Lab Manual Appendix

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab 11
Lab 12

Lab Demonstrations

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10

Introduction

   In this lab we have placed several small (but very important) groups of nematodes. With the exception of the Strongyloides spp., and a few Spirurids, these worms live outside the GI tract.

     The lungworms, as the name indicates, live in the lungs (parenchyma or bronchi/trachea) of their host. Most are members of the Superfamily Metastrongyloidea and their L1 have a "kink" in the tail. However, Dictyocaulus spp. are in the family Trichostrongyloidea and their L1 have a straight tail.

    The genus Strongyloides contains many species that are important parasites of neonatal animals. The small parasitic adult is a parthenogenic female that lives in the crypts of the small intestine. All species of this genus have one or more free-living generations. 

     The Spirurids and Filarids are nematode parasites that live in the tissues or vasculature of their hosts (with exception of some Spirurids that live in the stomach) . The Spirurids lay larvated eggs, while the Filarids females are viviparous and "give birth" to actively motile embryos called microfilariae. The Filarid life cycles involve an intermediate host (generally a blood sucking arthropod) and, as a rule, the Spirurid life cycle involves an arthropod intermediate host.

Objectives Checklist

Be able to do (and explain the theory behind) the following techniques:

9 The Knott concentration technique

9 The Filtration technique

9 An assay for heartworm antigen

Be able to identify the following:

9 The typical lungworm L1 ("kinked" tail) and be able to identify to species (by host).

9 Dictyocaulus spp. L1 from cattle (larva with straight tail in fresh feces).

9 The eggs and L1 of Strongyloides spp.

9 Distinguish between and recognize the L1 of Strongyloides stercoralis, Ancylostoma sp., and Oslerus (Filaroides) sp. from the feces of dogs and Aelurostrongylus sp. from cat feces.

9 Adult Dirofilaria immitis (by size and location in host).

9 Adult Spirocerca lupi (by size and location in host).

9 microfilaria (the pre-L1 stage of filariids).

9 a spiruid egg (all similar to S. lupi eggs).

Lab Exercises

Lungworms

Dogs: Oslerus (Filaroides) osleri

Nodules at the bifurcation of the trachea.

Adult worms removed from a nodule.

Cats: Aelurostrongylus sp.

Sheep: Muellerius capillaris

Cattle: Dictyocaulus viviparous

Adult Dictyocaulus viviparous worms in the lung of a cow.

Click here to link to the Dictyocaulus viviparus life cycle.

Pigs: Metastongylus apri

Cross section of adult Metastrongylus apri worms in the bronchi of a pig's lung.

Strongyloides Sheep: Strongyloides papillosus Horse: Strongyloides westeri Pigs: Strongyloides ransomi Dogs: Strongyloides stercoralis   Third stage larva (L3)Strongyloides stercoralis Note the long esophagus.   First stage Strongyloides stercoralis larva (L1). Note the large genital promodium. Click here to link to the Strongyloides stercoralis life cycle. Primary Diagnostic Features of Nematode Larvae Found in Canine Larvae   Ancylostoma caninum (L3) Stongyloides stercoralis (L3) sheath present absent esophagus bulbed and runs ~ 25% the length of the worm straight and runs ~ 40- 50% the length of the worm tail straight notched Top of Page

Spirurids

Physaloptera
Dogs and cats are occasionally infected with this nematode that is common in the stomachs of raccoons and opossums. Diagnosis is usually made by identifying the adult worm after it has been vomited or seen during endoscopy since the eggs may not float in a standard saturated salt flotation.

Dogs: Spirocerca lupi

Adult worms removed from an esophageal tumor of a dog.	Fibrous nodules caused by Spirocerca lupi in the wall of a dog's esophagus. Note the worms emerging from the nodule.	Cross section of the nodules shown in the previous image.   Note how the nodules occlude the lumen of the esophagus.

Spirocerca lupi Eggs measure 30 - 37 µm X 11 - 15 µm and are parallel-sided in appearance. Eggs are larvated.

Ruminants: Gongylonema sp.

Tracks made in the wall of a cow's esophagus by Gongylonema pulchrum.

Racoons (occasionally dogs and cats): Dracunculus insignis

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Filarids

A. Dirofilaria immitis (Heartworm)
These nematodes live as adults in the pulmonary arteries of dogs, cats, ferrets and seals. Microfilariae are in the peripheral circulation and are used for parasitologic diagnosis.

Adult Dirofilaria immitis worms in the heart of a dog.

Adult D. immitisworms removed from the pulmonary arteries of a dog.

	Microfilaria of Dirofilaria immitis Note the zonal staining pattern when stained with acid phosphatase method.

Click here to link to the Dirofilaria immitis life cycle.

B. Dipetalonema reconditum
This non-pathogenic nematode is transmitted by fleas and is found in the subcutaneous tissues of the dog. Its microfilariae are located in the peripheral blood and thus can confound the diagnosis of D. immitis infection based solely on presence of microfilariae per se. However, the antigen-capture serologic tests we have discussed are specific for Dirofilaria and will not cross react with Dipetalonema.

	Microfilaria of Dipetalonema reconditum Note the blunt anterior end, the bent body and the hooked tail.
	D. reconditum stained by the acid phosphatase method.  Note the general staining pattern.

Comparison of the 2 microfilaria which may be found in dog blood.	Comparison of the acid phosphatase staining patterns of the 2 microfilaria which may be found in dog blood.

C. Onchocerca sp.
In large animals adult onchocercid worms usually live in the large ligaments and microfilariae migrate through the skin.

Onchocerca cervicalis may cause a pruritic, non-seasonal dermatitis in horses.

Onchocerca volvulus causes onchocerciasis in humans, a leading cause of blindness in endemic areas.

D. Setaria equina
These nematodes are usually harmless and live in the peritoneal and pleural cavities of horses. They are transmitted by mosquitoes.

Setaria equina microfilaria. Note the sheath.
Adult Setaria equinaworms in the peritoneal cavity of a horse.

Answers to the questions in Lab 5.

    Lab5 pg 2:

1.  Direct wet mount - Finds many cases of patent (microfilariae positive) infections, but, because of the small amount of blood used, it misses light infections (or young infections) and misses the 30% of infections that are not patent. Can not determine the species of microfilariae, but allows a good guess based on movement and numbers.

2. Giemsa stained smears - uses less blood than the direct wet mount so is even less sensitive!  Again, no species ID, and many times no good guess.

3. Knott Test - Will allow you to find most patent infections (it has a sensitivity of 1 microfilaria per unit of blood (usually 1 ml) if done correctly and the entire pellet is examined).  Allows the identification of the microfilariae to species as the morphology of the microfilariae has been reported as fixed in 2% formalin. But 30% of infections are not patent.

4.  Filter test - just as sensitive as the Knott test.  However in practice it is easier to do as you only have to examine the one filter (it may take 2 or 3 slides to examine the Knott Test pellet).  Still does not allow the identification of the microfilariae as they are not fixed in 2% formalin.

Lab 5 pg 3:

The average feline heartworm infection has less than 2 worms (cats are very poor hosts).  Thus, half the infections are male only and can't be detected by the antigen test.  Also you need a mature female worm and most heartworm infections in cats do not develop as far as the mature adult.  Clinical cases of heartworm in cats are usually due to the intense response to the L4 and early L5 in the pulmonary arteries causing an asthmatic like condition know as HARD (heartworm associated respiratory disease).  The antibody test is an "exposure" test, it begins to become positive around 60 days of infection and will remain positive as long as a worm is present and then a few months after all worms have been cleared.  Thus the test does not tell you if the cat has a heartworm now, but if clinical signs are present you would move heartworm disease to the top of your differential list.

Lab 5 pg 6:

Unless you have identified the microfilaria as Dipetalonema, you must still consider Dirofilaria immitis.  Heartworm infections can be patent (have microfilariae in the blood) but antigen negative in 2 cases:   

1.  The small number of mature adult females have just started to produce microfilariae, but as yet haven't put enough antigen into the dog's blood to reach the level of detection.  This doesn't happen often, generally the antigen precedes the microfilariae or they develop at the same time. 

 2.  The adult worms have died and the antigen has been cleared from the blood, but the microfilariae have not yet died.  This will happen if the dog has been treated for adult worms but not for microfilariae or if the adult worms have died of old age (the microfilariae can live in the dog for up to 2 years after the adults have died).

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