Knott's Technique Research Articles

New insights onto cardiopulmonary nematodes of dogs in Sardinia, Italy

Dog heartworms Angiostrongylus vasorum and Dirofilaria immitis cause severe parasitological diseases; the importance of these parasitosis is growing due to their health impact on animals, the possible zoonotic implications and the recent spreading across several European countries and previously non-endemic areas. The aim of this study is to update the epidemiological scenario of cardiopulmonary nematodes A. vasorum and D. immitis in dogs of Sardinia island and to perform a morphological identification of larvae by the use of the Baermann and Knott techniques respectively and the molecular characterization of the mitochondrial cytochrome c oxidase subunit I (cox1) and the second ribosomal transcribed spacer region (ITS-2) of larvae L1 of A. vasorum. In the present study, 3.4% (5/146) of dogs resulted positive at Baermann technique for A. vasorum while 8.9% (61/684) to D. immitis. If on one side A. vasorum can be considered an emerging parasite in Sardinia, the parasitic pressure and the risk of infection for D. immitis in the island seems to be increased compared with the recent past.

Prevalence of canine heartworm (Dirofilaria immitis) disease in dogs of central Portugal

The aim of the present study was to determine the prevalence and risk factors concerning Dirofilaria immitis infection in dogs from Figueira da Foz, located in the central region of Portugal. In the period between November 2009 and January 2011, 304 blood samples were obtained from dogs over 1 year of age, with no previous history of heartworm prevention or diagnosis. Every blood sample was analyzed using varied laboratory techniques (direct microscopic evaluation of a fresh blood sample, the modified Knott technique, and the ELISA antigen detection test – IDEXX Snapp®). In the samples in which microfilaremia was detected, a histochemical technique using acid phosphatase staining was applied to identify the species of microfilariae. A total prevalence of 27.3% (83 out of 304) was found. We also found that 73.5% of all positive cases (61 out of 83) were microfilaremic, and 26.5% were occult infections (22 out of 83). By means of a histochemical technique Dirofilaria immitis was identified in 96.7% of microfilaremic samples. A multivariate model allowed us to identify the following risk factors for the presence of heartworm disease: age between 4 and 9 years, dogs living in a rural environment, large breed dogs, and living outdoors. This study shows for the first time the high prevalence of heartworm disease in a central area of Portugal and emphasizes the importance of systematic screening for this disease, as well as the need to prevent it in dogs in this area.

DIROFILARIOSIS CANINA EN LOS DISTRITOS COLINDANTES CON LA RIBERA DEL RÍO LURÍN

Heartworm is a parasitic disease caused by Dirofilaria immitis. The adult form of the parasite can be found in the right heart and pulmonary arteries, especially in canines. The objective of the study was to quantify the presence of D. immitis in districts located along the Lurín river (Cieneguilla, Pachacamac and Lurín). Blood samples were collected from 177 dogs (males and females) from January till July 2000. The age of the animals varied from 1 to 10 years. Three methods were conducted for diagnosing Dirofilaria: the microhaematocrit and the modified Knott techniques were used for the identification of microfilarias, and ELISA for the determination of secretion / excretion antigens of mature forms. Cieneguilla was the district with the highest frequency of heartworms. 6.4% of positive samples were obtained by the microhaematocrit and modified Knott techniques whereas 12.8% were obtained by ELISA. In Lurín was observed 2.5% using the microhaematocrit and modified Knott techniques whereas 7.5% by ELISA. In Pachacamac was only observed 2% of positive samples by the ELISA technique. In conclusion, the estimated prevalence in the studied area was 7.3% ± 3.8 CI when ELISA was used and 2.8 % ± 2.4 CI when microhaematocrit or the modified Knott techniques were used. The relative low prevalence of the disease in the area does not constitute a serious parasitic problem, however, proper monitoring should be considered.

DIAGNOSTIC STUDY ON MICROFILARIAE AND SOME BLOOD PROTOZOA IN QUAIL BIRDS (COTURNIX COTURNIX JAPONICA) IN NINEVEH GOVERNORATE

The study covered a diagnosis for detection of microfliaria in 90 blood samples obtained from quail birds. The results recorded that total infestion rate was (15.6%). Three species of microfliaria have been diagnosed in quail. Form A of detected larvae measured 106 – 125 µm in length and 2.5 –3.0 µm in width, while form B measured 97–145.4 µm in length and 4.0 – 6.0 µm in width , finally form C dimensions measured 120–150 µm and 4.5 – 5.0 µm in width respectively. Knott technique detected most positive cases with higher sensitivity rate 85.7%, than other techniques used in the study. The rate of infection recorded in adult birds 14.5% was little higher than it in young birds 10.7% with no significant statitiscal defferences between males and females. The study showed appearance of 7.7% of mixed infections of microfliaria with Aegyptianella spp. and 4.4% with plasmodium spp. Finally the study pointed that microfliaria appeared with high percentage in circulating blood than peripheral blood of 40 quail birds, as15% and 10%in circulating and peripheral blood respectively.

Identificación morfológica y molecular de filarías caninas en una comuna semi-rural de la Región Metropolitana, Chile

To date, there has been no definitive confirmation of the presence of zoonotic dirofilariasis in dogs in Chile. To study the presence of dirofilarias in blood samples from dogs collected in a semi-rural district near Santiago and to compare their frequency in dogs with and without dermatological manifestations. We examined 100 blood samples for dog filariae infections using microscopic methods (modified Knott technique). 50 dogs presented dermatological symptoms or signs compatible with filarial infections and 50 were asymptomatic. ITS-2 and 12s rDNA gene amplification by PCR and sequencing were performed in samples microscopically positive for microfilariae. Results. We observed microfilariae in 22 dogs (22%). Of these, 16/50 (32%) were symptomatic and 6/50 (12%) were asymptomatic (p = 0.02). Morphologically, the majority of micro-filariae were similar to Dirofilaria repens, although many had a bigger size than previously described. Nucleotide sequencing of the amplified genes showed no more than 95% homology with the D. repens sequences available for comparison. D. reconditum and D. dracunculoides infections were also identified. These features might indicate the presence of new species of Dirofilaria or a D. repens close related variant in Chile.

Diagnóstico diferencial de filariasis importada mediante técnicas moleculares (2006-2009)

The last few years has seen an increase in the number of immigrants and travellers from endemic areas where filariasis are mainly caused by Loa loa (L. loa), Mansonella perstans (M. perstans) and Wuchereria bancrofti (W. bancrofti) species. These demographic changes has led to the need for better filariae species-specific molecular diagnostic tests to solve problems, as alternatives to the more time consuming classic parasitology methods. Thus, the objective of the present work was the implementation of optimised molecular protocols (nested-PCR and ITS1-RFLP) developed in our laboratory, for the differential diagnosis of filarial parasites. The results obtained were compared with those obtained using the conventional parasitological methods. A total of 523 samples (517 peripheral blood, 5 adult worms and one vitreous body) were sent to Parasitology Department of the National Microbiology Centre, Carlos II Research Institute (ISCIII), from 47 Health Centres in the Autonomous Regions of Spain, from 2006 to 2009. The samples were studied by the Knott technique, nested-PCR and ITS1-RFLP. The molecular techniques applied on blood samples showed to be more sensitive that Knott's concentration technique in the diagnosis of both L. loa (n=12 versus n=4) and M. perstans (n=57 versus n=25) infections. The nested-PCR and ITS1-RFLP are potential diagnostic tools for daily routine laboratory species-specific and sensitive detection of L. loa and M. perstans filarial species in immigrant population and travellers from endemic areas where these filarial species are co-endemic. Knott's concentration technique was less sensitive than molecular methods and should be carried out as a complementary diagnostic assay.

Prevalence of Dirofilaria immitis in dogs from Hatay province, Turkey

The present study was conducted to determine the prevalence of canine dirofilariosis in Hatay province, south of Turkey. A total of 269 blood samples were collected from owned dogs in this multi-centre survey between March and July 2006. Blood samples were examined by modified Knott and enzyme-linked immunosorbent assay (ELISA) techniques to detect circulating microfilariae and antigens of Dirofilaria immitis, respectively. Seventy out of 269 dogs (26.0%) were positive for D. immitis. However, 61.4% of positive dogs had occult infection. The prevalence of canine dirofilariosis was heterogeneous in Hatay province, with higher values in shoreline (33.0%) and riverside (30.9%) areas followed by the lowland (25.5%) or mountainous (15.2%) areas. No statistically significant differences were observed in relation to sex (females, 33.3%; males, 24.4%, P>0.05). When evaluating the prevalence of D. immitis by age, the highest prevalence was observed in dogs older than 4 years of age. Large breeds (29.6%) and the dogs living outdoors (30.2%) showed a higher prevalence in comparison to small breeds (21.4%) and the dogs living indoors (10.5%), respectively. In conclusion, according to the results of this study canine dirofilariosis had a high prevalence in Hatay province. Therefore, prophylaxis against heartworm is advisable to decrease the incidence of canine dirofilariosis.

GIS modeling for canine dirofilariosis risk assessment in central Italy

A survey was conducted in an area of central Italy in order to study the prevalence of Dirofilaria immitis and D. repens in dogs. Blood samples were collected from 283 dogs and examined using a modified Knott's technique. In addition, in order to detect D. immitis occult infection, 203 serum samples were also analysed for D. immitis antigen detection. The results were analyzed in order to evaluate the behavioural and attitudinal risk factors. A geographical information system (GIS) for the study area was constructed, utilizing the following data layers: administrative boundaries, elevation, temperature, rainfall and humidity. Microfilariae were detected in 32 of the 283 dogs surveyed, constituting a total Dirofilaria prevalence of 11.3%. In particular, 20 dogs (7.1%) were positive for D. immitis and 12 dogs (4.2%) for D. repens microfilariae. One case of D. immitis occult infection was also detected. Choroplethic municipal maps were drawn within the GIS in order to display the distribution of each Dirofilaria species in the study area. Statistical analysis showed a significant association between Dirofilaria infection and animal attitude (hunting/truffle dogs showed a higher prevalence compared to guard/pet dogs). A higher prevalence was also recorded in 2 to 5-years old dogs. Furthermore a GIS-based modelling of climatic data, collected from 5 meteorological stations in the study area, was performed to estimate the yearly number of D. immitis generations in the mosquito vector. The results of the model as depicted by GIS analysis was highly concordant with the territorial distribution of positive dogs and showed that D. immitis spreading is markedly influenced by season. The potential transmission period in the study area was found to be confined to summer months with a peak in July and August, as expected for a temperate region where summer season is the most favourable period for the parasite.

Prevalence of Setaria equine microfilaraemia in horses in Hungary

Peripheral blood samples were collected randomly from 195 horses in various parts of Hungary, and the presence of microfilariae was evaluated by the Knott technique. On the basis of morphological identification 18 of the horses (9.2 per cent) were infected with Setaria equina, and the infection was confirmed in 10 animals by pcr and sequencing. The level of microfilaraemia was between 1 and 1138 larvae in 2 ml of blood. There was no correlation between the time of sampling or the sex of the animals (stallions versus mares) and the prevalence of infection, but the prevalence decreased with age. There was a significant association between the prevalence of microfilaraemia and the presence of still waters; positive samples were collected either in the region of Lake Balaton, the largest lake in the country, or at places with nearby ponds.

Filarial Worm Infections in Shelter Dogs and Cats from Northwestern South Carolina, U.S.A

During 1991–1992 and 1999–2000, domestic dogs (n = 611) and cats (n = 71) from animal shelters in Anderson, Pickens, and Oconee counties in northwestern South Carolina, U.S.A., were examined for the filarial heartworm, Dirofilaria immitis. For both dogs and cats, whole blood was collected and tested for microfilaria using the Modified Knott's technique. In an effort to find adult heartworms in cats only, a gross necropsy of heart, lungs, and pulmonary vessels was performed immediately after municipal euthanasia. Plasma from each cat was tested for anti–D. immitis antibodies and D. immitis antigens using in-house antibody and antigen tests. In the 1991–1992 study, 21 of 242 dogs (8.7%) were positive for D. immitis. The prevalence of heartworm in dogs increased to 12.7% (47 of 369 dogs) in 1999–2000. Another species of filarial worm, Dipetalonema reconditum, also was observed in blood samples from dogs (18 of 242 dogs [7.44%] and 28 of 369 dogs [7.6%] in 1991–1992 and 1999–2000, respectively). The prevalence of the nonpathogenic D. reconditum was not significantly different from that of D. immitis, emphasizing the importance of correct diagnosis of circulating microfilaria. Modified Knotts' and gross necropsy of cats revealed no heartworms, but 5 of 71 (7%) plasma samples were positive for heartworm antibodies.

Frequency of canine heartworm disease (D. immitis) in some regions of São Paulo State using a circulating antigen indicator test

The frequency of canine heartworm disease was determined in 310 dogs (158 males and 152 females) from São Paulo city (154) and several towns along the São Paulo state coast (156). The survey was carried out through the Knott's technique (direct presence of microfilariae in the blood) and ELISA for soluble circulating antigen detection. Twenty five (8.0%) samples were positive to D. immitis using both methods being 6 dogs (3.9%) from the city and 19 (12.2%) from the coastal area. Nineteen dogs (6.1%) were positive to the Knott's technique (4 from the city and 15 from the coastal area) and 12 dogs (3.9%) to the ELISA (4 from the city and 8 from the coastal area); six dogs (1.9%) were positive for both methods being two from the city and four from the coastal area. The frequency of positive dogs by the Knott's technique in the coastal area was higher than that in São Paulo city (P<0.05), but equivalent frequency was found in dogs from both areas when the ELISA test was used. Higher number of dogs with a patent infection (microfilaremic) was found than those with occult infection (amicrofilaremic). This survey has shown that the dogs were not infected with high frequency probably because the studied areas were not hiperenzootic endemic (higher than 10% of frequency).

Environmental factors and the distribution of mansonelliases in southern Venezuela.

The distribution of mansonelliases and their relation to various quantitative criteria were determined through the study of 1,057 subjects in 17 localities in ten regions of Amazonas State and Bolívar State. The total prevalence among the blood samples, determined through the Knott technique, was 18.54%. 11.26% were parasited by Mansonella perstans, 9.93% by Mansonella ozzardi, and 2.63% by both species. The average of microfilaremia was 48.19 mf/mL of blood in M. perstans and 13.79 mf/mL in M. ozzardi. In the regions studied, M. ozzardi has a wider area of distribution than M. perstans. Prevalence, average number of parasites per host, and the infection index have a positive and statistically significant correlation with the total annual precipitation mean for each region for M. perstans; in the case of M. ozzardi the quantitative parameters are positively correlated with the altitude of each region, this correlation being statistically significant. With respect to type of vegetation, M. perstans had a higher infection index in Amazonian caatinga transition in pluvial lowland forest, and M. ozzardi in semideciduous forest of the alisio type. Therefore two types of transmission, M. ozzardi-Simulium and M. perstans-Culicoides are suggested.

A survey on the prevalence of internal parasites of equines in macedonia and Thessalia-Greece

A 3-year survey was conducted to determine the incidence of endoparasitic infection of equines in several regions of Macedonia and Thessalia, Greece. Three hundred fecal and 252 blood samples were examined. Fecal examination was carried out using. Teleman and Faust techniques while blood was examined according to the modified Knott technique. The Graham test was also performed. The prevalence of infection found was 62.4% for horses, 75.7% for donkeys and 89.2% for mules. Strongyles (large and small) were the most common parasitic species found, with a corresponding prevalence of 42.5% and 45.6% for horses, 73.0% and 37.8% for donkeys, and 89.2% for mules. Other parasites found were: Eimeria leuckarti (2.6% in horses and 8.1% in donkeys), Anoplocephala perfoliata (0.4% in horses), Parascaris equorum (1.7% in horses), Oxyuris equi (4.1% in horses and 8.7% in mules), Strongyloides westeri (2.2% in horses, 5.4% in donkeys and 10.8% in mules), Dictyocaulus arnfieldi (0.9% in horses and 2.7% in donkeys) and Setaria equina (2.2% in horses and 2.7% in donkeys).

A survey of the prevalence of canine filariasis in Spain

A survey was conducted in Spain to update and expand information currently available on the prevalence of infection by filarids in dogs. Between August 1987 and March 1988, blood samples and epizootiological data from 1723 dogs were submitted by 72 veterinarians. Samples were examined by the modified Knott technique and microfilariae were identified on the basis of specific morphological characteristics. The prevalence of filarial parasites was determined for dogs sampled in each of 34 provinces as well as in the Canary and the Balearic Islands. Dirofilaria immitis microfilariae were detected in 3.7% of the dogs sampled. Microfilariae of Dipetalonema reconditum and Dipetalonema dracunculoides were identified in 1.0% and 1.5% of the samples, respectively. Mixed infections of D. immitis and Dip. dracunculoides were observed in seven dogs. Dirofilaria immitis was prevalent in the southern part of the country and on the Canary and Balearic Islands. This parasite was not observed in dogs from the northernmost provinces, but was patent in dogs from the Ebro Valley (located in the northeast) and in dogs from some large urban areas.

"Jump Starting" the Immune System

Letters15 December 1990"Jump Starting" the Immune SystemFredric S. Young, PhDFredric S. Young, PhDSearch for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-113-12-995_1 SectionsAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail ExcerptTo the Editors:I read with great interest the article on extracorporeal photopheresis in a recent issue (1). As pointed out in the article, it is not known how this therapy stimulates the immune system. Although it came from the field of photodynamic cancer therapy, it is possible, and perhaps even likely, that the immune system stimulation results from mechanisms unrelated to the photoactivation of the psoralen compounds. In the 1930s and 1940s, the Knott technique, an extracorporeal irradiation of blood with ultraviolet light, was widely reported to be effective in the therapy of viral infections (2). Further, a new...

The acid phosphatase activity and morphological characteristics of Dipetalonema dracunculoides (cobbold, 1870) microfilariae

The acid phosphatase activity and some morphological characteristics of Dipetalonema dracunculoides microfilariae are described. Their morphological features are closely related to those of the pathogenic Dirofilaria immitis when Knott's technique is used for the microfilarial diagnosis. The acid phosphatase activity pattern found in Dip. dracunculoides microfilariae is clearly different from those previously described for D. immitis, D. repens and Dip. reconditum.

Canine filariasis in Salamanca (northwest Spain).

A survey of dogs in four areas of Salamanca province (northwest Spain) was carried out to determine the prevalence of filariids in the canine population. Blood samples from 293 dogs were tested for the presence of microfilariae, using a modification of the Knott technique. For correct identification of filariid species, blood smears associated with the 43 positive Knott tests were histochemically stained for demonstration of the somatic distribution of acid phosphatase activity. The survey revealed the presence in the northwest of Spain of Dirofilaria immitis, D. repens and Dipetalonema reconditum, with an overall prevalence in Salamanca of 12.3%, 0.3% and 2.1% respectively. The greater prevalence of canine heartworm was found in the irrigated area, where 33.3% of the dogs harboured microfilariae. The relationship between the prevalence of D. immitis and mosquito populations, and the influence of host sex and age in this infection, are analysed.

Tetrapetalonema (T.) colombiensis sp. n. (Nematoda: Filarioidea) from Colombian Primates

Tetrapetalonema (T.) colombiensis sp. n., a filarial nematode from the subcutaneous and intermuscular connective tissues of squirrel monkeys (Saimiri sciureus) and tufted ringtail monkeys (Cebus apella) from central Colombia is described. Tetrapetalonema colombiensis is a very small species (male 8 mm, females 11-21 mm), and within the subgenus most closely resembles T. panamensis and T. saimiri in dimensions and in general appearance. It can be distinguished from these species by the following major features: smoothly rounded profile of the anterior extremity; single pair of lateral caudal petals in the male; vagina vera directed immediately posteriad in the female; and a large (295-335 by 4.3-6.7 micron) microfilaria that assumes a gently curved attitude in 2% aqueous formalin preparations (Knott's technique), and which has a conspicuous inner body.

Tetrapetalonema (T.) saimiri sp. n. (Nematoda: Filarioidea) from Colombian Squirrel Monkeys, Saimiri sciureus

Tetrapetalonema (T.) saimiri sp. n., a filarial nematode from the subcutaneous tissues of squirrel monkeys (Saimiri sciureus) from central Colombia, is described. Within the subgenus, the small size of this worm (females 19-25 mm long) readily distinguishes it from several of the other species in which the minimum length exceeds 40 mm. Tetrapetalonema saimiri can be separated further from nearly all the other small species (T. dunni, T. barbascalensis, T. parvum, and T. obtusa) by its lack of annular body swellings, its low spicular ratio, the location of the vulva near the midportion of the esophagus, the shape and length of the tail in the female, and in details of spicular structure. Tetrapetalonema saimiri closely resembles T. panamensis, but is separated from T. panamensis by the salient buccal region, the acute flexure of the female tail, the shorter right spicule, and the configuration of the vagina of the former species. The microfilaria of T. saimiri is distinguished from that of all other known species of the subgenus by its consistent attitude (loose helical coil or closed C-shape) in 2% aqueous formalin preparations (Knott's technique). During an investigation of the filarial para- sites of Neotropical primates in Colombia, a distinctive microfilaria, easily recognized by its helical attitude in wet, 2% aqueous for- malin concentrates of blood (Knott's tech- nique), was observed in squirrel monkeys (Saimiri sciureus). Of 42 monkeys examined from Barbascal, Meta, 30 were infected with this microfilaria. Only one of 10 Saimiri sci-

Studies on naturally occurring filarial infections in dogs in Lebanon I.Dipetalonema recondition

A survey for filarial parasites was carried out on dogs in Lebanon. The peripheral blood was examined for microfilariae and the skins and carcasses for adult worms. Three methods were used for blood examination: (a) thick blood films stained with Giemsa; (b) a modified Knott's technique (Methylene Blue stained); (c) sodium citrate technique. Two species of filarial worm were found, Dipetalonema reconditum and another species of Dipetalonema which will be described in a later paper. The morphology of microfilariae and adults of D. reconditum is described. The microfilariae of D. reconditum showed a nocturnal sub-periodicity. Attempts were made to find vectors by feeding Aedes aegypti, Aedes phoeniciae, Culex pipiens molestus and Clenocephalides felis on an infected dog. Developing and infective stages were recovered only from C. felis.