Molecular diagnosis of granulocytic anaplasmosis and infectious cyclic thrombocytopenia by PCR-RFLP.

The research article describes a molecular diagnosis approach for identifying anaplasmosis, a tick-borne disease, using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). The study took place in Sardinia and explored the prevalence of this disease in humans, dogs, and horses, later comparing findings with other global regions using a phylogenetic analysis.

Understanding the Disease

• Anaplasma phagocytophilum is the tick-borne pathogen causing granulocytic anaplasmosis in various animals and humans. Symptoms and pathogenicity can vary widely.
• This disease is responsible for tick-borne fever in animals such as ruminants (like cows and sheep), equine granulocytic ehrlichiosis in horses, canine granulocytic ehrlichiosis in dogs, and human granulocytic ehrlichiosis. Cases have been registered in many countries.

The Study and its Methodology

• This study focused on Sardinia as it had a significantly higher prevalence of the disease among humans compared to the rest of Italy.
• Researchers collected blood samples from dogs and horses on the island and extracted genomic DNA from the buffy coat – the layer of separated blood components that contain a rich concentration of white blood cells and platelets.
• The genetic material was then amplified using a heminested polymerase chain reaction (PCR) which used the groEL gene primers.
• The first PCR reaction created a 624-base pair fragment for both Anaplasma phagocytophilum and Anaplasma platys. The second reaction created different fragments for the two pathogens, that were 573-base pairs and 515-base pairs long respectively.

Findings and Analysis

• In the samples tested, six proved to be PCR positive for Anaplasma phagocytophilum (3 each from dogs and horses), while one dog was PCR positive for Anaplasma platys
• A phylogenetic analysis of Anaplasma phagocytophilum sequences gathered from the United States, Slovenia, Switzerland, Germany, the United Kingdom, Austria, and the Czech Republic was conducted for comparison purposes.
• Surprisingly, the analysis showed that the strains of the bacteria found in Sardinia were closer to the American strains, which have higher mortality rates, rather than the other European strains.

Significance of Research

• This finding has the potential to impact the understanding of the disease’s transmission, genetic diversity, and the potential impact on public health across different regions.