This study compared the course of infection by Ancylostoma caninum and Ancylostoma braziliense in mixed-breed dogs infected with L3 larvae. Dogs infected with A. caninum eliminated more eggs than did those infected with A. braziliense. A total of 38 % of A. caninum and 44 % of A. braziliense larvae were recovered as adult worms. There were no marked clinical abnormalities in dogs with either infection. A. caninum was associated with anemia and an increased number of circulating neutrophils, whereas infection with A. braziliense led to a decrease in the number of leukocytes. The humoral response against excreted and secreted antigens from adult worms was more sensitive and specific than the response induced with the crude antigen. No immune response was observed for either crude or excreted-secreted (ES) antigens from larvae of either species. A nonspecific response against the crude antigen of A. braziliense was found at 0 and 7 days postinfection and maintained throughout the infection period. However, antibody titers against ES antigens were elevated in A. caninum infection at patency and death, showing that this antigen has a higher specificity. The immune response elicited by infection with A. braziliense in dogs has not been described previously. No significant differences were observed in the infection processes of the two Ancylostoma species, except for the higher number of eggs eliminated from dogs infected with A. caninum, which may indicate a better evolutionary adaptation of the parasite to its host in comparison with A. braziliense.

The objective of this randomized, blinded, placebo controlled laboratory study was to confirm the efficacy of a single oral administration of two marketed formulations of milbemycin oxime (Interceptor(®) Flavor Tabs(®) and Sentinel(®) Flavor Tabs(®)) at a minimum dose of 0.5mg/kg (0.23mg/lb) against natural infections of Ancylostoma braziliense in dogs. Thirty-six hookworm infected dogs, a minimum of 10 weeks of age and of various breeds and genders were used. Fecal egg counts were done on three separate days prior to treatment for randomization purposes. Dogs were ranked by descending order of the fecal egg count arithmetic means and randomly assigned to either the two milbemycin treatment groups or the placebo control group in blocks of three dogs each, 12 dogs per group. Dogs were dosed according to the product label with blinding maintained by separation of function. Worm counts were done at necropsy 7 days after treatment. Reduction in A. braziliense worm counts in the milbemycin groups were compared to the placebo control group using analysis of variance of the A. braziliense logarithmic mean worm counts and percent efficacy was based on geometric means. Efficacy was defined as the ability of the test products to significantly (p≤0.05) reduce parasite load by 90% or greater in treated dogs when compared to adequately infected placebo control dogs. The placebo control group had a geometric mean worm count of 19.2. The Interceptor treated group had a geometric mean worm count of 0.38 representing a 98% reduction in parasite load and the Sentinel treated group had a geometric mean worm count of 0.98 representing a 95% reduction in parasite load. Both reductions were highly significant (p<0.0001). In this study, milbemycin oxime, when administered as two marketed formulations at a minimum dose of 0.5mg/kg (0.23mg/lb), was efficacious for removing adult A. braziliense in naturally infected dogs.

Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species.Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples.The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.

Cutaneous larva migrans (CLM) is a common endemic disease in tropical and subtropical countries. This condition is caused by skin-penetrating larvae of nematodes, mainly of the hookworm Ancylostoma braziliense and other nematodes of the family Ancylostomidae. We report three cases of CLM acquired during vacations in different regions of Brazil.

We report a case of hookworm-related cutaneous larva migrans diagnosed microscopically. Viable hookworm larvae were found by microscopic examination of a skin scraping from follicular lesions. Amplification and sequencing of the internal transcribed spacer 2 allowed the specific identification of the larvae as Ancylostoma braziliense.

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).

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.

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.

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.

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.