ORIGINAL ARTICLE

Clinical, hematological, biochemical, and ultrasonographic
aspects of Platynosomum sp. (Trematoda: Dicrocoeliidae) infection
of captive Callithrix penicillata

Mariana Portugal Mattioli1 • Juliana dos Santos Batista1 • Marlon Ferrari2 •

Giane Regina Paludo3 • Cecı́lia Azevedo Dias4 • Estevam G. L. Hoppe5 •

Gino Chaves da Rocha3 • Rafael Verı́ssimo Monteiro3,6

Received: 7 July 2015 / Accepted: 5 February 2016 / Published online: 27 February 2016

� Japan Monkey Centre and Springer Japan 2016

Abstract Trematodes from the genus Platynosomum have

been found to infect Neotropical primates in captivity, but

little is known about their pathogeny in such hosts. This study

evaluated the physiological effects of natural infection by the

liver-dwelling trematodePlatynosomum sp. in tenmales and

ten females of Callithrix penicillata kept in captivity at the

Primate Center of the University of Brası́lia. The marmosets

were examined twice, 6 months apart. The following

parameters were analyzed: complete blood count, bleeding

time, serum total protein, albumin, and the liver enzymes

AST andALT, and both a stool analysis and a liver ultrasonic

evaluation were performed. We were able to characterize a

group of abnormalities associated with this trematode

infection which were mainly derived from the hepatitis

caused by it: coagulation disorders, abnormal red blood cells,

hypoalbuminemia, and abnormal levels of liver-linked

serum enzymes. Eosinophilia and thrombocytopenia were

also commonly seen. All of the aforementioned abnormali-

ties were in good accordwith typical effects of trematodes on

liver parenchyma.We suggest that this set of abnormalities is

characteristic of the infection of C. penicillata with

Platynosomum sp., and should be among themost prominent

aspects that the veterinary surgeon considers when suspect-

ing such an infection. We also suggest that these clinical

signs and abnormalities will be similar in other liver-dwell-

ing trematode-infected primate species.

Keywords Black-tufted marmoset � Health rank � Liver
disease � Hepatic ultrasound � Trematode hepatopathy

Introduction

Breeding groups of captive neotropical primates are com-

monly maintained in large cages or open enclosures to

promote a social structure closer to natural conditions,

thereby enhancing welfare and the reproductive success of

the colony. However, the more natural the enclosure, the

greater the risk of exposure of the housed primate to

undesirable external parasites. These parasites may harm

the primate, causing diseases ranging from low-impact

illnesses which are easily treated and controlled to severe

clinical impairments which may even result in death. In

any case, the primate may develop physiological abnor-

malities significant enough to preclude it from being used

as an experimental subject. Reproductive impairment is

another common consequence of disease, and one that is

also detrimental to colony viability. Therefore, the spread

of external parasites in a primate colony may cause med-

ical and reproductive problems, not to mention the eco-

nomic losses incurred due to the significant investment

involved in housing each animal in such research facilities.

& Rafael Verı́ssimo Monteiro

rvmonteiro@unb.br

1 Hospital Veterinário, Faculdade de Agronomia e Medicina

Veterinária, Universidade de Brası́lia, Brası́lia, DF, Brazil

2 Serviço de Ultrassonografia, Hospital Veterinário, Faculdade

de Agronomia e Medicina Veterinária, Universidade de

Brası́lia, Brası́lia, DF, Brazil

3 Faculdade de Agronomia e Medicina Veterinária,

Universidade de Brası́lia, Brası́lia, DF, Brazil

4 Centro de Primatologia, Universidade de Brası́lia, Brası́lia,

DF, Brazil

5 Faculdade de Medicina Veterinária, UNESP Jaboticabal,

Jaboticabal, SP, Brazil

6 Hospital Veterinário, Faculdade de Agronomia e Medicina

Veterinária, Universidade de Brası́lia, Av. L4 Norte/Asa

Norte, Brası́lia, DF 70910-900, Brazil

123

Primates (2016) 57:279–287

DOI 10.1007/s10329-016-0520-8

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The Centro de Primatologia da UnB (CPUnB) is a

Brazilian primate research center which houses neotropical

species, mainly capuchin monkeys (Sapajus apella), black-

tufted marmosets (Callithrix penicillata), and squirrel

monkeys (Saimiri sciureus). The CPUnB is located inside

the Brası́lia University experimental farm in a well-con-

served Cerrado area (Brazilian savanna), away from any

agricultural activities. Wild populations of C. penicillata

and Alouatta caraya also roam free over the farm’s Cer-

rado conservation area. This location permits very calm,

nature-integrated enclosures, but it also allows parasites to

reach the captive monkeys. Wild-borne parasitic disease

may arise from two commonly observed situations: (1)

invertebrates and small vertebrates—potential intermediate

hosts or carriers of many parasites—enter the cages and are

eaten by captive primates; (2) wild, free-roaming primates

interact with those in captivity.

At the end of 2006, liver-dwelling trematode parasites

were identified during the necropsies of two deceased

marmosets. From 2006 to 2012, marmosets died from

abnormalities which were tentatively linked to trematode

infection. Based on a comparison of the specimens col-

lected from necropsied marmosets with descriptions pro-

vided by Kingston and Cosgrove (1967) and Travassos

et al. (1969), the parasite was identified as Platynosomum

sp. (Fig. 1). Little is known about trematodes that infect

nonhuman primates, but they are commonly found in the

pancreas, gall bladder, and biliary ducts (Toft 1982; Sousa

et al. 2008). Light infestations are mostly asymptomatic,

but high burdens cause thickening of the biliary ducts and

traumatic compression of the surrounding tissue, resulting

in hepatic cell degeneration and vacuolization (Marcos

et al. 2008; Rojo-Vázquez et al. 2012). The histological

modifications caused by Platynosomum sp. in the liver of a

congeneric primate species, C. jacchus, were shown to be

portal fibrosis, ascending cholangitis, cholestasis, and even

secondary biliary cirrhosis (Sousa et al. 2008). Infections

with this trematode genus currently pose a problem for

other primate centers throughout Brazil (Sousa et al. 2008;

Silva et al. 2012).

Hepatic disease may be characterized clinically and by

laboratory tests. Clinical signs such as jaundice and an

enlarged and palpable liver are typical. These signs are

correlated with high serum levels of the liver-associated

enzymes AST and ALT (Tygstrup 1990; Marcos et al.

2008). Liver disease may cause other clinical signs, such as

coagulation disorder and edema, both of which are related

to impairment of coagulation cascade precursors and

albumin synthesis (Kavanagh et al. 2011). Ultrasounds and

other image-based exams may also be performed in order

to evaluate liver abnormality, severity, and localization.

This research aimed to analyze clinical signs, blood counts,

biochemical profiles, and liver ultrasound images of black-

tufted marmosets infected by Platynosomum sp.

Methods

Human care guidelines

All of the procedures described below were approved by

the Ethical Committee for the Use of Animals of UnB,

under the number 53242/2011. The Primatology Center

granted their full consent to collect samples from the

animals.

Marmoset characteristics and restraint technique

The 20 adult marmosets (ten males and ten females) of

species Callithrix penicillata were housed at the CPUnB,

and their body weights ranged from 245 to 410 g (321 g on

average). The marmosets were fed with a small-primate-

specific commercial chow which was supplemented daily

with a mix of fruits and vegetables. Twelve of these mar-

mosets (eight males, four females) were born at the

Fig. 1 Specimen of Platynosomum sp. collected from the liver of a

dead marmoset from the CPUnB. Authorship: G.C. Rocha

280 Primates (2016) 57:279–287

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CPUnB. At the time of the sampling, the youngest was

1 year and 4 months old while the oldest was 8 years and

5 months old. The other eight individuals came from zoo

captivity or were provided by the governmental agency.

The most recently arrived had spent 6 months at the

CPUnB, and the longest housed marmoset was there for a

year and a month. Except for the trematode infection, no

other helminths were characterized in flotation and sedi-

mentation stool examination techniques. Moreover, no

other parasites or infections were characterized in the

necropsies routinely performed on dead marmosets from

the CPUnB—whether they were based on macroscopic

examinations or histological slides—that could be associ-

ated with hepatic disease.

During the year before our first sample, the twenty

marmosets were submitted to 12–15 stool examinations in

blocks of three successive days of stool collection. All

marmosets provided at least one trematode-positive stool

sample; 14 of them had four or more positive stool sam-

ples. The marmosets had been deverminated on two

occasions during this previous year with praziquantel,

150 mg/kg, PO, SID or nitazoxanide, 150 mg/kg, PO, BID,

with both drugs administered on three successive days. All

marmosets were trematode-free at least once after dever-

mination. This recursive trematode infection cycle is

putatively attributed to the constant reinfection of mar-

mosets after the ingestion of unidentified invertebrate

intermediate hosts originating from outside the cages.

Two samples were taken 6 months apart (these two

samplings are referred to in the text below as ‘‘S1’’ and

‘‘S2’’). Two females were pregnant and so no blood sam-

ples were taken from them at S1, resulting in a total of 38

blood samples. The same deworming schedule described

above for nitazoxanide was repeated between the two

samplings. The frequency of occurrence of Platynosomum

sp. in the marmoset stools ranged from 90 % at S1 to 35 %

at S2. Leather gloves and nets were used during sampling

to briefly restrain all individuals in order to facilitate the

intramuscular administration of a combination of 20 mg/kg

of ketamine (Cetamin, Syntec, Brazil) and 1 mg/kg xyla-

zine (Calmium, Agener Uniao, Brazil) (Joslin 2003). While

unconscious, each subject was weighed, and its body

temperature (�C) as well as their cardiac and respiratory

frequencies were monitored at 5-min intervals throughout

the trans-anesthetic period.

Blood collection

Peripheral blood was collected from the femoral-inguinal

vein with a 3-mL syringe attached to a 20 mm 9 0.6 mm

needle (PrecisionGlide, BD, Brazil). The blood was divi-

ded into two vials: a dry one which allowed the blood to

clot for serum separation, and another containing sodium

heparin (Hemofol, Cristalia Ltda, Brazil) as an anticoagu-

lant to enable a blood count.

Health parameter analysis

Bleeding time evaluation

The bleeding time (BT) after blood collection was used as

an indirect measure of the primary hemostasis of the

marmosets. Subjects with no sign of bleeding 3 min after

venipuncture were classified as having good primary

hemostasis. Any bleeding after this time classified the

animal as having delayed primary hemostasis. The pro-

portion of marmosets with a delayed bleeding time was

compared to the expected distribution (10 % of the animals

would present this abnormality) through a chi-square test,

considering an alpha of 0.05.

Blood analysis

Red and white blood cells (RBCs and WBCs, respectively),

together with thrombocytes (Thromb), were counted using

the classical protocol with a Neubauer chamber. Hemat-

ocrit (HT) was determined by centrifugation of blood-filled

capillary tubes at 13000 rcf for 5 min (Celm MH, Celm,

Brazil). Leukocyte types were counted in blood smears

stained with rapid panoptic stain (Instant Prov, NewProv,

Brazil). Blood hemoglobin (HB) content was determined

by spectrophotometry. The mean cell hemoglobin con-

centration (MCHC), mean cell hemoglobin (MCH), and

mean cell volume (MCV) were calculated from the afore-

mentioned values. The following serum enzymes and

proteins were evaluated using spectrophotometry: AST,

ALT, total protein (TP), and albumin (ALB). These ana-

lyzes were conducted with commercial kits (Labtest, Bra-

zil) and a semi-automatic spectrophotometer (BIO 2000,

BIOPLUS, Brazil).

Statistical evaluation of the Platynosomum sp.

infection pattern

We used two different approaches to evaluate the

Platynosomum sp. infection pattern in our marmoset sam-

ple. First, we compared the mean health parameter values

of our trematode-infected marmosets with the corre-

sponding values reported by Yarbrough et al. (1984). The

mean point between the adult male and female values as

displayed in Yarbrough et al. (1984) was considered for the

average and standard deviation calculations. We compared

the mean values of the two studies using a t test considering

an alpha level of 0.05. We looked for significant differ-

ences which would indicate a general effect of trematode

infection on the marmoset population.

Primates (2016) 57:279–287 281

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Second, to identify common characteristics of the most

severely trematode-sick animals, we compared the value of

each health parameter for each trematode-infected marmoset

with a reference range also created based on Yarbrough et al.

(1984). The reference range corresponded to the average plus

or minus two standard deviations. We counted the number of

marmoset individuals above or below the established limits

(of the reference range) for each health parameter. We

employed a chi-square test (alpha = 0.05) to investigate any

statistical difference from the expected distribution of 5, 90,

and 5 % of the primates with values below, within, and above

the reference range, respectively.

Finally, using a t test (p\ 0.05), we compared the mean

health parameter values of the trematode-positive (23

individuals, 18 from S1) to those of the trematode-negative

(15 individuals, 13 from S2) marmosets in order to char-

acterize which parameter values were significantly modi-

fied after the worms had been removed from the

marmosets. All tests were performed with the software R.

Ranking marmosets by health

A health rank (HR) was obtained for each animal sampled

based on the results of the clinical and laboratory exami-

nations. The residual z score for each parameter was cal-

culated for each animal. Our health rank was derived from

the sum of the z scores, as shown below:

HR = zscore (HT ? RBC ? HB ? ALB) - zscore (BT

? WBC ? EOS ? AST ? ALT)

We used the HR for two purposes. First, we evaluated how

many marmosets were at the lower 2.5 % limit of the HR, and

which characteristics they had in common. Second, we com-

pared theHRvalues atS1 to those at S2.One standarddeviation

of the HRwas used to define reference thresholds that could be

used to identify the animals that showed an improvement or a

deterioration in health at the second sampling.

Hepatic ultrasonography

The ultrasound examinations (US-E) were conducted with

a MyLab 30 VET ultrasound machine (Universal Ultra-

sound, USA) wired to a 12-MHz linear probe. All exami-

nations (n = 39) were performed by a single investigator

(M. Ferrari). One marmoset from S1 was not submitted to

US-E. The animals were restrained in dorsal recumbency

and the fur over the abdominal area was completely

shaved. A generous amount of ultrasound gel was applied

over the area to be scanned. The best liver images obtained

were utilized to describe the shape and size of the liver and

the echotexture and echogenicity of the liver parenchyma

and gall bladder. The gall bladder was analyzed in terms of

its contour, relative size, wall thickness, and content. The

distance from the liver to the sternum (DLS; in cm) was

compared between animals considered to have normal

livers and those considered to have hepatitis or both hep-

atitis and hepatomegaly using a one-way ANOVA; sig-

nificance was set at p\ 0.05.

Results

Bleeding time

Table 1 presents the observed and expected distributions of

marmosets with normal or delayed bleeding times. The

proportion of animals that presented problems with pri-

mary hemostasis was significantly higher than expected at

both S1 and S2.

Effects of Platynosomum sp. infection on overall

population health

Table 2 shows that some of the parameters were statisti-

cally different from their reference means. The trematode-

infected marmoset population presented higher hematocrits

because of higher RBC counts, but had lower mean cell

hemoglobin concentrations and albumin serum levels. The

leukocyte profile for the marmoset population indicates

that they have significantly lower mean neutrophils, eosi-

nophils, lymphocytes, and thrombocytes than the corre-

sponding reference means.

Marmosets with out-of-range health parameter

values

Table 3 shows that Platynosomum sp. infection modified

black-tufted marmoset health parameters to the point that

many of them could be considered unhealthy. The

Table 1 Classification of a group of twenty black-tufted marmosets

based on bleeding time at two different sampling sessions S1 and S2

Sampling season Bleeding time status p

Normal Delayed

S1 12 6 0.03

S2 15 5 0.03

Expected distribution 16/18a 2

Total 27 11 0.03

Expected distribution 34 4

Expected frequencies were calculated based on the assumption that

90 % of the marmosets would have normal bleeding times and 10 %

would have delayed bleeding times. Observed frequencies were

compared to the expected ones using a chi-square test. Samplings

were performed 6 months apart
a Expected frequencies for S1 and S2, respectively

282 Primates (2016) 57:279–287

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modifications occurred to four groups of parameters:

(a) red blood cell size and hemoglobin content character-

istics; (b) leukocyte profile; (c) serum albumin levels; and

(d) levels of serum proteins that are dependent on liver

metabolism.

Modifications to health parameter values

after deworming

Table 4 presents the health parameters for which the

trematode-positive marmosets (mostly at S1) and

trematode-negative marmosets (mostly at S2) presented

statistically significantly different values. We consider

these differences to indicate the parameters that

improved the most after the marmosets were dewormed.

Health rank analysis

The average HR of the 38 animals was -0.29 ± 4.79

(mean ± SD) and the rank distribution was skewed

towards the worst ranks (skew = -0.68). One animal had

an HR (-15.11) that was below the lower limit (2.5 %) of

the reference range (-9.87). This marmoset was one of the

pregnant females in S1. The other female pregnant at S1

also had a very low HR (-9.53) at S2.

When comparing the 18 individual HRs at S1 and S2

(excluding the two females that were not sampled at S1),

we noticed that the health of two marmosets (one male, one

female) had deteriorated, but only the male was Platyno-

somum sp.-negative in the stool exam. The aforementioned

female showed the most pronounced health deterioration,

with an HR reduction of 8.41 units. Five marmosets

showed health improvements (one male, four females),

with three animals becoming free of trematode infection.

The aforementioned male marmoset displayed the best

health improvement between the two sampling sessions, as

its HR rose by 10.39 units. The HRs of the other animals

fluctuated within the 1 SD limit. The HR did not differ

statistically significantly depending on stool exam status.

Liver ultrasonographic characteristics

during trematode infection

US-E of the livers that were considered normal showed the

usual appearance: preserved organ margins and size toge-

ther with a hypoechoic homogeneous echotexture. The

biliary bladder may have been be filled with anechoic

homogeneous bile and may have had a hyperechoic wall.

In contrast, the liver images that were considered charac-

teristic of inflammatory hepatitis (49 % of the marmosets)

Table 2 A t test comparison of

the mean health parameter

values obtained in this work

with those reported by

Yarbrough (1984)

Health parameter n This research From Yarbrough (1984)

Mean (SD) Reference mean (SD) 95 % Reference range

Blood analysis

HT (%)** 38 48 (5) 42 (8) 26–58

HB (g/dL) 34 14.2 (2.6) 15.6 (2.7) 10.2–21.0

RBC (9106/mL) 38 5.5 (1.8) 5.7 (1.1) 3.5–7.9

MCV (fL)** 38 94 (30) 73 (5) 63–83

MCH (pg) 34 28 (11) 27.5 (4) 19.5–35.5

MCHC (%)** 34 29.9 (4.4) 38 (4.7) 28.6–47.4

WBC (9103/mL) 38 8.5 (5.4) 7.8 (3.0) 1.8–13.8

Neutrophils (/mL)* 38 5196 (3960) 3822 (1248) 1326–6318

Band cells (/mL)** 38 400 (371) 23 (69) 0–161

Eosinophils (/mL)** 38 321 (429) 39 (78) 0–195

Basophils (/mL) 38 77 (24) 94 (133) 0–360

Lymphocytes (/mL)* 38 2667 (2130) 3588 (1170) 1248–5928

Monocytes (/mL) 38 412 (478) 273 (226) 0–725

Thrombocytes (9103/mL)** 25 144 (104) 281 (111) 59–503

Serum biochemistry

Total protein (g/dL) 35 6.6 (1.5) 6.9 (0.8) 5.3–8.5

Albumin (g/dL)** 38 3.4 (0.6) 4.8 (0.7) 3.4–6.2

AST (U/L) 35 119 (94) 149 (44) 61–237

ALT (U/L) 36 52 (107) 55 (20) 15–95

SD standard deviation

* p\ 0.05; ** p\ 0.01

Primates (2016) 57:279–287 283

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showed a hyperechoic heterogeneous aspect (granular;

Fig. 2). Anechoic areas (hepatic cysts) were commonly

found next to the biliary bladder, which also showed wall

thickening in one marmoset (Fig. 3). Hepatitis was present

in 47 % (9/19) of the S1 marmosets, while 50 % (10/20) of

the marmosets displayed this abnormality at S2. An

enlarged liver was observed in 47 % of the marmosets with

hepatitis, but not in the marmosets with a normal US-E.

Hepatitis-associated hepatomegaly occurred in 33 % (3/9)

and 60 % (6/10) of the cases at S1 and S2, respectively.

The DLS was statistically different (p\ 0.001) among the

marmosets with a normal US-E (1.60 cm), those with

hepatitis (1.86 cm), and those with hepatitis-associated

hepatomegaly (2.58 cm). Hepatitis was not correlated with

the parasitological results for the stool, as 42 % (8/15) of

the trematode-negative marmosets presented it.

Discussion

The results presented in this work indicate a Platyno-

somum sp. infection pattern in the Callithrix penicillata

individuals sampled, which is compatible with its par-

asitic mode of action. The typical pathogenesis of liver-

dwelling trematodes results from their invasion of the

liver and the establishment of parasitic cysts which

affect the bile ducts and surrounding tissue (Marcos

et al. 2008; Rojo-Vázquez et al. 2012). Liver cell death

Table 3 Number of lack-tufted

marmosets that were infected

with Platynosomum sp. and had

health parameter values that

were outside the corresponding

reference ranges

Health parameter n Frequency of individuals with out-of-range values

Below Above p value

Blood analysis

HT (%) 23 0 0

HB (g/dL) 21 1 0

RBC (9106/mL) 23 5* 2 \0.001

MCV (fl) 23 2 13* \0.001

MCH (pg) 21 3 4* \0.005

MCHC (%) 21 12* 0 \0.001

WBC (9103/mL) 23 2 1

Neutrophils (9103/mL) 23 2 5* \0.001

Band cells (9103/mL) 23 0 1

Eosinophils (9103/mL) 23 0 6* \0.001

Basophils (9103/mL) 23 0 0

Lymphocytes (9103/mL) 23 9* 0 \0.001

Monocytes (9103/mL) 23 0 3

Thrombocytes (9103/mL) 18 5* 0 \0.001

Serum biochemistry

Total protein (g/dL) 20 2 1

Albumin (g/dL) 21 9* 0 \0.001

AST (U/L) 21 4* 2 \0.01

ALT (U/L) 22 6* 5* \0.001

Expected frequencies for chi-square calculations were 5 % of marmosets below the lower limit of the

reference range, 90 % between the upper and lower limits of the reference range, and 5 % above the upper

limit. The number of marmosets that were within the reference range is not shown for the sake of clarity.

The reference ranges used are shown in Table 2, and were calculated based on the average plus or minus

two standard deviations as presented by Yarbrough (1984)

* Statistically significant deviation from the expected value

Table 4 Health parameters for

which the trematode-positive

and -negative marmosets

presented statistically

significantly different

values after deworming

Health parameter Platynosomum sp. infection status p

Positive Negative

WBC 9103/mL 6.56 11.57 0.004

Neutrophils 9103/mL-1 3.91 7.17 0.011

Lymphocytes 9103/mL-1 2.11 3.53 0.040

284 Primates (2016) 57:279–287

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leads to fibrosis and liver enlargement, or even a

reduction in size in severely ill animals (Sousa et al.

2008). All of these effects impair liver function,

potentially causing primate death when the trematode

burden is high and the resulting liver damage is sig-

nificant. In our work, Platynosomum sp. infection was

Fig. 2 Liver ultrasonographic

image for a black-tufted

marmoset; the image shows the

granular aspect characteristic of

inflammatory hepatitis.

Ultrasound performed by M.

Ferrari

Fig. 3 Abnormalities found in

the liver ultrasonographic image

of a black-tufted marmoset

(Callithrix penicillata) infected

with Platynosomum sp. CH

hepatic parasitic cysts, Fi liver

parenchyma fibrosis spots, PE

thickened gall bladder wall, GB

gall bladder. Ultrasound

performed by M. Ferrari

Primates (2016) 57:279–287 285

123



found to compromise hepatic functions in three ways:

(a) impairment of bleeding control; (b) impairment of

red blood cell production; and (c) impairment of albu-

min synthesis.

We found that our experimental Platynosomum sp.-in-

fected marmosets had a significantly higher frequency of

delayed bleeding (Table 1). Low platelet counts were also

seen not only at the population level (Table 2) but also at

the individual level (Table 3). Coagulopathies are a com-

mon consequence of hepatic disease, and thrombocytope-

nia is characteristically found in trematode infections.

Liver parenchymal cells synthesize most of the coagulation

factors, while the liver’s reticuloendothelial system helps to

clear activation products. Local decreased platelet aggre-

gation, disseminated intravascular coagulation, occult

thrombosis, autoimmune destruction of platelets, and gas-

trointestinal hemorrhage may all contribute to driving the

patient into a hypocoagulatory state. It is noteworthy that

even in S2, when most of the marmosets were trematode-

free, a high proportion of the marmosets still had bleeding

abnormalities (Kavanagh et al. 2011).

The trematode-infected marmoset population of the

present study, while not, on average, anemic (Table 2), had

individuals with macrocytic hypochromic anemia

(Table 3), a common characteristic observed in the

pathogeny of other liver-dwelling trematodes (Valero et al.

2008). This morphologic classification is consistent with a

regenerative anemia, reflecting the increased size and

decreased hemoglobin of the reticulocytes (Barger 2003).

This anemia may have multiple causes. The blood-sucking

trematodes (which cause hemolysis) provoke increased

hematopoiesis turnover, meaning that insufficient RBCs

many be produced due to progressive hepatic destruction

and low iron storage. The proline released during trema-

tode metabolism also induces anemia in rat fasciolosis

(Spengler and Isseroff 1981; Coffin et al. 1984). Extrapo-

lating from the proposed classification of Valero et al.

(2008) for trematode-associated anemia, all of our mar-

mosets were in the early to advanced chronic phases of

infection.

The hypoalbuminemia demonstrated by the data shown

in Tables 2 and 3 is commonly associated with peripheral

or generalized edema, which was not diagnosed in our

experimental marmosets. This does not mean that these

clinical signs could not appear afterwards, with liver

deterioration and prolongation of the disease. In this case,

we cannot rule out the possible appearance of other clinical

signs linked to hypoalbuminemia, such as fur loss, dysp-

nea, muscle weakness, and/or wasting (Tygstrup 1990).

Hepatic parenchyma abnormalities can also be inferred

by evaluating the typical liver-associated enzymes AST

and ALT. Among our trematode-infected marmosets, some

displayed high serum levels of AST and ALT, signaling

recent hepatic damage (Table 3). Other marmosets which

displayed lower-than-normal AST and ALT levels were

probably animals with a prior chronic infection that led to a

cirrhotic, diminished liver. It is interesting to note that the

average values of these two health parameters did not differ

from their respective reference values. This was because

both excessively high and excessively low values were

observed in this work, meaning that the liver enzyme

averages did not shift markedly from the reference values.

Population health analysis should always consider the

extreme values and the data distribution.

Table 2 also indicates that trematode infection modified

the leukocyte profiles of the infected hosts. On average,

these marmosets displayed neutrophilia, lymphopenia, and

eosinophilia; some individuals also displayed leukocytosis.

This is a typical leukocyte profile for hosts infected with

trematodes (Marcos et al. 2008; Robinson and Dalton

2009). Table 3 reiterates this profile, showing more indi-

viduals than expected in the extreme reference range for

these WBC types. Chronic inflammation, such as the

hepatitis observed in these marmosets, in conjunction with

a stress response (Barger 2003) mediated by glucocorticoid

released during manual restraint of the marmosets, may

account for this profile. We also noticed that the dewormed

marmosets presented significantly increased neutrophil and

lymphocyte counts (Table 4). Immunosuppression is a

possible outcome of hepatic disease. Another hypothesis is

that it could be a shift of leukocytes from the liver par-

enchyma to the systemic circulation following deworming.

Regardless of the explanation, an improved immune

response can be expected from dewormed marmosets.

Our health rank analysis showed that, overall, the mar-

moset population affected by Platynosomum sp. was dis-

eased, but some animals were in worse health than others,

making these individuals more likely to die. The two

females that were pregnant at the first sampling had very

low HRs in the second sampling. This highlights that the

combination of a compromised liver and the physiological

demands of pregnancy will compromise female health and

may result in death. We believe that this may well also be

the case for other physiologically demanding activities,

such as growth and the immune response. In these cases,

marmoset health may be impaired, potentially reducing

growth rates and weakening the immune response of the

marmoset to parasites.

US-E confirmed that invasion of the hepatic parenchyma

by Platynosomum sp. results in inflammatory hepatitis and

parasitic cyst development, and may lead to hepatomegaly.

The different DLSs of marmosets with different hepatic

parenchyma inflammatory statuses is an expression of this

inflammation. However, given that nearly all of our mar-

mosets were infected at some point during the experimental

period, we consider that the presence (or absence) of these

286 Primates (2016) 57:279–287

123



abnormalities is dependent on the parasite burden and the

timing and length of trematode infection for each mar-

moset. The histopathological modifications that were pre-

viously observed in C. penicillata infected by

Platynosomum sp. (Sousa et al. 2008) are in good accord

with the US-E images obtained for the trematode-infected

marmosets in the present study.

Comparison of the health ranks at S1 and S2 showed

that removing the trematode infection marks the begin-

ning of recovery. The veterinary clinician should expect

liver function impairment to last longer than the infection

due to the liver fibrosis resulting from infection, which

will persist for the rest of the marmoset’s life. Veteri-

narians should consider this when contemplating the

marmoset’s feed and general care needs, as well as

eventual therapeutics.

Many of the abnormalities observed in the trematode-

infected marmosets studied here (anemia, neutrophilia,

lymphopenia, eosinophilia, increased or decreased AST

and ALT, and hepatomegalia) are also seen in cats

infected with P. fastosum (Basu and Charles 2014). As

also occurs with cats, marmosets may forage on cer-

cariae-infected molluscs and isopods, as well as small

lizards. In a free-range situation, such marmosets would

be unlikely to encounter and ingest cercariae-infected

intermediate hosts because they are much more likely to

feed on other insects and even other types of food.

However, with captive marmosets, a local cycle can

establish. Invertebrates feed inside the enclosures on the

trematode-infected marmoset feces, and marmosets eat

these new intermediate hosts of Platynosomum sp. This

positive feedback may lead to a large proportion of the

potential invertebrate intermediate hosts in the enclosure

becoming infected with Platynosomum sp. cercariae,

causing a high reinfection rate in the marmosets, as seen

in the CPUnB.

Finally, we suggest that our clinical examination

panel can be used to diagnose Platynosomum sp. infec-

tion in black-tufted marmosets, and that this panel

should also work in other primate species and for other

liver-dwelling trematodes. Also, the health rank we used

is able to identify which of the infected marmoset

individuals were in the worst condition, and this method

is replicable in other animal models. Marmosets with a

low health status should be more intensively treated and

cared for. Health rank may thus act as a red flag for

animals with particularly pronounced impairment of

physiological function.

Acknowledgments Mariana P. Mattioli and Juliana dos S. Batista

were supported by the PIBIC program of UnB.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict

of interest.

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	Clinical, hematological, biochemical, and ultrasonographic aspects of Platynosomum sp. (Trematoda: Dicrocoeliidae) infection of captive Callithrix penicillata
	Abstract
	Introduction
	Methods
	Human care guidelines
	Marmoset characteristics and restraint technique
	Blood collection
	Health parameter analysis
	Bleeding time evaluation
	Blood analysis

	Statistical evaluation of the Platynosomum sp. infection pattern
	Ranking marmosets by health
	Hepatic ultrasonography

	Results
	Bleeding time
	Effects of Platynosomum sp. infection on overall population health
	Marmosets with out-of-range health parameter values
	Modifications to health parameter values after deworming
	Health rank analysis
	Liver ultrasonographic characteristics during trematode infection

	Discussion
	Acknowledgments
	References