SEROLOGICAL DETECTION OF HEPATITIS A VIRUS IN FREE-RANGING NEOTROPICAL PRIMATES (Sapajus spp., Alouatta caraya) FROM THE PARANÁ RIVER BASIN, BRAZIL.

Nonhuman primates are considered as the natural hosts of Hepatitis A virus (HAV), as well as other pathogens, and can serve as natural sentinels to investigate epizootics and endemic diseases that are of public health importance. During this study, blood samples were collected from 112 Neotropical primates (NTPs) (Sapajus nigritus and S. cay, n = 75; Alouatta caraya, n = 37) trap-captured at the Paraná River basin, Brazil, located between the States of Paraná and Mato Grosso do Sul. Anti-HAV IgG antibodies were detected in 4.5% (5/112) of NTPs, specifically in 6.7% (5/75) of Sapajus spp. and 0% (0/37) of A. caraya. In addition, all samples were negative for the presence of IgM anti-HAV antibodies. These results suggest that free-ranging NTPs were exposed to HAV within the geographical regions evaluated.

INTRODUCTION

Hepatitis A is an acute viral infectious disease caused by Hepatitis A virus (HAV), a single-stranded unenveloped RNA virus that is member of the genus,Hepatovirus, family Picornaviridae(1). Although HAV has one known serotype and seven genotypes, denominated I-VII1 , 2, recent studies have reclassified HAV into six genotypes based on the molecular analysis of the VP1 gene3 ,. Nevertheless, genotypes I-III are more related with disease in humans2, while genotypes IV-VI are associated with simian hepatitis, and were originally identified in Old World monkeys4. Consequently, humans and nonhuman primates (NHPs) are the natural reservoirs of HAV1.

HAV-induced infection occurs primarily via the oral-fecal route due to the ingestion of contaminated food and water, or contact with infected hosts1 , 2. This disease is endemic in some developing countries4, including those of South America5, but particularly in regions of poor socioeconomic conditions, where adequate sanitation and hygiene practices are absent 6. A serological investigation of the presence of anti-HAV antibodies in patients from different geographical regions of Brazil demonstrated an incidence of 77.2% (19,507/25,270), with most elevated levels occurring in northern Brazil7. However, a study conducted during an outbreak of hepatitis A in Águas Lindas de Goiás, Midwestern Brazil, revealed an incidence of 46.4% (326/865)6; data collected from the Ministry of Health between 1999 and 2010 has demonstrated an increase in the number of cases of HAV between 1999 and 2005 in Midwestern Brazil, with a gradual decrease in the number of notifications from 2005 to 2010, finishing with less than 5% in 20108. Additionally, molecular analyses have demonstrated that the subgenotype IA is predominant within all geographical regions of Brazil5, suggesting that HAV is probably endemic9. Nevertheless, recent data have supported the shift of Brazil towards intermediate (North, Northeast and Central-West regions) and low (South and Southeast regions) endemicity levels of HAV, with the shift of the infection risk towards older age groups8 , 10.

NHPs are considered natural reservoirs of HAV1 , 3 and zoonotic diseases such as toxoplasmosis11 and yellow fever12, and can serve as natural sentinels for investigation of epizootics and endemic diseases of public health importance. Old World Primates are known to be very susceptible to HAV infection, with descriptions in species such as the Macaca fascicularis,Cercopithecus aethiops 13, M. mulatta 14,Nomascus leucogenys, Hylobates lar, andH. pileatus 15, however, reports of HAV in free-ranging Neotropical primates (NTPs) are sparse, with only one description in a collection of captive NTPs from northern Brazil16 and in several Owl monkeys (Aotus trivirgatus) from the USA17 , 18. There is also an experimental confirmation of HAV-induced hepatitis in the Callithrix jacchus 19. Additionally, the detection of HAV in primates within Brazil is significantly reduced16 , 19; while descriptions of HAV in free-ranging NTPs were not located when major online databases were consulted. This report investigated the occurrence of HAV in populations of free-ranging NTPs from the Paraná River basin, southern Brazil.

MATERIALS AND METHODS

Study location: All NTPs evaluated during this study were trap-captured as previously described20 , 21 within the Paraná River basin, southern Brazil, between June 2004 and April 2006. TheParaná River basin consists of several islands and riparian forest reserves that are environmentally protected by the Instituto Chico Mendes de Conservação da Biodiversidade (APA Ilhas e Varzeas do Rio Paraná; Dec s/no. September 30, 1997), in the Atlantic Forest domain, and are classified as Seasonal Semideciduous Forests. The NTPs evaluated during this study were from two forested islands (Mutum and Porto Rico) that are seasonally flooded and from forests of both riverbanks located within the outskirts of the city of Porto Rico. This city is located in the northwest ofParaná State at latitude 22°46'20"S and longitude 53°16'01"W; divides the border with the state of Mato Grosso do Sul, and is approximately 336 km away from Foz do Iguaçu. Therefore, the serum samples were obtained from NTPs that resided within the states of Paraná and Mato Grosso do Sul.

Neotropical primates (NTPs): Serum samples were obtained from 112 free-ranging NTPs: Capuchin monkeys (Sapajus nigritus from the left margin of the Paraná River; S. cay from the right bank), n = 75; and Black-and-Gold Howler monkeys (Alouatta carayafrom the two islands), n = 37. These primates lived within the forested regions of the Paraná River basin, where dense populations of NTPs are known to reside22. Permissions to capture (license No 104/04) and examine these NTPs were obtained from the Brazilian Institute of Environment and Renewable Natural Resources (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis - IBAMA). All NTPs were anesthetized by using Zoletil(r) (HILST et al. unpublished data), after which blood samples were obtained by jugular venipuncture; serum was extracted and maintained at -20 °C until used for serological assay. Biological and clinical parameters of all captured NTPs were collected (data not shown), after which all animals were released.

Serological investigation: All serum samples were evaluated by the enzyme-linked immunosorbent assay (ELISA) for the presence of total anti-HAV (Boehring Laboratory; Hessen, Germany) and anti-HAV IgM antibodies (DiaSorin; Saluggia Vercelli, Italy), using commercially available kits. The presence of anti-HAV IgG antibodies was determined by the criterion of total serum sample anti-HAV (IgG + IgM) reactive and nonreactive anti-HAV IgM antibodies. Additionally, for the presence of total anti-HAV antibodies (competitive test principle), samples whose absorbance readings yield a range of +/- 10% of the cut-off were considered negative or positive, respectively. While for the presence of anti-HAV IgM antibodies (noncompetitive test principle), samples whose assays yield absorbance readings within the range of +/- 20% of the cut-off were considered positive or negative, respectively.

Statistical analyses: Statistical analyses were performed by using the Chi-square and Fisher exact Test to establish differences between the characteristics evaluated; associations were determined by using the odds ratio (OR) at a 95% confidence interval. A r-value of < 0.05 was considered as statistically significant. All statistical analyses were performed by using the software Epi Info 6.0423.

Humane Care Guidelines: This study was approved by The Ethics Committee for Animal Experiments of the Universidade Estadual de Londrina(process No. 17115/2005), Paraná State, Brazil.

RESULTS

Only 4.5% (5/112) of the NTPs evaluated demonstrated positive seroreactivity to anti-HAV IgG antibodies; four of these were S. nigritus captured at the left bank of the Paraná River, state ofParaná, and the other was a S. cay captured at the border of Mato Grosso doSul. Only Sapajus spp. (6.7%; 5/75) individuals reacted positively; all serum samples from A. caraya were anti-HAV negative (Table 1). Additionally, all samples evaluated were negative to anti-HAV IgM antibodies. Although no significant differences were observed when the sex (r = 0.67) and the age (ρ = 0.50) of the NTPs were evaluated (Table 1), more adult NTPs (5.2%; 4/77) were anti-HAV seropositive relative to their younger counterparts (2.9%; 1/35), while the rate of anti-HAV seropositivity in males (4.5%; 3/67) was similar to that found in females (4.4%; 2/45). Additionally, no significant differences were observed based on species (ρ = 0.12) and the states where primates were captured (ρ = 0.65).

Table 1

Association between the characteristics studied (sex, age, species, and the States where primates were captured) and the presence of anti-hepatitis A virus IgG antibodies in free-ranging Neotropical primates

	ELISA	OR (95% Cl)a	p-values b
	Positive (%)	Negative (%)	Total (%)
Sex
Male	3 (4.5)	64 (95.5)	67 (59.8)	1	0.67
Female	2 (4.4)	43 (95.6)	45 (40.2)	0.16<OR<6.28
Age
Young	1 (2.9)	34 (97.1)	35 (31.3)	0.54	0.5
Adult	4 (5.2)	73 (94.8)	77 (68.8)	0.06<OR<4.99
Specie
Sapajus spp.	5 (6.7)	70 (93.3)	75 (67.0)	Not determined	0.12
Alouatta caraya	0 (0)	37 (100)	37 (33.0)	Not determined
State captured
Mato Grosso do Sul	1 (4.8)	20 (95.2)	21 (18.8)	0.92	0.65
Paraná	4 (4.4)	87 (95.6)	91 (81.3)	0.09<OR<8.67
Total	5 (4.5)	107 (95.5)	112 (100)

aOdds ratio and 95% confidence limits. bFisher exact

DISCUSSION

The results from this study have shown that 4.5% (5/107) of the serum samples derived from free-ranging NTPs were positive for anti-HAV IgG antibodies, and represent the first published investigation that evaluated the risk of NTPs being infected by HAV in Brazil. However, the results of a study that determined the occurrence of HAV in captive non-human primates (NHPs) demonstrated that 4.5% (5/125) of NHPs from the State of Riode Janeiro and 7.6% (17/233) of NHPs maintained within zoological parks of the State of São Paulo were seropositive for anti-HAV IgG24. These results suggest that captive and free-ranging NTPs from distinct geographical regions of Brazil were exposed to HAV.

Alternatively, elevated serum positivity was observed in a study performed in Thailand that evaluated the presence of HAV in 11 species of captive NHPs, where seroreactivity to HAV was demonstrated in 64.7% (11/17) of Macaques, 85.7% (6/7) in Langurs, and 94.6% (35/37) of Gibbons15. Consequently, seropositivity to HAV in captive NHPs seems to vary in different geographical locations. Additionally, SETZER24 evaluated the presence of HAV in captive and free-ranging NHPs, and demonstrated that only captive NHPs were seropositive to IgG-anti HAV antibodies. Moreover, a recent study that evaluated the presence of zoonotic disease in captive NTPs from the state of Pará, northern Brazil, demonstrated that 12% of the 29 NTPs evaluated were seropositive for anti-HAV antibodies, but without specific reaction to IgG or IgM anti-HAV antibodies16.

In conclusion, the results of this study show the circulation of HAV in free-ranging NTPs within the Paraná River basin, Brazil. However, additional studies are required to evaluate the possible role of wildlife to act as "sentinels" for the monitoring, control, and prevention of this zoonotic disease of public health importance.