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Study Of Molecular Diagnosis, Associated Risk Factors And Treatment Of Anaplasmosis In Bovines

By: Muhammah Hassan Haider (2008-VA-252) | Dr. Jawaria Ali Khan.
Contributor(s): Dr. Muhammad Avais | Dr. Ali Ahmad Sheikh.
Material type: materialTypeLabelBookPublisher: 2015Description: 50p.Subject(s): Department of Clinical Medicine & SurgeryDDC classification: 2228-T Dissertation note: According to the economic survey of Pakistan 2013-14, livestock sector contributed about 55.9 and 11.8% to the agriculture and national GDP, respectively.Total cattle population in the year 2013-14 is 39.7 million. Total milk and meat production contributed by cattle is 18,027000 tons and 18,87000 tons respectively (Anonymous 2013-14) . In Pakistan, the chiefhindrance in performance and health of animals are parasitic diseases including tick born. Ticks have been easily grown and reproduce in the optimal climate of tropical weather in Pakistan. In Pakistan, there are huge number of species and genera of tick fauna (Durrani and Shakoori, 2009). Tick-borne diseases include babesiosis, theileriosis, anaplasmosis, lymedisease . Among these tick-borne diseases anaplasmosis is one of the vital diseases which is responsible for the substantial economic losses in term of high morbidity, mortality and production losses that are decreased in milk, meat and other important livestock productions. Throughout the world, the “tick-borne diseases” are extensivelyspread in sub-tropic and tropicareasand Pakistan also includes in these areas(Khan et al. 2004). Moreover, anaplsamosis is non-contagious disease but also known to be infectious, transmission of which occur through mechanical means that includes the flies or tick bites. As well as different equipment that are using during tattooing or castration, in dehorning and needles also causes transmission (Aubry & Geale. 2011). Anaplasma marginale (A. marginale) is the major cause of Bovine anaplasmosis and this speciesbelongs to the genus Anaplasma (“Rickettsiales, Anaplasmataceae”). The most important 20 ticks species which cause transmission are Argaspersicus, Boophiludecoloratus, B. microplus, B. annulatus, Dermacentoroccidentalis, D. andersoni, D. variabilis, D.albipictus, Hyalommaexcavatum, Ixodesricinus, Ornithodoroslahorensis, Rhipicephalussimus, R. saniguineus, and R. bursa(Marchette & Stiller. 1982), on the other hand, Boophilusmicroplus found as a major contributor in cause of anaplasmosis (Tick Fever Research Centre, 1996). The family anaplasmatacease was classified in 1957 of the order Rickettsiale. In the genera Anaplasma, Aegyptianella, Haemobartonella and Eperythrozoon, this family was rationalized (Ristic&Kreier. 1984), and projected a combination based on analysis of genes sequences for protein on surface and ribosomal RNA (16S)(Dumler et al. 2001; Walker & Dumler. 1996). Anaplasma is a rickettsial, obligate intracellular bacteria found in red blood cells. Disease is characterized by fever, anemia and jaundice. Infection is transmitted by ticks or mechanically by biting insects or contaminated hypodermic needles or surgical instruments (Tylor et al. 2007).The most significant parasite is Anaplasma speciesthat is transmitted by at least twenty different species of ticks. But among those all mostly Bmicroplus causes Anaplasmosis (Rajput et al.2005). A. marginale is etiological agent of bovine anaplasmosis (Bram, 1957; Dumler et al., 2001; Kocan et al., 2000). A. marginale persistently infect both cattle and ticks and help as infection reservoirs(Kocan et al. 2003). Bovine erythrocyte membrane bound parasitophorous vacuoles is the site for replication of this obligate intracellular organisms. Dogs, humans, wild and domesticated ruminants are the species effected by A. marginale. This anaplasma genus is an obligate intracellular parasite of vertebrate hosts. In cattle, theA. marginal, A. phagocytophilum, A. central, and A. bovisare major pathogenic species producing diseases (Inokuma et al. 2007). Researchers suggestedthat in cattle, some unidentified Ehrlichia or Anaplasma species are present (Awadia et al. 2006). For this pathogen, which cause the persistent infection, the reservoirs are generally present in different host like tick or mammalian (Kocan et al. 2004). The wide range of different ruminant can be affected by Anaplasmabecause the species of anaplasma are not considered as strictly specific for any particular host specific(Kuttler. 1984). The A. marginalecan only be developed inside the RBCs of bovine (Richey et al. 1981). After invading the erythrocyte the tick multiplies and form almost eight initial bodies, they then get enlarge in the outer membrane and give it an appearance of a large dot. The organism gets mature in the outer membrane of RBC and force the infected RBC to get rupture due to infection. By rupturing the outer membrane where it previously multiplied come into the blood stream to infect the other RBCs. On the progression of progresses, more number of erythrocytes is infected by this organism and afterward destruction of erythrocytes occur (Stewart et al. 1981). Upon the clinical signs and symptoms are appear when the 15 percent of the RBCsare affected by the parasite(Radostits et al. 2006). The symptoms of the disease are pyrexia, anemia, icterus, hematuria, anorexia, muscular tremors, dyspnea, depression, lethargy, constipation and yellow colour of mucous membrane (Bram et al. 1983), low milk production, miscarriage and sometime mortality (Alderink and Dietrich. 1981). The mature RBCs are the site for multiplication after the invading by A.marginale. In the acute case of Anaplasmosis the higher number of RBCs are affected by the parasite for example more the 109 red blood cells per ml and after that the disease is categorized by death, abortion, loss of weight and anemia, and the survival cattle that are infected act as reservoirs for transmission of the disease in the herd level(De Echaide et al. 1998).The haematological profile can be used as useful diagnostic tool in anaplasma infection(Van Wyk et al. 2013).There is significant decrease in packed cell volume (PCV), Hemoglobin (Hb) and erythrocytes count.Serum biochemical analysis showed increased total protein,bilirubin and alanine aminotransferase enzyme (Sharma et al. 2013). A diagnosis ofcattle anaplasmosis may be made tentatively based on geographical location, seasonal variation and presenting clinical signs or necropsy finding in infected animals (Kocan et al. 2010). A.marginale,can affect throughout the life of cattle but disease severity is dependent on the age of the cattle. The clinical disease is less occurs in the calves and disease is very rare under the age of 6 month. The mild type of the disease can occur in the animal, which are between the ages of six month to one year. The acute type of the disease occurs in the animals between the ages ofone to twoyears but this disease is never fatal for the animal between these age groups.Whereas if the acute type of the disease occur in the adult animal that are more than two year aged can face the serious consequences and this disease can cause the death of the cattle with higher percentage (29 to 49%) of mortality (Kocan et al. 2003; Richey. 1991). Beside the animal age at the infection time, onceA. marginale infect animals, the animals become carrier for the infection throughout of animal life, with or without the clinical signs of the infection (Richey et al. 1991). The animal with the strong immune system are recovered from the acute type of anaplasmosis (Palmer et al. 1989). The regular cycle of ten to fourteen days are present in the carrier animals and in this cycle there are more or less number of RBCs are infected with the pathogen(Kocan et al. 2003;Kieser et al. 1990; Viseshakul et al. 2000). For the proper diagnosis of the disease and quality control in livestock, the most important aspect is the preciseor correct detection of pathogen that is spread by the tick.In 1990, with the beginning of the diagnosis with the techniques on the molecular bases the researcher produced more accurate and sensitive techniquesfor pathogenic species determination and these techniques are still used till to date (Ahantarig et al. 2008). The Giemsa staining method is used for the diagnosis of anaplasmosis in bovinesthat can be achievedby finding ofA.marginalein smear of blood from animals that are affected clinically. For the carrier animals or pre symptomatic animals thistechnique is not reliable. In these types of conditions, the serological tests are used for general diagnosis of infection by detection of antibodies. This is confirmed by molecular detection methods. A.marginaleappears as “dark staining blue purple bodies” measuring about 0.3 to 1 µm in length. A. centrale inclusion bodies are well differentiated by their location. Serological diagnosis can be done for the identification of infected cattle, DNA based test can be used for the molecular diagnosis(Stuen et al. 2011). The conventional parasitological technique like Giemsa staining always remained gold standard for diagnosis of Bovine anaplasmosis. Light microscopy of thin blood smears stained with giemsa stain may facilitate demonstration of A. marginale organisms in the erythrocytes (Kocan et al. 2004) .As giemsa staining method is not applicable for the detection of subclinical infection. Therefore PCR can also be used as a diagnostic tool for the detection of A.marginale in this technique primer pairs are used which resulted in the amplification of only their target DNA i.e. A.marginale(Munderloh et al. 2004). In the United states, for the treatment of anaplasmosis, the specific approved compound is the tetracyclines that include chlortetracycline or oxytetracycline(Kuttler. 1980). Oxytetracycline, Chlortetracycline and Tetracycline can be used to treat anaplasma infection in cattle .Among these Oxytetracycline is the most used drug at the dose rate of 10mg/kg body weight. While single dose of imidocarb dipropionte can be used at the dose rate 3mg/kg body weight (De Waal 2000).Imidocarb dipropionate with the dose of 3 milligram/kilogram body weight repidly treat A. marginale(anaplsmosis), on the other hand, same drug with double dose (6 milligram/kilogram body weight) and each dose has gape of two weeks,fail to cure A. marginalecarrier heifers(McHardy & Simpson. 1974). The animals recovered completely with imidocarb dipropionate (Akhter et al. 2010).The E elephantina and Aloemarlothiileaf extracts demonstrated good activity against rickettsia. These 2 plants along with Usanguineaand Rtridentata in certain concoctions are believed to be effective against anaplasmosis (Naidoo 2004).
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According to the economic survey of Pakistan 2013-14, livestock sector contributed about 55.9 and 11.8% to the agriculture and national GDP, respectively.Total cattle population in the year 2013-14 is 39.7 million. Total milk and meat production contributed by cattle is 18,027000 tons and 18,87000 tons respectively (Anonymous 2013-14) . In Pakistan, the chiefhindrance in performance and health of animals are parasitic diseases including tick born. Ticks have been easily grown and reproduce in the optimal climate of tropical weather in Pakistan. In Pakistan, there are huge number of species and genera of tick fauna (Durrani and Shakoori, 2009).
Tick-borne diseases include babesiosis, theileriosis, anaplasmosis, lymedisease . Among these tick-borne diseases anaplasmosis is one of the vital diseases which is responsible for the substantial economic losses in term of high morbidity, mortality and production losses that are decreased in milk, meat and other important livestock productions. Throughout the world, the “tick-borne diseases” are extensivelyspread in sub-tropic and tropicareasand Pakistan also includes in these areas(Khan et al. 2004). Moreover, anaplsamosis is non-contagious disease but also known to be infectious, transmission of which occur through mechanical means that includes the flies or tick bites. As well as different equipment that are using during tattooing or castration, in dehorning and needles also causes transmission (Aubry & Geale. 2011).
Anaplasma marginale (A. marginale) is the major cause of Bovine anaplasmosis and this speciesbelongs to the genus Anaplasma (“Rickettsiales, Anaplasmataceae”). The most important 20 ticks species which cause transmission are Argaspersicus, Boophiludecoloratus, B. microplus, B. annulatus, Dermacentoroccidentalis, D. andersoni, D. variabilis, D.albipictus, Hyalommaexcavatum, Ixodesricinus, Ornithodoroslahorensis, Rhipicephalussimus, R. saniguineus, and R. bursa(Marchette & Stiller. 1982), on the other hand, Boophilusmicroplus found as a major contributor in cause of anaplasmosis (Tick Fever Research Centre, 1996).
The family anaplasmatacease was classified in 1957 of the order Rickettsiale. In the genera Anaplasma, Aegyptianella, Haemobartonella and Eperythrozoon, this family was rationalized (Ristic&Kreier. 1984), and projected a combination based on analysis of genes sequences for protein on surface and ribosomal RNA (16S)(Dumler et al. 2001; Walker & Dumler. 1996). Anaplasma is a rickettsial, obligate intracellular bacteria found in red blood cells. Disease is characterized by fever, anemia and jaundice. Infection is transmitted by ticks or mechanically by biting insects or contaminated hypodermic needles or surgical instruments (Tylor et al. 2007).The most significant parasite is Anaplasma speciesthat is transmitted by at least twenty different species of ticks. But among those all mostly Bmicroplus causes Anaplasmosis (Rajput et al.2005).
A. marginale is etiological agent of bovine anaplasmosis (Bram, 1957; Dumler et al., 2001; Kocan et al., 2000). A. marginale persistently infect both cattle and ticks and help as infection reservoirs(Kocan et al. 2003). Bovine erythrocyte membrane bound parasitophorous vacuoles is the site for replication of this obligate intracellular organisms. Dogs, humans, wild and domesticated ruminants are the species effected by A. marginale. This anaplasma genus is an obligate intracellular parasite of vertebrate hosts. In cattle, theA. marginal, A. phagocytophilum, A. central, and A. bovisare major pathogenic species producing diseases (Inokuma et al. 2007). Researchers suggestedthat in cattle, some unidentified Ehrlichia or Anaplasma species are present (Awadia et al. 2006). For this pathogen, which cause the persistent infection, the reservoirs are generally present in different host like tick or mammalian (Kocan et al. 2004). The wide range of different ruminant can be affected by Anaplasmabecause the species of anaplasma are not considered as strictly specific for any particular host specific(Kuttler. 1984).
The A. marginalecan only be developed inside the RBCs of bovine (Richey et al. 1981). After invading the erythrocyte the tick multiplies and form almost eight initial bodies, they then get enlarge in the outer membrane and give it an appearance of a large dot. The organism gets mature in the outer membrane of RBC and force the infected RBC to get rupture due to infection. By rupturing the outer membrane where it previously multiplied come into the blood stream to infect the other RBCs. On the progression of progresses, more number of erythrocytes is infected by this organism and afterward destruction of erythrocytes occur (Stewart et al. 1981). Upon the clinical signs and symptoms are appear when the 15 percent of the RBCsare affected by the parasite(Radostits et al. 2006). The symptoms of the disease are pyrexia, anemia, icterus, hematuria, anorexia, muscular tremors, dyspnea, depression, lethargy, constipation and yellow colour of mucous membrane (Bram et al. 1983), low milk production, miscarriage and sometime mortality (Alderink and Dietrich. 1981).
The mature RBCs are the site for multiplication after the invading by A.marginale. In the acute case of Anaplasmosis the higher number of RBCs are affected by the parasite for example more the 109 red blood cells per ml and after that the disease is categorized by death, abortion, loss of weight and anemia, and the survival cattle that are infected act as reservoirs for transmission of the disease in the herd level(De Echaide et al. 1998).The haematological profile can be used as useful diagnostic tool in anaplasma infection(Van Wyk et al. 2013).There is significant decrease in packed cell volume (PCV), Hemoglobin (Hb) and erythrocytes count.Serum biochemical analysis showed increased total protein,bilirubin and alanine aminotransferase enzyme (Sharma et al. 2013). A diagnosis ofcattle anaplasmosis may be made tentatively based on geographical location, seasonal variation and presenting clinical signs or necropsy finding in infected animals (Kocan et al. 2010).
A.marginale,can affect throughout the life of cattle but disease severity is dependent on the age of the cattle. The clinical disease is less occurs in the calves and disease is very rare under the age of 6 month. The mild type of the disease can occur in the animal, which are between the ages of six month to one year. The acute type of the disease occurs in the animals between the ages ofone to twoyears but this disease is never fatal for the animal between these age groups.Whereas if the acute type of the disease occur in the adult animal that are more than two year aged can face the serious consequences and this disease can cause the death of the cattle with higher percentage (29 to 49%) of mortality (Kocan et al. 2003; Richey. 1991). Beside the animal age at the infection time, onceA. marginale infect animals, the animals become carrier for the infection throughout of animal life, with or without the clinical signs of the infection (Richey et al. 1991). The animal with the strong immune system are recovered from the acute type of anaplasmosis (Palmer et al. 1989). The regular cycle of ten to fourteen days are present in the carrier animals and in this cycle there are more or less number of RBCs are infected with the pathogen(Kocan et al. 2003;Kieser et al. 1990; Viseshakul et al. 2000).
For the proper diagnosis of the disease and quality control in livestock, the most important aspect is the preciseor correct detection of pathogen that is spread by the tick.In 1990, with the beginning of the diagnosis with the techniques on the molecular bases the researcher produced more accurate and sensitive techniquesfor pathogenic species determination and these techniques are still used till to date (Ahantarig et al. 2008). The Giemsa staining method is used for the diagnosis of anaplasmosis in bovinesthat can be achievedby finding ofA.marginalein smear of blood from animals that are affected clinically. For the carrier animals or pre symptomatic animals thistechnique is not reliable. In these types of conditions, the serological tests are used for general diagnosis of infection by detection of antibodies. This is confirmed by molecular detection methods. A.marginaleappears as “dark staining blue purple bodies” measuring about 0.3 to 1 µm in length. A. centrale inclusion bodies are well differentiated by their location. Serological diagnosis can be done for the identification of infected cattle, DNA based test can be used for the molecular diagnosis(Stuen et al. 2011).
The conventional parasitological technique like Giemsa staining always remained gold standard for diagnosis of Bovine anaplasmosis. Light microscopy of thin blood smears stained with giemsa stain may facilitate demonstration of A. marginale organisms in the erythrocytes (Kocan et al. 2004) .As giemsa staining method is not applicable for the detection of subclinical infection. Therefore PCR can also be used as a diagnostic tool for the detection of A.marginale in this technique primer pairs are used which resulted in the amplification of only their target DNA i.e. A.marginale(Munderloh et al. 2004).
In the United states, for the treatment of anaplasmosis, the specific approved compound is the tetracyclines that include chlortetracycline or oxytetracycline(Kuttler. 1980). Oxytetracycline, Chlortetracycline and Tetracycline can be used to treat anaplasma infection in cattle .Among these Oxytetracycline is the most used drug at the dose rate of 10mg/kg body weight. While single dose of imidocarb dipropionte can be used at the dose rate 3mg/kg body weight (De Waal 2000).Imidocarb dipropionate with the dose of 3 milligram/kilogram body weight repidly treat A. marginale(anaplsmosis), on the other hand, same drug with double dose (6 milligram/kilogram body weight) and each dose has gape of two weeks,fail to cure A. marginalecarrier heifers(McHardy & Simpson. 1974). The animals recovered completely with imidocarb dipropionate (Akhter et al. 2010).The E elephantina and Aloemarlothiileaf extracts demonstrated good activity against rickettsia. These 2 plants along with Usanguineaand Rtridentata in certain concoctions are believed to be effective against anaplasmosis (Naidoo 2004).

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