Use of filter papers to determine seroprevalence of Toxoplasma gondii among hunted ungulates in remote Peruvian Amazon.

Toxoplasmosis is a zoonosis caused by the protozoan Toxoplasma gondii, and it is found worldwide. To determine whether ungulates are reservoirs of T. gondii in an isolated and remote region of the northeastern Peruvian Amazon, antibodies to T. gondii were determined in 5 species of ungulates by the modified agglutination test (MAT). These animals were hunted by subsistence hunters along the Yavarí-Mirín River, in the northeastern Peruvian Amazon. Blood samples were collected by hunters on filter papers. For determination of T. gondii antibodies, blood was eluted from filter papers, and a titer of 1:25 was considered indicative of exposure to T. gondii. Antibodies to T. gondii were found in 26 (31.0%) peccaries (Pecari tajacu, Tayassu pecari), six (17.1%) brocket deer (Mazama americana, Mazama gouazoubira), and four (40.0%) lowland tapir (Tapirus terrestris). We also introduced a modification to the MAT protocol that allows the extraction of fluid samples from several types of laboratory-grade filter paper, thus enabling researchers to easily adapt their approaches to the materials presented to them.

Prevalence of Ancylostoma braziliense in dogs from Alachua and Marion Counties, Florida, United States.

A convenience collection of fecal samples from 148 dogs in northern Florida was examined for the presence of Ancylostoma braziliense eggs by using centrifugal sugar flotation and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Of the 148 samples, 64 (43.2%) contained hookworm eggs. DNA from 42 samples was successfully amplified using PCR; using RFLP, 2 samples were identified as containing DNA of A. braziliense (4.8% of the 42 successfully amplified samples).

Prevalence of Ancylostoma braziliense in cats in three northern counties of Florida, United States.

A convenience sampling of fecal specimens from 40 cats in northern Florida was examined for the presence of Ancylostoma braziliense eggs by using centrifugal sugar flotation and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Of the 40 samples, 26 (65%) contained hookworm eggs. DNA from 24 samples was successfully amplified using PCR; using RFLP, 10 samples were identified as containing DNA of A. braziliense (41.7% of the 24 samples that successfully amplified). Of these, 6 samples contained DNA of both Ancylostoma tubaeforme and A. braziliense, and 4 samples contained only DNA of A. braziliense. The remaining samples (n  =  14) contained only the DNA of A. tubaeforme, except for 1 sample that had no discernible bands after RFLP.

Obtaining an isolate of Ancylostoma braziliense from dogs without the need for necropsy.

Isolation of a specific Ancylostoma species typically requires death of the source animal, or holding an animal long enough to collect feces after treatment, for worm recovery and identification. The reason for collecting worms is that the eggs are not easy to distinguish morphologically. In keeping with the 3 Rs of laboratory animal research (reduction, refinement, replacement), the objective of this study was to obtain an isolate of Ancylostoma braziliense from 1-time field-collected samples of canine feces without the need for killing the host. During a collection trip to Florida, fecal samples (n  =  148) were collected and identified as containing eggs of Ancylostoma species (n  =  64) using centrifugal sugar flotation. Eggs from hookworm-positive slides were washed into tubes, DNA was extracted, and 2 samples were identified as A. braziliense using restriction fragment length polymorphism (RFLP) with Hinf1. Larval cultures were initiated from these samples, and larvae from the cultures were returned to New York and used to inoculate a purpose-bred kitten with the goal of inhibiting the growth of any contaminating Ancylostoma caninum that might be present in the culture. The infection was patent at 15 days, and eggs were identified as A. braziliense by RFLP and DNA sequencing. Using forceps during endoscopy, 2 adult worms (1 male, 1 female) were recovered from the cat and identified morphologically as A. braziliense . Larvae were cultured from the feces of this cat and used to infect a laboratory-reared beagle dog. Additionally, worms recovered from the feces of the cat post-treatment were confirmed to be A. braziliense , except for 1 female A. caninum containing infertile eggs. The dog (patent 14 days post-infection) was also infected with A. braziliense as determined by RFLP and DNA sequencing of eggs and cultured larvae. Both the cat and dog were treated, verified to be no longer shedding eggs, and then placed into adoptive homes.

Obtaining an isolate of Ancylostoma braziliense from cats without the need for necropsy.

Because the eggs of Ancylostoma species of dogs and cats are difficult to readily distinguish morphologically, isolation of a certain species often requires the humane death of the source animal or holding an animal after treatment to obtain worms for specific identification or to harvest ex utero eggs. The objective of this study was to obtain an isolate of Ancylostoma braziliense from 1-time, field-collected samples of feline feces without the need for the killing of any animals. During a collection trip to Florida, fecal samples (n  =  40) were collected and identified as containing A. braziliense eggs (n  =  26) using centrifugal sugar flotation. Eggs from hookworm-positive slides were washed into tubes, DNA was extracted, and 10 samples were identified as containing A. braziliense using restriction fragment length polymorphism (RFLP) with Hinf1. Six of these samples also contained DNA of Ancylostoma tubaeforme and, thus, only 4 samples were from cats infected only with A. braziliense. Larvae cultured from two of the latter samples were used to subcutaneously inoculate a purpose-bred puppy with the intention to inhibit the growth of any potentially contaminating A. tubaeforme larvae in the culture. The infection was patent at 14 days after inoculation, and the eggs were identified as A. braziliense by RFLP and DNA sequencing. Larvae were cultured from the feces of this dog and used to infect a laboratory-reared, specific-pathogen-free cat; the eggs and larvae produced by the cat were also identified molecularly as those of A. braziliense. The larvae from this cat were used to infect other cats to maintain the isolate for further research. Both the puppy and the first cat used in this study were treated to clear their infections and have since been adopted by new owners.

Morphological differentiation of eggs of Ancylostoma caninum, Ancylostoma tubaeforme, and Ancylostoma braziliense from dogs and cats in the United States.

The establishment of cat- and dog-derived laboratory strains of Ancylostoma braziliense allowed for a morphological comparison of the eggs of A. braziliense, Ancylostoma caninum, and Ancylostoma tubaeforme. The length, width, and perimeter were determined for images of 10 eggs each of A. braziliense from the feces of a dog infected with a canine isolate and a cat infected with a feline isolate, A. caninum from dog feces, and A. tubaeforme from cat feces. The specific identity of the eggs was verified by polymerase chain reaction and restriction fragment length polymorphism by using HinfI and RsaI restriction digests followed by gel electrophoresis and sequencing. The mean (±SD) length, width, and perimeter and the length-to-width ratio (±SD) (all measurements are in micrometers) for the eggs of each species were as follows: A. braziliense eggs (combined cat and dog source), 53.03 ± 2.33, 36.37 ± 1.35, 140.43 ± 2.56, and 1.46 ± 0.11; A. caninum eggs, 63.92 ± 5.28, 39.21 ± 1.52, 161.99 ± 9.30, and 1.63 ± 0.13; and A. tubaeforme eggs, 61.44 ± 3.05, 39.14 ± 1.40, 157.98 ± 5.81, and 1.57 ± 0.08. The eggs of A. braziliense were significantly (P < 0.001) smaller than the eggs of A. caninum and A. tubaeforme in all dimensions. Thus, the eggs seem to be readily distinguishable using light microscopy, thereby aiding in species identification in fecal samples for a more comprehensive clinical picture and assessment of zoonotic risk.