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Dec 17, 2008 - well as fluctuation (Willis, 1927) and sedimentation. (Hoffman et al., 1934) ..... Felt, S. & White, C. (2005) Evaluation of a timed and repeated perianal tape ... Wrobleski, S.K., Hawley, A.E. & Myers, D.D. Jr (2006). Biology and ...
Journal of Helminthology (2009) 83, 225–229

doi:10.1017/S0022149X08156772

Biochemical and haematological parameters in owl monkeys infected and uninfected with Trypanoxyuris sp. F.O. Barros Monteiro*, L.N. Coutinho, K.F. de Arau´jo, M.V. Barros Monteiro, P.H.G. de Castro, K.S.M. da Silva, R.N.M. Benigno and W.R.R. Vicente Universidade Federal Rural da Amazoˆnia (UFRA)/Instituto da Sau´de e Produc¸a˜o Animal (ISPA), Avenida Presidente Tancredo Neves, No. 2501, Bairro, Terra Firme Cep: 66.077-530 Caixa Postal: 917, Bele´m-Para´-Brasil Abstract The objective of the present study was to report the occurrence of Trypanoxyuris in owl monkeys, using data from clinical and haematological examinations, as well as clinical chemistry (blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) of infected and uninfected animals. Twenty animals in apparently good clinical health were studied. The coproparasitological examinations showed eggs compatible with Trypanoxyuris sp. in 50% of animals. The number of red blood cells, haematocrit and haemoglobin levels were significantly higher in the males, compared to the females, irrespective of parasitism. However, comparing segmented neutrophils in infected males and females, a significant difference (P , 0.05) was observed. All blood chemistry values were considered normal for the species pattern, even though significant differences were observed for BUN and ALT in infected males. The infection by Trypanoxyuris sp. did not appear to interfere with the clinical condition of animals.

Introduction Given the evolutionary proximity and phylogenetic similarities to humans, non-human primates (NHP) have the potential to transmit several parasitic and infectious diseases (Andrade, 2002). As members of biologically diverse habitats, they act as sentinels for pathogen investigation and models for basic research about the dynamics of natural transmission. As such, the NHP populations have contributed to information about emerging diseases (AIDS, Ebola, hantavirus and dengue). Studies related to these species may also benefit conservation efforts as well as fulfilling the gaps between laboratory studies and the already recognized need for epidemiological investigations (Wolfe et al., 2000). The genus Aotus is widely distributed throughout South America, from Panama to the north of Argentina (Wright, 1994). The Aotidae family comprises 13 species *Fax: þ 55 91 32105256 E-mail: [email protected]

and subspecies (Rylands et al., 2000). The geographic range of Aotus azarai infulatus is wide, occurring in the west and south-western Amazon, east of the rivers Tapajo´s and Juruena in Brazil, west of the rivers Gurupı´ and Araguaia. It was also recently identified in the state of Amapa´, north of the lower Amazonas river. There is much forest destruction and fragmentation in the south of its range in southern Para´, but this subspecies is not currently considered to be threatened (Rylands et al., 2003). Historically, Aotus monkeys have been utilized in biomedical research to address a diverse array of scientific questions. These moderately small, nocturnal Neotropical primates possess several unique features that make them ideal for use as animal models for human diseases. These include susceptibility to infection with Plasmodium spp., large eyes with easily visualized retinas, adaptability to captivity and relative ease of handling (Baer, 1994). Many NHP species are susceptible to infection by nematodes of the Oxyuridae family, which show

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F.O. Barros Monteiro et al.

preference for the intestinal colon. The genera known to parasitize NHP are: Enterobius, Trypanoxyuris and Lemuricola (Prieto et al., 2002). According to Urquhart et al. (1998), oxiurid pathogenic effects result in small lesions of the intestinal mucosa, which may become diffuse, followed by an inflammatory response in severe infestations. The life cycle is direct and infection occurs through the ingestion of the larval eggs, which are deposited on the perianal skin. In some species of pinworms, it is speculated that retroinfection may occur whereby the newly hatched larvae enter the host directly through the rectum (Felt & White, 2005). Trypanoxyuris sp. was described as non-pathogenic and common in owl monkeys (Tantalea´n & Gozalo, 1994). However, there are reports of death in spider monkeys (Ateles sp.) and owl monkeys (A. nancymaae) severely infested by this nematode (Fiennes, 1972; Sa´nchez et al., 2006). In general, the main sign of infection is the perineal irritation caused by adult females during the egglaying stage (Urquhart et al., 1998). In other NHP species, tail and perineal rubbing can be a result of pruritus and associated oxyuriasis. However, Aotus use both urine and glandular secretions to convey olfactory information. Rubbing of the tail and perineum, a behaviour usually attributed to scent marking, is well recognized as an important form of olfactory communication in this genus (Wright, 1994). Therefore, according to Felt & White (2005), if perineal rubbing occurred as a result of pruritus from pinworms in owl monkeys this behaviour might be indistinguishable from scent marking. Parasitic diseases constitute a serious public health problem in developing countries. Poor sanitation, poverty and tropical climate all contribute to the development, spread and perpetuation of gastrointestinal diseases (Michaud et al., 2003). Research centres that maintain and work with NHP need to control the sanitation of colony animals. This is important to promote quality of research and avoid pathogen transmission to keepers and researchers who work with these animals. Clinical examination, coproparasitological exams, biochemical and haematological exams, as well as disinfection of the facility and fomites are essential tools to diagnose and control helminth dissemination in captivity, especially in the Amazon, where climatic conditions favour pathogen multiplication. In spite of the existence of abundant literature on parasites in Neotropical primates, there is little information on parasites in the Aotus monkey (Tantalea´n & Gozalo, 1994). Given the epidemiological relevance related to the incidence of parasitic diseases in captivity, the objective of this study was to report the Trypanoxyuris sp. occurrence in captive owl monkeys, comparing clinical and laboratory examinations of infected and uninfected animals.

Materials and methods The study was performed with 20 animals (10 couples previously established), where females were 4 – 10 years old, and males were 5 – 13 years old. Animals were

captive born and belonged to the National Primate Center (CENP) breeding colony, located in the Ananindeua municipality, state of Para´, Brazil (latitude 18380 2600 and longitude 488380 2200 ). Each animal was identified with a three-letter code tattooed on the inside of the right thigh and a microchip placed in the interscapular area. Animals were housed indoors, in Sector I (for monogamic species), in brick-built enclosures covered with tile and wire netting. This sector was projected in a north – south direction, in order to receive up to 12 h of natural light, enabling regular access to solar radiation throughout the year. The climate in the region was classified as humid, Megathermic with regular rainfall from January to June and pluviometric indices of 2250– 2500 mm. The average temperature was 258C (max. 328C and min. 228C), with air relative humidity around 85%. The cages measured 3.85 m in length £ 1.2 m width £ 2.40 m height. Externally, the enclosures had a 35 cm shelter box of 35 cm £ 30 cm £ 30 cm, with a guillotine type door and a water bottle for drinking. Internally, there were two wooden platforms for walking and access to the food bowls. Animals were fed according to CENP husbandry practice: a variety of fruits, vegetables, milk, eggs, dry dog food, vitamin and mineral supplements and water ad libitum. Animal health was assessed through clinical examinations, clinical and laboratory testing, including coproparasitological examinations, haemogram and clinical chemistry (creatinine, blood urea nitrogen (BUN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST)). Samples for coproparasitological exams were collected and stored in sterile and identified plastic bottles, shortly after each animal defecated. Samples were analysed according to a standard protocol established by the CENP using direct examination, as well as fluctuation (Willis, 1927) and sedimentation (Hoffman et al., 1934) techniques. According to this protocol, levels of gastrointestinal helminth parasite infections were determined when calculating the number of positive microscopic fields. Values that did not exceed 25% of the examined fields were considered low. Results identified between 25 and 50% were considered as medium and above 50% as high level of infection. Haematological exams were also performed, and the blood samples (3 ml) were collected from the femoral vein under manual restraint, using sterile syringes and needles. The haemogram was performed using an automatic counter Celm CC-550 (CELM -Cia. Equipadora de Laborato´rios Modernos Alameda Amazonas, Sa˜o Paulo, Brazil), and clinical chemistry with the Vitros Systemw DTSC II, DT60 and DTE2 (Johnson & Johnson Medical Argentina, San Isidro, Argentina). The haemogram and clinical chemistry results were submitted to analysis of variance (ANOVA). Comparisons of individual means were examined with the Tukey test (Steel et al., 1997). For differences between males and females and between parasitized and non-parasitized animals, two-sided P values of 0.05 or less were considered to be statistically significant.

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Trypanoxyuris sp. infection of owl monkeys

Results

Discussion

During the clinical examinations, all animals were apparently healthy. In the coproparasitological examinations, eggs compatible with Trypanoxyuris sp. were identified in 50% of the couples. It was observed that, when one of animals was infected, the other one was also infected. However, the levels of infection were considered to be low, as the number of positive fields was lower than 25% of the total examined microscopic fields. Positive animals were treated orally with 9.5 mg oxantel pamoate together with 14.5 mg pirantel pamoate per kg of body weight (Basken suspensa˜ow, Ko¨nig do Brasil, Ltd), and the same treatment was repeated after 15 days. As a prophylactic measure, a blowtorch was utilized in order to eliminate foci of nematoid contamination in the enclosures. The haematological results (table 1) indicated that the number of red blood cells, the haematocrit and the haemoglobin levels were influenced by the sex, as the males evidenced values significantly higher than the females (P , 0.05), irrespective of parasitism. As for the mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC), no significant differences were observed. The white blood cell analysis did not reveal significant changes in the number of eosinophils, basophils, lymphocytes and monocytes from infected and uninfected animals. However, there was a significant difference in the segmented neutrophils when comparing infected males and females. Table 2 lists the blood chemistry values (BUN, creatinine, ALT and AST) in male and female A. azarai infulatus parasitized or not by Trypanoxyuris sp. Infected males had BUN levels significantly higher when compared to other groups (P , 0.05). The same occurred with the ALT values.

Coproparasitological examinations were not performed with the specific objective of investigating oxyurids. Therefore, the exams were sporadic in the CENP’s Aotus colony. Repeating the examinations utilizing a perianal tape test was not possible due to the fact that therapeutic and prophylactic protocols were initiated soon after the results were confirmed. The identification of parasitism by Trypanoxyuris sp. is supported by data from Tantalea´n & Gozalo (1994) who reported this to be the most frequently identified pinworm species in Aotus sp. Research on New World non-human primates in the wild indicated infection rates of 54.7% in the species Aotus nancymaae and lower (8.7%) in Aotus vociferans (Michaud et al., 2003). Prevalence determination enables the comprehension of whether a disease is common or rare in a given population. Therefore, comparing prevalence data of free-ranging animals to our study indicates that these parasites can also be identified in captive populations. The considerable number of infected animals observed in this study (50%) may be related to repeated selfinfection events or typical oxiurid cross-infection (Flynn, 1973). It was also observed that all parasitized animals were previously established couples, suggesting that infection may have occurred among cagemates. This is compatible with the results from Felt & White (2005), as they determined that the risk of infection by T. microon in A. nancymaae was 14 times greater when animals were housed with an infected cagemate. Data presented in table 1 indicated significant differences for red blood cells, haemoglobin and haematocrit values, despite the fact that the same did not occur in the MCV, MCH and MCHC. Sex-related differences observed in the haematological examinations were reported in the literature (Malaga et al., 1990) and confirmed by our results. This may be explained by

Table 1. Haematological analyses of Aotus azarai infulatus (n ¼ 20) infected or uninfected by Trypanoxyuris sp. (CENP, Ananindeua/Para´/Brazil, 2007). Females (mean ^ SD) Parameters 6

23

Red blood cells ( £ 10 mm ) Haemoglobin (g dl21) Haematocrit (%) MCV (fl) MCH (pg) MCHC (%) Leucocytes ( £ 103 mm23) Neutrophils (mm23) Eosinophils (mm23) Basophils (mm23) Lymphocytes (mm23) Monocytes (mm23)

Males (mean ^ SD)

Infected

Uninfected

Infected

Uninfected

4.78 ^ 0.5A 13.12 ^ 1.2A 40.44 ^ 4.7A 84.6 ^ 2.3A 27.8 ^ 1.3A 32.86 ^ 1.3A 11.06 ^ 3.11A 2508 ^ 553.6A 607.0 ^ 377.8A 0 ^ 0A 7848 ^ 2933A 97.00 ^ 97.73A

4.69 ^ 0.8A 13.64 ^ 1.6A 41.72 ^ 4.9A 89.38 ^ 4.7A 29.4 ^ 1.6A 33.0 ^ 1.0A 11.76 ^ 1.77A 3214 ^ 1909AB 645.6 ^ 339.7A 18 ^ 40A 7859 ^ 2568A 23.80 ^ 53.22A

5.88 ^ 0.5B 15.82 ^ 0.73B 50.4 ^ 3.7B 85.5 ^ 1.9A 27.0 ^ 1.8A 31.5 ^ 2.0A 11.84 ^ 3.10A 6189 ^ 1994B 375.6 ^ 192.6A 0 ^ 0A 5225 ^ 1158A 50.20 ^ 72.26A

5.6 ^ 0.2B 15.56 ^ 0.45B 49.52 ^ 2.9B 87.4 ^ 3.6A 27.5 ^ 0.7A 31.5 ^ 1.5A 11.28 ^ 2.67A 4910 ^ 1858AB 625.4 ^ 326.9A 0 ^ 0A 5695 ^ 2597A 49.60 ^ 69.68A

MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration. Different letters in the same row indicate a significant difference (P , 0.05).

228

F.O. Barros Monteiro et al. Table 2. Values of blood chemistry analyses for Aotus azarai infulatus (n ¼ 20) infected and uninfected by Trypanoxyuris sp. (CENP, Ananindeua/Para´/Brazil, 2007). Reference values

Parameters 21

BUN (mg dl ) Creatinine (mg dl21) 21

ALT (U l ) AST (U l21)

4–25* 15 ^ 5.4† 0.1–0.6* 1.0 ^ 0.4† 23–135* 47 ^ 37† 70–250*

Females (mean ^ SD)

Males (mean ^ SD)

Infected

Uninfected

Infected

Uninfected

4.6 ^ 1.5A

4.2 ^ 0.8A

8.8 ^ 3.2B

6.2 ^ 2.8A

0.66 ^ 0.05A

0.76 ^ 0.05A

0.76 ^ 0.16A

0.68 ^ 0.14A

34 ^ 11.2A

24.4 ^ 15.7A

65.6 ^ 15.2B

48.2 ^ 12.6A

88.2 ^ 29.4A

131.5 ^ 23.2A

100 ^ 22.1A

111.2 ^ 40A

BUN, blood urea nitrogen; ALT, alanine aminotransferase; AST, aspartate aminotransferase. Different letters in the same row indicate a significant difference (P , 0.05). * Reference interval for Aotus spp. using the products Vitros Systemw according to Ortho-Clinical Diagnostics. † Linn et al., 2006.

physiological variation in erythrocyte parameters, as testosterone stimulates erythropoiesis, while oestrogen is an inhibitor (Jain, 1993). Malaga et al. (1990) described significant differences in the number of red blood cells, haemoglobin, MCV and platelet values between female and male owl monkeys. In the present study, platelet information was not obtained. Therefore, the differences in the relative values of erythrocytes are due to sexual differences rather than infection. According to Jain (1993), evaluation of erythrocytic values can reveal disorders such as haemorrhagic syndromes, anaemia and decreased vascular fluid due to haemoconcentration, dehydration and hypovolaemia. Leucocyte values can also indicate infectious, inflammatory, toxic and stress conditions. Eosinophils correspond to 1 – 3% of the total leucocytes in the peripheral bloodstream, varying, in absolute terms, from 0 to 0.45 £ 103 ml21 in most NHP. The number of these cells increases in the tissues and in the peripheral blood as a response to parasitic infections and hypersensitivity reactions (Albert et al., 1996). The same authors observed that in Aotus azarai boliviensis the number of eosinophils was significantly higher than the number observed in A. nancymaae, suggesting that there is variation in the count within the genus Aotus. In the present study no significant alterations were observed in the eosinophil count for infected and uninfected animals, probably due to the low infection level identified in animals. The absence of this alteration corroborates the findings by Felt & White (2005) when diagnosing parasitism by Trypanoxyuris microon in A. nancymaae. When infected male and female data were compared, the significant difference identified in the segmented neutrophils was within the normal values when compared to clinically healthy individuals, as described by Baer (1994). Therefore, we believe that this result was not biologically meaningful. However, an increase in the catecholamine numbers in animals submitted to acute stress promotes the transfer of neutrophils from the marginal pool to the circulating pool, resulting in increased number of these cells in the leucogram (Thrall, 2004; Cunha et al., 2005). Laboratory tests may serve many functions, including prognosis, response to therapy and diagnosis. In some

cases it may be necessary to run blood chemistry tests to assess different organ-systems, including the kidney and liver (Porter, 2005). In this study the blood chemistry (BUN, creatinine, ALT and AST) results were not clinically relevant, since the values were identified as normal when compared to reference values reported by Linn et al. (2006) and the Vitros Systemw as a pattern for the species (Aotus spp.). According to the literature, differences may exist in the haematology and serum chemistry data from different sources for owl monkeys, but it is not known whether these are related to the methods utilized for data collection, examination techniques, karyotype, husbandry or other environmental factors (Baer, 1994). Therefore, in the circumstances of the present study, it was possible to infer that the infection by Trypanoxyuris sp. did not influence the clinical and laboratory examination results. This also explains some differences identified in the haemogram and serum chemistry of animals utilized in this study, when compared to the literature (Malaga et al., 1990; Baer, 1994; Brieva et al., 2001; Linn et al., 2006). Therefore, a detailed clinical examination is very important to ensure an effective evaluation. The level of parasite infection did not appear to adversely effect the clinical condition of animals. This is emphasized by the combined analysis of the clinical and laboratory examinations, confirming the literature reports about Trypanoxyuris sp. as a parasite well tolerated by its host (Tantalea´n & Gozalo, 1994). However, it is a fact that some facilities experienced difficulty when attempting to eradicate the parasite (Felt & White, 2005). Therefore, control measures and therapeutic protocols must be routinely implemented in order to avoid transmission and dissemination of the parasite in the colony.

Acknowledgements We would like to thank the National Primate Center (CENP) keepers and technicians for their support during the laboratory work and handling of animals utilized in this study, and Dr Christina Wippich Whiteman, DVM, PhD, for translating this paper. We also would like to thank Dr James Else, DVM, MPVM (Yerkes National

Trypanoxyuris sp. infection of owl monkeys

Primate Research Center, Emory University, Atlanta, Georgia, USA) for suggestions and a technical English review. This study was approved by the Animal Research and Ethics Committee from the Evandro Chagas Institute (Health Vigilance Secretary of the Health Ministry of Brazil).

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