The response of the pituitary-adrenal and pituitary-thyroidal axes to the plasma glucose perturbations in Babesia canis rossi babesiosis

This prospective, cross-sectional, interventional study was designed to determine the association between the hormones of the pituitary-adrenal and pituitary-thyroid axes and other clinical parameters with the blood glucose perturbations in dogs with naturally occurring Babesia canis rossi babesiosis. Thirty-six dogs with canine babesiosis were studied. Blood samples were obtained from the jugular vein in each dog prior to treatment at admission to hospital and serum endogenous adrenocorticotrophic hormone (ACTH), pre-ACTH cortisol, thyroxine, free thyroxine and TSH concentrations were measured. Immediately thereafter each dog was injected intravenously with 5 microg/kg of ACTH (tetracosactrin). A 2nd blood sample was taken 1 hour later for serum post-ACTH cortisol measurement. Three patient groups were recruited: hypoglycaemic dogs (glucose < 3.3 mmol/l, n = 12); normoglycaemic dogs (glucose 3.3-5.5 mmol/l, n = 12); hyperglycaemic dogs (glucose > 5.5 mmol/l, n = 12). Basal and post-ACTH serum cortisol concentrations were significantly higher in hypoglycaemic dogs, whereas body temperature, serum thyroxine and free thyroxine were significantly lower in hypoglycaemic dogs. Haematocrit was significantly lower in both hypo-and hyperglycaemic dogs compared with normoglycaemic dogs. Low blood glucose concentrations were significantly associated with high basal and post-ACTH cortisol concentrations and with low serum thyroxine and free thyroxine concentrations in dogs suffering from B. canis rossi babesiosis.     

Adrenal response to the low dose ACTH stimulation test and the cortisol-to-adrenocorticotrophic hormone ratio in canine babesiosis

This prospective, interventional, case-controlled study sought to determine the association between adrenocortical function and mortality in dogs with naturally occurring Babesia rossi babesiosis. Sixty-eight dogs with canine babesiosis were studied and fifteen normal dogs were used as controls. Blood samples were obtained from the jugular vein in each dog prior to treatment, at admission to hospital, for the measurement of basal plasma ACTH (adrenocorticotrophic hormone) and serum cortisol concentrations. Immediately thereafter, each dog was injected intravenously with 5 microg/kg of ACTH (tetracosactrin). A second blood sample was taken 1h later for serum ACTH-stimulated cortisol measurement and the resultant calculation of delta cortisol by subtracting basal from ACTH-stimulated cortisol. Diagnosis of babesiosis was confirmed by polymerase chain reaction (PCR) and reverse line blot (RLB). Three outcomes were defined: hospitalization with subsequent death (n=4); hospitalization followed by recovery (n=48); and treatment as an outpatient (n=16). Basal cortisol, but not ACTH-stimulated cortisol, was significantly higher in patients compared to control dogs. Basal- and ACTH-stimulated serum cortisol concentrations were significantly higher in the dogs that died, compared to hospitalized dogs that survived and compared to dogs treated as outpatients. There was no significant difference in delta cortisol concentrations or cortisol to ACTH ratios across outcome groups in dogs suffering from B. rossi babesiosis However, dogs with delta cortisol concentrations below 83 nmol/l had significantly higher cortisol to ACTH ratios compared to dogs with delta cortisol concentrations above 83 nmol/l. These findings of increased basal- and ACTH-stimulated cortisol and increased cortisol to ACTH ratios confirm the absence of adrenal insufficiency and concur with those in human malaria.     

Serum concentration of some acute phase proteins in naturally occurring canine babesiosis: a preliminary study

BACKGROUND: Serum concentrations of acute phase proteins can provide valuable diagnostic information in the detection, prognosis, or monitoring of disease. Information available on the acute phase response in naturally occurring canine babesiosis is limited. OBJECTIVE: The purpose of this investigation was to retrospectively evaluate serum concentrations of haptoglobin, C-reactive protein, and ceruloplasmin in dogs naturally infected with Babesia canis. METHODS: Haptoglobin, C-reactive protein, and ceruloplasmin concentrations were measured in serum samples from dogs with uncomplicated (n = 6) and complicated (n = 1) babesiosis and compared with 6 healthy dogs. RESULTS: Serum C-reactive protein and ceruloplasmin concentrations were significantly higher in dogs with babesiosis; however, serum haptoglobin concentration was significantly lower compared with control dogs (P <.01). CONCLUSIONS: Results of this study suggest that acute phase protein concentrations could be beneficial in the diagnosis and determination of the severity of babesiosis in dogs.     

A field trial evaluation of the prophylactic efficacy of amitraz-impregnated collars against canine babesiosis (Babesia canis rossi) in South Africa

South African canine babesiosis caused by Babesia canis rossi is a common clinical disease in dogs in South Africa and remains a significant cause of domestic dog mortality. To determine whether tick-repellent, 9% amitraz-impregnated tick collars (Preventic-Virbac) could prevent tick-borne exposure to B. canis rossi, 50 dogs were assigned to two groups. Group 1 (20 dogs), polymerase chain reaction (PCR)--and reverse line blot (RLB)-negative for B. canis rossi, were fitted with amitraz collars and blood samples collected monthly, over a 6-month period, and analysed for B. canis rossi. Group 2 (30 dogs) included 5 dogs selected on a month-by-month basis from a population of dogs from the same geographical area as the group 1 dogs, but with no history of previous tick control, which were blood-sampled together with the treatment group and analysed for B. canis rossi by PCR and RLB, to serve as the control group. Eight of the 30 control dogs (26.6%) were PCR/RLB positive for B. canis rossi, indicating high pathogen exposure during the trial period. All twenty of the treatment group dogs remained negative for B. canis rossi throughout the 6 months of the trial. These results suggest that the use of amitraz-impregnated collars had a significant effect on reducing infection with B. canis rossi.     

Confirmation of occurrence of Babesia canis vogeli in domestic dogs in South Africa

The prevalence of Babesia infections in domestic dogs in South Africa was studied using reverse line blot hybridization and 18S sequence analysis. Babesia canis vogeli was confirmed for the first time in domestic dogs in South Africa. Out of a total of 297 blood samples collected from domestic dogs in Bloemfontein, East London, Johannesburg, Durban and from the Onderstepoort Veterinary Academic Hospital, 31 were positive for Babesia canis rossi, whereas B. c. vogeli was detected in 13 dogs. None of the dogs carried both parasites. The detection of B. c. vogeli has implications with regard to prevalence and varied clinical manifestation of canine babesiosis in South Africa.     

Detection and molecular characterization of a novel large Babesia species in a dog

Babesia canis has generally been considered the only large Babesia to infect dogs. Here we describe the molecular characterization of a large Babesia species that was detected in the blood and bone marrow of a dog with clinical and hematological abnormalities consistent with babesiosis. Analysis of the 18S rRNA genes revealed a unique sequence that shared 93.9% sequence identity with B. bigemina and 93.5% sequence identity with B. caballi, compared to 91.2-91.6% identity with B. canis canis, B. c. vogeli, and B. c. rossi. Cross-reactive antibodies against B. canis, B. gibsoni (Asian genotype), or B. gibsoni (California genotype) antigens were not detected in acute or convalescent serum samples. The dog was treated with imidocarb diproprionate, which resulted in the resolution of clinical signs, and subsequently Babesia DNA was not detectable by PCR in post-treatment samples. The organism described in this report represents a genetically unique large Babesia sp. and is the eighth genetically distinct piroplasm capable of infecting the domestic dog.     

Determination of thyroxine, triiodothyronine, and cortisol changes during simultaneous adrenal and thyroid function tests in healthy dogs

Changes in thyroxine (T4), triiodothyronine (T3), and cortisol during a combined adrenal (dexamethasone suppression/adrenocorticotrophic hormone response test) and thyroid function tests (thyroid-stimulating hormone [TSH] response test) were determined in 20 healthy hospitalized pet dogs. The effect of dexamethasone on T4 and T3 changes was evaluated during a simultaneous TSH response/dexamethasone suppression adrenocorticotrophic hormone response test. Greater ranges in basal cortisol concentrations and slower changes after dexamethasone was administered were observed in healthy pet dogs kenneled in a hospital setting than those reported for conditioned laboratory dogs. Pet dogs were observed to demonstrate cortisol suppression more reliably at 4 hours than at 2 hours after dexamethasone was administered. Dexamethasone had no effect on the response to TSH as assessed by T4 and T3 assays, thus supporting the validity of combining adrenal and thyroid response tests in a 5-hour period.     

Vaccination of dogs against heterologous Babesia canis infection using antigens from culture supernatants

Soluble parasite antigens (SPA) from European Babesia canis can be used to protect dogs against a homologous but not heterologous challenge infection. In this study it is shown that when dogs are vaccinated with a mixture of SPA from both, a European B. canis isolate and a South African Babesia rossi isolate, protective immunity against heterologous B. canis infection is induced. Three groups of five beagle dogs each were vaccinated twice with graded doses of SPA derived from in vitro cultures of B. canis and B. rossi, with a 3-week interval. Saponin was used as adjuvant. Three weeks after booster vaccination immunological responsiveness against heterologous B. canis antigen was measured by seroconversion against infected erythrocytes and lymphocyte transformation using SPA. Upon vaccination dogs produced antibodies against infected erythrocytes and lymphoblastogenic responses against SPA in a dose-dependent manner. Dogs were then challenged with heterologous B. canis parasites. Dogs appeared to be protected against challenge infection, which was reflected in less severe decrease of packed cell volume (PCV) and reduced clinical signs. The level of protection to clinical signs (but not excessive PCV drop) was related to the level of SPA in plasma and spleen size, and not related to peripheral parasitaemia. The results suggest that vaccination with this bivalent vaccine primes T-helper cells that recognise common epitopes on SPA from an antigenically distinct B. canis isolate. These cells provide the essential Th signal to mount an effective and timely antibody response against SPA and parasites or parasitised erythrocytes, which prevents the further development of clinical babesiosis.     

Immunity against Babesia rossi infection in dogs vaccinated with antigens from culture supernatants

Soluble parasite antigens (SPA) from different Babesia species have been shown earlier to induce protective immunity when used as vaccine. However, initial attempts to produce such vaccine against Babesia rossi infection using SPA from B. rossi culture supernatants were not or only partially successful. Here we show that when dogs were vaccinated with a vaccine comprising SPA from B. rossi combined with SPA from Babesia canis protective immunity against experimental challenge infection was induced. Immunity was reflected in reduced clinical signs that resolved spontaneously, and reduction of parasitaemia and SPA in the blood. Not a single infected erythrocyte could be found in blood smears of dogs that had been repeatedly boosted (three vaccinations in total). In contrast, three out of four control dogs required chemotherapeutic treatment to prevent death. The fourth control dog showed a transient parasitaemia that resolved spontaneously. Vaccination did not prevent the development of a transient anaemia. It is concluded that a vaccine containing a mixture of SPA obtained from in vitro culture supernatants of B. rossi and B. canis induces protection in dogs against heterologous challenge infection with B. canis (as shown before) or B. rossi.     

Three groups of Babesia canis distinguished and a proposal for nomenclature

Summary

Two stocks of large Babesiae from dogs originating in France, transmitted by Dermacentor reticulatus, two from North Africa, having Rhipicephalus sanguineus as vector, and one from South Africa, transmitted by Haemaphysalis leachi, were compared in cross‐immunity tests in dogs and in the indirect fluorescent antibody test (IFAT).

The French and North African stocks did not immunise against the South African one, while the North African stocks did not protect against a French one. The South African stock partially protected against a French one.

The three groups could be clearly distinguished in the IFAT These differences have practical implications for existing and future vaccines against canine babesiosis and for the serological diagnosis of atypical and chronic cases.

It is proposed to use a trinomial system of nomenclature for these groups: Babesia canis canis (Piana and Galli‐Valerio, 1895), Babesia canis vogeli Reichenow, 1937, and Babesia canis rossi (Nuttall, 1910), having Dermacentor, Rhipicephalus and Haemaphysalis ticks as their vectors respectively.     

Nitric oxide metabolites in naturally occurring canine babesiosis.

Babesiosis, caused by the virulent haemoprotozoan parasite Babesia canis rossi, is an important disease of dogs in South Africa. The nitric oxide metabolites, nitrate and nitrite (collectively termed reactive nitrogen intermediates or RNIs) were measured in admission sera from dogs in a babesiosis-endemic area. Five groups were prospectively studied: mild uncomplicated (n=9), severe uncomplicated (severe anaemia) (n=10) and complicated babesiosis (n=11); and two groups of healthy aparasitaemic dogs: endemic controls from the study area (n=10) and experimental dogs kept in tick-free conditions (n=10). Four measures of RNI production were studied: (i) serum RNI; (ii) serum RNI/creatinine ratio; (iii) fractional clearance of RNI (FC(RNI)); (iv) fractional excretion of RNI (FE(RNI)). Marked elevations of serum RNI occurred in only two dogs, both in the severe uncomplicated group. The highest concentration (log value 5.29 micromol/l) was in a dog that died, but concentrations in the other four dogs that died were unremarkable (0, 0.34, 1.66 and 2.64 micromol/l). Age, appetite and free serum haemoglobin were significant covariates for measures of RNI production. There were no significant differences among the babesiosis groups for serum RNI. Adjustment for creatinine had minor effects on the results. All babesiosis groups had significantly higher serum RNI and RNI/creatinine than the tick-free control group, but did not differ from the endemic controls except for the severe uncomplicated group, which had higher RNI/creatinine. The complicated group had significantly lower FC(RNI) and FE(RNI) than all other groups, except for the tick-free control group, which had similar FE(RNI). The results indicate that, in an endemic area, measures of RNI production are unlikely to be useful indicators of severity or outcome in canine babesiosis.     

Plasma insulin concentrations in hypoglycaemic dogs with Babesia canis rossi infection

Hypoglycaemia has been identified as a life-threatening metabolic complication in almost 20% of severely ill dogs suffering from babesiosis due to Babesia canis rossi infection, and has been correlated with mortality. Hyperinsulinaemia as a result of inappropriate insulin secretion may precipitate hypoglycaemia, and has been suggested as a possible cause of hypoglycaemia in human and murine malaria. This prospective, cross-sectional, observational study, including 94 dogs with naturally occurring virulent babesiosis, sought to identify the presence of inappropriate insulin secretion in hypoglycaemic canine babesiosis. Pre-treatment jugular blood samples were collected for simultaneous determination of plasma glucose and insulin concentrations. Animals were retrospectively divided into three groups: hypoglycaemic (BG<3.3 mmol/L; n=16), normoglycaemic (BG 3.3-5.5 mmol/L; n=62), and hyperglycaemic (BG>5.5 mmol/L; n=16). The median insulin concentrations for the hypoglycaemic, normoglycaemic, and hyperglycaemic groups were 10.7 pmol/L, 10.7 pmol/L, and 21.7 pmol/L, respectively. Statistical analysis revealed no significant difference in insulin concentration between the three groups. Additionally, the median insulin concentration in the hypoglycaemic and normoglycaemic groups was below the detection limit of the assay, suggesting that insulin secretion was appropriately low (i.e. undetectable) in these cases. Only two dogs had inappropriately elevated insulin concentrations. One of these dogs was hypoglycaemic. We conclude that hyperinsulinaemia is an infrequent cause of hypoglycaemia in virulent canine babesiosis. Other causes of hypoglycaemia, such as increased glucose consumption, depletion of hepatic glycogen stores, and hepatic dysfunction with impaired gluconeogenesis, are speculated to play more important roles in the pathophysiology of hypoglycaemia in canine babesiosis.     

Detection and molecular characterization of Babesia canis vogeli from naturally infected dogs and Rhipicephalus sanguineus ticks in Tunisia

Canine babesiosis, caused by intra-erythrocytic Babesia, is a tick-borne disease of worldwide importance. No information on canine babesiosis has been documented in Tunisia. Detection and analysis of Babesia species from naturally infected dogs and ticks recovered from dogs were attempted by reverse line blot hybridization and nucleotide sequence analysis based on 18S rRNA gene. Out of 180 blood samples collected from domestic dogs in 4 villages situated in different bioclimatic zones, 12 were positive for Babesia canis vogeli. In addition, a total of 160 Rhipicephalus sanguineus were analysed; only one male was infected by B. canis vogeli. This is the first report on the detection of DNA belonging to B. canis vogeli in domestic dogs and in R. sanguineus in Tunisia.     

Changes in serum thyroxine and thyroid-stimulating hormone concentrations in epileptic dogs receiving phenobarbital for one year

A multicentric prospective study was conducted to monitor the effect of phenobarbital on serum total thyroxine (T4) and thyroid-stimulating hormone (TSH) concentrations in epileptic dogs. Serum T4 concentrations were determined for 22 epileptic dogs prior to initiation of phenobarbital therapy (time 0), and 3 weeks, 6 months, and 12 months after the start of phenobarbital. Median T4 concentration was significantly lower at 3 weeks and 6 months compared to time 0. Thirty-two percent of dogs had T4 concentrations below the reference range at 6 and 12 months. Nineteen of the 22 dogs had serum TSH concentrations determined at all sampling times. A significant upward trend in median TSH concentration was found. No associations were found between T4 concentration, dose of phenobarbital, or serum phenobarbital concentration. No signs of overt hypothyroidism were evident in dogs with low T4, with one exception. TSH stimulation tests were performed on six of seven dogs with low T4 concentrations at 12 months, and all but one had normal responses. In conclusion, phenobarbital therapy decreased serum T4 concentration but did not appear to cause clinical signs of hypothyroidism. Serum TSH concentrations and TSH stimulation tests suggest that the hypothalamic-pituitary-thyroid axis is functioning appropriately.     

The scintigraphic evaluation of the pulmonary perfusion pattern of dogs hospitalised with babesiosis

The possibility of coagulopathy in Babesia canis rossi infections in the canine patient has been suggested in the literature, but minimal work has been done to evaluate the clinicopathological nature of it in further detail. Pulmonary thromboembolism (PTE) has not yet been implicated in canine babesiosis (CB), but may also be one of the causes of the sudden dyspnoea and tachypnoea that are frequently seen in complicated CB patients. The objective of this study was to prospectively evaluate the scintigraphic pulmonary perfusion pattern in hospitalised dogs with babesiosis in an attempt to ascertain whether a scintigraphic pattern consistent with clinically relevant PTE does indeed occur in these patients. The study consisted of a normal control group of 9 mature healthy Beagle dogs (group 1) and a Babesia group with 14 dogs of a variety of breeds that were naturally infected with Babesia (group 2). Pulmonary perfusion scintigraphy was performed after making thoracic radiographs and performing a blood gas analysis in both groups. The scintigraphic images were visually inspected for changes suggestive of PTE, but not a single dog in group 2 had pleural-based, wedge-shaped perfusion defects which would have resulted in a high probability for clinically relevant PTE. The scintigraphic pulmonary perfusion pattern demonstrated was not significantly different between the 2 groups (P = 1.00).     

Molecular survey of Babesia canis in dogs in Nigeria

An epidemiological study of Babesia canis in dogs in Nigeria was performed. Four hundred blood samples collected from dogs in Nigeria were investigated using nested PCR and sequence analysis. On nested PCR screening, nine samples (2.3%) produced a band corresponding to a 698-bp fragment indicative of B. canis infection. Sequence analysis of the PCR products identified eight samples (2.0%) as B. canis rossi and the ninth (0.3%) as B. canis vogeli. This is the first report of the prevalence of B. canis rossi and B. canis vogeli in dogs in Nigeria.     

Metabolic and electrolyte disturbances in acute canine babesiosis.

Arterial blood pH, PCO2, bicarbonate, base excess/deficit, and lactate, as well as serum sodium, potassium, and chloride were measured in clinically normal dogs and in dogs with acute canine babesiosis. Metabolic acidosis developed in dogs with fatal as well as nonfatal Babesia canis infection. In the fatal group, the acidosis was uncompensated; among survivors, base deficit and blood lactate were significantly lower, and pH, PCO2, and bicarbonate values were significantly higher. Serum potassium values were significantly lower, and serum chloride values were significantly higher in dogs with acute babesiosis than in clinically normal dogs. The shock resulting from acute canine babesiosis is best viewed as anemic shock. Treatment should include an alkalizing agent, a blood transfusion, fluid therapy, and a babesicidal drug.     

Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay

Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.     

Thyroid function testing in Greyhounds.

OBJECTIVE: To evaluate thyroid function in healthy Greyhounds, compared with healthy non-Greyhound pet dogs, and to establish appropriate reference range values for Greyhounds. ANIMALS: 98 clinically normal Greyhounds and 19 clinically normal non-Greyhounds. PROCEDURES: Greyhounds were in 2 groups as follows: those receiving testosterone for estrus suppression (T-group Greyhounds) and those not receiving estrus suppressive medication (NT-group Greyhounds). Serum thyroxine (T4) and free thyroxine (fT4) concentrations were determined before and after administration of thyroid-stimulating hormone (TSH) and thyroid-releasing hormone (TRH). Basal serum canine thyroid stimulating hormone (cTSH) concentrations were determined on available stored sera. RESULTS: Basal serum T4 and fT4 concentrations were significantly lower in Greyhounds than in non-Greyhounds. Serum T4 concentrations after TSH and TRH administration were significantly lower in Greyhounds than in non-Greyhounds. Serum fT4 concentrations after TSH and TRH administration were significantly lower in NT-group than T-group Greyhounds and non-Greyhounds. Mean cTSH concentrations were not different between Greyhounds and non-Greyhounds. CONCLUSIONS AND CLINICAL RELEVANCE: Previously established canine reference range values for basal serum T4 and fT4 may not be appropriate for use in Greyhounds. Greyhound-specific reference range values for basal serum T4 and fT4 concentrations should be applied when evaluating thyroid function in Greyhounds. Basal cTSH concentrations in Greyhounds are similar to non-Greyhound pet dogs.     

Serum total thyroxine, total triiodothyronine, free thyroxine, and thyrotropin concentrations in epileptic dogs treated with anticonvulsants.

OBJECTIVE: To determine whether administration of phenobarbital, potassium bromide, or both drugs concurrently was associated with abnormalities in baseline serum total thyroxine (T4), triiodothyronine (T3), free T4, or thyrotropin (thyroid-stimulating hormone; TSH) concentrations in epileptic dogs. DESIGN: Prospective case series. ANIMALS: 78 dogs with seizure disorders that did not have any evidence of a thyroid disorder (55 treated with phenobarbital alone, 15 treated with phenobarbital and bromide, and 8 treated with bromide alone) and 150 clinically normal dogs that were not receiving any medication. PROCEDURE: Serum total T4, total T3, free T4, and TSH concentrations, as well as serum concentrations of anticonvulsant drugs, were measured in the 78 dogs with seizure disorders. Reference ranges for hormone concentrations were established on the basis of results from the 150 clinically normal dogs. RESULTS: Total and free T4 concentrations were significantly lower in dogs receiving phenobarbital (alone or with bromide), compared with concentrations in clinically normal dogs. Administration of bromide alone was not associated with low total or free T4 concentration. Total T3 and TSH concentrations did not differ among groups of dogs. CLINICAL IMPLICATIONS: Results indicate that serum total and free T4 concentrations may be low (i.e., in the range typical for dogs with hypothyroidism) in dogs treated with phenobarbital. Serum total T3 and TSH concentrations were not changed significantly in association with phenobarbital administration. Bromide treatment was not associated with any significant change in these serum thyroid hormone concentrations.