Robert B. Burrows

Comparative morphology of Ancylostoma tubaeforme (Zeder, 1800) and Ancylostoma caninum (Ercolani, 1859).

As a result of studies of Ancylostoma from European, African, and Australian dogs and cats, it was found that dogs harbored A. caninum and cats A. tubaeforme. A detailed study of these hookworms from dogs and cats in the United States confirmed the observations made by other investigators that A. caninum is not the common species in cats. A. tubaeforme differs from A. caninum in the following features: smaller size, larger ventral and esophageal teeth, shorter esophagus, thicker cuticle, smaller bursa, different arrangement of lateral bursal rays, smaller costa dorsalis, longer spicules, and shorter, narrower female tail. The two species apparently have world-wide distribution and A. caninum is seldom or never found as a natural infection of cats or A. tubaeforme as a natural infection of dogs. In 1800 Zeder (Biocca, 1954) described hookworms he found in a cat in 1783, giving them the name Strongylus tubaeformis. Then in 1859 Ercolani (Biocca, 1954) published a description of the dog hookworm, naming it Sclerostoma caninum. Later S. caninum was transferred to the genus Ancylostoma, but S. tubaeformis apparently was overlooked by most investigators. For nearly 100 years the common hookworm of both dogs and cats was referred to as Ancylostoma caninum. A number of investigators (Scott, 1928, 1929; McCoy, 1931; Foster and Cort, 1932; and others) found that it was difficult to infect dogs with larvae from cats and cats with larvae from dogs. However, the differences were believed to be physiological rather than morphological, and the two were considered different strains of the same species. In 1954 Biocca published the results of his detailed study of Ancylostoma from dogs and cats in Italy. He found that there were two species rather than two strains of one species. In his paper he gave the original descriptions of both Zeder and Ercolani, a redescription of A. tubaeforme, and the major morphological differences between the two species. Bohm (1955) in Austria, Rohde (1959) in Germany, and Fitzsimmons (1961a, b), who studied hookworms from both African (Nyasaland) and Australian dogs and cats, agreed with the findings of Biocca. In the United States the writer has noticed in chemotherapeutic trials of several active compounds that the activity is not always the Received for publication 4 June 1962. same against Ancylostoma from dogs and those from cats. After consulting the papers by Biocca and Rohde, a detailed examination was made of hookworms obtained from dogs and cats in this country. As the morphological differences agree with those found by other investigators in Europe, Africa, and Australia, it is evident that we are dealing with two differ nt species, each of which appears to have a world-wide distribution. The major differences are described and illustrated below. MATERIALS AND METHODS Ancylostoma were obtained from 12 dogs and 9 cats used in chemotherapeutic trials in this laboratory. Numerous measurements were made from relaxed worms obtained either as a result of treatment or at autopsy. Numbers of worms were fixed; the anterior and posterior ends cut off; the middle sections discarded; and the ends dehydrated, cleared, and mounted for detailed study. The best of the numerous mounted ends were photographed to illustrate the major differences between the two species. Ancylostoma from a number of other dogs and cats were examined to determine whether natural infections of A. caninum occurred in cats or A. tubaeforme in dogs. DIFFERENTIATION OF THE TWO SPECIES (All measurements in millimeters, unless otherwise stated) Sizes of worms: Measurements of lengths and widths of 20 mature males and 20 mature females of each species gave the following ranges and averages: A. tubaeforme: males, 9.5 to 11.0 by 0.30 to 0.35 (avg. 10.3 by 0.32); females, 12.0 to 15.0 by 0.38 to 0.43 (avg. 13.2 by 0.41); A. caninum: males, 11.0 to 13.0 by

The Whipworm as a Blood Sucker

Numerous Trichuris vulpis engorged with reddish or pinkish fluid have been found at necropsies of dogs. These worms, when cut, gave strong positive reactions to the benzidine test. Of hundreds of hookworms and whipworms, washed and cut, nearly all were positive with this test. Longitudinal and transverse sections of the slender anterior end of whipworms, when stained with the dilute trichrome stain, gave unmistakable evidence of having blood cells in the esophageal lumen; and a detailed study of the anterior end of the worms indicated the means whereby whipworms can penetrate blood vessels and suck blood. Though less voracious than hookworms, whipworms are, nonetheless, avid blood suckers. Inasmuch as anemia is associated frequently with whipworm infections, a number of investi- gators have sought evidence of blood in these worms. Askanazy (1896), using a special stain- ing technique on over 60 Trichuris trichiura from 12 human cadavers, demonstrated iron- containing pigment in the intestinal epithelial cells, but not in other organs. One whipworm had a doubtful positive; all others were def- initely positive. In addition, he reported a binucleate leukocyte in the worm esophagus in one section. Guiart and Garin (1909) and Garin (1913) obtained positive Weber reac- tions for blood in feces of Trichuris-infected patients, reported blood-engorged whipworms

The anthelmintic effect of bephenium on Ancylostoma caninum.

A new series of anthelmintics, designated by the generic name, bephenium, has shown activity against a large number of intestinal nematodes of laboratory and domestic animals. These compounds appear to be more active against nematodes attached to the mucosa than to those living free within the lumen (Copp et al, 1958). One of the compounds, bephenium embonate, was found effective against larval and adult forms of Nematodirus in lambs (Rawes and Scarnell, 1958). Bephenium chloride has the following formula:

Trematodes of New Jersey dogs and cats.

During the period from 1959 to 1964 the following nine species of trematodes were found in New Jersey cats and dogs: Eurytrema procyonis, Parametorchis complexus, Phagicola longus, Cryptocotyle concava, C. lingua, Echinochasmus schwartzi, Paragonimus kellicotti, Alaria canis, and A. marcianae. E. schwartzi, and A. marcianae are redescribed. Trematodes are reported relatively infrequently from dogs and cats in North America, whereas nematode and cestode infections are rather common. During the period from 1959 to 1964 we found 23 fluke infections in animals examined in our laboratory. We probably missed others, because we used only the zinc sulfate flotation technique in stool examinations in our search for nematode infections, and operculated eggs fail to float in many cases. The dogs and cats came from central New Jersey, particularly Monmouth and Burlington counties. We cannot determine the exact locality of any specific animal, for the dealer who supplies us collects strays throughout the area. Living flukes from these animals were relaxed in menthol solution prior to fixation, with the result that relatively few were distorted. Permanent preparations of a number of these flukes were deposited in the USNM Helm. Coll., Beltsville, Maryland, and their accession numbers are given below. The dates after the accession numbers refer to the dates the flukes

Stilbazium iodide as an anthelmintic. II. Against various nematodes of dogs and cats.

Stilbazium iodide, which was effective against the mouse pinworm Syphacia obvelata, was tested against three species of hookworms, three of roundworms, and one of whipworm in 120 dogs and cats. Two or three 25 mg base/kg doses removed most Ancylostoma caninum, A. tubaeforme, Uncinaria stenocephala, Toxocara canis, and T. cati, but Toxascaris leonina was more resistant. The drug is active against Trichuris vulpis in dogs when it is given in courses of 3 to 6 doses of 25 mg base/kg or higher. Two or more courses may be necessary for puppies and for dogs having narrow cecal openings or extensively coiled ceca. Except for five dogs with atypical ceca, 24 of 29 dogs given these multiple doses averaged over 90% elimination of whipworms, with 18 of the 24 having complete clearance. Vomiting is negligible in these animals provided the dose is less than 50 mg base/kg. This compound is in human clinical trial against hookworms, ascaris, whipworms, and pinworms. Some of the pyridines found active against the mouse pinworm Syphacia obvelata were tested against various nematodes of dogs and cats and were found effective against a number of species (Phillips and Burrows, 1961; Burrows, Hunt, and Lillis, 1961). The most active against Syphacia, stilbazium iodide, is relatively nontoxic (Burrows and Hunt, 1962) and it was found also to be the best against dog and cat nematodes. A series of trials was set up to test the effectiveness of stilbazium against three species of hookworms, three of roundworms, and one of whipworm in these animals. Several different dosage levels were used in these experiments in an endeavor to find the optimum dosage for the various species. MATERIALS AND METHODS From stool examinations done on all stray dogs and cats and from registered beagle puppies received by this laboratory, those animals showing moderate to large numbers of eggs of one or more species of nematodes in zinc sulfate flotation preparations were selected for trial. One hundred and twenty animals (55 dogs, 32 puppies, and 33 cats) were used in the trials. There were 207 infections (106 in the dogs, 56 in the puppies, and 45 in the cats) in these animals against which stilbazium was tested. Dogs and puppies selected for treatment were switched from a Gaines meal diet to a canned dog food diet the day prior to treatment as it is less time-consuming to search for passed worms in the canned food debris. Cats were kept on canned cat food before and during treatment. The compound was administered orally in gelatin Received for publication 1 August 1962. capsules according to mg of base per kg of body weight. As the iodide salt makes up nearly onefourth of the weight of the compound, it was necessary, for example, to give 32 mg of the compound per kg in order to have 25 mg of base per kg. Animals given a single dose in a day received the drug between 0830 and 0930 and were fed between 1230 and 1330. Those given 2 doses a day had the second dose at about 1600. All fecal specimens were collected from all animals after treatment began, and this was continued until the animals were autopsied. Those given a single dose were autopsied after 48 hr; those dosed for 2 or more days were autopsied 48 hr after the last dose, except that those treated for whipworm infection were followed for 7 days after the initial dose. Animals were killed with a lethal dose of Nembutal given in the heart and were autopsied i mediately. Fecal material was washed through a 40-mesh sieve and the sieve contents examined, a little at a time, in a black-bottomed pan for worms passed. At autopsy the stomach and intestines were examined carefully for worms remaining. The contents of the large intestine were considered part of the last fecal specimen, except for living whipworms still attached to the cecum or large intestine. The percentage of elimination of worms was computed for each species of helminth of each animal by dividing the number passed by the number harbored. The average percentage of elimination of a species, to the nearest whole digit, was found by averaging the percentages of all animals harboring that species and having been given the same dosage level. It was not possible to autopsy six of the dogs or the 32 puppies used in these experiments, but the percentage of elimination was determined from the averages of three preand three post-treatment Stoll egg counts, the former being done the week prior to treatment and the latter beginning 7 or 8 days after the initial dose.

Eurytrema procyonis Denton, 1942 (Trematoda: Dicrocoeliidae), from the domestic cat.

Eurytrema procyonis was described by Denton (1942) from racoons in Texas. Stunkard (1947) found a species in a red fox from New York State which he (lescribed and named Eurytrema vulpis. Later, on comparing his specimens with some received from Denton, Stunkard concluded that they were of the same species and that E. vulpis is a synonym of E. procyonis (Stunkard and Goss, 1950). Since then E. procyonis has been found in a racoon in Connecticut (Penner, et al, 1954) and in racoons and gray foxes in Maryland (Herman, et al, 1957). On July 31, 1959, a stray cat from New Jersey was examined in this Laboratory for intestinal helminths and dicrocoeliid-type eggs were found in the feces. At autopsy 67 small flukes were found in the bile ducts and gall bladder and over 300 were tightly packed in the pancreatic ducts. (Another stray cat from New Jersey taken on February 29, 1960, was found at autopsy to have 28 Eurytrema procyonis in the distal portion of the pancreatic duct.)

An abnormal proglottid of Taenia saginata.

AMEEL, D. J. 1942 Two larval cestodes from the muskrat. Tr. Am. Micro. Soc. 61: 267-271. BAYLIS, H. A. 1935 Some parasitic worms from muskrats in Great Britain. Ann. and Mag. of Nat. Hist. 15: 543-549. JOYEUX, C. E. and BAER, J. G. 1936 Faune de France Cestodes, Fed. Franc. Soc. Sc. Nat. 30, 613 pp. RAUSCH, R. 1952 Studies on the helminth fauna of Alaska XI. Helminth parasites of microtine rodents-taxonomic considerations. J. Parasit. 38: 415-444. RUDOLPHI, C. A. 1819 Entozoorum synopsis cui accedunt mantissa duplex et indices locupletissimi. X + 811 pp., Berolini. SKINKER, M. S. 1935 A redescription of Taenia tenuicollis Rudolphi, 1819, and its larva, Cysticercus talpae Rudolphi, 1819. Parasitology 27: 175-185. WARDLE, R. A. AND MCLEOD, J. A. 1952 The zoology of tapeworms. 780 pp., The Univ. Minn. Press, Minneapolis. WARWICK, T. 1936 Parasites of the muskrat (Ondatra zibethica L.) in the British Isles. Parasitology 28: 395-402.

A new dihydrotriazine effective against Syphacia obvelata in mice.

Various piperazine compounds show excellent activity against Syphacia obvelata in mice (Mouriquand, et al, 1951; Brown, et al, 1954) and Brown and his coworkers have found that, in general, compounds active against Syphacia were also active against the human pinworm, Enterobius vermicularis. As the piperazines are more effective against mature pinworms than against immature forms, it is necessary to give repeated doses in order to eliminate the worms as they mature. Several years ago a number of dihydrotriazines were given us for anthelmintic screening and were found active against Syphacia. Since then Roth (Roth, et al, 1960) has prepared a large number of these compounds, all of which showed some pinworm activity in single doses. One compound, B.W. 58-232, was found to be among the most active and, at the same time, the least toxic of the series. The oral LD50 of this compound was determined by Mr. Rocco Fanelli, of this Laboratory, who found that 20 g per kg of body weight failed to kill any of the mice receiving this dose.

Stilbazium iodide in the mass treatment of mice infected with Syphacia obvelata.

Since stilbazium iodide is an effective drug in minute amounts for the elimination of the mouse pinworm, Syphacia obvelata, but is relatively insoluble, a series of tests was made of its activity when mixed with food. Even with the drug as low as 25 ppm of food, it was so effective during a 2-day trial that a plan for mass treatment of mouse colonies is proposed. On several occasions we have received requests for a method of eliminating the mouse pinworm, Syphacia obvelata, from mouse colonies used in special types of research. We recommended that piperazine citrate be dissolved in drinking water and given to the mice for some time. This drug is more effective against the adult than against the immature worms, and by using it in the water over a period of weeks, the new infections are killed off as the worms reach the right stage. The discovery that stilbazium iodide [2,6Bis(p-pyrrolidinostyryl) pyridine ethiodide] or Monopar? is more active than piperazine citrate, that much smaller doses are required, and that it is equally effective against the mature and immature forms (Burrows and Hunt, 1962), suggested its use in mass treatment of mouse colonies. Since the drug is almost insoluble in water, it has to be given in food. MATERIALS AND METHODS Four levels of drug in food were selected for trial: 25, 50, 100, and 150 mg drug/kilo of Purina lab chow. Food without drug was given to the controls. Because the active principle (drug base) is 78.1% of the drug, the actual effective amounts were 19.5, 39.1, 78.1, and 117.2 mg base/kilo of lab chow. Each batch of drug-food mixture was prepared by thoroughly mixing the two while dry; adding water while mixing until the dough was moist enough not to crumble; preparing a cake of the mixture and cutting it into blocks about 3 to 4 cm long by about 1.5 to 2.0 cm wide and high; drying these blocks in an oven at 60 C for 18 to 24 hr, turning them several times for uniform drying; and storing in containers until needed. Mice used in these experiments were male Ha/ ICR Swiss (from Millerton Research Farms), averReceived for publication 26 June 1963. aging about 25 to 30 g in weight, and all had natural infections of Syphacia obvelata. During the course of the experiment each mouse was housed in a small, individual, wire-bottomed cage which contained a feeding rack designed to prevent food particles from falling out of the rack, and a water bottle. Beneath each cage was a pan of water to collect worms and feces. At the start of the experiment each mouse was switched from the regular Purina dog chow to one of the prepared mixtures. At the end of 24 hr the pan was removed from under the cage and another pan of water substituted. All worms in the pan were counted. At the end of 48 hr the mouse was necropsied, all worms remaining alive in the cecum and large intestine were counted, any dead or disintegrated worms were added to the second 24-hr pan, the worms in the second 24-hr pan were counted, and the percentage of elimination for the mouse was computed by dividing the total number passed or killed in the intestine by the total number harbored. The average percentage of elimination for a group (i.e., all mice given the same dosage level) was determined by averaging all percentages obtained for the mice in that group. The food containers, as well as the mice, were weighed before and at the end of the experiment, so that the amount of drug taken in and the amount of drug/kilo of body weight could be computed. As the mouse weight changed somewhat during the 48 hr of the experiment, the average of the two weights was used to compute the number of mg base/kg body weight.

Stilbazium iodide as an anthelmintic. I. Against Syphacia obvelata in mice.

As the result of comparative trials of two active pyridines, each at 6 dosage levels, one was selected on the basis of greater activity and lesser toxicity. With the better compound all Syphacia obvelata were eliminated from mice at 10 mg base/kg or higher and over 90% at levels down to 2.5 mg base/kg. The selected compound with the generic name stilbazium iodide is relatively nontoxic, having a lethal level over 100 times the curative level. This compound is now in clinical trial against the human pinworm Enterobius vermicularis. A number of pyridines have been found active against the mouse pinworm Syphacia obvelata (Phillips and Burrows, 1961; Burrows, Hunt, and Lillis, 1961). Of the more than 20 compounds with good to excellent activity at 20 mg/kg or less, two produced superior and somewhat comparable activity. The two compounds have the following basic structure: R -CH= CH\ 7 CH = CH R I N