Babesia microti diagnosis methods

       As stated in the previous post Babesia microti can sometimes be difficult to screen for.  In the past the only reliable method for screening for Babesia microti was through microscopic examinations of blood smears.  However, this form of testing is extremely limited because it can only detect down to the genus level based on morphological features (Teal et. al, 2011).  This means that Babesia microti cannot be distinguished from other Babesia species.  Another common problem with using microscopic examinations of blood smears is that Babesia microti is often confused with plasmodium in the trophozoite stage (Teal et. al, 2011). 

            This article developed a new method to testing for Babesia microti.  The new method uses a real time PCR assay that targets 1 8S rRNA gene of Babesia microti.  This test is preformed on the DNA that has been extracted from whole blood samples (Teal et. al, 2011).  This method of testing has many benefits compared to the old standard of testing.  First, real time PCR is 100% effective in distinguishing species in the genus Babesia.  In addition to this it also distinguishes between Babesia microti and Plasmodium species that it is commonly confused with (Teal et. al, 2011).  Real time PCR can detect very low parasitemia.  When using microscopic examinations of blood smears its possible to make an incorrect diagnosis if the parasitemia is too low (Teal et. al, 2011).  Using the new PCR assay this will no longer be a problem. 

            It is essential to use sensitive and accurate testing.  Incidents of Babesia microti have had a high increase over the past ten years.  Many times Babesia microti infections will clear over time and will remain asymptomatic in healthy individuals (Teal et. al, 2011).  One of the difficulties with diagnosis through microscopic observations of blood smears is that asymptomatic hosts would not be tested because they showed no symptoms.  This results in asymptomatic carries donating contaminated blood for transfusions.  Usually the recipients of blood transfusions are immune compromised and are more vulnerable to the parasite.  Infections of Babesia microti can become more severe when the host is also infected with Lyme disease (Teal et. al, 2011).  This is a common occurrence because Ixodid ticks transmit both parasites.  Using real time PCR allows for a rapid and accurate test so people donating blood can now be easily screened.  

            The reason I choose this article was because in my previous post I discussed difficulties with diagnosing Babesia microti infections.  I wanted to provide a more in depth look at the problems that occur with the current methods of diagnosis and what methods were likely to be used in the future.  The real time PCR analysis has been developed with in the past year and is not yet a common clinical testing method.  However, due to the success seen in this article I would anticipate it would start to become more common. 

Teal, A. E. Habura, A. Ennis, J. Keithly, J.S. Antenucci S.M. 2011. A new real-time PCR assay for improved detection of the parasite Babesia microti. Journal of Clinical Microbiology. 50: 903-908

All The Genes In One Big VAT

Thus far we have reviewed the influence of the specificity of vectors, as well as the habitat and age of the hosts, and even the season of the year on the infection capabilities and density of infection of different species of Trypanosoma. Today we will delve even deeper into the stores of knowledge on trypanosomes. More specifically, we will learn how the length of the telomeres on the end of the genes in Trypanosoma brucei can affect the frequency of antigenic variation, which is the parasite's main means of avoiding its hosts immune system (Galadriel et al., 2012). 

First, let us recap. Recall from our Parasitology class that one way for the immune system to recognize a pathogen is by the antigens present on the surface of the invading cell.  Trypanosomes are capable of evading the defenses of a host by varying the antigens present on the surface of the parasite. This antigenic variation can be coded for by any one of approximately 1,000 Variant Surface Glycoprotein (VSG) genes within Trypanosoma brucei. Also recall from Bio 230 (Genetics) that telomeres protect a chromosome from deterioration by repeating sequences of nucleotides on the end of the chromosome. It is also important to keep in mind that telomeres become shorter over time due to cell division.  

The authors had hypothesized that the length of telomeres would directly affect the frequency of VSG switching. The method of study was relatively simple: the correlation which the authors proposed was examined by comparing the frequency at which "wild-type" or T. brucei with long telomeres switched the expression of glycoproteins to the frequency at which T. brucei with shortened telomeres switched the expression of glycoproteins. Ultimately, it was concluded that the length of telomeres did indeed influence the rate at which VSGs switched. 

 Most notably I have realized that the above information can be translated to the cycle of infection. Keep in mind that while infected with T. brucei, there are approximately 3 or 4 Variable Antigen Types (VATs) present within the human body with one VAT being dominant over all the rest. These VATs cause infections to cycle between high density and low density which also means that symptoms cycle from very severe to moderate. It is therefore entirely possible for the cycle to occur at a faster rate if there are populations of T. brucei present in the body which possess shortened telomeres! Scary stuff to say the least. 

Literature Cited

Hovel-Miner, G.A., C.E. Boothroyd, M. Mugnier, O. Dreesen, G.A.M. Cross, F.N. Papavasilou. 2012. Telomere length affects the frequency and mechanism of antigenic variation in Trypanosoma brucei. PLOS Pathogens. 8(8): pp. 1-10.

Continuous in vitro propagation of the malaria parasite Plasmodium vivax

Claudia F. Golenda, Jun Li, Roland Rosenburg

            The control of Plasmodium vivax, one of the more severe strains of the devastating disease Malaria, has been very complicated due to its potential drug resistance factors. The culturing of P. vivax not only will give scientist a broader scope into the morphology of the parasite, but it will also help develop vaccines, test drugs, and clone parasites for genome sequencing. The cultivation of the P, vivax parasite was done by using Chesson strains of the parasite from owl monkeys, and passages them to human reticulocytes. The Chesson strain was isolated shortly after WWII from an American soldier, which has been maintained in Aotus monkeys since 1994.  Peripheral blood from a hemochromatosis patient was sued to separate the human reticulocytes by differential centrifugation (Golenda, Li, Rosenburg 1997).

 The parasites were grown in 48 hour durations (considered one cycle) in a static candle jar, until the beginning of schizogony. At about hour 36-40 human reticulocytes were added and the cultures were transferred to a shaker for 10-12 hours. The success of the human reticulocytes binding to the susceptible red blood cells and causing an infection was merely based on the availability of sufficient red blood cell numbers. Malaria treatment usually includes the drug hloroquine, but P. vivax has recently become more resistant in Southeast Asia (Golenda, Li, Rosenburg 1997).

 Two karyotypes of the Aotus monkeys were used: 1. Autos nancymai and 2. Aotus lemurinus griseimembra. The A. nancymai species was inoculated with the Chesson strain intravenously and blood was tested and every other day until the monkey self- cured or was treated with Chloroquine. The A. griseimembra species was inoculated with the ring, and young trophozoite stage of the parasite. Both species were monitored the same. The reticulocytes used were separated from the positive Duffy Antigen group from a patient undergoing hemochromatosis. Reticulocytes were enriched by differential centrifugation in homologous plasma. The results of the experiment showed a successful passage of owl monkey to human erythrocytes, when recycled in a culture for 6-8 generation. The addition of human reticulocytes during schizogony provided merozoites with the targets cells needed for invasion of the cell. In both cultures (both monkey species) the parasite density increased at least 2-fold by cycle 3 (Golenda, Li, Rosenburg 1997).

            By alternating between the static candle jar and the shaker, as well as periodically adding reticulocytes, P. vivax was maintained in a culture for 6-8 cycles. The parasites were mostly in the ring and young trophozoite stage and were confirmed by the analysis of the 18S rRNA gene, a extremely sensitive indicator for Plasmodium. Although the asexual in vitro recycling of P. vivax has been achieved, it leads to some limitations. For one the lack of gametocyte formation in the cultures prohibits the infection of mosquitoes which is directly related to the control of the parasite. Secondly the procedure is labor and time sensitive, and you need access to patients that are suffering from hemochromatosis to do the experiment (Golenda, Li, Rosenburg 1997).

            Exposure to P. vivax is strongly correlated with poverty and low- income living. Those in third world countries at most risk are working aged men. In regions where P. vivax is the most prevalent, effective immunity to the parasite is usually never achieved because it occurs at low incident rates in certain regions (Mendis et. Al 2001). The cultivation of P. vivax will hopefully expose the biochemistry, immunology, biology, physiology, and pharmacology of the parasite for the ultimate regulation of the parasite (Golenda, Li, Rosenburg 1997).

Literature Cited

1.      Golenda CF, Li J., Rosenberg, R. 1997. Continuous in vitro propagation of the malaria parasite plasmodium vivax. Developmental Biology. 94: 6786-6791.

2.      Mendis, K., Sina BJ, Marchesini P, Carter R. 200. The neglected burden of plasmodium vivax malaria. The American Society of Tropical Medicine and Hygien. 64:97-106.

Ultrastructure of Endotrypanum

In my previous articles my main focus was on Leishmania equatorensis and the genus Leishmania.  The last article I looked at suggested that Leishmania equatorensis was more related to the Genus Endotrypanum.  I decided to focus on the Genus Endotrypanum for this third article to see what I could find out.

             In this study, Soares et. al. (1991) looked at the structures within species of the genus Endotrypanum.  The main focus of the researchers was the mitochondria, glycosomes, lipid inclusions, and membrane bound vacuoles.  Measurements were taken of each organelle so that the dimensions and volumes could be compared among species.  Only two species of Endotrypanum have been described, Endotrypanum monterogeii and Endotrypanum schaudinni.  Four strains of these parasites were looked at under an electron microscope, and electron micrographs were taken to get measurements of the organelles.  The results of this study showed that the four strains had the same morphological structures, and it was also stated that all the structures looked at are common among all trypanosomatids, which includes the genus Leishmania.  The study showed that there were no differences between the two species of Endotrypanum that could be used to tell them apart.  The glycosomes and lipid inclusions were larger in E. monterogeii than in E. schaudinni, however researchers suggested that the small differences could be due to physiological conditions.  However there was a strain, in this study labeled as IM201 (Endotrypanum sp.), that could be differentiated from the others due to the size of the kinetoplastid, and from the larger volume that its mitochondria and glycosome took up in the cell.  The data are more in agreement with data collected from individuals of the species Leptomonas samueli and Leishmania donovani.  As a result of differences seen in strain IM201, researchers suggest that this strain may not be of the genus Endotrypanum. 

As stated before, all members of the family trypanosomatidae have the morphological structures that were looked at in this study, and as mentioned before this family includes the genus Leishmania.  Knowing all of this, can these structures be looked at in what is now known as Leishmania equatorensis and be compared to the two species of Endotrypanum to determine if there are any differences between the two genera?  The papers I have found on L. equatorensis only focus on the molecular make up of the parasite, and not on the size and volume of its morphological features.  The sizes of the internal organelles are in no way going to definitively tell us what L. equatorensis is more closely related to but in conjunction with what is known about its molecular make up it may help give us a better idea of where it belongs.  One question I have after reading this paper is, are there any morphological structures that are specific to the genus Leishmania or to the genus Endotrypanum that could be used to help in identification of new species?  Going along with this question, I’m curious as to how Leishmania and Endotrypanum have been divided into two different genera.  Is it based only on molecular differences?    There is so little known about Endotrypanum and of L. equatorensis that there are so many unanswered questions. 

Soares, M. J.,  A. Lopes, and W. De Souza.  1991.  Ultrastructural and Stereological Analysis of Trypanosomatids of the Genus Endotrypanum.  Mem. Inst. Oswaldo Cruz. 86(2):  175-180.  

Ixodes scapularis in: Tick Pickin'

My last Ixodes blog discussed some of the interesting biology that hard-bodied ticks use to attach themselves to their hosts. Also discussed were some implications of limiting tick pathogen transmission by genetically interfering with salivary anticoagulants and adhesives. But an obviously simpler preventive measure exists: manual tick removal. A paper published in 2001 by the CDC and Yale’s department of Epidemiology and Public Health reported on the efficacy of tick removal on pathogen transmission in tick vectors. By analyzing transmission of Lyme disease between mice and ticks, computer models were used to predict the benefits of simple tick removal in preventing transmission of Borellia burgdorferi (Lyme) spirochetes. 

Examining both laboratory and wild strains of B. burgdorferi in Ixodes scapularis, infected ticks were collected from three endemic sites in the Northeast: Lyme, Connecticut, Colts Neck, New Jersey, and Armonk, New York. The researchers tested specimens for infection with both B. burgdorferi and Ehrlichia phagocytophilia, two common zoonotic pathogens. Of those collected, 21% were infected with E. phagocytophilia, 26% were infected with B. burgdorferi, and 6% were infected with both (des Vignes et al., 2001). The wild Lyme strains were isolated and cultured in I. scapularis, but only 30% of field-collected ticks were infected with B. burgdorferi were usable - good news for humans.


To measure Lyme transmission, 4-week-old mice were fed on by nymphal ticks infected with each strain. The nymphs were removed after 24, 48, 72, and ≤ 96 hours (or until replete) of feeding. One month later, the same mice were fed on by uninfected nymphal ticks, and the transmission rates of B. burgdorferi from mouse to tick was determined via dark microscopy. Mice that were fed on by infected ticks were held for approximately a month before biopsies of ear, urinary bladder, and heart tissues were performed. These tissues were tested for the presence of spirochetes to confirm infection.


Des Vignes, et al. (2001) reported 0% transmission within 24 hours, and only 12.5% within 48 hours of feeding. By the third day, however, transmission rates climbed to 78.9% and peaked at 93.8% infection within 96 hours (or until the ticks fed to full engorgement). Based on their experimental results, and those previously reported in the literature, the researchers used statistical algorithms to estimate the probability of infection per hour of feeding. The models estimate that only 4.6% of all nymphal I. scapularis in Lyme endemic regions will transmit the spirochetes if removed before the ticks can feed to repletion. The researchers note that this frequency is largely based off of the 30% transmission capacity of field-collected nymphs, though the model’s estimates are similar to those previously published (des Vignes et al., 2001). 

It is important to note that these frequencies are specific to I. scapularis, and that Eurasian, Pacific, and European species are well known to infect hosts within 24 hours of feeding. Nevertheless, this research supports the idea that mechanical removal of larval I. scapularis is effective in preventing transmission of Lyme spirochetes to humans in the Northeastern United States. Additionally, this report emphasizes the importance of regular tick checks, though simple mechanical removal alone is not  completely effective in combatting all tick-transmitted disease. 

des Vignes, F., Piesman, J., Heffernan, R., Schulze, T., Stafford, K., Fish, D. 2001. Effect of Tick Removal on Transmission of Borrelia burgdorferi and Ehrlichia phagocytophilia by Ixodes scapularis Nymphs. Journal of Infectious Diseases. Vol. 183(5): 773-778. 

Prevalence of Eimeria Species in Lambs in Antakya Province

            Eimeria species can cause the disease called coccidiosis in many types of animals, as well as humans. Pre-weaned and recently weaned lambs are of particular importance when it comes to this disease, because if they have subclinical coccidiosis it is likely that the lambs will have a reduction in weight gain and feed efficiency and be more likely to become infected with other diseases (Kaya, 2004). Subclinical coccidiosis occurs when an animal is infected but does not show obvious signs of infection. Another stage of infection, clinical coccidiosis, has even higher economic losses due to the cost of medical treatment, more severe growth impediment, and death of the animal. Mortality in sheep during an outbreak of coccidiosis can range from 10 to 40%, but is rarely over 10% (Kaya, 2004). Clinical coccidiosis only became an economically important issue when intensive animal rearing systems were introduced, and outbreaks of the disease occurred in dense animal populations and in poor weather conditions (Kaya, 2004). At the time this article was published, it was believed that only fourteen species of Eimeria were capable of infecting sheep, however as stated in the previous article reviewed (which was published in 2011) the number of species that can infect sheep is now at fifteen.

            The province of Antakya has very different conditions from the rest of Turkey, being mild and rainy during winter and spring, and very hot and humid in the summer and fall, with sheep grazing outside in pastures year round on sunny days (Kaya, 2004). The researchers wanted to determine what the prevalence and intensity of Eimeria infection was in lambs in the province because of the climatic difference of the area.

            A total of 248 samples were taken from 34 lamb flocks (aged 2 weeks to 6 months) from the locations of Antakya, Reyhanh, Serinyol, Kirikhan, Hassa, and Harbiye. Lambs were chosen randomly and the sample was taken directly from the lamb’s rectum, and stored in a lidded container (Kaya, 2004). All samples were infected with 2 to 8 of the 15 Eimeria species capable of infecting sheep. The species of Eimeria with the greatest prevalence of infection were E.crandallis with an infection rate of 64.91% and E.ovinoidallis with an infection rate of 55.24% (Kaya, 2004). One hundred percent of the lambs sampled were found to be infected with one of 10 different Eimeria speicies, while other areas in Turkey range from 29.9% infected with 9 species, to 94.8% with 9 species as well (Kaya, 2004). Except for the Van province, the infection rate in Antakya province was higher than that in other provinces of Turkey, with the number of identified Eimeria species being similar to other studies, but the actual identified species differed.

            The difference in the presence of particular Eimeria species and the prevalence of each species depends on factors such as environment, animal factors (age, species, etc.), farm management and other factors such as other illnesses or stress (Kaya, 2004). Eimerian parasites can co-exist with the host sheep without negative consequences so long as other illnesses or stress does not upset the balance between the host and the parasite. Farm management can be altered or introduced to shepherds in order to cut economic losses caused by clinical or subclinical cases of coccidiosis in the herd (Kaya, 2004).

Reference:

Kaya, G. 2004. Prevalence of Eimeria species in lambs in Antakya province. Turkish Journal of Veterinary & Animal Sciences. 28: 687-692.

Filarial Worm or Tumor?

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Filarial worms cause a serious issue in the world. Filariasis usually refers to Wucheria bancrofti and Brugia species; lymphatic filariasis. Filariasis mainly involves the lymphatic system. As of December 2006 1.254 million people in 83 endemic countries were at risk, 64% of which was contributed to South-East Asia Region. When dealing with lumps in breasts of persons from India, it is prudent, then, to keep filariasis (though generally rare in breasts) in mind.

This was exactly the case in the work done by Behera et al., (2008). A 40 year old female presented a ‘peanut sized’ nodule that did not hurt, but was increasing in size. She was clinically diagnosed with a  fibradenoma breast, essentially a benign tumor. The mass was removed from her breast and examined. The examination revealed an adult worm cut in several planes, surrounded by dense inflammatory cells; a granuloma. The granuloma, as we would expect, consisted of lymphocytes, eosinphils, histiocytes, and plasma cells. Behera et al., were able to find the uterus and intestines in a cross section of the worm.

Of all the filariasis cases in the world, W. bancrofti accounts for 90%. Adult worms can be found in lymphatics, subcutaneous tissue, peritoneal and pleural cavities, heart, brain, scrotum, and breast. Very few cases have ever been reported of worms in the breast such as in the case studied by Behera et al., (2008).  The painless breast lumps we know are granulomas, and if left to calcify can damage a decent portion of the breast tissue. Clinically, these granulomas are indistinguishable from carcinoma. In the case of the 40-year-old woman in India, microfilaria were not detected in blood smears, so only the histopathology (microscopic examination) can confirm the presence of the adult worm.  Figure 1 from the article shows the adult worm and granuloma in a photomicrograph. 

Works Cited

Biren Kumar Sarkar, et al. "Adult Filarial Worm In The Tissue Section Of A Breast Lump." Indian Journal Of Surgery 71.4 (2009): 210-212. Academic Search Premier. Web. 1 Nov. 2012.

http://0-web.ebscohost.com.www.consuls.org/ehost/pdfviewer/pdfviewer?sid=2f7f4364-38bc-46d4-9ebc-bffb090a0a9e%40sessionmgr115&vid=2&hid=107

Toxoplasma gondii: Mouse parasite that makes it attracted to cats

            Toxoplasma gondii, like the other parasites that I have researched and talked about on this blog, somehow effect or modify the behavior of it host. Toxoplasma gondii is a protozoan parasite that affects mammals (Berenreiterova´ et al. 2011).  Although the parasite is study mainly through mice, it can infect humans and must reproduce sexually in the intestines of a cat (Berenreiterova´ et al. 2011).  Toxoplasma gondii develops in the brain of a mouse where it works to alter the mouse’s behavior (Berenreiterova´ et al. 2011).  One way the parasite manipulates the behavior of the mouse is by signaling the mouse to be attracted to the smell of a cat, whereas a mouse would normally be repulsed by the scent of the cat in fear of being attacked (Berenreiterova´ et al. 2011). This change is behavior results in the mouse being caught and eaten by the cat and thus allows the parasite to reproduce in the cat’s intestines thus renewing the cycle (Berenreiterova´ et al. 2011). Scientists have not been able to figure out how exactly Toxoplasma gondii is able to manipulate the mouse’s behavior this way (Berenreiterova´ et al. 2011).

            It is believed that Toxoplasma gondii affects a quarter of the human population (“How Different Strains...” 2011). Scientist believes that it alters the human brain effecting behavior and is responsible for the psychological disorder schizophrenia (“How Different Strains...” 2011).  Scientists believe that the parasite affects the brain causing hallucinations however; more research needs to be done on this subject (“How Different Strains...” 2011).

Berenreiterova´, Miroslava, Jaroslav Flegr, Ales A. Kube, and Pavel Neˇmec . The Distribution of Toxoplasma gondii Cysts in the Brain of a Mouse with Latent Toxoplasmosis: Implications for the Behavioral Manipulation Hypothesis . PLos One. 2011. P 1-14.

“How Different Strains of Parasite Infection Affect Behavior Differently”. Science Daily. March 22 2011. Available: http://www.sciencedaily.com/releases/2011/03/110321203437.htm.