the soil (Guimarães et al. 2011).

Caseous lymphadenitis is characterized by the formation of caseous abscesses in internal or superficial lymph nodes and organs (Sood et al. 2012). As previously reported by Guimarães et al. (2011), abscesses can be found in the lungs, liver, spleen, kidneys, internal lymph nodes, and the uterus. According to Adza-Rina et al. (2013), intradermal infection causes increased alanine transaminase and leukocyte counts, and also induces severe abscesses in the lymph node of affected goats. Additionally, pulmonary congestion, splegnomegally, liver abscesses, and renal congestion were also found following infection with the bacteria. Moreover, recent findings by Othman et al. (2016) following inoculation of C. pseudotuberculosis for 30 days showed various histopathological changes in the reproductive system which includes congestion, necrosis, degeneration, and inflammatory cell infiltration. These findings suggest the ability of C. pseudotuberculosis to induce pathology in the reproductive organs of female goats. However, there is currently a paucity of data on the chronic effect of the disease on the reproductive organs and associated lymph node following infection. Therefore, this study was designed in order to determine the histopathological changes in the reproductive organs and iliac lymph node of Katjang does following chronic infection with C. pseudotuberculosis.

Materials and methods

Inoculum preparation of C. pseudotuberculosis

C. pseudotuberculosis colony was previously isolated from an outbreak of clinical caseous lymphadenitis cases in goats at Taman Pertanian Universiti, Putra, Malaysia. The C. pseudotuberculosis organism were isolated and subcultured onto newly prepared blood agar media and incubated at 37 °C for 48 h. Twenty colonies were inoculated into 500 ml of brain heart infusion (BHI) broth and incubated at 37 °C for 48 h. The bacteria concentration was determined using a plate count method and 107 cfu/ml was used in this study.

Animal management, bacterial inoculation, and sample collection

A total of 18 does were randomly divided into two groups (groups 1 and 2) of nine does each. The first group inoculated with 1 ml of 107 cfu/ml live C. Pseudotuberculosis via the intradermal route around the neck region, whereas, the second group was administered 1 ml of PBS pH (Dorella et al. 2006) intradermally. Three does from each group were randomly selected and euthanized at 1, 2, and 3 months. Tissue samples were collected from the ovary, uterine horn, uterus, cervix, vagina, and iliac lymph node and preserved in 10 % buffered formalin. Tissue were dehydrated using different alcohol concentrations, processed, embedded, sectioned and stained with H&E for evaluation of histopathological lesions under light microscopy (Osman et al. 2012).

Lesion scoring

The histopathological lesion scoring was based on methods previously described by Jesse et al. (2015a). Six microscopic areas from each slide were observed at a magnification of 200 and 400×, respectively, and lesions were scored on a scale of 0 to 3 based on the presence/distribution of congestion, infiltration of neutrophils and macrophages, tissue degeneration, and necrosis. Slide with no lesion observed was indicated as score 0, 1 for mild, 2 for moderate, and 3 for severe (Othman et al. 2016).

Statistical analysis

Data were analyzed using statistical software IBM SPSS Statistic 20.0. A non-parametric analysis (Kruskal Wallis) was used to compare the lesion severity in all organs between the three sampling points. Data were expressed as mean ± SE with a significance at p < 0.05.

Results

The summary of histopathological lesion scores observed in different organs at 1, 2, and 3 months post inoculation are shown in Table 1. Generally, inflammatory changes in all tissues are characterized by leucocytic infiltration and vascular response. Macrophages and neutrophils were the predominant cells observed, while vacuolar degeneration was the most common form of degeneration observed. Cellular necrosis was typified by presence of nuclear pyknosis, while microabscesses contained neutrophils and necrotic debris, surrounded by a layer of fibrocytes.

Table 1 Pathological lesions scoring in reproductive organs and iliac lymph nodes of non-pregnant does intradermally inoculated with C. pseudotuberculosis in chronic state
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The histopathological changes observed in the ovaries are shown in Fig. 1. In this tissue, macrophage infiltration was lower (p < 0.05) at 1 month and higher at 2 and 4 months. However, the neutrophil response was higher (p < 0.05) at 1 and 2 months, and lower at 3 months. Cellular degeneration was lower (p < 0.05) at 1 month and higher at 2 and 3 months post inoculation. Necrosis was mild to moderate at 1 and 2 months, and moderate to severe at 3 months post inoculation. Vascular congestion was mild to moderate at all time points.

Fig. 1
figure 1

Photomicrograph of the ovarian section of Boer does intradermally inoculated with C. pseudotuberculosis a 1 month after infection and b 2 months after infection, showing mild vascular congestion (C), inflammatory cell infiltration (IC) typified by neutrophils and macrophages and degenerating follicles showing vacuolar degeneration (D), and c 3 months after infection, showing vascular congestion (C), numerous degenerating follicles with adjacent necrotic cells showing nuclear pyknosis (N) and inflammatory cells (neutrophils and macrophages) (IC), H&E ×100

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The histopathological changes observed in the uterine horns are shown in Fig. 2. In this tissue, while macropage infiltration was observed to be lower (p < 0.05) at 1 month, it was higher (p < 0.05) at 2 and 3 months post inoculation. However, neutrophil infiltration was higher (p < 0.05) at 1 and 2 months, and lower at 3 months post inoculation. Degeneration was mild (p < 0.05) at 1 month and moderate to severe (p < 0.05) at 2 and 3 months. Necrosis was lower (p < 0.05) at 1 and 2 months, and higher at 3 months post inoculation. Congestion was moderate to severe at all time points.

Fig. 2
figure 2

Photomicrograph of the uterine horn section of Boer does intradermally inoculated with C. pseudotuberculosis a 1 month after infection and b 2 months after infection, showing mild vascular congestion (C), inflammatory cell infiltration (predominantly neutrophils with macrophages) (IC) and degenerating and necrotic endometrial glands (D), and c 3 months after infection, showing numerous degenerating and necrotic endometrial glands showing vacuolar degeneration and nuclear pyknosis (G), H&E ×100

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The histopathological changes observed in the uterus are shown in Fig. 3. Here, macrophage infiltration and cellular degeneration were both lower at 1 month and higher (p < 0.05) at 2 and 3 months post inoculation. However, cellular necrosis and vascular congestion were both lower at 1 and 2 months, and higher at 3 months post inoculation. Neutrophil infiltration was moderate at all time points.

Fig. 3
figure 3

Photomicrograph of the uterine body section of Boer does intradermally inoculated with C. pseudotuberculosis a 1 month after infection and b 2 months after infection, showing inflammatory cell infiltration (neutrophils and macrophages) (IC) surrounding degenerating endometrial glands having cytoplasmic vacuolations (G) c 3 months after infection, showing numerous degenerating and necrotic endometrial glands (G) with presence of leucocytic infiltration (neutrophils and macrophages) around the glands (IC), H&E × 100

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The histopathological changes in the cervix are shown in Fig. 4. Here, macrophage infiltration and cellular degeneration were both lower (p < 0.05) at 1 and 2 months post inoculation and higher at 3 months post inoculation. Neutrophil infiltration was moderate to severe at all time points, while cellular necrosis was lower (p < 0.05) at 1 month and higher at 2 and 3 months post inoculation. Congestion was mild to moderate at all time points. Multifocal presence of microabscesses, which were mild in distribution, were observed in the lamina propria of the cervix at 3 months post inoculation.

Fig. 4
figure 4

Photomicrograph of the cervical section of Boer does intradermally inoculated with C. pseudotuberculosis a 1 month after infection, showing moderate leucocytic infiltration (predominantly neutrophils with macrophages) in the lamina propria, b 2 months after infection, showing epithelial vacuolation (V), and c 3 months after infection, showing moderate leucocytic infiltration (macrophages and neutrophils) (IC) in the lamina propria with multifocal micro abscesses (A ), H&E ×100.

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The histopathological changes in the vagina are shown in Fig. 5. In this tissue, the distribution of macrophage infiltration and cellular degeneration were lower (p < 0.05) at 1 and 2 months and higher (p < 0.05) at 3 months of post inoculation. Neutrophil infiltration was mild to moderate throughout the study period, while cellular necrosis was mild to moderate at 1 month and moderate to severe at 2 and 3 months post inoculation. Congestion was sustained at moderate to severe throughout the study period.

Fig. 5
figure 5

Photomicrograph of the vaginal section of Boer does intradermally inoculated with C. pseudotuberculosis a 1 month after infection, showing inflammatory cell infiltration (predominantly neutrophils) (IC), b 2 months after infection, showing mild vascular congestion (C), inflammatory cell infiltration (macrophages and neutrophils) (IC) and degenerating and necrotic vaginal glands evidenced by glandular hypoplasia, vacuolation, and cytoplasmic eosinophilia with nuclear pyknosis (G), and c 3 months after infection, showing vacuolar degeneration and necrosis of the vaginal glands (G) with leucocytic infiltration (neutrophils and macrophages) (IC), H&E ×100

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The histopathological response observed in the iliac lymph node is shown in Fig. 6. While congestion and degenerative changes were lower (mild to moderate; p < 0.05), extensive areas of lymphocyte depletion were observed in the white pulp. The distribution of neutrophils was comparable to macrophages. Numerous microabscesses with mild to moderate distribution were observed multifocally at 1 and 2 months of infection. At 3 months, the abscesses had incomplete walls surrounded by fibrocytes, and contained necrotic debris and leucocytes.

Fig. 6
figure 6

Photomicrograph of the iliac lymph node section of Boer does intradermally inoculated with C. pseudotuberculosis a 1 month after infection, showing increased neutrophil aggregation (N) and presence of microabscesses (A), b 2 months after infection, showing lymphocyte depletion in the white pulp (LD), and c 3 months after infection, showing moderate sized abscesses with incomplete walls (A) made up of fibrocytes with necrotic debris, neutrophils with lymphocyte depletion in the white pulp (LD), H&E ×100.

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Discussion

In recent years, there has been an increasing research interest in caseous lymphadenitis, especially as it to the Malaysian livestock industry. Most studies have been focusing on the bacterial characteristics, morphology, eradication program, and vaccine development; however, there is little concern in studying its cellular changes in the reproductive organs and associated lymph nodes in goats (Brown & Olander, 1987; Dorella et al. 2006; Baird & Fontaine, 2007; Guimarães et al. 2011; Osman et al. 2012; Adza-Rina et al. 2013; Jesse et al. 2013). Hence, there is the need to investigate the effects of C. pseudotuberculosis in the reproductive organs and iliac lymph nodes of non-pregnant goats over a chronic infection course.

Our results indicate that the iliac lymph node showed the presence of inflammatory cells, vascular congestion and necrosis, and degeneration during each sampling interval; and the severity increased as the disease became chronic. In an earlier study, Othman et al. (2016) observed similar findings following inoculation of the bacteria through different routes. However, since the study lasted only 1 month, the lesions observed in this study after two and 3 months of post infection is the first to be reported. The lymph node is known to be the major organ of blood filtration, and bacteria present in the blood are engulfed by macrophages and phagocytosed. However, since some intracellular bacteria such as Salmonella and C. pseudotuberculosis cannot be eliminated by the macrophages, they reside within the phagocytic cell for a long time, thus making eradication nearly impossible (Baird & Fontaine, 2007; Fontaine & Baird, 2008).

The histopathological changes observed in the ovaries, uterus, uterine horns, cervix, and vagina at 1 and 2 months of post infection were in agreement with what was observed by Othman et al. (2016). However, the lesions at 3 months post infection was more severe. Similarly, Khuder et al. (2012) observed infiltration of polymorphonuclear cells, leukocytes in the lumen of ovulated follicles and generalized thrombosis, congestion, and necrosis and degeneration of mouse stromal cells inoculated with C. pseudotuberculosis and its phospholipase D. In a related study, Mahmood et al. (2015) observed severe abscess formation, congestion, hemorrhage, degeneration and necrosis, and inflammatory cellular infiltration in Boer goats subcutaneously inoculated with C. pseudotuberculosis as compared to those inoculated intravenously with its phospholipase D. In this study, the predominant inflammatory cells observed were neutrophils and macrophages. While the distribution of macrophages was mild to moderate in all the tissues after 1 month of infection, neutrophil infiltration was moderate to severe during the same period. This shows that there is still an active inflammatory response due to the presence of the bacteria in these tissues. According to Bastos et al. (2012), C. pseudotuberculosis infection induces the migration of neutrophils to the area of infection as early as 1–4 days, followed by formation of pyogranuloma from days 5–10. However, because the bacteria is capable of surviving within the macrophages, the infection is prolonged and becomes chronic, thus resulting in an increase population of macrophage in the tissue. In this study, an increase in the distribution of macrophages were observed in all tissues with a higher distribution in the iliac lymph node. Similarly, evidence of pyogranuloma (microabscess) formation was observed at 1 month post infection. Bastos et al. (2012) further explained that the failure of the macrophages to eliminate the bacteria results in the formation of the pyogranuloma, which is intended to wall of the infected macrophages from spreading the infection further.

More recent studies have confirmed that bacterial infection in the ovary affects the production of progesterone hormone, which is crucial for implantation of the embryo and maintenance of pregnancies; hence, it may lead to early abortion and infertility (Othman et al. 2014). In another related study, Jesse et al. (2015b), reported a significant gradual decrease in progesterone concentration from the first (0.31 ± 0.12 pg/ml), second (0.29 ± 0.12 pg/ml), and third months (0.24 ± 0.14 pg/ml) post-infections with C. pseudotuberculosis. This suggests that the does may be predisposed to high incidence of abortion or stillbirth if pregnancy occurs. According to Williams et al. (2005), bacterial infection in the vagina reflects uterine infection. This, however, needs to be confirmed by biochemical testing method. A study previously conducted by LeBlanc et al. (2002) reported an association between the color and consistency of vaginal mucus and the presence of A. pyogenes, E. coli, P. melaninogenicus, and F. necrophorum. Apparently, the consequence of bacterial infection observed in the uterine horn is similar with the ovary, which suggests a possible exotoxemia. This might be due to the exotoxin (PLD) that causes severe biological damage such as complete capillary destruction, dermanecrosis, and cell death (Brodgen, 1984; Muckle and Gyles 1982). Furthermore, bacterial disruption of uterine and ovarian function leads to cervicitis and mucopurulent vaginitis which causes disruption in reproductive hormonal level in does and ewes, which results in decreased reproductive efficiency, infertility, and abortion in CLA-affected does (Sheldon et al., 2009; Palmieri et al., 2011).

Conclusion

In conclusion, the histopathological changes observed in the reproductive tract of Katjang does proves that C. pseudotuberculosis can successfully ascend to the reproductive tract and penetrate the physical barrier of the genital tract causing tissue lesions in the vagina, cervix, uterus, uterine horns, ovaries, and iliac lymph nodes. Our results also showed that, the lesions observed in these organs were more severe after 3 months of infection. Severe infection of the reproductive tract can lead to infertility and abortion in farm animals resulting in huge economic losses.