Research Article

Biochemical and Histopathological Changes Associated with Fasciola spp. in Slaughtered Buffaloes at Al-Muthanna Province, Iraq

Hussein Jabar Jasim1*, Amer Murhum Al-Amery2

1College of Veterinary Medicine, Al-Muthanna University, Iraq; 2College of Veterinary Medicine, University of Baghdad, Iraq.

Received | January 07, 2023; Accepted | February 08, 2023; Published | February 20, 2023

*Correspondence | Hussein Jabar Jasim, College of Veterinary Medicine, Al-Muthanna University, Iraq; Email: husseinjasem2014@gmail.com

Citation | Jasim HJ, Al-Amery AM (2023). Biochemical and histopathological changes associated with Fasciola spp. in slaughtered buffaloes at Al-Muthanna Province, Iraq. Adv. Anim. Vet. Sci. 11(3):446-452.

DOI | https://dx.doi.org/10.17582/journal.aavs/2023/11.3.446.452

ISSN (Online) | 2307-8316

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Water buffaloes are physiologically and anatomically adapted to survive rivers, marshes, and areas subjected to flooding. Thus, they are most well suited for the rivers and marshes, particularly in the Baghdad countryside and the southern marshes in Basrah, Missan, and Thi-Qar provinces of southern Iraq (AlSaedy, 2007). Besides, buffaloes play an important role in the lives of people who live along the marshes of central and Southern Iraq because they are primarily dairy animals that produce thick butter fat and are never used for work, have high production rates in milk and meat conversion from low-quality forage (Minervino et al., 2020). Fasciolasis is one of the most important parasitic zoonotic diseases in the world, posing a serious health threat and causing enormous economic losses in livestock industries through death of infected animals, decrease in production, growth retardation, condemnation of affected livers, costs of treatment, and expense for control measures in ruminants caused by endoparasitic trematodes of the genus Fasciola, which belongs to the Phylum Platyhelminthes Class Trematoda, with two important species, F. hepatica and F. gigantica (Al-Sultan et al., 1999; Abdulwahed and Al-Amery, 2019). Moreover, Fasciolasis has a wide geographic spread as food-borne zoonotic disease. F. gigantica is responsible for the large liver fluke in tropical areas, which is particularly prevalent in Asia and Africa due to the wide distribution of its intermediate host Lemnia natalensis. while, F. hepatica is found in temperate regions like Europe, Americas and Australia, with limited distribution of its intermediate host Lemnia truncatula (Kanyari et al., 2010; Hassone and Salah, 2019). On the other hand, the invasive or acute stage, the prepatent stage, and the chronic or obstructive stage are the three clinical manifestations of Fasciolasis in animals (Mikaeel, 2020). Acute Fasciolosis result into high mortality especially in sheep, but sub-acute and chronic Fasciolasis cause fever, anemia, icterus, bottle jaw and reduction in production and fertility (Anne and Gary, 2006). A variety of laboratory tests based on biochemical analysis of serum parameters are available includes determination of serum alanine transaminase, aspartate transaminase (ALT, AST), which are the most sensitive indicators of hepatocellular injury. The degree of cholestasis and the liver’s synthetic capacity are also assessed by using Alkaline Phosphatase (ALP), gamma-glutamyl tranferase (GGT), and bilirubin (Okoye et al., 2013; Oleiwi et al., 2017).

Materials and Methods

Study area and sample collection

The study was conducted randomly on buffaloes slaughtered at the abattoirs of Rumaitha and Samawah in Al-Muthanna province, south of Iraq. The sample collection time was extended from March 2022 to December 2022. Three times a week, the abattoirs were visited, and the number of buffaloes slaughtered range from 2 to 5 every day. Visceral organs infected with Fasciolosis were put in plastic bags and were brought to Parasitology Laboratory, College of Veterinary Medicine, University of Al-Muthanna to identify any gross pathology changes by visualization and palpation by making numerous incisions in various parts to confirm the presence of Fasciola spp. in the liver and gall bladder.

Blood samples and biochemical examination

Blood samples were drawn from two groups of buffaloes from different age groups and both sexes. Fasciolosis-infected buffaloes make up the first group, while the second group includes healthy buffaloes as a control group. Before slaughtering buffaloes, blood samples were collected by puncturing of the jugular vein, after cleaning and disinfectant the area by the using ethyl alcohol (70%) through using sterilized 10 ml syringes. The blood samples were put in tubes without EDTA and send directly to laboratory by cooled box to separate the sera according to the method previously described (Singh et al., 2011), and stored at -20 °C in Eppendorf tubes for subsequent biochemical tests including total Bilirubin(TB), Creatinine (Cr), Total Albumin (ALB), alanine transaminase (ALT), aspartate transaminase (AST), Alkaline Phosphatase (ALP) and Gamma-Glutamyl Transferase (GGT), were estimated using commercial kit provided by BIOLABO (France).

Histopathological examinations

After recording the gross pathology changes, a pieces of liver and gall bladder from the infected buffaloes with Fasciola spp. were collected and wash several times with normal saline (0.9%) and then place immediately into 10% formalin for histopathological examination. The histopathological sections were processed according to (Al-Kubaisee et al., 1999).

Results and Discussion

The biochemical results showed statistically significant increases in the serum total bilirubin, creatinine and active serum enzymes ALT, AST, ALP and GGT when compared to healthy control animals. In contrast, the serum total albumin showed significant decrease in infected buffaloes during the period post infection, as shown in the Table 1. The biochemical results of the current study affirmed many previous studies by Hashem and Mohamed (2017), Nasreldin and Zaki (2020), Brahmbhatt et al. (2021), concerning effects of Fasciolosis on serum total Bilirubin, Albumin, Creatinine level and active serum enzyme ALT, AST, GGT and ALP during the period post infection. But our results study were conflict with the findings obtains by Rajamanickam et al. (1987), Matanović et al. (2007), who found a significant decrease in biochemical parameters in infected animals such as creatinine and active serum enzymes AST and ALP during the post-infection period, as well as disagreements with the findings of Ganguly et al. (2016), that found a significant increase in the albumin level of infected animals. This variation might be attributed to the migration of juvenile flukes through the liver parenchyma, which causes disruption of hepatocellular integrity, fibrosis, and necrosis, thus, enzymes from damaged cells are released into the blood and their concentration increases in serum AST and ALT in the infected animals (Brahmbhatt et al., 2021). Also, the cellular changes from parasitism increased the permeability of the hepatic cells, which in turn resulted in the release of the enzymes into the serum (Hashem and Mohamed, 2017). In addition, the serum GGT levels found in our study is an indicator of chronic changes, cholestasis, and epithelial damage in bile ducts that may be due to the presence of adult flukes in biliary tract (Hodzic et al., 2013). Our findings, on the other hand, revealed a significant increase in ALP, which is indicative of parenchymal and hepatobiliary damage or obstruction of the hepatobiliary system and may be caused by liver flukes present within the biliary network, resulting in hyperplastic obstructive and cholangitis (Edith et al., 2010). While the decrease in albumin concentrations can be attributed to either the fluke’s loss in the gastro-intestinal tract via the bile duct or the parasite’s destructive effects on hepatic parenchyma caused by migratory flukes, which impair liver function and insufficiencies the ability to retain and use nitrogen and produce albumin (Oleiwi et al., 2017), In contrast, elevations of creatinine levels in our study may be attributed to renal problems according to the previous study by Nasreldin and Zaki (2020), who reported that glomerulopathy is associated with fasciolosis, which may be due to the deposition of granular and pesudolinear immunoglobulin (IgG) in the mesangial region of the glomeruli that results in membranoproliferative and mesangioproliferative glomerulonephritis. Furthermore, a significant increase in bilirubin was found in our study, which could be caused by bile duct obstruction, which causes bilirubin to return to the hepatocyte and then increase in serum (Kiladze et al., 2000).

 

Table 1: The effects of Fasciolosis in buffaloes on some biochemical parameter by T test (Mean±SE).

Biochemical parameters	Group	Mean ± SE	P value
Albumin (g/dl)	Control	3.395±0.009*	0.00
	Infected	2.899±0.045
Creatinine (mg/dl)	Control	0.740±0.107	0.00
	Infected	1.841±0.032*
Bilirubin TSB(mg/dl)	Control	0.201±0.026	0.05
	Infected	1.310±0.069*
AST/GOT (IU/L)	Control	101.916±0.340	0.00
	Infected	190.304±3.219*
ALT / GPT (IU/L)	Control	20.508±0.166	0.04
	Infected	77.214±8.605*
ALP (IU/L)	Control	118.303±0.147	0.03
	Infected	181.230±0.298*
GGT (IU/L)	Control	26.317±1.155	0.00
	Infected	71.281±0.849*
No. of animals	Control	15	*= p ≤0.05
	Infected	30

 

On the other hand, the results of gross pathological examination showed that the most commonly found gross lesion was cirrhosis, paleness of the liver, and thickness of the bile ducts associated with the presence of an adult parasite, as shown in Figure 1. Besides, the liver were appear to be tougher with irregular surface due to parenchymal congestion with multiples abscesses formation appear as a pale necrotic areas, as showed in Figure 2. While advanced lesions lead to nodule formation and fibrosis, as well as enlargement, thickening, and calcification of the bile duct walls, all these changes have been noticed in buffaloes infected with chronic fasciolosis, as seen in Figure 3. Besides, the gallbladder was congested and dilated, full of dense matter of bile juice, as showed in Figure 4.

 

 

Furthermore, Fasciola spp. infection caused varying degrees of histopathological changes in infected buffaloes liver and gallbladder. The liver histopathological changes in the present study included hyperplasia, fibrous proliferation in the portal area, and coagulative necrosis of hepatocytes with large numbers of inflammatory cell infiltration, such as

 

 

eosinophils and macrophages, with scattered lymphocytes in the area previously migrated by young flukes, as shown in Figure 5. In addition, hepatocytes displayed a fatty change that was distinguished from other hepatocytes by

 

 

 

separate vacuoles that developed in the cytoplasm and had nuclei that were placed peripherally, as showed in Figure 6. Besides, hepatic sinusoids dilated as a result of their cell walls degenerating, nuclei deforming, and cytoplasmic contents leaking into the sinusoids as showed in Figure 7. Moreover, the histopathological examination showed that the hepatocytes were hypertrophied (swollen), which is demonstrated by an increase in size, opaque cytoplasm, and centrally located nuclei. It was also accompanied by periportal vein cellular infiltration, which was primarily composed of eosinophils, Kupffer cells, and lymphocytes, as shown in Figure 8. In addition, our results revealed that the bile ducts displayed hyperplasia, which is accompanied by fibrous cholangitis and an abundance of inflammatory cells; as well, bile pigment was accumulated as yellowish brown concretions in the dilated bile ducts (hemosiderin pigmentation), as seen in Figure 9. In contrast, histopathological lesions of the gallbladder showed the same features already described for the liver lesions. Various lesions were found during the histopathological investigation of the gallbladder that had Fasciola spp. infection, such as epithelial necrosis, hemorrhage, inflammatory cell infiltration, and hypertrophy and hyperplasia of the gallbladder’s epithelium as showed in Figure 10.

 

 

 

The histopathological findings in the current study were similar to those in the study conducted by Kovorodin et al. (2021), who reported that the histopathological changes in the liver infected with fasciolosis showed atrophy and mild hepatocellular degeneration in the liver tissue around the capsule, infiltration of eosinophils, fibroblasts, and lymphocytes, and dilatation in the sinusoids. The main histopathological changes might be due to the juvenile flukes migrating through the liver capsule and hepatic tissue, which results in necrosis, hemorrhages, fibrosis, and cirrhosis in the liver, or the chemical effects either by these parasites or by the hosts’ inflammatory and immune responses, which involve a strong Th2 response, which in turn is associated with the production of cytokines, thereby contributing to the overall pathophysiological condition (Al-Mahmood et al., 2017). Furthermore, the atrophy, necrosis, and fatty changes in the liver tissue may be because of the digestion of the host components by the flukes through the release of proteases, which facilitates their migration, feeding, as well as immune evasion (McGavin et al., 2001). Also, the infiltration of inflammatory cells may occur either due to the migration of immature liver flukes or due to the release of proteases by the flukes, which cause continuous irritation, that leads to hemorrhages and the infiltration of inflammatory cells (Okoye et al., 2015).In addition, the results of the bile duct histopathological examination were similar to those obtained by Talukder et al. (2010), who noted epithelial hyperplasia, ductal fibrosis, and inflammatory cell infiltration with eosinophils, macrophages, and lymphocytes. These lesions might be because suppressing the early peripheral inflammatory response and delaying the hepatic inflammatory response following infection, juvenile flukes change the immune response (Sadeghi-Hashjin and Naem, 2001). On the other hand, the histopathological changes of the gallbladder isolated of the buffaloes infected with Fasciola spp. were in agreement with those reported by Kardena et al. (2017), who revealed that the most common pathological changes were epithelial necrosis, hemorrhage, inflammatory cell infiltration, and hyperplasia of the epithelium of the gall bladder. These lesions may be caused by the migration activity and spiny tegument of Fasciola spp. which can irritate the mucous membranes of the gall bladder and is considered to be an indication of a chronic trauma because persistent worms sucking blood at the same location may lead to blood vessel ruptures, which result in bleeding and tissue damage (Lotfollahzadeh et al., 2008). Moreover, the increase thickening the mucous membranes in the lamina propria of the gallbladder which may be associated with the stimulation of proline secreted by worms Fasciola spp. (Kardena et al., 2017).

Conclusions and Recommendations

According to the data, the present study concluded that fasciolosis is an important veterinary disease that infects buffaloes and causes a significant economic loss. Also, this study revealed that infected buffaloes with fasciolosis experience significant changes in their biochemical parameters. Furthermore, the study found that fasciolosis infection resulted in a high level of pathological changes in the liver and gallbladder. 

Acknowledgements

The authors are thankful to manager of veterinary hospital, the doctors and the workers of the slaughterhouse of Al-Rumathia and Al-Samawah in Muthanna province for their help during the collection of samples and many thanks to staff of departments’ parasitology, college of veterinary medicine, University of Bagdad, Iraq for their support and providing the facilities during the samples processing.

Novelty Statement

The novelty of the study is focus on determining the biochemical changes in the infected buffaloes with fasciolosis and also, recognized the gross and histopathological changes resulting from the invasive Fasciola spp. in the liver and gallbladder, thus, they pose serious health risk and has a significant negative economic impact on the livestock industry due to animal deaths, decreased production, growth retardation, liver damage, treatment costs, and the high cost of control measures.

Author’s Contribution

All authors contributed equally.

Conflict of interest

The authors have not declared any conflict of interests.

References

Abdulwahed TK, Al-Amery AM (2019). Morphological and molecular study of Fasciola spp. in sheep in Alkut city. Int. J. Biosci., 14(1): 121-130. https://doi.org/10.12692/ijb/14.1.121-130

Al-Kubaisee RY, Alwan MJ, Al-Kaisee B (1999). Ecotopic infection of cattle with Fasciola gigantica: Parasitological and pathological studies. Iraqi J. Vet. Med., 23(1): 113–123. https://doi.org/10.30539/ijvm.v23i1.1198

Al-Mahmood SS, Farhan AM, Daoud ZS, Hamed OS (2017). Pathological study of liver lesions in cattle slaughtered at Kirkuk province abattoir. Iraqi J. Vet. Sci., 31(1): 7-16. https://doi.org/10.33899/ijvs.2017.126713

Al-Saedy JK (2007). Iraqi buffalo now. Ital. J. Anim. Sci., 6(2): 1234-1236. https://doi.org/10.4081/ijas.2007.s2.1234

Al-Sultan II, Youkhana SO, Mahran OM (1999). Study on the pathology and parasitic affection of liver in sheep and cattle at Mosul area. Iraqi J. Vet. Med., 23(1): 50-58. https://doi.org/10.30539/ijvm.v23i1.1191

Anne MZ, Gray MC (2006). Veterinary clinical Parasitol. 7th ed. American Association of the Proctolohist. pp. 185-210.

Brahmbhatt NN, Kumar B, Thakre BJ, Bilwal AK (2021). Haemato-biochemical characterization of fasciolosis in Gir cattle and Jaffrabadi buffaloes. J. Parasit. Dis., 45(3): 683-688. https://doi.org/10.1007/s12639-020-01342-1

Edith R, Godara R, Sharma RL, Thilagar MB (2010). Serum enzyme and hematological profile of Fasciola gigantica immunized and experimentally infected riverine buffaloes. Parasitol. Res., 106(4): 947-956. https://doi.org/10.1007/s00436-010-1741-1

Ganguly A, Bisla RS, Chaudhri SS (2016). Haematological and biochemical changes in ovine fasciolosis. Haryana Vet., 55(1): 27-30.

Hashem MA, Mohamed SS (2017). Hazard assessments of cattle fascioliasis with special reference to hemato-biochemical biomarkers. Vet. Med. Open J., 2(1): 12-18. https://doi.org/10.17140/VMOJ-2-111

Hassone WS, Salah MHK (2019). Phylogenetics of Fasciola hepatica in cattle (Iraq). Online J. Vet. Res., 23(8): 776-781.

Hodzic A, Zuko A, Avdic R, Alic A, Omeragic J, Jazic A (2013). Influence of Fasciola hepatica on serum biochemical parameters and vascular and biliary system of sheep liver. Iraq J. Parasitol., 8(1): 92-98.

Kanyari PWN, Kagira JM, Mhoma JRL (2010). Prevalence of endoparasites in cattle within urban and peri-urban areas of Lake Victoria Basin, Kenya with special reference to zoonotic potential. Sci. J. Parasitol., 11: 171–178.

Kardena IM, Winaya IBO, Adi AAAM, Berata IK, Adnyana IBW, Sukada IM, Agustina KK, Antara PATK (2017). Patological changes in liver and gall bladder of Bali cattle infected by fasciolosis. J. Vet. Anim. Sci., 1(1): 6-10.

Kovorodin E, Gimranov V, Bagautdinov A, Zhukov A (2021). Pathomorphological and morphometric studies of bovine liver infected with fascioles. Adv. Anim. Vet. Sci., 9(3): 372-378. https://doi.org/10.17582/journal.aavs/2021/9.3.372.378

Kiladze M, Chipashvili L, Abuladze D, Jatchvliani D (2000). Obstruction of common bile duct caused by liver fluke Fasciola hepatica. Sbornik Lekarsky, 101(3): 255-259.

Lotfollahzadeh S, Mohri M, Bahadori SR, Dezfouly MM, Tajik P (2008). The relationship between normocytic, hypochromic anaemia and iron concentration together with hepatic enzyme activities in cattle infected with Fasciola hepatica. J. Helminthol., 82(1): 85-88. https://doi.org/10.1017/S0022149X07874232

Matanović K, Severin K, Martinković F, Šimpraga M, Janicki Z, Barišić J (2007). Hematological and biochemical changes in organically farmed sheep naturally infected with Fasciola hepatica. Parasitol. Res., 101(6): 1657-1661. https://doi.org/10.1007/s00436-007-0709-2

McGavin MD, Carlton WW, Zachary JF (2001). Thomson’s special veterinary pathology. 3rd St Louis. Mosby, pp. 325-245.

Mikaeel FB (2020). Prevalence of Fasciola hepatica in goats and sheep by using ELISA in sera and milk in Duhok, Iraq. Iraqi J. Vet. Med., 44(2): 113–119. https://crerativecommons.org/licenses/by/4, https://doi.org/10.30539/ijvm.v44i2.983

Minervino AHH, Zava M, Vecchio D, Borghese A (2020). Bubalus bubalis: A short story. Front. Vet. Sci., 7: 1-15. https://doi.org/10.3389/fvets.2020.570413

Nasreldin N, Zaki RS (2020). Biochemical and immunological investigation of fascioliasis in cattle in Egypt. Vet. World, 13(5): 923-930. https://doi.org/10.14202/vetworld.2020.923-930

Okoye IC, Egbu FMI, Ubachukwu PO, Obiezue NR (2015). Liver histopathology in bovine Fascioliasis. African Journal of Biotechnology, 14(33): 2576-2582. https://doi.org/10.5897/AJB2014.13756

Okoye IC, Egbu FMI, Ubachukwu P, Okafor F (2013). Biochemical alterations due to Bovine Fascioliasis. Int. J. Sci. Res., 2(11): 503-507. https://doi.org/10.15373/22778179/NOV2013/169

Oleiwi KI, Hussein ZS, Salman KO (2017). Detection of Fasciola hepatica in Abu-Ghraib district (Iraq). J. Entomol. Zool. Stud., 5(6): 1067-1072.

Rajamanickam C, Muniandy N, Yahya M, Cheah TS, Paramasvaran S (1987). Haematological and biochemical parameters of cattle naturally infested with Fasciola gigantica in Perak, Malaysia. Kajian Vet., 19(1): 143-151.

Sadeghi-Hashjin G, Naem S (2001). Parasiticidal effects of peroxynitrite on ovine liver flukes in vitro. J. Helminthol., 75(1): 73-76. https://doi.org/10.1079/JOH200032

Singh P, Verma AK, Jacob AB, Gupta SC, Mehra UR (2011). Haematological and biochemical changes in Fasciola gigantica infected buffaloes fed on diet containing deoiled mahua (Bassia latifolia) seed cake. J. Appl. Anim. Res., 39(3): 185-188. https://doi.org/10.1080/09712119.2011.607703

Talukder S, Bhuiyan MJ, Hossain MM, Uddin MM, Paul S, Howlader MMR (2010). Pathological investigation of liver fluke infection of slaughtered black Bengal goat in a selected area of Bangladesh. Bangladesh J. Vet. Med., 8(1): 35-40. https://doi.org/10.3329/bjvm.v8i1.7717