Clinico-biochemical Responses of Dogs to Experimental Infection with Babesia Canis

Introduction species of Babesia parasite present in an area, the common tick vectors reported to serve in transmission Canine babesiosis caused by different Babesia of canine babesiosis include Rhipicephalus species, species is a protozoan tick-borne disease with Haemaphysalis species and Dermacentor species [7]. worldwide distribution and of global significance. A non-vectored transmission through blood exchange Historically, Babesia infection in dogs was identified during fighting and biting has also been reported, as based on morphologic appearance in the erythrocyte, seen with some aggressive breeds of dogs like the Pit until recently revolutionized by the introduction of Bull terriers [8, 9]. Other route of transmission is by newer techniques like serological [1, 2] and molecular transplacental route, as reported in puppies as young as techniques like polymerase chain reaction [1-3]. 3 days old [ 8, 10]. Morphological analysis has demonstrated three Most of the abnormalities seen in Babesia canis distinct sub species of Babesia canis viz., Babesia infection are related both to the severity of the disease canis rossi, Babesia canis canis and Babesia canis and the degree of hypoxia. The severity ranges from a vogeli [4]. The three sub species are morphologically mild asymptomatic infection to a severe hyperacute indistinguishable and demonstrate tremendous shock-related haemolytic crisis [11] with generalized variations in clinical signs, geographical distribution organ failure resulting to death within few days of and vector specificity [5, 6]. infection. The disease is generally characterized with The parasite has been reported to be transmitted regenerative haemolytic anaemia [12] and/or throm-by an array of well-described Ixodid tick vectors from bocytopenia [13,14], and usually accompanied with different geographical locations. Depending on the inappetence to complete anorexia, fever, increase in pulse and respiratory rates, pallor of the mucous membranes of the mouth and eyelids, lethargy, spleno-megaly, water-hammer pulse, icterus and pigmenturia, Abstract Aim: A study on the clinical and biochemical parameters of Nigerian dogs experimentally infected with Babesia canis was conducted. Materials and Methods: A total of ten naive dogs of both sex and aged between 6 months to 1 year, were used for the study. They were divided into two groups of five each-A (control) and B (infected). Dogs in group B were infected with 1ml of Babesia canis positive infectious inoculum, while those in group A were left as uninfected control. Following infection, clinical and biochemical responses were analyzed in group B and compared with those in group A. Results: Clinical signs …


Introduction
species of Babesia parasite present in an area, the common tick vectors reported to serve in transmission Canine babesiosis caused by different Babesia of canine babesiosis include Rhipicephalus species, species is a protozoan tick-borne disease with Haemaphysalis species and Dermacentor species [7].worldwide distribution and of global significance.
A non-vectored transmission through blood exchange Historically, Babesia infection in dogs was identified during fighting and biting has also been reported, as based on morphologic appearance in the erythrocyte, seen with some aggressive breeds of dogs like the Pit until recently revolutionized by the introduction of Bull terriers [8,9].Other route of transmission is by newer techniques like serological [1, 2] and molecular transplacental route, as reported in puppies as young as techniques like polymerase chain reaction [1-3].
3 days old [ 8,10].Morphological analysis has demonstrated three Most of the abnormalities seen in Babesia canis distinct sub species of Babesia canis viz., Babesia infection are related both to the severity of the disease canis rossi, Babesia canis canis and Babesia canis and the degree of hypoxia.The severity ranges from a vogeli [4].The three sub species are morphologically mild asymptomatic infection to a severe hyperacute indistinguishable and demonstrate tremendous shock-related haemolytic crisis [11] with generalized variations in clinical signs, geographical distribution organ failure resulting to death within few days of and vector specificity [5,6].
infection.The disease is generally characterized with The parasite has been reported to be transmitted regenerative haemolytic anaemia [12] and/or thromby an array of well-described Ixodid tick vectors from bocytopenia [13,14], and usually accompanied with different geographical locations.Depending on the inappetence to complete anorexia, fever, increase in pulse and respiratory rates, pallor of the mucous membranes of the mouth and eyelids, lethargy, splenomegaly, water-hammer pulse, icterus and pigmenturia, depending on the specie of parasite.Clinico-pathologic Experimental animal inoculation: A total of ten dogs abnormalities reported before include hypoglycemia, (mongrels) of both sex were randomly separated into acid-base disturbance, azoturia and alterations in the two experimental groups (A & B) of five dogs each.levels of the liver and kidney enzymes and the acute Group B were inoculated with 1ml of the inoculums 4 phase protein [15,16].Common biochemical findings containing 10 parasitized erythrocytes per ml (1-4 are increase in the serum activity of aspertate parasites per microscopic field), while those in group A aminotransferase (AST) and alanine aminotransferase were left as uninfected (control).(ALT), hyperbilirubinaemia, hypoalbuminaemia, On the day of challenge infection, and for a period electrolyte and acid-base abnormalities (mostly of 14 days, the vital parameters (temperature, pulse and hypokalemia, hypercloremia and metabolic acidosis) respiratory rates) were taken on alternate days.The [17][18][19][20].This study was designed to determine the dogs were observed daily for clinical signs.Special clinico-biochemical changes associated with canine attention was given to behaviour, spleen size, size of babesiosis under experimental condition using African lymph nodes and colour of the mucous membranes of (Nigeria) dogs as models.
mouth and eyelid as described previously [22].Blood samples for biochemical analysis were collected daily

Materials and Methods
from dogs in group B (infected) and compared with Ethical consideration: All procedures were performed those in group A (uninfected).Plain sample bottles with the approval of the Institutional Animal Care and were used for biochemical analysis, fluoride bottles Use Committee of the University of Maiduguri, were used for glucose determination, while EDTA Nigeria.
bottle was used for haematological analysis.A total of ten naive dogs (Mongrels) of age 6 month Biochemical parameters were determined using a to 1 year and of both sex were used for the experiment, biochemical auto-analyzer (Vital Scientific Microlab following physical and clinical examination to 300, Netherland).Analyses were also performed using ascertain that the dogs are clinically free from Babesia the standard methods and original reagents of the infection before experimental infection with Babesia manufacturers.canis.The experimental dogs were fed daily with Following manifestation of clinical sign between day 2-14 and after the experiment at day 21; dogs in standard dog feed and a potable drinking water ad group B (infected) were treated with a single deep libitum.
intramuscular injection of Diminazeneaceturate at a Preparation of infection inoculum: A puppy of less dose rate of 3.5mg/kg body weight.than six months of age was splenectomised following Statistical analysis: Data collected were subjected to physical and clinical examination to ascertain that the one-way analysis of variance (ANOVA) and p < 0.05 dog is free from any haemoparasites.Thereafter, regarded as significant [23].screened B. canis positive blood from an infected dog was used to inoculate the splenectomised puppy.

Results
Heparinized blood samples were obtained daily for the Following infection, the major clinical signs seen determination of PCV and parasitaemia.During patent were inappetence (100%) two days post infection, parasitaemia, peripheral blood was collected in citrate followed by anorexia (40%) and lethargy (100%) on solution and used to prepare the challenge inoculum third day post infection.On the fourth day, the major according to the protocol described earlier [21].The clinical signs recorded in addition to the former ones puppy then served as the donor animal/parasite bank were pallor of mucous membranes of the mouth and for subsequent inoculation into the experimental eyelid (100%) and emaciation (100%); on day five, animals.muscle tremor (20%), respiratory distress (10%); on infection, reaching its peak between day 6 and14 post day six, nervousness (20%), drooling salivation (20%) infection (Fig. 1b) as compared with the uninfected and haemoglobinuria (80%); on day seven, mucoid (control) group which showed with undulating pattern (Fig. 1a).The body temperature of three dogs in group ocular discharge (40%); followed by death of one dog B (infected) was restored to normal values within 2-3 (1%) at day eight post infection.Other clinical signs day post chemotherapeutic treatment, even though recorded between day 1-14 post infection included temperature in some dogs had started resolving prior ascites, edematous swelling of the whole body and hair to treatment.However one dog still showed a erection (Table-1).
relapse/undulating fever post treatment.There was a significant rise (p < 0.05) for all the There was a transient rise in the pulse reaching a vital parameters during the early course of infection peak of 196 per minute in 4 (80%) dogs (Fig. 2b); while compared with the control (Table-2).All infected dogs 1 dog (20%) recorded fall in pulse to as low as 27 per developed fever and it was first recorded two days post     Each minute; as compared with the uninfected (control) in appetite and lethargy may be as a result of fever.The group that showed undulating patterns ranging from fever was as a result of the presence of parasites in the 64-90 beats per minute (fig.2a).Following Chemo-circulating blood as foreign bodies thereby triggering therapeutic treatment, there was a sharp drop in pulse the release of vasoactive amines like cytokines.rate in dogs recording higher pulse to as low as 55 per Edematous body swelling caused by buildup and minute then followed by a transient raise to normal retention of abnormal amounts of fluid in the body may value within 3-4 days.
be due to hypoproteinaemia and hypoalbuminaemia Generally, there was a transient raise in the caused by a decrease in plasma proteins and the lack of respiratory rate of all infected dogs reaching a peak of capillary colloidal osmotic pressure or the pressure 90 breaths per minute (figure 3b) when compared with needed to pull back the fluid into the vascular space.the uninfected (control) group that ranges between 12-Usually, the plasma protein is what causes the fluid to 34 cycles per minutes (figure 3a).But, following be pulled back into the capillaries from the tissue chemotherapeutic treatment an undulating pattern of spaces.So, when there is a decrease in plasma proteins breath has been recorded with an initial raise, then as a result of an increased intravascular hemolysis of followed by a drop in most (90%) of the dogs within 2-red blood cells, this pressure decreases and fluid starts 4 days post treatment, before restoring to normal.
to accumulate in the tissue spaces.The mean ± standard deviation (range) values of On the other hand, the liver cannot synthesize serum enzymes and metabolites of the experimental these plasma proteins without certain amino acids dogs are shown in Table-3.There was a significant (p < which can only be achieved by dietary intake.With 0.05) rise in alkaline phosphatase (ALP) in infected anorexia and mal-absorption, there is insufficient dogs (58.50±1.4)compared with the control group intake of these amino acids resulting to malnutrition.(51.67±1.6).Also, there was a significant rise (p < Although many cases of malnutrition in dogs are 0.05) in the alanine amino transferase (ALAT) values connected to neglect or an insufficient diet, parasites of infected group compared with the control values may also create malnutrition in canines resulting to (8.27±2.0).However, the mean± standard deviation emaciation, which is the easiest sign of malnutrition as values for creatinine of infected group (78.10±1.2) was seen in this research.significantly lower (p < 0.05) than that of the control Biochemical profile abnormalities in Babesia (91.73±1.3).Similarly, the glucose values for infected canis infection are related to both the severity of the group (3.80±2.3)were significantly (p < 0.05) lower disease and the degree of hypoxia as previously than that of control (5.35±2.1).

Discussion
increased serum activity of alkaline phosphatase and This study clearly shows that canine babesiosis alanine amino transferase activity in majority of the associated with Babesia canis runs in two courses infected dogs.This finding concurs with the previous (acute and chronic) in West African dogs with the first reports [17][18][19][20]25].Alkaline phosphatase (ALP) values clinical sign being recorded two days post infection, in plasma were affected after challenge infection with a followed by death 8 days post infection, and others significant (p < 0.05) rise in ALP values of infected show chronic convalescent forms.The major clinical dogs compared to the uninfected (control) group.signs seen in this study were associated with intra-There was a clear trend of increasing values of vascular haemolysis of the red blood cells in circulation, ALP from 2-4 folds in most of the infected dogs (80%) resulting in anaemia depicted as the pallor of mucous starting at day four post infection.While 20% of the membrane of the mouth and eyelid.The sudden change dogs showed decrease in values below normal, though the difference is not much.This finding agrees with the script; AWM revised the manuscript; JL analyzed the report by [24] from Warsaw, Poland, who reported that data.All authors read and approved the final the primary abnormalities found in affected dogs have manuscript.increased serum activity of transaminases and alkaline isolation; Dr. O.O.Okubanjo for assisting in parasite and biochemical changes in blood samples of some identification, the entire staff of the Veterinary Teaching naturally infected dogs and reported that the bioche-Hospital ABU Zaria and Protozoology laboratory, mical values were within normal range but serum Department of Veterinary Parasitology and Entomobiochemistry varied for each dog.Even though, report logy, Ahmadu University Zaria for providing all the from India was based on natural infection and that from necessary assistance during parasite isolation.The Thailand was a retrospective study, the reason for such authors are also thankful to Management of Veterinary difference is not clear.
Teaching Hospital, University of Maiduguri and There was a decrease in serum activity of Parasitology Laboratory, Department of Veterinary creatinine in most of the infected dogs as opposed to the Microbiology and Parasitology, Faculty of Veterinary report by [25] whose work was conducted under Medicine, University of Maiduguri for their assistance natural condition.The initial decrease in creatinine during the course of the research and partial funding.values may be as a result of dilution of blood.However, some few dogs showed an increase.

A
= Control, B = infected, m = minutes, SD= standard deviation.Column variables with different superscripts (a & b) are statistically significant (p<0.05).Mean ± SD (range) of serum enzymes and metabolites of experimental dogs.ALP = Alkaline phosphatase, ALAT = Alanine amino transferase.Group A = Uninfected untreated (control), Group B = Infected group, SD = Standard deviation.Column variables with different superscripts (a & b) are statistically significant (p<0.05).

Figure- 1a .
Figure-1a.Temperature of dogs in group A (control).Figure-1b: Temperature responses of dogs in group B (infected).Each point on the horizontal axis represent 2 days.

Figure- 1b :
Figure-1a.Temperature of dogs in group A (control).Figure-1b: Temperature responses of dogs in group B (infected).Each point on the horizontal axis represent 2 days.

Figure- 2a .
Figure-2a.Pulse rates of dogs in Group A (Control) Figure-2b: Pulse rates of dogs in Group B (infected). point on the horizontal axis represent 2 days.

Figure- 3a .
Figure-3a.Respiratory rates of dogs in Group A (Control) Figure-3b: Respiratory rates of dogs in Group B (infected).Each point on the horizontal axis represent 2 days.

Table - 2
. Mean± SD (range) of vital parameters of experimental dogs.