Epidemiological Aspects of Canine Dirofilariasis in the North of Iran

Background

Dirofilaria immitis is an important parasite in dog and other carnivores. Our objective was study on incidence and periodicity of heartworm in north of Iran and using other methods for its diagnosis in addition to Parasitology exam.

Methods

This survey spanned two years, between 2006 and 2008. Blood samples were collected from 431 stray dogs distributed along north of Iran, the coastal areas of the Caspian Sea. The Knott''s modified test was used for diagnosis of D. immitis and other filariae. Meanwhile, the periodicity of microfilaria in peripheral blood circulation was calculated and the imaging diagnosis techniques of four dogs that had positive results were done.

Results

Diagnostic parasitology results indicated that 16.01% of stray dogs were microfilaremic. Two different microfilariae were diagnosed: D. immitis in 13.69%, Dipetalonema reconditum in 1.86% and in 0.46% both of them. There was no statistically significant between infection to fiariae with sex and age of dogs. Also study on the periodicity of the presence of microfilaria in peripheral circulation showed that the highest rate of those was at 1 am and the lowest rate at 12 pm. Radiographic study showed distinctive signs with varied degrees of severity included: Tortuous and enlargement of main and lobar pulmonary artery, pulmonary parenchymal lesions and Right side heart enlargement that confirmed in electrocardiography. Also in echocardiographic images observed short parallel-sided images with the appearance of equal signs that indicated the presence of the heartworm.

Conclusion

These results showed that to obtain a reliable diagnosis of heartworm infection, imaging tests could support parasitological exams.     

Histopathologic Effects of Dirofilaria immitis Microfilaria on Internal Organs of Dog Confirming by PCR Technique

Background

The heartworm disease is an infectious disease of dogs with Dirofilaria immitis combined with cardiovascular and circulatory abnormalities. The heartworm disease can become a serious health risk when associated with a severe infection. In this study, a male, 8 year-old dog that died suddenly was necropsied and all tissues were examined grossly.

Methods

Major organs including heart, lungs, liver, spleen, kidneys, brain, eyes, and testis were fixed in 10% neutral formalin, embedded in paraffin, sectioned at 5-µm thickness, stained with hematoxylin and eosin, and examined with a light microscope. For each examined organ, paraffin-embedded tissues were cut and placed in eppendorf tubes for genomic DNA extraction. PCR was performed using two sets of primers for amplification of a 302 bp ITS-2 gene fragment and a 203 bp cytochrome oxidase subunit 1 (CO1) gene fragment of D. immitis.

Results

During the necropsy examination, 46 adult D. immitis were found in the portal vein, right ventricle, and atrium of the heart and pulmonary trunk. Microscopically, microfilarias were found throughout the vessels of different organs including lungs, kidneys, liver, heart, brain, and spleen. All tissues examined by PCR were positive for D. immitis ITS-2 and CO1.

Conclusion

PCR technique now represents an effective method for identification of D. immitis from formalin-fixed samples.     

Canine Babesiosis in China Caused by Babesia gibsoni: A Molecular Approach

Background

To provide a point of reference to study the epidemiology and clinical expression of canine babesiosis in China.

Methods

A total of 30 dogs infected with canine babesiosis were evaluated by mean of clinical history, physical examination, hematological, restriction fragment length polymorphism of PCR products (PCR-RFLP) and sequencing analysis.

Result

The most prevalent clinical abnormalities were lethargy (100%), anorexia (100%), pale or icteric mucous membranes (80%), fever (70%) and dark urine (70%). Hematology parameters revealed that anemia and thrombocytopenia were the major abnormalities in blood of dogs infected with canine babesia. The results of PCR-RFLP and sequencing analysis indicated that B. gibsoni was the main species responsible for canine babesiosis cases at the time of the study in Nanjing, China.

Conclusions

The results provide valuable information for better understanding of the epidemiology of canine babesiosis in China.     

Trichinella Infection in Wildlife of Northeast of Iran

Background

The objective of this investigation was to detect the presence of Trichinella in some carnivores of Mashhad in northeast of Iran and to identify Trichinella species circulating in this area.

Methods

The present study was carried out using muscle tissue collected from 120 stray dogs, 26 wild boars, 25 rodents, two foxes and two hyenas captured in Mashhad City, province of Khorasan Razavi, Iran.

Results

Trichinella larvae were detected in three stray dogs by artificial digestion and compression. All larvae were identified as T. britovi using multiplex PCR.

Conclusion

This is the first report of identification of T. britovi in stray dog in Iran.     

A Preliminary Parasitological Survey of Hepatozoon Spp. Infection in Dogs in Mashhad,Iran

Background

We attempted to determine the prevalence of Hepatozoon spp. infection in Mashhad, northeast of Iran, via blood smear parasitology.

Methods

The prevalence was investigated by examination of blood smear parasitology, using blood samples collected from 254 dogs (51 strays and 203 privately owned-dogs).

Results

Two stray dogs (2/51; 3.92%) and two privately-owned dogs (2/203; 0.98%) were infected with Hepatozoon spp. Therefore, as per blood smear parasitology, the prevalence of Hepatozoon spp. infection was 1.57% (4/254). Sixteen out of 254 dogs (6.29%) were infested with ticks; all of which were Rhipicephalus sanguineus. One of the dogs infected with Hepatozoon spp. exhibited ticks at the time of examination. Concurrent infection with Ehrlichia canis and Leishmania infantum was not detected in the four Hepatozoon spp. infected dogs.

Conclusion

This is the first epidemiological study on the prevalence of Hepatozoon spp. infection in dogs in Iran.     

Immunotherapy Using Autoclaved L. major Antigens and M. vaccae with Meglumine Antimoniate,for the Treatment of Experimental Canine Visceral Leishmaniasis

Background

To evaluate immunotherapy against canine visceral leishmaniasis, Leishmania major antigen and heat-killed Mycobacterium vaccae (SRL172) were used as stimulators of immune defense mechanisms and the results were compared with standard chemotherapy meglumine antimoniate.

Methods

Nineteen mongrel dogs aging 1-3 years old were used in this experiment. Infection was carried out in 15 out of 19 dogs using L. infantum, isolated from a naturally infected poly-symptomatic dog.

Results

All the cases showed positive serologic results by direct agglutination test during 30-60 days following inoculation. In the first group, which was under chemotherapy (Glucantime^R), one of the members showed recurrence of the disease despite rapid effect of the therapeutic protocol. Immunotherapy using SRL172 caused complete cleaning of the parasite in group 2, but the speed was less than Glucantime. Immunotherapy using L. major antigen combined with M. vaccae in group 3 and combine administration of immunotherapy and chemotherapy in group 4 both were with relapsing of one case in each group. Group 5 and 6 were consisted of positive and negative control dogs, respectively.

Conclusion

Immunotherapy seems to be an adjuvant in treatment of canine leishmaniasis but it needs more investigation for final confirmation.     

Canine Visceral Leishmaniasis in Kerman,Southeast of Iran: A Seroepidemiological,Histopathological and Molecular Study

Background

Canine visceral leishmaniasis (CVL) is a systemic disease with a high mortality rate, caused by a diphasic protozoan parasite, Leishmania infantum/chagasi in the world. The objective of the present study was to determine the presence of CVL in the city and suburbs of Kerman, using a range of serological, histopathological and molecular methods.

Methods

Blood samples were taken from 80 clinically symptomatic stray dogs All the collected blood samples were tested by direct agglutination test (DAT) to detect the anti-Leishmania antibodies in dogs, using a cut-off value of ≥1:320. Pathological specimens including spleen, liver and lymph nodes were prepared for paraffin blocks, sectioning, staining and final microscopic examination in the pathology laboratory. PCR amplification of kDNA from 9 samples of DAT positive stray dogs was studied.

Results

The anti-Leishmania antibody was detected in 9 dogs (11.25 %) of the total 80 studied dogs. No significant difference was found between VL infection and gender. In contrast, there was a significant difference between seropositivity and age (P<0.05). Pathological samples showed changes including hyperplasia of infected macrophages and inflammatory cells that occupied sinusoids and splenic cords. Among the samples which was characterized by PCR, only one specimen revealed to be mixed infection between L. infantum and L. tropica.

Conclusion

The results revealed a high prevalence of L. infantum infection in stray dogs in Kerman. This kind of information is needed for implementation of future control programs.     

Seroprevalence of Toxoplasma gondii Infection in Dogs in Tehran,Iran

Background

Toxoplasma gondii infects a wide range of animals; felines are definitive hosts and other animals including the dogs are intermediate hosts. The aim of this study was to determine the seroprevalence of T. gondii infection in dogs in Tehran, capital of Iran and to investigate possible associated risk factors.

Methods

Three hundreds ninety six serum samples were collected during 2007–8 from the dogs. Collected samples were tested using an indirect fluorescent antibody test (IFAT) in dilutions of 1:16 and more. All procedures were carried out in Shahrekord University, Iran. All the data were analyzed using SPSS software, qui square test with confidence interval of 0.95.

Results

From evaluated samples, 89 (22.47%) were positive in titers of at least 1:16. further evaluations in other dilutions showed positive results in dilutions of maximum 1:16, 1:32, 1:64, 1:128 and 1:256 in 38, 29, 15, 2 and 5 dogs respectively. Investigation of the role of risk factors showed no sex predisposition while infection rate was significantly higher in dogs older than one year old. Living places were of significant importance; infection rate was significantly higher in stray or guard dogs in compare with household dogs (P<0.05).

Conclusion

Relatively high seroprevalence of T. gondii infection in dogs in Tehran shows high environmental contamination. It is recommended that the dogs with suspected clinical signs be tested for T. gondii infection.     

Hair Contamination of Sheepdog and Pet Dogs with Toxocara canis Eggs

Background

We tried to investigate the hair contamination of pet dogs and farm sheepdog with Toxocara eggs in terms of the different sex and age groups in north-west of Iran (Urmia and its suburbs).

Methods

Hair samples were collected from a total of 138 pet and farm sheepdogs from November 2008 to June 2009 in Urmia City and the suburb (West Azerbaijan-Iran) and examined for the presence of T. canis eggs.

Results

T. canis eggs found in 60 samples altogether (pet and shepherd dogs) showed a contamination rate of 36.2%. The number of observed T. canis eggs in each microscope field was varied from 1 to > 400. The age of the dog was found a significant factor to influence the prevalence and intensity of contamination, with 82% of all the eggs recovered from puppies (six months and younger). Additionally, the numbers of eggs in farm sheepdogs were significantly higher than pet dogs (P<0.05).

Conclusion

This report shows that direct contact with T. canis infected dogs, particularly puppies from shepherd dogs, may pose a serious hazard to human. Besides, as they may harbor a considerable number of eggs on their hair, they can contaminate the soil and the environment.     

A Histopathology Study of Caspian Seal (Pusa caspica) (Phocidae,Mammalia) Liver Infected with Trematode,Pseudamphistomum truncatum (Rudolphi, 1819) (Opisthorchidae,Trematoda)

Background

Main objective of this study was to investigate the invasive activity of the liver fluke, Pseudamphistomom truncatum against the Caspian seal (Pusa caspica) and was exemplified at the gross, light microscopy (LM) and electron microscopy (EM) levels.

Methods

The study was done on a freshly dead Caspian Seal in the southern coast of Caspian Sea. The checked Caspian seal probably being died of canine distemper virus and was found host to numerous parasites of four helminth species.

Results

P. truncatum caused edematous foci on the surface of the liver with prominent fluid accumulation. Sections of the liver viewed with LM had multiple necrotic areas with extensive hemorrhaging and disorganized hepatic lobules. Granulocytes and invasion of connective tissue were prominent. Whole worms were visible with invasive pathways through the host tissue. Damage to both hepatic ducts and blood vessels were prominent. At the EM level, organelles within the impacted hepatocytes were disorganized as exemplified by the cristae of the mitochondria and the endoplasmic reticulum. Parasite eggs were scattered throughout the tissue.

Conclusion

It was shown that this trematode can be very pathogenic to Caspian Seal and as this only mammal of Caspian Sea is an endangered species; this needs more investigation toward control or possible treatment of this helminth.     

Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody

Background:

Cystic echinococcosis is an important zoonosis caused by the Echinococcus granulosus with a substantial impact in human and animal health in endemic areas. The purpose of the present study was serodiagnosis optimizing of dog echinococcosis in order to achieve a rapid diagnostic method.

Methods:

Eight dogs were challenged with protoscoleces in order to have positive echinococcosis serum and 2 two-month old puppies were used as uninfected controls. Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1. Dot immunogold filtration assay (DIGFA) was developed by coating rEPC1 labeled colloidal gold on nitrocellulose membrane. The canine sera, taken three times including, 15, 28 and 35 days post infection were tested. A total of 30 serum samples including 24 sera from 8 infected dogs and 6 sera from 2 puppies were comparatively detected with both DIGFA and ELISA.

Results:

Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera. Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA.

Conclusion:

DIGFA as a rapid and simple procedure could be utilized in quickly diagnosis of echinococcosis.     

Protoscolecidal Effect of Berberis vulgaris Root Extract and Its Main Compound,Berberine in Cystic Echinococcosis

Background

Cystic echinococcosis (CE), a zoonotic parasitic infection caused by the metacestode (larvae) stage of dog tapeworm Echinococcus granulosus and recognized as a major economic and public health concern in the world. This study aimed to investigate the in vitro scolicidal effect of methanolic extract of Berberis vulgaris L. roots and its main compound, berberine against protoscoleces of hydatid cysts.

Methods

For this purpose, protoscoleces were aseptically aspirated from sheep livers having hydatid cysts. Various concentrations of the methanolic extract (0.25-2 mg/ml) and berberine (0.062- 0.5 mg/ml) were used for 5 to 30 min. Viability of protoscoleces was confirmed by eosin exclusive test.

Results

In the present study, all of the various concentrations of the B. vulgaris methanolic extract (0.25, 0.5, 1 and 2 mg/ml) and berberine (0.062, 0.125, 0.25 and 0.5 mg/ml) revealed significant (P<0.05) scolicidal effects against protoscoleces of E. granulosus in a dose-dependent manner. Both berberine and methanolic extract exhibited 100% inhibition against protoscoleces of E. granulosus at the concentration of 2.0 and 0.5 mg/ml after 10 min incubation, respectively.

Conclusion

According to the results, both B. vulgaris methanolic extract and berberine alone demonstrated high scolicidal activities against protoscoleces of hydatid cysts in low concentration and short exposure time on in vitro model. However, in vivo efficacy of B. vulgaris and berberine also requires to be evaluated using an animal model with hydatid infection.     

An Analysis of Clinical Characteristics of Strongyloides stercoralis in 70 indigenous patients in Iran

Background

Clinical manifestations of Strongyloides stercoralis are variable from asymptomatic to hyperinfection and devastating disseminated infections. Hereby, clinical characteristics of a large series of Iranian strongyloidiasis indigenous cases are described.

Methods

The records of people referred to the Helminthological Diagnostic Laboratory of School of Public Health, Tehran University of Medical Sciences and School of Medicine, Gilan University of Medical Sciences, during 2009-2013 were reviewed. For those patients that were infected with S. stercoralis and their clinical manifestations and demographic data were available (70 cases) a checklist was prepared and data analyzed.

Results

Forty-three patients (61.4%) were male and 27 (38.6%) female. Gastrointestinal, cutaneous and pulmonary symptoms were present in 71.4%, 25.7%, and 15.7% of patients, respectively. None of them had larva currens eruption. Eosinophilia was the most prevalent reason for suspicious on S. stercoralis, but the mean was lower in elderly patients. Hyperinfection were recorded in 8 patients (11.4%), and 2 cases had disseminated infection.

Conclusion

Eosinophilia is common both in asymptomatic and symptomatic cases of strongyloidiasis, but the mean tend to lower with increase in age.     

Seroprevalence of Neospora caninum Infection in Dairy Cattle in Tabriz,Northwest Iran

Background

The aim of this study was to determine the seroprevalence of antibody to Neospora caninum in healthy and aborted dairy cattle in Tabriz, capital of East-Azarbaijan in northwest of Iran.

Methods

In this cross-sectional study serum samples were collected from 266 healthy and aborted Holestein-Feriesisnc cows from September 2008 to August 2009. The sera were analyzed to detect of antibody against N. caninum using the commercially ELISA kit.

Results

Seroprevalence of antibody to N. caninum was 10.5% in Tabriz dairy cattle. Also the abortion rate in all cattle sampled was 33.6% but percentage of seropositive aborted cattle was 18.4%.

Conclusion

Neosporosis could be one of the possible causes of abortion in dairy cattle in Tabriz and regarding the distribution in dogs as definitive host for the parasite, further studies in dog and cattle are recommended.     

Prevalence of Balantidium coli Infection in Bred Rhesus Monkeys (Macaca mulatta) in Guangxi,southern China

Background

Balantidium coli infects humans, primates and pigs, causing serious diarrhea and dysentery. Little information on the prevalence of B. coli in primates is available in China. This investigation was conducted to determine the prevalence of B. coli infection in bred rhesus monkeys in Guangxi Zhuang Nationality Autonomous Region (GZNAR), southern China.

Methods

A total of 120 fecal samples were collected from rhesus monkeys bred in cages in GZNAR and B. coli cysts and/or trophozoites were examined microscopically after sedimentation with water in May 2013.

Results

(64.2%) samples were tested positive. The prevalence was 65% (39/60) and 63.3% (38/60) in female and male monkeys, respectively. 80% (48/60) cages in this nonhuman primate center were positive for B. coli.

Conclusion

The present survey revealed high circulation of B. coli in bred rhesus monkeys in GZNAR, which poses potential threats to animal and human health.     

Toxocara Nematodes in Stray Cats from Shiraz,Southern Iran: Intensity of Infection and Molecular Identification of the Isolates

Background

Toxocara is a common nematode of cats in different parts of Iran. Despite the close association of cats with human, no attempt has been done so far for molecular identification of this nematode in the country. Therefore, current study was performed on identification of some isolates of Toxocara from stray cats in Shiraz, Fars Province, Southern Iran, based on morphological and molecular approaches, and also determination of intensity of infection.

Methods

This cross-sectional study was carried out on 30 stray cats trapped from different geographical areas of Shiraz in 2011. Adult male and female worms were recovered from digestive tract after dissection of cats. Morphological features using existing keys and PCR-sequencing of ITS-rDNA region and pcox1 mitochondrial l gene were applied for the delineating the species of the parasites.

Results

Eight out of 30 cats (26.7%) were found infected with Toxocara nematodes. All the isolates were confirmed as Toxocara cati based on morphological features and the sequence of ribosomal and mitochondrial targets. Intensity of infection ranged from one to a maximum of 39 worms per cat, with a mean of 10.25±12.36, and higher abundance of female nematodes.

Conclusion

The most prevalent ascaridoid nematode of stray cats in the study area was T. cati and female nematodes were more abundant than that of males. This issue has important role in spreading of eggs in the environment and impact on human toxocariasis.     

Still and Moving Image Evidences for Mating of Echinococcus granulosus Reared in Culture Media

Background

Echinococcus granulosus cultivation is very important for improvement of different aspect of medical and veterinary researches. Despite many advances in this case, there is a missing link for in vitro life cycle of adult worms and it is fertilization. Regarding the researchers’ observations, self-fertilization can be done in worms living in dog intestine, but despite all sorts of experimental techniques, this phenomenon has never been observed in reared worms in culture media. Furthermore, cross fertilization has not been observed in vitro and even in parasites with dog intestinal origin; although it theoretically is possible. During a follow-up of cultivated adult worms, evidences of behaviors similar to self-mating (Type 2) and cross-mating were observed in our lab which will be presented here.

Methods

Protoscoleces were aseptically removed from sheep hydatid cysts, washed twice with PBS and then cultivated in S.10E.H culture medium. The stages of parasite growth were observed using an inverted microscope for two months and all stages and behaviors were microscopically photographed. Different movies have also been made from these behavioral features.

Results

After around 55 days post cultivation, some evidences of behaviors similar to self-mating (Type 2) and cross-mating were observed in some of the mature adult worms. However, fertile eggs in these parasites have never been observed.

Conclusion

Regarding the above observations, these parasites show tendency to unsuccessful self-mating/fertilization (type 2) which failure could be due to anatomical position and physiological maturation. Also lack of suitable conditions for self-fertilization causes the worms try to do unsuccessful cross- mating/fertilization in culture media     

Seroprevalence of Toxoplasma gondii Infections in Pregnant Women in Gorgan City,Golestan Province,Northern Iran-2012

Background

Toxoplasma gondii is one of the most prevalent parasites of human and warm- blooded animals. Toxoplasmosis is important especially in two groups: pregnant women and immunocompromised patients. If women acquire the primary infection during the pregnancy, it would be life threatening or remains severe disorders for the fetus. This study was performed to evaluate the seroprevalence of T. gondii infection in pregnant women referred to Health Center in Gorgan City, Golestan Province, northern Iran.

Methods

Serum samples were collected from pregnant women referred to Health Center in Gorgan City, south eastern Caspian Sea. Anti- Toxoplasma IgG and IgM antibodies were determined by commercially ELISA kits and the relation of infection with socio-demographic and risk factors such as age, education, occupation, cat ownership, soil contact and some other factors was studied.

Results

From 555 tested sera of pregnant women referred to Health Center in Gorgan, 39.8% had IgG antibodies against T. gondii and 3.4% were positive for IgM antibodies. A significant correlation was seen between T. gondii infection with age and soil contact.

Conclusion

About 60% of pregnant women in Gorgan City are seronegative against T. gondii, so they should considered as at risk persons.     

Genetic Diversity of Human Blastocystis Isolates in Khorramabad,Central Iran

Background

There are some genetic differences in Blastocystis that show the existence of species or genotypes. One of these genes that help in identifying Blastocystis is SSUrRNA. The aim of this study was assessment of genetic diversity of Blastocystis by PCR with seven pairs of STS primers.

Methods

This study was done on 511 stool samples collected from patients referred to the health care centers of Khorramabad, Central Iran, in 2012. Genomic DNA was extracted and in order to determine the Blastocystis subtype in contaminated samples, seven pairs of primers STS (subtype specific sequence-tagged site) were used.

Results

Out of 511 samples, 33 (6.5%) samples were infected with Blastocystis. Subtype (ST) of 30 samples was identified and three subtypes 2, 3 and 4 were determined. Mix infection was reported 10% which 3.33% of the infection was for the mixture of ST 3 and ST5 and 6.67% was for the mixture of ST 2 and ST 3.

Conclusion

The predominant subtype was ST3 that is the main human subtype. The dominance of ST2 and 5 are important in this study. This superiority has been reported in some of the studies in ST 2 which is different from the studies in other countries, because they have announced priorities of the ST1 and ST6 after ST3.