Prevalence of Toxoplasma gondii,Bartonella species and haemoplasma infection in cats in South Africa

Abstract

Vector-borne agents and Toxoplasma gondii are common in cats, with many being zoonotic. The current study investigated the prevalence of selected infectious agents in cats from Johannesburg, South Africa, for which no published data exists. Whole blood and sera were obtained from 102 cats with a variety of disease conditions. Total DNA was extracted from the blood and assayed using PCR techniques for Mycoplasma haemofelis, Candidatus M haemominutum, Candidatus M turicensis, Bartonella species, Ehrlichia species and Anaplasma species. Enzyme-linked immunosorbent assays were used to detect IgG and IgM serum antibodies to T gondii and IgG serum antibodies to Bartonella species. Associations between test results, patient characteristics and haematological values were also evaluated. Overall, 56 cats (55%) were positive in one or more of the assays. Haemoplasma DNA was amplified from 26 cats [M haemofelis: four cats (3.9%); Candidatus M haemominutum from 22 cats (21.6%)] and Bartonella species DNA was amplified from eight cats [Bartonella henselae: five cats (4.9%); Bartonella clarridgeieae: three cats (2.9%)]; DNA of Ehrlichia species or Anaplasma species were not amplified. Of the cats, 24 (23.5%) were seropositive for Bartonella IgG and 18 (17.6%) were positive for T gondii IgM (12 cats), IgG (eight cats), or both (two cats). The study concluded that Bartonella species haemoplasmas and T gondii are common in client-owned cats in the region and the diagnosis of feline vector-borne agents and T gondii is difficult without the use of specific diagnostic tests, as there are minimal patient characteristics or haematological changes that indicate infection.

Introduction

Although vector-borne disease agents and Toxoplasma gondii are common in cats, little is known about the prevalence of select agents in client-owned cats in South Africa. Fleas, ticks and mosquitoes are plentiful in South Africa and so vector-borne diseases may be common. A number of infectious agents are important, not only because of the diseases they induce in cats, but also because they may be potentially zoonotic or may be carried silently and transmitted to other cats through blood transfusion.

Cats are the definitive hosts for T gondii, which is a zoonotic disease in humans and may cause significant disease in both species. Although transmission is usually thought to occur following ingestion of undercooked meat, oocysts passed into the environment from infected cats also can be a significant source of infection for humans.¹ As cats in South Africa are commonly allowed outdoors and transport hosts for T gondii, such as rodents, are common, the cat can be an important source of infection.

Cats and fleas (Ctenocephalides felis) are considered important for transmission of Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae to humans.² Bartonella species have a worldwide distribution in domestic cats with varying antibody (5–80%) and infection (up to 50%) prevalence rates depending on geographical location, status (pet or stray) and the assays used.³ Ctenocephalides felis and B henselae are known to exist in South Africa, but prevalence rates for other C felis-associated Bartonella species are unknown.⁴

In cats, Mycoplasma haemofelis is considered a significant primary pathogen. Although Candidatus M haemominutum be a complicating factor of infection with retroviruses and other debilitating diseases, some clinically ill cats have had no identifiable cofactors.⁵ Of intermediate pathogenicity compared with the other two, Candidatus M turincensis is known to occur in some parts of Europe, the USA, South Africa, Australia and Japan.^6–8 In previous studies^5,6,9 the prevalence of Candidatus M haemominutum infection was 23.1% cats in Australia,¹⁰ 12.7% in the USA,¹¹ 16.9% in the UK¹² and 32.1% in South Africa.¹³ Mycoplasma haemofelis infection was less common: 4.1% cats in Australia,¹⁰ 4.5% in the USA,¹¹ 1.4% in the UK¹² and 6.4% in South Africa.¹³

DNA of Ehrlichia canis, E canis-like organism, and Anaplasma phagocytophilum have been amplified from the blood of cats in some countries.^9,14–16 To date, these organisms have not been reported in cats of South Africa. However, dogs in the region are known to be infected by E canis.^17,18

The purpose of this study was to determine the prevalence of infectious agents in convenience-collected blood samples from cats in the Johannesburg area. The agents were selected base on the commonality of known vectors in the region and for Bartonella species, Ehrlichia species and Anaplasma species to provide information on agents minimally studied in the region previously. Data on prevalence of these organisms in cats would provide useful information on the likelihood of infection and risks associated with blood donation, as well as the possibility of transmission to humans.

Materials and methods

Surplus ethylenediamine tetra-acetic acid (EDTA)-anticoagulated blood and serum from 102 feline samples submitted to a private veterinary laboratory (Idexx Laboratories) for routine testing was stored at -20ºC. These were convenience samples that had been obtained from cats with a variety of disease conditions; thus, specific disease conditions, clinical presentation and final diagnosis results were unavailable. The batched blood samples were then couriered on ice packs to the Center for Companion Animal Studies in the Department of Clinical Sciences, Colorado State University (Fort Collins, CO, USA).

All haematology, serum biochemistry tests and feline immunodeficiency virus (FIV)/feline leukaemia virus (FeLV) assays were done at Idexx laboratories in Johannesburg. Haematology was performed using a LaserCyte Hematology Analyzer and serum biochemistry was done using a on VetTest Chemistry Analyze (Idexx). FIV/FeLV assays were done using the Idexx Snap FIV/FeLV combo test. For each sample, the cat’s age, haematological data and any available biochemical data were recorded.

Total DNA was extracted from the blood and assayed for DNA of M haemofelis, Candidatus M haemominutum, Candidatus M turicensis, Bartonella species, Ehrlichia species and Anaplasma species using previously reported conventional polymerase chain reaction (PCR) assays.^11,16,19 Positive amplicons were sequencing to confirm the results. Enzyme-linked immunosorbent assays were used to detect IgG and IgM serum antibodies to T gondii and IgG serum antibodies to Bartonella species.^20,21 The details of the assays are provided in the referenced articles. The Center for Companion Animal Studies provides these assays commercially through the Colorado State University Veterinary Diagnostic Laboratory which is accredited (full service, all species) by the American Association of Veterinary Laboratory Diagnosticians meeting standards based on ISO17025/OIE (http://dlab.colostate.edu/). The laboratory is also a member of the National Animal Health Laboratory Network.

In addition, associations between test results, patient characteristics and haematological values were evaluated. Descriptive statistics were obtained for age, haematological parameters and biochemical parameters. Normally distributed data were analysed using analysis of variance (ANOVA) and non-normally distributed data were analysed using Kruskal-Wallis ANOVA. Statistical evaluation was carried out using NCSS programme. When a statistically significant difference was identified between the groups, the nature of the difference between individual groups was determined using the Tukey post hoc multiple comparisons test for normally distributed data and the Mann-Whitney U test for non-normally distributed data. A significant association was taken to be one with a P value ≤0.05.

Results

Results are summarised in Table 1. A total of 102 cats were sampled (50 males and 52 females) with a median age of 120 months (range 5–216 months). Of the 102 cats, 46 (45%) were negative in all tests whereas 56 (55%) were positive in one or more assays. In the negative group there were 21 males and 25 females, with a median age of 108 months (range 5–216 months). In the positive group there were 29 males and 27 females, with a median age of 120 months (range 9–216 months). There were no significant age or sex differences between the positive and negative groups. However, in the haemoplasma PCR-positive cats there were more males (18 cats) than females (eight cats). Although the group with M haemofelis DNA was younger, this was not statistically significant.

Table 1

Serology and PCR results for Toxoplasma species and Bartonella species, and Bartonella species and mycoplasmosis PCR from 102 blood samples

	Number (%)
Total number	102
Negative results	46 (45)
Positive results	56 (55)
Toxoplasma IgM	12 (11.7)
Toxoplasma IgG	8 (7.8)
Toxoplasma M and G	2 (1.9)
Toxoplasma M or G	18 (17.6)
Bartonella IgG	24 (23.5)
Bartonella PCR	8 (7.8)
Bartonella IgG and PCR	3 (2.9)
Bartonella IgG positive/PCR negative	21 (20.6)
Bartonella IgG negative/PCR positive	5 (4.9)
Any haemoplasma	26 (25.5)
Mycoplasma haemofelis	4 (3.9)
Candidatus M haemominutum	22 (21.6)
Mycoplasma haemofelis and Candidatus M haemominutum	0 (0)
Any Bartonella or haemoplasma PCR-positive	32 (31.4)
Bartonella and haemoplasma PCR-positive	2 (1.9)

While haemoplasma DNA was amplified from 26 cats [M haemofelis: four cats (3.9%); Candidatus M haemominutum: 22 cats (21.6%)] and Bartonella species. DNA was amplified from eight cats [B henselae: five cats (4.9%); B clarridgeieae: three cats (2.9%)], DNA of Ehrlichia species, Anaplasma species, B koehlerae or Candidatus M turicensis were not amplified.

Of the cats, 24 (23.5%) were seropositive for Bartonella species IgG and 18 (17.6%) were positive for T gondii IgM (12 cats, 11.8%), IgG (eight cats, 7.8%) or both (two cats, 1.9%). Of the cats with Bartonella species IgG in serum, eight were concurrently PCR-positive. The median T gondii IgM antibody titre was 1:64 (range 64–128), the median T gondii IgG antibody titre was 1:128 (range 64–512) and the median Bartonella species IgG antibody titre was 1:128 (range 64–512).

Haematological parameters are summarised in Table 2. In the negative group the median haematocrit was 36% (range 6–54), the median white cell count was 9.84 × 10⁹/l (range 1.49–34.00) and the median thrombocyte count was 148 × 10⁹/l, range 0–918. In the T gondii IgM group median haematocrit was 34% (range 23–37), median white cell count was 14.9 × 10⁹/l (range 5.01–44.00) and the median thrombocyte count was 130 × 10⁹/l (range 24–794). In the T gondii IgG group median haematocrit was 36% (range 28–43), the median white cell count was 7.47 × 10⁹/l (range 3.01–44.00) and the median thrombocyte count was 209 × 10⁹/l (range 40–794). In the Bartonella species IgG group median haematocrit was 32% (range 10–44), the median white cell count was 7.87 × 10⁹/l (range 2.75–27.00) and the median thrombocyte count was 168 × 10⁹/l (range 35–754). In the Bartonella species PCR-positive group median haematocrit was 35% (range 24–43), the median white cell count 7.95 × 10⁹/l (range 4.4–21.0) and the median thrombocyte count was 224 × 10⁹/l (range 13–436). In the M haemofelis PCR-positive group median haematocrit was 12% (range 11–42), the median white cell count was 4.13 × 10⁹/l (range 2.29–17.50) and the median thrombocyte count was 7 × 10⁹/l (range 15–177). In the Candidatus M haemominutum PCR-positive group median haematocrit was 35% (range 16–45), the median white cell count was 10.65 × 10⁹/l (range 3.01–44.00) and median thrombocyte count was 135 × 10⁹/l (range 38–794).

Table 2

Haematological and serum biochemistry results from 102 cats screened for Toxoplasma gondii, Bartonella species or haemoplasma infection

Parameter		Negative	Tox IgM	Tox IgG	Bart Ig	Bart PCR	Haem	M Hf	M Hm
Haematocrit (%)	Median	36	34	36	32	35	35	12	35
Haematocrit (%)	Range	6–54	23–37	28–43	10–44	24–43	11–45	11–42	16–45
White cell count (×10⁹/l)	Median	9.84	14.9	7.47	7.87	7.95	10.4	4.13	10.65
White cell count (×10⁹/l)	Range	1.49–34.00	5.01–44.00	3.01–44.00	2.75–27.00	4.4–21.0	2.29–44.00	2.29–17.50	3.01–44.00
Thrombocytes (×10⁹/l)	Median	148	130	209	168	224	132	7	135
Thrombocytes (×10⁹/l)	Range	0–918	24–794	40–794	35–754	13–436	5–794	5–177	38–794
FeLV	Total	1	1	2	2	1	1	0	1
FIV	Total	0	0	1	0	0	5	1	4
ALT (U/l)	Median	59	62	75	56	110	67	102	58
	Range	15–388	25–1515	38–143	31–294	49–147	38–147	98–129	38–147
ALP (U/l)	Median	31	58	40	31	26	54	44	35
	Range	10–215	10–700	26–147	14–300	10–147	18–203	10–198	18–203
Total serum proteins (g/l)	Median	79	66	68	67	68	74	72	75
Total serum proteins (g/l)	Range	45–97	55–77	56–83	54–85	59–79	56–83	59–76	56–83
Albumin (g/l)	Median	32	30	30	30	32	31	34	31
	Range	15–50	24–35	24–37	23–42	25–38	24–31	24–36	24–41
Globulin (g/l)	Median	38	37	36	38	32	40	36	41
	Range	30–59	31–47	32–56	28–50	24–50	31–56	35–42	31–56
Urea (mmol/l)	Median	8.4	8.7	8.3	9.2	8.8	8.4	7.4	8.7
	Range	4.3–67.4	3.8–45.00	6.7–23.0	4.5–80.0	6.7–19.7	5.5–45.0	6–7.6	5.5–45.0
Creatinine (µmol/l)	Median	118	132	108	113	171	120	88	122
Creatinine (µmol/l)	Range	40–935	71–516	92–197	86–1542	77–241	54–516	770–160	54–516

FeLV = feline leukaemia virus, FIV = feline immunodeficiency virus, ALT = alanine aminotransferase, ALP = alkaline phosphatase, Tox = Toxoplasma gondii, Bart = Bartonella species, Haem = any haemoplasma, M Hf = Mycoplasma haemofelis, M Hm = Candidatus M haemominutum

The only group that showed a statistically significant difference within haematological parameters was the M haemofelis PCR-positive group that had a statistically lower haematocrit, white cell count and thrombocyte count when compared with the rest of the groups. The rate of FIV/FeLV infection in all groups was low (Table 2).

The only biochemical parameters (Table 2) that were statistically different among groups was an association of increased ALT and ALP activity in cats with a positive T gondii IgM. There were no statistical significant associations amongst infectious agent test results and total serum protein, albumin, globulins, urea or creatinine.

DNA of Ehrlichia species or Anaplasma species were not amplified in any of the samples.

Discussion

DNA of M haemofelis, Candidatus M haemominutum, B henselae and B clarridgeieae was amplified from the blood of client-owned cats in the Johannesburg area of South Africa. While the travel history for these cats was unknown, it was believed that the majority of the cats originated in the region and so we believe these results suggest the infections should be on the differential diagnoses lists from appropriate problems for cats in the area.

The prevalence of M haemofelis and Candidatus M haemominutum was very similar to that reported in a previous study of cats in South Africa, where the prevalence rates for Candidatus M haemominutum and for M haemofelis were 32% and 6.4%, respectively.¹³ The prevalence of haemoplasmas was higher in male than in female cats, and in younger cats, which is in accordance with previous studies.^1,7,12 None of the cats tested positive for infection with Candidatus M turicensis, which is in contrast to a prevalence of 26% reported in a previous study.⁷ In most studies around the world, Candidatus M turicensis has the lowest prevalence rate and so the failure to amplify the DNA of this organism could possibly reflect the relatively low sample size. Alternately, in the conventional PCR used, it is possible that the other haemoplasmas were amplified preferentially, leading to falsely negative Candidatus M turicensis results if only low levels of DNA were present.

The primary statistical difference between test results, patient characteristics and haematological values was seen in the M haemofelis-positive cats where all affected animals were young males and showed severe anaemia and thrombocytopenia with more positive males than females. These findings are similar to those from a previous study.¹³

Bartonella henselae infection of HIV-infected humans was shown to be 11% in one study in South Africa; therefore, the infection is relatively common in high-risk population groups.²² To our knowledge, B henselae bacteraemia has only been documented in one cat housed in South Africa in previous studies.²³ While the prevalence of Bartonella species bacteraemia was relatively low (7.8%) in the cats described here, this is the first study to document B clarridgeiae infection of cats in South Africa. Overall, Bartonella species bacteraemia rates vary considerably among studies and generally relate to the risk of flea exposure.^1,2,21,24,25

Seroprevalences of 21% and 24% Bartonella species antibodies have previously been reported in cats from South Africa and Zimbabwe, respectively,⁴ which is similar to the prevalence in this current study. This prevalence falls in the range of what has been reported previously in cats of other countries: 26.5% in Ireland,¹ 29.6% in Spain,²⁴ 50% in the Netherlands,²⁵ 54.6% in south-eastern USA, 47.4% in Hawaii and 5% in Alaska.²⁶ In this study there were 24 Bartonella species-seropositive cats, but only eight PCR-positive cats. Serum antibodies against Bartonella species can be present for months to years after bacteraemia has resolved; thus, a positive antibody titre test does not prove current infection but merely prior exposure. This would explain the discrepancy between the seroprevalence of B henselae and/or B clarridgeiae detected by PCR and the antibody prevalence demonstrated by the serological test in this study. A previous South African study showed a seroprevalence of 21% but only a 3% culture-positive result.⁴

Although evidence of infection has been recently reported in cats elsewhere in the world,^9,14–16 DNA from Ehrlichia species and Anaplasma species was not amplified from cats in the present study, which would suggest that the agents are not present in the region of study, the prevalence is very low, the organisms were not in the blood or the organisms were present at levels below the detectable limits of the assays. However, before concluding that these infectious agents do not occur in South Africa, larger studies including a greater number of cats and from different geographical areas within South Africa is warranted, especially as they occur in other species and potential vectors are present.

The primary statistical difference between test results, patient characteristics and biochemical values was seen in the cats positive for T gondii IgM that showed elevated liver enzyme activities. There were, however, no age or sex differences. Elevated liver enzyme activity is most likely from underlying hepatic infection, which has been well described in feline clinical toxoplasmosis.²⁷ The prevalence of T gondii antibodies in this study is similar to those reported in previous studies.^20,28–31 The high prevalence of T gondii antibodies in this study is most likely a reflection of the free access that the cats had to the outdoors and thus to the increased likelihood of hunting and ingestion of intermediate hosts.³²

We conclude that Bartonella species, haemoplasmas and T gondii are common in client-owned cats in the study area region. The results also emphasise that the diagnosis of feline vector-borne agents and T gondii is difficult without the use of specific diagnostic tests as there are no obvious patient characteristics, haematological or biochemical changes that indicated infection. Further study of associations between these infections and clinical syndromes is, however, indicated. With the prevalence of potentially zoonotic agents in cats described in this study appropriate care with the handling of cat faeces and regular flea control remain important prevention strategies.

Footnotes

Acknowledgements

The authors would like to thank Nicol Schneider of Idexx Laboratories, Johannesburg, South Africa for all her assistance with the samples.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors declare that there is no conflict of interest.

Accepted: 30 May 2012

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