Collection of Specimens

From March 2002 through August 2003, bats were collected from 8 provinces throughout central, eastern, and southern Thailand (Figure 1). Sites were chosen on the basis of local reports of known bat colonies or after investigation by the Royal Department of Forestry, Ministry of Agriculture. Insectivorous bats in caves were captured during the day by using fine-mesh, long-handled butterfly nets. Larger fruit bats were captured with nets near sunset, as the bats flew for foraging activities, or before dawn when returning to their roosts (Figure 2). Thick leather gloves were worn when bats were handled and transferred into individual cotton pouches for transportation and processing.

Of the 932 bats collected, all were identified to 11 different species of both insectivorous and frugivorous bats (Table 1). Forty percent were female. All bats appeared healthy. At least 110 bat species (>20 million) are believed to be present in Thailand, according to estimates from a Royal Department of Forestry survey in 2003. Eighty five percent are insectivorous; the rest are frugivorous.

Bats were anesthetized by administering a 0.2- to 0.5-mg intramuscular injection of ketamine hydrochloride. Animals were identified to sex and by species, based on gross morphology, as described (9). Animals were marked by hair or claw clipping. Blood, obtained from wing veins or by direct cardiac puncture, was transferred from the collecting syringe into 1.5-mL microtubes (Axygen Scientific, Union City, CA, USA) and stored in an icebox until centrifugation. Serum was frozen at –20°C during transportation and stored in a freezer at –70C. After recovery from sedation, bats were allowed to fly to their roosts. Sixteen of 932 died during the capture process. Inspection of the capture sites 1–2 months later included an assessment of whether the local ecology was disturbed. No additional bats died after the procedure, according to residents living near roosts.

Serologic Testing for Neutralizing Antibodies

Serum specimens were obtained from blood samples after clotting. In general, 394 samples from 4 different species were of sufficient volume and quality (Table 2). The samples originated from Chonburi (n = 167), Ayuttaya (n = 105), Chachoengsao (n = 36), Singburi (n = 81), and Surattani (n = 5). For adequate volume during testing, they were diluted 1:5 in Eagle’s minimum essential medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2% fetal bovine serum (Invitrogen). Serum samples were heat-inactivated for 30 min at 56°C before testing.

Initially, all 394 samples were screened in a modified rapid fluorescent focus inhibition test (6) against rabies virus (RABV, strain CVS-11) and ABLV (pteropid subtype; 40 50% tissue culture infective dose), with World Health Organization standard serum as a source for positive control antibody with 50% endpoint dilution of 1 IU = 1:20. Approximately 50 μL of diluted serum at 1:5, 1:10, and 1:20 dilutions was incubated with 50 μL of ABLV in 96-well microtiter plates for 90 min at 37°C in a CO₂ incubator. Murine neuroblastoma cells (50 μL) were added to each serum-virus mixture, which was incubated for 20 h. Culture medium was removed after incubation, and the plates were fixed with 90% acetone, air-dried, and then stained with fluorescein isothiocyanate–conjugated anti-rabies monoclonal antibodies (Fujirebio Diagnostic, Inc, Malvern, PA, USA). Samples were considered positive if the number of fluorescent foci was reduced by 50% at the 1:5 dilution.

Those samples that demonstrated positive or suspicious activity were additionally tested against a broader panel of other lyssaviruses, including Aravan, Khujand, and Irkut virus isolates. Twofold serum dilutions, from 1:25 to 1:100, were tested, and virus doses varied from 32 to 100 infectious units. These reactions were performed by using drops of cell culture medium on 4-well (6-mm) Teflon-coated glass slides (Cell-line/Erie Scientific Co., Portsmouth, NH, USA), incubated in a moist chamber for 48 h.

Direct Fluorescent Antibody (DFA) and Mouse Inoculation (MI) Testing of Brains

Brains from 16 dead bats (2 P. lylei and 14 P. hypomelanus) were collected in iceboxes at the capture sites for transportation and then were stored at –70°C until testing. Each brain was tested for lyssavirus antigen by DFA. Multiple impressions were prepared, and slides were fixed in acetone, allowed to dry at room temperature, and stained with commercial fluorescein isothiocyanate–conjugated anti-rabies monoclonal antibodies (Fujirebio Diagnostic, Inc). These brain impressions were examined with a fluorescent microscope.

For MI testing, pooled 20% brain suspensions from all 16 bats were prepared by mixing ≈0.5 g of each bat brain in 32 mL of normal saline solution. No antimicrobial preparations were added. The mixture was left to sediment at room temperature for 30 min, and the supernatant was used to inject into the brains of 1-month-old Swiss albino strain mice. Approximately 0.03 mL of each suspension was injected into each of 30 mouse brains. They were kept in 6 glass jars (5 in each) with a diameter of 15 cm and were observed for 60 days.

Serologic Testing

All 394 serum samples were negative against RABV, but 16 (4%) were positive or suggestive of ABLV (Table 2). Further tests of these samples demonstrated neutralizing activity against Aravan, Khujand, or Irkut viruses or ABLV (Table 3). These 16 samples originated from 2 species, P. lylei (n = 15) and Eonycteris spelaea (n = 1), collected at Chonburi (n = 9), Singburi (n = 4), Ayuttaya (n = 2), and Chachoengsao (n =1) Provinces (Table 2).

Chonburi is adjacent to Chachoengsao Province in the east, whereas Singburi and Ayuttaya are both located in the central part of the country (Figure 1). Approximately 5% of positive bat serum specimens were found in 2 eastern provinces (Chonburi, 9/158 and Chachoengsao, 1/36) versus 3% in 2 central provinces (Singburi, 4/81, and Ayuttaya, 2/105). Antibody-positive bats were dispersed throughout the collection period (March 2002 through August 2003). Most (15 of 16) positive samples came from P. lylei. One of 45 E. spelaea (versus 15 of 335 P. lylei) tested positive.

DFA and MI Testing

Sixteen bat brains tested by DFA had no detectable lyssavirus antigen. After intracerebral injection, 4 of 30 mice died, on days 11, 12, 14, and 21, respectively. None of these 4 brains tested positive with DFA for evidence of lyssavirus antigens.