Albanian j. agric. sci. 2017; (Special edition)

Agricultural University of Tirana

(Open Access)

RESEARCH ARTICLE

Larval Ascaris suum migration and diagnostic value in pigs YLLKA (MIJA) ÇANI1*, BEJO BIZHGA2 1

PhD Candidate, Faculty of Veterinary Medicine, Agricultural University, Tirana, Albania

2

Faculty of Veterinary Medicine, Agricultural University, Tirana, Albania

*Corresponding author; E-mail: [email protected]

Abstract Study focused on diagnostic alternatives of Ascaris suum infection in 162 pigs in slaughterhouse. The range and infestation intensities of Ascaris suum infection depend on age, period and method of examination. Coproscopic examinations from 162 samples showed 124 of them (76.54%) positive for Ascaris suum eggs. Coproscopic examinations resulted the most efficient and offer the possibility of epidemiological estimates. From the samples were found with injuries as a result of migration process 49 pigs or 30.24% of the sample surveyed. During the macroscopic examination of the intestine of pigs were found with the adult parasite in 78 heads or 48.14% of the examined pigs. This value was about 20% lower than the value of coproscopic examination, but about 20% higher than the value of the examination of the milk liver spots. During of the macroscopic and microscopic examination in lungs were found with signs of pneumonia and parasitic larva migration in the lung parenchyma and the bronchi, 58 heads or 35,8% of the examined pig lungs. In pigs when parasitic pneumonia detected, the nose leaks were examined for the presence and number of larvae. From 162 nose tampons examined resulted positive for the presence of Ascaris suum migration larvae 26 pigs (16.04%) from the total of examined heads. Post-mortem examinations in slaughterhouses at pigs resulted efficient, low cost and provide sufficient data for Ascaris suum infection. Keywords: Ascariasis; swine; method; larvae; diagnose.

1. Introduction

in their place is created the second stage larvae. At the temperature 20

0

C they become second-stage

Ascaris suum is a nematode that infects pigs and

larvae and they infest pigs when they eat food or

randomly it can affect people. A. suum is present

swallow water and invasive eggs, from which the

across in our country and causing major damage to the

small intestine out of inexpensive stage larvae II, to

growth economies of pigs and the disease causes

begin phased hepato-entero-pulmonary migration [10,

called ascariasis [4, 6, 13, 17]. Adult ascarids living in

12, 15, 17]. The stage II larvae pierce the intestines

the small intestine and the mature females produce

and enter the blood vessels (veins), beginning their

100,000-200,000 a day eggs that come along with the

migration to the liver, where they stay 4-5 days and

feces in the external environment. The male is 15-25

transformed into L 3 larvae that reach the liver

cm long and 0.3-0.4 cm wide, while the female is 25-

through the hepatic veins, pass in vena cava caudalis

41 cm long and 0.3-0.6 cm wide. It is a long worm,

and the right half of the heart, from which through the

the shaft-shaped yellow. Mouth are surrounded by

pulmonary artery into the lungs arrive 4-7 days after

three edge equipped with scarring little theeth. Eggs

infestation [3, 7, 14]. Pierce blood vessels and walls of

are 50-70x40-50μ, they have ellipsoidal shape, thin-

the alveoli, hit the alveoli and begin to climb in the

walled and small granules. They have brown to

airways, continuing movement and assisted by the

yellow colour, are non-segmented and very resistant.

mucociliary apparatus, the larvae emerge in the

Eggs maintain vitality in the land, but can be

pharynx, where together with saliva and bronchial

destroyed by the direct rays of the sun [4, 6, 8, 13,

fluids ingested and down to the intestine where it

27]. Eggs newly emerging with the feces are not

begins the second phase or intestinal stages. The

infestive, they can be transformed invasive eggs when

larvae reappear on the day of the casing 8 and 9 after

369

Çani et al., 2017

infestation there doing stripping III and transformed

migration of the ascarids larvae [6, 12, 16, 18, 22].

into L4 and around day 30 to make stripping IV and

To differentiate parasitic stains from stains caused by

transformed into L5. The entire cycle of pig organs

fungi and bacteria samples from the liver in the

lasts 60-70 days and the life of A. suum lasts about

laboratory were stained with Wright Gimsae and Ziehl

one year [1, 9, 21]. A. suum exists everywhere where

Neelsen [16]. In the same piglets were observed also

pigs grow and the level of infestation ranges routine

the

from 20-70%. Infestation can be caused at all ages,

pneumonia or suspected cases of migration of larvae

but more serious are age 1-5 months. More infested

in the lungs when the hemorrhagic lesions were noted

are the pigs who do not eat well, and when lacking

and eosinophils infiltration around alveoli (the larvae

hygiene is bigger [12, 23, 27]. The source of the

migrate to the bronchial tree) were noted hemorrhage,

disease are infested pigs. The greatest danger comes

edema and emphysema [3, 8, 13, 25].

from manure from stalls without leave and employees

resulting suspicious or affected, were sampled from

who can spread the eggs wherever they move [1, 4, 6].

damaged areas, or to be fully examined in the perfusion method [17, 25, 28].

The examinations were conducted in 162 pigs in the slaughterhouse. Scope of work during coproscopic examination was to determine the prevalence and parasite load of A.suum in pigs in the according

to

epidemiological

alignment criteria, sampling and evaluation [2, 4, 6, 17, 23]. Fecal samples were taken directly to the right intestine which often were completely campionated. In this case the right hose broke away from the rest connected on both sides and the plastic bag was transported

in

laboratory.

For

coprologic

examinations were used qualitative and quantitative methods of sedimentation and flotation [4, 6, 17]. Examinations post mortun to the peculiarities of biology A.suum highlighted changes in the lungs and the liver and damages during the phase of hepatopulmonary migration of slaughtered pigs in the slaughterhouse. These surveys were carried out in these organs which often were sampled completely. We routinely by these organs were sampled and samples

for

macroscopic,

Lungs

laboratory. In lab full lungs were examined with

2. Material and Methods

slaughterhouse

lungs. After an observation of parasitic

histological

and

As for the debris

became microscopic examination of smears prepared by the

fluid in the bronchial

tree. At

the

slaughterhouse was prepared swabs from the nose leaks from all piglets that were used in the experiment [3, 14, 19].

Tampons were observed to find

migratory larvae. In cases when larvae were found calculates their number. To observe, intestines were divided into several parts by ligature and were cut into pieces 1-2 meters long and trasported introduced in plastic boxes. For examination of the intestine parts were washed in pure water and the water was collected and was surveyed after sedimentation [2, 7, 21].

After each rinse portion of the intestine was

opened by enteretom was surveyed and the interior to discover fixed worms mucus. To be separated from the mucous membrane do not retreat with pliers but torn mucous fixed around the country to favor secession. Content and mixed water filtered and the remaining filter disposed in a container to be examined [7, 9]. In the lab was raided all bowels and sediment rinse was tested on a glass tray located on a black sfod. For detection of small nematode was used

microscopic examination [11, 12, 15].

artificial digestion and accomplished overnight in 2.1. Procedures at slaughter

and post

thermostat at 37°C. After that was observed with low

mortum examinations

magnification dissolved material from which were

In slaughterhouses in livers of piglets was conducted

separated parasites [6, 17, 21, 26]. During micro and

evaluation of the milk spots. Livers were carefully

stereomicroscopic

observed for the evaluation of hot spots due to

collected, differentiated and counted young and adult

370

examination

was

observed,

Larval Ascaris suum migration and diagnostic value in pigs

parasites in the intestines of examined piglets [1, 3, 5,

Coproscopic examinations were conducted

24, 28]. Coproscopic examinations, the nasal and the

with alterations of the method of sedimentation and

majority of the organs were performed in the

flotation (Mc Master). In 72 (44%) stoll samples from

laboratory of veterinary parasitology, FVM.

the intestine (rectum and right intestine) were applied to both quantitative methods for the same sample [2,

3. Results and Discussion

4, 17, 23].

Table 1. Results of post-mortem examinations of pigs in the slaughterhouse.

No

Examination method

Sample piglets no

Positive no

Positive %

1

Coprology

162

124

76.54

2

Milk spots

162

49

30.24

3

Larval pulmone migration

162

58

35.8

4

Nasal swabs

162

26

16.04

5

Ascaids in intestine

162

78

48.14

Table 2. Parasitic loud under quantitative coproscopic examinations.

Examination method Parasitic loud

Sample piglets no 162

Average e/g/f 168

Variations e/g/f 10-1800

From coproscopic examinations were found

injuries were were noted in about one third of the pigs

the highest values of the infestation of Ascaris suum

examined. Liver examination for "milk spots" is valid

in piglets. The data confirm that coproscopic study

and provides enough information for the particular

regardless of the method applied is most successful

observations may be made in the slaughterhouse [4, 6,

methods for diagnosing ascariasis in piglets. It is fast,

12, 16, 26]. Examination is easy and free of cost and

easy and low cost. From 162 samples examined

status is sufficient to approvement conclusions for the

(coproscopy) 124 of them (76.54%) resulted positive

ascarids pathology in piglets. The opposite applies to

for Ascaris suum infection [4, 6].

the

From

coproscopic

observations

method

negativity.

Negativity

should

be

average

reconsidered and evaluated carefully, as not all

parasitic load in piglets resulted in 168 e/g/f with 10-

affected piglets may have the presence of milk spots

1800 e/g/f variations. During post-mortem inspection

in the liver. The data from the study showed that only

knowing the peculiarities of A. suum biology,

about a third of pigs affected by milk spots in their

examinations were carried out in the liver, lungs, nose

liver. These milk spots are classified in three models

and intestines. These surveys were carried out in

based on the macroscopic appearance. Histologically

slaughterhouses and by the same piglets that served as

the following three types of lesions are seen:

the basis of this study. From samples, were found with

interstitial hepatitis and eosinophilic intralobular

injuries as a result of liver migration 49 piglets or

necrosis, arteriolar degeneration and granuloma, and

30.24% of the observed samples. These injuries are

lymphofolicular

known as "milk spots" in the liver. This was the main

relationship between macroscopic and histological

characteristic of a chronic hepatitis caused by parasitic

patterns,

Ascaris suum larvae migrans. The spots are the result

manufactured by interstitial eosinophilic hepatitis [6,

of larval migration within the liver. These migratory

12, 16, 17]. With the naked eye, microscopic

371

compact

hyperplasia. milk

spots

Regarding are

the

generally

Çani et al., 2017

observations and histopathology were observed milk

This is followed by an intensive infiltration of

spots in the livers of pigs in the slaughterhouse. Livers

eosinophils. These lesions are visible at necropsy on

of piglets positive for milk spots were brought to the

the lung surface as white areas and are clearly

laboratory for thorough examination. Liver samples

separated from the healthy part of the body. In these

resulting with milk spots belonging to all ages of pigs

cases were found migrating larvae of A. suum [6, 12,

2-7 months. Lesions were in diameter between 0.5 and

19]. In piglets from leaks nose in 162 piglets were

1 cm distributed in whole liver parenchyma and

taken tampons in their noses and were prepared to

infiltration in hepatic parenchyma. Often larvae were

microscopic

noted in the parenchyma, or migrate freely. In cases of

observed in stereomicroscopes and microscopes as

chronic forms of larvae resulted in dead and blocked

wet and dry preparations. The technique proved to be

abscesses or granulomas. For differentiated parasitic

very present when it is known that the larvae appear

stains, or stains caused by fungi and bacteria samples

on the nose of the pig infested on day 7 and 9 after

from the liver were stained with Wright Gimsae and

infestation. Larvae in the nose and mouth swallowed

Ziehl Neelsen [12, 16].

During entero-hepato-

into the gut or leaving by sneezing or runny nose in

cardio-pnemo-intestinal migration in tissues where

the external environment. All samples proved positive

larvae pass, causing bleeding and increased volume of

for migratory larvae of A. suum. Swabs diagnostic

organs and necrotic spots. Lung larvae are reared and

technique proved to be very simple, extremely

cause multiple hemorrhages in the alveoli, the

efficient and the very fruitful outcome [14, 19, 23].

brokiola, east of edema and parasitic pneumonia in

Work is underway to convert into quantitative

pigs [3, 6, 12, 19]. The larvae produce toxins that act

technique for estimating the number of larvae and

and other organs of piglets. Lung larvae are reared and

other parasitic estimates.

cause

alveoli,

tampons tested positive for the presence of larvae 19

bronchioles, east of edema, formation of parasitic

piglets or 15.83% of the total examined tampons. This

pneumonia which is present in piglets. From samples

is the lowest value recorded for the same piglets

examined were found to damage as a result of

category. The reason is the method fragility and

migration in the lungs 58 piglets or 35.8% of the

biological features of Ascaris suum which only for a

observed samples. These migratory lung damage

short period of time can be found in the nasal leakage.

resulted in about 5% higher than milk spots that were

This is the period when he comes in after the

noted in about one third of the pigs examined. Lung

migration bronchioles and mucociliary apparatus

examination for the presence of migratory pneumonia

comes in the mouth to swallow or nose to be removed

resulted more efficient examination of the liver and

with leaks in the external environment [6, 19]. This

provide

in

time is limited and therefore values and will be

slaughterhouses [6, 12, 19, 21, 22, 26]. The larvae

smaller. The advantage of the method is that the

migration to the lungs are capable of causing an

examination method may bring valuable data and the

inflammatory reaction, destruction of pulmonary

alive piglets (not necessarily killed) and status and

tissue and parasitic pneumonia with hemorrhagic foci.

parasitic loads are more value [6].

multiple

hemorrhages

sufficient

data

for

in

the

observations

smears.

Microscopic

swabs

were

From 162 examined

Table 3. Parasitic loud under quantitative nasal tampon examinations.

Examination method

Samples no

Average l/ml

Variations l/ml

Nasal swabs parasitic loud

162

2.5

1-12

In our case average parasitic loud was 2.5 larva per ml found that the method was converted into 372

quantitative method and the number of larvae per ml ranged in values 1-12 l/ml.

In these piglets was

Çani et al., 2017

performed and intestinal observation to find and count

monitoring of post-mortem swine ascaridiasis. While

increased ascarids. While mature parasites were found

nasal examinations in young piglets can be used as an

in the intestines of 78 piglets or 48.14% of the

alternative method for the diagnosis and monitoring of

observed samples [6, 13].

ascaridiasis.

To resist intestinal

descending flow they stare ends of their body in the intestinal mucosa and the damage it mechanically. When numerous they block the movement of food and cause measures extensions, twisting, gut cracks and east of peritonitis. They can climb in the stomach and cause nausea. They absorb large amounts of protein and food stuffs [1, 6, 12, 19, 21, 23, 28]. 4. Conclusions Coproscopy resulted in successful methods for diagnosing ascaridiasis in piglets. It is the fast, easy and low cost. Of 162 samples examined 124 of them (76.54%) resulted positive for Ascaris suum eggs. From coproscopic observations average parasitic load in piglets resulted in 168 e/g/f with 10-1800 e/g/f variations. The migratory damage (milk spot) was seen in about 1/3 of the liver of the piglets examined, while in coproscopy were identified 76.5% of the samples taken in the same piglets. The difference is explained by the features of the biological cycle of the parasite and the specificity of the immune piglets. Lung examination for the presence of migratory pneumonia resulted in the efficient examination of the liver and provide sufficient data for observations in slaughterhouses. From samples examined were found to damage as a result of migration in the lungs 58 piglets or 35.8% of the observed samples. These migratory lung damage resulted in about 5% higher than milk spots. From 162 examined tampons tested positive for the presence of larvae 19 piglets or 15.83% of the total examined tampons. This is the lowest value recorded for the same piglets category. The reason is the fragility of method and biological features of Ascaris suum. While were found positive for askarids in the small intestine about 50% of the examinated piglets. Control of liver and lung where they are visible signs of pneumonia migration and the presence of invasive larvae, can be applied as successful diagnostic techniques for the diagnosis and 373

6. References

1. Anderson TJ: The dangers of using single locus markers in parasite epidemiology: Ascaris as a case study. Trends in parasitology 2001, 17, 183–188. 2. Arizono N, Yoshimura Y, Tohzaka N, Yamada M, Tegoshi T: Ascariasis in Japan: is pig-derived Ascaris infecting humans? Japanese journal of infectious diseases 2010, 63, 447–448. 3. Bernardo T. M, Dohoo I. R, Donald A: Effect of ascariasis and respiratory diseases on growth rates in swine. Canadian Journal of Veterinary Research 1990, 54, 278–284. 4. Bizhga B.: Parazitologjia veterinare. Pg. 391-419. Gear Tirane, 2013. 5. Boes J, Coates S, Medley G. F, Varady M, Eriksen L, Roepstorff A, Nansen P: Exposure of sows to Ascaris suum influences worm burden distributions in experimentally infected suckling piglets. Veterinary Parasitology 1999, 119, 509–520. 6. Çani M.Y, Bizhga B: Ascaris suum Infection Estimate. Anglisticum Journal (AJ), 2016, 5 (9), 8-13. 7. Carstensen L, Vaarst M, Roepstorff A: Helminth infections in Danish organic swine herds. Veterinary Parasitology 2002, 106, 253–264. 8. Coates S.: Modelling the population dynamics of Ascaris suum in pigs. PhD Dissertation. Danish Centre for Experimental Parasitology, Copenhagen, Denmark and the University of Warwick, Coventry, UK. 2000. 9. Criscione CD, Anderson JD, Sudimack D, Peng W, Jha B: Disentangling hybridization and host colonization in parasitic roundworms of humans and pigs. Proc Biol Sci 2007, 274, 2669–2677. 10. Crompton DW: Ascaris and ascariasis. Adv Parasitol 2001, 48, 285–375.

Larval Ascaris suum migration and diagnostic value in pigs

11. Dold C, Holland CV: Investigating the underlying mechanism of resistance to Ascaris infection. Microbes and infection/Institut Pasteur 2011, 13, 624–631. 12. Geenen P L, Bresciani J, Boes J, Pedersen A, Eriksen L, Fagerholm H.P, Nansen P: The morphogenesis of Ascaris suum to the infective third-stage larvae within the egg. Journal of Parasitology 1999, 85, 616–622. 13. Holland CV: Predisposition to ascariasis: patterns, mechanisms and implications. Parasitology 2009, 136, 1537–1547. 14. Jolie R, Backstrom L, Pinckney R, Olson L: Ascarid infection and respiratory health in feeder pigs raised on pasture or in confinement. Swine Health and Production 1998, 6, 115–120. 15. Jungersen G: Immunity and immune responses to Ascaris suum in pigs. In The Geohelminths: Ascaris, Trichuris and Hookworm (ed. Holland, C. V. and Kennedy, M. W.): Kluwer Academic Publishers. Boston, Dordrecht, London; 2002: 105–124. 16. Kakihara D, Yoshimitsu K, Ishigami K, Irie H, Aibe H, Tajima T, Shinozaki K, Nishie A, Nakayama T, Hayashida K, Nakamuta M, Nawata H, Honda H: Liver lesions of visceral larva migrans due to Ascaris suum infection: CT findings. Abdom Imaging, 2004, 29, 598-602.

21. Napravnik J, Berka T: Cause and effect of ascariosis in pig herds. Helminthologia 2002, 39, 171-183. 22. Ngowi HA, Kassuku AA, Maeda GE, Boa ME, Willingham AL: A slaughter slab survey for extra-intestinal porchine helminth infections in northern Tanzania. Trop Anim Health Prod, 2004, 36, 335-340. 23. Roepstorff A, Eriksen L, Slotved HC, Nansen P: Experimental Ascaris suum infection in the pig: worm population kinetics following single inoculations with three doses of infective eggs. Parasitology 1997, 115 (4), 443–452. 24. Roepstorff A.: Ascaris suum in pigs: population biology and epidemiology. Doctorate thesis, the Royal Veterinary and Agricultural University, Copenhagen, Denmark. 2003. 25. Slotved H.C, Barnes E.H, Eriksen L, Roepstorff A, Nansen P, Bjorn H: Use of an agar-gel technique for large-scale application to recover Ascaris suum larvae from intestinal contents of pigs. Acta Veterinaria Scandinavica 1997, 38, 207–212.

17. Kassai T.: Veterinary Helminthology. Butterworth-Heinemann, Oxford, UK. 1999.

26. Wang T, Van Steendam K, Dhaenens M, Vlaminck J, Deforce D, Jex A.R, Gasser R.B, Geldhof P: Proteomic analysis of the excretory-secretory products from larval stages of Ascaris suum reveals high abundance of glycosyl hydrolases. PLoS Neglected Tropical Diseases 2013, 7(10):e2467.

18. Masure D, Vlaminck J, Wang T, Chiers K, Van den Broeck W, Vercruysse J, Geldhof P: A role for eosinophils in the intestinal immunity against infective Ascaris suum larvae. Plos Neglected Tropical Diseases 2013, 7(3), e2138.

27. Weng YB, Hu YJ, Li Y, Li BS, Lin RQ, Xie DH, Gasser RB, Zhu XQ: Survey of intestinal parasites in pigs from intensive farms in Guandong province, Peoples Republic of China. Veterinary Parasitology 2005, 127(3-4), 333-336.

19. Mejer H, Wendt S, Thomsen L. E, Roepstorff A, Hindsbo O: Nose-rings and transmission of helminth parasites in outdoor pigs. Acta Veterinaria Scandinavica 2000, 41, 153–165.

28. Zajac A.M, Comboy G. A.: Veterinary clinical parasitology. Blackwell publishing. State Avenue, Ames, USA. 2006.

20. Miquel N, Roepstorff A, Bailey M, Eriksen L: Host immune reactions and worm kinetics during the expulsion of Ascaris suum in pigs. Parasite Immunology 2005, 27, 79–88.

374