Spirocercosis in stray dogs of Chittagong Metropolitan area of Bangladesh : an epidemiological and pathological investigation

A total of sixty adult stray dogs (32 male and 28 female) randomly captured from different locality of Chittagong metropolitan area were euthanized during the period of May 2010 to January 2011. The dogs were necropsized to observe the presence of Spirocerca lupi in their oesophagus producing characteristic nodular lesions. The associated haematological changes were also determined. Twenty four (40%) of the captured dogs were positive (40.62 % male and 39.28 % female) for spirocercosis having oesophageal nodular lesions. One to three broad based nodular lesions with an average diameter of 2.104±0.139 cm were observed in the caudal oesophagus of spirocerca positive dogs. Minimum 1 and maximum 12 worms found harboring in each nodule with an average number of 3.75± 0.686. The association between the oesophageal nodular lesions and body condition of the dogs was inconclusive and locationwise prevalence was also not clear. Anaemia was observed in infected dogs with significant reduction in ESR values and MCHC%. Leukocytosis, particularly neutrophilia was common in infected dogs in comparison to the noninfected. Histopathology revealed extensive fibrous tissue proliferation around the adult worms in most of the nodular lesions with large numbers of newly formed blood vessels resembling granulation tissues. A considerable degree of fibroplasia was evident with predominant accumulation of fibroblasts and irregular layers collagen fibers in mature nodules. Accumulation of large numbers of neutrophil was common in nodular lesions, whereas, infiltration of lymphoplasmacytic cells also found in some cases. One lesion showed evidence of fibrosarcoma characterized by proliferation of undifferentiated atypical fibroblasts having abnormal mitotic figures.


Spirocercosis is a cosmopolitan parasitic disease
The Spirocerca lupi associated lesions are of carnivores caused by a spirurid nematode dependent upon its indirect lifecycle which involves Spirocerca lupi (Mazaki-Tovi et al. 2002, Dvir et al. an intermediate (coprophagus beetle) and a variety of 2001, and Lobetti, 2000).It is mostly prevalent in paratenic hosts (amphibians, reptiles, lizards, warmer climatic conditions particularly in tropical and domestic, birds and small mammals) (Anderson, subtropical countries (Van der Merwe et al. 2008).In 2000, Soulsby, 1986 andSharpilo, 1983).Infective infected dogs, spirocercosis produce variety of clinical larvae within the intermediate host or paratenic host manifestations depending on the stage of the disease, follow a specific migratory route when ingested by aberrant migrations and complications.Vomition or final hosts (Dogs); penetrating the gastric mucosa of regurgitation, dysphagia, dyspnoea, pyrexia and the dog, migrating along arteries, maturing in the anorexia are the commonly found manifestations thoracic aorta and then migrate to the caudal (Lobetti, 2000, Jubb et al. 1993and Bailey, 1972).No oesophagus to form a nodular or tumor-like growth sex or age predilection for this infection have been (Urquhart et al. 1996 andSoulsby, 1986).Larvae recorded, but due to the nematodal lifecycle, dogs cause necrosis, hemorrhage and neutrophil exudation below 6 months of age do not develop oesophageal within the vessel walls in which they migrate.In the disease or exhibit classic clinical signs (Fox et al. 1988 thoracic aorta, degenerate elastic and muscle tissue and Wandera, 1976).There seem to be an increased becomes fibroid and mineralized to form permanent susceptibility for infection in larger breed dogs like intimal scars with aneurysms of varying size and populated metropolitan as an endemic place for number.Metaplastic ossification of the aorta, to the spirocercosis.A very few reports on spirocercosis in extent of bone marrow formation, has also been Bangladesh are available; but no epidemiological or reported (Kumar et al. 1981).Parasites containing pathological investigation has been conducted yet.oesophageal nodular lesions are the most common Therefore this study was undertaken to find out the findings for definitive diagnosis of spirocercosis in prevalence of spirocercosis in the stray dogs of dogs which can be observed by necropsy, radiography Chittagong metropolitan area emphasizing on and endoscopy.The detection of typical embryonated epidemiology and pathology of this disease.eggs in fecal smears is difficult and requires special Materials and Methods laboratory techniques and facilities for visualization (Fox et al. 1988;Cabrera and Bailey, 1964).One to Collection of animals and Necropsy: A total of four worm-containing nodules are usually found in the sixty (60) stray dogs from different thanas of submucosa of the wall of the oesophagus located a few Chittagong metropolitan area were randomly captured centimeters cranial to the diaphragm (Bailey, 1963 and and humanely euthanized during the period from May-Chandrasekharon et al. 1958).The nodules may vary 2010 to February-2011.The capture was conducted from <1 to >4 cm in diameter and they can distort the under permission of Chittagong City Corporation.The oesophageal wall and extend to the surrounding necropsy was conducted in the Pathology and mediastinal tissues.The worms present in a nodule Parasitology laboratory of CVASU at the earliest may vary from a few to greater than 30 in number.The possible time of euthanasia following standard adults are large spiraled pink worm with males up to 54 method described in Coles (1986).For studying the mm and females up to 80 mm in length (Soulsby, oesophageal lesions the junction of the stomach and 1982).
duodenum was ligated and oesophagus was separated Histologically Spirocerca lupi associated from other parts of gastro-intestinal tract.Gross oesophageal nodules are composed predominantly of pathological changes in oesophagus were carefully fibrocytes with ample mature collagen at initial stage recorded, photographed and parasites were collected.
From the gross lesions, smaller tissue sections of 4 where with the time of nodular maturation; actively 3 dividing fibroblasts become predominant.These mm thickness were fixed in 10% neutral buffered fibroblasts are located between numerous immature formalin for histopathological study.capillaries, peripheral to the worms and their Histopathological study : Only one nodule preferably migratory tracts.The development of oesophageal the most mature one was selected for each dog for osteosarcomas and fibrosarcomas has been reported in histopathology.Formalin fixed tissue samples were dogs infected with Spirocerca lupi, particularly in washed and dehydrated in graded ethanol and embedded endemic areas (Moulton, 2002 andBailey, 1972).The in paraffin wax.Fixed tissues were sectioned at 5 µm infected dogs usually show mild anaemia in almost thickness and stained with hematoxylin and eosin as 50% cases (normocytic normochromic anaemia) of per standard method (Luna, 1968).
Haematological study: Haematological parameters early disease conditions (Ranen et al. 2004 and such as Red blood cells (RBCs), white blood cells Mazaki-Tovi et al. 2002).In advanced conditions (WBCs) count and haemoglobin concentration (Hb) when nodules transforms into neoplasia microcytic were manually done according to Campbell (1995).hypochromic anaemia become evident.Leukocytosis Packed cell volume (PCV) was determined according is more likely found associated with oesophageal to Howlett et al. (2002).Mean corpuscular volume osteosarcomas though monocytosis is also reported in (MCV), mean corpuscular haemoglobin (MCH) and some cases.In some recent studies no correlation mean corpuscular haemoglobin concentration found between the white cell counts and the (MCHC) were calculated as mentioned by Campbell spirocercosis associated inflammatory conditions (1995).Thin blood smears were immediately prepared such as pneumonia or spondylitis (Ranen et al. 2004 and stained with Diff Quick stain (EMD Chemicals, and Mazaki-Tovi et al. 2002).
Inc., Gibbstown, New Jersey 08027, USA) and 200 Stray dogs are free roaming ownerless or feral leukocytes were differentiated in smears prepared dogs that are not dependent upon human for from each animal.supervision.Chittagong is a port city located on the Statistical analysis : The obtained data was banks of the Karnaphuli River in the southeast part of imported, stored and coded accordingly using Bangladesh.Tropical monsoon climate and thousands Microsoft Excel-2000.Chi Square test was done for of free roaming stray dogs attributes this densely determination of association between the variable by Necropsy Findings: Minimum One to maximum using statistical software STATA/IC-11.The three oesophageal nodules were found in each infected associations of spirocercosis with Haematological dog having a broad firm nipple like protuberance values were measured by One way ANOVA using bulging from the oesophageal wall.Most of the statistical software SPSS-11.5.Significance was nodular lesions had smooth surface with a window like determined in 95% confidence interval when p<0.05.
orifice at the center and adult parasites were seen protruding out through that opening (Figure I).The

Results
nodules were also seen distorting the oesophageal Based on oesophageal lesions spirocercosis were lumen.The nodular lesions varied in size from less diagnosed in 24 (out of 60) captured stray dogs.Among than 1 cm (0.7 cm) to more than 3 cm in diameter and the positive dogs 13 (40.62%)were male and 11 (39.28%)usually situated at the distal portion (less than 4 cm were female.The prevalence of the infections was from the cardia of stomach) of the thoracic oesophagus various in different areas and ranged from 35.29% in (Figure II).The average size of the oesophageal Pahartali to 47.05% in Halishahor.Prevalence of nodules was 2.104±0.139cm (Table 2).Red colored spirocercosis in Pachlaish and West khulshi area was spirally coiled adult S. lupi were seen embedded 36.36% and 40.00% respectively.The prevalence was within the nodular lesions.Several sinus pockets and considerably higher (50.00%) in summer than (31.00%) parasitic migratory tracts were also seen in the incised in winter (Table 1).In respect to the body condition the nodules.The number of worms per nodule varied from prevalence of spirocercosis observed highest (47.36%) one to twelve with an average of 3.750±.686(Table in dogs with poor body condition, where only 30.00% 2).dogs with fair health showed infection.Dogs with The haematological tests exhibits reduction in good health also showed infection in 42.82% dogs in Hb (g/dl) concentration and RBC counts in 66% this study (Table 1).In the present study no significant spirocerca positive cases although no significant difference observed between spirocercosis with the gender variation, seasonality, locality and body condition of the stray dogs.As because dogs below 6-9 month age do not exhibit any typical oesophageal lesion and in this study the age of the dogs were not considered.Again, it was practically impossible to study the breed predilection of the disease as all captured dogs were of indiscriminate native types.3).lesions showed different histological pictures Microscopic features: Histopathological examina-according to their constituents and their stage of tion in all formalin-fixed oesophageal lesions revealed development.Nodules showed diffuse thickening in the was statistically insignificant.This result is comparable oesophageal wall with fibrous tissue accumulation with the finding of other researchers who showed no having abundant collagen fiber, mature fibrocytes and sex predilection for this infection (Fox et al. 1988 andWandera, 1976).Irrespective to the body condition the intervening lymphoplasmacytic cell.Large numbers higher prevalence of spirocercosis was observed in of newly formed blood vessel was seen in the loosely dogs with both poor health (47.36%) and good health arranged fibrous layer giving the appearance of granulation (42.82%) status.Thus the effect of in tissue (Figure V).Large numbers of neutrophils, fewer body condition remains inconclusive which supports lymphocytes, plasma cells and macrophages were the idea that Spirocerca lupi infection is mostly sub commonly found in between the diffusely arranged clinical and only shows clinical manifestation in connective tissue layers (Figure V).

Table-2. The biometric values of the oesophageal nodules
severe oesophageal obstruction (Bailey, 1972, Hu and A considerable degree of fibroplasia was also Hoeppli, 1936).evident in some mature nodules with predominant The Haematological picture of all infected dogs fibroblasts having plump atypical nuclei and irregular showed lower haemoglobin concentration than the collagen layers.Accumulation of multiple clumps of reference value.Among the infected dogs a significant lymphocytes, plasma cells, macrophages and fewer reduction in MCHC% and ESR values was observed neutrophils were observed in those lesions.Only one but the MCV% remained within the normal range.oesophageal lesion showed clear evidence of fibrosar-Therefore, a variable degree of normocytic normocoma characterized by overgrowth of poorly differenchromic to normocytic hypochromic anaemia is evidently tiated spindle to polygonal fibroblasts and abnormal associated with spirocercosis.In previous studies mild mitotic figures.The neoplastic cells residing in the anaemia was well described in Spirocerca lupi intervening fibrillar collagenous matrix showed higher infected dogs (Ranen et al. 2004  High prevalence of spirocercosis among the highly vascularized fibrous connective tissue layers.stray dogs of Chittagong metropolitan describes it as Unlike many previously reported cases the term an endemic region for this parasite that has capability granuloma was not applicable for the lesions as there to produce neoplasia.Further research should be was no dense gathering of macrophages in any of these attempted for detecting the probable paratenic host nodules.Van der Merwe et al. (2008) also noted that and to understanding the complex host-parasite the nodules are usually incorrectly referred as granurelationship might be helpful for preventive measures.lomas.Because of the high degree of neovascula-A new serological technique to confirm S. lupi rization, the lesions can rather be described as infection prior developing incurable nodules will be granulation tissue; and it has been previously observed helpful for treatment in such endemic areas.by (Baily, 1963).

Figure - I .
Figure -I.Large nodular lesion with smooth surface in the wall of esophagus protruding outwards from the serosal surface (Lt); broad base nodule with window like opening (aero head) for releasing eggs; note the red colored coiled adult parasite emerged from the opening (Rt)

Figure-II.
Figure-II.Three nodules (N) of different size simultaneously present within the wall of the caudal esophagus.Inset Adult Spirocerca lupi reside in the esophageal wall and fibrous nodule forms around the worms in the wall of the esophagus Figure-III.Cross section of the adult S. lupi in the sinus tract surrounded by multiple layers of proliferating fibroblasts (F).The parasite contains typical egg (E) (300X)Figure-IV.S. lupi (S) surrounded by polymorphonuclear cells and purulent cellular debris (D) (1200 X).

Figure- V .
Figure-V.Section from esophageal mass showing neovascularization (arrowhead) within dense fibrous bundles (80X).Accumulation of neutrophils, fewer lymphocytes, plasma cells and macrophages between the layers of fibroblasts (Inset 1200X).Figure-VI.Fibrosarcoma indicated by plump, spindle-shaped (S) to undifferentiated polyhedral (P) fibroblasts with a large nucleus-cytoplasm ratio and increased mitotic figures.(2000X) and Mazaki-Tovi et degrees of atypia having plump ovoid multiple nuclei al. 2000) and hence our finding is highly compa-rable which are often overlapping within the scanty cytoplasm with their reports.Mylonakis et al. (2006) and Mazaki-(Figure VI).Tovi et al. (2002) also reported normocytic normo-Histopathological features of oesophageal nodules Conclusion showed worm containing sinus tracts surrounded by