Histopathological and Serological Analysis of Aborted Ewes and Neonatal Death with Toxoplasma gondii in Duhok City, Kurdistan-Iraq

Introduction

Toxoplasmosis is an important zoonotic disease caused by Toxoplasma gondii (T. gondii), a ubiquitous protozoan parasite that uses felids as definitive hosts and several species of vertebrate animals as intermediate hosts ( Dubey, 2009 ). Toxoplasmosis is a major cause of reproductive failure associated with abortion in sheep and goats ( Tenter et al., 2000 ). In sheep, the abortion or prenatal mortality of lambs occurs when ewes suffer a primary infection during pregnancy ( Tenter et al., 2000 ). Abortion and neonatal mortality are the most important economic losses of sheep and goat industries worldwide ( Dubey, 1996 ). The lambs or kids may die in the uterus and after delivery (i.e., stillbirth) or succumb within a few days of birth. Sheep are globally considered important in the epidemiology of T. gondii infection, especially in Europe ( Buxton et al., 2007 ). Sheep are also essential to the economy of most countries because they are considered sources of food for human consumption. The T. gondii infection of sheep and goat poses a risk to public health and causes economic losses due to reproductive failure (Edwards and Dubey, 2013). The diagnosis of toxoplasmosis may be established through a primary exposure history of animals to Toxoplasma infection during pregnancy or white foci or nodules visible on the cotyledons, livers, or tissues of aborted fetuses ( Schlafer and Miller, 2007 ). Histologically, necrotic foci are observed in the white matter of the brain and cotyledons. The results of serology indicate exposure to the parasites, and elevated immunoglobulin M (IgM) or rising immunoglobulin G (IgG) titers suggest Toxoplasma abortion. The serologic activity of the fetus shows the organisms that cross the placenta because the maternal antibody (i.e., IgG) does not cross the ruminant placenta; accordingly, the observation of the organism in tissue confirms infection. Several serological diagnostic tests, including indirect immunofluorescence ( van der Puije et al., 2000 ), indirect haemagglutination test ( Nieto and Melendez, 1998 ), latex agglutination test ( Hashemi-Fesharki, 1996 ), and enzyme-linked immunosorbent assay ( Subedi et al., 2018 ), are used to establish Toxoplasma infection. With this background in mind, the present study was conducted to estimate the extent to which abortion and neonatal mortality in ewes were associated with T. gondii infection using histopathological and serological investigations.

Material and Methods

Study Design and Sampling. This study was carried out in different areas of Duhok province, Kurdistan Region, Iraq, within October 2016 to November 2017. The serum samples were collected from a total of 211 aborted ewes in seven different flocks of Duhok province. The sampled ewes constituted about one-third of all the ewes in which abortion or stillbirth has recently occurred. For the separation of serum, a blood sample of 5 ml was obtained from each animal by jugular venipuncture in nonheparinized vacutainer tubes (Becton, Dickinson and Co., Franklin Lakes, N.J., USA). The sera were then separated and stored at -20oC for the analysis. Five of the aborted fetuses were necropsied from each flock of the sheep to examine the gross lesions; however, the samples of their placentae, brains, livers, and lungs were directly submitted for microscopic (i.e., Giemsa-stained impression smear) and histopathological examinations.

Experimental infection of mice with T. gondii. Albino laboratory mice about 6 weeks old and weighing 20-25 g were investigated from the College of Veterinary Medicine, University of Duhok. All the mice were acclimatized under laboratory conditions for a week before the initiation of the experiment. The mice were kept in clean cages and fed on pelleted food and water. These mice were used for the bioassay of the aborted infected tissues in order to isolate T. gondii tachyzoites from the peritoneal cavity or obtain T. gondii tissue cysts containing bradyzoites from their brains, lungs, and livers. The inoculation was performed on the tissues taken from the small samples of the liver, lung, and brain of the aborted fetuses as described by Dubey et al. (2002) , with several modifications. The tissues were homogenized in a sterile mortar and then suspended in normal saline solution at a ratio of 1 gm tissue: 2.5 ml normal saline. To prevent bacterial contamination, 1000 and 100 mg of penicillin and streptomycin were added per milliliter of tissue homogenate, respectively. The heavy material was removed by standing at room temperature at least for 1 hr , and supernatant fluid (0.5 ml) was intraperitoneally inoculated into every five mice. The mice were then daily monitored for illness. In the control group, every five mice were inoculated with 0.5 ml of phosphate-buffered saline. Afterward, the peritoneal ascites were examined by direct wet smears from the infected mice for T. gondii isolates immediately following the observation of clear clinical signs. Imprinted smears were also obtained from the livers, lungs, or brains and hearts of slaughtered mice and then stained by 10% Giemsa in order to demonstrate tachyzoites and/or tissue cysts. The inoculated mice were considered infected with T. gondii since the observation of tachyzoites or tissue cysts in the examined tissues.

Microscopic examination (i.e., tissue impression smears).T. gondii infection was initially diagnosed by Giemsa-stained impression smears obtained from different organs of the fetus. The samples were collected from the cut surface of the brain, lung, heart, and liver of the aborted fetus for the identification of T. gondii infection stages, including cyst, tachyzoite, and bradyzoite ( Soulsby, 1986 ). Briefly, the impression smears were fixed with 70% methanol for 5 min, directly stained with 10% Giemsa for 30 min, and then examined for the presence of T. gondii stages under oil immersion lens (100×).

Serological examination (i.e., latex agglutination test).T. gondii antibody was detected in the infected sera by latex agglutination test using a Toxoplasmosis Latex Test Kit (Plasmatec Laboratory Products Ltd, UK). The TOXO-Latex reagent is a suspension of polystyrene latex particles coated with soluble T. gondii antigen. Latex particles allow the observation of the antigen-antibody reaction. If specific anti-Toxoplasma antibodies are present in the tested sera, they will react to soluble T. gondii antigen in the latex reagent and be visualized by latex particles as a clear agglutination after 5 min of the slide rotation due to the presence of Toxoplasma antibodies ˃ 4 IU/ml.

Histopathological examination. The portions of different organs (including the liver, lung, heart, and brain) obtained from the aborted fetuses were firstly fixed with 10% formalin and then dehydrated in ethyl alcohol with increasing concentration (from 70% to 100%). Afterward, the samples were embedded in paraffin wax using routine tissue processing. From each block, the tissue sections of 4 µm thicknesses were cut using a rotary microtome (Leica RM2235, Biosystems, Leica Biosystem Company, Germany), deparaffinized, rehydrated, and stained with hematoxylin and eosin in order to observe histopathological changes in the placentae, brain, heart, liver, and lungs. The entire slide was also investigated for T. gondii tissue cysts, including bradyzoites and tachyzoites, for the presence of histopathological section (Olympus BX40, objective 40X).

Results

Clinical symptoms. Within October to April 2017, the ewes in different flocks of Duhok province witnessed the successive waves of abortion and stillbirth in the late stage of gestation during the breeding seasons. Most abortion cases occurred during the late stage of gestation (>4 months) in the ewes of 1-3 years of age, and some of the aborted ewes suffered from depression, dyspnea, pyrexia, and vaginal discharge shortly before the abortion.

Abortion rate due to toxoplasmosis in sheep of different flocks. Out of 1,516 ewes, 211 (13.9%) sheep were aborted in seven different flocks of Duhok province (Table 1). This incidence indicated an infectious cause of active toxoplasmosis. All the aborted animals of different flocks were confirmed positive for T. gondii antibody infection immunologically in the primary diagnosis of toxoplasmosis using latex agglutination test but with different degrees of agglutination and microscopically by Giemsa-stained impression smears of the aborted organs.

Microscopic examination. The microscopic examination of Giemsa-stained impression smears obtained from the lungs, livers, brains, and hearts of the aborted fetuses in different flocks of sheep infected with toxoplasmosis showed the stages of T. gondii including cysts, bradyzoites, and tachyzoites (Figure 1). According to morphological characteristics, the isolated tachyzoites appeared dark-purple crescent or banana-shaped with nucleus and cytoplasm, pointed at one end and round or blunt at another one (Figure 1-C).

Isolation of T. gondii infection stages in mice tissues. The bioassay of the suspected infected tissues was conducted in mice to isolate T. gondii tachyzoites from the peritoneal cavity or obtain the T. gondii tissue cysts containing bradyzoites from their organs. T. gondii tachyzoites were successfully isolated from the peritoneal ascites of mice inoculated 12 days after the infection with the organs of the homogenized aborted ewe. However, T. gondii tissue cysts containing bradyzoites were detected in the brain tissues of the mice 32 days following the infection.

Figure 1. Giemsa staining of Toxoplasma gondii; A) and B) tissue cyst; C) tachyzoites (typically crescent-shaped with a prominent centrally-placed nucleus); D) bradyzoites (tissue cyst separated from host tissue by homogenization of infected brain, lung, and liver).

Pathological and Histopathological Findings. The postmortem examination of the aborted fetuses in the late gestational stage showed the generalized blood-stained fluid in their thoracic and abdominal cavities. Most of the aborted fetuses were reported with enlarged, congested, and friable livers and lungs. Furthermore, the placenta was swollen, reddish, and friable, and its cotyledons were speckled with whitish foci or nodules of necrosis. In most flocks, the lambs born within 1 to 3 days were dead (i.e., stillbirth). Intracellular T. gondii tachyzoites were demonstrated in the placental sections of some aborted fetus. Histopathological examinations showed severe fatty change (i.e., fatty droplets) and congestion of the liver in aborted animals. There was also the infiltration of multinucleated inflammatory cells, including Kupffer cells surrounding the portal vein and artery. Moreover, the liver showed disseminated irregular multiple necrotic foci throughout the hepatic lobules associated with the dissociation of hepatic cords. Toxoplasmosis stages, especially tissue cyst, were also presented and distributed through the liver. Furthermore, the histopathological investigation also revealed severe congestion and necrosis of the brain, lung, and heart of the aborted fetus with the mild infiltration of inflammatory cells and presented different stages of toxoplasmosis, including tissue cysts.

Discussion

Toxoplasmosis is a global zoonosis caused by T. gondii as an intracellular apicomplexan parasite. Sheep are economically important in many countries due to the production of meat and milk for human consumption ( Ragozo et al., 2010 ). Sheep rearing constitutes an important agricultural industry with a huge economy in Kurdistan Region. Sheep are commonly infected with tissue protozoa parasite; in this regard, the T. gondii infection of sheep and goat poses a risk to public health in the world and causes significant economic losses to farm animals due to reproductive failure ( Buxton et al., 2007 ; Edwards and Dubey, 2013 ). Reproductive failure has a negative influence on animal production, welfare, health, and rural economy. Abortion and neonatal mortality are among the most important economic losses to sheep and goat industries worldwide ( Buxton et al., 2007 ). Such cases were observed among the studied area in different farm animals due to T. gondii infection. Therefore, the present study was performed to confirm toxoplasmosis as the main etiological cause of abortion in flocks of sheep using serological, microscopic, and histopathological examinations. In the present study, the abortion rate of different sheep flocks in Duhok province was 13.9% (n=211) due to T. gondii infection that showed a serious economic effect on the fertility and overall reproductive failure (i.e., abortion) of the involved sheep flocks. All the serum samples of the aborted ewes were also confirmed positive for agglutination to T. gondii antibodies by latex agglutination test and Giemsa-stained impression smears of the organs. Infection is commonly diagnosed by the presence of specific IgM and IgG antibodies to T. gondii antigens in the sera of infected animals. The IgM antibodies usually become detectable within several days after infection; nevertheless, IgG antibodies become identified after 1-2 weeks and may have lifelong persistence. The presence of IgM antibodies indicates acute infection; however, the presence of IgG antibodies demonstrates a chronic infection ( Petersen et al., 2005 ). The general diagnosis of toxoplasmosis in animals is established through serological, histopathological, or molecular techniques or combination of the aforementioned methods. The clinical signs of toxoplasmosis are nonspecific and not adequately characteristic of a definite diagnosis. The diagnosis of toxoplasmosis can also be reached by the identification of the T. gondii stages in host tissue removed by biopsy or necropsy. A rapid diagnosis can be reached through the microscopic examination of the impression smears of the aborted fetus organs. After drying the smears, they are fixed in methanol and stained with Giemsa in order to identify the infection stages of T. gondii. Well-preserved T. gondii organisms are dark purple and crescent-shaped (or banana-shaped). In tissue sections, the tachyzoites usually appear round to oval ( Hill and Dubey, 2002 ). The stages of toxoplasmosis, predominantly tissue cysts, were presented in the livers, lungs, and brains of the aborted cases during the microscopic examination of Giemsa-stained impression smears. Several studies reported that the rate of abortion due to T. gondii infection may vary from 10-100% in different parts of northern Iraq ( Issa and Omer, 2011 ; Al-Barwary and Mikail, 2014 ). This finding is partly similar to the results of the present study depending on the immunity of animals, dose of oocyst, and time of exposure to infection. Neonatal mortality in sleep is more likely due to the multiplication of T. gondii in the placenta rather than the invasion of the parasite ( Dubey, 1996 ). When T. gondii infection starts in the placenta, the multiplication of tissue protozoa causes the necrotic foci of the placenta and some inflammatory changes which develop throughout the rest of the gestation up to birth or abortion ( Buxton et al., 2007 ). The persistent multiplication of parasite in the fetus and placenta is more likely due to local immune mechanism suppression in the maternal placenta and maturity of the fetal immune system. It was previously reported that the ovine fetus is able to mount an immune response to T. gondii infection for about 2 months of gestation; however, the immunocompetence is not adequate to protect up to the last month before birth ( Buxton et al., 2007 ). It was also noted that most of the aborted ewes in the abortion group subsequently conceived and lambed normally; accordingly, it was confirmed that ewes usually develop immunity against the parasite which protects them from disease during subsequent pregnancies ( Dubey, 2009 ). Therefore, T. gondii infection before 40 days of gestation can cause embryonic mortality and fetal resorption. In addition, the infection within 40 to 120 days of gestation can lead to fetal mummification and maceration or may result in abortion; however, the infection after 120 days of gestation causes stillbirth and/or weak birth ( Dubey, 2009 ). In the current study, most abortion cases among the ewes were observed during the late stage of gestation (>4 months), and some of them suffered from dyspnea, depression, pyrexia, and vaginal discharge shortly before the abortion. In the present study, T. gondii tachyzoites were detected in the peritoneal fluids of the inoculated mice with the homogenized infected tissue of the aborted ewes 12 days following the infection. Nevertheless, T. gondii tissue cysts comprising bradyzoites were successfully isolated from the brains of the mice 32 days after the infection. These results are inconsistent with the findings of another study ( Shaapan and Ghazy, 2007 ) which reported T. gondii tachyzoites in the impression smears were obtained from the peritoneal exudate on inoculated mice 6-8 days following the infection. They also observed that T. gondii tissue cysts containing bradyzoites in the impression smears were taken from the brains of mice 45 days after the inoculation. Dubey (1997) reported that T. gondii tachyzoites and cysts were noticed in the mesenteric lymph nodes, lungs, and brains of mice 3, 7, and 60 days after the infection, respectively. The discrepancy between expected and obtained results may be due to the differences in the route of inoculation, inoculum dose, strain of the parasite, age of inoculated mice, and development of their immune system. The present study also investigated the postmortem examination of the ovine aborted fetus and demonstrated widespread subcutaneous edema associated with the accumulation of the blood-stained fluid in the abdominal and thoracic cavities. Most of the aborted fetuses showed enlarged, congested, reddish, and friable livers; however, the lungs were also congested and hemorrhagic. In addition, the cotyledons of the aborted cases were spotted with whitish foci or nodules of necrosis. Consistent with the results of the current study, several previous studies reported naturally occurring and experimentally induced toxoplasmosis in fetal kids ( Ahmed et al., 2008 ). The manifestation of such pathological changes and identification of T. gondii stages in fetal heart, brain, and liver indicated transplacental infection. In the current study, the placenta, lung, brain, and heart are the most commonly affected organs in Toxoplasma-induced abortion among sheep. The presence of T. gondii cysts in these organs was also reported by other studies ( Dubey and Jones, 2008 ). In the present study, it was also observed that necrotic foci followed by the infiltration of multinucleated inflammatory reactions were the most frequent changes in toxoplasmosis in organs severely infected with tissue cysts and tachyzoites; this finding seems to be commonly associated with the rapid intracellular replication of T. gondii in such organs. It was previously confirmed that the demonstration of the tissue parasites with the characteristic histopathological images in the placenta, liver, lung, and the brain of the aborted fetus is important for the diagnosis of ovine toxoplasmosis ( Ahmed et al., 2008 ). It was also confirmed that interstitial nephritis, myocarditis, hepatitis, nonsupportive encephalitis, and diffuse interstitial pneumonia are the most significant histopathological changes of an aborted fetus. In conclusion, the infection by the protozoan parasite T. gondii is highly prevalent in animals. Toxoplasmosis in the sheep industry has a potential risk of transmission to humans through the consumption of contaminated meat with the tissue cysts of T. gondii in Iraq. Most of the sheep breeding farms in Kurdistan Region are traditional, and animals are directly in contact with cats. The presence of cats and history of abortion in farm sheep are the main risk factors associated with T. gondii infection. In another study ( Dubey and Jones, 2008 ), it was reported that tissue oocyst-contaminated pastures and drinking water are the major sources of postnatal infection in sheep. The results of the present study provided evidence of the T. gondii involvement in abortion and neonatal mortality among the naturally infected ewes in Duhok province. Accordingly, it is essential to identify different stages of T. gondii infection with some characteristic histopathological changes in the organs of an aborted fetus and perform serological and microscopic examinations for the diagnosis of toxoplasmosis in ewes.

Ethics

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Authors’ Contribution

Study concept and design: Naqid, I.A.; Zangana, I.Q.

Acquisition of data: Khanamir, R.A.; Zangana, I.Q.

Analysis and interpretation of data: Naqid, I.A.; Khanamir, R.A.

Drafting of the manuscript: Naqid, I.A.

Critical revision of the manuscript for important intellectual content: Khanamir R.A.; Naqid, I.A.; Zangana, I.Q.

Statistical analysis: Naqid, I.A.

Administrative, technical, and material support: Khanamir, R.A.; Zangana, I.Q.

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