Molecular detection of Hammondia heydorni in dogs in Mosul city

Through this study 122 individual fecal samples were collected from dogs, with different ages, sexes, and breeding management (stray and pet house hold). Some dogs were with healthy appearances while others were suffered from diarrhea, anorexia, depression, dehydration. Infection was investigated by light microscope after flotation technique by using Sheather's sugar solution. if the sample gives positive to oocyst (H. heydorni or other relative group), were mixed with potassium dichromate solution a rate of 2.5% for the stimulation of sporulation. DNA extraction was done using 2% sarcosyl, pronase E followed by phenol/chloroform extraction then precipitation attempt using ethanol. DNA amplification was attempt using H. heydorni primers JS4 and JS5 n microscope examination give 78 sample positives to the presence of oocyst, while polymerase chain reaction showed total percentage of infection with H. heydorni was 34.6% (27 case give positive reaction to PCR). Dogs less than 6 months of age show high infective rate 17.9%, dogs which suffer from gastrointestinal tract troubles (diarrhea, anorexia and dehydration) gave high infective rate 24.4%. Stray dogs gave high prevalence of infection to H. heydorni 25.6% when compared with pet house hold dogs which recorded 9% percentage of infection. This study is the first recorded H. heydorni in dogs in Mosul city.


‫طفيل‬ ‫عن‬ ‫الجزيئي‬ ‫الكشف‬ ‫ي‬
Definitive hosts become infected only after eating tissue cysts (2,3). Oocyst can excrete by definitive hosts and sporulated within 3 days (4). The infection with H. heydorni of the definitive hosts is characterized by subclinical course, some times the animals suffer from anorexia, severe diarrhea or recurrent diarrhea, unresponsive specially in puppies and immune compromised dogs, sometimes neurological signs have been observed (5).
Distinguishing of oocyst of the relative group of H. heydorni which include Neospora caninum and Toxoplasma gondii is so difficult (6,7), so that the differentiation between oocyst of these species using routine microscopic technique give inexact results (8). In order to detect the infection of H. heydorni a variable technique can be used which include serological and molecular technique (9,10), polymerase chain reaction is the best method for the detection and identification this coccidian parasite (11). The spacer internal transcribed spacer (ITS) can be used as genetic markers to detect this species of parasites (8).
This spacer is varied between H. heydorni and other coccidian parasites so this spacer can be used to discrimination between them (12).

Fecal samples and morphological study of oocysts
This study was achieved in Mosul University, College of Veterinary Medicine, during the period from January 2019 to June 2019. A One-hundred and twenty individual fecal samples were collected from dogs with different ages, sexes, and breeds management (stray and house hold). Some animals healthy in their appearance and while others were suffering from diarrhea, anorexia, depression, dehydration.
The samples were examined to investigate the presence of oocysts by using light microscope after flotation technique was done using Sheather's sugar solution. if the sample gives positive to oocysts (H. heydorni relative group), were add to potassium dichromate solution a rate of 2.5% for the stimulation the sporulation in petri dishes at a room temperature for 10 days, sporulated oocyst were collected and purified by centrifugation and preserved at 4 º C (8).

DNA extraction
A 0.5 ml of each sample was blended with 1.5mL of phosphate-buffered saline, then centrifuged at 14.000 g for 3 minutes. the Precipitate materials were washed 3 times in phosphate buffer saline and combined with 1.5 ml in micro tubes half hour. DNA was extracted with 2% sarcosyl (Merck), pronase E (Roche and with phenol/chloroform extraction then sedimentation attempt accomplished by using ethanol (13), DNA dissolved in RNA, DNA free water and preserve at -20 º C.

DNA amplification
H. heydorni primers designed according to ITS1 sequences. The primers JS4 (5 -CGA AAT GGG AAG TTT TGT GAA C-3) and JS5 (5-CAG CAG CTA CAT ACG TAG A-3) (Sigma Aldrich, Germany). reactions was done at 25 uL, the reaction include 25-100 pmol of two primers, dNTPs, buffer 1-2U Taq polymerase reaction mix contain 25-100 pmol of each primer, reaction buffer, dNTPs, and 0.5-2U of Taq DNA polymerase (D 1806 Sigma-Aldrich) and 1 ul of DNA sample (8), ten amplification attempt in thermocyclers at the program listed in table 1 were applied, size of the amplification products was 270 bp, then those products detected by using electrophoresis in a 2% agarose gel and ethidium bromide.

Results
The results of sporulation and light microscope examination give 78 sample positive to the presence of oocyst, the DNA amplification were positive which revealed as band in 270 bp (Figure 1), the results showed total percentage of infection with H. heydorni is 34.6 % (27 case give positive reaction to PCR), animals less than 6 months of age recorded high infective rate 17.9 % while the lowest percentage of infection showed in animals more than 1 year of age ( Table 2). The dogs which suffer from gastrointestinal tract troubles included diarrhea, anorexia and dehydration gives high infective rate 24.4% when compare with healthy dogs which showed 10.2% percentage of infection (Table 3).    (Table 4).

Discussion
There are no studies indicating the presence H. heydorni in dogs in Mosul city, so this study was done to determine the H. heydorni based on the detection of parasite DNA.  (16) reported high prevalence of infection in dogs 1-4 years old and lower percentage in older dogs, (17) reported affected dogs are generally less than six months old, the reasons of increase the percentage of infection younger animals is the nature infection of H. heydorni in the definitive hosts which take subclinical form specially in younger and immunosuppressed animals (5), younger dogs immune system can exposed to several etiological microorganisms which cause immune suppression which lead to allow H. heydorni to invade gastrointestinal tracts infection and appearing clinical signs.
High infective rate in animals suffer from gastrointestinal tract troubles, Abel (1) report that the infection of H. heydorni is increasingly becoming associated with diarrhea and gastrointestinal tract problem, Webb (18) found the oocyst of H. heydorni in feces of a dog suffer from intermittent bouts diarrhea this dog already been treated with immunosuppressive treatment. Stray dogs give higher prevalence of infection with H. heydorni when compared with pet house hold dogs, the reason of that is due to periodic vaccination program and preventive medicine to another parasitic disease in pet housed dogs, which lead to decrease the possibility of immune suppression, another reason is the use of commercial dog food to feeding the pet dogs while stray dogs are free feeding (19). In contrast to the stray dogs, pet dog health care is crucial in terms of veterinary follow-up with periodic antiparasitic treatment (20).
The role of veterinarian visits has an important role in the reduction of disease occurrence. Our results confirm this hypothesis, since recurrent visits to the veterinary clinics by pet dog owners has a positive impact on the health of their animals.

Conclusions
Hammondia heydorni was affected dogs more than 1 year of age and in dogs suffer from GIT troubles and this parasite was first recorded in dogs in Mosul city.